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Recent Articles in Molecular Biology of the Cell

Badowski C, Pawlak G, Grichine A, Chabadel A, Oddou C, Jurdic P, Pfaff M, Albig�s-Rizo C, Block MR
Paxillin Phosphorylation Controls Invadopodia/Podosomes Spatiotemporal Organization.
Mol Biol Cell. 2007 Nov 28; .
Monitoring Editor: Mark Ginsberg In RSV-transformed BHK cells, invadopodia can self-organize into rings and belts, similarly to podosome distribution during osteoclast differentiation. The composition of individual invadopodia is spatiotemporally regulated and depends on invadopodia localization along the ring section: the actin core assembly precedes the recruitment of surrounding integrins and integrin-linked proteins while the loss of the actin core was a prerequisite to invadopodia disassembly. We have shown that invadopodia ring expansion is controlled by paxillin phosphorylations on tyrosine 31 and 118 which allows invadopodia disassembly. In BHK-RSV cells, ectopic expression of the paxillin mutant Y31F-Y118F induces a delay in invadopodia disassembly and impairs their self organization. Similar mechanism is unraveled in osteoclasts using paxillin knockdown. Lack of paxillin phosphorylation, calpain or Erk inhibition, result in similar phenotype suggesting that these proteins belong to the same regulatory pathways. Indeed, we have shown that paxillin phosphorylation promotes Erk activation that in turn activates calpain. Finally, we observed that invadopodia/podosomes ring expansion is required for efficient extracellular matrix degradation both in BHK-RSV cells and primary osteoclasts, and transmigration through a cell monolayer. [Abstract/Link to Full Text]

Casey L, Patterson EE, M�ller U, Fox CA
Conversion of a Replication Origin to a Silencer through a Pathway Shared by a Forkhead Transcription Factor and an S-Phase Cyclin.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Orna Cohen-Fix Silencing of the mating-type locus HMR in S. cerevisiae requires DNA elements called silencers. To establish HMR silencing the Origin Recognition Complex binds the HMR-E silencer and recruits the Sir1 protein. Sir1 in turn helps establish silencing by stabilizing binding of the other Sir proteins, Sir2-4. However, silencing is semistable even in sir1Delta cells, indicating that SIR1-independent establishment mechanisms exist. Furthermore, the requirement for SIR1 in silencing a sensitized version of HMR can be bypassed by high-copy expression of FKH1 (FKH1(hc)), a conserved forkhead transcription factor, or by deletion of the S-phase cyclin CLB5 (clb5Delta). FKH1(hc) caused only a modest increase in Fkh1 levels but effectively reestablished Sir2-4 chromatin at HMR as determined by Sir3-directed chromatin immunoprecipitation. In addition, FKH1(hc) prolonged the cell cycle in a manner distinct from deletion of its close paralog FKH2, and created a cell cycle phenotype more reminiscent to that caused by a clb5Delta. Unexpectedly, and in contrast to SIR1, both FKH1(hc) and clb5Delta established silencing at HMR using the replication origins, ARS1 or ARSH4, as complete substitutes for HMR-E (HMRDeltaE::ARS). HMRDeltaE::ARS1 was a robust origin in CLB5 cells. However, initiation by HMRDeltaE::ARS1 was reduced by clb5Delta or FKH1(hc), while ARS1 at its native locus was unaffected. The CLB5-sensitivity of HMRDeltaE::ARS1 did not result from formation of Sir2-4 chromatin since sir2Delta did not rescue origin firing in clb5Delta cells. These and other data supported a model in which FKH1 and CLB5 modulated Sir2-4 chromatin and late-origin firing through opposing regulation of a common pathway. [Abstract/Link to Full Text]

