• Don't forget to check out Neurotransmitter.net's research index for a list of dozens of other resources created by Shawn Thomas.
• The abstracts are retrieved from PubMed Central and PubMed on a daily basis.
• Free full text articles are also available for the following subjects: biology, neuroscience, genetics and proteomics, psychiatry, pharmacology, endocrinology, microbiology, cancer, biotechnology, informatics, and miscellaneous.
  
• You can jump directly to articles from Molecular Biology of the Cell, Molecular and Cellular Biology, Journal of Cell Science, Journal of Cellular and Molecular Medicine, Molecules and Cells, Cell Research, Cellular & Molecular Biology Letters, BMC Cell Biology, Cell Communication and Signaling, Eukaryotic Cell, The Journal of Biological Chemistry, Bioscience, Biotechnology, Biochemistry, Experimental & Molecular Medicine, BMC Biochemistry, BMC Chemical Biology, and BMC Molecular Biology.
• You can view earlier articles from these journals as well: skip 0, skip 30, skip 60, or skip 90 of the most recent articles.
• Confused by the terminology? Try searching The On-Line Medical Dictionary (OMD).

Web www.neurotransmitter.net

---

Recent Articles in Molecular Biology of the Cell

---

Kazemi S, Mounir Z, Baltzis D, Raven JF, Wang S, Krishnamoorthy JL, Pluquet O, Pelletier J, Koromilas AE
A novel function of eIF2alpha kinases as inducers of the phosphoinositide-3 kinase signaling pathway.
Mol Biol Cell. 2007 Sep;18(9):3635-44.
Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the alpha subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2alpha kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR(-/-) or PERK(-/-) mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2alpha kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2alpha phosphorylation as demonstrated by the inactivation of endogenous eIF2alpha by small interfering RNA or utilization of MEFs bearing the eIF2alpha Ser51Ala mutation. Our data reveal a novel property of eIF2alpha kinases as activators of PI3K signaling and cell survival. [Abstract/Link to Full Text]

Lian S, Fritzler MJ, Katz J, Hamazaki T, Terada N, Satoh M, Chan EK
Small interfering RNA-mediated silencing induces target-dependent assembly of GW/P bodies.
Mol Biol Cell. 2007 Sep;18(9):3375-87.
Gene silencing using small interfering RNA (siRNA) is a valuable laboratory tool and a promising approach to therapeutics for a variety of human diseases. Recently, RNA interference (RNAi) has been linked to cytoplasmic GW bodies (GWB). However, the correlation between RNAi and the formation of GWB, also known as mammalian processing bodies, remains unclear. In this report, we show that transfection of functional siRNA induced larger and greater numbers of GWB. This siRNA-induced increase of GWB depended on the endogenous expression of the target mRNA. Knockdown of GW182 or Ago2 demonstrated that the siRNA-induced increase of GWB required these two proteins and correlated with RNAi. Furthermore, knockdown of rck/p54 or LSm1 did not prevent the reassembly of GWB that were induced by and correlated with siRNA-mediated RNA silencing. We propose that RNAi is a key regulatory mechanism for the assembly of GWB, and in some cases, GWB may serve as markers for RNAi in mammalian cells. [Abstract/Link to Full Text]

Bhangoo MK, Tzankov S, Fan AC, Dejgaard K, Thomas DY, Young JC
Multiple 40-kDa heat-shock protein chaperones function in Tom70-dependent mitochondrial import.
Mol Biol Cell. 2007 Sep;18(9):3414-28.
Mitochondrial preproteins that are imported via the translocase of the mitochondrial outer membrane (Tom)70 receptor are complexed with cytosolic chaperones before targeting to the mitochondrial outer membrane. The adenine nucleotide transporter (ANT) follows this pathway, and its purified mature form is identical to the preprotein. Purified ANT was reconstituted with chaperones in reticulocyte lysate, and bound proteins were identified by mass spectrometry. In addition to 70-kDa heat-shock cognate protein (Hsc70) and 90-kDa heat-shock protein (Hsp90), a specific subset of cochaperones were found, but no mitochondria-specific targeting factors were found. Interestingly, three different Hsp40-related J-domain proteins were identified: DJA1, DJA2, and DJA4. The DJAs bound preproteins to different extents through their C-terminal regions. DJA dominant-negative mutants lacking the N-terminal J-domains impaired mitochondrial import. The mutants blocked the binding of Hsc70 to preprotein, but with varying efficiency. The DJAs also showed significant differences in activation of the Hsc70 ATPase and Hsc70-dependent protein refolding. In HeLa cells, the DJAs increased new protein folding and mitochondrial import, although to different extents. No single DJA was superior to the others in all aspects, but each had a profile of partial specialization. The Hsp90 cochaperones p23 and Aha1 also regulated Hsp90-preprotein interactions. We suggest that multiple cochaperones with similar yet partially specialized properties cooperate in optimal chaperone-preprotein complexes. [Abstract/Link to Full Text]

Ye Y, Fujii M, Hirata A, Kawamukai M, Shimoda C, Nakamura T
Geranylgeranyl diphosphate synthase in fission yeast is a heteromer of farnesyl diphosphate synthase (FPS), Fps1, and an FPS-like protein, Spo9, essential for sporulation.
Mol Biol Cell. 2007 Sep;18(9):3568-81.
Both farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) are key enzymes in the synthesis of various isoprenoid-containing compounds and proteins. Here, we describe two novel Schizosaccharomyces pombe genes, fps1(+) and spo9(+), whose products are similar to FPS in primary structure, but whose functions differ from one another. Fps1 is essential for vegetative growth, whereas, a spo9 null mutant exhibits temperature-sensitive growth. Expression of fps1(+), but not spo9(+), suppresses the lethality of a Saccharomyces cerevisiae FPS-deficient mutant and also restores ubiquinone synthesis in an Escherichia coli ispA mutant, which lacks FPS activity, indicating that S. pombe Fps1 in fact functions as an FPS. In contrast to a typical FPS gene, no apparent GGPS homologues have been found in the S. pombe genome. Interestingly, although neither fps1(+) nor spo9(+) expression alone in E. coli confers clear GGPS activity, coexpression of both genes induces such activity. Moreover, the GGPS activity is significantly reduced in the spo9 mutant. In addition, the spo9 mutation perturbs the membrane association of a geranylgeranylated protein, but not that of a farnesylated protein. Yeast two-hybrid and coimmunoprecipitation analyses indicate that Fps1 and Spo9 physically interact. Thus, neither Fps1 nor Spo9 alone functions as a GGPS, but the two proteins together form a complex with GGPS activity. Because spo9 was originally identified as a sporulation-deficient mutant, we show here that expansion of the forespore membrane is severely inhibited in spo9Delta cells. Electron microscopy revealed significant accumulation membrane vesicles in spo9Delta cells. We suggest that lack of GGPS activity in a spo9 mutant results in impaired protein prenylation in certain proteins responsible for secretory function, thereby inhibiting forespore membrane formation. [Abstract/Link to Full Text]

Deraison C, Bonnart C, Lopez F, Besson C, Robinson R, Jayakumar A, Wagberg F, Brattsand M, Hachem JP, Leonardsson G, Hovnanian A
LEKTI fragments specifically inhibit KLK5, KLK7, and KLK14 and control desquamation through a pH-dependent interaction.
Mol Biol Cell. 2007 Sep;18(9):3607-19.
LEKTI is a 15-domain serine proteinase inhibitor whose defective expression underlies the severe autosomal recessive ichthyosiform skin disease, Netherton syndrome. Here, we show that LEKTI is produced as a precursor rapidly cleaved by furin, generating a variety of single or multidomain LEKTI fragments secreted in cultured keratinocytes and in the epidermis. The identity of these biological fragments (D1, D5, D6, D8-D11, and D9-D15) was inferred from biochemical analysis, using a panel of LEKTI antibodies. The functional inhibitory capacity of each fragment was tested on a panel of serine proteases. All LEKTI fragments, except D1, showed specific and differential inhibition of human kallikreins 5, 7, and 14. The strongest inhibition was observed with D8-D11, toward KLK5. Kinetics analysis revealed that this interaction is rapid and irreversible, reflecting an extremely tight binding complex. We demonstrated that pH variations govern this interaction, leading to the release of active KLK5 from the complex at acidic pH. These results identify KLK5, a key actor of the desquamation process, as the major target of LEKTI. They disclose a new mechanism of skin homeostasis by which the epidermal pH gradient allows precisely regulated KLK5 activity and corneodesmosomal cleavage in the most superficial layers of the stratum corneum. [Abstract/Link to Full Text]

Mardones GA, Burgos PV, Brooks DA, Parkinson-Lawrence E, Mattera R, Bonifacino JS
The trans-Golgi network accessory protein p56 promotes long-range movement of GGA/clathrin-containing transport carriers and lysosomal enzyme sorting.
Mol Biol Cell. 2007 Sep;18(9):3486-501.
The sorting of acid hydrolase precursors at the trans-Golgi network (TGN) is mediated by binding to mannose 6-phosphate receptors (MPRs) and subsequent capture of the hydrolase-MPR complexes into clathrin-coated vesicles or transport carriers (TCs) destined for delivery to endosomes. This capture depends on the function of three monomeric clathrin adaptors named GGAs. The GGAs comprise a C-terminal "ear" domain that binds a specific set of accessory proteins. Herein we show that one of these accessory proteins, p56, colocalizes and physically interacts with the three GGAs at the TGN. Moreover, overexpression of the GGAs enhances the association of p56 with the TGN, and RNA interference (RNAi)-mediated depletion of the GGAs decreases the TGN association and total levels of p56. RNAi-mediated depletion of p56 or the GGAs causes various degrees of missorting of the precursor of the acid hydrolase, cathepsin D. In the case of p56 depletion, this missorting correlates with decreased mobility of GGA-containing TCs. Transfection with an RNAi-resistant p56 construct, but not with a p56 construct lacking the GGA-ear-interacting motif, restores the mobility of the TCs. We conclude that p56 tightly cooperates with the GGAs in the sorting of cathepsin D to lysosomes, probably by enabling the movement of GGA-containing TCs. [Abstract/Link to Full Text]

Kloepper TH, Kienle CN, Fasshauer D
An elaborate classification of SNARE proteins sheds light on the conservation of the eukaryotic endomembrane system.
Mol Biol Cell. 2007 Sep;18(9):3463-71.
Proteins of the SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) family are essential for the fusion of transport vesicles with an acceptor membrane. Despite considerable sequence divergence, their mechanism of action is conserved: heterologous sets assemble into membrane-bridging SNARE complexes, in effect driving membrane fusion. Within the cell, distinct functional SNARE units are involved in different trafficking steps. These functional units are conserved across species and probably reflect the conservation of the particular transport step. Here, we have systematically analyzed SNARE sequences from 145 different species and have established a highly accurate classification for all SNARE proteins. Principally, all SNAREs split into four basic types, reflecting their position in the four-helix bundle complex. Among these four basic types, we established 20 SNARE subclasses that probably represent the original repertoire of a eukaryotic cenancestor. This repertoire has been modulated independently in different lines of organisms. Our data are in line with the notion that the ur-eukaryotic cell was already equipped with the various compartments found in contemporary cells. Possibly, the development of these compartments is closely intertwined with episodes of duplication and divergence of a prototypic SNARE unit. [Abstract/Link to Full Text]

Samarin SN, Ivanov AI, Flatau G, Parkos CA, Nusrat A
Rho/Rho-associated kinase-II signaling mediates disassembly of epithelial apical junctions.
Mol Biol Cell. 2007 Sep;18(9):3429-39.
Apical junctional complex (AJC) plays a vital role in regulation of epithelial barrier function. Disassembly of the AJC is observed in diverse physiological and pathological states; however, mechanisms governing this process are not well understood. We previously reported that the AJC disassembly is driven by the formation of apical contractile acto-myosin rings. In the present study, we analyzed the signaling pathways regulating acto-myosin-dependent disruption of AJC by using a model of extracellular calcium depletion. Pharmacological inhibition analysis revealed a critical role of Rho-associated kinase (ROCK) in AJC disassembly in calcium-depleted epithelial cells. Furthermore, small interfering RNA (siRNA)-mediated knockdown of ROCK-II, but not ROCK-I, attenuated the disruption of the AJC. Interestingly, AJC disassembly was not dependent on myosin light chain kinase and myosin phosphatase. Calcium depletion resulted in activation of Rho GTPase and transient colocalization of Rho with internalized AJC proteins. Pharmacological inhibition of Rho prevented AJC disassembly. Additionally, Rho guanine nucleotide exchange factor (GEF)-H1 translocated to contractile F-actin rings after calcium depletion, and siRNA-mediated depletion of GEF-H1 inhibited AJC disassembly. Thus, our findings demonstrate a central role of the GEF-H1/Rho/ROCK-II signaling pathway in the disassembly of AJC in epithelial cells. [Abstract/Link to Full Text]

Lubben NB, Sahlender DA, Motley AM, Lehner PJ, Benaroch P, Robinson MS
HIV-1 Nef-induced down-regulation of MHC class I requires AP-1 and clathrin but not PACS-1 and is impeded by AP-2.
Mol Biol Cell. 2007 Sep;18(9):3351-65.
Major histocompatibility complex class I is down-regulated from the surface of human immunodeficiency virus (HIV)-1-infected cells by Nef, a virally encoded protein that is thought to reroute MHC-I to the trans-Golgi network (TGN) in a phosphofurin acidic cluster sorting protein (PACS) 1, adaptor protein (AP)-1, and clathrin-dependent manner. More recently, an alternative model has been proposed, in which Nef uses AP-1 to direct MHC-I to endosomes and lysosomes. Here, we show that knocking down either AP-1 or clathrin with small interfering RNA inhibits the down-regulation of HLA-A2 (an MHC-I isotype) by Nef in HeLa cells. However, knocking down PACS-1 has no effect, not only on Nef-induced down-regulation of HLA-A2 but also on the localization of other proteins containing acidic cluster motifs. Surprisingly, knocking down AP-2 actually enhances Nef activity. Immuno-electron microscopy labeling of Nef-expressing cells indicates that HLA-A2 is rerouted not to the TGN, but to endosomes. In AP-2-depleted cells, more of the HLA-A2 localizes to the inner vesicles of multivesicular bodies. We propose that depleting AP-2 potentiates Nef activity by altering the membrane composition and dynamics of endosomes and causing increased delivery of HLA-A2 to a prelysosomal compartment. [Abstract/Link to Full Text]

Hu J, Wittekind SG, Barr MM
STAM and Hrs down-regulate ciliary TRP receptors.
Mol Biol Cell. 2007 Sep;18(9):3277-89.
Cilia are endowed with membrane receptors, channels, and signaling components whose localization and function must be tightly controlled. In primary cilia of mammalian kidney epithelia and sensory cilia of Caenorhabditis elegans neurons, polycystin-1 (PC1) and transient receptor polycystin-2 channel (TRPP2 or PC2), function together as a mechanosensory receptor-channel complex. Despite the importance of the polycystins in sensory transduction, the mechanisms that regulate polycystin activity and localization, or ciliary membrane receptors in general, remain poorly understood. We demonstrate that signal transduction adaptor molecule STAM-1A interacts with C. elegans LOV-1 (PC1), and that STAM functions with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) on early endosomes to direct the LOV-1-PKD-2 complex for lysosomal degradation. In a stam-1 mutant, both LOV-1 and PKD-2 improperly accumulate at the ciliary base. Conversely, overexpression of STAM or Hrs promotes the removal of PKD-2 from cilia, culminating in sensory behavioral defects. These data reveal that the STAM-Hrs complex, which down-regulates ligand-activated growth factor receptors from the cell surface of yeast and mammalian cells, also regulates the localization and signaling of a ciliary PC1 receptor-TRPP2 complex. [Abstract/Link to Full Text]

Kaufman BA, Durisic N, Mativetsky JM, Costantino S, Hancock MA, Grutter P, Shoubridge EA
The mitochondrial transcription factor TFAM coordinates the assembly of multiple DNA molecules into nucleoid-like structures.
Mol Biol Cell. 2007 Sep;18(9):3225-36.
Packaging DNA into condensed structures is integral to the transmission of genomes. The mammalian mitochondrial genome (mtDNA) is a high copy, maternally inherited genome in which mutations cause a variety of multisystem disorders. In all eukaryotic cells, multiple mtDNAs are packaged with protein into spheroid bodies called nucleoids, which are the fundamental units of mtDNA segregation. The mechanism of nucleoid formation, however, remains unknown. Here, we show that the mitochondrial transcription factor TFAM, an abundant and highly conserved High Mobility Group box protein, binds DNA cooperatively with nanomolar affinity as a homodimer and that it is capable of coordinating and fully compacting several DNA molecules together to form spheroid structures. We use noncontact atomic force microscopy, which achieves near cryo-electron microscope resolution, to reveal the structural details of protein-DNA compaction intermediates. The formation of these complexes involves the bending of the DNA backbone, and DNA loop formation, followed by the filling in of proximal available DNA sites until the DNA is compacted. These results indicate that TFAM alone is sufficient to organize mitochondrial chromatin and provide a mechanism for nucleoid formation. [Abstract/Link to Full Text]

Krappmann AB, Taheri N, Heinrich M, Mösch HU
Distinct domains of yeast cortical tag proteins Bud8p and Bud9p confer polar localization and functionality.
Mol Biol Cell. 2007 Sep;18(9):3323-39.
In Saccharomyces cerevisiae, diploid yeast cells follow a bipolar budding program, which depends on the two transmembrane glycoproteins Bud8p and Bud9p that potentially act as cortical tags to mark the cell poles. Here, we have performed systematic structure-function analyses of Bud8p and Bud9p to identify functional domains. We find that polar transport of Bud8p and Bud9p does not depend on N-terminal sequences but instead on sequences in the median part of the proteins and on the C-terminal parts that contain the transmembrane domains. We show that the guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange factor Bud5p, which is essential for bud site selection and physically interacts with Bud8p, also interacts with Bud9p. Regions of Bud8p and Bud9p predicted to reside in the extracellular space are likely to confer interaction with the N-terminal region of Bud5p, implicating indirect interactions between the cortical tags and the GDP/GTP exchange factor. Finally, we have identified regions of Bud8p and Bud9p that are required for interaction with the cortical tag protein Rax1p. In summary, our study suggests that Bud8p and Bud9p carry distinct domains for delivery of the proteins to the cell poles, for interaction with the general budding machinery and for association with other cortical tag proteins. [Abstract/Link to Full Text]

Borthwick LA, McGaw J, Conner G, Taylor CJ, Gerke V, Mehta A, Robson L, Muimo R
The formation of the cAMP/protein kinase A-dependent annexin 2-S100A10 complex with cystic fibrosis conductance regulator protein (CFTR) regulates CFTR channel function.
Mol Biol Cell. 2007 Sep;18(9):3388-97.
Cystic fibrosis results from mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/protein kinase A (PKA) and ATP-regulated Cl(-) channel. CFTR is increasingly recognized as a component of multiprotein complexes and although several inhibitory proteins to CFTR have been identified, protein complexes that stimulate CFTR function remain less well characterized. We report that annexin 2 (anx 2)-S100A10 forms a functional cAMP/PKA/calcineurin (CaN)-dependent complex with CFTR. Cell stimulation with forskolin/3-isobutyl-1-methylxanthine significantly increases the amount of anx 2-S100A10 that reciprocally coimmunoprecipitates with cell surface CFTR and calyculin A. Preinhibition with PKA or CaN inhibitors attenuates the interaction. Furthermore, we find that the acetylated peptide (STVHEILCKLSLEG, Ac1-14), but not the nonacetylated equivalent N1-14, corresponding to the S100A10 binding site on anx 2, disrupts the anx 2-S100A10/CFTR complex. Analysis of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and CFTR(inh172)-sensitive currents, taken as indication of the outwardly rectifying Cl(-) channels (ORCC) and CFTR-mediated currents, respectively, showed that Ac1-14, but not N1-14, inhibits both the cAMP/PKA-dependent ORCC and CFTR activities. CaN inhibitors (cypermethrin, cyclosporin A) discriminated between ORCC/CFTR by inhibiting the CFTR(inh172)-, but not the DIDS-sensitive currents, by >70%. Furthermore, peptide Ac1-14 inhibited acetylcholine-induced short-circuit current measured across a sheet of intact intestinal biopsy. Our data suggests that the anx 2-S100A10/CFTR complex is important for CFTR function across epithelia. [Abstract/Link to Full Text]

Hsueh YP, Xue C, Heitman J
G protein signaling governing cell fate decisions involves opposing Galpha subunits in Cryptococcus neoformans.
Mol Biol Cell. 2007 Sep;18(9):3237-49.
Communication between cells and their environments is often mediated by G protein-coupled receptors and cognate G proteins. In fungi, one such signaling cascade is the mating pathway triggered by pheromone/pheromone receptor recognition. Unlike Saccharomyces cerevisiae, which expresses two Galpha subunits, most filamentous ascomycetes and basidiomycetes have three Galpha subunits. Previous studies have defined the Galpha subunit acting upstream of the cAMP-protein kinase A pathway, but it has been unclear which Galpha subunit is coupled to the pheromone receptor and response pathway. Here we report that in the pathogenic basidiomycetous yeast Cryptococcus neoformans, two Galpha subunits (Gpa2, Gpa3) sense pheromone and govern mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. By contrast, deletion of GPA3 (but not GPA2) constitutively activates pheromone response and filamentation. Expression of GPA2 and GPA3 is differentially regulated: GPA3 expression is induced by nutrient-limitation, whereas GPA2 is induced during mating. Based on the phenotype of dominant active alleles, Gpa2 and Gpa3 signal in opposition: Gpa2 promotes mating, whereas Gpa3 inhibits. The incorporation of an additional Galpha into the regulatory circuit enabled increased signaling complexity and facilitated cell fate decisions involving choice between yeast growth and filamentous asexual/sexual development. [Abstract/Link to Full Text]

Riggs B, Fasulo B, Royou A, Mische S, Cao J, Hays TS, Sullivan W
The concentration of Nuf, a Rab11 effector, at the microtubule-organizing center is cell cycle regulated, dynein-dependent, and coincides with furrow formation.
Mol Biol Cell. 2007 Sep;18(9):3313-22.
Animal cytokinesis relies on membrane addition as well as acto-myosin-based constriction. Recycling endosome (RE)-derived vesicles are a key source of this membrane. Rab11, a small GTPase associated with the RE and involved in vesicle targeting, is required for elongation of the cytokinetic furrow. In the early Drosophila embryo, Nuclear-fallout (Nuf), a Rab11 effector, promotes vesicle-mediated membrane delivery and actin organization at the invaginating furrow. Although Rab11 maintains a relatively constant localization at the microtubule-organizing center (MTOC), Nuf is present at the MTOC only during the phases of the cell cycle in which furrow invagination occurs. We demonstrate that Nuf protein levels remain relatively constant throughout the cell cycle, suggesting that Nuf is undergoing cycles of concentration and dispersion from the MTOC. Microtubules, but not microfilaments, are required for proper MTOC localization of Nuf and Rab11. The MTOC localization of Nuf also relies on Dynein. Immunoprecipitation experiments demonstrate that Nuf and Dynein physically interact. In accord with these findings, and in contrast to previous reports, we demonstrate that microtubules are required for proper metaphase furrow formation. We propose that the cell cycle-regulated, Dynein-dependent recruitment of Nuf to the MTOC influences the timing of RE-based vesicle delivery to the invaginating furrows. [Abstract/Link to Full Text]

Chen Q, Lakshmikanth GS, Spudich JA, De Lozanne A
The localization of inner centromeric protein (INCENP) at the cleavage furrow is dependent on Kif12 and involves interactions of the N terminus of INCENP with the actin cytoskeleton.
Mol Biol Cell. 2007 Sep;18(9):3366-74.
The inner centromeric protein (INCENP) and other chromosomal passenger proteins are known to localize on the cleavage furrow and to play a role in cytokinesis. However, it is not known how INCENP localizes on the furrow or whether this localization is separable from that at the midbody. Here, we show that the association of Dictyostelium INCENP (DdINCENP) with the cortex of the cleavage furrow involves interactions with the actin cytoskeleton and depends on the presence of the kinesin-6-related protein Kif12. We found that Kif12 is found on the central spindle and the cleavage furrow during cytokinesis. Kif12 is not required for the redistribution of DdINCENP from centromeres to the central spindle. However, in the absence of Kif12, DdINCENP fails to localize on the cleavage furrow. Domain analysis indicates that the N terminus of DdINCENP is necessary and sufficient for furrow localization and that it binds directly to the actin cytoskeleton. Our data suggest that INCENP moves from the central spindle to the furrow of a dividing cell by a Kif12-dependent pathway. Once INCENP reaches the equatorial cortex, it associates with the actin cytoskeleton where it then concentrates toward the end of cytokinesis. [Abstract/Link to Full Text]

Beaulieu N, Zahedi B, Goulding RE, Tazmini G, Anthony KV, Omeis SL, de Jong DR, Kay RJ
Regulation of RasGRP1 by B cell antigen receptor requires cooperativity between three domains controlling translocation to the plasma membrane.
Mol Biol Cell. 2007 Aug;18(8):3156-68.
RasGRP1 is a Ras-activating exchange factor that is positively regulated by translocation to membranes. RasGRP1 contains a diacylglycerol-binding C1 domain, and it has been assumed that this domain is entirely responsible for RasGRP1 translocation. We found that the C1 domain can contribute to plasma membrane-targeted translocation of RasGRP1 induced by ligation of the B cell antigen receptor (BCR). However, this reflects cooperativity of the C1 domain with the previously unrecognized Plasma membrane Targeter (PT) domain, which is sufficient and essential for plasma membrane targeting of RasGRP1. The adjacent suppressor of PT (SuPT) domain attenuates the plasma membrane-targeting activity of the PT domain, thus preventing constitutive plasma membrane localization of RasGRP1. By binding to diacylglycerol generated by BCR-coupled phospholipase Cgamma2, the C1 domain counteracts the SuPT domain and enables efficient RasGRP1 translocation to the plasma membrane. In fibroblasts, the PT domain is inactive as a plasma membrane targeter, and the C1 domain specifies constitutive targeting of RasGRP1 to internal membranes where it can be activated and trigger oncogenic transformation. Selective use of the C1, PT, and SuPT domains may contribute to the differential targeting of RasGRP1 to the plasma membrane versus internal membranes, which has been observed in lymphocytes and other cell types. [Abstract/Link to Full Text]

Dulyaninova NG, House RP, Betapudi V, Bresnick AR
Myosin-IIA heavy-chain phosphorylation regulates the motility of MDA-MB-231 carcinoma cells.
Mol Biol Cell. 2007 Aug;18(8):3144-55.
In mammalian nonmuscle cells, the mechanisms controlling the localized formation of myosin-II filaments are not well defined. To investigate the mechanisms mediating filament assembly and disassembly during generalized motility and chemotaxis, we examined the EGF-dependent phosphorylation of the myosin-IIA heavy chain in human breast cancer cells. EGF stimulation of MDA-MB-231 cells resulted in transient increases in both the assembly and phosphorylation of the myosin-IIA heavy chains. In EGF-stimulated cells, the myosin-IIA heavy chain is phosphorylated on the casein kinase 2 site (S1943). Cells expressing green fluorescent protein-myosin-IIA heavy-chain S1943E and S1943D mutants displayed increased migration into a wound and enhanced EGF-stimulated lamellipod extension compared with cells expressing wild-type myosin-IIA. In contrast, cells expressing the S1943A mutant exhibited reduced migration and lamellipod extension. These observations support a direct role for myosin-IIA heavy-chain phosphorylation in mediating motility and chemotaxis. [Abstract/Link to Full Text]

Selvapandiyan A, Kumar P, Morris JC, Salisbury JL, Wang CC, Nakhasi HL
Centrin1 is required for organelle segregation and cytokinesis in Trypanosoma brucei.
Mol Biol Cell. 2007 Sep;18(9):3290-301.
Centrin is a calcium-binding centrosome/basal body-associated protein involved in duplication and segregation of these organelles in eukaryotes. We had shown that disruption of one of the centrin genes (centrin1) in Leishmania amastigotes resulted in failure of both basal body duplication and cytokinesis. Here, we undertook to define the role of centrin1 (TbCen1) in the duplication and segregation of basal body and its associated organelles kinetoplast and Golgi, as well as its role in cytokinesis of the procyclic form of Trypanosoma brucei by depleting its protein using RNA inhibition methodology. TbCen1-depleted cells showed significant reduction in growth compared with control cells. Morphological analysis of these cells showed they were large and pleomorphic with multiple detached flagella. Both immunofluorescence assays using organelle-specific antibodies and electron microscopic analysis showed that TbCen1-deficient cells contained multiple basal bodies, kinetoplasts, Golgi, and nuclei. These multiple organelles were, however, closely clustered together, indicating duplication without segregation in the absence of centrin. This failure in organelle segregation may be the likely cause of inhibition of cytokinesis, suggesting for the first time a new and unique role for centrin in the segregation of organelles without affecting their multiplication in the procyclic form of T. brucei. [Abstract/Link to Full Text]

Moseley GW, Roth DM, DeJesus MA, Leyton DL, Filmer RP, Pouton CW, Jans DA
Dynein light chain association sequences can facilitate nuclear protein import.
Mol Biol Cell. 2007 Aug;18(8):3204-13.
Nuclear localization sequence (NLS)-dependent nuclear protein import is not conventionally held to require interaction with microtubules (MTs) or components of the MT motor, dynein. Here we report for the first time the role of sequences conferring association with dynein light chains (DLCs) in NLS-dependent nuclear accumulation of the rabies virus P-protein. We find that P-protein nuclear accumulation is significantly enhanced by its dynein light chain association sequence (DLC-AS), dependent on MT integrity and association with DLCs, and that P-protein-DLC complexes can associate with MT cytoskeletal structures. We also find that P-protein DLC-AS, as well as analogous sequences from other proteins, acts as an independent module that can confer enhancement of nuclear accumulation to proteins carrying the P-protein NLS, as well as several heterologous NLSs. Photobleaching experiments in live cells demonstrate that the MT-dependent enhancement of NLS-mediated nuclear accumulation by the P-protein DLC-AS involves an increased rate of nuclear import. This is the first report of DLC-AS enhancement of NLS function, identifying a novel mechanism regulating nuclear transport with relevance to viral and cellular protein biology. Importantly, this data indicates that DLC-ASs represent versatile modules to enhance nuclear delivery with potential therapeutic application. [Abstract/Link to Full Text]

Zhang X, Lan W, Ems-McClung SC, Stukenberg PT, Walczak CE
Aurora B phosphorylates multiple sites on mitotic centromere-associated kinesin to spatially and temporally regulate its function.
Mol Biol Cell. 2007 Sep;18(9):3264-76.
Chromosome congression and segregation require the proper attachment of microtubules to the two sister kinetochores. Disruption of either Aurora B kinase or the Kinesin-13 mitotic centromere-associated kinesin (MCAK) increases chromosome misalignment and missegregation due to improper kinetochore-microtubule attachments. MCAK localization and activity are regulated by Aurora B, but how Aurora B phosphorylation of MCAK affects spindle assembly is unclear. Here, we show that the binding of MCAK to chromosome arms is also regulated by Aurora B and that Aurora B-dependent chromosome arm and centromere localization is regulated by distinct two-site phosphoregulatory mechanisms. MCAK association with chromosome arms is promoted by phosphorylation of T95 on MCAK, whereas phosphorylation of S196 on MCAK promotes dissociation from the arms. Although targeting of MCAK to centromeres requires phosphorylation of S110 on MCAK, dephosphorylation of T95 on MCAK increases the binding of MCAK to centromeres. Our study reveals a new role for Aurora B, which is to prevent excess MCAK binding to chromatin to facilitate chromatin-nucleated spindle assembly. Our study also shows that the interplay between multiple phosphorylation sites of MCAK may be critical to temporally and spatially control MCAK function. [Abstract/Link to Full Text]

Venegas V, Zhou Z
Two alternative mechanisms that regulate the presentation of apoptotic cell engulfment signal in Caenorhabditis elegans.
Mol Biol Cell. 2007 Aug;18(8):3180-92.
Phosphatidylserine exposed on the surface of apoptotic mammalian cells is considered an "eat-me" signal that attracts phagocytes. The generality of using phosphatidylserine as a clearance signal for apoptotic cells in animals and the regulation of this event remain uncertain. Using ectopically expressed mouse MFG-E8, a secreted phosphatidylserine-binding protein, we detected specific exposure of phosphatidylserine on the surface of apoptotic cells in Caenorhabditis elegans. Masking the surface phosphatidylserine inhibits apoptotic cell engulfment. CED-7, an ATP-binding cassette (ABC) transporter, is necessary for the efficient exposure of phosphatidylserine on apoptotic somatic cells, and for the recognition of these cells by phagocytic receptor CED-1. Alternatively, phosphatidylserine exposure on apoptotic germ cells is not CED-7 dependent, but instead requires phospholipid scramblase PLSC-1, a homologue of mammalian phospholipid scramblases. Moreover, deleting plsc-1 results in the accumulation of apoptotic germ cells but not apoptotic somatic cells. These observations suggest that phosphatidylserine might be recognized by CED-1 and act as a conserved eat-me signal from nematodes to mammals. Furthermore, the two different biochemical activities used in somatic cells (ABC transporter) and germ cells (phospholipid scramblase) suggest an increased complexity in the regulation of phosphatidylserine presentation in response to apoptotic signals in different tissues and during different developmental stages. [Abstract/Link to Full Text]

Hall JR, Kow E, Nevis KR, Lu CK, Luce KS, Zhong Q, Cook JG
Cdc6 stability is regulated by the Huwe1 ubiquitin ligase after DNA damage.
Mol Biol Cell. 2007 Sep;18(9):3340-50.
The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC(Cdh1)) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC(Cdh1) ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage. [Abstract/Link to Full Text]

Rutkowski DT, Kang SW, Goodman AG, Garrison JL, Taunton J, Katze MG, Kaufman RJ, Hegde RS
The role of p58IPK in protecting the stressed endoplasmic reticulum.
Mol Biol Cell. 2007 Sep;18(9):3681-91.
The preemptive quality control (pQC) pathway protects cells from acute endoplasmic reticulum (ER) stress by attenuating translocation of nascent proteins despite their targeting to translocons at the ER membrane. Here, we investigate the hypothesis that the DnaJ protein p58(IPK) plays an essential role in this process via HSP70 recruitment to the cytosolic face of translocons for extraction of translocationally attenuated nascent chains. Our analyses revealed that the heightened stress sensitivity of p58-/- cells was not due to an impairment of the pQC pathway or elevated ER substrate burden during acute stress. Instead, the lesion was in the protein processing capacity of the ER lumen, where p58(IPK) was found to normally reside in association with BiP. ER lumenal p58(IPK) could be coimmunoprecipitated with a newly synthesized secretory protein in vitro and stimulated protein maturation upon overexpression in cells. These results identify a previously unanticipated location for p58(IPK) in the ER lumen where its putative function as a cochaperone explains the stress-sensitivity phenotype of knockout cells and mice. [Abstract/Link to Full Text]

Basu S, Mohan ML, Luo X, Kundu B, Kong Q, Singh N
Modulation of proteinase K-resistant prion protein in cells and infectious brain homogenate by redox iron: implications for prion replication and disease pathogenesis.
Mol Biol Cell. 2007 Sep;18(9):3302-12.
The principal infectious and pathogenic agent in all prion disorders is a beta-sheet-rich isoform of the cellular prion protein (PrP(C)) termed PrP-scrapie (PrP(Sc)). Once initiated, PrP(Sc) is self-replicating and toxic to neuronal cells, but the underlying mechanisms remain unclear. In this report, we demonstrate that PrP(C) binds iron and transforms to a PrP(Sc)-like form (*PrP(Sc)) when human neuroblastoma cells are exposed to an inorganic source of redox iron. The *PrP(Sc) thus generated is itself redox active, and it induces the transformation of additional PrP(C), simulating *PrP(Sc) propagation in the absence of brain-derived PrP(Sc). Moreover, limited depletion of iron from prion disease-affected human and mouse brain homogenates and scrapie-infected mouse neuroblastoma cells results in 4- to 10-fold reduction in proteinase K (PK)-resistant PrP(Sc), implicating redox iron in the generation, propagation, and stability of PK-resistant PrP(Sc). Furthermore, we demonstrate increased redox-active ferrous iron levels in prion disease-affected brains, suggesting that accumulation of PrP(Sc) is modulated by the combined effect of imbalance in brain iron homeostasis and the redox-active nature of PrP(Sc). These data provide information on the mechanism of replication and toxicity by PrP(Sc), and they evoke predictable and therapeutically amenable ways of modulating PrP(Sc) load. [Abstract/Link to Full Text]

Fernández-Ulibarri I, Vilella M, Lázaro-Diéguez F, Sarri E, Martínez SE, Jiménez N, Claro E, Mérida I, Burger KN, Egea G
Diacylglycerol is required for the formation of COPI vesicles in the Golgi-to-ER transport pathway.
Mol Biol Cell. 2007 Sep;18(9):3250-63.
Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation. [Abstract/Link to Full Text]

Joch M, Ase AR, Chen CX, MacDonald PA, Kontogiannea M, Corera AT, Brice A, Séguéla P, Fon EA
Parkin-mediated monoubiquitination of the PDZ protein PICK1 regulates the activity of acid-sensing ion channels.
Mol Biol Cell. 2007 Aug;18(8):3105-18.
Mutations in the parkin gene result in an autosomal recessive juvenile-onset form of Parkinson's disease. As an E3 ubiquitin-ligase, parkin promotes the attachment of ubiquitin onto specific substrate proteins. Defects in the ubiquitination of parkin substrates are therefore believed to lead to neurodegeneration in Parkinson's disease. Here, we identify the PSD-95/Discs-large/Zona Occludens-1 (PDZ) protein PICK1 as a novel parkin substrate. We find that parkin binds PICK1 via a PDZ-mediated interaction, which predominantly promotes PICK1 monoubiquitination rather than polyubiquitination. Consistent with monoubiquitination and recent work implicating parkin in proteasome-independent pathways, parkin does not promote PICK1 degradation. However, parkin regulates the effects of PICK1 on one of its other PDZ partners, the acid-sensing ion channel (ASIC). Overexpression of wild-type, but not PDZ binding- or E3 ubiquitin-ligase-defective parkin abolishes the previously described, protein kinase C-induced, PICK1-dependent potentiation of ASIC2a currents in non-neuronal cells. Conversely, the loss of parkin in hippocampal neurons from parkin knockout mice unmasks prominent potentiation of native ASIC currents, which is normally suppressed by endogenous parkin in wild-type neurons. Given that ASIC channels contribute to excitotoxicity, our work provides a mechanism explaining how defects in parkin-mediated PICK1 monoubiquitination could enhance ASIC activity and thereby promote neurodegeneration in Parkinson's disease. [Abstract/Link to Full Text]

Buster DW, Zhang D, Sharp DJ
Poleward tubulin flux in spindles: regulation and function in mitotic cells.
Mol Biol Cell. 2007 Aug;18(8):3094-104.
The poleward flux of tubulin subunits through spindle microtubules is a striking and conserved phenomenon whose function and molecular components remain poorly understood. To screen for novel components of the flux machinery, we utilized RNA interference to deplete regulators of microtubule dynamics, individually and in various combinations, from S2 cells and examined the resulting impact on flux rate. This led to the identification of two previously unknown flux inhibitors, KLP59C and KLP67A, and a flux promoter, Mini-spindles. Furthermore, we find that flux rate is regulated by functional antagonism among microtubule stabilizers and destabilizers specifically at plus ends. Finally, by examining mitosis on spindles in which flux has been up- or down-regulated or restored after the codepletion of antagonistic flux regulators, we show that flux is an integral contributor to anaphase A but is not responsible for chromosome congression, interkinetochore tension, or the establishment of normal spindle length during prometaphase/metaphase. [Abstract/Link to Full Text]

McLachlan RW, Kraemer A, Helwani FM, Kovacs EM, Yap AS
E-cadherin adhesion activates c-Src signaling at cell-cell contacts.
Mol Biol Cell. 2007 Aug;18(8):3214-23.
Cadherin-based cell-cell contacts are prominent sites for phosphotyrosine signaling, being enriched in tyrosine-phosphorylated proteins and tyrosine kinases and phosphatases. The functional interplay between cadherin adhesion and tyrosine kinase signaling, however, is complex and incompletely understood. In this report we tested the hypothesis that cadherin adhesion activates c-Src signaling and sought to assess its impact on cadherin function. We identified c-Src as part of a cadherin-activated cell signaling pathway that is stimulated by ligation of the adhesion receptor. However, c-Src has a biphasic impact on cadherin function, exerting a positive supportive role at lower signal strengths, but inhibiting function at high signal strengths. Inhibiting c-Src under circumstances when it is activated by cadherin adhesion decreased several measures of cadherin function. This suggests that the cadherin-activated c-Src signaling pathway serves positively to support cadherin function. Finally, our data implicate PI3-kinase signaling as a target for cadherin-activated c-Src signaling that contributes to its positive impact on cadherin function. We conclude that E-cadherin signaling is an important activator of c-Src at cell-cell contacts, providing a key input into a signaling pathway where quantitative changes in signal strength may result in qualitative differences in functional outcome. [Abstract/Link to Full Text]

Cheshenko N, Liu W, Satlin LM, Herold BC
Multiple receptor interactions trigger release of membrane and intracellular calcium stores critical for herpes simplex virus entry.
Mol Biol Cell. 2007 Aug;18(8):3119-30.
Herpes simplex viruses (HSV) harness cellular calcium signaling pathways to facilitate viral entry. Confocal microscopy and small interfering RNA (siRNA) were used to identify the source of the calcium and to dissect the requisite viral-cell interactions. Binding of HSV to human epithelial cells induced no calcium response, but shifting the cells to temperatures permissive for penetration triggered increases in plasma membrane calcium followed by a global release of intracellular calcium. Transfection with siRNA targeting the proteoglycan syndecan-2 blocked viral binding and abrogated any calcium response. Transfection with siRNA targeting nectin-1, a glycoprotein D receptor, also prevented both membrane and intracellular calcium responses. In contrast, the membrane response was preserved after transfection with siRNA targeting integrinalphav, a novel glycoprotein H receptor. The membrane response, however, was not sufficient for viral entry, which required interactions with integrinalphav and release of inositol-triphosphate receptor-dependent intracellular calcium stores. Thus, calcium plays a critical, complex role in HSV entry. [Abstract/Link to Full Text]

---

Recent Articles in Molecular and Cellular Biology

---

Kim SI, Bultman SJ, Jing H, Blobel GA, Bresnick EH
Dissecting molecular steps in chromatin domain activation during hematopoietic differentiation.
Mol Cell Biol. 2007 Jun;27(12):4551-65.
GATA factors orchestrate hematopoiesis via multistep transcriptional mechanisms, but the interrelationships and importance of individual steps are poorly understood. Using complementation analysis with GATA-1-null cells and mice containing a hypomorphic allele of the chromatin remodeler BRG1, we dissected the pathway from GATA-1 binding to cofactor recruitment, chromatin loop formation, and transcriptional activation. Analysis of GATA-1-mediated activation of the beta-globin locus, in which GATA-1 assembles dispersed complexes at the promoters and the distal locus control region (LCR), revealed molecular intermediates, including GATA-1-independent and GATA-1-containing LCR subcomplexes, both defective in promoting loop formation. An additional intermediate consisted of an apparently normal LCR complex and a promoter complex with reduced levels of total RNA polymerase II (Pol II) and Pol II phosphorylated at serine 5 of the carboxy-terminal domain. Reduced BRG1 activity solely compromised Pol II and serine 5-phosphorylated Pol II occupancy at the promoter, phenocopying the LCR-deleted mouse. These studies defined a hierarchical order of GATA-1-triggered events at a complex locus and establish a novel mechanism of long-range gene regulation. [Abstract/Link to Full Text]