Granell S, Baldini G, Mohammad S, Nicolin V, Narducci P, Storrie B, Baldini G
Sequestration of Mutated {alpha}1-Antitrypsin Into Inclusion Bodies Is a Cell Protective Mechanism to Maintain Endoplasmic Reticulum Function.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Thomas Sommer A variant alpha1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers and is associated with liver disease. In the hepatocyte of individuals carrying the mutation, alpha1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or are protective to the cell. We found that in hepatoma cells, mutated alpha1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, contained several ER components, but not calnexin. Mutated alpha1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell-type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulphide isomerase, inhibited formation of IBs and lead to retention of mutated alpha1-antitrypsin in the ER. In hepatoma cells, shift of mutated alpha1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated alpha1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway. [Abstract/Link to Full Text]

Ansbach AB, Noguchi C, Klansek IW, Heidlebaugh M, Nakamura TM, Noguchi E
RFCCtf18 and the Swi1-Swi3 Complex Function in Separate and Redundant Pathways Required for the Stabilization of Replication Forks to Facilitate Sister Chromatid Cohesion in Schizosaccharomyces pombe.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Mark Solomon Sister chromatid cohesion is established during S-phase near the replication fork. However, how DNA replication is coordinated with chromosomal cohesion pathway is largely unknown. Here we report studies of fission yeast Ctf18, a subunit of the RFC(Ctf18) replication factor C complex, and Chl1, a putative DNA helicase. We show that RFC(Ctf18) is essential in the absence of the Swi1-Swi3 replication fork protection complex required for the S-phase stress response. Loss of Ctf18 leads to an increased sensitivity to S-phase stressing agents, a decreased level of Cds1 kinase activity, and accumulation of DNA damage during S-phase. Ctf18 associates with chromatin during S-phase and is required for the proper resumption of replication after fork arrest. We also show that chl1Delta is synthetically lethal with ctf18Delta and that a dosage increase of chl1(+) rescues sensitivities of swi1Delta to S-phase stressing agents, indicating that Chl1 is involved in the S-phase stress response. Finally, we demonstrate that inactivation of Ctf18, Chl1 or Swi1-Swi3 leads to defective centromere cohesion, suggesting the role of these proteins in chromosome segregation. We propose that RFC(Ctf18) and the Swi1-Swi3 complex function in separate and redundant pathways essential for replication fork stabilization to facilitate sister chromatid cohesion in fission yeast. [Abstract/Link to Full Text]

Paroni G, Cernotta N, Russo CD, Gallinari P, Pallaoro M, Foti C, Talamo F, Orsatti L, Steink�hler C, Brancolini C
PP2A Regulates HDAC4 Nuclear Import.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Karsten Weis Different signal-regulated serine/threonine kinases phosphorylate class II HDACs to promote nuclear export, cytosolic accumulation, and activation of gene transcription. However, little is known about mechanisms operating in the opposite direction, which, possibly through phosphatases, should promote class II HDACs nuclear entry and subsequent gene repression. Here we show that HDAC4 forms a complex with the PP2A holoenzyme Calpha, Aalpha, B/PR55alpha. In vitro and in vivo binding studies demonstrate that the N-terminus of HDAC4 interacts with the catalytic subunit of PP2A. HDAC4 is dephosphorylated by PP2A and experiments using okadaic acid (OA) or RNAi have revealed that PP2A controls HDAC4 nuclear import. Moreover, we identified serine 298 as a putative phosphorylation site important for HDAC4 nuclear import. The HDAC4 mutant mimicking phosphorylation of serine 298 is defective in nuclear import. Mutation of serine 298 to alanine partially rescues the defect in HDAC4 nuclear import observed in cells with down-regulated PP2A. These observations suggest that PP2A, via the dephosphorylation of multiple serines including the 14-3-3 binding sites and serine 298, controls HDAC4 nuclear import. [Abstract/Link to Full Text]