Azare J, Leslie K, Al-Ahmadie H, Gerald W, Weinreb PH, Violette SM, Bromberg J
Constitutively activated Stat3 induces tumorigenesis and enhances cell motility of prostate epithelial cells through integrin beta 6.
Mol Cell Biol. 2007 Jun;27(12):4444-53.
The persistent activation of signal transducer and activator of transcription 3 (Stat3) is a common feature of prostate cancer. However, little is known about the Stat3 targets that may mediate prostate tumorigenesis. The introduction of an activating mutant form of Stat3 (Stat3-C) into immortalized prostate epithelial cells resulted in tumorigenesis. Stat3-C-expressing cells had decreased E-cadherin levels, increased numbers of lamellipodia and stress fibers, and enhanced migratory capacities compared to vector control-expressing cells, with a concomitant increase in the expression of integrin beta6 and its ligand, fibronectin (FN). Exogenously added FN increased cellular migration, with a concomitant loss of E-cadherin expression. The blockade of integrin alphavbeta6 in Stat3-C-expressing cells inhibited migration, increased E-cadherin levels, and reduced colony formation in soft agar. These results demonstrate the sufficiency of constitutively activated Stat3 in mediating prostate tumorigenesis and identify novel Stat3 targets that are involved in promoting cell migration and transformation. [Abstract/Link to Full Text]

Lovgren AK, Kovarova M, Koller BH
cPGES/p23 is required for glucocorticoid receptor function and embryonic growth but not prostaglandin E2 synthesis.
Mol Cell Biol. 2007 Jun;27(12):4416-30.
A number of studies have identified cytosolic prostaglandin E(2) synthase (cPGES)/p23 as a cytoplasmic protein capable of metabolism of prostaglandin E(2) (PGE(2)) from the cyclooxygenase metabolite prostaglandin endoperoxide (PGH(2)). However, this protein has also been implicated in a number of other pathways, including stabilization of the glucocorticoid receptor (GR) complex. To define the importance of the functions assigned to this protein, mice lacking detectible cPGES/p23 expression were generated. cPGES/p23(-/-) pups die during the perinatal period and display retarded lung development reminiscent of the phenotype of GR-deficient neonates. Furthermore, GR-sensitive gluconeogenic enzymes are not induced in the prenatal period. However, unlike GR-deficient embryos, cPGES/p23(-/-) embryos are small and a proliferation defect is observed in cPGES/p23(-/-) fibroblasts. Analysis of arachidonic acid metabolites in embryonic tissues and primary fibroblasts failed to support a function for this protein in PGE(2) biosynthesis. Thus, while the growth retardation of the cPGES/p23(-/-) pups and decreased proliferation of primary fibroblasts identify functions for this protein in addition to GR stabilization, it is unlikely that these functions include metabolism of PGH(2) to PGE(2). [Abstract/Link to Full Text]

Tshori S, Sonnenblick A, Yannay-Cohen N, Kay G, Nechushtan H, Razin E
Microphthalmia transcription factor isoforms in mast cells and the heart.
Mol Cell Biol. 2007 Jun;27(11):3911-9.
The microphthalmia transcription factor (Mitf) is critical for the survival and differentiation of a variety of cell types. While on the transcript level it has been noted that melanocytes and cardiomyocytes express specific Mitf isoforms, mast cells express several isoforms, mainly Mitf-H and Mitf-MC, whose function has not been thoroughly investigated. We found that in mast cells the expression of the specific Mitf isoforms is dependent on physiological stimuli that cause a major shifting of promoter usage and internal splicing. For example, activation of the c-kit signaling pathway almost totally abolished one of the main splice isoforms. Since cardiomyocytes express only the Mitf-H isoform, they were an ideal system to determine this isoform's physiological role. We identified that the expression of myosin light-chain 1a (MLC-1a) is regulated by Mitf-H. Interestingly, the transactivation of MLC-1a by Mitf-H in cardiomyocytes is decreased by overexpression of the splice form with exon 6a. In conclusion, we found that there is physiological switching of Mitf isoforms and that the promoter context and the cell context have a combined influence on gene expression programs. [Abstract/Link to Full Text]

Prickett TD, Brautigan DL
Cytokine activation of p38 mitogen-activated protein kinase and apoptosis is opposed by alpha-4 targeting of protein phosphatase 2A for site-specific dephosphorylation of MEK3.
Mol Cell Biol. 2007 Jun;27(12):4217-27.
alpha-4 is an essential gene and is a dominant antiapoptotic factor in various tissues that is a regulatory subunit for type 2A protein phosphatases. A multiplexed phosphorylation site screen revealed that knockdown of alpha-4 by small interfering RNA (siRNA) increased p38 mitogen-activated protein kinase (MAPK) and c-Jun phosphorylation without changes in JNK or ERK. FLAG-alpha-4 coprecipitated hemagglutinin-MEK3 plus endogenous protein phosphatase 2A (PP2A) and selectively enhanced dephosphorylation of Thr193, but not Ser189, in the activation loop of MEK3. Overexpression of alpha-4 suppressed p38 MAPK activation in response to tumor necrosis factor alpha (TNF-alpha). The alpha-4 dominant-negative domain (DND) (residues 220 to 340) associated with MEK3, but not PP2A, and its overexpression sensitized cells to activation of p38 MAPK by TNF-alpha and interleukin-1beta, but not by ansiomycin or sorbitol. The response was diminished by nocodazole or by siRNA knockdown of the Opitz syndrome protein Mid1 that binds alpha-4 to microtubules. Interference by alpha-4 DND or alpha-4 siRNA increased caspase 3/7 activation in response to TNF-alpha. Growth of transformed cells in soft agar was enhanced by alpha-4 and suppressed by alpha-4 DND. The results show that alpha-4 targets PP2A activity to MEK3 to suppress p38 MAPK activation by cytokines, thereby inhibiting apoptosis and anoikis. [Abstract/Link to Full Text]

Shah YM, Morimura K, Yang Q, Tanabe T, Takagi M, Gonzalez FJ
Peroxisome proliferator-activated receptor alpha regulates a microRNA-mediated signaling cascade responsible for hepatocellular proliferation.
Mol Cell Biol. 2007 Jun;27(12):4238-47.
Activation of peroxisome proliferator-activated receptor alpha (PPARalpha) leads to hepatocellular proliferation and liver carcinomas. The early events mediating these effects are unknown. A novel mechanism by which PPARalpha regulates gene expression and hepatocellular proliferation was uncovered. MicroRNA (miRNA) expression profiling demonstrated that activated PPARalpha was a major regulator of hepatic miRNA expression. Of particular interest, let-7C, an miRNA important in cell growth, was inhibited following 4-h treatment and 2-week and 11-month sustained treatment with the potent PPARalpha agonist Wy-14,643 in wild-type mice. let-7C was shown to target c-myc via direct interaction with the 3' untranslated region of c-myc. The PPARalpha-mediated induction of c-myc via let-7C subsequently increased expression of the oncogenic mir-17-92 cluster; these events did not occur in Pparalpha-null mice. Overexpression of let-7C decreased c-myc and mir-17 and suppressed the growth of Hepa-1 cells. Furthermore, using the human PPARalpha-expressing mouse model, which is responsive to Wy-14,643 effects on beta-oxidation and serum triglycerides but resistant to hepatocellular proliferation and tumorigenesis, we demonstrated a critical role for let-7C in liver oncogenesis. Wy-14,643 treatment did not inhibit let-7C or induce c-myc and mir-17 expression. These observations reveal a let-7C signaling cascade critical for PPARalpha agonist-induced liver proliferation and tumorigenesis. [Abstract/Link to Full Text]

Yoshiko Y, Candeliere GA, Maeda N, Aubin JE
Osteoblast autonomous Pi regulation via Pit1 plays a role in bone mineralization.
Mol Cell Biol. 2007 Jun;27(12):4465-74.
The complex pathogenesis of mineralization defects seen in inherited and/or acquired hypophosphatemic disorders suggests that local inorganic phosphate (P(i)) regulation by osteoblasts may be a rate-limiting step in physiological bone mineralization. To test whether an osteoblast autonomous phosphate regulatory system regulates mineralization, we manipulated well-established in vivo and in vitro models to study mineralization stages separately from cellular proliferation/differentiation stages of osteogenesis. Foscarnet, an inhibitor of NaP(i) transport, blocked mineralization of osteoid formation in osteoblast cultures and local mineralization after injection over the calvariae of newborn rats. Mineralization was also down- and upregulated, respectively, with under- and overexpression of the type III NaP(i) transporter Pit1 in osteoblast cultures. Among molecules expressed in osteoblasts and known to be related to P(i) handling, stanniocalcin 1 was identified as an early response gene after foscarnet treatment; it was also regulated by extracellular P(i), and itself increased Pit1 accumulation in both osteoblast cultures and in vivo. These results provide new insights into the functional role of osteoblast autonomous P(i) handling in normal bone mineralization and the abnormalities seen in skeletal tissue in hypophosphatemic disorders. [Abstract/Link to Full Text]

Chang MY, Sun W, Ochiai W, Nakashima K, Kim SY, Park CH, Kang JS, Shim JW, Jo AY, Kang CS, Lee YS, Kim JS, Lee SH
Bcl-XL/Bax proteins direct the fate of embryonic cortical precursor cells.
Mol Cell Biol. 2007 Jun;27(12):4293-305.
In the developing mouse brain, the highest Bcl-X(L) expression is seen at the peak of neurogenesis, whereas the peak of Bax expression coincides with the astrogenic period. While such observations suggest an active role of the Bcl-2 family proteins in the generation of neurons and astrocytes, no definitive demonstration has been provided to date. Using combinations of gain- and loss-of-function assays in vivo and in vitro, we provide evidence for instructive roles of these proteins in neuronal and astrocytic fate specification. Specifically, in Bax knockout mice, astrocyte formation was decreased in the developing cortices. Overexpression of Bcl-X(L) and Bax in embryonic cortical precursors induced neural and astrocytic differentiation, respectively, while inhibitory RNAs led to the opposite results. Importantly, inhibition of caspase activity, dimerization, or mitochondrial localization of Bcl-X(L)/Bax proteins indicated that the differentiation effects of Bcl-X(L)/Bax are separable from their roles in cell survival and apoptosis. Lastly, we describe activation of intracellular signaling pathways and expression of basic helix-loop-helix transcriptional factors specific for the Bcl-2 protein-mediated differentiation. [Abstract/Link to Full Text]

Lazarou M, McKenzie M, Ohtake A, Thorburn DR, Ryan MT
Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I.
Mol Cell Biol. 2007 Jun;27(12):4228-37.
Complex I of the respiratory chain is composed of at least 45 subunits that assemble together at the mitochondrial inner membrane. Defects in human complex I result in energy generation disorders and are also implicated in Parkinson's disease and altered apoptotic signaling. The assembly of this complex is poorly understood and is complicated by its large size and its regulation by two genomes, with seven subunits encoded by mitochondrial DNA (mtDNA) and the remainder encoded by nuclear genes. Here we analyzed the assembly of a number of mtDNA- and nuclear-gene-encoded subunits into complex I. We found that mtDNA-encoded subunits first assemble into intermediate complexes and require significant chase times for their integration into the holoenzyme. In contrast, a set of newly imported nuclear-gene-encoded subunits integrate with preexisting complex I subunits to form intermediates and/or the fully assembly holoenzyme. One of the intermediate complexes represents a subassembly associated with the chaperone B17.2L. By using isolated patient mitochondria, we show that this subassembly is a productive intermediate in complex I assembly since import of the missing subunit restores complex I assembly. Our studies point to a mechanism of complex I biogenesis involving two complementary processes, (i) synthesis of mtDNA-encoded subunits to seed de novo assembly and (ii) exchange of preexisting subunits with newly imported ones to maintain complex I homeostasis. Subunit exchange may also act as an efficient mechanism to prevent the accumulation of oxidatively damaged subunits that would otherwise be detrimental to mitochondrial oxidative phosphorylation and have the potential to cause disease. [Abstract/Link to Full Text]

Wang H, Hertlein E, Bakkar N, Sun H, Acharyya S, Wang J, Carathers M, Davuluri R, Guttridge DC
NF-kappaB regulation of YY1 inhibits skeletal myogenesis through transcriptional silencing of myofibrillar genes.
Mol Cell Biol. 2007 Jun;27(12):4374-87.
NF-kappaB signaling is implicated as an important regulator of skeletal muscle homeostasis, but the mechanisms by which this transcription factor contributes to muscle maturation and turnover remain unclear. To gain insight into these mechanisms, gene expression profiling was examined in C2C12 myoblasts devoid of NF-kappaB activity. Interestingly, even in proliferating myoblasts, the absence of NF-kappaB caused the pronounced induction of several myofibrillar genes, suggesting that NF-kappaB functions as a negative regulator of late-stage muscle differentiation. Although several myofibrillar promoters contain predicted NF-kappaB binding sites, functional analysis using the troponin-I2 gene as a model revealed that NF-kappaB-mediated repression does not occur through direct DNA binding. In the search for an indirect mediator, the transcriptional repressor YinYang1 (YY1) was identified. While inducers of NF-kappaB stimulated YY1 expression in multiple cell types, genetic ablation of the RelA/p65 subunit of NF-kappaB in both cultured cells and adult skeletal muscle correlated with reduced YY1 transcripts and protein. NF-kappaB regulation of YY1 occurred at the transcriptional level, mediated by direct binding of the p50/p65 heterodimer complex to the YY1 promoter. Furthermore, YY1 was found associated with multiple myofibrillar promoters in C2C12 myoblasts containing NF-kappaB activity. Based on these results, we propose that NF-kappaB regulation of YY1 and transcriptional silencing of myofibrillar genes represent a new mechanism by which NF-kappaB functions in myoblasts to modulate skeletal muscle differentiation. [Abstract/Link to Full Text]

Cline EG, Nelson WJ
Characterization of mammalian Par 6 as a dual-location protein.
Mol Cell Biol. 2007 Jun;27(12):4431-43.
Par 6 acts as a scaffold protein to facilitate atypical protein kinase C-mediated phosphorylation of cytoplasmic protein complexes, leading to epithelial and neuronal cell polarization. In addition to its location in the cytoplasm, Par 6 is localized to the nucleus. However, its organization and potential functions in the nucleus have not been examined. Using an affinity-purified Par 6 antibody and a chimera of Par 6 and green fluorescent protein, we show that Par 6 localizes precisely to nuclear speckles, but not to other nuclear structures, and displays characteristics of speckle proteins. We show that Par 6 colocalizes in the nucleus with Tax, a transcriptional activator of the human T-cell leukemia virus type 1 long terminal repeat, but multiple lines of evidence show that Par 6 is not directly involved in known functions of speckle proteins, including general transcription, splicing, or mRNA transport. Significantly, however, the structure of nuclear speckles is lost when Par 6 levels are reduced by Par 6-specific small interfering RNA. Therefore, we hypothesize that Par 6 in the nucleus acts as a scaffolding protein in nuclear speckle complexes, similar to its role in the cytoplasm. [Abstract/Link to Full Text]

Popova SN, Barczyk M, Tiger CF, Beertsen W, Zigrino P, Aszodi A, Miosge N, Forsberg E, Gullberg D
Alpha11 beta1 integrin-dependent regulation of periodontal ligament function in the erupting mouse incisor.
Mol Cell Biol. 2007 Jun;27(12):4306-16.
The fibroblast integrin alpha11beta1 is a key receptor for fibrillar collagens. To study the potential function of alpha11 in vivo, we generated a null allele of the alpha11 gene. Integrin alpha11(-/-) mice are viable and fertile but display dwarfism with increased mortality, most probably due to severely defective incisors. Mutant incisors are characterized by disorganized periodontal ligaments, whereas molar ligaments appear normal. The primary defect in the incisor ligament leads to halted tooth eruption. alpha11beta1-defective embryonic fibroblasts displayed severe defects in vitro, characterized by (i) greatly reduced cell adhesion and spreading on collagen I, (ii) reduced ability to retract collagen lattices, and (iii) reduced cell proliferation. Analysis of matrix metalloproteinase in vitro and in vivo revealed disturbed MMP13 and MMP14 synthesis in alpha11(-/-) cells. We show that alpha11beta1 is the major receptor for collagen I on mouse embryonic fibroblasts and suggest that alpha11beta1 integrin is specifically required on periodontal ligament fibroblasts for cell migration and collagen reorganization to help generate the forces needed for axial tooth movement. Our data show a unique role for alpha11beta1 integrin during tooth eruption. [Abstract/Link to Full Text]

Suzuki A, Okamoto S, Lee S, Saito K, Shiuchi T, Minokoshi Y
Leptin stimulates fatty acid oxidation and peroxisome proliferator-activated receptor alpha gene expression in mouse C2C12 myoblasts by changing the subcellular localization of the alpha2 form of AMP-activated protein kinase.
Mol Cell Biol. 2007 Jun;27(12):4317-27.
Leptin stimulates fatty acid oxidation in skeletal muscle through the activation of AMP-activated protein kinase (AMPK) and the induction of gene expression, such as that for peroxisome proliferator-activated receptor alpha (PPARalpha). We now show that leptin stimulates fatty acid oxidation and PPARalpha gene expression in the C2C12 muscle cell line through the activation of AMPK containing the alpha2 subunit (alpha2AMPK) and through changes in the subcellular localization of this enzyme. Activated alpha2AMPK containing the beta1 subunit was shown to be retained in the cytoplasm, where it phosphorylated acetyl coenzyme A carboxylase and thereby stimulated fatty acid oxidation. In contrast, alpha2AMPK containing the beta2 subunit transiently increased fatty acid oxidation but underwent rapid translocation to the nucleus, where it induced PPARalpha gene transcription. A nuclear localization signal and Thr(172) phosphorylation of alpha2 were found to be essential for nuclear translocation of alpha2AMPK, whereas the myristoylation of beta1 anchors alpha2AMPK in the cytoplasm. The prevention of alpha2AMPK activation and the change in its subcellular localization inhibited the metabolic effects of leptin. Our data thus suggest that the activation of and changes in the subcellular localization of alpha2AMPK are required for leptin-induced stimulation of fatty acid oxidation and PPARalpha gene expression in muscle cells. [Abstract/Link to Full Text]

Sobeck A, Stone S, Hoatlin ME
DNA structure-induced recruitment and activation of the Fanconi anemia pathway protein FANCD2.
Mol Cell Biol. 2007 Jun;27(12):4283-92.
The Fanconi anemia (FA) pathway proteins are thought to be involved in the repair of irregular DNA structures including those encountered by the moving replication fork. However, the nature of the DNA structures that recruit and activate the FA proteins is not known. Because FA proteins function within an extended network of proteins, some of which are still unknown, we recently established cell-free assays in Xenopus laevis egg extracts to deconstruct the FA pathway in a fully replication-competent context. Here we show that the central FA pathway protein, xFANCD2, is monoubiquitinated (xFANCD2-L) rapidly in the presence of linear and branched double-stranded DNA (dsDNA) structures but not single-stranded or Y-shaped DNA. xFANCD2-L associates with dsDNA structures in an FA core complex-dependent manner but independently of xATRIP, the regulatory subunit of xATR. Formation of xFANCD2-L is also triggered in response to circular dsDNA, suggesting that dsDNA ends are not required to trigger monoubiquitination of FANCD2. The induction of xFANCD2-L in response to circular dsDNA is replication and checkpoint independent. Our results provide new evidence that the FA pathway discriminates among DNA structures and demonstrate that triggering the FA pathway can be uncoupled from DNA replication and ATRIP-dependent activation. [Abstract/Link to Full Text]

Klattig J, Sierig R, Kruspe D, Besenbeck B, Englert C
Wilms' tumor protein Wt1 is an activator of the anti-Müllerian hormone receptor gene Amhr2.
Mol Cell Biol. 2007 Jun;27(12):4355-64.
The Wilms' tumor protein Wt1 plays an essential role in mammalian urogenital development. WT1 mutations in humans lead to a variety of disorders, including Wilms' tumor, a pediatric kidney cancer, as well as Frasier and Denys-Drash syndromes. Phenotypic anomalies in Denys-Drash syndrome include pseudohermaphroditism and sex reversal in extreme cases. We have used cDNA microarray analyses on Wt1 knockout mice to identify Wt1-dependent genes involved in sexual development. The gene most dramatically affected by Wt1 inactivation was Amhr2, encoding the anti-Müllerian hormone (Amh) receptor 2. Amhr2 is an essential factor for the regression of the Müllerian duct in males, and mutations in AMHR2 lead to the persistent Müllerian duct syndrome, a rare form of male pseudohermaphroditism. Here we show that Wt1 and Amhr2 are coexpressed during urogenital development and that the Wt1 protein binds to the promoter region of the Amhr2 gene. Inactivation and overexpression of Wt1 in cell lines was followed by immediate changes of Amhr2 expression. The identification of Amhr2 as a Wt1 target provides new insights into the role of Wt1 in sexual differentiation and indicates, in addition to its function in early gonad development and sex determination, a novel function for Wt1, namely, in Müllerian duct regression. [Abstract/Link to Full Text]

Hedlund M, Tangvoranuntakul P, Takematsu H, Long JM, Housley GD, Kozutsumi Y, Suzuki A, Wynshaw-Boris A, Ryan AF, Gallo RL, Varki N, Varki A
N-glycolylneuraminic acid deficiency in mice: implications for human biology and evolution.
Mol Cell Biol. 2007 Jun;27(12):4340-6.
Humans and chimpanzees share >99% identity in most proteins. One rare difference is a human-specific inactivating deletion in the CMAH gene, which determines biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc). Since Neu5Gc is prominent on most chimpanzee cell surfaces, this mutation could have affected multiple systems. However, Neu5Gc is found in human cancers and fetuses and in trace amounts in normal human tissues, suggesting an alternate biosynthetic pathway. We inactivated the mouse Cmah gene and studied the in vivo consequences. There was no evidence for an alternate pathway in normal, fetal, or malignant tissue. Rather, null fetuses accumulated Neu5Gc from heterozygous mothers and dietary Neu5Gc was incorporated into oncogene-induced tumors. As with humans, there were accumulation of the precursor N-acetylneuraminic acid and increases in sialic acid O acetylation. Null mice showed other abnormalities reminiscent of the human condition. Adult mice showed a diminished acoustic startle response and required higher acoustic stimuli to increase responses above the baseline level. In this regard, histological abnormalities of the inner ear occurred in older mice, which had impaired hearing. Adult animals also showed delayed skin wound healing. Loss of Neu5Gc in hominid ancestors approximately 2 to 3 million years ago likely had immediate and long-term consequences for human biology. [Abstract/Link to Full Text]

Kuo YY, Chang ZF
GATA-1 and Gfi-1B interplay to regulate Bcl-xL transcription.
Mol Cell Biol. 2007 Jun;27(12):4261-72.
The induction of Bcl-x(L) is critical for the survival of late proerythroblasts. The erythroid-specific transcriptional network that regulates Bcl-x(L) expression in erythropoiesis remains unclear. The activation of the central erythropoietic transcriptional factor, GATA-1, leads to the early, transient induction of a transcription repressor, Gfi-1B, followed by the late induction of Bcl-x(L) during erythroid maturation in G1ER cells. Chromatin immunoprecipitation assays demonstrated that a constant level of GATA-1 binds to the Bcl-x promoter throughout the entire induction period, while Gfi-1B is transiently associated with the promoter in the early phase. The sustained expression of Gfi-1B abolished GATA-1-induced Bcl-x(L) expression. Here, we present evidence that GATA-1 binds to the noncanonical GATT motif of the Bcl-x promoter for trans-activation. Gfi-1B expressed at increased levels is recruited to the Bcl-x promoter through its association with GATA-1, suppressing Bcl-x(L) transcription. Therefore, the down-regulation of Gfi-1B in the late phase of erythroid maturation is necessary for Bcl-x(L) induction. Furthermore, we show that the inhibition of Bcr-Abl kinase by treatment with imatinib caused the up-regulation of Gfi-1B in K562 cells, where Gfi-1B also cooperated with GATA-1 to repress Bcl-x(L) transcription. Gfi-1B knockdown by RNA interference diminished imatinib-induced apoptosis, while the overexpression of Gfi-1B sensitized K562 cells to arsenic-induced death. These findings illuminate the role of Gfi-1B in GATA-1-mediated transcription in the survival aspect of erythroid cells. [Abstract/Link to Full Text]

Goetze S, Mateos-Langerak J, Gierman HJ, de Leeuw W, Giromus O, Indemans MH, Koster J, Ondrej V, Versteeg R, van Driel R
The three-dimensional structure of human interphase chromosomes is related to the transcriptome map.
Mol Cell Biol. 2007 Jun;27(12):4475-87.
The three-dimensional (3D) organization of the chromosomal fiber in the human interphase nucleus is an important but poorly understood aspect of gene regulation. Here we quantitatively analyze and compare the 3D structures of two types of genomic domains as defined by the human transcriptome map. While ridges are gene dense and show high expression levels, antiridges, on the other hand, are gene poor and carry genes that are expressed at low levels. We show that ridges are in general less condensed, more irregularly shaped, and located more closely to the nuclear center than antiridges. Six human cell lines that display different gene expression patterns and karyotypes share these structural parameters of chromatin. This shows that the chromatin structures of these two types of genomic domains are largely independent of tissue-specific variations in gene expression and differentiation state. Moreover, we show that there is remarkably little intermingling of chromatin from different parts of the same chromosome in a chromosome territory, neither from adjacent nor from distant parts. This suggests that the chromosomal fiber has a compact structure that sterically suppresses intermingling. Together, our results reveal novel general aspects of 3D chromosome architecture that are related to genome structure and function. [Abstract/Link to Full Text]

Ruas M, Gregory F, Jones R, Poolman R, Starborg M, Rowe J, Brookes S, Peters G
CDK4 and CDK6 delay senescence by kinase-dependent and p16INK4a-independent mechanisms.
Mol Cell Biol. 2007 Jun;27(12):4273-82.
Replicative senescence of human diploid fibroblasts (HDFs) is largely implemented by the cyclin-dependent kinase (CDK) inhibitors p16(INK4a) and p21(CIP1). Their accumulation results in a loss of CDK2 activity, and cells arrest with the retinoblastoma protein (pRb) in its hypophosphorylated state. It has become standard practice to bypass the effects of p16(INK4a) by overexpressing CDK4 or a variant form that is unable to bind to INK4 proteins. Although CDK4 and CDK6 and their INK4-insensitive variants can extend the life span of HDFs, they also cause a substantial increase in the levels of endogenous p16(INK4a). Here we show that CDK4 and CDK6 can extend the life span of HDFs that have inactivating mutations in both alleles of INK4a or in which INK4a levels are repressed, indicating that overexpression of CDK4/6 is not equivalent to ablation of p16(INK4a). However, catalytically inactive versions of these kinases are unable to extend the replicative life span, suggesting that the impact of ectopic CDK4/6 depends on their ability to phosphorylate as yet unidentified substrates rather than to sequester CDK inhibitors. Since p16(INK4a) deficiency, CDK4 expression, and p53 or p21(CIP1) ablation have additive effects on replicative life span, our results underscore the idea that senescence is an integrated response to diverse signals. [Abstract/Link to Full Text]

Yamasaki S, Ishikawa E, Sakuma M, Kanagawa O, Cheng AM, Malissen B, Saito T
LAT and NTAL mediate immunoglobulin E-induced sustained extracellular signal-regulated kinase activation critical for mast cell survival.
Mol Cell Biol. 2007 Jun;27(12):4406-15.
Immunoglobulin E (IgE) induces mast cell survival in the absence of antigen (Ag) through the high-affinity IgE receptor, Fcepsilon receptor I (FcepsilonRI). Although we have shown that protein tyrosine kinase Syk and sustained extracellular signal-regulated kinase (Erk) activation are required for IgE-induced mast cell survival, how Syk couples with sustained Erk activation is still unclear. Here, we report that the transmembrane adaptors LAT and NTAL are phosphorylated slowly upon IgE stimulation and that sustained but not transient Erk activation induced by IgE was inhibited in LAT(-/-) NTAL(-/-) bone marrow-derived mast cells (BMMCs). IgE-induced survival requires Ras activation, and both were impaired in LAT(-/-) NTAL(-/-) BMMCs. Sos was preferentially required for FcepsilonRI signals by IgE rather than IgE plus Ag. Survival impaired in LAT(-/-) NTAL(-/-) BMMCs was restored to levels comparable to those of the wild type by membrane-targeted Sos, which bypasses the Grb2-mediated membrane recruitment of Sos. The IgE-induced survival of BMMCs lacking Gads, an adaptor critical for the formation of the LAT-SLP-76-phospholipase Cgamma (PLCgamma) complex, was observed to be normal. IgE stimulation induced the membrane retention of Grb2-green fluorescent protein fusion proteins in wild-type but not LAT(-/-) NTAL(-/-) BMMCs. These results suggest that LAT and NTAL contribute to the maintenance of Erk activation and survival through the membrane retention of the Ras-activating complex Grb2-Sos and, further, that the LAT-Gads-SLP-76-PLCgamma and LAT/NTAL-Grb2-Sos pathways are differentially required for degranulation and survival, respectively. [Abstract/Link to Full Text]

Bonn S, Seeburg PH, Schwarz MK
Combinatorial expression of alpha- and gamma-protocadherins alters their presenilin-dependent processing.
Mol Cell Biol. 2007 Jun;27(11):4121-32.
Alpha- and gamma-protocadherins (Pcdhs) are type I transmembrane receptors expressed predominantly in the central nervous system and located in part in synapses. They are transcribed from complex genomic loci, giving rise in the mouse to 14 alpha-Pcdh and 22 gamma-Pcdh isoforms consisting of variable domains, each encompassing the extracellular region, the transmembrane region, and part of the intracellular region harboring the alpha- or gamma-Pcdh-specific invariant cytoplasmic domain. Presenilin-dependent intramembrane proteolysis (PS-IP) of gamma-Pcdhs and the formation of alpha/gamma-Pcdh heteromers led us to investigate the effects of homo- and heteromer formation on gamma- and putative alpha-Pcdh membrane processing and signaling. We find that upon surface delivery, alpha-Pcdhs, like gamma-Pcdhs, are subject to matrix metallo-protease cleavage followed by PS-IP in neurons. We further demonstrate that the combinatorial expression of alpha- and gamma-Pcdhs modulates the extent of their PS-IP, indicating the formation of alpha/gamma-Pcdh heteromers with an altered susceptibility to processing. Cell-specific expression of alpha/gamma-Pcdh isoforms could thus determine cell and synapse adhesive properties as well as intracellular and nuclear signaling by their soluble cytoplasmic cleavage products, alpha C-terminal fragment 2 (alpha-CTF-2) and gamma-CTF-2. [Abstract/Link to Full Text]

Eulalio A, Behm-Ansmant I, Schweizer D, Izaurralde E
P-body formation is a consequence, not the cause, of RNA-mediated gene silencing.
Mol Cell Biol. 2007 Jun;27(11):3970-81.
P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. We show that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, we show that releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity. [Abstract/Link to Full Text]

Padron-Barthe L, Leprêtre C, Martin E, Counis MF, Torriglia A
Conformational modification of serpins transforms leukocyte elastase inhibitor into an endonuclease involved in apoptosis.
Mol Cell Biol. 2007 Jun;27(11):4028-36.
The best-characterized biochemical feature of apoptosis is degradation of genomic DNA into oligonucleosomes. The endonuclease responsible for DNA degradation in caspase-dependent apoptosis is caspase-activated DNase. In caspase-independent apoptosis, different endonucleases may be activated according to the cell line and the original insult. Among the known effectors of caspase-independent cell death, L-DNase II (LEI [leukocyte elastase inhibitor]-derived DNase II) has been previously characterized by our laboratory. We have thus shown that this endonuclease derives from the serpin superfamily member LEI by posttranslational modification (A. Torriglia, P. Perani, J. Y. Brossas, E. Chaudun, J. Treton, Y. Courtois, and M. F. Counis, Mol. Cell. Biol. 18:3612-3619, 1998). In this work, we assessed the molecular mechanism involved in the change in the enzymatic activity of this molecule from an antiprotease to an endonuclease. We report that the cleavage of LEI by elastase at its reactive center loop abolishes its antiprotease activity and leads to a conformational modification that exposes an endonuclease active site and a nuclear localization signal. This represents a novel molecular mechanism for a complete functional conversion induced by changing the conformation of a serpin. We also show that this molecular transformation affects cellular fate and that both endonuclease activity and nuclear translocation of L-DNase II are needed to induce cell death. [Abstract/Link to Full Text]

Ménasché G, Kliche S, Chen EJ, Stradal TE, Schraven B, Koretzky G
RIAM links the ADAP/SKAP-55 signaling module to Rap1, facilitating T-cell-receptor-mediated integrin activation.
Mol Cell Biol. 2007 Jun;27(11):4070-81.
One outcome of T-cell receptor (TCR) signaling is increased affinity and avidity of integrins for their ligands. This occurs through a process known as inside-out signaling, which has been shown to require several molecular components including the adapter proteins ADAP (adhesion and degranulation-promoting adapter protein) and SKAP-55 (55-kDa src kinase-associated phosphoprotein) and the small GTPase Rap1. Herein, we provide evidence linking ADAP and SKAP-55 to RIAM, a recently described adapter protein that binds selectively to active Rap1. We identified RIAM as a key component linking the ADAP/SKAP-55 module to the small GTPase Rap1, facilitating TCR-mediated integrin activation. We show that RIAM constitutively interacts with SKAP-55 in both a heterologous transfection system and primary T cells and map the region essential for this interaction. Additionally, we find that the SKAP-55/RIAM complex is essential both for TCR-mediated adhesion and for efficient conjugate formation between T cells and antigen-presenting cells. Mechanistic studies revealed that the ADAP/SKAP-55 module relocalized RIAM and Rap1 to the plasma membrane following TCR activation to facilitate integrin activation. These results describe for the first time a link between ADAP/SKAP-55 and the Rap1/RIAM complex and provide a potential new mechanism for TCR-mediated integrin activation. [Abstract/Link to Full Text]

Carneiro T, Carvalho C, Braga J, Rino J, Milligan L, Tollervey D, Carmo-Fonseca M
Depletion of the yeast nuclear exosome subunit Rrp6 results in accumulation of polyadenylated RNAs in a discrete domain within the nucleolus.
Mol Cell Biol. 2007 Jun;27(11):4157-65.
Recent data reveal that a substantial fraction of transcripts generated by RNA polymerases I, II, and III are rapidly degraded in the nucleus by the combined action of the exosome and a noncanonical poly(A) polymerase activity. This work identifies a domain within the yeast nucleolus that is enriched in polyadenylated RNAs in the absence of the nuclear exosome RNase Rrp6 or the exosome cofactor Mtr4. In normal yeast cells, poly(A)(+) RNA was undetectable in the nucleolus but the depletion of either Rrp6 or Mtr4 led to the accumulation of polyadenylated RNAs in a discrete subnucleolar region. This nucleolar poly(A) domain is enriched for the U14 snoRNA and the snoRNP protein Nop1 but is distinct from the nucleolar body that functions in snoRNA maturation. In strains lacking both Rrp6 and the poly(A) polymerase Trf4, the accumulation of poly(A)(+) RNA was suppressed, suggesting the involvement of the Trf4-Air1/2-Mtr4 polyadenylation (TRAMP) complex. The accumulation of polyadenylated snoRNAs in a discrete nucleolar domain may promote their recognition as substrates for the exosome. [Abstract/Link to Full Text]

Pham CG, Bubici C, Zazzeroni F, Knabb JR, Papa S, Kuntzen C, Franzoso G
Upregulation of Twist-1 by NF-kappaB blocks cytotoxicity induced by chemotherapeutic drugs.
Mol Cell Biol. 2007 Jun;27(11):3920-35.
NF-kappaB/Rel transcription factors are central to controlling programmed cell death (PCD). Activation of NF-kappaB blocks PCD induced by numerous triggers, including ligand engagement of tumor necrosis factor receptor (TNF-R) family receptors. The protective activity of NF-kappaB is also crucial for oncogenesis and cancer chemoresistance. Downstream of TNF-Rs, this activity of NF-kappaB has been linked to the suppression of reactive oxygen species and the c-Jun-N-terminal-kinase (JNK) cascade. The mechanism by which NF-kappaB inhibits PCD triggered by chemotherapeutic drugs, however, remains poorly understood. To understand this mechanism, we sought to identify unrecognized protective genes that are regulated by NF-kappaB. Using an unbiased screen, we identified the basic-helix-loop-helix factor Twist-1 as a new mediator of the protective function of NF-kappaB. Twist-1 is an evolutionarily conserved target of NF-kappaB, blocks PCD induced by chemotherapeutic drugs and TNF-alpha in NF-kappaB-deficient cells, and is essential to counter this PCD in cancer cells. The protective activity of Twist-1 seemingly halts PCD independently of interference with cytotoxic JNK, p53, and p19(ARF) signaling, suggesting that it mediates a novel protective mechanism activated by NF-kappaB. Indeed, our data indicate that this activity involves a control of inhibitory Bcl-2 phosphorylation. The data also suggest that Twist-1 and -2 play an important role in NF-kappaB-dependent chemoresistance. [Abstract/Link to Full Text]

Wiebe PO, Kormish JD, Roper VT, Fujitani Y, Alston NI, Zaret KS, Wright CV, Stein RW, Gannon M
Ptf1a binds to and activates area III, a highly conserved region of the Pdx1 promoter that mediates early pancreas-wide Pdx1 expression.
Mol Cell Biol. 2007 Jun;27(11):4093-104.
The critical pancreatic transcription factor Pdx1 is expressed throughout the pancreas early but enriched in insulin-producing beta cells postnatally. Previous studies showed that the 5' conserved promoter regions areas I and II (Pdx1(PB)) direct endocrine cell expression, while an adjacent region (Pdx1(XB)) containing conserved area III directs transient beta-cell expression. In this study, we used Cre-mediated lineage tracing to track cells that activated these regions. Pdx1(PB)Cre mediated only endocrine cell recombination, while Pdx1(XB)Cre directed broad and early recombination in the developing pancreas. Also, a reporter transgene containing areas I, II, and III was expressed throughout the embryonic day 10.5 (E10.5) pancreas and gradually became beta cell enriched, similar to endogenous Pdx1. These data suggested that sequences within area III mediate early pancreas-wide Pdx1 expression. Area III contains a binding site for PTF1, a transcription factor complex essential for pancreas development. This site contributed to area III-dependent reporter gene expression in the acinar AR42J cell line, while PTF1 specifically trans-activated area III-containing reporter expression in a nonpancreatic cell line. Importantly, Ptf1a occupied sequences spanning the endogenous PTF1 site in area III of E11.5 pancreatic buds. These data strongly suggest that PTF1 is an important early activator of Pdx1 in acinar and endocrine progenitor cells during pancreas development. [Abstract/Link to Full Text]

Sumi K, Tanaka T, Uchida A, Magoori K, Urashima Y, Ohashi R, Ohguchi H, Okamura M, Kudo H, Daigo K, Maejima T, Kojima N, Sakakibara I, Jiang S, Hasegawa G, Kim I, Osborne TF, Naito M, Gonzalez FJ, Hamakubo T, Kodama T, Sakai J
Cooperative interaction between hepatocyte nuclear factor 4 alpha and GATA transcription factors regulates ATP-binding cassette sterol transporters ABCG5 and ABCG8.
Mol Cell Biol. 2007 Jun;27(12):4248-60.
Cholesterol homeostasis is maintained by coordinate regulation of cholesterol synthesis and its conversion to bile acids in the liver. The excretion of cholesterol from liver and intestine is regulated by ATP-binding cassette half-transporters ABCG5 and ABCG8. The genes for these two proteins are closely linked and divergently transcribed from a common intergenic promoter region. Here, we identified a binding site for hepatocyte nuclear factor 4alpha (HNF4alpha) in the ABCG5/ABCG8 intergenic promoter, through which HNF4alpha strongly activated the expression of a reporter gene in both directions. The HNF4alpha-responsive element is flanked by two conserved GATA boxes that were also required for stimulation by HNF4alpha. GATA4 and GATA6 bind to the GATA boxes, coexpression of GATA4 and HNF4alpha leads to a striking synergistic activation of both the ABCG5 and the ABCG8 promoters, and binding sites for HNF4alpha and GATA were essential for maximal synergism. We also show that HNF4alpha, GATA4, and GATA6 colocalize in the nuclei of HepG2 cells and that a physical interaction between HNF4alpha and GATA4 is critical for the synergistic response. This is the first demonstration that HNF4alpha acts synergistically with GATA factors to activate gene expression in a bidirectional fashion. [Abstract/Link to Full Text]

Luke-Glaser S, Roy M, Larsen B, Le Bihan T, Metalnikov P, Tyers M, Peter M, Pintard L
CIF-1, a shared subunit of the COP9/signalosome and eukaryotic initiation factor 3 complexes, regulates MEL-26 levels in the Caenorhabditis elegans embryo.
Mol Cell Biol. 2007 Jun;27(12):4526-40.
The COP9/signalosome (CSN) is an evolutionarily conserved macromolecular complex that regulates the cullin-RING ligase (CRL) class of E3 ubiquitin ligases, primarily by removing the ubiquitin-like protein Nedd8 from the cullin subunit. In the Caenorhabditis elegans embryo, the CSN controls the degradation of the microtubule-severing protein MEI-1 through CUL-3 deneddylation. However, the molecular mechanisms of CSN function and its subunit composition remain to be elucidated. Here, using a proteomic approach, we have characterized the CSN and CUL-3 complexes from C. elegans embryos. We show that the CSN physically interacts with the CUL-3-based CRL and regulates its activity by counteracting the autocatalytic instability of the substrate-specific adaptor MEL-26. Importantly, we identified the uncharacterized protein K08F11.3/CIF-1 (for CSN-eukaryotic initiation factor 3 [eIF3]) as a stoichiometric and functionally important subunit of the CSN complex. CIF-1 appears to be the only ortholog of Csn7 encoded by the C. elegans genome, but it also exhibits extensive sequence similarity to eIF3m family members, which are required for the initiation of protein translation. Indeed, CIF-1 binds eIF-3.F and inactivation of cif-1 impairs translation in vivo. Taken together, our results indicate that CIF-1 is a shared subunit of the CSN and eIF3 complexes and may therefore link protein translation and degradation. [Abstract/Link to Full Text]

Zapater M, Sohrmann M, Peter M, Posas F, de Nadal E
Selective requirement for SAGA in Hog1-mediated gene expression depending on the severity of the external osmostress conditions.
Mol Cell Biol. 2007 Jun;27(11):3900-10.
Regulation of gene expression by the Hog1 stress-activated protein kinase is essential for proper cell adaptation to osmostress. Hog1 coordinates an extensive transcriptional program through the modulation of transcription. To identify systematically novel components of the transcriptional machinery required for osmostress-mediated gene expression, we performed an exhaustive genome-wide genetic screening, searching for mutations that render cells osmosensitive at high osmolarity and that are associated with reduced expression of osmoresponsive genes. The SAGA and Mediator complexes were identified as putative novel regulators of osmostress-mediated transcription. Interestingly, whereas Mediator is essential for osmostress gene expression, the requirement for SAGA is different depending on the strength of the extracellular osmotic conditions. At mild osmolarity, SAGA mutants show only very slight defects on RNA polymerase II (Pol II) recruitment and gene expression, whereas at severe osmotic conditions, SAGA mutants show completely impaired RNA Pol II recruitment and transcription of osmoresponsive genes. Thus, our results define an essential role for Mediator in osmostress gene expression and a selective role for SAGA under severe osmostress. Our results indicate that the requirement for a transcriptional complex to regulate a promoter might be determined by the strength of the stimuli perceived by the cell through the regulation of interactions between transcriptional complexes. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Cell Science

---

Schober JM, Komarova YA, Chaga OY, Akhmanova A, Borisy GG
Microtubule-targeting-dependent reorganization of filopodia.
J Cell Sci. 2007 Apr 1;120(Pt 7):1235-44.
Interaction between the microtubule system and actin cytoskeleton has emerged as a fundamental process required for spatial regulation of cell protrusion and retraction activities. In our current studies, analysis of digital fluorescence images revealed targeting of microtubules to filopodia in B16F1 melanoma cells and fibroblasts. We investigated the functional consequence of targeting on filopodia reorganization and examined mechanisms by which microtubules may be guided to, or interact with, filopodia. Live cell imaging studies show that targeting events in lamellipodia wings temporally correlated with filopodia turning toward the lamellipodium midline and with filopodia merging. Rapid uncoupling of targeting with nocodazole decreased filopodia merging events and increased filopodia density. Total internal reflection fluorescence microscopy identified microtubules near the ventral surface and upward movement of targeted filopodia. The role of adhesion sites and microtubule plus-end proteins in targeting was investigated. Correlation of adhesion sites with microtubule targeting to filopodia was not observed and depletion of microtubule plus-end proteins did not significantly alter targeting frequency. We propose that microtubules target filopodia, independent of focal adhesions and plus-end proteins, causing filopodia movement and microtubules regulate filopodia density in lamellipodia wings through filopodia merging events. [Abstract/Link to Full Text]


The more the merrier. By Caveman.
J Cell Sci. 2007 Jan 15;120(Pt 2):201-3. [Abstract/Link to Full Text]

Keady BT, Kuo P, Martínez SE, Yuan L, Hake LE
MAPK interacts with XGef and is required for CPEB activation during meiosis in Xenopus oocytes.
J Cell Sci. 2007 Mar 15;120(Pt 6):1093-103.
Meiotic progression in Xenopus oocytes, and all other oocytes investigated, is dependent on polyadenylation-induced translation of stockpiled maternal mRNAs. Early during meiotic resumption, phosphorylation of CPE-binding protein (CPEB) is required for polyadenylation-induced translation of mRNAs encoding cell cycle regulators. Xenopus Gef (XGef), a Rho-family guanine-exchange factor, influences the activating phosphorylation of CPEB. An exchange-deficient version of XGef does not, therefore implicating Rho-family GTPase function in early meiosis. We show here that Clostridium difficile Toxin B, a Rho-family GTPase inhibitor, does not impair early CPEB phosphorylation or progression to germinal vesicle breakdown, indicating that XGef does not influence these events through activation of a Toxin-B-sensitive GTPase. Using the inhibitors U0126 for mitogen-activated protein kinase (MAPK), and ZM447439 for Aurora kinase A and Aurora kinase B, we found that MAPK is required for phosphorylation of CPEB, whereas Aurora kinases are not. Furthermore, we do not detect active Aurora kinase A in early meiosis. By contrast, we observe an early, transient activation of MAPK, independent of Mos protein expression. MAPK directly phosphorylates CPEB on four residues (T22, T164, S184, S248), but not on S174, a key residue for activating CPEB function. Notably, XGef immunoprecipitates contain MAPK, and this complex can phosphorylate CPEB. MAPK may prime CPEB for phosphorylation on S174 by an as-yet-unidentified kinase or may activate this kinase. [Abstract/Link to Full Text]

Moens PB, Marcon E, Shore JS, Kochakpour N, Spyropoulos B
Initiation and resolution of interhomolog connections: crossover and non-crossover sites along mouse synaptonemal complexes.
J Cell Sci. 2007 Mar 15;120(Pt 6):1017-27.
Programmed double-strand breaks at prophase of meiosis acquire immunologically detectable RAD51-DMC1 foci or early nodules (ENs) that are associated with developing chromosome core segments; each focus is surrounded by a gammaH2AX-modified chromosome domain. The 250-300 ENs per nucleus decline in numbers during the development of full-length cores and the remaining foci are relatively evenly distributed along the mature cores (gamma distribution of nu=2.97). The ENs become transformed nodules (TNs) by the acquisition of RPA, BLM, MSH4 and topoisomerases that function in repair and Holliday junction resolution. At the leptotene-zygotene transition, TNs orient to positions between the aligned cores where they initiate structural interhomolog contacts prior to synaptonemal complex (SC) formation, possibly future crossover sites. Subsequently, TNs are associated with SC extension at the synaptic forks. Dephosphorylation of TN-associated histone gammaH2AX chromatin suggests annealing of single strands or repair of double-strand breaks DSBs at this time. Some 200 TNs per pachytene nucleus are distributed proportional to SC length and are evenly distributed along the SCs (nu= approximately 4). At this stage, gammaH2AX-modified chromatin domains are associated with transcriptionally silenced sex chromosomes and autosomal sites. Immunogold electron microscope evidence shows that one or two TNs of the 10-15 TNs per SC acquire MLH1 protein, the hallmark of reciprocal recombination, whereas the TNs that do not acquire MLH1 protein relocate from their positions along the midline of the SCs to the periphery of the SCs. Relocation of TNs may be associated with the conversion of potential crossovers into non-crossovers. [Abstract/Link to Full Text]

Kawaguchi N, Horiuchi K, Becherer JD, Toyama Y, Besmer P, Blobel CP
Different ADAMs have distinct influences on Kit ligand processing: phorbol-ester-stimulated ectodomain shedding of Kitl1 by ADAM17 is reduced by ADAM19.
J Cell Sci. 2007 Mar 15;120(Pt 6):943-52.
Kit ligand (Kitl), the ligand for the Kit receptor tyrosine kinase, plays important roles in hematopoiesis, gametogenesis and melanogenesis. Kitl is synthesized as a membrane-anchored precursor that can be processed to produce the soluble growth factor. Here, we evaluated the role of ADAM (a disintegrin and metalloprotease) metalloproteases in ectodomain shedding of Kitl. We found that both ADAM17 and ADAM19 affect Kitl1 shedding, albeit in different ways. Overexpression of ADAM19 resulted in decreased levels of Endo-H-resistant mature Kitl1, thereby reducing the amount of Kitl that is shed from cells following stimulation with phorbol esters. ADAM17 was identified as the major phorbol-ester-stimulated sheddase of Kitl1, whereas ADAMs 8, 9, 10, 12 and 15 were not required for this process. ADAM17 also emerged as the major constitutive and phorbol-ester-stimulated sheddase of Kitl2 in mouse embryonic fibroblasts. Mutagenesis of the juxtamembrane domain of Kitl2 showed no stringent sequence requirement for cleavage by ADAM17, although two nonadjacent stretches of four amino acid residues were identified that are required for Kitl2 shedding. Taken together, this study identifies a novel sheddase, ADAM17, for Kitl1 and Kitl2, and demonstrates that ADAM19 can reduce ADAM17-dependent phorbol-ester-stimulated Kitl1 ectodomain shedding. [Abstract/Link to Full Text]

Arora P, Ricks TK, Trejo J
Protease-activated receptor signalling, endocytic sorting and dysregulation in cancer.
J Cell Sci. 2007 Mar 15;120(Pt 6):921-8.
Protease-activated receptors (PARs) are G-protein-coupled receptors (GPCRs) that are activated by a unique proteolytic mechanism. PARs play crucial roles in hemostasis and thrombosis, as well as in inflammation and vascular development. Coagulant proteases, which are generated at sites of vascular injury, act mainly through PARs to elicit signalling in a variety of cell types. Since PARs are irreversibly activated signalling must be tightly regulated. Desensitization and trafficking of proteolytically activated PARs control the magnitude, duration and spatial aspects of receptor signalling. Recent studies have revealed novel endocytic sorting mechanisms that regulate PAR signalling. PARs have also been implicated in tumor progression. PARs are overexpressed in several types of malignant cancer, transmit signals in response to tumor-generated proteases and promote tumor growth, invasion and metastasis. Recent work also indicates that matrix metalloprotease 1 (MMP-1) signals through PAR1 to promote tumor growth and invasion. In addition to PAR overexpression, tumor cells display aberrant PAR1 trafficking, which causes persistent signalling and cellular invasion. Thus, a novel type of gain-of-function in GPCR signalling in cancer can be acquired through dysregulation of receptor trafficking. [Abstract/Link to Full Text]

Chen CL, Hsieh YT, Chen HC
Phosphorylation of adducin by protein kinase Cdelta promotes cell motility.
J Cell Sci. 2007 Apr 1;120(Pt 7):1157-67.
Protein kinase Cdelta (PKCdelta) has been implicated to play a crucial role in cell proliferation, differentiation and apoptosis. In this study, we have investigated the role of PKCdelta in cell motility using Madin-Darby canine kidney cells. Overexpression of PKCdelta promoted membrane protrusions, concomitant with increased cell motility. By contrast, suppression of PKCdelta expression by RNA interference inhibited cell motility. Moreover, a fraction of PKCdelta was detected at the edge of membrane protrusions in which it colocalized with adducin, a membrane skeletal protein whose phosphorylation state is important for remodeling of the cortical actin cytoskeleton. Elevated expression of PKCdelta correlated with increased phosphorylation of adducin at Ser726 in intact cells. In vitro, PKCdelta, but not PKCalpha, directly phosphorylated the Ser726 of adducin. Finally, we demonstrated that overexpression of both adducin and PKCdelta could generate a synergistic effect on promoting cell spreading and cell migration. Our results support a positive role for PKCdelta in cell motility and strongly suggest a link between PKCdelta activity, adducin phosphorylation and cell motility. [Abstract/Link to Full Text]

Muretta JM, Romenskaia I, Cassiday PA, Mastick CC
Expression of a synapsin IIb site 1 phosphorylation mutant in 3T3-L1 adipocytes inhibits basal intracellular retention of Glut4.
J Cell Sci. 2007 Apr 1;120(Pt 7):1168-77.
Glut4 exocytosis in adipocytes uses protein machinery that is shared with other regulated secretory processes. Synapsins are phosphoproteins that regulate a ;reserve pool' of vesicles clustered behind the active zone in neurons. We found that adipocytes (primary cells and the 3T3-L1 cell line) express synapsin IIb mRNA and protein. Synapsin IIb co-localizes with Glut4 in perinuclear vesicle clusters. To test whether synapsin plays a role in Glut4 traffic, a site 1 phosphorylation mutant (S10A synapsin) was expressed in 3T3-L1 adipocytes. Interestingly, expression of S10A synapsin increased basal cell surface Glut4 almost fourfold (50% maximal insulin effect). Insulin caused a further twofold translocation of Glut4 in these cells. Expression of the N-terminus of S10A synapsin (amino acids 1-118) was sufficient to inhibit basal Glut4 retention. Neither wild-type nor S10D synapsin redistributed Glut4. S10A synapsin did not elevate surface levels of the transferrin receptor in adipocytes or Glut4 in fibroblasts. Therefore, S10A synapsin is inhibiting the specialized process of basal intracellular retention of Glut4 in adipocytes, without affecting general endocytic cycling. While mutant forms of many proteins inhibit Glut4 exocytosis in response to insulin, S10A synapsin is one of only a few that specifically inhibits Glut4 retention in basal adipocytes. These data indicate that the synapsins are important regulators of membrane traffic in many cell types. [Abstract/Link to Full Text]

Primrose DA, Chaudhry S, Johnson AG, Hrdlicka A, Schindler A, Tran D, Foley E
Interactions of DNR1 with the apoptotic machinery of Drosophila melanogaster.
J Cell Sci. 2007 Apr 1;120(Pt 7):1189-99.
Caspases are crucial activators of apoptosis and NF-kappaB signaling in vertebrates and invertebrates. In Drosophila, the caspase-9 counterpart Dronc is essential for most apoptotic death, whereas the caspase-8 homolog Dredd activates NF-kappaB signaling in response to gram-negative bacterial infection. The mechanics of caspase regulation are conserved and include the activities of a family of inhibitor of apoptosis (IAP) proteins. The RING-domain-bearing protein Defense repressor 1 (Dnr1), blocks ectopic Dredd-mediated induction of an NF-kappaB reporter in the Drosophila S2 cell line. In this study, we present novel data indicating that Dnr1 impacts on Dronc-dependent regulation of the apoptotic program. We show that depletion of Dnr1 results in elevated Dronc protein levels, which translates to increased caspase activation and activity upon induction of apoptosis. Conversely, we demonstrate that overexpression of Dnr1 blocks apoptotic caspase activity and prevents induction of apoptosis in tissue culture assays. Furthermore, we show that Dnr1 overexpression significantly reduces Dronc protein levels and identify the domains of Dnr1 necessary for these effects. From these data, we propose that Dnr1 inhibits initiator caspases in S2 cells. [Abstract/Link to Full Text]

Zunino R, Schauss A, Rippstein P, Andrade-Navarro M, McBride HM
The SUMO protease SENP5 is required to maintain mitochondrial morphology and function.
J Cell Sci. 2007 Apr 1;120(Pt 7):1178-88.
Mitochondria are dynamic organelles that undergo regulated fission and fusion events that are essential to maintain metabolic stability. We previously demonstrated that the mitochondrial fission GTPase DRP1 is a substrate for SUMOylation. To further understand how SUMOylation impacts mitochondrial function, we searched for a SUMO protease that may affect mitochondrial dynamics. We demonstrate that the cytosolic pool of SENP5 catalyzes the cleavage of SUMO1 from a number of mitochondrial substrates. Overexpression of SENP5 rescues SUMO1-induced mitochondrial fragmentation that is partly due to the downregulation of DRP1. By contrast, silencing of SENP5 results in a fragmented and altered morphology. DRP1 was stably mono-SUMOylated in these cells, suggesting that SUMOylation leads to increased DRP1 mediated fission. In addition, the reduction of SENP5 levels resulted in a significant increase in the production of free radicals. Reformation of the mitochondrial tubules by expressing the dominant interfering DRP1 or by RNA silencing of endogenous DRP1 protein rescued both the morphological aberrations and the increased production of ROS induced by downregulation of SENP5. These data demonstrate the importance of SENP5 as a new regulator of SUMO1 proteolysis from mitochondrial targets, impacting mitochondrial morphology and metabolism. [Abstract/Link to Full Text]

Wojciak-Stothard B, Torondel B, Tsang LY, Fleming I, Fisslthaler B, Leiper JM, Vallance P
The ADMA/DDAH pathway is a critical regulator of endothelial cell motility.
J Cell Sci. 2007 Mar 15;120(Pt 6):929-42.
Asymmetric dimethylarginine (ADMA) is an inhibitor of nitric oxide production associated with abnormal blood vessel growth and repair, however, the mechanism of action of ADMA is not well understood. We studied the role of exogenous and endogenous ADMA in the regulation of cell motility and actin cytoskeleton in porcine pulmonary endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMECs) from knockout mice that lack one of the enzyme metabolising ADMA, dimethylarginine dimethylaminohydrolase I (DDAHI) as well as endothelial cells overexpressing DDAH in vitro. We show that ADMA induced stress fibre and focal adhesion formation and inhibited cell motility in primary pulmonary endothelial cells. The effects of ADMA depended on the activity of RhoA and Rho kinase and were reversed by overexpression of DDAH, nitric oxide donors and protein kinase G activator, 8-bromo-cGMP. ADMA also inhibited the activities of Rac1 and Cdc42 in cells but these changes had a minor effect on cell motility. Endogenous ADMA increased RhoA activity and inhibited cell motility in PMECs from DDAHI knockout mice and inhibited angiogenesis in vitro. These results are the first demonstration that metabolism of cardiovascular risk factor ADMA regulates endothelial cell motility, an important factor in angiogenesis and vascular repair. [Abstract/Link to Full Text]

Hojilla CV, Kim I, Kassiri Z, Fata JE, Fang H, Khokha R
Metalloproteinase axes increase beta-catenin signaling in primary mouse mammary epithelial cells lacking TIMP3.
J Cell Sci. 2007 Mar 15;120(Pt 6):1050-60.
Multiple cancers exhibit mutations in beta-catenin that lead to increased stability, altered localization or amplified activity. beta-catenin is situated at the junction between the cadherin-mediated cell adhesion and Wnt signaling pathways, and TIMP3 functions to alter beta-catenin signaling. Here we demonstrate that primary mouse embryonic fibroblasts (MEFs) and mammary epithelial cells (MECs) deficient in Timp3 have increased beta-catenin signaling. Functionally, the loss of TIMP3 exerted cell-type-specific effects, with Timp3(-/-) MEFs being more sensitive and Timp3(-/-) MECs more resistant to EGTA-induced cell detachment than the wild type. Timp3(-/-) MECs had higher dephosphorylated beta-catenin levels and increased beta-catenin transcriptional activity as measured by TCF/LEF-responsive reporter assays. Real-time PCR analysis of beta-catenin target genes in MEFs and MECs showed no alteration in Myc, decreased Ccnd1 (cyclin D1) and increased Mmp7 mRNA levels upon loss of TIMP3, with the latter occurring only in epithelial cells. Recombinant TIMP3 and synthetic metalloproteinase inhibitors reverted the increase in dephosphorylated beta-catenin, decrease in Ccnd1 gene expression and increase in Mmp7 gene expression. Physiologically, Timp3(-/-) mammary glands displayed accelerated mammary ductal elongation during pubertal morphogenesis. Gain-of-function studies using slow-release TIMP-containing pellets revealed distinct effects of individual TIMPs on ductal morphogenesis. Recombinant TIMP1, TIMP3 and TIMP4 inhibited ductal elongation whereas TIMP2 promoted this process. [Abstract/Link to Full Text]

Mongiu AK, Weitzke EL, Chaga OY, Borisy GG
Kinetic-structural analysis of neuronal growth cone veil motility.
J Cell Sci. 2007 Mar 15;120(Pt 6):1113-25.
Neuronal growth cone advance was investigated by correlative light and electron microscopy carried out on chick dorsal root ganglion cells. Advance was analyzed in terms of the two principal organelles responsible for protrusive motility in the growth cone - namely, veils and filopodia. Veils alternated between rapid phases of protrusion and retraction. Electron microscopy revealed characteristic structural differences between the phases. Our results provide a significant advance in three respects: first, protruding veils are comprised of a densely branched network of actin filaments that is lamellipodial in appearance and includes the Arp2/3 complex. On the basis of this structural and biomarker evidence, we infer that the dendritic nucleation and/or array-treadmilling mechanism of protrusive motility is conserved in veil protrusion of growth cones as in the motility of fibroblasts; second, retracting veils lack dendritic organization but contain a sparse network of long filaments; and third, growth cone filopodia have the capacity to nucleate dendritic networks along their length, a property consistent with veil formation seen at the light microscopic level but not previously understood in supramolecular terms. These elements of veil and filopodial organization, when taken together, provide a conceptual framework for understanding the structural basis of growth cone advance. [Abstract/Link to Full Text]

Tran TH, Zeng Q, Hong W
VAMP4 cycles from the cell surface to the trans-Golgi network via sorting and recycling endosomes.
J Cell Sci. 2007 Mar 15;120(Pt 6):1028-41.
VAMP4 is enriched in the trans-Golgi network (TGN) and functions in traffic from the early and recycling endosomes to the TGN, but its trafficking itinerary is unknown. Cells stably expressing TGN-enriched VAMP4 C-terminally-tagged with EGFP (VAMP4-EGFP) are able to internalize and transport EGFP antibody efficiently to the TGN, suggesting that VAMP4-EGFP cycles between the cell surface and the TGN. The N-terminal extension of VAMP4 endows a chimeric VAMP5 with the ability to cycle from the surface to the TGN. Detailed time-course analysis of EGFP antibody transport to the TGN as well as pharmacological and thermal perturbation experiments suggest that VAMP4-EGFP is endocytosed by clathrin-dependent pathways and is delivered to the sorting and then recycling endosomes. This is followed by a direct transport to the TGN, without going through the late endosome. The di-Leu motif of the TGN-targeting signal is important for internalization, whereas the acidic cluster is crucial for efficient delivery of internalized antibody from the endosome to the TGN. These results suggest that the TGN-targeting signal of VAMP4 mediates the efficient recycling of VAMP4 from the cell surface to the TGN via the sorting and recycling endosomes, thus conferring steady-state enrichment of VAMP4 at the TGN. [Abstract/Link to Full Text]

Matsumoto M, Yaginuma K, Igarashi A, Imura M, Hasegawa M, Iwabuchi K, Date T, Mori T, Ishizaki K, Yamashita K, Inobe M, Matsunaga T
Perturbed gap-filling synthesis in nucleotide excision repair causes histone H2AX phosphorylation in human quiescent cells.
J Cell Sci. 2007 Mar 15;120(Pt 6):1104-12.
Human histone H2AX is rapidly phosphorylated on serine 139 in response to DNA double-strand breaks and plays a crucial role in tethering the factors involved in DNA repair and damage signaling. Replication stress caused by hydroxyurea or UV also initiates H2AX phosphorylation in S-phase cells, although UV-induced H2AX phosphorylation in non-cycling cells has recently been observed. Here we study the UV-induced H2AX phosphorylation in human primary fibroblasts under growth-arrested conditions. This reaction absolutely depends on nucleotide excision repair (NER) and is mechanistically distinct from the replication stress-induced phosphorylation. The treatment of cytosine-beta-D-arabinofuranoside strikingly enhances the NER-dependent H2AX phosphorylation and induces the accumulation of replication protein A (RPA) and ATR-interacting protein (ATRIP) at locally UV-damaged subnuclear regions. Consistently, the phosphorylation appears to be mainly mediated by ataxia-telangiectasia mutated and Rad3-related (ATR), although Chk1 (Ser345) is not phosphorylated by the activated ATR. The cellular levels of DNA polymerases delta and epsilon and proliferating cell nuclear antigen are markedly reduced in quiescent cells. We propose a model that perturbed gap-filling synthesis following dual incision in NER generates single-strand DNA gaps and hence initiates H2AX phosphorylation by ATR with the aid of RPA and ATRIP. [Abstract/Link to Full Text]

Tóth ML, Simon P, Kovács AL, Vellai T
Influence of autophagy genes on ion-channel-dependent neuronal degeneration in Caenorhabditis elegans.
J Cell Sci. 2007 Mar 15;120(Pt 6):1134-41.
Necrotic cell death is a common feature in numerous human neurodegenerative disorders. In the nematode Caenorhabditis elegans, gain-of-function mutations in genes that encode specific ion channel subunits such as the degenerins DEG-1 and MEC-4, and the acetylcholine receptor subunit DEG-3 lead to necrotic-like degeneration of a subset of neurons. Neuronal demise caused by ion channel hyperactivity is accompanied by intense degradation of cytoplasmic contents, dramatic membrane infolding and vacuole formation; however, the cellular pathways underlying such processes remain largely unknown. Here we show that the function of three autophagy genes, whose yeast and mammalian orthologs are implicated in cytoplasmic self-degradation, membrane trafficking and the cellular response to starvation, contributes to ion-channel-dependent neurotoxicity in C. elegans. Inactivation of unc-51, bec-1 and lgg-1, the worm counterparts of the yeast autophagy genes Atg1, Atg6 and Atg8 respectively, partially suppresses degeneration of neurons with toxic ion channel variants. We also demonstrate that the TOR-kinase-mediated signaling pathway, a nutrient sensing system that downregulates the autophagy gene cascade, protects neurons from undergoing necrotic cell death, whereas nutrient deprivation promotes necrosis. Our findings reveal a role for autophagy genes in neuronal cell loss in C. elegans. [Abstract/Link to Full Text]

Domínguez-Giménez P, Brown NH, Martín-Bermudo MD
Integrin-ECM interactions regulate the changes in cell shape driving the morphogenesis of the Drosophila wing epithelium.
J Cell Sci. 2007 Mar 15;120(Pt 6):1061-71.
During development, morphogenesis involves migration and changes in the shape of epithelial sheets, both of which require coordination of cell adhesion. Thus, while modulation of integrin-mediated adhesion to the ECM regulates epithelial motility, cell-cell adhesion via cadherins controls the remodelling of epithelial sheets. We have used the Drosophila wing epithelium to demonstrate that cell-ECM interactions mediated by integrins also regulate the changes in cell shape that underly epithelial morphogenesis. We show that integrins control the transitions from columnar to cuboidal cell shape underlying wing formation, and we demonstrate that eliminating the ECM has the same effect on cell shape as inhibiting integrin function. Furthermore, lack of integrin activity also induces detachment of the basal lamina and failure to assemble the basal matrix. Hence, we propose that integrins control epithelial cell shape by mediating adherence of these cells to the ECM. Finally, we show that the ECM has an instructive rather than a structural role, because inhibition of Raf reverses the cell shape changes caused by perturbing integrins. [Abstract/Link to Full Text]

Röper K
Rtnl1 is enriched in a specialized germline ER that associates with ribonucleoprotein granule components.
J Cell Sci. 2007 Mar 15;120(Pt 6):1081-92.
During oogenesis in Drosophila an organelle called the fusome plays a crucial role in germline cyst development and oocyte selection. The fusome consists of cytoskeletal proteins and intracellular membranes and, whereas many cytoskeletal components have been characterized, the nature and function of the membrane component is poorly understood. I have found the reticulon-like 1 (Rtnl1) protein, a membrane protein resident in the endoplasmic reticulum (ER), to be highly enriched in the fusome. In other Drosophila tissues Rtnl1 marks a subset of ER membranes often derived from smooth ER. During oogenesis, Rtnl1-containing membranes are recruited to the fusome by the cytoskeletal components and become concentrated into the forming oocyte. On the central part of the fusome, which is contained within the future oocyte and also at later stages in the growing oocyte and the nurse cells, Rtnl1-containing membranes colocalize with components of ribonucleoprotein complexes that store translationally repressed mRNAs. As the ER is actively transported into the oocyte, this colocalization suggests a role for the Rtnl1-containing subdomain in anchoring the ribonucleoprotein complexes within and/or transporting them into the oocyte. [Abstract/Link to Full Text]

Bongiorni S, Pasqualini B, Taranta M, Singh PB, Prantera G
Epigenetic regulation of facultative heterochromatinisation in Planococcus citri via the Me(3)K9H3-HP1-Me(3)K20H4 pathway.
J Cell Sci. 2007 Mar 15;120(Pt 6):1072-80.
Using RNA interference (RNAi) we have conducted a functional analysis of the HP1-like chromobox gene pchet2 during embryogenesis of the mealybug Planococcus citri. Knocking down pchet2 expression results in decondensation of the male-specific chromocenter that normally arises from the developmentally-regulated facultative heterochromatinisation of the paternal chromosome complement. Together with the disappearance of the chromocenter the staining levels of two associated histone modifications, tri-methylated lysine 9 of histone H3 [Me(3)K9H3] and tri-methylated lysine 20 of histone H4 [Me(3)K20H4], are reduced to undetectable levels. Embryos treated with double-stranded RNA (dsRNA) targeting pchet2 also exhibit chromosome abnormalities, such as aberrant chromosome condensation, and also the presence of metaphases that contain 'lagging' chromosomes. We conclude that PCHET2 regulates chromosome behavior during metaphase and is a crucial component of a Me(3)K9H3-HP1-Me(3)K20H4 pathway involved in the facultative heterochromatinisation of the (imprinted) paternal chromosome set. [Abstract/Link to Full Text]

Abdu U, Klovstad M, Butin-Israeli V, Bakhrat A, Schüpbach T
An essential role for Drosophila hus1 in somatic and meiotic DNA damage responses.
J Cell Sci. 2007 Mar 15;120(Pt 6):1042-9.
The checkpoint proteins Rad9, Rad1 and Hus1 form a clamp-like complex which plays a central role in the DNA-damage-induced checkpoint response. Here we address the function of the 9-1-1 complex in Drosophila. We decided to focus our analysis on the meiotic and somatic requirements of hus1. For that purpose, we created a null allele of hus1 by imprecise excision of a P element found 2 kb from the 3' of the hus1 gene. We found that hus1 mutant flies are viable, but the females are sterile. We determined that hus1 mutant flies are sensitive to hydroxyurea and methyl methanesulfonate but not to X-rays, suggesting that hus1 is required for the activation of an S-phase checkpoint. We also found that hus1 is not required for the G2-M checkpoint and for post-irradiation induction of apoptosis. We subsequently studied the role of hus1 in activation of the meiotic checkpoint and found that the hus1 mutation suppresses the dorsal-ventral pattering defects caused by mutants in DNA repair enzymes. Interestingly, we found that the hus1 mutant exhibits similar oocyte nuclear defects as those produced by mutations in DNA repair enzymes. These results demonstrate that hus1 is essential for the activation of the meiotic checkpoint and that hus1 is also required for the organization of the oocyte DNA, a function that might be independent of the meiotic checkpoint. [Abstract/Link to Full Text]

Rey M, Valenzuela-Fernández A, Urzainqui A, Yáñez-Mó M, Pérez-Martínez M, Penela P, Mayor F, Sánchez-Madrid F
Myosin IIA is involved in the endocytosis of CXCR4 induced by SDF-1alpha.
J Cell Sci. 2007 Mar 15;120(Pt 6):1126-33.
Endocytosis of chemokine receptors regulates signal transduction initiated by chemokines, but the molecular mechanisms underlying this process are not fully defined. In this work, we assessed the involvement of the motor protein nonmuscle myosin heavy chain IIA (MIIA) in the endocytosis of CXCR4 induced by SDF-1alpha (also known as CXCL12) in T lymphocytes. Overexpression of the C-terminal half of MIIA inhibited the ligand-induced endocytosis of CXCR4, but not that of transferrin receptor. Targeting MIIA either by silencing its expression with small interfering RNA (siRNA) or by blebbistatin treatment also inhibited endocytosis of CXCR4. Inhibition of endocytosis of CXCR4 by targeting endogenous MIIA resulted in an increased migration of T cells induced by SDF-1alpha, and in the inhibition of the HIV-1-Env antifusogenic activity of this chemokine. Coimmunoprecipitation and protein-protein binding studies demonstrated that MIIA interacts with both the cytoplasmic tail of CXCR4 and beta-arrestin. Moreover, SDF-1alpha promotes a rapid MIIA-beta-arrestin dissociation. Our data reveal a novel role for MIIA in CXCR4 endocytosis, which involves its dynamic association with beta-arrestin and highlights the role of endogenous MIIA as a regulator of CXCR4 internalization and, therefore, the onset of SDF-1alpha signaling. [Abstract/Link to Full Text]

Pedersen LB, Rompolas P, Christensen ST, Rosenbaum JL, King SM
The lissencephaly protein Lis1 is present in motile mammalian cilia and requires outer arm dynein for targeting to Chlamydomonas flagella.
J Cell Sci. 2007 Mar 1;120(Pt 5):858-67.
Lissencephaly is a developmental brain disorder characterized by a smooth cerebral surface, thickened cortex and misplaced neurons. Classical lissencephaly is caused by mutations in LIS1, which encodes a WD-repeat protein involved in cytoplasmic dynein regulation, mitosis and nuclear migration. Several proteins required for nuclear migration in Aspergillus bind directly to Lis1, including NudC. Mammalian NudC is highly expressed in ciliated epithelia, and localizes to motile cilia in various tissues. Moreover, a NudC ortholog is upregulated upon deflagellation in Chlamydomonas. We found that mammalian Lis1 localizes to motile cilia in trachea and oviduct, but is absent from non-motile primary cilia. Furthermore, we cloned a gene encoding a Lis1-like protein (CrLis1) from Chlamydomonas. CrLis1 is a approximately 37 kDa protein that contains seven WD-repeat domains, similar to Lis1 proteins from other organisms. Immunoblotting using an anti-CrLis1 antibody revealed that this protein is present in the flagellum and is depleted from flagella of mutants with defective outer dynein arm assembly, including one strain that lacks only the alpha heavy chain/light chain 5 thioredoxin complex. Biochemical experiments confirmed that CrLis1 associates with outer dynein arm components and revealed that CrLis1 binds directly to rat NudC. Our results suggest that Lis1 and NudC are present in cilia and flagella and may regulate outer dynein arm activity. [Abstract/Link to Full Text]

Huang Y, Burkhardt JK
T-cell-receptor-dependent actin regulatory mechanisms.
J Cell Sci. 2007 Mar 1;120(Pt 5):723-30.
Following stimulation, T cells undergo marked changes in actin architecture that are required for productive immune responses. T-cell-receptor-dependent reorganization of the actin cytoskeleton is necessary for the formation of the immunological synapse at the T-cell-antigen-presenting-cell contact site and the distal pole complex at the opposite face of the T cell. Convergence of specific signaling molecules within these two plasma membrane domains facilitates downstream signaling events leading to full T-cell activation. Recent studies have identified many of the relevant actin-regulatory proteins, and significant progress has been made in our understanding of how these proteins choreograph molecular movements associated with T-cell activation. Proteins such as WASp, WAVE2, HS1 and cofilin direct the formation of a cortical actin scaffold at the immune synapse, while actin-binding proteins such as ezrin and moesin direct binding of signaling molecules to actin filaments within the distal pole complex. [Abstract/Link to Full Text]

Multani AS, Chang S
WRN at telomeres: implications for aging and cancer.
J Cell Sci. 2007 Mar 1;120(Pt 5):713-21.
Werner Syndrome (WS) is a premature aging syndrome characterized by early onset of age-related pathologies and cancer. Since WS is due to a single gene defect, it has attracted much interest from researchers seeking to understand pathways that contribute to cancer and aging at cellular and molecular levels. The protein mutated in WS, WRN, appears to play a major role in genome stability, particularly during DNA replication and telomere metabolism. Much of the pathophysiology associated with WS, including the rapid onset of cellular senescence, early cancer onset and premature aging, can be attributed to a defect in telomere maintenance. Recent genetic evidence from the mTerc(-/-) Wrn(-/-) mouse demonstrates that mice with critically shortened telomeres display aging phenotypes reminiscent of human WS, further reinforcing the notion that telomere dysfunction is required for the manifestation of aging pathophysiologies in the setting of WRN deficiency. [Abstract/Link to Full Text]

Delaunay JL, Breton M, Goding JW, Trugnan G, Maurice M
Differential detergent resistance of the apical and basolateral NPPases: relationship with polarized targeting.
J Cell Sci. 2007 Mar 15;120(Pt 6):1009-16.
Targeting of glycosylphosphatidylinositol-anchored proteins to the apical surface of epithelial cells involves clustering in Triton X-100-resistant membrane microdomains or rafts. The role of these microdomains in sorting transmembrane proteins is more questionable because, unlike glycosylphosphatidylinositol-anchored proteins, apical transmembrane proteins are rather soluble in Triton X-100. They are, however, resistant to milder detergents such as Lubrol WX or Tween 20. It has been proposed that specific membrane microdomains, defined by resistance to these detergents, would carry transmembrane proteins to the apical surface. We have used MDCK cells stably transfected with the apical and basolateral pyrophosphatases/phosphodiesterases, NPP3 and NPP1, to examine the relationship between detergent resistance and apical targeting. The apically expressed wild-type NPP3 was insoluble in Lubrol WX whereas wild-type NPP1, which is expressed basolaterally, was essentially soluble. By using tail mutants and chimeric constructs that combine the cytoplasmic, transmembrane and extracellular domains of NPP1 and NPP3, we show that there is not a strict correlation between detergent resistance and apical targeting. Lubrol resistance is an intrinsic property of NPP3, which is acquired early during the biosynthetic process irrespective of its final destination, and depends on positively charged residues in its cytoplasmic tail. [Abstract/Link to Full Text]

Scharpfenecker M, van Dinther M, Liu Z, van Bezooijen RL, Zhao Q, Pukac L, Löwik CW, ten Dijke P
BMP-9 signals via ALK1 and inhibits bFGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis.
J Cell Sci. 2007 Mar 15;120(Pt 6):964-72.
Genetic studies in mice and humans have shown that the transforming growth factor-beta (TGF-beta) type-I receptor activin receptor-like kinase 1 (ALK1) and its co-receptor endoglin play an important role in vascular development and angiogenesis. Here, we demonstrate that ALK1 is a signalling receptor for bone morphogenetic protein-9 (BMP-9) in endothelial cells (ECs). BMP-9 bound with high affinity to ALK1 and endoglin, and weakly to the type-I receptor ALK2 and to the BMP type-II receptor (BMPR-II) and activin type-II receptor (ActR-II) in transfected COS cells. Binding of BMP-9 to ALK2 was greatly facilitated when BMPR-II or ActR-II were co-expressed. Whereas BMP-9 predominantly bound to ALK1 and BMPR-II in ECs, it bound to ALK2 and BMPR-II in myoblasts. In addition, we observed binding of BMP-9 to ALK1 and endoglin in glioblastoma cells. BMP-9 activated Smad1 and/or Smad5, and induced ID1 protein and endoglin mRNA expression in ECs. Furthermore, BMP-9 was found to inhibit basic fibroblast growth factor (bFGF)-stimulated proliferation and migration of bovine aortic ECs (BAECs) and to block vascular endothelial growth factor (VEGF)-induced angiogenesis. Taken together, these results suggest that BMP-9 is a physiological ALK1 ligand that plays an important role in the regulation of angiogenesis. [Abstract/Link to Full Text]

Peter AK, Miller G, Crosbie RH
Disrupted mechanical stability of the dystrophin-glycoprotein complex causes severe muscular dystrophy in sarcospan transgenic mice.
J Cell Sci. 2007 Mar 15;120(Pt 6):996-1008.
The dystrophin-glycoprotein complex spans the muscle plasma membrane and provides a mechanical linkage between laminin in the extracellular matrix and actin in the intracellular cytoskeleton. Within the dystrophin-glycoprotein complex, the sarcoglycans and sarcospan constitute a subcomplex of transmembrane proteins that stabilize alpha-dystroglycan, a receptor for laminin and other components of the extracellular matrix. In order to elucidate the function of sarcospan, we generated transgenic mice that overexpress sarcospan in skeletal muscle. Sarcospan transgenic mice with moderate (tenfold) levels of sarcospan overexpression exhibit a severe phenotype that is similar to mouse models of laminin-deficient congenital muscular dystrophy (MD). Sarcospan transgenic mice display severe kyphosis and die prematurely between 6 and 10 weeks of age. Histological analysis reveals that sarcospan expression causes muscle pathology marked by increased muscle fiber degeneration and/or regeneration. Sarcospan transgenic muscle does not display sarcolemma damage, which is distinct from dystrophin- and sarcoglycan-deficient muscular dystrophies. We show that sarcospan clusters the sarcoglycans into insoluble protein aggregates and causes destabilization of alpha-dystroglycan. Evidence is provided to demonstrate abnormal extracellular matrix assembly, which represents a probable pathological mechanism for the severe and lethal dystrophic phenotype. Taken together, these data suggest that sarcospan plays an important mechanical role in stabilizing the dystrophin-glycoprotein complex. [Abstract/Link to Full Text]

Rasmussen HB, Frøkjaer-Jensen C, Jensen CS, Jensen HS, Jørgensen NK, Misonou H, Trimmer JS, Olesen SP, Schmitt N
Requirement of subunit co-assembly and ankyrin-G for M-channel localization at the axon initial segment.
J Cell Sci. 2007 Mar 15;120(Pt 6):953-63.
The potassium channel subunits KCNQ2 and KCNQ3 are believed to underlie the M current of hippocampal neurons. The M-type potassium current plays a key role in the regulation of neuronal excitability; however, the subcellular location of the ion channels underlying this regulation has been controversial. We report here that KCNQ2 and KCNQ3 subunits are localized to the axon initial segment of pyramidal neurons of adult rat hippocampus and in cultured hippocampal neurons. We demonstrate that the localization of the KCNQ2/3 channel complex to the axon initial segment is favored by co-expression of the two channel subunits. Deletion of the ankyrin-G-binding motif in both the KCNQ2 and KCNQ3 C-terminals leads to the disappearance of the complex from the axon initial segment, albeit the channel complex remains functional and still reaches the plasma membrane. We further show that although heteromeric assembly of the channel complex favours localization to the axon initial segment, deletion of the ankyrin-G-binding motif in KCNQ2 alone does not alter the subcellular localization of KCNQ2/3 heteromers. By contrast, deletion of the ankyrin-G-binding motif in KCNQ3 significantly reduces AIS enrichment of the complex, implicating KCNQ3 as a major determinant of M channel localization to the AIS. [Abstract/Link to Full Text]

Loane DJ, Lima PA, Marrion NV
Co-assembly of N-type Ca2+ and BK channels underlies functional coupling in rat brain.
J Cell Sci. 2007 Mar 15;120(Pt 6):985-95.
Activation of large conductance Ca(2+)-activated potassium (BK) channels hastens action potential repolarisation and generates the fast afterhyperpolarisation in hippocampal pyramidal neurons. A rapid coupling of Ca(2+) entry with BK channel activation is necessary for this to occur, which might result from an identified coupling of Ca(2+) entry through N-type Ca(2+) channels to BK channel activation. This selective coupling was extremely rapid and resistant to intracellular BAPTA, suggesting that the two channel types are close. Using reciprocal co-immunoprecipitation, we found that N-type channels were more abundantly associated with BK channels than L-type channels (Ca(V)1.2) in rat brain. Expression of only the pore-forming alpha-subunits of the N-type (Ca(V)2.2) and BK (Slo(27)) channels in a non-neuronal cell-line gave robust macroscopic currents and reproduced the interaction. Co-expression of Ca(V)2.2/Ca(V)beta(3) subunits with Slo(27) channels revealed rapid functional coupling. By contrast, extremely rare examples of rapid functional coupling were observed with co-expression of Ca(V)1.2/Ca(V)beta(3) and Slo(27) channels. Action potential repolarisation in hippocampal pyramidal neurons was slowed by the N-type channel blocker omega-conotoxin GVIA, but not by the L-type channel blocker isradipine. These data showed that selective functional coupling between N-type Ca(2+) and BK channels provided rapid activation of BK channels in central neurons. [Abstract/Link to Full Text]

Handley MT, Haynes LP, Burgoyne RD
Differential dynamics of Rab3A and Rab27A on secretory granules.
J Cell Sci. 2007 Mar 15;120(Pt 6):973-84.
We have assessed the dynamics of the association of Rab3A and Rab27A with secretory granules at various stages of their life in PC12 cells. Endogenous Rab3A colocalised with the secretory granule marker secretogranin II (SGII) and expressed EGFP-Rab3A and ECFP-Rab27A colocalised with one another. The extent of colocalisation between EGFP-Rab3A or EGFP-Rab27 and SGII increased after longer times post transfection suggesting that these Rab proteins are preferentially recruited to newly synthesised granules. Following the release of immature secretory granules from the trans-Golgi network, Rab3A and Rab27A became associated with the immature granules after a lag period of around 20 minutes. Rab dynamics on granules were analysed in fluorescence recovery after photobleaching (FRAP) experiments. The recovery profile of EGFP-Rab27A was comparable to that of ppANF-EGFP, whereas the recovery profile of EGFP-Rab3A was significantly faster, indicating that Rab3A but not Rab27A might be rapidly exchanged between granules and cytosol. Inhibition of heat-shock protein 90 with 10 muM geldanamycin did not affect the exchange process or regulated exocytosis. Rab dynamics during stimulation with 300 muM ATP were analysed in live cells. Loss of granular ppANF-EGFP fluorescence was seen at the cell periphery after stimulation but only limited changes in EGFP-Rab3A and EGFP-Rab27A fluorescence was observed, indicating that the Rab proteins do not immediately dissociate or disperse on stimulation. The data suggest potentially distinct roles for Rab3A and Rab27A and we suggest that the finding that young secretory granules have a higher capacity for binding Rab3A and Rab27A is functionally important for preferential exocytosis from these granules. [Abstract/Link to Full Text]

de Boer E, Dietrich AJ, Höög C, Stam P, Heyting C
Meiotic interference among MLH1 foci requires neither an intact axial element structure nor full synapsis.
J Cell Sci. 2007 Mar 1;120(Pt 5):731-6.
During meiosis, homologous chromosomes (homologs) perform reciprocal exchanges (crossovers) at a high frequency. Crossovers display interference, i.e. their spacing is more even than would be expected if they were placed randomly along the chromosomes. Concomitantly with crossover formation, synaptonemal complexes (SCs) appear between homologs: each chromosome forms an axial structure, the axial element (AE); the AEs of homologs align, and numerous transverse filaments connect the AEs to form an SC. Both the AE and the SC have been implicated in the imposition of interference. We investigated whether intact AEs or SCs are required for crossover interference in the mouse, using a mutant lacking AE protein SYCP3, which displays structurally abnormal AEs and incomplete synapsis. We estimated the level of interference from the spacing of immunofluorescent MLH1 foci, which mark almost all crossover sites in the mouse, along the SCs. The levels of interference among MLH1 foci in wild-type and Sycp3(-/-) mice were comparable, implying that neither an intact AE structure nor full synapsis is required for wild-type levels of interference. [Abstract/Link to Full Text]

Weston CA, Teressa G, Weeks BS, Prives J
Agrin and laminin induce acetylcholine receptor clustering by convergent, Rho GTPase-dependent signaling pathways.
J Cell Sci. 2007 Mar 1;120(Pt 5):868-75.
During neuromuscular junction formation, extracellular matrix-mediated signals cause muscle surface acetylcholine receptors (AChRs) to aggregate at synaptic sites. Two extracellular matrix proteins, agrin and laminin, have each been shown to initiate signaling pathways that culminate in AChR clustering in cultured muscle cells. Here we present evidence that laminin-induced AChR clustering is mediated by the activation of the Rho GTPases Cdc42, Rac and Rho. Clustering in response to laminin is blocked by the dominant negative mutants Cdc42N17, RacN17 and RhoN19, as well as by the Rho inhibitor C3 transferase. Moreover, laminin-induced AChR clustering is impaired by the Rho kinase inhibitor Y-27632. Agrin-induced AChR clustering has previously been shown to require activation of Cdc42, Rac and Rho. Therefore, although agrin and laminin use distinct transmembrane receptors to initiate AChR clustering, their signaling pathways converge at the level of Rho GTPase activation. [Abstract/Link to Full Text]