Roth L, Nasarre C, Dirrig-Grosch S, Aunis D, Cr�mel G, Hubert P, Bagnard D
Transmembrane Domain Interactions Control Biological Functions Neuropilin-1.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Carl-Henrik Heldin Neuropilin-1 (NRP1) is a transmembrane receptor playing a pivotal role in the control of semaphorins and VEGF signaling pathways. The exact mechanism controlling semaphorin receptor complex formation is unknown. A structural analysis and modeling of NRP1 revealed a putative dimerization GxxxG motif potentially important for NRP1 dimerization and oligomerization. Our data show that this motif mediates the dimerization of the transmembrane domain of NRP1 as demonstrated by a dimerization assay (ToxLuc assay) performed in natural membrane and FRET analysis. A synthetic peptide derived from the transmembrane segment of NRP1 abolished the inhibitory effect of Sema3A. This effect depends on the capacity of the peptide to interfere with NRP1 dimerization and the formation of oligomeric complexes. Mutation of the GxxxG dimerization motif in the transmembrane domain of NRP1 confirmed its biological importance for Sema3A signaling. Overall, our results shed light on an essential step required for semaphorin signaling and provide novel evidence for the crucial role of transmembrane domain of bitopic protein containing GxxxG motif in the formation of receptor complexes that are a prerequisite for cell signaling. [Abstract/Link to Full Text]

Peng Y, Liu X, Schoenberg DR
Hsp90 Stabilizes the PMR1 mRNA Endonuclease to Degradation by the 26S Proteasome.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: A. Gregory Matera The PMR1 mRNA endonuclease forms a selective complex with its translating substrate mRNAs where it is activated to initiate mRNA decay. Previous work showed tyrosine phosphorylation is required for PMR1 targeting to this polysome-bound complex, and identified c-Src as the responsible kinase. c-Src phosphorylation occurs in a distinct complex, and the current study shows that Hsp90 is also recovered with PMR1 and c-Src. Hsp90 binding to PMR1 is inhibited by geldanamycin, and geldanamycin stabilizes substrate mRNA to PMR1-mediated decay. PMR1 is inherently unstable and geldanamycin causes PMR1 to rapidly disappear in a process that is catalyzed by the 26S proteasome. We present a model where Hsp90 interacts transiently to stabilize PMR1 in a manner similar to its interaction with c-Src, thus facilitating the tyrosine phosphorylation and targeting of PMR1 to polysomes. [Abstract/Link to Full Text]

Kodani A, S�tterlin C
The Golgi Protein GM130 Regulates Centrosome Morphology and Function.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Vivek Malhotra The Golgi apparatus (GA) of mammalian cells is positioned in the vicinity of the centrosome, the major microtubule organizing center of the cell. The significance of this physical proximity for organelle function and cell cycle progression is only beginning to being understood. We have identified a novel function for the GA protein, GM130, in the regulation of centrosome morphology, position and function during interphase. RNAi-mediated depletion of GM130 from five human cell lines revealed abnormal interphase centrosomes that were mispositioned and defective with respect to microtubule organization and cell migration. When GM130-depleted cells entered mitosis, they formed multipolar spindles, arrested in metaphase and died. We also detected aberrant centrosomes during interphase and multipolar spindles during mitosis in ldlG cells, which do not contain detectable GM130. While GA proteins have been described to regulate mitotic centrosomes and spindle formation, this is the first report of a role for a GA protein in the regulation of centrosomes during interphase. [Abstract/Link to Full Text]

Cammarato A, Dambacher CM, Knowles AF, Kronert WA, Bodmer R, Ocorr K, Bernstein SI
Myosin Transducer Mutations Differentially Affect Motor Function, Myofibril Structure, and the Performance of Skeletal and Cardiac Muscles.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Thomas Pollard Striated muscle myosin is a multi-domain ATP-dependent molecular motor. Alterations to various domains affect the motor's chemomechanical properties and are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here we helped define the transducer's role by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc(5) affect myosin function as well as skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility while Mhc(5) (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc(5) myosin function allows normal skeletal myofibril assembly, but induces degradation of the myofibrillar apparatus, likely as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc(5) mutations illustrate the transducer's role in influencing myosin's chemomechanical properties and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders. [Abstract/Link to Full Text]