Benard G, Bellance N, James D, Parrone P, Fernandez H, Letellier T, Rossignol R
Mitochondrial bioenergetics and structural network organization.
J Cell Sci. 2007 Mar 1;120(Pt 5):838-48.
Mitochondria form a dynamic network, and it remains unclear how the alternate configurations interact with bioenergetics properties. The metabolic signals that link mitochondrial structure to its functional states have not been fully characterized. In this report, we analyze the bidirectional relationships between mitochondrial morphology and function in living human cells. First, we determined the effect of mitochondrial fission on energy production by using small interfering RNA (siRNA) targeting DRP1, which revealed the importance of membrane fluidity on the control of bioenergetics. Second, we followed the effect of rotenone, a specific inhibitor of respiratory chain complex I, which causes large structural perturbations, once a threshold was reached. Last, we followed changes in the mitochondrial network configuration in human cells that had been treated with modulators of oxidative phosphorylation, and in fibroblasts from two patients with mitochondrial disease where the respiratory rate, DeltaPsi and the generation of reactive oxygen species (ROS) were measured. Our data demonstrate that the relationship between mitochondrial network organization and bioenergetics is bidirectional, and we provide a model for analyzing the metabolic signals involved in this crosstalk. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Cellular and Molecular Medicine

---

Kriebardis AG, Antonelou MH, Stamoulis KE, Economou-Petersen E, Margaritis LH, Papassideri IS
Progressive oxidation of cytoskeletal proteins and accumulation of denatured hemoglobin in stored red cells.
J Cell Mol Med. 2007 Jan-Feb;11(1):148-55.
Red blood cell (RBC) membrane proteins undergo progressive pathological alterations during storage. In conditions of increased cellular stress, the cytoskeleton also sustains certain modifications. The hemoglobin (Hb) content and oxidative status of the RBC cytoskeletons as a function of the storage period remain unclear. The possible Hb content and oxidative alterations occurring in the cytoskeletons in the course of storage were monitored in six units, by means of electrophoresis, immunoblotting and protein carbonylation assays. A proportion of the ghost-bound Hb consists of non-reducible crosslinkings of probably oxidized(denatured Hb or hemichromes.The defective Hb-membrane association was strongly affected by the prolonged storage. A progressive accumulation of Hb monomers, multimers and high molecular weight aggregates to corresponding cytoskeletons were also evident. The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins. The reported data corroborate the evidence for oxidative damage in membrane proteins with emphasis to the cytoskeletal components. They partially address the pathophysiological mechanisms underlying the RBC storage lesion, add some new insight in the field of RBC storage as a hemoglobin- and cytoskeleton-associated pathology and suggest the possible use of antioxidants in the units intended for transfusion. [Abstract/Link to Full Text]

Walker T, Wendel HP, Tetzloff L, Raabe C, Heidenreich O, Simon P, Scheule AM, Ziemer G
Inhibition of adhesion molecule expression on human venous endothelial cells by non-viral siRNA transfection.
J Cell Mol Med. 2007 Jan-Feb;11(1):139-47.
OBJECTIVE: Expression of adhesion molecule receptors on venous endothelial cells crucially influences the fate of venous grafts by mediating leukocyte-endothelium interactions. These interactions include adhesion of leukocytes to the endothelium, followed by transendothelial migration, leading to neointimal hyperplasia (NIH) and finally graft occlusion. Therefore, inhibition of adhesion molecule expression may be a promising strategy to improve the quality of venous grafts.We tested the efficiency of non-viral transfection of human venous endothelial cells (HVEC) with short interfering RNA (siRNA) to specifically down-regulate adhesion molecule expression. METHODS: Primary cultures of HVEC were examined for expression of the adhesion molecules ICAM1, VCAM1 and E-selectin (SELE) after non viral siRNA transfection. Adhesion molecule expression was measured by flow cytometry, real-time polymerase chain reaction and immunoblotting after stimulation with TNF-alpha, an inflammatory cytokine. RESULTS: Non-transfected cells showed a strong increase of adhesion molecule expression following cytokine stimulation (P < 0.01). Upon transfection with specific siRNAs a sixfold decrease in ICAM1 (P < 0.001) and SELE expression and cell positivity (P < 0.05) and a twofold decrease in VCAM1 expression and cell positivity (P < 0.01) P could be observed. SiRNA-mediated gene suppression of adhesion molecules was also reflected by corresponding decreases in adhesion protein and transcript levels. CONCLUSIONS: The expression of adhesion molecules on HVECs can be effectively inhibited by specific siRNAs using a safe, non-viral transfection approach. This is a promising tool to pre-condition venous bypass grafts in order to interfere with endothelium-leukocyte interactions and to prohibit neointima thickening or atherosclerosis, which are regarded to be the most important causes of venous graft failure. [Abstract/Link to Full Text]

Szewczyk MM, Davis KA, Samson SE, Simpson F, Rangachari PK, Grover AK
Ca2+-pumps and Na2+-Ca2+-exchangers in coronary artery endothelium versus smooth muscle.
J Cell Mol Med. 2007 Jan-Feb;11(1):129-38.
Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+-Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases. [Abstract/Link to Full Text]

Gonzalez FJ, Quesada AR, Sevilla I, Baca JJ, Medina MA, Amores J, Diaz JM, Rius-Diaz F, Marques E, Alba E
Prognostic value of serum angiogenic activity in colorectal cancer patients.
J Cell Mol Med. 2007 Jan-Feb;11(1):120-8.
Angiogenesis, resulting from an imbalance between angiogenic activator factors and inhibitors, is required for tumour growth and metastasis. The determination of the circulating concentration of all angiogenic factors (activators and inhibitors) is not feasible at present. We have evaluated diagnostic and prognostic values of the measurement of serum angiogenic activity in colorectal carcinoma (CRC) patients. Serum proliferative activity (PA) on human umbilical vein endothelial cells (HUVEC) in vitro, and serum vascular endothelial growth factor (VEGF) levels were determined by ELISA in 53 patients with primary CRC, 16 subjects with non-neoplastic gastrointestinal disease (SC) and 34 healthy individuals. Data were compared with clinical outcome of the patients. Although serum from CRC patients significantly increased the PA of HUVEC, compared to culture control (HUVEC in medium + 10% foetal bovine serum (FBS); P < 0.001); our results indicate that serum PA in CRC patients was similar to that of SC or healthy individuals. There was no correlation between serum PA and circulating VEGF concentrations. Surgery produced a decrease of PA at 8 hrs after tumour resection in CRC patients compared to pre-surgery values (186 +/- 47 versus 213 +/- 41, P < 0.001). However, an increase in serum VEGF values was observed after surgery (280 [176-450] versus 251 [160-357] pg/ml, P = 0.004). Patients with lower PA values after surgery showed a worse outcome that those with higher PA values. Therefore, this study does not support a diagnostic value for serum angiogenic activity measured by proliferative activity on HUVEC but suggests it could have a prognostic value in CRC patients. [Abstract/Link to Full Text]

Zhang Z, Fauser U, Schluesener HJ
Expression of RhoA by inflammatory macrophages and T cells in rat experimental autoimmune neuritis.
J Cell Mol Med. 2007 Jan-Feb;11(1):111-9.
RhoA is one of the best-studied members of Rho GTPases. Experimental autoimmune neuritis (EAN), which is characterized by infiltration of T cells and macrophages into the peripheral nervous system, is an autoantigen-specific T-cell-mediated animal model of human Guillain-Barré Syndrome. In this study, RhoA expression has been investigated in the dorsal/ventral roots of EAN rats by immunohistochemistry. A significant accumulation of RhoA+ cells was observed on Day 12, with a maximum around Day 15, correlating to the clinical severity of EAN. In dorsal/ventral roots of EAN, RhoA+ cells were seen in perivascular areas but also in the parenchyma. Furthermore, double-labelling experiments showed that the major cellular sources of RhoA were reactive macrophages and T cells. In conclusion, this is the first demonstration of the presence of RhoA in the dorsal/ventral roots of EAN. The time courses and cellular sources of RhoA together with the functions of RhoA indicate that RhoA may function to facilitate macrophage and T-cell infiltration in EAN and therefore could be a potential therapeutic target. [Abstract/Link to Full Text]

Kinsey CG, Bussolati G, Bosco M, Kimura T, Pizzorno MC, Chernin MI, Cassoni P, Novak JF
Constitutive and ligand-induced nuclear localization of oxytocin receptor.
J Cell Mol Med. 2007 Jan-Feb;11(1):96-110.
Oxytocin receptor (OTR) is a membrane protein known to mediate oxytocin (OT) effects, in both normal and neoplastic cells. We report here that human osteosarcoma (U2OS, MG63, OS15 and SaOS2), breast cancer (MCF7), and primary human fibroblastic cells (HFF) all exhibit OTR not only on the cell membrane, but also in the various nuclear compartments including the nucleolus. Both an OTR-GFP fusion protein and the native OTR appear to be localized to the nucleus as detected by transfection and/or confocal immunofluorescence, respectively. Treatment with oxytocin causes internalization of OTR and the resulting vesicles accumulate in the vicinity of the nucleus and some of the perinuclear OTR enters the nucleus. Western blots indicate that OTR in the nucleus and on the plasma membrane are likely to be the same biochemical and immunological entities. It appears that OTR is first visible in the nucleoli and subsequently disperses within the nucleus into 4-20 spots while some of the OTR diffuses throughout the nucleoplasm.The behaviour and kinetics of OTR-GFP and OTR are different, indicating interference by GFP in both OTR entrance into the nucleus and subsequent relocalization of OTR within the nucleus. There are important differences among the tested cells, such as the requirement of a ligand for transfer of OTR in nuclei. A constitutive internalization of OTR was found only in osteosarcoma cells, while the nuclear localization in all other tested cells was dependent on ligand binding. The amount of OTR-positive material within and in the vicinity of the nucleus increased following a treatment with oxytocin in both constitutive and ligand-dependent type of cells. The evidence of OTR compartmentalization at the cell nucleus (either ligand-dependent or constitutive) in different cell types suggests still unknown biological functions of this protein or its ligand and adds this G-protein-coupled receptor to other heptahelical receptors displaying this atypical and unexpected nuclear localization. [Abstract/Link to Full Text]

Li H, Sun B
Toll-like receptor 4 in atherosclerosis.
J Cell Mol Med. 2007 Jan-Feb;11(1):88-95.
Toll-like receptor 4 (TLR4) is key regulators of both innate and adaptive immune responses. TLR4 recognizes pathogen-associated molecular patterns (PAMPs) and activates the inflammatory cells. The function of TLR4 in atherosclerosis has been investigated in mouse knockout studies and epidemiological studies of human TLR4 polymorphisms. These studies have shown that TLR4 function affects the initiation and progression of atherosclerosis. This article reviews the biological functions and clinical implications of TLR4 in atherosclerosis. [Abstract/Link to Full Text]

Jarrar MH, Baranova A
PPARgamma activation by thiazolidinediones (TZDs) may modulate breast carcinoma outcome: the importance of interplay with TGFbeta signalling.
J Cell Mol Med. 2007 Jan-Feb;11(1):71-87.
The thiazolidinediones (TZDs) are a class of synthetic antidiabetic drugs exerting its action primarily upon activation of the peroxisome proliferator-activated receptor-gamma (PPARgamma). Given the widespread incidence of diabetes type II and lifelong exposure of these patients to TZDs, there is a possibility that chronic treatment with TZD modifies clinical phenotypes of other common human diseases, for example breast carcinoma. There is evidence that TZDs act as breast carcinoma suppression agents, at least in the in vitro and animal models. Stimulation of the PPARgamma by TZDs interferes with oestrogen receptor signalling, STAT5B and NF-kappaB signalling cascades. On the other hand, TZDs repress TGFbeta signalling, a well-known suppressor of the initial stages of breast carcinoma development. Another layer of complexity arises at the later stages of tumour development, when TGFbeta acts as a tumour promoter: its overexpression is associated with poor prognosis, higher degree of tumour vascularization and metastasis. Longitudinal studies of breast carcinoma development in chronic TZD users are needed. In this review, we dissect possible interplays between chronic exposure of breast tis-sue to TZDs and TGFbeta signalling and predict influence of TZD exposure on cancer-related clinical outcome. [Abstract/Link to Full Text]

Böldicke T
Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER.
J Cell Mol Med. 2007 Jan-Feb;11(1):54-70.
Intracellular antibodies (intrabodies) constitute a potent tool to neutralize the function of target proteins inside specific cell compartments (cytosol, nucleus, mitochondria and ER). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals and complements or replaces knockdown techniques such as antisense-RNA, RNAi and RNA aptamers. This article focuses on intrabodies targeted to the ER. Intracellular anti-bodies expressed and retained inside the ER (ER intrabodies) are shown to be highly efficient in blocking the translocation of secreted and cell surface molecules from the ER to the cell surface.The advantage of ER intrabodies over cytoplasmic intrabodies is that they are correctly folded and easier to select. A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins.The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells.In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing.In the future, endosomal localized receptors involved in bacterial and viral infections, intracellular oncogenic receptors and enzymes involved in glycosylation of tumour antigens might be new targets for ER intrabodies. [Abstract/Link to Full Text]

Chan CB, Ye K
PIKE GTPase are phosphoinositide-3-kinase enhancers, suppressing programmed cell death.
J Cell Mol Med. 2007 Jan-Feb;11(1):39-53.
Phosphoinositide-3-kinase enhancers (PIKE) are GTP-binding proteins that posses anti-apoptotic functions. The PIKE family includes three members, PIKE-L, PIKE-S and PIKE-A, which are originated from a single gene (CENTG1) through alternative splicing or differential transcription initiation. Both PIKE-S and PIKE-L bind to phosphoinositide-3-kinase (PI3K) and enhance its activity. PIKE-A does not interplay with PI3K. Instead, it interacts with the downstream effector Akt and promotes its activity. These actions are mediated by their GTPase activity. Because both PI3K and Akt are important effectors in the growth factor-mediated signaling which triggers cellular growth and acts against apoptosis, PIKEs therefore serve as the molecular switch that their activation are crucial for growth factors to exert their physiological functions. In this review, the current understanding of different PIKE isoforms in growth factors-induced anti-apoptotic function will be discussed. Moreover, the role of PIKE in the survival and invasion activity of cancer cells will also be introduced. [Abstract/Link to Full Text]

Docheva D, Popov C, Mutschler W, Schieker M
Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system.
J Cell Mol Med. 2007 Jan-Feb;11(1):21-38.
The identification of mesenchymal stem cells (MSCs) in adult human tissues and the disclosure of their self-renew-al and multi-lineage differentiation capabilities have provided exciting prospects for cell-based regeneration and tis-sue engineering. Although a considerable amount of data is available describing MSCs, there is still lack of information regarding the molecular mechanisms that govern their adhesion and migration. In this work, we will review the current state of knowledge on integrins and other adhesion molecules found to be expressed on MSCs. The discussed topics include the characteristics of MSCs and their clinical applications, integrins and their central role in cell-matrix attachment and migration, and comments on mobilization, differentiation and contribution to tumour development. Finally, by understanding the complex and fundamental pathways by which MSCs attach and migrate, it might be possible to fine-tune the strategies for effective and safe use of MSCs in regenerative therapies. [Abstract/Link to Full Text]

Polykandriotis E, Arkudas A, Horch RE, Stürzl M, Kneser U
Autonomously vascularized cellular constructs in tissue engineering: opening a new perspective for biomedical science.
J Cell Mol Med. 2007 Jan-Feb;11(1):6-20.
In tissue engineering cell cultures play a crucial role besides the matrix materials for the end of substituting lost tissue functions. The cell itself is situated at the cross-roads leading to different orders of scale, from molecule to organism and different levels of function, from biochemistry to macrophysiology. Extensive in vitro investigations have dissected a vast amount of cellular phenomena and the role of a number of bioactive substances has been elucidated in the past. Further, recombinant DNA technologies allow modulation of the expression profiles of virtually all kinds of cells. However, issues of vascularization in vivo limit transferability of these observations and restrict upscaling into clinical applications. Novel in vivo models of vascularization have evolved inspired from reconstructive microsurgical concepts and they encompass axial neovascularization by means of vascular induction. This work represents a brief description of latest developments and potential applications of neovascularization and angiogenesis in tissue engineering. [Abstract/Link to Full Text]


Tribute to Professor George E. Palade.
J Cell Mol Med. 2007 Jan-Feb;11(1):2-3. [Abstract/Link to Full Text]

Min KW, Leabu M
Interstitial cells of Cajal (ICC) and gastrointestinal stromal tumor (GIST): facts, speculations, and myths.
J Cell Mol Med. 2006 Oct-Dec;10(4):995-1013.
Interstitial cells of Cajal (ICC) is a peculiar cell network composed of cells having processes described by the eminent Spanish neuroanatomist of the 19th century, S. Ramon y Cajal. ICC became a fascinating subject to many investigators and it is estimated that there are over 100 publications yearly on the subject related to ICC, in the last three years. Now it is widely accepted that ICC are pace maker cells of the gut and probable progenitor cells of gastrointestinal stromal tumors (GIST). Lately, interstitial Cajal-like cells (ICLC) are being found in various organs and their physiological role is still to be defined. We have reviewed the literature trying to evaluate the validity of the current concept and found that there are a few salient points to be considered. 1) There has been some important departure in defining the identity of ICC from the original criteria of Cajal. In particular, ICC with myoid feafures in intestinal smooth muscle layers (ICC-DPM) do not seem to fit to the original description of interstitial cell network by Cajal. We have also pointed out that the current reports assigning a pace maker role to ICC vastly depend on the scientific data on "ICC with myoid features", not on "fibroblast-like ICC", which are more abundant and easier to identify. 2) There seem to be an overwhelming amount of data proving the relationship between ICC and GIST. Both are known to express c-Kit and the ultrastructural characteristics seen in GIST roughly parallel those of ICC including minimal myoid differentiation seen in the majority of GIST, supporting the current concept that GIST are ICC tumors. 3) According to the original description of Cajal, ICC was not limited to the gut, suggesting an existence of ICC in other organs. The list of organs reported to contain ICC (currently identified by immunohistochemistry and electron microscopy) is ever growing and further studies are needed to define their identity and pathophysiologic role. 4). Recent data concerning gut development suggest that both c-Kit expressing ICC (fibroblasts-like as well as muscle-like) and gut muscle cells derive from the common progenitor cells of the embryonic gut unifying the histogenetic concept of all GIST with heterogeneous cytomorphologic features. In this review we attempted to incorporate recent information on interstitial Cajal-like cells (ICLC) found in other organs to broaden our understanding of ICC in general in terms of their ultrastructure, physiology, and neoplasia. [Abstract/Link to Full Text]

Simionescu O, Costache M, Testori A
Cutaneous melanoma: digital dermoscopy-essential tool for positive diagnosis.
J Cell Mol Med. 2006 Oct-Dec;10(4):991-4.
Cutaneous melanoma is a "perfid", aggressive and hard to be treated malignant tumor in case of delayed diagnosis. However, patients still have a chance to escape progressive disease if the lesion is recognized early, when the surgical approach is curative. Dermoscopy has the important advantage of rapidity and non-invasivity in a field with (still) contradictory algorithms of diagnosis and treatment. The recognition of the elementary dermoscopic lesions enables accurate diagnosis for cutaneous melanoma. In our opinion, dermoscopy appears compulsory in the routine dermatologic examination. In vivo microscopy (dermoscopy) together with histopathology (plus or minus immunohistochemistry) seem, at present, to provide the most reliable diagnosis of melanoma. [Abstract/Link to Full Text]

Popescu LM, Gherghiceanu M, Mandache E, Cretoiu D
Caveolae in smooth muscles: nanocontacts.
J Cell Mol Med. 2006 Oct-Dec;10(4):960-90.
Smooth muscle cell (SMC) caveolae have been investigated by quantitative and qualitative analysis of transmission electron microscopy (TEM) images of rat stomach, bladder and myometrium, guinea pig taenia coli, human ileum, and rat aortic SMCs. Ultrathin (below 30 nm) serial sections were used for examination of caveolar morphology and their connections with SMC organelles. Average caveolar diameter was smaller in vascular SMCs (70 nm, n=50) than in visceral SMCs (77 nm, n=100), but with the same morphology. Most of the caveolae, featured as flask-shaped plasma membrane (PM) invaginations, opened to the extracellular space through a 20 nm stoma (21 +/- 3 nm) having a 7 nm thick diaphragm. A small percentage of caveolae (3%), gathered as grape-like clusters, did not open directly to the extracellular space, but to irregular PM pockets having a 20-30 nm opening to the extracellular space. In visceral SMCs, caveolae were disposed in 4-6 rows, parallel to myofilaments, whilst aortic SMCs caveolae were arranged as clusters. This caveolar organization in rows or clusters minimizes the occupied volume, providing more space for the contractile compartment. The morphometric analysis of relative volumes (% of cell volume) showed that caveolae were more conspicuous in visceral than in vascular SMCs (myometrium - 2.40%; bladder - 3.66%, stomach - 2.61%, aorta - 1.43%). We also observed a higher number of caveolae per length unit of cellular membrane in most visceral SMCs compared to vascular SMCs (myometrium - 1.06/microm, bladder - 0.74/microm, aorta - 0.57/microm, stomach - 0.48/microm). Caveolae increase the cellular perimeter up to 15% and enlarge the surface area of the plasma membrane about 80% in SMCs. Threedimensional reconstructions (15micro(3)) showed that most caveolae, in both visceral and vascular SMCs, have nanocontacts with SR (87%), other with mitochondria (10%) and 3% apparently have no contact with these organelles. Usually, 15 nm wide junctional spaces exist between caveolae and SR, some of them with nanostructural links between each other or with mitochondria: direct contacts (space <2 nm or none) and molecular links, so called 'feet' (about 12 nm electron dense structures between organellar membranes). Direct contacts possibly allow molecular translocation between the two membranes. Electron-dense 'feet'-like structures suggest a molecular link between these organelles responsible for intracellular Ca(2+) homeostasis (excitation-contraction coupling or pharmaco-mechanical coupling). Close appositions (approximately 15 nm) have also been observed between caveolae and perinuclear SR cisternae, suggesting that caveolae might be directly implicated in excitation-transcription coupling. [Abstract/Link to Full Text]

Sfrent-Cornateanu R, Mihai C, Balan S, Ionescu R, Moldoveanu E
The IL-6 promoter polymorphism is associated with disease activity and disability in systemic sclerosis.
J Cell Mol Med. 2006 Oct-Dec;10(4):955-9.
Systemic sclerosis (SSc) is a rare, autoimmune disease characterized by cutaneous and visceral fibrosis. Interleukin- 6 (IL-6) is involved in the pathogenesis of many immune-mediated diseases. IL-6 plays an important role in the initiation and promotion of fibrosis. The polymorphism in the position -174 (G/C) of the promoter region of the IL-6 gene (IL-6pr) may alter the expression of the gene. Complete linkage disequilibrium was observed between the -174 and -597 alleles. The aim of this study is to investigate the possible influence of -597 (-174) IL-6pr polymorphism on the susceptibility and/or the clinical course of SSc in Romanian population. Genotyping of -597 variant was performed by an RFLP method on 20 SSc patients and 26 healthy subjects. Patients having the homozygous GG (-597) genotype had higher disease activity and disability scores than heterozygous GA patients: the European Scleroderma Study Group (EScSG) disease activity score was 5.0 +/- 3.3 in homozygous GG subjects vs. 2.4 +/- 3.6 in heterozygous GA patients (p < 0.05), and the Disability Index of the Health Assessment Questionnaire (HAQ-DI) was 1.42 +/- 1.04 in homozygous GG subjects vs. 0.53 +/- 0.55 in heterozygous GA patients (p < 0.05). No difference was observed in the distribution of allele frequencies between SSc patients and healthy controls. Conclusions: The GG homozygosis was found to be associated with a higher degree of illness activity and disability in SSc patients. No statistically significant differences were found between SSc patients and healthy controls with respect to the -597 allele distribution. [Abstract/Link to Full Text]

Beretta L, Santaniello A, Cappiello F, Barili M, Scorza R
No evidence for a role of the proximal IL-6 G/C -174 single nucleotide polymorphism in Italian patients with systemic sclerosis.
J Cell Mol Med. 2007 Jul-Aug;11(4):896-8; author reply 898-9. [Abstract/Link to Full Text]

Rodella LF, Rezzani R, Buffoli B, Bonomini F, Tengattini S, Laffranchi L, Paganelli C, Sapelli PL, Bianchi R
Role of mast cells in wound healing process after glass-fiber composite implant in rats.
J Cell Mol Med. 2006 Oct-Dec;10(4):946-54.
Glass-fiber composites are frequently used in dentistry. In order to evaluate their biocompatibility we tested, in an experimental model "in vivo", their tissue response pointing our attention on presence of mast cells (MCs) and fibrotic process. Sprague Dawley rats were used for the experimental design. The fibers were introduced in a subcutaneous pocket along the middle dorsal line between the two scapulas for 7, 14 or 21 days. At the end of the treatments the skins were excised and then processed for Toluidine Blue, to determine the presence of MCs, and Picrosirius Red staining, to evaluate the presence of fibrotic tissue. Our preliminary results showed and increase of both MC number and deposition of collagen type I, which characterized the fibrotic tissue. So, subsequent aims of our study were to evaluate the role played by MCs in tissue fibrosis and to give a possible explanation regarding the mechanisms that were responsible of biological response observed, through the analyses of some proteins, such as metalloproteinase-2 (MMP-2), its inhibitor (TIMP-2) and transforming growth factor-beta (TGF-beta). Our data confirmed the involvement of TGF-beta, released by MCs, in the disruption of the equilibrium between MMP-2 and TIMP-2 that were implicated in the enhancement of fibrosis. In summary, this study demonstrate that this type of materials induced an inflammatory response at the site of implant and help to clarify what type of mechanism and which proteins are involved in this biological response. Nevertheless, more extensive investigations are in progress to better evaluate the inflammatory process. [Abstract/Link to Full Text]

Niiranen K, Keinänen TA, Pirinen E, Heikkinen S, Tusa M, Fatrai S, Suppola S, Pietilä M, Uimari A, Laakso M, Alhonen L, Jänne J
Mice with targeted disruption of spermidine/spermine N1-acetyltransferase gene maintain nearly normal tissue polyamine homeostasis but show signs of insulin resistance upon aging.
J Cell Mol Med. 2006 Oct-Dec;10(4):933-45.
The N(1)-acetylation of spermidine or spermine by spermidine/spermine N(1)-acetyltransferase (SSAT) is the ratecontrolling enzymatic step in the polyamine catabolism. We have now generated SSAT knockout (SSAT-KO) mice, which confirmed our earlier results with SSATdeficient embryonic stem (ES) cells showing only slightly affected polyamine homeostasis, mainly manifested as an elevated molar ratio of spermidine to spermine in most tissues indicating the indispensability of SSAT for the spermidine backconversion.Contrary to SSAT deficient ES cells, polyamine pools in SSAT-KO mice remained almost unchanged in response to N(1),N(11)-diethylnorspermine (DENSPM) treatment compared to a significant reduction of the polyamine pools in the wild-type animals and ES cells. Furthermore, SSATKO mice were more sensitive to the toxicity exerted by DENSPM in comparison with wild-type mice. The latter finding indicates that inducible SSAT plays an essential role in vivo in DENSPM treatmentevoked polyamine depletion, but a controversial role in toxicity of DENSPM. Surprisingly, liver polyamine pools were depleted similarly in wild-type and SSAT-KO mice in response to carbon tetrachloride treatment. Further characterization of SSAT knockout mice revealed insulin resistance at old age which supported the role of polyamine catabolism in glucose metabolism detected earlier with our SSAT overexpressing mice displaying enhanced basal metabolic rate, high insulin sensitivity and improved glucose tolerance. Therefore SSAT knockout mice might serve as a novel mouse model for type 2 diabetes. [Abstract/Link to Full Text]

Seyhan H, Hamzavi J, Wiercinska E, Gressner AM, Mertens PR, Kopp J, Horch RE, Breitkopf K, Dooley S
Liver fibrogenesis due to cholestasis is associated with increased Smad7 expression and Smad3 signaling.
J Cell Mol Med. 2006 Oct-Dec;10(4):922-32.
BACKGROUND/AIMS: Profibrogenic TGF-beta signaling in hepatic stellate cells is modulated during transdifferentiation. Strategies to abrogate TGF-beta effects provide promising antifibrotic results, however, in vivo data regarding Smad activation during fibrogenesis are scarce. METHODS: Here, liver fibrosis was assessed subsequent to bile duct ligation by determining liver enzymes in serum and collagen deposition in liver tissue. Activated hepatic stellate cells were identified by immunohistochemistry and immunoblots for alpha smooth muscle actin. Cellular localization of Smad3 and Smad7 proteins was demonstrated by immunohistochemistry. RTPCR for Smad4 and Smad7 was conducted with total RNA and Northern blot analysis for Smad7 with mRNA. Whole liver lysates were prepared to detect Smad2/3/4 and phospho- Smad2/3 by Western blotting. RESULTS: Cholestasis induces TGF-beta signaling via Smad3 in vivo, whereas Smad2 phosphorylation was only marginally increased. Smad4 expression levels were unchanged. Smad7 expression was continuously increasing with duration of cholestasis. Hepatocytes of fibrotic lesions exhibited nuclear staining Smad3. In contrast to this, Smad7 expression was localized to activated hepatic stellate cells. CONCLUSIONS: Hepatocytes of damaged liver tissue display increased TGF-beta signaling via Smad3. Further, negative feedback regulation of TGF-beta signaling by increased Smad7 expression in activated hepatic stellate cells occurs, however does not interfere with fibrogenesis. [Abstract/Link to Full Text]

Kassimatis TI, Giannopoulou I, Koumoundourou D, Theodorakopoulou E, Varakis I, Nakopoulou L
Immunohistochemical evaluation of phosphorylated SMAD2/SMAD3 and the co-activator P300 in human glomerulonephritis: correlation with renal injury.
J Cell Mol Med. 2006 Oct-Dec;10(4):908-21.
BACKGROUND: Smad2 and Smad3 are transcription factors that mediate transforming growth factor beta (TGF-beta) signals. Upon their activation, phosphorylated Smad2/Smad3 (pSmad2/Smad3), translocate to the nucleus and associate with co-activators such as p300, regulating the transcription of genes that contribute to the fibrotic processes. METHODS: We investigated the immunohistochemical expression of pSmad2/Smad3 and the co-activator p300 in 152 renal biopsy specimens from patients with various types of glomerulonephritides (GNs) and in 15 normal kidney specimens. Patients' clinical data (serum creatinine levels and proteinuria) had been collected. RESULTS: There was a dramatic increase in the expression of pSmad2/3 and p300 in all glomerular cell types in all GNs. pSmad2/3 expression was increased in all tubular segments (except for the proximal tubules in nonproliferative GNs), while p300 expression was significantly increased only in the proximal tubular cells in all GNs. Glomerular and tubular pSmad2/Smad3 and p300 were significantly increased in proliferative GNs (compared to the nonproliferative), particularly in the secondary group. The expression profile of p300 correlated positively with the expression of pSmad2/Smad3 in the diseased glomeruli and proximal tubules. pSmad2/3 and p300 were very often detected in segmental hyperplastic lesions, cellular crescents, microadhesions and segmental or global sclerotic areas. Glomerular and proximal tubular pSmad2/Smad3 was positively correlated with serum creatinine levels, while distal and collecting tubular pSmad2/3 and p300 correlated positively with tubular atrophy. Glomerular and proximal tubular pSmad2/3 expression and glomerular p300 expression correlated positively with lupus nephritis activity. CONCLUSION: Our results suggest that pSmad2/3-p300 pathway may play a pivotal role in the pathogenesis and progression of human glomerulonephritis. [Abstract/Link to Full Text]

Bezstarosti K, Das S, Lamers JM, Das DK
Differential proteomic profiling to study the mechanism of cardiac pharmacological preconditioning by resveratrol.
J Cell Mol Med. 2006 Oct-Dec;10(4):896-907.
Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning of the heart through a NO-dependent mechanism. To further explore the molecular mechanisms involved in resveratrol-mediated cardioprotection, we monitored the effects of resveratrol treatment after ischemia-reperfusion on the protein profile by implementation of proteomic analysis. Two groups of rats were studied; one group of animals was fed resveratrol for 7 days, while the other group was given vehicle only. The rats were sacrificed for the isolated working heart preparation and for isolation of cytoplasmic fraction from left ventricle homogenates to carry out the proteomic as well as immunoblot at baseline and at the end of 30 min ischemia/2-h perfusion. The results demonstrate significant cardioprotection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apoptosis. The left ventricular cytoplasmic fractions were separated by two-dimensional electrophoresis (2-DE). Differentially regulated proteins were detected with quantitative computer analysis of the Coomassie blue stained 2-DE images and identified by MALDI-TOF (MS) and nanoLC-ESI-Q-TOF mass spectrometry (MS/MS). Five redox-regulated and preconditioning- related proteins were identified that were all upregulated by resveratrol: MAPKK, two different alphaB-crystallin species, HSP 27 and PE binding protein. Another HSP27 species and aldose reductase were downregulated and peroxiredoxin- 2 remained constant. The results of the immunoblot analysis of phosphorylated MAPKK, -HSP27 and -alphaB-crystallin and PE binding protein were consistent with the proteomic findings, but not with peroxiredoxin-2. The proteomic analysis showed also downregulation of some proteins in the mitochondrial respiratory chain and matrix and the myofilament regulating protein MLC kinase-2. The results of the present study demonstrate that proteomic profiling enables the identification of resveratrol induced preconditioning-associated proteins which reflects not only changes in their expression level but also isoforms, post-translational modifications and regulating binding or activating partner proteins. [Abstract/Link to Full Text]

Brock P, Sparmann G, Ritter T, Jaster R, Liebe S, Emmrich J
Adenovirus-mediated gene transfer of interleukin-4 into pancreatic stellate cells promotes interleukin-10 expression.
J Cell Mol Med. 2006 Oct-Dec;10(4):884-95.
Pancreatic stellate cells (PSC) are crucially involved in the development of fibrosis, a hallmark of chronic pancreatitis. Therefore, PSC represent an attractive target for the modulation of cellular functions providing the prerequisite for the establishment of novel therapeutic strategies like transfer of genetic material to the cells. Based on recent studies suggesting that the chronic course of pancreatitis is associated with immune deviation towards a Th1 cytokine profile, we have investigated the applicability of primary PSC to an adenovirus-mediated transfer of the cDNA encoding the Th2 cytokine interleukin (IL) 4 and the autocrine-acting effects of IL 4 on the cells in vitro. The transduction of primary PSC with a replication-incompetent adenovirus type 5 vector carrying the cDNA encoding rat IL- 4 resulted in a distinct expression of the cytokine on mRNA and protein level for two weeks. Similar to recombinant IL 4, effects of the endogenously synthesized cytokine were mediated by the signal transducer and activator of transcription (STAT)6. Interestingly, beside the increase of PSC proliferation, IL 4 transduction was accompanied by an up-regulation in the endogenous expression of the anti-inflammatory cytokine IL 10. In summary, our data suggest that PSC are suitable targets for gene therapy modulating cellular interactions in the pancreas. [Abstract/Link to Full Text]

Santana A, Enseñat-Waser R, Arribas MI, Reig JA, Roche E
Insulin-producing cells derived from stem cells: recent progress and future directions.
J Cell Mol Med. 2006 Oct-Dec;10(4):866-83.
Type 1 diabetes is characterized by the selective destruction of pancreatic beta-cells caused by an autoimmune attack. Type 2 diabetes is a more complex pathology which, in addition to beta-cell loss caused by apoptotic programs, includes beta-cell dedifferentiation and peripheric insulin resistance. beta-Cells are responsible for insulin production, storage and secretion in accordance to the demanding concentrations of glucose and fatty acids. The absence of insulin results in death and therefore diabetic patients require daily injections of the hormone for survival. However, they cannot avoid the appearance of secondary complications affecting the peripheral nerves as well as the eyes, kidneys and cardiovascular system. These afflictions are caused by the fact that external insulin injection does not mimic the tight control that pancreatic-derived insulin secretion exerts on the body's glycemia. Restoration of damaged beta-cells by transplantation from exogenous sources or by endocrine pancreas regeneration would be ideal therapeutic options. In this context, stem cells of both embryonic and adult origin (including beta-cell/islet progenitors) offer some interesting alternatives, taking into account the recent data indicating that these cells could be the building blocks from which insulin secreting cells could be generated in vitro under appropriate culture conditions. Although in many cases insulin-producing cells derived from stem cells have been shown to reverse experimentally induced diabetes in animal models, several concerns need to be solved before finding a definite medical application. These refer mainly to the obtainment of a cell population as similar as possible to pancreatic beta-cells, and to the problems related with the immune compatibility and tumor formation. This review will summarize the different approaches that have been used to obtain insulin-producing cells from embryonic and adult stem cells, and the main problems that hamper the clinical applications of this technology. [Abstract/Link to Full Text]

Ganss R
Tumor stroma fosters neovascularization by recruitment of progenitor cells into the tumor bed.
J Cell Mol Med. 2006 Oct-Dec;10(4):857-65.
The tumor stroma is an active player during carcinogenesis and contains a variety of cell types such as vascular cells, fibroblasts and inflammatory cells which directly or indirectly foster neovascularization. During tumor progression stromal cells, in particular the neovasculature, acquire new characteristics distinct from their normal counterparts and display a high degree of plasticity to meet the tumor's demands. The local environment may, to some extent, shape pre-existing, tumor-resident stromal cells. However, there is accumulating evidence that new endothelial and other stromal cells are actively recruited into tumors, and that this recruitment is essential for a unique and tumor-specific proangiogenic environment. [Abstract/Link to Full Text]

Allison DP, Doktycz MJ
Cellular secretion studied by force microscopy.
J Cell Mol Med. 2006 Oct-Dec;10(4):847-56.
Using the optical microscope, real adventures in cellular research began in earnest in the latter half of the nineteenth century. With the development of the electron microscope, ultramicroscopy, and improved cell staining techniques, significant advances were made in defining intracellular structures at the nanometer level. The invention of force microscopy, the atomic force microscope (AFM) in the mid 1980s, and the photonic force microscope (PFM) in the mid 1990s, finally provided the opportunity to study live cellular structure-function at the nanometer level. Working with the AFM, dynamic cellular and subcellular events at the molecular level were captured in the mid 1990s, and a new cellular structure 'the porosome' in the plasma membrane of all secretory cells has been defined, where specific docking and fusion of secretory vesicles occur. The molecular mechanism of fusion of the secretory vesicle membrane at the base of the porosome membrane in cells, and the regulated release of intravesicular contents through the porosome opening to the extracellular space, has been determined. These seminal discoveries provide for the first time a molecular mechanism of cell secretion, and the possibility to ameliorate secretory defects in disease states. [Abstract/Link to Full Text]

Chua CE, Tang BL
alpha-synuclein and Parkinson's disease: the first roadblock.
J Cell Mol Med. 2006 Oct-Dec;10(4):837-46.
alpha-synuclein gene mutations are major underlying genetic defects known in familial juvenile onset Parkinson's disease (PD), and alpha-synuclein is a major constituent of Lewy Bodies, the pathological hallmark of PD. The normal cellular function of alpha-synuclein has been elusive, and its exact etiological mechanism in causing dopaminergic neuronal death in PD is also not clearly understood. Very recent reports now indicate that mutant or simply over-expressed alpha- synuclein could cause damage by interfering with particular steps of neuronal membrane traffic. alpha-synuclein selectively blocks endoplamic reticulum-to-Golgi transport, thus causing ER stress. A screen in a yeast revealed that alpha- synuclein toxicity could be suppressed by over-expression of the small GTPase Ypt1/Rab1, and that over-expression of the latter rescues neuron loss in invertebrate and mammalian models of alpha-synuclein-induced neurodegeneration. alpha-synuclein may also serve a chaperone function for the proper folding of synaptic SNAREs that are important for neurotransmitter release. We discuss these recent results and the emerging pathophysiological interaction of alpha-synuclein with components of neuronal membrane traffic. [Abstract/Link to Full Text]

Verkhratsky A, Toescu EC
Neuronal-glial networks as substrate for CNS integration.
J Cell Mol Med. 2006 Oct-Dec;10(4):826-36.
Astrocytes have been considered, for a long time, as the support and house-keeping cells of the nervous system. Indeed, the astrocytes play very important metabolic roles in the brain, but the catalogue of nervous system functions or activities that involve directly glial participation has extended dramatically in the last decade. In addition to the further refining of the signalling capacity of the neuroglial networks and the detailed reassessment of the interactions between glia and vascular bed in the brain, one of the important salient features of the increased glioscience activity in the last few years was the morphological and functional demonstration that protoplasmic astrocytes occupy well defined spatial territories, with only limited areas of morphological overlapping, but still able to communicate with adjacent neighbours through intercellular junctions. All these features form the basis for a possible reassessment of the nature of integration of activity in the central nervous system that could raise glia to a role of central integrator. [Abstract/Link to Full Text]

Froyen G, Bauters M, Voet T, Marynen P
X-linked mental retardation and epigenetics.
J Cell Mol Med. 2006 Oct-Dec;10(4):808-25.
The search for the genetic defects in constitutional diseases has so far been restricted to direct methods for the identification of genetic mutations in the patients' genome. Traditional methods such as karyotyping, FISH, mutation screening, positional cloning and CGH, have been complemented with newer methods including array-CGH and PCR-based approaches (MLPA, qPCR). These methods have revealed a high number of genetic or genomic aberrations that result in an altered expression or reduced functional activity of key proteins. For a significant percentage of patients with congenital disease however, the underlying cause has not been resolved strongly suggesting that yet other mechanisms could play important roles in their etiology. Alterations of the 'native' epigenetic imprint might constitute such a novel mechanism. Epigenetics, heritable changes that do not rely on the nucleotide sequence, has already been shown to play a determining role in embryonic development, X-inactivation, and cell differentiation in mammals. Recent progress in the development of techniques to study these processes on full genome scale has stimulated researchers to investigate the role of epigenetic modifications in cancer as well as in constitutional diseases. We will focus on mental impairment because of the growing evidence for the contribution of epigenetics in memory formation and cognition. Disturbance of the epigenetic profile due to direct alterations at genomic regions, or failure of the epigenetic machinery due to genetic mutations in one of its components, has been demonstrated in cognitive derangements in a number of neurological disorders now. It is therefore tempting to speculate that the cognitive deficit in a significant percentage of patients with unexplained mental retardation results from epigenetic modifications. [Abstract/Link to Full Text]

Forero DA, Casadesus G, Perry G, Arboleda H
Synaptic dysfunction and oxidative stress in Alzheimer's disease: emerging mechanisms.
J Cell Mol Med. 2006 Jul-Sep;10(3):796-805.
In this paper, we review experimental advances in molecular neurobiology of Alzheimer's disease (AD), with special emphasis on analysis of neural function of proteins involved in AD pathogenesis, their relation with several signaling pathways and with oxidative stress in neurons. Molecular genetic studies have found that mutations in APP, PS1 and PS2 genes and polymorphisms in APOE gene are implicated in AD pathogenesis. Recent studies show that these proteins, in addition to its role in beta-amyloid processing, are involved in several neuroplasticity-signaling pathways (NMDA-PKA-CREB-BDNF, reelin, wingless, notch, among others). Genomic and proteomic studies show early synaptic protein alterations in AD brains and animal models. DNA damage caused by oxidative stress is not completely repaired in neurons and is accumulated in the genes of synaptic proteins. Several functional SNPs in synaptic genes may be interesting candidates to explore in AD as genetic correlates of this synaptopathy in a "synaptogenomics" approach. Thus, experimental evidence shows that proteins implicated in AD pathogenesis have differential roles in several signaling pathways related to neuromodulation and neurotransmission in adult and developing brain. Genomic and proteomic studies support these results. We suggest that oxidative stress effects on DNA and inherited variations in synaptic genes may explain in part the synaptic dysfunction seen in AD. [Abstract/Link to Full Text]

Cretoiu D, Ciontea SM, Popescu LM, Ceafalan L, Ardeleanu C
Interstitial Cajal-like cells (ICLC) as steroid hormone sensors in human myometrium: immunocytochemical approach.
J Cell Mol Med. 2006 Jul-Sep;10(3):789-95.
Expression of estrogen (ER) and progesterone (PR) receptors was investigated in cultured human normal myometrial cells (non-pregnant uterus, fertile period). The ER and PR expression was studied by immunohistochemistry and immunofluorescence on either myocytes or interstitial Cajal-like cells (ICLC). Only those cells double immunostained for c-kit and steroid receptors were considered as ICLC. ER and/or PR immunoreactivity was localized in ICLC, primarily concentrated at the nucleus level, but it was also observed in the cell body (cytoplasm) and processes. Stronger immunopositive reaction in the ICLC nucleus for PR than for ER was noted. Under our experimental conditions, a clear positive repeatable reaction for steroid receptors could not be detected in myocytes. In conclusion, these data suggest that ICLC could be true hormonal 'sensors', possibly participating in the regulation of human myometrial contractions (via gap junctions with myocytes and/or by paracrine signaling). [Abstract/Link to Full Text]

---

Recent Articles in Molecules and Cells

---

Lee EW, Oh W, Song J
Jab1 as a mediator of nuclear export and cytoplasmic degradation of p53.
Mol Cells. 2006 Oct 31;22(2):133-40.
Jun activation domain-binding protein 1 (Jab1) is involved in various cellular mechanisms including development in Drosophila and mouse, cell cycle control and signal transduction pathways. Recent studies also determined that Jab1 functions as a nuclear exporter and inducer of cytoplasmic degradation for several proteins including p53, p27, capsid of West Nile virus, and Smad4/7 proteins. In particular, p53 is shown to bind to and to be exported into the cytoplasm by Jab1, which helps to maintain low levels of p53 under normal conditions. This review was undertaken in an effort to understand the biological significance of the homeostasis of p53 as maintained in the presence of Jab1. Based on our observations, we have provided potential mechanistic hypotheses for the nuclear export of p53 in coordination with Jab1 and the role of other factors in these processes. [Abstract/Link to Full Text]

Yannay-Cohen N, Razin E
Translation and transcription: the dual functionality of LysRS in mast cells.
Mol Cells. 2006 Oct 31;22(2):127-32.
In the post genome project era, it is well established that the human genome contains a smaller number of genes than expected. The complexity found in higher organisms can be explained if proteins are multifunctional. Indeed, recent studies are continuing to reveal proteins that are capable of a broad repertoire of functions. A good paradigm for multifunctionality can be found in the amino-acyl tRNA synthetases (aaRSs), an ancient conserved family of proteins. This unique family, which is comprised of 20 different enzymes, is well known for its participation in protein synthesis. Several studies have described numerous examples of these "housekeeping" proteins taking part in extensive critical cellular activities. In this review, we focus on a member of that family, lysyl-tRNA synthetase (LysRS), which has been shown to have a dual functionality. In addition to its contribution to the translation process, LysRS also takes part in the regulation of MITF and USF2 target genes. This phenomenon was first described in mast cells. [Abstract/Link to Full Text]

Hirano M, Rakwal R, Shibato J, Agrawal GK, Jwa NS, Iwahashi H, Masuo Y
New protein extraction/solubilization protocol for gel-based proteomics of rat (female) whole brain and brain regions.
Mol Cells. 2006 Aug 31;22(1):119-25.
The rat is an accepted model for studying human psychiatric/neurological disorders. We provide a protocol for total soluble protein extraction using trichloroacetic acid/acetone (TCA/A) from rat (female) whole brain, 10 brain regions and the pituitary gland, and show that two-dimensional gel electrophoresis (2-DGE) using pre-cast immobilized pH (4-7) gradient (IPG) strip gels (13 cm) in the first dimension yields clean silver nitrate stained protein profiles. Though TCA/A precipitation may not be "ideal", the important choice here is the selection of an appropriate lysis buffer (LB) for solubilizing precipitated proteins. Our results reveal enrichment of protein spots by use of individual brain regions rather than whole brain, as well as the presence of differentially expressed spots in their proteomes. Thus individual brain regions provide improved protein coverage and are better suited for differential protein detection. Moreover, using a phosphoprotein-specific dye, in-gel detection of phosphoproteins was demonstrated. Representative high-resolution silver nitrate stained proteome profiles of rat whole brain total soluble protein are presented. Shortcomings apart (failure to separate membrane proteins), gel-based proteomics remains a viable option, and 2-DGE is the method of choice for generating high-resolution proteome maps of rat brain and brain regions. [Abstract/Link to Full Text]

Kang HJ, Hong SM, Kim BC, Park EH, Ahn K, Lim CJ
Effects of heterologous expression of thioredoxin reductase on the level of reactive oxygen species in COS-7 cells.
Mol Cells. 2006 Aug 31;22(1):113-8.
Thioredoxin reductase (TrxR), a component of the redox control system involving thioredoxin (Trx), is implicated in defense against oxidative stress, control of cell growth and proliferation, and regulation of apoptosis. In the present study a stable transfectant was made by introducing the vector pcDNA3.0 harboring the fission yeast TrxR gene into COS-7 African green monkey kidney fibroblast cells. The exogenous TrxR gene led to an increase in TrxR activity of up to 3.2-fold but did not affect glutathione (GSH) content, or glutaredoxin and caspase-3 activities. Levels of reactive oxygen species (ROS), but not those of nitric oxide (NO), were reduced. Conversely, 1-chloro-2,4-dinitrobezene (CDNB), an irreversible inhibitor of mammalian TrxR, enhanced ROS levels in the COS-7 cells. After treatment with hydrogen peroxide, the level of intracellular ROS was lower in the transfectants than in the vector control cells. These results confirm that TrxR is a crucial determinant of the level of cellular ROS during oxidative stress as well as in the normal state. [Abstract/Link to Full Text]

Kim JY, Kim DY, Lee YS, Lee BK, Lee KH, Ro JY
DA-9601, Artemisia asiatica herbal extract, ameliorates airway inflammation of allergic asthma in mice.
Mol Cells. 2006 Aug 31;22(1):104-12.
We previously reported that DA-9601, ethanol herbal extract of Artemisia asiatica, inhibited histamine and leukotriene releases in guinea pig lung mast cells activated with specific antigen/antibody reaction. This study aimed to evaluate the inhibitory effect of DA-9601 on the OVA-induced airway inflammation in allergic asthma mouse model. BALB/c mice were sensitized and challenged with OVA. DA-9601 was administered orally 1 h before every local OVA-challenge. OVA-specific serum IgE was measured by ELISA, recruitment of inflammatory cells in BAL fluids and lung tissues by Diff-Quik and H&E staining, respectively, the expressions of CD40, CD40L and VCAM-1 by immunohistochemistry, goblet cell hyperplasia by PAS staining, activities of MMPs by gelatin zymography, expressions of mRNA and proteins of cytokines by RT-PCR and ELISA, activities of MAP kinases by western blot, and activity of NF-KappaB by EMSA. DA-9601 reduced IgE level, recruitment of inflammatory cells into the BAL fluid and lung tissues, expressions of CD40, CD40L and VCAM-1 molecules, goblet cell hyperplasia, MMPs activity, expressions of mRNA and productions of various cytokines, activities of MAP kinases and NK-KappaB increased from OVA-challenged mice. These data suggest that DA-9601 may be developed as a clinical therapeutic agent in allergic diseases due to suppressing the airway allergic inflammation via regulation of various cellular molecules expressed by MAP kinases/NF-KappaB pathway. [Abstract/Link to Full Text]

Cho H, Kim YA, Ho WK
Phosphate number and acyl chain length determine the subcellular location and lateral mobility of phosphoinositides.
Mol Cells. 2006 Aug 31;22(1):97-103.
Phosphoinositides are critical regulators of ion channel and transporter activity. There are multiple isomers of biologically active phosphoinositides in the plasma membrane and the different lipid species are non-randomly distributed. However, the mechanism by which cells impose selectivity and directionality on lipid movements and so generate a non-random lipid distribution remains unclear. In the present study we investigated which structural elements of phosphoinositides are responsible for their subcellular location and movement. We incubated phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) with short or long acyl chains in CHO and HEK cells. We show that phosphate number and acyl chain length determine cellular location and translocation movement. In CHO cells, PI(4,5)P2 with a long acyl chain was released into the cytosol easily because of a low partition coefficient whereas long chain PI was released more slowly because of a high partition coefficient. In HEK cells, the cellular location and translocation movement of PI were similar to those of PI in CHO cells, whereas those of PI(4,5)P2 were different; some mechanism restricted the translocation movement of PI(4,5)P2, and this is in good agreement with the extremely low lateral diffusion of PI(4,5)P2. In contrast to the dependence on the number of phosphates of the phospholipid head group of long acyl chain analogs, short acyl chain phospholipids easily undergo translocation movement regardless of cell type and number of phosphates in the lipid headgroup. [Abstract/Link to Full Text]

Kim DH, Han MS, Cho HW, Jo YD, Cho MC, Kim BD
Molecular cloning of a pepper gene that is homologous to SELF-PRUNING.
Mol Cells. 2006 Aug 31;22(1):89-96.
"Determinate" and "indeterminate" inflorescences in plants are controlled by a single recessive gene, for example, SELF-PRUNING (SP) in Solanum lycopersicum, TERMINAL FLOWER1 in Arabidopsis, CENTRORADI-ALIS in Antirrhinum, and CENTRORADIALIS-like gene in tobacco. Pepper (Capsicum annuum L.) is an indeterminate species in which shoots grow indefinitely. In this study, we cloned and characterized the pepper SP-like gene (CaSP). RT-PCR revealed that the CaSP transcript accumulates to higher levels in floral buds than in other organs. Comparison of genomic DNA and cDNA sequences from indeterminate and determinate pepper plants revealed the insertion of a single base in the first exon of CaSP in the determinate pepper plants. CaSP is annotated in linkage group 8 (chromosome 6) of the SNU2 pepper genetic map and showed similar synteny to SP in tomato. Transgenic tobacco plants overexpressing CaSP displayed late-flowering phenotypes similar to the phenotypes caused by overexpression of CaSP orthologs in other plants. Collectively, these results suggest that pepper CaSP is an ortholog of SP in tomato. [Abstract/Link to Full Text]

Han HY, Ro KE, McPheron BA
Molecular phylogeny of the subfamily Tephritinae (Diptera: Tephritidae) based on mitochondrial 16S rDNA sequences.
Mol Cells. 2006 Aug 31;22(1):78-88.
The phylogeny of the subfamily Tephritinae (Diptera: Tephritidae) was reconstructed from mitochondrial 16S ribosomal RNA gene sequences using 53 species representing 11 currently recognized tribes of the Tephritinae and 10 outgroup species. The minimum evolution and Bayesian trees suggested the following phylogenetic relationships: (1) monophyly of the Tephritinae was strongly supported; (2) a sister group relationship between the Tephritinae and Plioreocepta was supported by the Bayesian tree; (3) the tribes Tephrellini, Myopitini, and Terelliini (excluding Neaspilota) were supported as monophyletic groups; (4) the non-monophyletic nature of the tribes Dithrycini, Eutretini, Noeetini, Tephritini, Cecidocharini, and Xyphosiini; and (5) recognition of 10 putative tribal groups, most of which were supported strongly by the statistical tests of the interior branches. Our results, therefore, convincingly suggest that an extensive rearrangement of the tribal classification of the Tephritinae is necessary. Since our sampling of taxa heavily relied on the current accepted classification, some lineages identified by the present study were severely under-sampled and other possible major lineages of the Tephritinae were probably not even represented in our dataset. We believe that our results provide baseline information for a more rigorous sampling of additional taxa representing all possible major lineages of the subfamily, which is essential for a comprehensive revision of the tephritine tribal classification. [Abstract/Link to Full Text]

Ko EM, Lee IY, Cheon IS, Kim J, Choi JS, Hwang JY, Cho JS, Lee DH, Kang D, Kim SH, Choe J
Monoclonal antibody to CD9 inhibits platelet-induced human endothelial cell proliferation.
Mol Cells. 2006 Aug 31;22(1):70-7.
Platelets are anucleate cytoplasmic fragments derived from bone marrow megakaryocytes, and endothelial cells constitute the barrier between bloodstream and adjacent tissues. Although platelets are thought to regulate the biological functions of endothelial cells, the molecular mechanisms involved are poorly understood. With human umbilical vein endothelial cells and freshly isolated platelets, we established an in vitro model of platelet-induced endothelial cell proliferation. Platelets stimulated endothelial cell proliferation in a dose-dependent manner and transwell experiments with semi-permeable membranes suggested that direct cell-to-cell contacts were required. We developed mAbs against platelets and selected a mAb that blocks their proliferative effect. We purified the antigen by immunoprecipitation and identified it by Q-TOF MS analysis as the tetraspanin CD9. Since both platelets and endothelial cells expressed CD9 strongly on their surfaces we carried out a pre-treatment experiment that showed that CD9 molecules on the endothelial cells participate in the mitogenic effect of the platelets. The inhibitory effect of our mAb was comparable to that of a well-known functional anti-CD9 mAb. These results suggest that the tetraspanin CD9 plays an important role in endothelial regeneration. [Abstract/Link to Full Text]

Kim BJ, So I, Kim KW
Involvement of the phospholipase C beta1 pathway in desensitization of the carbachol-activated nonselective cationic current in murine gastric myocytes.
Mol Cells. 2006 Aug 31;22(1):65-9.
In murine gastrointestinal myocytes muscarinic stimulation activates nonselective cation channels via a G-protein and Ca2+-dependent pathway. We recorded inward cationic currents following application of carbachol (ICCh) to murine gastric myocytes held at -60 mV, using the whole-cell patch-clamp method. The properties of the inward cationic currents were similar to those of the nonselective cation channels activated by muscarinic stimulation in other gastrointestinal smooth muscle cells. CCh-induced ICCh and spontaneous decay of ICCh (desensitization of ICCh) occurred. Unlike the situation in guinea pig gastric myocytes, desensitization was not affected by varying [EGTA]i. Pretreatment with the PLC inhibitor (U73122) blocked the activation of ICCh, and desensitization of ICCh was attenuated in PLC beta1 knock-out mice. These results suggest that the desensitization of ICCh in murine gastric myocytes is not due to a pathway dependent on intracellular Ca2+ but to the PLC beta1 pathway. [Abstract/Link to Full Text]

Oh SK, Yi SY, Yu SH, Moon JS, Park JM, Choi D
CaWRKY2, a chili pepper transcription factor, is rapidly induced by incompatible plant pathogens.
Mol Cells. 2006 Aug 31;22(1):58-64.
WRKY family proteins are a class of plant-specific transcription factors involved in stress response signaling pathways. In this study a gene encoding a putative WRKY protein was isolated from a pepper EST database (http://genepool.kribb.re.kr). The cDNA, named Capsicum annuum WRKY2 (CaWRKY2), encodes a putative polypeptide of 548 amino acids, containing two WRKY domains with zinc finger motifs and two potential nuclear localization signals. Northern blot analyses showed that CaWRKY2 mRNA was preferentially induced during incompatible interactions of pepper plants with PMMoV, Pseudomonas syringae pv. syringae 61, and Xanthomonas axonopodis pv. vesicatoria race 3. Furthermore, CaWRKY2 transcripts were strongly induced by wounding and ethephon treatment, whereas only moderate expression was detected following treatment with salicylic acid and jasmonic acid. CaWRKY2 was translocated to the nucleus when a CaWRKY2-smGFP fusion construct was expressed in onion epidermal cells. CaWRKY2 also had transcriptional activation activity in yeast. Taken together our data suggest that CaWRKY2 is a pathogen-inducible transcription factor that may have a role in early defense responses to biotic and abiotic stresses. [Abstract/Link to Full Text]

Kim HS, Yumkham S, Choi JH, Kim EK, Kim YS, Ryu SH, Suh PG
Haloperidol induces calcium ion influx via L-type calcium channels in hippocampal HN33 cells and renders the neurons more susceptible to oxidative stress.
Mol Cells. 2006 Aug 31;22(1):51-7.
Haloperidol is a classical neuroleptic drug that is still in clinical use and can lead to abnormal motor activity following repeated administration. However, there is little knowledge of how it triggers neuronal impairment. In this study, we report that it induced calcium ion influx via L-type calcium channels and that the elevation of calcium ions induced by haloperidol appeared to render hippocampal cells more susceptible to oxidative stress. Indeed, the level of cytotoxic reactive oxygen species (ROS) and the expression of pro-apoptotic Bax increased in response to oxidative stress in haloperidol-treated cells, and these effects were inhibited by verapamil, a specific L-type calcium channel blocker, but not by the T-type calcium channel blocker, mibefradil. These findings indicate that haloperidol induces calcium ion influx via L-type calcium channels and that this calcium influx influences neuronal fate. [Abstract/Link to Full Text]

Song HJ, Min YS, Shin CY, Jeong JH, Sohn UD
Activation of p38 MAPK is involved in endothelin-1-stimulated COX-2 expression in cultured Feline esophageal smooth muscle cells.
Mol Cells. 2006 Aug 31;22(1):44-50.
We investigated the possible role of p38 MAPK and ETB receptors in ET-1 induction of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in cultured feline esophageal smooth muscle cells (ESMC). Confluent layers of ESMC were stimulated with 10 nM ET-1 and expression of COX-1 and COX-2, involvement of receptors, and activation of p38 MAPK, were examined by Western blot analysis. Levels of PGE2 induced by ET-1 were measured by Elisa. Using ETA and ETB antagonists (BQ-123 and BQ-788, respectively), the contribution of the ET receptors to COX-1 and COX-2 expression induced by ET-1 was determined. Western blot analysis revealed that treatment of ESMC with ET-1 resulted in transient expression of COX-2 and activation of p38 MAPK. Activation of p38 MAPK was maximal after 1 h. SB202190, a p38 MAPK inhibitor, reduced expression of COX-2, but not COX-1. ET-1-induced release of PGE2 was also blocked by SB202190. COX-2 expression was upregulated only via the ETB receptor, and COX-1 expression was not affected by either antagonist. Taken together, our data suggest that ET-1 causes p38 MAPK-dependent expression of COX-2 by interacting with ETB receptors on ESMC. [Abstract/Link to Full Text]

Jeong JA, Lee Y, Lee W, Jung S, Lee DS, Jeong N, Lee HS, Bae Y, Jeon CJ, Kim H
Proteomic analysis of the hydrophobic fraction of mesenchymal stem cells derived from human umbilical cord blood.
Mol Cells. 2006 Aug 31;22(1):36-43.
Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering, but their application has been impeded by lack of knowledge of their core biological properties. In order to identify MSC-specific proteins, the hydrophobic protein fraction was individually prepared from two different umbilical cord blood (UCB)-derived MSC populations; these were then subjected to two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). Although the 2D gel patterns differed somewhat between the two samples, computer-assisted image analysis identified shared protein spots. 35 spots were reliably identified corresponding to 32 different proteins, many of which were chaperones. Based on their primary sub-cellular locations the proteins could be grouped into 6 categories: extracellular, cell surface, endoplasmic reticular, mitochondrial, cytoplasmic and cytoskeletal proteins. This map of the water-insoluble proteome may provide valuable insights into the biology of the cell surface and other compartments of human MSCs. [Abstract/Link to Full Text]

Kim BU, Choi J, Ahn KH, Jeong JK, Ha CM, Jeong CS, Lee CK, Kang SG, Lee BJ
Munc18 plays an important role in the regulation of glutamate release during female puberty onset.
Mol Cells. 2006 Aug 31;22(1):30-5.
Munc18, a mammalian homolog of C. elegans Unc, is essential for neurotransmitter release. The aim of this study was to identify estrogen-dependent expression of Munc18-1 and its role in the regulation of glutamate release for puberty onset. Hypothalamic munc18-1 mRNA levels were significantly increased by estrogen treatment in ovariectomized, immature female rats. During pubertal development, the munc18-1 mRNA levels dramatically increased between the juvenile period and the anestrous phase of puberty. Intracerebroventricular administration of an antisense oligodeoxynucleotide against munc18-1 mRNA significantly decreased glutamate release and delayed the day of puberty onset. These results suggest that Munc18-1, expressed in an estrogen-dependent manner, plays an important role in the onset of female puberty via the regulation of glutamate release. [Abstract/Link to Full Text]

Lee JC, Son YO, Choi KC, Jang YS
Hydrogen peroxide induces apoptosis of BJAB cells due to formation of hydroxyl radicals via intracellular iron-mediated Fenton chemistry in glucose oxidase-mediated oxidative stress.
Mol Cells. 2006 Aug 31;22(1):21-9.
The aim of this study was to determine if hydrogen peroxide (H2O2) generated by glucose oxidase (GO) induces apoptosis or necrosis of BJAB cells and which radical is the direct mediator of cell death. We found that GO produced H2O2 continuously in low concentrations, similar to in vivo conditions, and decreased proliferation and cell viability in a dose-dependent manner. The GO-mediated cytotoxicity resulted from apoptosis, and was confirmed by monitoring the cells after H33342/Annexin V/propidium iodide staining. Decreases of mitochondrial membrane potential and intracellular glutathione level were found to be critical events in the H2O2-mediated apoptosis. Additional experiments revealed that H2O2 exerted its apoptotic action through the formation of hydroxyl radicals via the Fenton rather than the Haber-Weiss reaction. Moreover, intracellular redox-active iron, but not copper, participated in the H2O2-mediated apoptosis. [Abstract/Link to Full Text]

Liang Y, Kang CB, Yoon HS
Molecular and structural characterization of the domain 2 of hepatitis C virus non-structural protein 5A.
Mol Cells. 2006 Aug 31;22(1):13-20.
Hepatitis C virus (HCV) non-structural protein 5A protein (NS5A), which consists of three functional domains, is involved in regulating viral replication, interferon resistance, and apoptosis. Recently, the three-dimensional structure of the domain 1 was determined. However, currently the molecular basis for the domains 2 and 3 of HCV NS5A is yet to be defined. Toward this end, we expressed, purified the domain 2 of the NS5A (NS5A-D2), and then performed biochemical and structural studies. The purified domain 2 was active and was able to bind NS5B and PKR, biological partners of NS5A. The results from gel filtration, CD analysis, 1D 1H NMR and 2D 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy indicate that the domain 2 of NS5A appears to be flexible and disordered. [Abstract/Link to Full Text]

Kim Y, Hong S, Noh MR, Kim SY, Huh PW, Park SH, Sun W, Kim H
Inductin of neuron-derived orphan receptor-1 in the dentate gyrus of the hippocampal formation following transient global ischemia in the rat.
Mol Cells. 2006 Aug 31;22(1):8-12.
Neuron-derived orphan receptor (NOR-1) is a member of the thyroid/steroid receptor superfamily that was originally identified in forebrain neuronal cells undergoing apoptosis. In addition to apoptotic stimuli, activation of several signal transduction pathways including direct neuronal depolarization regulates the expression of NOR-1. In this study we tested whether the expression of NOR-1 is changed following transient ischemic injury in the adult rat brain. NOR-1 mRNA increased rapidly in the dentate gyrus of the hippocampal formation and piriform cortex 3 h after transient global ischemia and returned to basal level at 6 h. On the other hand, oxygen-glucose deprivation of cultured cerebral cortical neurons did not alter the expression of NOR-1. These results suggest that expression of NOR-1 is differentially regulated in different brain regions in response to globally applied brain ischemia, but that hypoxia is not sufficient to induce its expression. [Abstract/Link to Full Text]

Lee M, Park J
Regulation of NFAT activation: a potential therapeutic target for immunosuppression.
Mol Cells. 2006 Aug 31;22(1):1-7.
The NFAT family of transcription factors plays pivotal roles in the development and function of the immune system. Their activation process is tightly regulated by calcium-dependent phosphatase calcineurin and has been a target of the immunosuppressive drugs cyclosporin A and FK-506. Although the clinical use of these drugs has dramatically increased the success of organ transplantation, their therapeutic use is limited by severe side effects. Recent studies for the calcineurin/NFAT signaling pathway have identified a number of cellular proteins that inhibit calcineurin function. Specific peptide sequences that interfere with the interaction between calcineurin and NFAT have also been characterized. Moreover, diverse approaches to identify small organic molecules that modulate NFAT function have been performed. This review focuses on the recent advances in our understanding of the inhibitory modulation of NFAT function, which may open up the additional avenues for immunosuppressive therapy. [Abstract/Link to Full Text]

Kang SY, Kim SN, Kim SH, Jeon SH
Temporal and spatial expression of homeotic genes is important for segment-specific neuroblast 6-4 lineage formation in Drosophila.
Mol Cells. 2006 Jun 30;21(3):436-42.
Different proliferation of neuroblast 6-4 (NB6-4) in the thorax and abdomen produces segmental specific expression pattern of several neuroblast marker genes. NB6-4 is divided to form four medialmost cell body glia (MM-CBG) per segment in thorax and two MM-CBG per segment in abdomen. As homeotic genes determine the identities of embryonic segments along theA/P axis, we investigated if temporal and specific expression of homeotic genes affects MM-CBG patterns in thorax and abdomen. A Ubx loss-of-function mutation was found to hardly affect MM-CBG formation, whereas abd-A and Abd-B caused the transformation of abdominal MM-CBG to their thoracic counterparts. On the other hand, gain-of-function mutants of Ubx, abd-A and Abd-B genes reduced the number of thoracic MM-CBG, indicating that thoracic MM-CBG resembled abdominal MM-CBG. However, mutations in Polycomb group (PcG) genes, which are negative transregulators of homeotic genes, did not cause the thoracic to abdominal MM-CBG pattern transformation although the number of MM-CBG in a few per-cent of embryos were partially reduced or abnormally patterned. Our results indicate that temporal and spa-tial expression of the homeotic genes is important to determine segmental-specificity of NB6-4 daughter cells along the anterior-posterior (A/P) axis. [Abstract/Link to Full Text]

Kweon DH, Shin YK, Shin JY, Lee JH, Lee JB, Seo JH, Kim YS
Membrane topology of helix 0 of the Epsin N-terminal homology domain.
Mol Cells. 2006 Jun 30;21(3):428-35.
Specific interaction of the epsin N-terminal homology (ENTH) domain with the plasma membrane appears to bridge other related proteins to the specific regions of the membrane that are invaginated to form endocytic vesicles. An additional a-helix, referred to as helix 0 (H0), is formed in the presence of the soluble ligand inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] at the N terminus of the ENTH domain (amino acid residues 3-15). The ENTH domain alone and full-length epsin cause tubulation of liposomes made of brain lipids. Thus, it is believed that H0 is membrane-inserted when it is coordinated with the phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], resulting in membrane deformation as well as recruitment of accessory factors to the membrane. However, formation of H0 in a real biological membrane has not been demonstrated. In the present study, the membrane structure of H0 was determined by measurement of electron paramagnetic resonance (EPR) nitroxide accessibility. H0 was located at the phosphate head-group region of the membrane. Moreover, EPR line-shape analysis indicated that no pre-formed H0-like structure were present on normal acidic membranes. PtdIns(4,5)P2 was necessary and sufficient for interaction of the H0 region with the membrane. H0 was stable only in the membrane. In conclusion, the H0 region of the ENTH domain has an intrinsic ability to form H0 in a PtdIns(4,5)P2-containing membrane, perhaps functioning as a sensor of membrane patches enriched with PtdIns(4,5)P2 that will initiate curvature to form endocytic vesicles. [Abstract/Link to Full Text]

Kang S, Kim HB, Lee H, Choi JY, Heu S, Oh CJ, Kwon SI, An CS
Overexpression in Arabidopsis of a plasma membrane-targeting glutamate receptor from small radish increases glutamate-mediated Ca2+ influx and delays fungal infection.
Mol Cells. 2006 Jun 30;21(3):418-27.
Ionotropic glutamate receptors (iGluRs) are ligand-gated nonselective cation channels that mediate fast excitatory neurotransmission. Although homologues of the iGluRs have been identified in higher plants, their roles are largely unknown. In this work we isolated a full-length cDNA clone (RsGluR) encoding a putative glutamate receptor from small radish. An RsGluR: mGFP fusion protein was localized to the plasma membrane. In Arabidopsis thaliana overexpressing the full-length cDNA, glutamate treatment triggered greater Ca2+ influx in the root cells of transgenic seedlings than in those of the wild type. Transgenic plants exhibited multiple morphological changes such as necrosis at their tips and the margins of developing leaves, dwarf stature with multiple secondary inflorescences, and retarded growth, as previously observed in transgenic Arabidopsis overexpressing AtGluR3.2 [Kim et al. (2001)]. Microarray analysis showed that jasmonic acid (JA)-responsive genes including defensins and JA-biosynthetic genes were up-regulated. RsGluR overexpression also inhibited growth of a necrotic fungal pathogen Botrytis cinerea possibly due to up-regulation of the defensins. Based on these results, we suggest that RsGluR is a glutamate-gated Ca2+ channel located in the plasma membrane of higher plants and plays a direct or indirect role in defense against pathogen infection by triggering JA biosynthesis. [Abstract/Link to Full Text]

Cho KS, Yang TJ, Hong SY, Kwon YS, Woo JG, Park HG
Determination of cytoplasmic male sterile factors in onion plants (Allium cepa L.) using PCR-RFLP and SNP markers.
Mol Cells. 2006 Jun 30;21(3):411-7.
We have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker that can distinguish male-fertile (N) and male-sterile (S) cytoplasm in onions. The PCR-RFLP marker was located in a chloroplast psbA gene amplicon. Digesting the amplicons from different cytoplasm-containing varieties with the restriction enzyme MspI revealed that N-cytoplasm plants have a functional MspI site (CCGG), whereas the S-cytoplasm plants has a substitution in that site (CTGG), and thus no MspI target. The results obtained using this PCR-RFLP marker to distinguish between cytoplasmic male sterile factors in 35 onion varieties corresponded with those using a CMS-specific sequence-characterized amplified region (SCAR) marker. Moreover, the PCR-RFLP marker can identify N- ot S-cytoplasms in DNA sample mixtures in which they are in up to a 10-fold minority, indicating that use of the marker has high diagnostic precision. We also demonstrated the usefulness of the SNP detected in the psbA gene for high-throughput discrimination of CMS factors using Real-time PCR and a TaqMan probe assay. [Abstract/Link to Full Text]

Lee SM, Kang K, Chung H, Yoo SH, Xu XM, Lee SB, Cheong JJ, Daniell H, Kim M
Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny.
Mol Cells. 2006 Jun 30;21(3):401-10.
The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastid-expressed green fluorescent protein (GFP) and aminoglycoside 3'-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops. [Abstract/Link to Full Text]

Zhao X, Fan Y, Shen J, Wu Y, Yin Z
Human glutathione S-transferase P1 suppresses MEKK1-mediated apoptosis by regulating MEKK1 kinase activity in HEK293 cells.
Mol Cells. 2006 Jun 30;21(3):395-400.
Glutathione S-transferase P1 (GSTP1) plays an important role in detoxification and the metabolism of xenobiotics. Here we show that GSTP1 also regulates the MEKK1-MKK7 signaling pathway. Over-expression of GSTP1 in HEK293 cells inhibited both DMEKK1- and etoposide-induced apoptosis, and inhibited pro-caspase-3 activation and PARP cleavage. MEKK1-induced apoptosis requires both its kinase activity and proteolytic cleavage. DMEKK1 activity was inhibited by over-expression of GSTP1 in vivo and MEKK1 kinase activity was also inhibited by GSTP1 in vitro when assayed with bacterially-expressed MKK7(KM) protein as substrate. GSTP1 inhibition of etoposide-induced cell apoptosis was mainly due to its ability to suppress MEKK1 kinase activity. The glutathione-conjugating activity of GSTP1 was essential for the above effects. These findings provide insight into the mechanism by which GSTP1 protects cells from genotoxin-induced apoptosis. [Abstract/Link to Full Text]

Kim YS, Ham BK, Paek KH, Park CM, Chua NH
An Arabidopsis homologue of human seven-in-absentia-interacting protein is involved in pathogen resistance.
Mol Cells. 2006 Jun 30;21(3):389-94.
Human seven-in-absentia (SIAH)-interacting protein (SIP) is a component of the E3 ligase complex targeting beta-catenin for destruction. Arabidopsis has one SIP protein (AtSIP) with 32% amino acid sequence identity to SIP. To investigate the functions of AtSIP, we isolated an atsip knockout mutant, and generated transgenic plants overexpressing AtSIP. The growth rates and morphologies of the atsip and transgenic plants were indistinguishable from those of wild type. However, atsip plants were more susceptible to Pseudomonas syringae infection, and the transgenic plants overexpressing AtSIP were more resistant. Consistent with this, RNA blot analysis showed that the AtSIP gene is strongly induced by wounding and hydrogen peroxide treatment. In addition, when plants were infected with P. syringae, AtSIP was transiently induced prior to PR-1 induction. These observations show that Arabidopsis AtSIP plays a role in resistance to pathogenic infection. [Abstract/Link to Full Text]

Matsumoto R, Rakwal R, Agrawal GK, Jung YH, Jwa NS, Yonekura M, Iwahashi H, Akama K
Search for novel stress-responsive protein components using a yeast mutant lacking two cytosolic Hsp70 genes, SSA1 and SSA2.
Mol Cells. 2006 Jun 30;21(3):381-8.
Heat shock proteins (Hsp) 70 are a ubiquitous family of molecular chaperones involved in many cellular processes. A yeast strain, ssa1/2, with two functionally redundant cytosolic Hsp70s (SSA1 and SSA2) deleted shows thermotolerance comparable to mildly heat-shocked wild type yeast, as well as increased protein synthesis and ubiquitin-proteasome protein degradation. Since mRNA abundance does not always correlate well with protein expression levels it is essential to study proteins directly. We used a gel-based approach to identify stress-responsive proteins in the ssa1/2 mutant and identified 43 differentially expressed spots. These were trypsin-digested and analyzed by nano electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). A total of 22 non-redundant proteins were identified, 11 of which were confirmed by N-terminal sequencing. Nine proteins, most of which were up-regulated (2-fold or more) in the ssa1/2 mutant, proved to be stress-inducible proteins such as molecular chaperones and anti-oxidant proteins, or proteins related to carbohydrate metabolism. Interestingly, a translational factor Hyp2p up-regulated in the mutant was also found to be highly phosphorylated. These results indicate that the cytosolic Hsp70s, Ssa1p and Ssa2p, regulate an abundance of proteins mainly involved in stress responses and protein synthesis. [Abstract/Link to Full Text]

Bae KH, Kim JS
One-step selection of artificial transcription factors using an in vivo screening system.
Mol Cells. 2006 Jun 30;21(3):376-80.
Gene expression is regulated in large part at the level of transcription under the control of sequence-specific transcriptional regulatory proteins. Therefore, the ability to affect gene expression at will using sequence-specific artificial transcription factors would provide researchers with a powerful tool for biotechnology research and drug discovery. Previously, we isolated 56 novel sequence-specific DNA-binding domains from the human genome by in vivo selection. We hypothesized that these domains might be more useful for regulating gene expression in higher eukaryotic cells than those selected in vitro using phage display. However, an unpredictable factor, termed the "context effect", is associated with the construction of novel zinc finger transcription factors--- DNA-binding proteins that bind specifically to 9-base pair target sequences. In this study, we directly selected active artificial zinc finger proteins from a zinc finger protein library. Direct in vivo selection of constituents of a zinc finger protein library may be an efficient method for isolating multi-finger DNA binding proteins while avoiding the context effect. [Abstract/Link to Full Text]

Shim H, Shim E, Lee H, Hahn J, Kang D, Lee YS, Jeoung D
CAGE, a novel cancer/testis antigen gene, promotes cell motility by activation ERK and p38 MAPK and downregulating ROS.
Mol Cells. 2006 Jun 30;21(3):367-75.
We previously identified a novel cancer/testis antigen gene CAGE by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera of gastric cancer patients. CAGE is expressed in many cancers and cancer cell lines, but not in normal tissues apart from the testis. In the present study, we investigated its role in the motility of cells of two human cancer cell lines: HeLa and the human hepatic cancer cell line, SNU387. Induction of CAGE by tetracycline or transient transfection enhanced the migration and invasiveness of HeLa cells, but not the adhesiveness of either cell line. Overexpression of CAGE led to activation of ERK and p38 MAPK but not Akt, and inhibition of ERK by PD98059 or p38 MAPK by SB203580 counteracted the CAGE-promoted increase in motility in both cell lines. Overexpression of CAGE also resulted in a reduction of ROS and an increase of ROS scavenging, associated with induction of catalase activity. Inhibition of ERK and p38 MAPK increased ROS levels in cells transfected with CAGE, suggesting that ROS reduce the motility of both cell lines. Inhibition of ERK and p38 MAPK reduced the induction of catalase activity resulting from overexpression of CAGE, and inhibition of catalase reduced CAGE-promoted motility. We conclude that CAGE enhances the motility of cancer cells by activating ERK and p38 MAPK, inducing catalase activity, and reducing ROS levels. [Abstract/Link to Full Text]

Kwon SJ, Hong SW, Son JH, Lee JK, Cha YS, Eun MY, Kim NS
CACTA and MITE transposon distributions on a genetic map of rice using F15 RILs derived from Milyang 23 and Gihobyeo hybrids.
Mol Cells. 2006 Jun 30;21(3):360-6.
Up to 35% of the rice genome consists of various kinds of transposons, and CACTA and MITE are two of the major class 2 DNA transposons in the genome. We have employed the consensus sequences of Rim2/Hipa CACTA, Stowaway MITE Pangrangja, and Tourist MITE Ditto for transposon display (TD) analysis to locate them on a genetic map, with 58 SSR markers used to anchor them. The TD analysis produced a high profile of the polymorphisms between the parental lines, Oryza sativa var. Gihobyeo/O. sativa var. Milyang, in intraspecific F15 RIL lines, locating 368 markers of Rim2/Hipa CACTA, 78 markers of Tourist MITE Ditto, and 22 markers of Stowaway MITE Pangrangja. In the segregation analysis, non-parental segregating bands and segregation distortion bands were observed. The recombinant genetic map spans 3023.9 cM, with 5.7 cM the average distance between markers. The TD markers were distributed unequally on the chromosomes because many TD markers were located in pericentric chromosomal regions except in the cases of chromosomes 2, 3, 6 and 9. Although the number of transposon markers was not sufficient to include all rice class 2 transposons, the current map of CACTA and MITE transposons should provide new insight into the genome organization of rice since no previous DNA transposon map is available. [Abstract/Link to Full Text]

---

Recent Articles in Cell Research

---

Yuan JD, Shi JX, Meng GX, An LG, Hu GX
Nuclear pseudogenes of mitochondrial DNA as a variable part of the human genome.
Cell Res. 1999 Dec;9(4):281-90.
Novel pseudogenes homologous to the mitochondrial (mt) 16S rRNA gene were detected via different approaches. Eight pseudogenes were sequenced. Copy number polymorphism of the mtDNA pseudogenes was observed among randomly chosen individuals, and even among siblings. A mtDNA pseudogene in the Y-chromosome was observed in a YAC clone carrying only repetitive sequence tag site (STS). PCR screening of human yeast artificial chromosome (YAC) libraries showed that there were at least 5.7 x 10(5) bp of the mtDNA pseudogenes in each haploid nuclear genome. Possible involvement of the mtDNA pseudogenes in the variable part of the human nuclear genome is discussed. [Abstract/Link to Full Text]

Zhu ZY, Zhong CP, Xu WF, Lin GM, Ye GQ, Ji YY, Sun B, Yeh M
PSMA mimotope isolated from phage displayed peptide library can induce PSMA specific immune response.
Cell Res. 1999 Dec;9(4):271-80.
Prostate-specific membrane antigen (PSMA) is a cell surface glycoprotein expressed predominantly in prostate secretory acinar epithelium and prostate cancer cells as well as in several extraprostatic tissues. Mouse monoclonal antibody 4G5 specific to the extracellular domain of PSMA was used to screen two phage displayed peptide libraries (9aa linear and 9aa cys library). Three 4G5-reactive phagotopes were identified. Sequence analysis of isolated clones demonstrated that the interaction motif "VDPA/SK" has high homology to 719-725aa on PSMA. Immunohistochemical staining of the prostate cancer sample with the PSMA-mimic phagotope (mimotope) immunized serum antibodies demonstrate that the mimotope isolated from the phage displayed peptide libraries can induce PSMA specific immune response in vivo. [Abstract/Link to Full Text]

Wadhwa R, Takano S, Mitsui Y, Kaul SC
NIH 3T3 cells malignantly transformed by mot-2 show inactivation and cytoplasmic sequestration of the p53 protein.
Cell Res. 1999 Dec;9(4):261-9.
In previous studies we have reported that a high level of expression of mot-2 protein results in malignant transformation of NIH 3T3 cells as analyzed by anchorage independent growth and nude mice assays [Kaul et al., Oncogene, 17, 907-11, 1998]. Mot-2 was found to interact with tumor suppressor protein p53. The transient overexpression of mot-2 was inhibitory to transcriptional activation function of p53 [Wadhwa et al., J. Biol. Chem., 273, 29586-91, 1998]. We demonstrate here that mot-2 transfected stable clone of NIH 3T3 that showed malignant properties indeed show inactivation of p53 function as assayed by exogenous p53 dependent reporter. The expression level of p53 in response to UV-irradiation was lower in NIH 3T3/mot-2 as compared to NIH 3T3 cells and also exhibited delay in reaching peak. Furthermore, upon serum starvation p53 was seen to translocate to the nucleus in NIH 3T3, but not in its mot-2 derivative. The data suggests that mot-2 mediated cytoplasmic sequestration and inactivation of p53 may operate, at least in part, for malignant phenotype of NIH 3T3/mot-2 cells. NIH 3T3/mot-2 cells show inactivation of p53 protein. [Abstract/Link to Full Text]

Zhao H, Zhang Y, Zhang SB, Jiang C, He QY, Li MQ, Qian RL
The structure of the nucleosome core particle of chromatin in chicken erythrocytes visualized by using atomic force microscopy.
Cell Res. 1999 Dec;9(4):255-60.
The structure of the nucleosome core particle of chromatin in chicken erythrocytes has been examined by using AFM. The 146 bp of DNA wrapped twice around the core histone octamer are clearly visualized. Both the ends of entry/exit of linker DNA are also demonstrated. The dimension of the nucleosome core particles is approximately 1-4 nm in height and approximately 13-22 nm in width. In addition, superbeads (width of approximately 48-57 nm, height of approximately 2-3 nm) are occasionally revealed, two turns of DNA around the core particles are also detected. [Abstract/Link to Full Text]

Yi J, Tang XM
The convergent point of the endocytic and autophagic pathways in leydig cells.
Cell Res. 1999 Dec;9(4):243-53.
Endocytic tracers and marker enzyme of lysosomes were used in the present study to analyze the processes of autophagocytosis and endocytosis, and the convergent point of these two pathways in Leydig cells. The endocytic and autophagic compartments can be easily identified in Leydig cells, which makes easier to define the stages of two pathways than was possible before. The evidences indicated that the late endosomes (dense MVBs) deliver their endocytosed gold tracers together with lysosomal enzymes to the early autophagosomes and they are the convergent point of the two pathways. During this convergent process, the early autophagosomes transform into late autophagosomes and the late endosomes transform into mature lysosomes. [Abstract/Link to Full Text]

Miao JY, Araki S, Han YR, Hayashi H
Involvement of gene expressions in apoptosis of vascular endothelial cells induced by rattlesnake venom.
Cell Res. 1999 Sep;9(3):237-42.
Formation of apoptotic bodies is a typical character of apoptotic cell death, but how the processes are controlled is not known. In this study, we compared two apoptosis inducing systems in vascular endothelial cells (VEC). We found that the formation of apoptotic bodies during apoptosis induced by rattlesnake venom, which is an unique and specific apoptosis inducer to vascular endothelial cells, was much faster than that induced by deprivation of survival factors (aFGF and serum). When we blocked the synthesis of mRNAs in cells treated with rattlesnake venom by DRB (5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole), an inhibitor of transcription, the formation of apoptotic bodies was dramatically inhibited. We examined the expression of p53 gene and found that its expression was much higher in apoptosis induced by rattlesnake venom than that in apoptosis induced by deprivation of aFGF and serum. Our results suggest that gene expression is important and p53 gene may play a major role in inducing the formation of apoptotic bodies in VEC. [Abstract/Link to Full Text]