Klein RM, Spofford LS, Abel EV, Ortiz A, Aplin AE
B-RAF Regulation of Rnd3 Participates in Actin Cytoskeletal and Focal Adhesion Organization.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Jean Schwarzbauer The actin cytoskeleton controls multiple cellular functions including cell morphology, movement and growth. Accumulating evidence indicates that oncogenic activation of the MEK/ERK1/2 pathway is accompanied by actin cytoskeletal reorganization. The signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation, however, are largely unknown. Mutant B-RAF is found in a wide variety of cancers including melanoma, and enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with siRNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/ROCK/LIM kinase-2 signaling pathway cumulating in the inactivation of the actin depolymerizing/severing protein, cofilin. The expression of Rnd3, a Rho antagonist, was attenuated following B-RAF knockdown or MEK inhibition, but was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Taken together our results identify Rnd3 as a regulator of cross-talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions. [Abstract/Link to Full Text]

Cheeseman IM, Hori T, Fukagawa T, Desai A
KNL1 and the CENP-H/I/K Complex Coordinately Direct Kinetochore Assembly in Vertebrates.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Kerry Bloom Chromosome segregation during mitosis requires the assembly of a large proteinaceous structure termed the kinetochore. In Caenorhabditis elegans, KNL-1 is required to target multiple outer kinetochore proteins. Here, we demonstrate that the vertebrate KNL1 counterpart is essential for chromosome segregation and is required to localize a subset of outer kinetochore proteins. However, unlike in C. elegans, depletion of vertebrate KNL1 does not abolish kinetochore localization of the microtubule-binding Ndc80 complex. Instead, we show that KNL1 and CENP-K, a subunit of a constitutively centromere-associated complex that is missing from C. elegans, coordinately direct Ndc80 complex localization. Simultaneously reducing both hKNL1 and CENP-K function abolishes all aspects of kinetochore assembly downstream of centromeric chromatin and causes catastrophic chromosome segregation defects. These findings explain discrepancies in kinetochore assembly pathways between different organisms and reveal a surprising plasticity in the assembly mechanism of an essential cell division organelle. [Abstract/Link to Full Text]

Blish KR, Wang W, Willingham MC, Du W, Birse CE, Krishnan SR, Brown JC, Hawkins GA, Garvin AJ, D'Agostino RB, Torti FM, Torti SV
A Human Bone Morphogenetic Protein Antagonist is Down-Regulated in Renal Cancer.
Mol Biol Cell. 2007 Nov 21;
Monitoring Editor: Carl-Henrik Heldin We analyzed expression of candidate genes encoding cell surface or secreted proteins in normal kidney and kidney cancer. This screen identified a BMP antagonist, SOSTDC1 (SclerOSTin Domain-Containing-1) as down-regulated in kidney tumors. To confirm screening results, we probed cDNA dot blots with SOSTDC1. SOSTDC1 message was decreased in 20/20 kidney tumors compared with normal kidney tissue. Immunohistochemistry confirmed significant decrease of SOSTDC1 protein in clear cell renal carcinomas relative to normal proximal renal tubule cells (p <0.001). Expression of SOSTDC1 was not decreased in papillary and chromophobe kidney tumors. SOSTDC1 was abundantly expressed in podocytes, distal tubules, and transitional epithelia of the normal kidney. Transfection experiments demonstrated that SOSTDC1 is secreted and binds to neighboring cells and/or the extracellular matrix. SOSTDC1 suppresses both BMP-7-induced phosphorylation of R-Smads-1, -5 and -8 and Wnt-3a signaling. Restoration of SOSTDC1 in renal clear carcinoma cells profoundly suppresses proliferation. Collectively, these results demonstrate that SOSTDC1 is expressed in the human kidney and decreased in renal clear cell carcinoma. Because SOSTDC1 suppresses proliferation of renal carcinoma cells, restoration of SOSTDC1 signaling may represent a novel target in treatment of renal clear cell carcinoma. [Abstract/Link to Full Text]

Galvan C, Camoletto PG, Cristofani F, Van Veldhoven PP, Ledesma MD
Anomalous Surface Distribution of GPI-anchored Proteins in Neurons Lacking Acid Sphingomyelinase.
Mol Biol Cell. 2007 Nov 21;
Monitoring Editor: Sean Munro Acid sphingomyelinase (ASM) converts sphingomyelin (SM) into ceramide. Mutations in the ASM gene cause the mental retardation syndrome Niemann Pick type A (NPA) characterized as a lysosomal disorder because of the SM accumulation in these organelles. We here report that neurons from mice lacking ASM (ASMKO) present increased plasma membrane SM levels evident in detergent resistant membranes. Paralleling this lipidic alteration, GPI-anchored proteins show an aberrant distribution in both axons and dendrites instead of the axonal enrichment observed in neurons from wild type mice. Trafficking analysis suggests that this is due to defective internalization from dendrites. Increasing the SM content in wild type neurons mimics these defects while SM reduction in ASMKO neurons prevents their occurrence. Moreover, expression of active RhoA, which membrane attachment is affected by SM accumulation, rescues internalization rates in ASMKO neurons. These data unveil an unexpected role for ASM in neuronal plasma membrane organization and trafficking providing insight on the molecular mechanisms involved. They also suggest that deficiencies in such processes could be key pathological events in NPA disease. Keywords: sphingomyelin; lipid microdomains; Niemann Pick type A; GPI-anchored proteins; endocytosis. [Abstract/Link to Full Text]

Oganesian A, Armstrong LC, Migliorini MM, Strickland DK, Bornstein P
Thrombospondins Use the VLDL Receptor and a Non-Apoptotic Pathway to Inhibit Cell Division in Microvascular Endothelial Cells.
Mol Biol Cell. 2007 Nov 21;
Monitoring Editor: Josephine Adams TSPs 1 and 2 function as endogenous inhibitors of angiogenesis. Although TSPs have been shown to induce apoptosis in HMVEC, we reasoned that a homeostatic mechanism would also be needed to inhibit EC growth without causing cell death, e.g., in the maintenance of a normal vascular endothelium. HMVEC, cultured in low serum, responded to VEGF with an increase in [(3)H]thymidine incorporation that was inhibited by TSPs and was accompanied by decreases in the phosphorylation of Akt and MAPK, without an increase in apoptosis. RAP, an inhibitor of the LDL family of endocytic receptors, and blocking antibodies to VLDLR, were as effective as TSPs in the inhibition of thymidine uptake in response to VEGF, and the effects of these agents were not additive. Supportive evidence for the role of the VLDLR in mediating this inhibition was provided by the demonstration of a high-affinity interaction between TSPs and the VLDLR. We propose that TSP1 and TSP2, together with the VLDLR, initiate a nonapoptotic pathway for maintenance of the normal adult vascular endothelium in a quiescent state, similar to that invoked for the regulation of mitogenesis by PDGF, but involving signaling via the VLDLR rather than LRP1. [Abstract/Link to Full Text]

Rue SM, Mattei S, Saksena S, Emr SD
Novel Ist1-Did2 Complex Functions at a Late Step in MVB Sorting.
Mol Biol Cell. 2007 Nov 21;
Monitoring Editor: Sean Munro In S. cerevisiae, integral plasma membrane proteins destined for degradation and certain vacuolar membrane proteins are sorted into the lumen of the vacuole via the multivesicular body (MVB) sorting pathway, which depends on the sequential action of three endosomal sorting complexes required for transport (ESCRTs). Here, we report the characterization of a new positive modulator of MVB sorting, Ist1. We show that endosomal recruitment of Ist1 depends on ESCRT-III. Deletion of IST1 alone does not cause cargo sorting defects. However, synthetic genetic analysis of double mutants of IST1 and positive modulators of MVB sorting showed that ist1Delta is synthetic with vta1Delta and vps60Delta, indicating that Ist1 is also a positive component of the MVB sorting pathway. Moreover, this approach revealed that Ist1-Did2 and Vta1-Vps60 compose two functional units. Ist1-Did2 and Vta1-Vps60 form specific physical complexes, and, like Did2 and Vta1, Ist1 binds to the AAA-ATPase Vps4. We provide evidence that the ist1Delta mutation exhibits a synthetic interaction with mutations in VPS2 (DID4) that compromise the Vps2-Vps4 interaction. We propose a model in which the Ist1-Did2 and Vta1-Vps60 complexes independently modulate late steps in the MVB sorting pathway. [Abstract/Link to Full Text]