Gao Ding Cheng W, Dai J
Retrovirus-mediated herpes simplex virus thymidine kinase gene therapy approach for hepatocellular carcinoma.
Cell Res. 1999 Sep;9(3):225-35.
The therapeutic effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system on hepatocellular carcinoma was studied in this experiment. The tk-containing retroviral recombinants were used to infect hepatoma cells (BEL-7402) and the cells were treated with ganciclovir (0-1000 microg/ml). The results showed that HSV-tk gene could be efficiently transferred in vitro into hepatoma cells and stably expressed. The growth potential of the tk-containing cells was significantly inhibited by GCV (P<0.01) as compared to the non-tk-containing cells. The antitumor effect of HSV-tk/GCV system was also produced ex vivo in tk-containing tumor of nude mice as characterized by a marked decrease in tumor growth after GCV treatment contrary to a progressive enlargement of non-tk-containing tumors. Although the histological examination demonstrated that the efficiency of the gene transfer was less than 30%, the killing effect of HSV-tk/GCV system on hepatocellular carcinoma was still significantly generated. The proper mechanism of HSV-tk gene therapy on hepatic tumor referred as "bystander effect" in therapeutic approach has not been found in this study and required to be explored further. [Abstract/Link to Full Text]

Qui JJ, Li YP
Random amplified polymorphic DNA analysis of eel genome.
Cell Res. 1999 Sep;9(3):217-23.
Eel family is a huge one, in which many kinds of eels especially some migratory eels, bear strong resemblance to each other, and are therefore difficult to be identified. In this study 29 random primers were used to make RAPD analysis for Japanese eel (Anguilla japonica), European eel (Anguilla anguilla) and Pike eel (Muraenesox cinereus). And totally 299 fragments were counted. Shared or specific fragments were counted and genetic similarity or genetic distance were calculated. The genetic similarity between Japanese eel and Pike eel is 0.68 and the genetic distance between them is 0.32; those between European eel and Pike eel are 0.72 and 0.28 respectively, and between Japanese eel and European eel are 0.74 and 0.25 respectively. The method has been shown to be suitable to molecular identification of eels. It provides an alternative approach to determine the relationship between species. [Abstract/Link to Full Text]

Tao QH, Yang J, Mei WY, Geng X, Ding XY
Cloning and analysing of 5' flanking region of Xenopus organizer gene noggin.
Cell Res. 1999 Sep;9(3):209-16.
Xenopus organizer specific gene Noggin possesses nearly all the characteristic properties of the action of organizer to specify the embryonic body axis. To analyze how the maternal inherited factors control its expression pattern, we cloned the 5' regulatory region of noggin gene. The 1.5 kb upstream sequence could direct reporter gene to express in vivo and data from deletion analysis indicated that a 229 base pair fragment is essential for activating noggin expression. We further demonstrated that the response elements within this regulatory region were indeed under the control of growth factor activin and Wnt signaling pathway components. [Abstract/Link to Full Text]

Xin X, Yu YS, Tsung HC, Sugano S, Yan YC
The developmental fate of green fluorescent mouse embryonic germ cells in chimeric embryos.
Cell Res. 1999 Sep;9(3):201-8.
Primordial germ cells (PGCs), as precursors of mammalian germ lineage, have been gaining more attention as a new resource of pluripotent stem cells, which bring a great possibility to study developmental events of germ cell in vitro and at animal level. EG4 cells derived from 10.5 days post coitum (dpc) PGCs of 129/svJ strain mouse were established and maintained in an undifferentiated state. With an attempt to study the differentiation capability of EG4 cells with a reporter protein: green fluorescence protein, and the possible application of EG4 cells in the research of germ cell development, we have generated several EG4-GFP cell lines expressing enhanced green fluorescence protein (EGFP) and still maintaining typical characteristics of pluripotent stem cells. Then, the differentiation of EG4-GFP cells in vitro as well as their developmental fate in chimeric embryos which were produced by aggregating EG4-GFP cells to 8-cell stage embryos were studied. The results showed that EG4 cells carrying green fluorescence have a potential use in the research of germ cell development and other related studies. [Abstract/Link to Full Text]

Hua GY, Wang P, Takagi K, Shimozato O, Yagita H, Okigaki T, Matasumura M
Expression of a soluble form of CTLA4 on macrophage and its biological activity.
Cell Res. 1999 Sep;9(3):189-99.
Interaction between cytotoxic T lymphocyte-associated antigen-4 (CTLA4, CD152) and B7 molecules (B7-1 and B7-2) is of importance in the cellular events of lymphocyte, including antigen-specific T-cell activation and induction of autoreactive T-cell. We describe here the first introduction of a murine soluble CTLA4 gene, CTLA4Ig, to Mm1 cells, a macrophagic cell line. CTLA4Ig was successfully expressed on Mml cells and the expressed CTLA4Ig was found to be functionally active in their binding to B7 molecules by flow cytometry and immunofluorescence studies. The biological activity of CTLA4Ig from the transfected Mm1 cells was studied and showed inhibitory activity on mixed lymphocyte culture. A high CTLA4Ig producing macrophagic cell line was obtained. As Mm1 cells were regarded as difficult for gene transfection and there had so far been no report on expression of CTLA4Ig gene on Mm1 cells, these results suggested that the CTLA4Ig expressing Mm1 cells could be useful for analysis of CTLA4 and B7 molecule interaction in both macrophage and T-cell. [Abstract/Link to Full Text]

Kageyama R, Ohtsuka T
The Notch-Hes pathway in mammalian neural development.
Cell Res. 1999 Sep;9(3):179-88.
A wide variety of neurons and glial cells differentiate from common precursor cells in the developing nervous system. During this process, Notch-mediated cell-cell interaction is essential for maintenance of dividing cells and subsequent generation of cell type diversity. Activation of Notch inhibits cellular differentiation, and abnormality of the Notch pathway leads to premature neuronal differentiation, the lack of some cell types, and severe defects of tissue morphogenesis. Recent data demonstrate that Notch fails to inhibit cellular differentiation in the absence of the bHLH genes Hes1 and Hes5, which functionally antagonize the neuronal bHLH genes such as Mash1. These results indicate that the two Hes genes are essential effectors for the Notch pathway and that neuronal differentiation is controlled by the pathway "Notch-Hes1/Hes5-|Mash1". [Abstract/Link to Full Text]

Li Z
The alphaMbeta2 integrin and its role in neutrophil function.
Cell Res. 1999 Sep;9(3):171-8.
Neutrophils are the first cell type to arrive at the injury sites and play a critical role in host defense, by virtue of its ability to adhere and transmigrate through endothelium, to phagocytose foreign pathogens, and to produce free oxygen radicals and proteolytic enzymes. Yet, inappropriate neutrophil activation causes tissue damage and various inflammatory diseases. These physiological and pathological functions of neutrophils depend on the engagement of certain surface receptors, especially alphaMbeta2, the major beta2 integrin receptor present on neutrophil surface. Understanding of the molecular mechanisms underlying ligand binding by alphaMbeta2, as well as the roles of alphaMbeta2-ligand interactions in neutrophil functions will enable us to regulate more precisely neutrophil activities: that is, to promote their host defense functions, and at the same time to minimize their deleterious effects on normal cells. [Abstract/Link to Full Text]

Rui WJ
Regulation of eukaryotic DNA replication and nuclear structure.
Cell Res. 1999 Sep;9(3):163-70.
In eukaryote, nuclear structure is a key component for the functions of eukaryotic cells. More and more evidences show that the nuclear structure plays important role in regulating DNA replication. The nuclear structure provides a physical barrier for the replication licensing, participates in the decision where DNA replication initiates, and organizes replication proteins as replication factory for DNA replication. Through these works, new concepts on the regulation of DNA replication have emerged, which will be discussed in this minireview. [Abstract/Link to Full Text]

Zhang R, Wang GL, Zhang PL, Xiong Y, Zhang WB, Wang XP, Yin DL, Jing Q
Suppression of angiotensin II stimulated responses in aortic vascular smooth muscle cells of experimental cirrhotic rats.
Cell Res. 1999 Jun;9(2):155-61.
Functional responses to angiotensin II (AT-II) were determined in aortic vascular smooth muscle cells (VSMCs) from experimental cirrhotic rats. Our data showed that AT-II-stimulated extracellular acidification rate (ECAR), which was measured by Cytosensor microphysiometry, was significantly reduced in the aortic VSMCs from the cirrhotic rats as compared to those from the control animals. The ability of AT-II to promote formation of inositol phosphates, the second messenger produced by the activation of Gq-coupled receptors, was also considerably suppressed in the cirrhotic VSMCs. Furthermore, the maximal p42/44 MAPK phosphorylation stimulated by AT-II was significantly reduced in the cirrhotic VSMCs in contrast to that in the normal VSMCs. Taken together, our data clearly demonstrated that the functional responses to AT-II was severely suppressed in aortic VSMCs in cirrhosis, indicating the impairment of general Gq-coupled receptor signaling and subsequent biological function in the cirrhotic VSMCs. [Abstract/Link to Full Text]

Yang ZA, Li QH, Wang YF, Gui JF
Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp.
Cell Res. 1999 Jun;9(2):145-54.
The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp. MPF activity was measured by using histone H1 as phosphorylation substrate. There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes, but there existed some subtle difference between them. The subtle difference was that the first peak of MPF kinase activity was kept to a longer-lasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp. It was suggested that the difference may be related to the spindle behavior changes, such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp. [Abstract/Link to Full Text]

Xu CS, Zhang WM, Techel D, Meyer M, Li YZ, Rensing L
Heat shock induction of a 65 kDa ATP-binding proteinase in rat C6 glioma cells.
Cell Res. 1999 Jun;9(2):135-44.
The 45, 55, 65 and 100 kDa ATP-binding proteinases (ATP-BPases) of the heat-shocked (44 degrees C for 30 min, recovery for 12 h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography. Their molecular masses, isoelectric points (pI), pH-optima and other properties were analyzed by native proteinase gels. It was shown that the 65 kDa ATP-BPase is specifically induced by heat shock and not detectable in control cells. Its N-terminal 1-9 amino acid sequence was determined by Edman degradation, but no homologies to other proteins in the protein data bases were found. 30 and 31 kDa proteinases can be cleaved from the 45, 55 and 65 kDa proteinases to which they are linked. A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed. [Abstract/Link to Full Text]

Zhu JD
Myeloid cell-lineage and premylocytic-stage-specific- expression of themouse myeloperoxidase gene is controlled at initiation as well as elongation levels of transcription.
Cell Res. 1999 Jun;9(2):107-34.
The myeloperoxidase (MPO) is an important microbicidal protein present at high concentration in the primary granule of mature granulocyte and its expression is regulated in both myeloidcell-lineage and premyelocytic-stage-specific manners. A better understanding of the underlying control mechanisms should provide insights into the temporal and co-ordinate regulation of the gene expression during granulopoiesis. We have identified its promoter by mapping the start(s) of transcription using various molecular approaches together with demonstrating the promoter function of the relevant DNA segment in a transient transfection reporter assay. Besides the major start of transcription mapped at G residue, 11 nucleotide upstream of the 3' end of exon 0, the usage of that is specific to the MPO expressing cell lines, we have shown that irrespective of the MPO-expression status of the hematopoietic cells, transcription occurs broadly within a two kb region upstream of the 5' proximity of the gene, and is largely terminated in intron 2. These data support a model of the premyelocytic-stage-specific MPO expression, the control of which is operated at initiation as well as elongation levels of transcription. [Abstract/Link to Full Text]

Damjanovski S, Ishizuya-Oka A, Shi YB
Spatial and temporal regulation of collagenases-3, -4, and stromelysin -3 implicates distinct functions in apoptosis and tissue remodeling during frog metamorphosis.
Cell Res. 1999 Jun;9(2):91-105.
Matrix metalloproteinases (MMPs) are a family of extracellular proteases capable of degrading various proteinaceous components of the extracellular matrix (ECM). They have been implicated to play important roles in a number of developmental and pathological processes, such as tumor metastasis and inflammation. Relatively few studies have been carried out to investigate the function of MMPs during postembryonic organ-development. Using Xenopus laevis development as a model system, we demonstrate here that three MMPs, stromelysin-3 (ST3), collagenases-3 (Col3), and Col4, have distinct spatial and temporal expression profiles during metamorphosis as the tadpole transforms into a frog. In situ hybridizations reveal a tight, but distinct, association of individual MMPs with tissue remodeling in the tail and intestine during metamorphosis. In particular, ST3 expression is strongly correlated with apoptosis in both organs as demonstrated by analyses of serial sections with in situ hybridization for ST3 mRNA and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling) for apoptosis, respectively. On the other hand, Col3 and Col4 are present in regions where extensive connective tissue remodeling take place. These results indicate that ST3 is likely to play a role in ECM-remodeling that facilitate apoptotic tissue remodeling or resorption while Col3 and Col4 appear to participate in connective tissue degradation during development. [Abstract/Link to Full Text]

Jiang ZF, Zhu S, Sun YL, Zhai ZH
Induction of apoptosis in purified animal and plant nuclei by Xenopus egg extracts.
Cell Res. 1999 Jun;9(2):79-90.
We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspension-cultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments. The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed. The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis. [Abstract/Link to Full Text]

Huang Y, Ding XY
Ni2+ treatment causes cement gland formation in ectoderm explants of Xenopus laevis embryo.
Cell Res. 1999 Mar;9(1):71-6.
We found T-type calcium channel blocker Ni2+ can efficiently induce the formation of cement gland in Xenopus laevis animal cap explants. Another T-type specific calcium channel blocker Amiloride can also induce the formation of cement gland, while L-type specific calcium channel blocker Nifedipine has no inductive effect. These results may offer us an new approach to study the differentiation of cement gland through the change of intracelluar calcium concentration. [Abstract/Link to Full Text]

Zeng XL, Jiao MD, Xing M, Wang XG, Hao S
Tropomyosin is localized in the nuclear matrix and chromosome scaffold of Physarum polycephalum.
Cell Res. 1999 Mar;9(1):61-9.
The nuclei and chromosomes were isolated from plasmodia of Physarum polycephalum. The nuclear matrix and chromosome scaffold were obtained after the DNA and most of the proteins were extracted with DNase I and 2 M NaCl. SDS-PAGE analyses revealed that the nuclear matrix and chromosome scaffold contained a 37 kD polypeptide which is equivalent to tropomyosin in molecular weight. Immunofluorescence observations upon slide preparations labeled with anti-tropomyosin antibody showed that the nuclear matrix and chromosome scaffold emanated bright fluorescence, suggesting the presence of the antigen in them. Immunodotting results confirmed the presence of tropomyosin in the nuclear matrix and chromosome scaffold. Immunoelectron microscopic observations further demonstrated that tropomyosin was dispersively distributed in the interphase nuclei and metaphase chromosomes. [Abstract/Link to Full Text]

Zhao H, Xu YH
Mad-overexpression down regulates the malignant growth and p53 mediated apoptosis in human hepatocellular carcinoma BEL-7404 cells.
Cell Res. 1999 Mar;9(1):51-9.
Mad protein has been shown as an antagonist of c-Myc protein in some cell lines. The effect of Mad protein to the malignant phenotype of human hepatoma BEL-7404 cell line was investigated experimentally. An eukarryotic vector pCDNA III containing full ORF fragment of mad cDNA was transfected into targeted cells. Under G418 selection, stable Mad-overexpressed cells were cloned. Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells. DNA synthesis, cell proliferation and anchorage-independent growth in soft-agar of the mad-transfected cells were partially inhibited in comparison to control cells. Flow Cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase, resulting in the retardation of cell proliferation. RT-PCR detected a marked inhibition of the expression of cdc25A, an important regulator gene of G0/G1 to S phase in cell cycle. It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-7404-M1 cells in the absence of serume. Thus, Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL-7404 cells. [Abstract/Link to Full Text]

An W, Liu XJ, Lei TG, Dai J, Du GG
Growth induction of hepatic stimulator substance in hepatocytes through its regulation on EGF receptors.
Cell Res. 1999 Mar;9(1):37-49.
The cytosolic liver-specific growth factor-hepatic stimulator substance (HSS) has been shown to be able to amplify the rat hepatocyte proliferation responded to EGF. In order to get more insight into the mechanism, the regulatory effect of HSS on EGF-receptor (EGF-R) and the receptor phosphorylation at molecular level was studied. HSS partially purified from weanling rat liver was given to cultured hepatocytes and its influence on EGF-R specific binding and internalization as well as mRNA expression were investigated. The results showed that preincubation of hepatocytes with HSS could lead to an increase in [125I]-EGF binding to its receptors and inhibit EGF-induced receptor down-regulation. Furthermore, the over-expression of EGF-R mRNA stimulated by HSS was seen during 2-12 h after the incubation. Additionally, it was demonstrated with human hepatoma SMMC-7721 cells in Western blot that the EGF-R expression and the receptor autophosphorylation were increased with dose/time-dependency after HSS treatment. These results strongly suggest that the mechanism of HSS action on hepatocyte growth might be related to its modulation on EGF-R and receptor-mediated signaling transduction. [Abstract/Link to Full Text]

Ni DA, Wang LJ, Xu ZH, Xia ZA
Foliar modifications induced by inhibition of polar transport of auxin.
Cell Res. 1999 Mar;9(1):27-35.
The effects of auxin polar transport inhibitors, 9-hydroxy-fluorene-9-carboxylic acid (HFCA); 2, 3, 5-triiodobenzoic acid (TIBA) and trans-cinnamic acid (CA) on leaf pattern formation were investigated with shoots formed from cultured leaf explants of tobacco and cultured pedicel explants of Orychophragmus violaceus, and the seedlings of tobacco and Brassica chinensis. Although the effective concentration varies with the inhibitors used, all of the inhibitors induced the formation of trumpet-shaped and/or fused leaves. The frequency of trumpet-shaped leaf formation was related to the concentration of inhibitors in the medium. Histological observation of tobacco seedlings showed that there was only one main vascular bundle and several minor vascular bundles in normal leaves of the control, but there were several vascular bundles of more or less the same size in the trumpet-shaped leaves of treated ones. These results indicated that auxin polar transport played an important role on bilateral symmetry of leaf growth. [Abstract/Link to Full Text]

Li JM, Han JS, Huang Y, Tain PK, Qu SM, Yao M, Jiang HQ, Wan DF, Luo JC, Gu CX, Gu JR
A novel gene delivery system targeting cells expressing VEGF receptors.
Cell Res. 1999 Mar;9(1):11-25.
Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors. GV1, GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex. Using pSV2-beta-galactosidase as a reporter gene, it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) and human malignant melanoma cell lines (A375) in vitro. In vivo experiments, exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO, human malignant melanoma A375 and human hepatoma graft in nude mice. This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice. These results are correlated with the relevant receptors (flt-1, flk-1/KDR) expression on the targeted cells and tissues. [Abstract/Link to Full Text]

Sharma K, Shi Y
The yins and yangs of ceramide.
Cell Res. 1999 Mar;9(1):1-10.
Since their discovery over 100 years ago, sphingolipids have caught the eyes and the imagination of scientists. Modern science has made many new insights on the cell biology and day-to-day functions of many integral sphingolipids, especially those of ceramide. Ceramide is recognized as a vital second messenger in the signal transduction process mediated by receptors of many cytokines and growth factors. A great part of our current understanding of ceramide has been achieved from apoptosis-related studies, however recent data in the fields of immunology, endocrinology and neurobiology, also suggest a fundamental involvement of ceramide in the onset of diseases. Therefore, understanding the biology of ceramide could be a key to unraveling many biological mechanisms and provide information for the treatment of some common diseases. [Abstract/Link to Full Text]

---

Recent Articles in Cellular & Molecular Biology Letters

---

Macioszek VK, Kononowicz AK
The evaluation of the genotoxicity of two commonly used food colors: Quinoline Yellow (E 104) and Brilliant Black BN (E 151).
Cell Mol Biol Lett. 2004;9(1):107-22.
Additives, especially colors, are in widespread use in the food industry. With the exception of the quinolines, food colors are relatively weak mutagens and are certified as safe additives despite reports that some people have allergic reactions to them. The number of food additives is still on the increase, and research on their potential mutagenic/carcinogenic activity in vivo is very expensive. Using two different cellular model systems, human lymphocytes in vitro and Vicia faba root tip meristems of in vivo, we evaluated the potential cytological and genotoxic effects of two dyes: Quinoline Yellow (E 104) and Brilliant Black BN (E 151). Two relatively new, very sensitive and rapid tests - the micronucleus and Comet assays - were used in this study. The data provided in this paper showed the genotoxic effects of the two analyzed food colors, and confirmed the diagnostic value of the MN and Comet assays for screening potentially genotoxic substances. [Abstract/Link to Full Text]

Narozna D, Pa? J, Schneider J, Madrzak CJ
Two sequences encoding chalcone synthase in yellow lupin (Lupinus luteus l.) may have evolved by gene duplication.
Cell Mol Biol Lett. 2004;9(1):95-105.
Two full copy cDNA sequences encoding chalcone synthase (CHS) were selected from a yellow lupin (Lupinus luteus L.) root and nodule cDNA library, and sequenced. Analysis of their open reading frames gave evidence that both encode the functional enzyme. Sequence alignment and phylogenetic studies on the DNA and protein level of these clones compared to the sequences of chalcone synthases from 54 other plant species reveal the possibility that lupin chalcone synthase is encoded by a multigene family consisting of at least two distinct genes that probably diverged by gene duplication. The duplication event is estimated to have taken place about 16 million years ago. [Abstract/Link to Full Text]

Wo?niak K, B?asiak J
Nickel impairs the repair of UV- and MNNG-damaged DNA.
Cell Mol Biol Lett. 2004;9(1):83-94.
Nickel(II) is reported to be genotoxic, but the mechanisms underlying its genotoxicity are largely unknown. It can interfere with DNA repair and this may contribute to its genotoxicity. We studied the effect of nickel chloride on the repair of DNA damaged by UV radiation or N-methyl-N-nitro-N-nitrosoguanidine (MNNG) in human lymphocytes using the alkaline comet assay. Nickel(II) at 1 microM caused an accumulation of DNA breaks during repair incubation, which could follow from the inhibition of the polymerization/ligation step of UV-damaged DNA repair. On the other hand, nickel(II) inhibited the formation of transient DNA breaks brought by the repair process after incubation with MNNG at 5 microM, which might follow from interference with the recognition/incision step of excision repair. Additionally, nickel at 1 microM inhibited the activity of formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (Alk A), enzymes involved in DNA excision repair. A decrease in endonuclease III (Endo III) activity was observed at 2 and 5 microM of nickel chloride. Our results suggest that nickel(II) at non-cytotoxic concentrations can inhibit various steps of DNA excision repair, and this may contribute to its genotoxicity. [Abstract/Link to Full Text]

Chechli?ska M, Duma A, Swierkowska K, Kami?ska J, Steffen J
Sera of lung cancer patients affect the release of Th1, Th2 and monocyte-derived cytokines, and the expression of IL-2Ralpha by normal, stimulated mononuclear cells.
Cell Mol Biol Lett. 2004;9(1):69-81.
We have shown that the sera of lung cancer patients affect the response of ConA-stimulated normal peripheral blood mononuclear cells by decreasing the expression of IL-2Ralpha and inhibiting the release of IL-1beta and IL-2. A tendency to enhance the release of IL-6 was also observed. We conclude that an imbalance in the Th1/Th2 cytokine response, typical for cancer patients, may at least partly be related to soluble factors circulating in the patients' blood. We discuss a putative role of serum IL-10, IL-1ra, and soluble IL-2Ralpha in the effects observed. [Abstract/Link to Full Text]

Mozrzymas JW, Barberis A
Changes of GABA(A)receptor activation kinetics in hippocampal neurons cultured for different periods of time.
Cell Mol Biol Lett. 2004;9(1):61-7.
Cell culture is a convenient model for pharmacokinetic studies, but during the culture period, GABA(A)receptors are likely to undergo different modulatory processes. In this study, the current responses to ultrafast GABA applications were recorded from patches excised from neurons cultured for either up to two days (short-term culture) or for more than two weeks (long-term culture). The dose-dependencies of the current rising phases revealed significant differences between the two groups. In the short-term cultures, the responses to both saturating and non-saturating GABA concentrations were slower than in the case of the long-term cultures. We conclude that the GABA(A)receptors in cultured neurons undergo profound kinetic changes involving the modulation of the binding reaction and transitions between bound states. [Abstract/Link to Full Text]

Nunes-Correia I, Eulálio A, Nir S, Pedroso de Lima MC
Caveolae as an additional route for influenza virus endocytosis in MDCK cells.
Cell Mol Biol Lett. 2004;9(1):47-60.
Clathrin-mediated endocytosis has been described as the primary internalization pathway for many viruses, including the influenza virus. However, caveolae, an alternative clathrin-independent endocytotic pathway, has also been described as mediating the entry of some molecules, including viruses. To address the question of pathway selection by the influenza virus, we have investigated whether the virus is internalized via clathrin-coated pits and/or caveolae in Madin Darby canine kidney (MDCK) cells. By applying pharmacological manipulations to selectively disrupt the cell internalization pathways, we found that, in MDCK cells, the influenza virus may be internalized via caveolae in addition to entry by clathrin-mediated endocytosis. However, a small contribution by another mode of entry, as recently proposed, cannot be excluded. [Abstract/Link to Full Text]

Wo?niak K, Arabski M, Ma?ecka-Panas E, Drzewoski J, B?asiak J
DNA damage in human colonic mucosa cells induced by bleomycin and the protective action of vitamin E.
Cell Mol Biol Lett. 2004;9(1):31-45.
Using the alkaline comet assay, we showed that bleomycin at 0.1-5 microg/ml induced DNA strand breaks and/or alkali-labile sites, measurable as the comet tail moment, in human colonic mucosa cells. This DNA damage was completely repaired during a 120-minute post-treatment incubation of the cells. Post-treatment of the bleomycin-damaged DNA with 3-methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing alkylated bases, gave rise to a significant increase in the extent of DNA damage, indicating that the drug could induce alkylative bases in DNA. We did not observe any change in the comet tail moment in the presence of catalase. Vitamin E ((+)-alpha -tocopherol) decreased DNA damage induced by bleomycin. The results obtained suggest that hydrogen peroxide might not be involved in the formation of DNA lesions induced by bleomycin in the colonic mucosa cells. [Abstract/Link to Full Text]

Sroka J, Kami?ski R, Michalik M, Madeja Z, Przestalski S, Korohoda W
The effect of triethyllead on the motile activity of walker 256 carcinosarcoma cells.
Cell Mol Biol Lett. 2004;9(1):15-30.
The effect of triethyllead (TriEL) on the morphology and motile activity of Walker 256 carcinosarcoma cells was investigated. It was found that both 2 and 5 microM TriEL affected the cellular motility in a dose- and time-dependent manner. Initially, 2 microM TriEL caused the formation of blebs instead of lamellipodia at the front of some cells and stimulated the migration of Walker cells, but after 2 hours of 2 microM TriEL treatment, a reduction of cellular motility was observed. In the presence of 5 microM TriEL, Walker 256 carcinosarcoma cells rounded up, and their rate of movement was reduced. Moreover, the treatment of Walker carcinosarcoma cells with TriEL caused the disruption of microtubules and affected the F-actin distribution at both concentrations. At a concentration of 2 microM TriEL, the actin staining intensity was greatest in the tail of front-tail polarised blebbing cells and the actin layer was very thin at the leading edge. The control cells showed linear cortical F-actin distribution and somewhat less intense cytoplasmic staining at the same TriEL concentration. Cells treated with 5 microM TriEL showed an under-membrane pattern of actin distribution. [Abstract/Link to Full Text]

Chaszczewska-Markowska M, Ugorski M, Langner M
Plasmid condensation induced by cationic compounds: hydrophilic polylysine and amphiphilic cationic lipid.
Cell Mol Biol Lett. 2004;9(1):3-13.
The construction of an efficient carrier for genetic material is a major research objective that needs to be achieved before gene therapy can become a viable pharmacological approach. Artificial aggregates containing nucleic acids are one of the options for the systemic delivery of genetic information. The diversity of functions the aggregate is expected to fulfill necessitates its complex architecture. In order to obtain a complex supramolecular aggregate, formed from elements that are themselves complex molecules, appropriate procedures based on the detailed understanding of processes at the molecular level are required. In this study, we investigated how the various properties of cationic compounds affect nucleic acid condensation. The combination of two condensing agents, differing in their affinity towards water, when mixed with plasmids, resulted in aggregates which are resistant to enzymatic digestion and which form particles with well-defined size distributions. Such uniform and well-defined complexes may subsequently be further modified in order to obtain a fully functional genetic material carrier. [Abstract/Link to Full Text]

---

Recent Articles in BMC Cell Biology

---

Xu H, Somers ZB, Robinson ML, Hebert MD
Tim50a, a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis.
BMC Cell Biol. 2005;6(1):29.
BACKGROUND: The Cajal body (CB) is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation. RESULTS: In this report, we identify a minor isoform of the mitochondrial Tim50, Tim50a, as a coilin interacting protein. The Tim50a transcript can be detected in some cancer cell lines and normal brain tissue. The Tim50a protein differs only from Tim50 in that it contains an additional 103 aa N-terminal to the translation start of Tim50. Importantly, a putative nuclear localization signal is found within these 103 residues. In contrast to Tim50, which localizes to the cytoplasm and mitochondria, Tim50a is strictly nuclear and is enriched in speckles with snRNPs. In addition to coilin, Tim50a interacts with snRNPs and SMN. Competition binding experiments demonstrate that coilin competes with Sm proteins of snRNPs and SMN for binding sites on Tim50a. CONCLUSION: Tim50a may play a role in snRNP biogenesis given its cellular localization and protein interaction characteristics. We hypothesize that Tim50a takes part in the release of snRNPs and SMN from the CB. [Abstract/Link to Full Text]

Mascarenhas J, Volkov AV, Rinn C, Schiener J, Guckenberger R, Graumann PL
Dynamic assembly, localization and proteolysis of the Bacillus subtilis SMC complex.
BMC Cell Biol. 2005;628.
BACKGROUND: SMC proteins are key components of several protein complexes that perform vital tasks in different chromosome dynamics. Bacterial SMC forms a complex with ScpA and ScpB that is essential for chromosome arrangement and segregation. The complex localizes to discrete centres on the nucleoids that during most of the time of the cell cycle localize in a bipolar manner. The complex binds to DNA and condenses DNA in an as yet unknown manner. RESULTS: We show that in vitro, ScpA and ScpB form different complexes with each other, among which the level of the putative 2 ScpA/4 ScpB complex showed a pronounced decrease in level upon addition of SMC protein. Different mutations of the ATPase-binding pocket of SMC reduced, but did not abolish interaction of mutant SMC with ScpA and ScpB. The loss of SMC ATPase activity led to a loss of function in vivo, and abolished proper localization of the SMC complex. The formation of bipolar SMC centres was also lost after repression of gyrase activity, and was abnormal during inhibition of replication, resulting in single central clusters. Resumption of replication quickly re-established bipolar SMC centres, showing that proper localization depends on ongoing replication. We also found that the SMC protein is subject to induced proteolysis, most strikingly as cells enter stationary phase, which is partly achieved by ClpX and LonA proteases. Atomic force microscopy revealed the existence of high order rosette-like SMC structures in vitro, which might explain the formation of the SMC centres in vivo. CONCLUSION: Our data suggest that a ScpA/ScpB sub-complex is directly recruited into the SMC complex. This process does not require SMC ATPase activity, which, however, appears to facilitate loading of ScpA and ScpB. Thus, the activity of SMC could be regulated through binding and release of ScpA and ScpB, which has been shown to affect SMC ATPase activity. The proper bipolar localization of the SMC complex depends on a variety of physiological aspects: ongoing replication, ATPase activity and chromosome supercoiling. Because the cellular concentration of SMC protein is also regulated at the posttranscriptional level, the activity of SMC is apparently regulated at multiple levels. [Abstract/Link to Full Text]

Paradisi M, McClintock D, Boguslavsky RL, Pedicelli C, Worman HJ, Djabali K
Dermal fibroblasts in Hutchinson-Gilford progeria syndrome with the lamin A G608G mutation have dysmorphic nuclei and are hypersensitive to heat stress.
BMC Cell Biol. 2005;627.
BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare sporadic disorder with an incidence of approximately 1 per 8 million live births. The phenotypic appearance consists of short stature, sculptured nose, alopecia, prominent scalp veins, small face, loss of subcutaneous fat, faint mid-facial cyanosis, and dystrophic nails. HGPS is caused by mutations in LMNA, the gene that encodes nuclear lamins A and C. The most common mutation in subjects with HGPS is a de novo single-base pair substitution, G608G (GGC>GGT), within exon 11 of LMNA. This creates an abnormal splice donor site, leading to expression of a truncated protein. RESULTS: We studied a new case of a 5 year-old girl with HGPS and found a heterozygous point mutation, G608G, in LMNA. Complementary DNA sequencing of RNA showed that this mutation resulted in the deletion of 50 amino acids in the carboxyl-terminal tail domain of prelamin A. We characterized a primary dermal fibroblast cell line derived from the subject's skin. These cells expressed the mutant protein and exhibited a normal growth rate at early passage in primary culture but showed alterations in nuclear morphology. Expression levels and overall distributions of nuclear lamins and emerin, an integral protein of the inner nuclear membrane, were not dramatically altered. Ultrastructural analysis of the nuclear envelope using electron microscopy showed that chromatin is in close association to the nuclear lamina, even in areas with abnormal nuclear envelope morphology. The fibroblasts were hypersensitive to heat shock, and demonstrated a delayed response to heat stress. CONCLUSION: Dermal fibroblasts from a subject with HGPS expressing a mutant truncated lamin A have dysmorphic nuclei, hypersensitivity to heat shock, and delayed response to heat stress. This suggests that the mutant protein, even when expressed at low levels, causes defective cell stability, which may be responsible for phenotypic abnormalities in the disease. [Abstract/Link to Full Text]

Band AM, Kuismanen E
Localization of plasma membrane t-SNAREs syntaxin 2 and 3 in intracellular compartments.
BMC Cell Biol. 2005;6(1):26.
BACKGROUND: Membrane fusion requires the formation of a complex between a vesicle protein (v-SNARE) and the target membrane proteins (t-SNAREs). Syntaxin 2 and 3 are t-SNAREs that, according to previous over-expression studies, are predominantly localized at the plasma membrane. In the present study we investigated localization of the endogenous syntaxin 2 and 3. RESULTS: Endogenous syntaxin 2 and 3 were found in NRK cells in intracellular vesicular structures in addition to regions of the plasma membrane. Treatment of these cells with N-ethylmaleimide (NEM), which is known to inactivate membrane fusion, caused syntaxin 3 to accumulate in the trans-Golgi network and syntaxin 2 in perinuclear membrane vesicles. Kinetic analysis in the presence of NEM indicated that this redistribution of syntaxin 2 and 3 takes place via actin containing structures. CONCLUSION: Our data suggest that syntaxin 2 cycles between the plasma membrane and the perinuclear compartment whereas syntaxin 3 cycles between the plasma membrane and the trans-Golgi network. It is possible that this cycling has an important role in the regulation of t-SNARE function. [Abstract/Link to Full Text]

Zhou J, Zhu P, Jiang JL, Zhang Q, Wu ZB, Yao XY, Tang H, Lu N, Yang Y, Chen ZN
Involvement of CD147 in overexpression of MMP-2 and MMP-9 and enhancement of invasive potential of PMA-differentiated THP-1.
BMC Cell Biol. 2005;6(1):25.
BACKGROUND: During infection and inflammation, circulating blood monocytes migrate from the intravascular compartments to the extravascular compartments, where they mature into tissue macrophages. The maturation process prepares the cells to actively participate in the inflammatory and immune responses, and many factors have been reported to be involved in the process. We found in our study that CD147 played a very important role in this process. RESULTS: By using PMA-differentiated human monocyte cells line THP-1, we found that CD147 mediated matrix metalloproteinases (MMPs) expression of the leukemic THP-1 cells and thus enhanced the invasiveness of THP-1 cells. After 24 hours of PMA-induced monocyte differentiation, the mean fluorescence intensity of CD147 in differentiated THP-1 cells (289.61 +/- 31.63) was higher than that of the undifferentiated THP-1 cells (205.1 +/- 19.25). There was a significant increase of the levels of proMMP-2, proMMP-9 and their activated forms in the differentiated THP-1 cells. Invasion assays using reconstituted basement membrane showed a good correlation between the invasiveness of THP-1 cells and the production of MMP-2 and MMP-9. The difference in the MMPs expression and the invasive ability was significantly blocked by HAb18G/CD147 antagonistic peptide AP-9. The inhibitory rate of the secretion of proMMP-9 in the undifferentiated THP-1 cells was 45.07%. The inhibitory rate of the secretion of proMMP-9, the activated MMP-9 and proMMP-2 in the differentiated THP-1 cells was 52.90%, 53.79% and 47.80%, respectively. The inhibitory rate of invasive potential in the undifferentiated cells and the differentiated THP-1 cells was 41.82 % and 25.15%, respectively. CONCLUSION: The results suggest that the expression of CD147 is upregulated during the differentiation of monocyte THP-1 cells to macrophage cells, and CD147 induces the secretion and activation of MMP-2 and MMP-9 and enhances the invasive ability of THP-1 cells. The matured monocytes / macrophages, via their high expression of CD147, may play an important role in promoting the tissue repair or tissue damage during their inflammatory response. [Abstract/Link to Full Text]

Sagot I, Schaeffer J, Daignan-Fornier B
Guanylic nucleotide starvation affects Saccharomyces cerevisiae mother-daughter separation and may be a signal for entry into quiescence.
BMC Cell Biol. 2005;6(1):24.
BACKGROUND: Guanylic nucleotides are both macromolecules constituents and crucial regulators for a variety of cellular processes. Therefore, their intracellular concentration must be strictly controlled. Consistently both yeast and mammalian cells tightly correlate the transcription of genes encoding enzymes critical for guanylic nucleotides biosynthesis with the proliferation state of the cell population. RESULTS: To gain insight into the molecular relationships connecting intracellular guanylic nucleotide levels and cellular proliferation, we have studied the consequences of guanylic nucleotide limitation on Saccharomyces cerevisiae cell cycle progression. We first utilized mycophenolic acid, an immunosuppressive drug that specifically inhibits inosine monophosphate dehydrogenase, the enzyme catalyzing the first committed step in de novo GMP biosynthesis. To approach this system physiologically, we next developed yeast mutants for which the intracellular guanylic nucleotide pools can be modulated through changes of growth conditions. In both the pharmacological and genetic approaches, we found that guanylic nucleotide limitation generated a mother-daughter separation defect, characterized by cells with two unseparated daughters. We then showed that this separation defect resulted from cell wall perturbations but not from impaired cytokinesis. Importantly, cells with similar separation defects were found in a wild type untreated yeast population entering quiescence upon nutrient limitation. CONCLUSION: Our results demonstrate that guanylic nucleotide limitation slows budding yeast cell cycle progression, with a severe pause in telophase. At the cellular level, guanylic nucleotide limitation causes the emergence of cells with two unseparated daughters. By fluorescence and electron microscopy, we demonstrate that this phenotype arises from defects in cell wall partition between mother and daughter cells. Because cells with two unseparated daughters are also observed in a wild type population entering quiescence, our results reinforce the hypothesis that guanylic nucleotide intracellular pools contribute to a signal regulating both cell proliferation and entry into quiescence. [Abstract/Link to Full Text]

Xu H, Hebert MD
A novel EB-1/AIDA-1 isoform, AIDA-1c, interacts with the Cajal body protein coilin.
BMC Cell Biol. 2005;6(1):23.
BACKGROUND: Cajal bodies (CBs) are nuclear suborganelles that play a role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are crucial for pre-mRNA splicing. Upon nuclear reentry, Sm-class snRNPs localize first to the CB, where the snRNA moiety of the snRNP is modified. It is not clear how snRNPs target to the CB and are released from this structure after their modification. Coilin, the CB marker protein, may participate in snRNP biogenesis given that it can interact with snRNPs and SMN. SMN is crucial for snRNP assembly and is the protein mutated in the neurodegenerative disease Spinal Muscular Atrophy. Coilin knockout mice display significant viability problems and altered CB formation. Thus characterization of the CB and its associated proteins will give insight into snRNP biogenesis and clarify the dynamic organization of the nucleus. RESULTS: In this report, we identify a novel protein isoform of EB-1/AIDA-1, termed AIDA-1c, that interacts with the CB marker protein, coilin. Northern and nested PCR experiments reveal that the AIDA-1c isoform is expressed in brain and several cancer cell lines. Competition binding experiments demonstrate that AIDA-1c competes with SmB' for coilin binding sites, but does not bind SMN. When ectopically expressed, AIDA-1c is predominantly nuclear with no obvious accumulations in CBs. Interestingly, another EB-1/AIDA-1 nuclear isoform, AIDA-1a, does not bind coilin in vivo as efficiently as AIDA-1c. Knockdown of EB-1/AIDA-1 isoforms by siRNA altered Cajal body organization and reduced cell viability. CONCLUSION: These data suggest that specific EB-1/AIDA-1 isoforms, such as AIDA-1c, may participate in the regulation of nucleoplasmic coilin protein interactions in neuronal and transformed cells. [Abstract/Link to Full Text]

Stubbs CD, Botchway SW, Slater SJ, Parker AW
The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cells.
BMC Cell Biol. 2005;6(1):22.
BACKGROUND: Two-photon-excitation fluorescence lifetime imaging (2P-FLIM) was used to investigate the association of protein kinase C alpha (PKCalpha) with caveolin in CHO cells. PKCalpha is found widely in the cytoplasm and nucleus in most cells. Upon activation, as a result of increased intracellular Ca2+ and production of DAG, through G-protein coupled-phospholipase C signalling, PKC translocates to a variety of regions in the cell where it phosphorylates and interacts with many signalling pathways. Due to its wide distribution, discerning a particular interaction from others within the cell is extremely difficult. RESULTS: Fluorescence energy transfer (FRET), between GFP-PKCalpha and DsRed-caveolin, was used to investigate the interaction between caveolin and PKC, an aspect of signalling that is poorly understood. Using 2P-FLIM measurements, the lifetime of GFP was found to decrease (quench) in certain regions of the cell from approximately 2.2 ns to approximately 1.5 ns when the GFP and DsRed were sufficiently close for FRET to occur. This only occurred when intracellular Ca2+ increased or in the presence of phorbol ester, and was an indication of PKC and caveolin co-localisation under these conditions. In the case of phorbol ester stimulated PKC translocation, as commonly used to model PKC activation, three PKC areas could be delineated. These included PKCalpha that was not associated with caveolin in the nucleus and cytoplasm, PKCalpha associated with caveolin in the cytoplasm/perinuclear regions and probably in endosomes, and PKC in the peripheral regions of the cell, possibly indirectly interacting with caveolin. CONCLUSION: Based on the extent of lifetime quenching observed, the results are consistent with a direct interaction between PKCalpha and caveolin in the endosomes, and possibly an indirect interaction in the peripheral regions of the cell. The results show that 2P-FLIM-FRET imaging offers an approach that can provide information not only confirming the occurrence of specific protein-protein interactions but where they occur within the cell. [Abstract/Link to Full Text]

Kokkola T, Savinainen JR, Mönkkönen KS, Retamal MD, Laitinen JT
S-nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner.
BMC Cell Biol. 2005;6(1):21.
BACKGROUND: Recent studies indicate that the G protein-coupled receptor (GPCR) signaling machinery can serve as a direct target of reactive oxygen species, including nitric oxide (NO) and S-nitrosothiols (RSNOs). To gain a broader view into the way that receptor-dependent G protein activation -- an early step in signal transduction -- might be affected by RSNOs, we have studied several receptors coupling to the Gi family of G proteins in their native cellular environment using the powerful functional approach of [35S]GTPgammaS autoradiography with brain cryostat sections in combination with classical G protein activation assays. RESULTS: We demonstrate that RSNOs, like S-nitrosoglutathione (GSNO) and S-nitrosocysteine (CysNO), can modulate GPCR signaling via reversible, thiol-sensitive mechanisms probably involving S-nitrosylation. RSNOs are capable of very targeted regulation, as they potentiate the signaling of some receptors (exemplified by the M2/M4 muscarinic cholinergic receptors), inhibit others (P2Y12 purinergic, LPA1lysophosphatidic acid, and cannabinoid CB1 receptors), but may only marginally affect signaling of others, such as adenosine A1, mu-opioid, and opiate related receptors. Amplification of M2/M4 muscarinic responses is explained by an accelerated rate of guanine nucleotide exchange, as well as an increased number of high-affinity [35S]GTPgammaS binding sites available for the agonist-activated receptor. GSNO amplified human M4 receptor signaling also under heterologous expression in CHO cells, but the effect diminished with increasing constitutive receptor activity. RSNOs markedly inhibited P2Y12 receptor signaling in native tissues (rat brain and human platelets), but failed to affect human P2Y12 receptor signaling under heterologous expression in CHO cells, indicating that the native cellular signaling partners, rather than the P2Y12 receptor protein, act as a molecular target for this action. CONCLUSION: These in vitro studies show for the first time in a broader general context that RSNOs are capable of modulating GPCR signaling in a reversible and highly receptor-specific manner. Given that the enzymatic machinery responsible for endogenous NO production is located in close proximity with the GPCR signaling complex, especially with that for several receptors whose signaling is shown here to be modulated by exogenous RSNOs, our data suggest that GPCR signaling in vivo is likely to be subject to substantial, and highly receptor-specific modulation by NO-derived RSNOs. [Abstract/Link to Full Text]

Kramer-Hämmerle S, Ceccherini-Silberstein F, Bickel C, Wolff H, Vincendeau M, Werner T, Erfle V, Brack-Werner R
Identification of a novel Rev-interacting cellular protein.
BMC Cell Biol. 2005;6(1):20.
BACKGROUND: Human cell types respond differently to infection by human immunodeficiency virus (HIV). Defining specific interactions between host cells and viral proteins is essential in understanding how viruses exploit cellular functions and the innate strategies underlying cellular control of HIV replication. The HIV Rev protein is a post-transcriptional inducer of HIV gene expression and an important target for interaction with cellular proteins. Identification of Rev-modulating cellular factors may eventually contribute to the design of novel antiviral therapies. RESULTS: Yeast-two hybrid screening of a T-cell cDNA library with Rev as bait led to isolation of a novel human cDNA product (16.4.1). 16.4.1-containing fusion proteins showed predominant cytoplasmic localization, which was dependent on CRM1-mediated export from the nucleus. Nuclear export activity of 16.4.1 was mapped to a 60 amino acid region and a novel transport signal identified. Interaction of 16.4.1 with Rev in human cells was shown in a mammalian two-hybrid assay and by colocalization of Rev and 16.4.1 in nucleoli, indicating that Rev can recruit 16.4.1 to the nucleus/nucleoli. Rev-dependent reporter expression was inhibited by overexpressing 16.4.1 and stimulated by siRNAs targeted to 16.4.1 sequences, demonstrating that 16.4.1 expression influences the transactivation function of Rev. CONCLUSION: These results suggest that 16.4.1 may act as a modulator of Rev activity. The experimental strategies outlined in this study are applicable to the identification and biological characterization of further novel Rev-interacting cellular factors. [Abstract/Link to Full Text]

Bahnson A, Athanassiou C, Koebler D, Qian L, Shun T, Shields D, Yu H, Wang H, Goff J, Cheng T, Houck R, Cowsert L
Automated measurement of cell motility and proliferation.
BMC Cell Biol. 2005;6(1):19.
BACKGROUND: Time-lapse microscopic imaging provides a powerful approach for following changes in cell phenotype over time. Visible responses of whole cells can yield insight into functional changes that underlie physiological processes in health and disease. For example, features of cell motility accompany molecular changes that are central to the immune response, to carcinogenesis and metastasis, to wound healing and tissue regeneration, and to the myriad developmental processes that generate an organism. Previously reported image processing methods for motility analysis required custom viewing devices and manual interactions that may introduce bias, that slow throughput, and that constrain the scope of experiments in terms of the number of treatment variables, time period of observation, replication and statistical options. Here we describe a fully automated system in which images are acquired 24/7 from 384 well plates and are automatically processed to yield high-content motility and morphological data. RESULTS: We have applied this technology to study the effects of different extracellular matrix compounds on human osteoblast-like cell lines to explore functional changes that may underlie processes involved in bone formation and maintenance. We show dose-response and kinetic data for induction of increased motility by laminin and collagen type I without significant effects on growth rate. Differential motility response was evident within 4 hours of plating cells; long-term responses differed depending upon cell type and surface coating. Average velocities were increased approximately 0.1 microm/min by ten-fold increases in laminin coating concentration in some cases. Comparison with manual tracking demonstrated the accuracy of the automated method and highlighted the comparative imprecision of human tracking for analysis of cell motility data. Quality statistics are reported that associate with stage noise, interference by non-cell objects, and uncertainty in the outlining and positioning of cells by automated image analysis. Exponential growth, as monitored by total cell area, did not linearly correlate with absolute cell number, but proved valuable for selection of reliable tracking data and for disclosing between-experiment variations in cell growth. CONCLUSION: These results demonstrate the applicability of a system that uses fully automated image acquisition and analysis to study cell motility and growth. Cellular motility response is determined in an unbiased and comparatively high throughput manner. Abundant ancillary data provide opportunities for uniform filtering according to criteria that select for biological relevance and for providing insight into features of system performance. Data quality measures have been developed that can serve as a basis for the design and quality control of experiments that are facilitated by automation and the 384 well plate format. This system is applicable to large-scale studies such as drug screening and research into effects of complex combinations of factors and matrices on cell phenotype. [Abstract/Link to Full Text]

Lavado A, Matheu A, Serrano M, Montoliu L
A strategy to study tyrosinase transgenes in mouse melanocytes.
BMC Cell Biol. 2005;6(1):18.
BACKGROUND: A number of transgenic mice carrying different deletions in the Locus Control Region (LCR) of the mouse tyrosinase (Tyr) gene have been developed and analysed in our laboratory. We require melanocytes from these mice, to further study, at the cellular level, the effect of these deletions on the expression of the Tyr transgene, without potential interference with or from the endogenous Tyr alleles. It has been previously reported that it is possible to obtain and immortalize melanocyte cell cultures from postnatal mouse skin. RESULTS: Here, we describe the efforts towards obtaining melanocyte cultures from our Tyr transgenic mice. We have bred our Tyr transgenic mice into Tyr c-32DSD mutant background, lacking the endogenous Tyr locus. In these conditions, we failed to obtain immortalized melanocytes. We decided to include the inactivation of the Ink4a-Arf locus to promote melanocyte immortalisation. For this purpose, we report the segregation of the Ink4a-Arf null allele from the brown (Tyrp1b) mutation in mice. Finally, we found that Ink4a-Arf +/- and Ink4a-Arf -/- melanocytes had undistinguishable tyrosine hydroxylase activities, although the latter showed reduced cellular pigmentation content. CONCLUSION: The simultaneous presence of precise genomic deletions that include the tyrosinase locus, such as the Tyr c-32DSD allele, the Tyr transgene itself and the inactivated Ink4a-Arf locus in Tyrp1B genetic background appear as the crucial combination to perform forthcoming experiments. We cannot exclude that Ink4a-Arf mutations could affect the melanin biosynthetic pathway. Therefore, subsequent experiments with melanocytes will have to be performed in a normalized genetic background regarding the Ink4a-Arf locus. [Abstract/Link to Full Text]

Riess NP, Milward K, Lee T, Adams M, Askham JM, Morrison EE
Trapping of normal EB1 ligands in aggresomes formed by an EB1 deletion mutant.
BMC Cell Biol. 2005;6(1):17.
BACKGROUND: EB1 is a microtubule tip-associated protein that interacts with the APC tumour suppressor protein and the p150glued subunit of dynactin. We previously reported that an EB1 deletion mutant that retains both of these interactions but does not directly associate with microtubules (EB1-DeltaN2-GFP) spontaneously formed perinuclear aggregates when expressed in COS-7 cells. RESULTS: In the present study live imaging indicated that EB1-DeltaN2-GFP aggregates underwent dynamic microtubule-dependent changes in morphology and appeared to be internally cohesive. EB1-DeltaN2-GFP aggregates were phase-dense structures that displayed microtubule-dependent accumulation around the centrosome, were immunoreactive for both the 20s subunit of the proteasome and ubiquitin, and induced the collapse of the vimentin cytoskeleton. Fractionation studies revealed that a proportion of EB1-DeltaN2-GFP was detergent-insoluble and ubiquitylated, indicating that EB1-DeltaN2-GFP aggregates are aggresomes. Immunostaining also revealed that APC and p150glued were present in EB1-DeltaN2-GFP aggregates, whereas EB3 was not. Furthermore, evidence for p150glued degradation was found in the insoluble fraction of EB1-DeltaN2-GFP transfected cultures. CONCLUSION: Our data indicate that aggresomes can be internally cohesive and may not represent a simple "aggregate of aggregates" assembled around the centrosome. Our observations also indicate that a partially misfolded protein may retain the ability to interact with its normal physiological ligands, leading to their co-assembly into aggresomes. This supports the idea that the trapping and degradation of co-aggregated proteins might contribute to human pathologies characterised by aggresome formation. [Abstract/Link to Full Text]

Bestvater F, Dallner C, Spiess E
The C-terminal subunit of artificially truncated human cathepsin B mediates its nuclear targeting and contributes to cell viability.
BMC Cell Biol. 2005;6(1):16.
BACKGROUND: Splicing variants of human cathepsinB primary transcripts (CB(-2,3)) result in an expression product product which lacks the signal peptide and parts of the propeptide. This naturally truncated Delta51CB is thus unable to follow the regular CB processing and sorting pathway. It is addressed to the mitochondria through an activated N-terminal mitochondrial targeting signal instead. Although Delta51CB is supposed to be devoid of the typical CB enzymatic activity, it might play a role in malignancies and trigger cell death/apoptosis independent from the function of the regular enzyme. Cytoplasmic presence of the mature CB might occur as a result of lysosomal damage. RESULTS: We investigated such "aberrant" proteins by artificial CB-GFP chimeras covering various sequence parts in respect to their enzymatic activity, their localization in different cell types, and the effects on the cell viability. Unlike the entire full length CB form, the artificial single chain form was not processed and did not reveal typical enzymatic CB activity during transient overexpression in large cell lung carcinoma cells. Delta51CB was found predominantly in mitochondria. In contrast, the shorter artificial CB constructs localized in the cytoplasm, inside the cell nucleus, and in the midbodies of dividing cells. Bleaching experiments revealed both mobile and immobile fractions of these constructs in the nucleus. Nuclear accumulation of artificially truncated CB variants led to disintegration of nuclei, followed by cell death. CONCLUSION: We propose that cell death associated with CB is not necessarily triggered by its regular enzymatic activity but alternatively by a yet unknown activity profile of truncated CB. Cytoplasmic CB might be able to enter the cell nucleus. According to a mutational analysis, the part of CB that mediates its nuclear import is a signal patch within its heavy chain domain. The results suggest that besides the N-terminal signal peptide also other CB domains contain patterns which are responsible for a differentiated targeting of the molecule, e.g. to the mitochondria, to the nucleus, or to vesicles. We propose a hierarchy of targeting signals depending on their strength and availability. This implies other possible transport mechanisms besides the usual trafficking via the mannose-6-sound recording copyright sign pathway. [Abstract/Link to Full Text]

Bayer M, Fischer J, Kremerskothen J, Ossendorf E, Matanis T, Konczal M, Weide T, Barnekow A
Identification and characterization of Iporin as a novel interaction partner for rab1.
BMC Cell Biol. 2005;6(1):15.
BACKGROUND: The small GTPase rab1a and its isoform rab1b are essential regulating components in the vesicle transport between the ER and the Golgi apparatus. Rab1 is thought to act as a molecular switch and can change between an active GTP-bound and an inactive GDP-bound conformation. To elucidate the function of rab1, several approaches have been established to isolate effector proteins, which interact with the activated conformation of rab1. To date p115, GM130, golgin-84 and MICAL have been identified as direct interacting partners. Together with rab1, these molecules are components of a protein complex, which mediates and regulates intracellular vesicle transport. RESULTS: Here, we report the characterization of Iporin, which is similar to KIAA0375 as a novel rab1-interacting protein. It was initially identified by yeast two-hybrid screening experiments with the active mutant of rab1b (rab1b Q67R) as bait. Iporin contains a SH3 domain and two polyproline stretches, which are known to play a role in protein/protein interactions. In addition, Iporin encloses a RUN domain, which seems to be a major part of the rab1binding domain (R1BD). Iporin is ubiquitously expressed and immunofluorescence staining displays a cytosolic punctual distribution. Interestingly, we also show that Iporin interacts with another rab1 interacting partner, the GM130 protein. CONCLUSION: Our results demonstrate that Iporin is a potential new interacting partner of rab1. Iporin is different from already identified rab1 interacting proteins concerning protein structure and cellular localization. We conclude that Iporin might function as a link between the targeting of ER derived vesicles, triggered by the rab1 GTPase and a signaling pathway regulated by molecules containing SH3 and/or poly-proline regions. The characterization of this novel intermolecular relation could help to elucidate how vesicles find their way from ER to the Golgi apparatus. [Abstract/Link to Full Text]

Windler-Hart SL, Chen KY, Chenn A
A cell behavior screen: identification, sorting, and enrichment of cells based on motility.
BMC Cell Biol. 2005;6(1):14.
BACKGROUND: Identifying and isolating cells with specific behavioral characteristics will facilitate the understanding of the molecular basis regulating these behaviors. Although many approaches exist to characterize cell motility, retrieving cells of specific motility following analysis remains challenging. RESULTS: Cells migrating on substrates coated with fluorescent microspheres generate non-fluorescent tracks as they move and ingest the spheres. The area cleared by each cell allows for quantitation of single cell and population motility; because individual cell fluorescence is proportional to motility, cells can be sorted according to their degree of movement. Using this approach, we sorted a glioblastoma cell line into high motility and low motility populations and found stable differences in motility following sorting. CONCLUSION: We describe an approach to identify, sort, and enrich populations of cells possessing specific levels of motility. Unlike existing assays of cell motility, this approach enables recovery of characterized cell populations, and can enable screens to identify factors that might regulate motility differences even within clonal population of cells. [Abstract/Link to Full Text]

Schaloske RH, Lusche DF, Bezares-Roder K, Happle K, Malchow D, Schlatterer C
Ca2+ regulation in the absence of the iplA gene product in Dictyostelium discoideum.
BMC Cell Biol. 2005;6(1):13.
BACKGROUND: Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i-change is composed of liberation of stored Ca2+ and extracellular Ca2+-entry. The significance of the [Ca2+]i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca2+-buffers in the cytosol indicates that a [Ca2+]i-increase is required for chemotaxis. Yet, the iplA- mutant disrupted in a gene bearing similarity to IP3-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca2+]i-transient which favours the view that [Ca2+]i-changes are insignificant for chemotaxis. RESULTS: We investigated Ca2+-fluxes and the effect of their disturbance on chemotaxis and development of iplA- cells. Differentiation was altered as compared to wild type amoebae and sensitive towards manipulation of the level of stored Ca2+. Chemotaxis was impaired when [Ca2+]i-transients were suppressed by the presence of a Ca2+-chelator in the cytosol of the cells. Analysis of ion fluxes revealed that capacitative Ca2+-entry was fully operative in the mutant. In suspensions of intact and permeabilized cells cAMP elicited extracellular Ca2+-influx and liberation of stored Ca2+, respectively, yet to a lesser extent than in wild type. In suspensions of partially purified storage vesicles ATP-induced Ca2+-uptake and Ca2+-release activated by fatty acids or Ca2+-ATPase inhibitors were similar to wild type. Mn2+-quenching of fura2 fluorescence allows to study Ca2+-influx indirectly and revealed that the responsiveness of mutant cells was shifted to higher concentrations: roughly 100 times more Mn2+ was necessary to observe agonist-induced Mn2+-influx. cAMP evoked a [Ca2+]i-elevation when stores were strongly loaded with Ca2+, again with a similar shift in sensitivity in the mutant. In addition, basal [Ca2+]i was significantly lower in iplA- than in wild type amoebae. CONCLUSION: These results support the view that [Ca2+]i-transients are essential for chemotaxis and differentiation. Moreover, capacitative and agonist-activated ion fluxes are regulated by separate pathways that are mediated either by two types of channels in the plasma membrane or by distinct mechanisms coupling Ca2+-release from stores to Ca2+-entry in Dictyostelium. The iplA- strain retains the capacitative Ca2+-entry pathway and an impaired agonist-activated pathway that operates with reduced efficiency or at higher ionic pressure. [Abstract/Link to Full Text]

Stovold CF, Millard TH, Machesky LM
Inclusion of Scar/WAVE3 in a similar complex to Scar/WAVE1 and 2.
BMC Cell Biol. 2005;6(1):11.
BACKGROUND: The Scar/WAVE family of proteins mediates signals to actin assembly by direct activation of the Arp2/3 complex. These proteins have been characterised as major regulators of lamellipodia formation downstream of Rac activation and as members of large protein complexes. RESULTS: We have investigated the interactions of the three human Scar/WAVE isoforms with several previously described binding partners for Scar/WAVE 1 or 2. We find that all three Scar/WAVE isoforms behave similarly and are likely to participate in the same kinds of protein complexes that regulate actin assembly. CONCLUSION: Differences between Scar/WAVE proteins are therefore likely to be at the level of tissue distribution or subtle differences in the affinity for specific binding partners. [Abstract/Link to Full Text]

Lusche DF, Bezares-Roder K, Happle K, Schlatterer C
cAMP controls cytosolic Ca2+ levels in Dictyostelium discoideum.
BMC Cell Biol. 2005;6(1):12.
BACKGROUND: Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) that is composed of liberation of stored Ca2+ and extracellular Ca2+-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca2+]i. RESULTS: We analyzed Ca2+-fluxes in a mutant that is devoid of the main cAMP-phosphodiesterase (PDE) RegA and displays an altered cAMP metabolism. In suspensions of developing cells cAMP-activated influx of extracellular Ca2+ was reduced as compared to wild type. Yet, single cell [Ca2+]i-imaging of regA- amoebae revealed a cAMP-induced [Ca2+]i increase even in the absence of extracellular Ca2+. The cytosolic presence of the cAMP PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced elevated basal [Ca2+]i in both, mutant and wild type cells. Under this condition wild type cells displayed cAMP-activated [Ca2+]i-transients also in nominally Ca2+-free medium. In the mutant strain the amplitude of light scattering oscillations and of accompanying cAMP oscillations were strongly reduced to almost basal levels. In addition, chemotactic performance during challenge with a cAMP-filled glass capillary was altered by EGTA-incubation. Cells were more sensitive to EGTA treatment than wild type: already at 2 mM EGTA only small pseudopods were extended and chemotactic speed was reduced. CONCLUSION: We conclude that there is a link between the second messengers cAMP and Ca2+. cAMP-dependent protein kinase (PKA) could provide for this link as a membrane-permeable PKA-activator also increased basal [Ca2+]i of regA- cells. Intracellular cAMP levels control [Ca2+]i by regulating Ca2+-fluxes of stores which in turn affect Ca2+-influx, light scattering oscillations and chemotactic performance. [Abstract/Link to Full Text]

Defeu Soufo HJ, Graumann PL
Bacillus subtilis actin-like protein MreB influences the positioning of the replication machinery and requires membrane proteins MreC/D and other actin-like proteins for proper localization.
BMC Cell Biol. 2005;6(1):10.
BACKGROUND: Bacterial actin-like proteins have been shown to perform essential functions in several aspects of cellular physiology. They affect cell growth, cell shape, chromosome segregation and polar localization of proteins, and localize as helical filaments underneath the cell membrane. Bacillus subtilis MreB and Mbl have been shown to perform dynamic motor like movements within cells, extending along helical tracks in a time scale of few seconds. RESULTS: In this work, we show that Bacillus subtilis MreB has a dual role, both in the formation of rod cell shape, and in chromosome segregation, however, its function in cell shape is distinct from that of MreC. Additionally, MreB is important for the localization of the replication machinery to the cell centre, which becomes aberrant soon after depletion of MreB. 3D image reconstructions suggest that frequently, MreB filaments consist of several discontinuous helical filaments with varying length. The localization of MreB was abnormal in cells with decondensed chromosomes, as well as during depletion of Mbl, MreBH and of the MreC/MreD proteins, which we show localize to the cell membrane. Thus, proper positioning of MreB filaments depends on and is affected by a variety of factors in the cell. CONCLUSION: Our data provide genetic and cytological links between MreB and the membrane, as well as with other actin like proteins, and further supports the connection of MreB with the chromosome. The functional dependence on MreB of the localization of the replication machinery suggests that the replisome is not anchored at the cell centre, but is positioned in a dynamic manner. [Abstract/Link to Full Text]

Qin J, Chittenden TW, Gao L, Pearlman JD
Automated migration analysis based on cell texture: method & reliability.
BMC Cell Biol. 2005;6(1):9.
BACKGROUND: In this paper, we present and validate a way to measure automatically the extent of cell migration based on automated examination of a series of digital photographs. It was designed specifically to identify the impact of Second Hand Smoke (SHS) on endothelial cell migration but has broader applications. The analysis has two stages: (1) preprocessing of image texture, and (2) migration analysis. RESULTS: The output is a graphic overlay that indicates the front lines of cell migration superimposed on each original image, with automated reporting of the distance traversed vs. time. Expert preference compares to manual placement of leading edge shows complete equivalence of automated vs. manual leading edge definition for cell migration measurement. CONCLUSION: Our method is indistinguishable from careful manual determinations of cell front lines, with the advantages of full automation, objectivity, and speed. [Abstract/Link to Full Text]

Rahman S, Patel Y, Murray J, Patel KV, Sumathipala R, Sobel M, Wijelath ES
Novel hepatocyte growth factor (HGF) binding domains on fibronectin and vitronectin coordinate a distinct and amplified Met-integrin induced signalling pathway in endothelial cells.
BMC Cell Biol. 2005;6(1):8.
BACKGROUND: The growth of new blood vessels in adult life requires the initiation of endothelial cell migration and proliferation from pre-existing vessels in addition to the recruitment and differentiation of circulating endothelial progenitor cells. Signals emanating from growth factors and the extracellular matrix are important in regulating these processes. RESULTS: Here we report that fibronectin (FN) and vitronectin (VN) modulate the responses of endothelial cells to HGF (Scatter Factor), an important pro-angiogenic mediator. Novel binding sites for HGF were identified on both FN and VN that generate molecular complexes with enhanced biological activity and these were identified in the supernatants of degranulated platelet suspensions implicating their release and formation in vivo. In the absence of co-stimulation with an ECM glycoprotein, HGF could not promote endothelial cell migration but retained the capacity to induce a proliferative response utilising the Map kinase pathway. Through promoting Met-Integrin association, HGF-FN and HGF-VN complexes coordinated and enhanced endothelial cell migration through activation of the PI-3 kinase pathway involving a Ras-dependent mechanism whereas a Ras-independent and attenuated migratory response was promoted by co-stimulation of cells with HGF and a non-binding partner ECM glycoprotein such as collagen-1. CONCLUSIONS: These studies identify a novel mechanism and pathway of HGF signalling in endothelial cells involving cooperation between Met and integrins in a Ras dependent manner. These findings have implications for the regulation of neovascularization in both health and disease. [Abstract/Link to Full Text]

Oliveira R, Christov C, Guillamo JS, de Boüard S, Palfi S, Venance L, Tardy M, Peschanski M
Contribution of gap junctional communication between tumor cells and astroglia to the invasion of the brain parenchyma by human glioblastomas.
BMC Cell Biol. 2005;6(1):7.
BACKGROUND: Gliomas are "intraparenchymally metastatic" tumors, invading the brain in a non-destructive way that suggests cooperation between glioma cells and their environment. Recent studies using an engineered rodent C6 tumor cell line have pointed to mechanisms of invasion that involved gap junctional communication (GJC), with connexin 43 as a substrate. We explored whether this concept may have clinical relevance by analyzing the participation of GJC in human glioblastoma invasion. RESULTS: Three complementary in vitro assays were used: (i) seeding on collagen IV, to analyze homocellular interactions between tumor cells (ii) co-cultures with astrocytes, to study glioblastoma/astrocytes relationships and (iii) implantation into organotypic brain slice cultures, that mimic the three-dimensional parenchymal environment. Carbenoxolone, a potent blocker of GJC, inhibited cell migration in the two latter models. It paradoxically increased it in the first one. These results showed that homocellular interaction between tumor cells supports intercellular adhesion, whereas heterocellular glioblastoma/astrocytes interactions through functional GJC conversely support tumor cell migration. As demonstrated for the rodent cell line, connexin 43 may be responsible for this heterocellular functional coupling. Its levels of expression, high in astrocytes, correlated positively with invasiveness in biopsied tumors. CONCLUSIONS: our results underscore the potential clinical relevance of the concept put forward by other authors based on experiments with a rodent cell line, that glioblastoma cells use astrocytes as a substrate for their migration by subverting communication through connexin 43-dependent gap junctions. [Abstract/Link to Full Text]

Wong C, Stearns T
Mammalian cells lack checkpoints for tetraploidy, aberrant centrosome number, and cytokinesis failure.
BMC Cell Biol. 2005;6(1):6.
BACKGROUND: Mammalian cells have been reported to have a p53-dependent tetraploidy checkpoint that blocks cell cycle progression in G1 in response to failure of cell division. In most cases where the tetraploidy checkpoint has been observed cell division was perturbed by anti-cytoskeleton drug treatments. However, other evidence argues against the existence of a tetraploidy checkpoint. Cells that have failed to divide differ from normal cells in having two nuclei, two centrosomes, a decreased surface to volume ratio, and having undergone an abortive cytokinesis. We tested each of these to determine which, if any, cause a G1 cell cycle arrest. RESULTS: Primary human diploid fibroblasts with intact cell cycle checkpoints were used in all experiments. Synchronized cells exhibited G1 arrest in response to division failure caused by treatment with either cytochalasin or the myosin II inhibitor blebbistatin. The role of tetraploidy, aberrant centrosome number, and increased cell size were tested by cell/cell and cell/cytoplast fusion experiments; none of these conditions resulted in G1 arrest. Instead we found that various drug treatments of the cells resulted in cellular damage, which was the likely cause of the arrest. When cytokinesis was blocked in the absence of damage-inducing drug treatments no G1 arrest was observed. CONCLUSIONS: We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause damage that is the likely cause of G1 arrest. Since tetraploid cells can cycle when created with minimal manipulation, previous reports of a tetraploidy checkpoint can probably be explained by side effects of the drug treatments used to observe them. [Abstract/Link to Full Text]

Hujová J, Sikora J, Dobrovolný R, Poupetová H, Ledvinová J, Kostrouchová M, Hrebícek M
Characterization of gana-1, a Caenorhabditis elegans gene encoding a single ortholog of vertebrate alpha-galactosidase and alpha-N-acetylgalactosaminidase.
BMC Cell Biol. 2005;6(1):5.
BACKGROUND: Human alpha-galactosidase A (alpha-GAL) and alpha-N-acetylgalactosaminidase (alpha-NAGA) are presumed to share a common ancestor. Deficiencies of these enzymes cause two well-characterized human lysosomal storage disorders (LSD)--Fabry (alpha-GAL deficiency) and Schindler (alpha-NAGA deficiency) diseases. Caenorhabditis elegans was previously shown to be a relevant model organism for several late endosomal/lysosomal membrane proteins associated with LSDs. The aim of this study was to identify and characterize C. elegans orthologs to both human lysosomal luminal proteins alpha-GAL and alpha-NAGA. RESULTS: BlastP searches for orthologs of human alpha-GAL and alpha-NAGA revealed a single C. elegans gene (R07B7.11) with homology to both human genes (alpha-galactosidase and alpha-N-acetylgalactosaminidase)--gana-1. We cloned and sequenced the complete gana-1 cDNA and elucidated the gene organization.Phylogenetic analyses and homology modeling of GANA-1 based on the 3D structure of chicken alpha-NAGA, rice alpha-GAL and human alpha-GAL suggest a close evolutionary relationship of GANA-1 to both human alpha-GAL and alpha-NAGA.Both alpha-GAL and alpha-NAGA enzymatic activities were detected in C. elegans mixed culture homogenates. However, alpha-GAL activity on an artificial substrate was completely inhibited by the alpha-NAGA inhibitor, N-acetyl-D-galactosamine.A GANA-1::GFP fusion protein expressed from a transgene, containing the complete gana-1 coding region and 3 kb of its hypothetical promoter, was not detectable under the standard laboratory conditions. The GFP signal was observed solely in a vesicular compartment of coelomocytes of the animals treated with Concanamycin A (CON A) or NH4Cl, agents that increase the pH of the cellular acidic compartment. Immunofluorescence detection of the fusion protein using polyclonal anti-GFP antibody showed a broader and coarsely granular cytoplasmic expression pattern in body wall muscle cells, intestinal cells, and a vesicular compartment of coelomocytes.Inhibition of gana-1 by RNA interference resulted in a decrease of both alpha-GAL and alpha-NAGA activities measured in mixed stage culture homogenates but did not cause any obvious phenotype. CONCLUSIONS: GANA-1 is a single C. elegans ortholog of both human alpha-GAL and alpha-NAGA proteins. Phylogenetic, homology modeling, biochemical and GFP expression analyses support the hypothesis that GANA-1 has dual enzymatic activity and is localized in an acidic cellular compartment. [Abstract/Link to Full Text]

Wu G, Olivecrona G, Olivecrona T
Extracellular degradation of lipoprotein lipase in rat adipose tissue.
BMC Cell Biol. 2005;6(1):4.
BACKGROUND: Recent studies in vivo indicate that short-term regulation of lipoprotein lipase (LPL) in rat adipose tissue is post-translational and occurs by a shift of the lipase protein towards an inactive form under the influence of another gene with short-lived message and product. It has not been possible to reproduce this process with isolated adipocytes suggesting that other cells are needed, and perhaps mediate the regulation. The objective of the present study was, therefore, to explore if explants of adipose tissue could be used for studies of the regulatory process. RESULTS: When explants of rat epididymal adipose tissue were incubated, LPL mass and activity decreased rapidly. Mass and activity within adipocytes remained constant for at least six hours, demonstrating that it was the extracellular portion of the enzyme that decreased. Adipocytes isolated from the explants after three or six hours of incubation retained their ability to secrete LPL to the medium. Addition of a cocktail of protease inhibitors to the incubation medium slowed down the decrease of LPL mass. Chloroquine was without effect, indicating that the degradation was not lysosomal. 125I-labeled LPL added to the medium was degraded to acid soluble products, indicating that the degradation occurred extracellularly. Fragmentation of the labelled lipase occurred in conditioned medium and this process was virtually abolished by two MMP inhibitors. CONCLUSIONS: The decrease of LPL mass and activity that occurs when explants of rat adipose tissue are incubated is due to proteolysis of extracellular LPL. The adipocytes continue to produce and secrete the enzyme. The effect of inhibitors indicates, but does not prove, that the degradation is mediated by MMPs. It appears that this process is accelerated in the tissue fragments compared to intact tissue. [Abstract/Link to Full Text]

Patel CA, Ghiselli G
Hinderin, a five-domains protein including coiled-coil motifs that binds to SMC3.
BMC Cell Biol. 2005;6(1):3.
BACKGROUND: The structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase. The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions. RESULTS: In order to identify proteins that by binding to SMC3 may interfere with the protein dimerization process, a human cDNA library was screened by the yeast two-hybrid system by using the hinge region of SMC3 as bait. This has lead to the identification of Hinderin, a novel five domains protein including two coiled-coil motifs and sharing a strikingly structural similarity to the SMC family of proteins. Hinderin is ubiquitously expressed in human tissues. Orthologue forms of the protein are present in other vertebrates but not in lower organisms. A mapping of the interaction sites revealed that the N- and C-terminal globular domains mediate the binding of Hinderin to SMC3. Hinderin/SMC3 complexes could be recovered by immunoprecipitation from cell lysates using an anti-SMC3 antibody, thus demonstrating that the two proteins interact in vivo. On the contrary, Hinderin did not interact with SMC1. In vivo the rate of SMC1/SMC3 interaction was decreased by the ectopic expression of Hinderin. CONCLUSIONS: Hinderin is a novel binding partner of SMC3. Based on its ability to modulate SMC1/SMC3 interaction we postulate that Hinderin affects the availability of SMC3 to engage in the formation of multimeric protein complexes. [Abstract/Link to Full Text]

Gordon PV, Paxton JB, Fox NS
A methodology for distinguishing divergent cell fates within a common progenitor population: adenoma- and neuroendocrine-like cells are confounders of rat ileal epithelial cell (IEC-18) culture.
BMC Cell Biol. 2005;6(1):2.
BACKGROUND: IEC-18 cells are a non-transformed, immortal cell line derived from juvenile rat ileal crypt cells. They may have experimental advantages over tumor-derived gastrointestinal lineages, including preservation of phenotype, normal endocrine responses and retention of differentiation potential. However, their proclivity for spontaneous differentiation/transformation may be stereotypical and could represent a more profound experimental confounder than previously realized. We hypothesized that IEC-18 cells spontaneously diverge towards a uniform mixture of epigenetic fates, with corresponding phenotypes, rather than persist as a single progenitor lineage. RESULTS: IEC-18 cells were cultured for 72 hours in serum free media (SFM), with and without various insulin-like growth factor agonists to differentially boost the basal rate of proliferation. A strategy was employed to identify constitutive genes as markers of divergent fates through gene array analysis by cross-referencing fold-change trends for individual genes against crypt cell abundance in each treatment. We then confirmed the cell-specific phenotype by immunolocalization of proteins corresponding to those genes. The majority of IEC-18 cells in SFM alone had a loss in expression of the adenomatous polyposis coli (APC) gene at the mRNA and protein levels, consistent with adenoma-like transformation. In addition, a small subset of cells expressed the serotonin receptor 2A gene and had neuroendocrine-like morphology. CONCLUSIONS: IEC-18 cells commonly undergo a change in cell fate prior to reaching confluence. The most common fate switch that we were able to detect correlates with a down regulation of the APC gene and transformation into an adenoma-like phenotype. [Abstract/Link to Full Text]

Petit MM, Meulemans SM, Alen P, Ayoubi TA, Jansen E, Van de Ven WJ
The tumor suppressor Scrib interacts with the zyxin-related protein LPP, which shuttles between cell adhesion sites and the nucleus.
BMC Cell Biol. 2005;6(1):1.
BACKGROUND: At sites of cell adhesion, proteins exist that not only perform structural tasks but also have a signaling function. Previously, we found that the Lipoma Preferred Partner (LPP) protein is localized at sites of cell adhesion such as focal adhesions and cell-cell contacts, and shuttles to the nucleus where it has transcriptional activation capacity. LPP is a member of the zyxin family of proteins, which contains five members: ajuba, LIMD1, LPP, TRIP6 and zyxin. LPP has three LIM domains (zinc-finger protein interaction domains) at its carboxy-terminus, which are preceded by a proline-rich pre-LIM region containing a number of protein interaction domains. RESULTS: To catch the role of LPP at sites of cell adhesion, we made an effort to identify binding partners of LPP. We found the tumor suppressor protein Scrib, which is a component of cell-cell contacts, as interaction partner of LPP. Human Scrib, which is a functional homologue of Drosophila scribble, is a member of the leucine-rich repeat and PDZ (LAP) family of proteins that is involved in the regulation of cell adhesion, cell shape and polarity. In addition, Scrib displays tumor suppressor activity. The binding between Scrib and LPP is mediated by the PDZ domains of Scrib and the carboxy-terminus of LPP. Both proteins localize in cell-cell contacts. Whereas LPP is also localized in focal adhesions and in the nucleus, Scrib could not be detected at these locations in MDCKII and CV-1 cells. Furthermore, our investigations indicate that Scrib is dispensable for targeting LPP to focal adhesions and to cell-cell contacts, and that LPP is not necessary for localizing Scrib in cell-cell contacts. We show that all four PDZ domains of Scrib are dispensable for localizing this protein in cell-cell contacts. CONCLUSIONS: Here, we identified an interaction between one of zyxin's family members, LPP, and the tumor suppressor protein Scrib. Both proteins localize in cell-cell contacts. This interaction links Scrib to a communication pathway between cell-cell contacts and the nucleus, and implicates LPP in Scrib-associated functions. [Abstract/Link to Full Text]

Fu H, Björkman L, Janmey P, Karlsson A, Karlsson J, Movitz C, Dahlgren C
The two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide.
BMC Cell Biol. 2004;5(1):50.
BACKGROUND: The formylpeptide receptor family members FPR and FPRL1, expressed in myeloid phagocytes, belong to the G-protein coupled seven transmembrane receptor family (GPCRs). They share a high degree of sequence similarity, particularly in the cytoplasmic domains involved in intracellular signaling. The established model of cell activation through GPCRs states that the receptors isomerize from an inactive to an active state upon ligand binding, and this receptor transformation subsequently activates the signal transducing G-protein. Accordingly, the activation of human neutrophil FPR and FPRL1 induces identical, pertussis toxin-sensitive functional responses and a transient increase in intracellular calcium is followed by a secretory response leading to mobilization of receptors from intracellular stores, as well as a release of reactive oxygen metabolites. RESULTS: We report that a cell permeable ten amino acid peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate (PIP2) binding region of gelsolin (an uncapper of actin filaments) blocks granule mobilization as well as secretion of oxygen radicals. The inhibitory effect of PBP10 is, however, receptor specific and affects the FPRL1-, but not the FPR-, induced cellular response. The transient rise in intracellular calcium induced by the active receptors is not affected by PBP10, suggesting that the blockage occurs in a parallel, novel signaling pathway used by FPRL1 to induce oxygen radical production and secretion. Also the FPR can activate neutrophils through a PBP10-sensitive signaling pathway, but this signal is normally blocked by the cytoskeleton. CONCLUSIONS: This study demonstrates that the two very closely related chemoattractant receptors, FPR and FPRL1, use distinct signaling pathways in activation of human neutrophils. The PIP2-binding peptide PBP10 selectively inhibits FPRL1-mediated superoxide production and granule mobilization. Furthermore, the activity of this novel PBP10 sensitive pathway in neutrophils is modulated by the actin cytoskeleton network. [Abstract/Link to Full Text]

---

Recent Articles in Cell Communication and Signaling

---

No recent articles are currently available.

---

Recent Articles in Eukaryotic Cell

---

Izumitsu K, Yoshimi A, Tanaka C
Two-component response regulators Ssk1p and Skn7p additively regulate high-osmolarity adaptation and fungicide sensitivity in Cochliobolus heterostrophus.
Eukaryot Cell. 2007 Feb;6(2):171-81.
Filamentous ascomycetous fungi possess many histidine kinases and two conserved response regulators, Ssk1p and Skn7p, in their two-component signaling systems. We previously reported that the fungus unique group III histidine kinase regulates high-osmolarity adaptation and iprodione/fludioxonil fungicide sensitivity by controlling the phosphorylation of Hog1-type mitogen-activated protein kinase (MAPK) in filamentous ascomycetes. Here, we have characterized the response regulator genes ChSsk1 and ChSkn7 in the southern corn leaf blight fungus Cochliobolus heterostrophus. Both ChSsk1- and ChSkn7-disrupted mutants showed little sensitivity to high-osmolarity stress and moderate resistance to the iprodione/fludioxonil fungicides. The phosphorylation of Hog1-type MAPK BmHog1p induced by high-osmolarity stress and fungicide treatments was only regulated by ChSsk1p, indicating that ChSkn7p has roles in high-osmolarity adaptation and fungicide sensitivity that are independent from the activation of BmHog1p. The Chssk1 Chskn7 double mutants clearly showed higher sensitivity to osmolar stress and higher resistance to fungicides than the single mutants. The dose responses of the double mutants fit well with those of the group III histidine kinase-deficient strain. These results suggest that in filamentous ascomycetes, the Ssk1- and Skn7-type response regulators control high-osmolarity adaptation and fungicide sensitivity additively with differential mechanisms under the regulation of the group III histidine kinase. This study provides evidence that filamentous fungi have a unique two-component signaling system that is different from that of yeast and is responsible for high-osmolarity adaptation and fungicide sensitivity. [Abstract/Link to Full Text]

Ishihara S, Hirata A, Nogami S, Beauvais A, Latge JP, Ohya Y
Homologous subunits of 1,3-beta-glucan synthase are important for spore wall assembly in Saccharomyces cerevisiae.
Eukaryot Cell. 2007 Feb;6(2):143-56.
During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-beta-glucan synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-beta-glucan synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of glucan synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-beta-glucan synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-beta-glucan synthase. [Abstract/Link to Full Text]

Gladfelter AS, Sustreanu N, Hungerbuehler AK, Voegeli S, Galati V, Philippsen P
The anaphase-promoting complex/cyclosome is required for anaphase progression in multinucleated Ashbya gossypii cells.
Eukaryot Cell. 2007 Feb;6(2):182-97.
Regulated protein degradation is essential for eukaryotic cell cycle progression. The anaphase-promoting complex/cyclosome (APC/C) is responsible for the protein destruction required for the initiation of anaphase and the exit from mitosis, including the degradation of securin and B-type cyclins. We initiated a study of the APC/C in the multinucleated, filamentous ascomycete Ashbya gossypii to understand the mechanisms underlying the asynchronous mitosis observed in these cells. These experiments were motivated by previous work which demonstrated that the mitotic cyclin AgClb1/2p persists through anaphase, suggesting that the APC/C may not be required for the division cycle in A. gossypii. We have now found that the predicted APC/C components AgCdc23p and AgDoc1p and the targeting factors AgCdc20p and AgCdh1p are essential for growth and nuclear division. Mutants lacking any of these factors arrest as germlings with nuclei blocked in mitosis. A likely substrate of the APC/C is the securin homologue AgPds1p, which is present in all nuclei in hyphae except those in anaphase. The destruction box sequence of AgPds1p is required for this timed disappearance. To investigate how the APC/C may function to degrade AgPds1p in only the subset of anaphase nuclei, we localized components and targeting subunits of the APC/C. Remarkably, AgCdc23p, AgDoc1p, and AgCdc16p were found in all nuclei in all cell cycle stages, as were the APC/C targeting factors AgCdc20p and AgCdh1p. These data suggest that the AgAPC/C may be constitutively active across the cell cycle and that proteolysis in these multinucleated cells may be regulated at the level of substrates rather than by the APC/C itself. [Abstract/Link to Full Text]

Ramírez MA, Lorenz MC
Mutations in alternative carbon utilization pathways in Candida albicans attenuate virulence and confer pleiotropic phenotypes.
Eukaryot Cell. 2007 Feb;6(2):280-90.
The interaction between Candida albicans and cells of the innate immune system is a key determinant of disease progression. Transcriptional profiling has revealed that C. albicans has a complex response to phagocytosis, much of which is similar to carbon starvation. This suggests that nutrient limitation is a significant stress in vivo, and we have shown that glyoxylate cycle mutants are less virulent in mice. To examine whether other aspects of carbon metabolism are important in vivo during an infection, we have constructed strains lacking FOX2 and FBP1, which encode key components of fatty acid beta-oxidation and gluconeogenesis, respectively. As expected, fox2Delta mutants failed to utilize several fatty acids as carbon sources. Surprisingly, however, these mutants also failed to grow in the presence of several other carbon sources, whose assimilation is independent of beta-oxidation, including ethanol and citric acid. Mutants lacking the glyoxylate enzyme ICL1 also had more severe carbon utilization phenotypes than were expected. These results suggest that the regulation of alternative carbon metabolism in C. albicans is significantly different from that in other fungi. In vivo, fox2Delta mutants show a moderate but significant reduction in virulence in a mouse model of disseminated candidiasis, while disruption of the glyoxylate cycle or gluconeogenesis confers a severe attenuation in this model. These data indicate that C. albicans often encounters carbon-poor conditions during growth in the host and that the ability to efficiently utilize multiple nonfermentable carbon sources is a virulence determinant. Consistent with this in vivo requirement, C. albicans uniquely regulates carbon metabolism in a more integrated manner than in Saccharomyces cerevisiae, such that defects in one part of the machinery have wider impacts than expected. These aspects of alternative carbon metabolism may then be useful as targets for therapeutic intervention. [Abstract/Link to Full Text]

Erb-Downward JR, Huffnagle GB
Cryptococcus neoformans produces authentic prostaglandin E2 without a cyclooxygenase.
Eukaryot Cell. 2007 Feb;6(2):346-50.
Many single-celled eukaryotes produce prostaglandin-like molecules, but these have not been absolutely verified by mass spectrometry. We have isolated, and identified by liquid chromatography-tandem mass spectrometry, authentic prostaglandin E(2) from Cryptococcus neoformans. Cyclooxygenase inhibitors did not inhibit prostaglandin synthesis, and the cryptococcal genome lacks a cyclooxygenase homolog. Thus, novel enzymes must exist. [Abstract/Link to Full Text]

Martchenko M, Levitin A, Whiteway M
Transcriptional activation domains of the Candida albicans Gcn4p and Gal4p homologs.
Eukaryot Cell. 2007 Feb;6(2):291-301.
Many putative transcription factors in the pathogenic fungus Candida albicans contain sequence similarity to well-defined transcriptional regulators in the budding yeast Saccharomyces cerevisiae, but this sequence similarity is often limited to the DNA binding domains of the molecules. The Gcn4p and Gal4p proteins of Saccharomyces cerevisiae are highly studied and well-understood eukaryotic transcription factors of the basic leucine zipper (Gcn4p) and C(6) zinc cluster (Gal4p) families; C. albicans has C. albicans Gcn4p (CaGcn4p) and CaGal4p with DNA binding domains highly similar to their S. cerevisiae counterparts. Deletion analysis of the CaGcn4p protein shows that the N' terminus is needed for transcriptional activation; an 81-amino-acid region is critical for this function, and this domain can be coupled to a lexA DNA binding module to provide transcription-activating function in a heterologous reporter system. Deletion analysis of the C. albicans Gal4p identifies a C-terminal 73-amino-acid-long transcription-activating domain that also can be transferred to a heterologous reporter construct to direct transcriptional activation. These two transcriptional activation regions show no sequence similarity to the respective domains in their S. cerevisiae homologs, and the two C. albicans transcription-activating domains themselves show little similarity. [Abstract/Link to Full Text]

Waki K, Dutta S, Ray D, Kolli BK, Akman L, Kawazu S, Lin CP, Chang KP
Transmembrane molecules for phylogenetic analyses of pathogenic protists: Leishmania-specific informative sites in hydrophilic loops of trans- endoplasmic reticulum N-acetylglucosamine-1-phosphate transferase.
Eukaryot Cell. 2007 Feb;6(2):198-210.
A sequence database was created for the Leishmania N-acetylglucosamine-1-phosphate transferase (nagt) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven nagt sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26 nagt sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus Leishmania (L. amazonensis-L. mexicana, L. donovani-L. infantum, L. tropica, L. major, and L. turanica-L. gerbilli), subgenus Viannia (L. braziliensis, L. panamensis), and one unclassified (L. enriettii) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of nagt varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to Leishmania evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in L. tropica but not in L. donovani-L. infantum. This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of nagt are restricted largely to several extended hydrophilic loops predicted from hypothetical models of Leishmania NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of nagt from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes. [Abstract/Link to Full Text]

Fudal I, Collemare J, Böhnert HU, Melayah D, Lebrun MH
Expression of Magnaporthe grisea avirulence gene ACE1 is connected to the initiation of appressorium-mediated penetration.
Eukaryot Cell. 2007 Mar;6(3):546-54.
Magnaporthe grisea is responsible for a devastating fungal disease of rice called blast. Current control of this disease relies on resistant rice cultivars that recognize M. grisea signals corresponding to specific secreted proteins encoded by avirulence genes. The M. grisea ACE1 avirulence gene differs from others, since it controls the biosynthesis of a secondary metabolite likely recognized by rice cultivars carrying the Pi33 resistance gene. Using a transcriptional fusion between ACE1 promoter and eGFP, we showed that ACE1 is only expressed in appressoria during fungal penetration into rice and barley leaves, onion skin, and cellophane membranes. ACE1 is almost not expressed in appressoria differentiated on Teflon and Mylar artificial membranes. ACE1 expression is not induced by cellophane and plant cell wall components, demonstrating that it does not require typical host plant compounds. Cyclic AMP (cAMP) signaling mutants delta cpkA and delta mac1 sum1-99 and tetraspanin mutant delta pls1::hph differentiate melanized appressoria with normal turgor but are unable to penetrate host plant leaves. ACE1 is normally expressed in these mutants, suggesting that it does not require cAMP signaling or a successful penetration event. ACE1 is not expressed in appressoria of the buf1::hph mutant defective for melanin biosynthesis and appressorial turgor. The addition of hyperosmotic solutes to buf1::hph appressoria restores appressorial development and ACE1 expression. Treatments of young wild-type appressoria with actin and tubulin inhibitors reduce both fungal penetration and ACE1 expression. These experiments suggest that ACE1 appressorium-specific expression does not depend on host plant signals but is connected to the onset of appressorium-mediated penetration. [Abstract/Link to Full Text]

Grabi?ska KA, Magnelli P, Robbins PW
Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III activity and chitin chain length.
Eukaryot Cell. 2007 Feb;6(2):328-36.
Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a approximately 60% decrease in CSIII activity, which is correlated with a approximately 30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro. [Abstract/Link to Full Text]

Olson GM, Fox DS, Wang P, Alspaugh JA, Buchanan KL
Role of protein O-mannosyltransferase Pmt4 in the morphogenesis and virulence of Cryptococcus neoformans.
Eukaryot Cell. 2007 Feb;6(2):222-34.
Protein O mannosylation is initiated in the endoplasmic reticulum by protein O-mannosyltransferases (Pmt proteins) and plays an important role in the secretion, localization, and function of many proteins, as well as in cell wall integrity and morphogenesis in fungi. Three Pmt proteins, each belonging to one of the three respective Pmt subfamilies, are encoded in the genome of the human fungal pathogen Cryptococcus neoformans. Disruption of the C. neoformans PMT4 gene resulted in abnormal growth morphology and defective cell separation. Transmission electron microscopy revealed defective cell wall septum degradation during mother-daughter cell separation in the pmt4 mutant compared to wild-type cells. The pmt4 mutant also demonstrated sensitivity to elevated temperature, sodium dodecyl sulfate, and amphotericin B, suggesting cell wall defects. Further analysis of cell wall protein composition revealed a cell wall proteome defect in the pmt4 mutant, as well as a global decrease in protein mannosylation. Heterologous expression of C. neoformans PMT4 in a Saccharomyces cerevisiae pmt1pmt4 mutant strain functionally complemented the deficient Pmt activity. Furthermore, Pmt4 activity in C. neoformans was required for full virulence in two murine models of disseminated cryptococcal infection. Taken together, these results indicate a central role for Pmt4-mediated protein O mannosylation in growth, cell wall integrity, and virulence of C. neoformans. [Abstract/Link to Full Text]

Hungerbuehler AK, Philippsen P, Gladfelter AS
Limited functional redundancy and oscillation of cyclins in multinucleated Ashbya gossypii fungal cells.
Eukaryot Cell. 2007 Mar;6(3):473-86.
Cyclin protein behavior has not been systematically investigated in multinucleated cells with asynchronous mitoses. Cyclins are canonical oscillating cell cycle proteins, but it is unclear how fluctuating protein gradients can be established in multinucleated cells where nuclei in different stages of the division cycle share the cytoplasm. Previous work in A. gossypii, a filamentous fungus in which nuclei divide asynchronously in a common cytoplasm, demonstrated that one G1 and one B-type cyclin do not fluctuate in abundance across the division cycle. We have undertaken a comprehensive analysis of all G1 and B-type cyclins in A. gossypii to determine whether any of the cyclins show periodic abundance across the cell cycle and to examine whether cyclins exhibit functional redundancy in such a cellular environment. We localized all G1 and B-type cyclins and notably found that only AgClb5/6p varies in subcellular localization during the division cycle. AgClb5/6p is lost from nuclei at the meta-anaphase transition in a D-box-dependent manner. These data demonstrate that efficient nuclear autonomous protein degradation can occur within multinucleated cells residing in a common cytoplasm. We have shown that three of the five cyclins in A. gossypii are essential genes, indicating that there is minimal functional redundancy in this multinucleated system. In addition, we have identified a cyclin, AgClb3/4p, that is essential only for sporulation. We propose that the cohabitation of different cyclins in nuclei has led to enhanced substrate specificity and limited functional redundancy within classes of cyclins in multinucleated cells. [Abstract/Link to Full Text]

Rodrigues ML, Nimrichter L, Oliveira DL, Frases S, Miranda K, Zaragoza O, Alvarez M, Nakouzi A, Feldmesser M, Casadevall A
Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport.
Eukaryot Cell. 2007 Jan;6(1):48-59.
The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment. [Abstract/Link to Full Text]

Deng F, Allen TD, Nuss DL
Ste12 transcription factor homologue CpST12 is down-regulated by hypovirus infection and required for virulence and female fertility of the chestnut blight fungus Cryphonectria parasitica.
Eukaryot Cell. 2007 Feb;6(2):235-44.
A putative homologue of the Saccharomyces cerevisiae Ste12 transcription factor was identified in a series of expressed sequence tag-based microarray analyses as being down-regulated in strains of the chestnut blight fungus, Cryphonectria parasitica, infected by virulence-attenuating hypoviruses. Cloning of the corresponding gene, cpst12, confirmed a high level of similarity to Ste12 homologues of other filamentous fungi. Disruption of cpst12 resulted in no alterations in in vitro growth characteristics or colony morphology and an increase in the production of asexual spores, indicating that CpST12 is dispensable for vegetative growth and conidiation on artificial medium. However, the disruption mutants showed a very substantial reduction in virulence on chestnut tissue and a complete loss of female fertility, two symptoms normally conferred by hypovirus infection. Both virulence and female fertility were restored by complementation with the wild-type cpst12 gene. Analysis of transcriptional changes caused by cpst12 gene disruption with a custom C. parastica cDNA microaray chip identified 152 responsive genes. A significant number of these putative CpST12-regulated genes were also responsive to hypovirus infection. Thus, cpst12 encodes a cellular transcription factor, CpST12, that is down-regulated by hypovirus infection and required for female fertility, virulence and regulated expression of a subset of hypovirus responsive host genes. [Abstract/Link to Full Text]

Maxson ME, Dadachova E, Casadevall A, Zaragoza O
Radial mass density, charge, and epitope distribution in the Cryptococcus neoformans capsule.
Eukaryot Cell. 2007 Jan;6(1):95-109.
Exposure of Cryptococcus neoformans cells to gamma radiation results in a gradual release of capsular polysaccharide, in a dose-dependent manner. This method allows the systematic exploration of different capsular regions. Using this methodology, capsule density was determined to change according to the radial distribution of glucuronoxylomannan and total polysaccharide, becoming denser at the inner regions of the capsule. Scanning electron microscopy of cells following gamma radiation treatment confirmed this finding. The zeta potential of the capsule also increased as the capsule size decreased. However, neither charge nor density differences were correlated with any change in sugar composition (xylose, mannose, and glucuronic acid) in the different capsular regions, since the proportions of these sugars remained constant throughout the capsule. Analysis of the capsular antigenic properties by monoclonal antibody binding and Scatchard analysis revealed fluctuations in the binding affinity within the capsule but not in the number of antibody binding sites, suggesting that the spatial organization of high- and low-affinity epitopes within the capsule changed according to radial position. Finally, evidence is presented that the structure of the capsule changes with capsule age, since the capsule of older cells became more resistant to gamma radiation-induced ablation. In summary, the capsule of C. neoformans is heterogeneous in its spatial distribution and changes with age. Furthermore, our results suggest several mechanisms by which the capsule may protect the fungal cell against exogenous environmental factors. [Abstract/Link to Full Text]

Dijksterhuis J, Nijsse J, Hoekstra FA, Golovina EA
High viscosity and anisotropy characterize the cytoplasm of fungal dormant stress-resistant spores.
Eukaryot Cell. 2007 Feb;6(2):157-70.
Ascospores of the fungus Talaromyces macrosporus are dormant and extremely stress resistant, whereas fungal conidia--the main airborne vehicles of distribution--are not. Here, physical parameters of the cytoplasm of these types of spores were compared. Cytoplasmic viscosity and level of anisotropy as judged by spin probe studies (electron spin resonance) were extremely high in dormant ascospores and during early germination and decreased only partly after trehalose degradation and glucose efflux. Upon prosilition (ejection of the spore), these parameters fell sharply to values characteristic of vegetative cells. These changes occurred without major volume changes that suggest dramatic changes in cytoplasmic organization. Azide reversibly inhibited prosilition as well as the decline in cytoplasmic parameters. No organelle structures were observed in etched, cryoplaned specimens of ascospores by low-temperature scanning electron microscopy (LTSEM), confirming the high cytoplasmic viscosity. However, cell structures became visible upon prosilition, indicating reduced viscosity. The viscosity of fresh conidia of different Penicillium species was lower, namely, 3.5 to 4.8 cP, than that of ascospores, near 15 cP. In addition the level of anisotropic motion was markedly lower in these cells (h(0)/h(+1) = 1.16 versus 1.4). This was confirmed by LTSEM images showing cell structures. The decline of cytoplasmic viscosity in conidia during germination was linked with a gradual increase in cell volume. These data show that mechanisms of cytoplasm conservation during germination differ markedly between ascospores and conidia. [Abstract/Link to Full Text]

Fleissner A, Glass NL
SO, a protein involved in hyphal fusion in Neurospora crassa, localizes to septal plugs.
Eukaryot Cell. 2007 Jan;6(1):84-94.
The colony of a filamentous ascomycete fungus typically grows as a multinucleate syncytium. While this syncytial organization has developmental advantages, it bears the risk of extensive damage caused by local injury of hyphae. Loss of cytoplasm in injured hyphae is restricted by the fast and efficient sealing of the central pores of hyphal crosswalls, or septa, by a peroxisome-derived organelle called the Woronin body. The formation of septal plugs is also associated with development and leads to separation of certain parts of the colony. Septal plugs associated with developmental processes or aging hyphae typically occur by the accumulation of sealing material. Here we report that in Neurospora crassa, a protein necessary for hyphal fusion and proper colony development called SO (SOFT) localizes to septal plugs. In response to injury, SO accumulates at the septal plug in a Woronin body-independent manner. However, the presence of the Woronin body affects the speed of accumulation of SO at the septal pore. We determined that SO contributes to, but is not essential for, septal plugging. SO accumulation was also observed at septal plugs formed during hyphal aging and during programmed cell death mediated by genetic differences at heterokaryon incompatibility (het) loci. [Abstract/Link to Full Text]

Wright LC, Santangelo RM, Ganendren R, Payne J, Djordjevic JT, Sorrell TC
Cryptococcal lipid metabolism: phospholipase B1 is implicated in transcellular metabolism of macrophage-derived lipids.
Eukaryot Cell. 2007 Jan;6(1):37-47.
Cryptococci survive and replicate within macrophages and can use exogenous arachidonic acid for the production of eicosanoids. Phospholipase B1 (PLB1) has a putative, but uninvestigated, role in these processes. We have shown that uptake and esterification of radiolabeled arachidonic, palmitic, and oleic acids by the Cryptococcus neoformans var. grubii H99 wild-type strain and its PLB1 deletion mutant strain (the Deltaplb1 strain) are independent of PLB1, except under hyperosmolar stress. Similarly, PLB1 was required for metabolism of 1-palmitoyl lysophosphatidylcholine (LysoPC), which is toxic to eukaryotic cell membranes, under hyperosmolar conditions. During both logarithmic and stationary phases of growth, the physiologically relevant phospholipids, dipalmitoyl phosphatidylcholine (DPPC) and dioleoyl phosphatidylcholine, were taken up and metabolized via PLB1. Exogenous DPPC did not enhance growth in the presence of glucose as a carbon source but could support it for at least 24 h in glucose-free medium. Detoxification of LysoPC by reacylation occurred in both the H99 wild-type and the Deltaplb1 strains in the presence of glucose, but PLB1 was required when LysoPC was the sole carbon source. This indicates that both energy-independent (via PLB1) and energy-dependent transacylation pathways are active in cryptococci. Phospholipase A(1) activity was identified by PLB1-independent degradation of 1-palmitoyl-2-arachidonoyl phosphatidylcholine, but the arachidonoyl LysoPC formed was not detoxified by reacylation. Using the human macrophage-like cell line THP-1, we demonstrated the PLB1-dependent incorporation of macrophage-derived arachidonic acid into cryptococcal lipids during cryptococcus-phagocyte interaction. This pool of arachidonate can be sequestered for eicosanoid production by the fungus and/or suppression of host phagocytic activity, thus diminishing the immune response. [Abstract/Link to Full Text]

Urbaniak MD, Turnock DC, Ferguson MA
Galactose starvation in a bloodstream form Trypanosoma brucei UDP-glucose 4'-epimerase conditional null mutant.
Eukaryot Cell. 2006 Nov;5(11):1906-13.
Galactose metabolism is essential for the survival of Trypanosoma brucei, the etiological agent of African sleeping sickness. T. brucei hexose transporters are unable to transport galactose, which is instead obtained through the epimerization of UDP-glucose to UDP-galactose catalyzed by UDP-glucose 4'-epimerase (galE). Here, we have characterized the phenotype of a bloodstream form T. brucei galE conditional null mutant under nonpermissive conditions that induced galactose starvation. Cellular levels of UDP-galactose dropped rapidly upon induction of galactose starvation, reaching undetectable levels after 72 h. Analysis of extracted glycoproteins by ricin and tomato lectin blotting showed that terminal beta-d-galactose was virtually eliminated and poly-N-acetyllactosamine structures were substantially reduced. Mass spectrometric analysis of variant surface glycoprotein confirmed complete loss of galactose from the glycosylphosphatidylinositol anchor. After 96 h, cell division ceased, and electron microscopy revealed that the cells had adopted a morphologically distinct stumpy-like form, concurrent with the appearance of aberrant vesicles close to the flagellar pocket. These data demonstrate that the UDP-glucose 4'-epimerase is essential for the production of UDP-galactose required for galactosylation of glycoproteins and that galactosylation of one or more glycoproteins, most likely in the lysosomal/endosomal system, is essential for the survival of bloodstream form T. brucei. [Abstract/Link to Full Text]

Gilbert LA, Ravindran S, Turetzky JM, Boothroyd JC, Bradley PJ
Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells.
Eukaryot Cell. 2007 Jan;6(1):73-83.
Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite. [Abstract/Link to Full Text]

Skovorodkin I, Pimenov A, Raykhel I, Schimanski B, Ammermann D, Günzl A
alpha-tubulin minichromosome promoters in the stichotrichous ciliate Stylonychia lemnae.
Eukaryot Cell. 2007 Jan;6(1):28-36.
Ciliated protists are model organisms for a number of molecular phenomena including telomerase function, self-splicing introns, and an RNA interference-related mechanism in programmed DNA elimination. Despite this relevance, our knowledge about promoters and transcriptional regulation in these organisms is very limited. The macronuclear genome of stichotrichous ciliates consists of minichromosomes which typically encode a single gene. The 5' nontranscribed spacers are usually no longer than 400 bp and highly suitable for promoter characterizations. We used microinjection of two artificial and differently tagged alpha1 tubulin minichromosomes into the macronucleus of Stylonychia lemnae as a means to characterize in detail the corresponding promoter. Clonal cell lines that stably maintained both minichromosomes were generated, enabling comparative expression analysis by primer extension assays. Deletion and block substitution mutations of one of the minichromosomes revealed a TATA-like element, a putative initiator element, and two distinct upstream sequence elements (USEs). Determination of transcription initiation sites and a sequence alignment indicated that both TATA-like and initiator elements are conserved components of S. lemnae minichromosomes, whereas the USEs appear to be specific for the alpha1 tubulin minichromosome. The alpha2 tubulin minichromosome promoter is very short, comprising the two proximal elements but not the USEs. Despite the latter finding, up-regulation of alpha-tubulin expression in cells treated with concanavalin A activated the alpha2 but not the alpha1 tubulin promoter. These results therefore show that gene expression regulation in S. lemnae occurs at the level of transcription initiation on the basis of structurally different promoters. [Abstract/Link to Full Text]

Saliola M, Scappucci G, De Maria I, Lodi T, Mancini P, Falcone C
Deletion of the glucose-6-phosphate dehydrogenase gene KlZWF1 affects both fermentative and respiratory metabolism in Kluyveromyces lactis.
Eukaryot Cell. 2007 Jan;6(1):19-27.
In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Delta strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield. [Abstract/Link to Full Text]

Cunningham FX, Lee H, Gantt E
Carotenoid biosynthesis in the primitive red alga Cyanidioschyzon merolae.
Eukaryot Cell. 2007 Mar;6(3):533-45.
Cyanidioschyzon merolae is considered to be one of the most primitive of eukaryotic photosynthetic organisms. To obtain insights into the origin and evolution of the pathway of carotenoid biosynthesis in eukaryotic plants, the carotenoid content of C. merolae was ascertained, genes encoding enzymes of carotenoid biosynthesis in this unicellular red alga were identified, and the activities of two candidate pathway enzymes of particular interest, lycopene cyclase and beta-carotene hydroxylase, were examined. C. merolae contains perhaps the simplest assortment of chlorophylls and carotenoids found in any eukaryotic photosynthetic organism: chlorophyll a, beta-carotene, and zeaxanthin. Carotenoids with epsilon-rings (e.g., lutein), found in many other red algae and in green algae and land plants, were not detected, and the lycopene cyclase of C. merolae quite specifically produced only beta-ringed carotenoids when provided with lycopene as the substrate in Escherichia coli. Lycopene beta-ring cyclases from several bacteria, cyanobacteria, and land plants also proved to be high-fidelity enzymes, whereas the structurally related epsilon-ring cyclases from several plant species were found to be less specific, yielding products with beta-rings as well as epsilon-rings. C. merolae lacks orthologs of genes that encode the two types of beta-carotene hydroxylase found in land plants, one a nonheme diiron oxygenase and the other a cytochrome P450. A C. merolae chloroplast gene specifies a polypeptide similar to members of a third class of beta-carotene hydroxylases, common in cyanobacteria, but this gene did not produce an active enzyme when expressed in E. coli. The identity of the C. merolae beta-carotene hydroxylase therefore remains uncertain. [Abstract/Link to Full Text]

Strmecki L, Bloomfield G, Araki T, Dalton E, Skelton J, Schilde C, Harwood A, Williams JG, Ivens A, Pears C
Proteomic and microarray analyses of the Dictyostelium Zak1-GSK-3 signaling pathway reveal a role in early development.
Eukaryot Cell. 2007 Feb;6(2):245-52.
GskA, the Dictyostelium GSK-3 orthologue, is modified and activated by the dual-specificity tyrosine kinase Zak1, and the two kinases form part of a signaling pathway that responds to extracellular cyclic AMP. We identify potential cellular effectors for the two kinases by analyzing the corresponding null mutants. There are proteins and mRNAs that are altered in abundance in only one or the other of the two mutants, indicating that each kinase has some unique functions. However, proteomic and microarray analyses identified a number of proteins and genes, respectively, that are similarly misregulated in both mutant strains. The positive correlation between the array data and the proteomic data is consistent with the Zak1-GskA signaling pathway's functioning by directly or indirectly regulating gene expression. The discoidin 1 genes are positively regulated by the pathway, while the abundance of the H5 protein is negatively regulated. Two of the targets, H5 and discoidin 1, are well-characterized markers for early development, indicating that the Zak1-GskA pathway plays a role in development earlier than previously observed. [Abstract/Link to Full Text]

Liapounova NA, Hampl V, Gordon PM, Sensen CW, Gedamu L, Dacks JB
Reconstructing the mosaic glycolytic pathway of the anaerobic eukaryote Monocercomonoides.
Eukaryot Cell. 2006 Dec;5(12):2138-46.
All eukaryotes carry out glycolysis, interestingly, not all using the same enzymes. Anaerobic eukaryotes face the challenge of fewer molecules of ATP extracted per molecule of glucose due to their lack of a complete tricarboxylic acid cycle. This may have pressured anaerobic eukaryotes to acquire the more ATP-efficient alternative glycolytic enzymes, such as pyrophosphate-fructose 6-phosphate phosphotransferase and pyruvate orthophosphate dikinase, through lateral gene transfers from bacteria and other eukaryotes. Most studies of these enzymes in eukaryotes involve pathogenic anaerobes; Monocercomonoides, an oxymonad belonging to the eukaryotic supergroup Excavata, is a nonpathogenic anaerobe representing an evolutionarily and ecologically distinct sampling of an anaerobic glycolytic pathway. We sequenced cDNA encoding glycolytic enzymes from a previously established cDNA library of Monocercomonoides and analyzed the relationships of these enzymes to those from other organisms spanning the major groups of Eukaryota, Bacteria, and Archaea. We established that, firstly, Monocercomonoides possesses alternative versions of glycolytic enzymes: fructose-6-phosphate phosphotransferase, both pyruvate kinase and pyruvate orthophosphate dikinase, cofactor-independent phosphoglycerate mutase, and fructose-bisphosphate aldolase (class II, type B). Secondly, we found evidence for the monophyly of oxymonads, kinetoplastids, diplomonads, and parabasalids, the major representatives of the Excavata. We also found several prokaryote-to-eukaryote as well as eukaryote-to-eukaryote lateral gene transfers involving glycolytic enzymes from anaerobic eukaryotes, further suggesting that lateral gene transfer was an important factor in the evolution of this pathway for denizens of this environment. [Abstract/Link to Full Text]

Dumas C, Chow C, Müller M, Papadopoulou B
A novel class of developmentally regulated noncoding RNAs in Leishmania.
Eukaryot Cell. 2006 Dec;5(12):2033-46.
Leishmania is a protozoan parasite that causes serious morbidity and mortality in humans worldwide. The ability of these parasites to survive within the phagolysosomes of mammalian macrophages is dependent on the developmental regulation of a variety of genes. Identifying genomic sequences that are preferentially expressed during the parasite's intracellular growth would provide new insights about the mechanisms controlling stage-specific gene regulation for intracellular development of the parasite. Using a genomic library that differentially hybridized to probes made from total RNA from Leishmania infantum amastigote or promastigote life cycle stages, we identified a new class of noncoding RNAs (ncRNAs) ranging from approximately 300 to 600 nucleotides in size that are expressed specifically in the intracellular amastigote stage. These ncRNAs are transcribed by RNA polymerase II from genomic clusters of tandem head-to-tail repeats, which are mainly located within subtelomeric regions. Remarkably, both the sense and antisense orientations of these ncRNAs are transcribed and are processed by trans splicing and polyadenylation. The levels of antisense transcripts are at least 10-fold lower than those of the sense transcripts and are tightly regulated. The sense and antisense ncRNAs are cytosolic as shown by fluorescence in situ hybridization studies and cosediment with a small ribonucleoprotein complex. Amastigote-specific regulation of these ncRNAs possibly occurs at the level of RNA stability. Interestingly, overexpression of these ncRNAs in promastigotes, as part of an episomal expression vector, failed to produce any transcript, which further highlights the instability of these RNAs in the promastigote stage. This is the first report describing developmentally regulated ncRNAs in protozoan parasites. [Abstract/Link to Full Text]

Dreesen O, Cross GA
Consequences of telomere shortening at an active VSG expression site in telomerase-deficient Trypanosoma brucei.
Eukaryot Cell. 2006 Dec;5(12):2114-9.
Trypanosoma brucei evades the host immune response by sequential expression of a large family of variant surface glycoproteins (VSG) from one of approximately 20 subtelomeric expression sites (ES). VSG transcription is monoallelic, and little is known about the regulation of antigenic switching. To explore whether telomere length could affect antigenic switching, we created a telomerase-deficient cell line, in which telomeres shortened at a rate of 3 to 6 bp at each cell division. Upon reaching a critical length, short silent ES telomeres were stabilized by a telomerase-independent mechanism. The active ES telomere progressively shortened and frequently broke. Upon reaching a critical length, the short active ES telomere stabilized, but the transcribed VSG was gradually lost from the population and replaced by a new VSG through duplicative gene conversion. We propose a model in which subtelomeric-break-induced replication-mediated repair at a short ES telomere leads to duplicative gene conversion and expression of a new VSG. [Abstract/Link to Full Text]

Hedbacker K, Carlson M
Regulation of the nucleocytoplasmic distribution of Snf1-Gal83 protein kinase.
Eukaryot Cell. 2006 Dec;5(12):1950-6.
Snf1 protein kinase containing the beta subunit Gal83 is localized in the cytoplasm during growth of Saccharomyces cerevisiae cells in abundant glucose and accumulates in the nucleus in response to glucose limitation. Nuclear localization of Snf1-Gal83 requires activation of the Snf1 catalytic subunit and depends on Gal83, but in the snf1Delta mutant, Gal83 exhibits glucose-regulated nuclear accumulation. We show here that the N terminus of Gal83, which is divergent from those of the other beta subunits, is necessary and sufficient for Snf1-independent, glucose-regulated localization. We identify a leucine-rich nuclear export signal in the N terminus and show that export depends on the Crm1 export receptor. We present evidence that catalytically inactive Snf1 promotes the cytoplasmic retention of Gal83 in glucose-grown cells through its interaction with the C terminus of Gal83; cytoplasmic localization of inactive Snf1-Gal83 maintains accessibility to the Snf1-activating kinases. Finally, we characterize the effects of glucose phosphorylation on localization. These studies define roles for Snf1 and Gal83 in determining the nucleocytoplasmic distribution of Snf1-Gal83 protein kinase. [Abstract/Link to Full Text]

Selitrennik M, Duek L, Lotan R, Choder M
Nucleocytoplasmic shuttling of the Rpb4p and Rpb7p subunits of Saccharomyces cerevisiae RNA polymerase II by two pathways.
Eukaryot Cell. 2006 Dec;5(12):2092-103.
Rpb4p and Rpb7p are subunits of the RNA polymerase II of Saccharomyces cerevisiae that form a dissociable heterodimeric complex. Whereas the only reported function of Rpb7p is related to transcription, Rpb4p has been found to also act in mRNA export and in the major mRNA decay pathway that operates in the cytoplasm, thus raising the possibility that Rpb4p links between the nuclear and cytoplasmic processes. Here we show that both Rpb4p and Rpb7p shuttle between the nucleus and the cytoplasm. Shuttling kinetics of the two proteins are similar as long as their interaction is possible, suggesting that they shuttle as a heterodimer. Under normal conditions, shuttling of Rpb4p and Rpb7p depends on ongoing transcription. However, during severe stresses of heat shock, ethanol, and starvation, the two proteins shuttle via a transcription-independent pathway. Thus, Rpb4p and Rpb7p shuttle via two pathways, depending on environmental conditions. [Abstract/Link to Full Text]

Cabral M, Anjard C, Loomis WF, Kuspa A
Genetic evidence that the acyl coenzyme A binding protein AcbA and the serine protease/ABC transporter TagA function together in Dictyostelium discoideum cell differentiation.
Eukaryot Cell. 2006 Dec;5(12):2024-32.
The acyl coenzyme A (CoA) binding protein AcbA is cleaved to form a peptide (SDF-2) that coordinates spore encapsulation during the morphogenesis of Dictyostelium discoideum fruiting bodies. We present genetic evidence that the misspecification of cell types seen in mutants of the serine protease/ABC transporter TagA results from the loss of normal interactions with AcbA. Developmental phenotypes resulting from aberrant expression of the TagA protease domain, such as the formation of supernumerary tips on aggregates and the production of excess prestalk cells, are suppressed by null mutations in the acbA gene. Phenotypes resulting from the deletion of tagA, such as overexpression of the prestalk gene ecmB and the misexpression of the prespore gene cotB in stalk cells, are also observed in acbA mutants. Moreover, tagA- mutants fail to produce SDF-2 during fruiting body morphogenesis but are able to do so if they are stimulated with exogenous SDF-2. These results indicate that the developmental program depends on TagA and AcbA working in concert with each other during cell type differentiation and suggest that TagA is required for normal SDF-2 signaling during spore encapsulation. [Abstract/Link to Full Text]

Romeralo M, Escalante R, Sastre L, Lado C
Molecular systematics of dictyostelids: 5.8S ribosomal DNA and internal transcribed spacer region analyses.
Eukaryot Cell. 2007 Jan;6(1):110-6.
The variability and adaptability of the amoebae from the class Dictyosteliomycetes greatly complicate their systematics. The nucleotide sequences of the ribosomal internal transcribed spacers and the 5.8S ribosomal DNA gene have been determined for 28 isolates, and their utility to discriminate between different species and genera has been shown. [Abstract/Link to Full Text]

---

Recent Articles in The Journal of Biological Chemistry

---

Radjabi AR, Sawada K, Jagadeeswaran S, Eichbichler A, Kenny HA, Montag A, Bruno K, Lengyel E
Thrombin induces tumor invasion through the induction and association of matrix metalloproteinase-9 and beta -1 integrin on the cell surface.
J Biol Chem. 2007 Nov 29; .
The procoagulatory serine protease, thrombin, is known to induce invasion and metastasis in various cancers. However, the mechanisms by which it promotes tumorigenesis are poorly understood. Since the 92 kDa gelatinase (MMP-9) is a known mediator of tumor cell invasion, we sought to determine if and how thrombin regulates MMP-9. The thrombin receptor, PAR-1 and MMP-9 are expressed in osteosarcomas, as determined by immunohistochemistry. Stimulation of U2-OS osteosarcoma cells with thrombin and a thrombin receptor activating peptide (TRAP) induced pro-MMP-9 secretion as well as cell surface associated pro-MMP-9 expression and proteolytic activity. This was paralleled by an increase in MMP-9 mRNA and MMP-9 promoter activity. Thrombin induced invasion of U2- OS cells through matrigel was mediated by the PI3-kinase signaling pathway and could be inhibited with an MMP-9 antibody. The stimulation of MMP-9 by thrombin was paralleled by an increase in ss1-integrin mRNA and ss1-integrin expression on the cell surface, which was also mediated by PI3- kinase and was required for invasion. Thrombin activation induced and co-localized both ss1-integrin and pro-MMP-9 on the cell membrane, as evidenced by co-precipitation, confocal microscopy and a protein binding assay. The thrombin mediated association of these two proteins, as well as thrombin mediated invasion of U2-OS cells, could be blocked with a cyclic peptide and with an antibody preventing binding of the MMP-9 hemopexin domain to ss1-integrin. These results suggest that thrombin induces expression and association of ss1-integrin with MMP-9, and that the cell surface localization of the protease by the integrin promotes tumor cell invasion. [Abstract/Link to Full Text]

Altman JK, Yoon P, Katsoulidis E, Krocszynska B, Sassano A, Redig AJ, Glaser H, Jordan A, Tallman MS, Hay N, Platanias LC
Regulatory effects of mTOR-mediated signals in the generation of arsenic trioxide responses.
J Biol Chem. 2007 Nov 29;
AArsenic trioxide (As2O3) is a potent inducer of apoptosis of leukemic cells in vitro and in vivo, but the mechanisms that mediate such effects are not well understood. We provide evidence that the Akt kinase is phosphorylated/ activated during treatment of leukemia cells with As2O3, to regulate downstream engagement of mTOR and its effectors. Using cells with targeted disruption of both the Akt1 and Akt2 genes, we found that induction of arsenic trioxide-dependent apoptosis is strongly enhanced in the absence of these kinases, suggesting that Akt1/Akt2 are activated in a negative feedback regulatory manner, to control generation of As2O3-responses. Consistent with this, As2O3-dependent pro-apoptotic effects are enhanced in double knockout cells for both isoforms of the p70 S6 kinase (S6k1/S6k2), a downstream effector of Akt and mTOR. On the other hand, As2O3-dependent induction of apoptosis is diminished in cells with targeted disruption of TSC2, a negative upstream effector of mTOR. In studies using primary hematopoietic progenitors from patients with AML we found that pharmacological inhibition of mTOR enhances the suppressive effects of arsenic trioxide on leukemic progenitor colony formation. Moreover, siRNA mediated inhibition of expression of the negative downstream effector, translational repressor 4E-BP1, partially reverses the effects of As2O3. Altoget-her, these data provide evidence for a key regulatory role of the Akt/mTOR pathway in the generation of the effects of As2O3, and suggest that targeting this signaling cascade may provide a novel therapeutic approach to enhance the antileukemic properties of As2O3. [Abstract/Link to Full Text]

Skinner SJ, Deleault KM, Fecteau R, Brooks SA
Extracellular signal-regulated kinase regulation of tumor necrosis factor-alpha mRNA nucleocytoplasmic transport requires TAP/NxT1 binding and the AU-Rich element.
J Biol Chem. 2007 Nov 29;
TNF-alpha production is regulated by transcriptional and post-transcriptional mechanism. LPS activates the NFkappaB pathway increasing TNF-alpha transcription. LPS also activates the MAP Kinase pathways resulting in stabilization and enhanced translation of the TNF-alpha message. In addition, nuclear export of the TNF-alpha mRNA is a regulated posttranscriptional process involving the Tpl2-ERK pathway and requiring the presence of the TNF- AU-Rich Element (ARE). We demonstrate that nuclear export of the TNF-alpha message requires not only the TNF-alpha AU-Rich Element but also the interaction of the proteins TAP and NxT1, both of which are involved in nucleo-cytoplasmic transport of mRNA. Through the use of dominant negative ERK1 and ERK2, we establish that control of TNF-alpha mRNA nuclear export operates specifically through ERK1. Finally, we examined the role of two established TNF-alpha ARE binding proteins, HuR and TTP, that shuttle between the nucleus and cytoplasm. These data demonstrate that neither TTP nor HuR is required for TNF-alpha mRNA export. It is unclear at this time if ARE binding protein(s) directly interact with the TAP/NxT1 complex or if each complex is independently targeted by ERK1. [Abstract/Link to Full Text]

Morris DP, Lei B, Wu YX, Michelotti GA, Schwinn DA
The alpha 1a-adrenergic receptor occupies membrane rafts with its G protein effectors but internalizes via clathrin coated pits.
J Biol Chem. 2007 Nov 29;
The alpha1a-adrenergic receptor (alpha1aAR) occupies intracellular and plasma membranes in both native and heterologous expression systems. Based on multiple independent lines of evidence, we demonstrate the alpha1aAR at the cell surface occupies membrane rafts but exits from rafts following stimulation. In non-detergent raft preparations, basal alpha1aAR is present in low density membrane rafts and co-localizes with its G protein effectors on density gradients. Raft disruption by cholesterol depletion with methyl-b-cyclodextrin (MCD) eliminates these light rafts. To confirm the presence of the alpha1aAR in plasma membrane rafts, FRET measurements were used to demonstrate colocalization of surface receptor and the raft marker, cholera toxin B. This colocalization was largely lost following alpha1aAR stimulation with phenylephrine. Similarly, receptor stimulation causes exit of the alpha1aAR from light rafts within 3 to 10 minutes in contrast to the G proteins, which largely remain in light rafts. Importantly, this delayed exit of the alpha1aAR suggests acute receptor signaling and desensitization occurs entirely within rafts. Interestingly, both confocal analysis and measurement of surface alpha1aAR levels indicate modest receptor internalization during the 10 minutes following stimulation, suggesting most receptor has entered non-raft plasma membrane. Nevertheless, activation does increase the rate of receptor internalization as does disruption of rafts with MCD, suggesting raft exit enables internalization. Confocal analysis of surface-labeled HA-alpha1aAR, reveals that basal and stimulated receptor occupies clathrin pits in fixed cells consistent with previous indirect evidence. The evidence presented here strongly suggests the alpha1aAR is a lipid raft protein under basal conditions and implies agonist mediated signaling occurs from rafts. [Abstract/Link to Full Text]

Mittra B, Zamudio JR, Bujnicki JM, Stepinski J, Darzynkiewicz E, Campbell DA, Sturm NR
The TbMTr1 spliced leader RNA cap 1 2'-O-ribose methyltransferase from trypanosoma brucei acts with substrate specificity.
J Biol Chem. 2007 Nov 29;
In metazoa cap 1 (m7GpppNmp-RNA) is linked to higher levels of translation, however the enzyme responsible remains unidentified. We have validated the first eukaryotic encoded cap 1 2 cent-O-ribose methyltransferase, TbMTr1, a member of a conserved family that modifies the first transcribed nucleotide of spliced leader and U1 small nuclear RNAs in the kinetoplastid protozoan Trypanosoma brucei. In addition to cap 0 (m7GpppNp-RNA), mRNA in these parasites has ribose methylations on the first four nucleotides with base methylations on the first and fourth (m7Gpppm6,6AmpAmpCmpm3Ump-SL RNA) conveyed via trans-splicing of a universal spliced leader. The function of this cap 4 is unclear. Spliced leader is the majority RNA polymerase II transcript; the RNA polymerase III-transcribed U1 small nuclear RNA has the same first four nucleotides as spliced leader, but it receives a m2,2,7G cap with hypermethylation at position one only (m2,2,7Gpppm6,6AmpApCpUp-U1 RNA). Here we examine the biochemical properties of recombinant TbMTr1. Active over a pH range of 6.0 to 9.5, TbMTr1 is sensitive to Mg2+. Positions K95-D204-K259-E285 constitute the conserved catalytic core. A guanosine cap on RNA independent of its N7 methylation status is required for substrate recognition, but an m2,2,7G-cap is not recognized. TbMTr1 favors the spliced leader 5 cent sequence, as reflected by a preference for A at position 1 and modulation of activity for substrates with base changes at positions 2 and 3. With similarities to human cap 1 methyltransferase activity, TbMTr1 is an excellent model for higher eukaryotic cap 1 methyltransferases and the consequences of cap 1 modification. [Abstract/Link to Full Text]

Li J, Chen LA, Townsend CM, Evers BM
PKD1, PKD2 and their substrate Kidins220 regulate neurotensin secretion in the BON human endocrine cell line.
J Biol Chem. 2007 Nov 29;
Neurotensin (NT) is a gut peptide that plays an important role in gastrointestinal secretion, motility, and growth as well as the proliferation of NT receptor positive cancers. Protein kinase D (PKD) family members (PKD1, 2 and 3) have been identified as important regulators of secretory transport at the trans-Golgi network (TGN). Previously, we showed that PKD1 contributes to stimulated NT secretion; however, the mechanisms are not entirely clear. Here, we show that Kidins220, which is a substrate of PKD proteins in neuroendocrine cells, is localized in the ends of the processes of BON cells, similar to the expression pattern of NT vesicles, and translocates to the membrane and large vesicle-like structures formed in response to phorbol ester (PMA) treatment. The short hairpin RNA targeting Kidins220 inhibits NT secretion in parental BON cells or BON cells stably expressing the gastrin releasing peptide (GRP) receptor treated with either PMA or bombesin, respectively. Furthermore, we demonstrate that endogenous PKD1, PKD2 and Kidins220 co-exist with NT-containing vesicles. Overexpression of the kinase dead PKD1 abrogates Kidins220 expression and NT vesicle formation. Our data establish a physiological link between the PKD/Kidins220 pathway and NT-containing vesicles and suggest the role of this pathway in the regulation of hormone secretion. Since NT is an important gut hormone which affects secretion, inflammation, and both normal and tumor cell growth, our findings identify a novel signaling pathway which may be amenable to drug targeting for clinical applications. [Abstract/Link to Full Text]

Primo ME, Klinke S, Sica MP, Goldbaum FA, Jakoncic J, Poskus E, Ermácora MR
Structure of the mature ectodomain of the human receptor-type protein-tyrosine phosphatase IA-2.
J Biol Chem. 2007 Nov 29;
IA-2 is a protein-tyrosine phosphatase receptor located in secretory granules of neuroendocrine cells. Initially, it attracted attention due to its involvement in the autoimmune response associated to diabetes. Later it was found that upon exocytosis, the cytoplasmic domain of IA-2 is cleaved and relocated to the nucleus, where it enhances the transcription of the insulin gene. A concerted functioning of the whole receptor is to be expected. However, very little is known about the structure and