Froget B, Blaisonneau J, Lambert S, Baldacci G
Cleavage of Stalled Forks by Fission Yeast Mus81/Eme1 in Absence of DNA Replication Checkpoint.
Mol Biol Cell. 2007 Nov 21;
Monitoring Editor: Wendy Bickmore During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed. [Abstract/Link to Full Text]

Dimaano C, Jones CB, Hanono A, Curtiss M, Babst M
Ist1 Regulates Vps4 Localization and Assembly.
Mol Biol Cell. 2007 Nov 21;
Monitoring Editor: Sandra Lemmon The ESCRT protein complexes are recruited from the cytoplasm and assemble on the endosomal membrane into a protein network that functions in sorting of ubiquitinated transmembrane proteins into the multivesicular body (MVB) pathway. This transport pathway packages cargo proteins into vesicles that bud from the MVB limiting membrane into the lumen of the compartment and delivers these vesicles to the lysosome/vacuole for degradation. The dissociation of ESCRT machinery by the AAA-type ATPase Vps4 is a necessary late step in the formation of MVB vesicles. This ATP-consuming step is regulated by several Vps4-interacting proteins, including the newly identified regulator Ist1. Our data suggest that Ist1 has a dual role in the regulation of Vps4 activity: it localizes to the ESCRT machinery via Did2 where it positively regulates recruitment of Vps4 and it negatively regulates Vps4 by forming an Ist1-Vps4 heterodimer, in which Vps4 cannot bind to the ESCRT machinery. The activity of the MVB pathway might be in part determined by outcome of these two competing activities. [Abstract/Link to Full Text]

Lam AD, Tryoen-Toth P, Tsai B, Vitale N, Stuenkel EL
SNARE-catalyzed Fusion Events Are Regulated by Syntaxin1A Lipid Interactions.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Patrick Brennwald Membrane fusion is a process that intimately involves both proteins and lipids. While the SNARE proteins, which ultimately overcome the energy barrier for fusion, have been extensively studied, regulation of the energy barrier itself, determined by specific membrane lipids, has been largely overlooked. Our findings reveal a novel function for SNARE proteins in reducing the energy barrier for fusion, by directly binding and sequestering fusogenic lipids to sites of fusion. We demonstrate a specific interaction between Syntaxin1A and the fusogenic lipid phosphatidic acid, in addition to multiple polyphosphoinositide lipids, and define a polybasic juxtamembrane region within Syntaxin1A as its lipid binding domain. In PC-12 cells, Syntaxin1A mutations that progressively reduced lipid binding resulted in a progressive reduction in evoked secretion. Moreover, amperometric analysis of fusion events driven by a lipid binding-deficient Syntaxin1A mutant (5RK/A) demonstrated alterations in fusion pore dynamics suggestive of an energetic defect in secretion. Overexpression of the phosphatidic acid-generating enzyme, phospholipase D1, completely rescued the secretory defect seen with the 5RK/A mutant. Moreover, knockdown of phospholipase D1 activity drastically reduced control secretion, while leaving 5RK/A-mediated secretion relatively unaffected. Altogether, these data suggest that Syntaxin1A-lipid interactions are a critical determinant of the energetics of SNARE-catalyzed fusion events. [Abstract/Link to Full Text]

Kim J, Shao Y, Kim SY, Kim S, Song HK, Jeon JH, Woo Suh H, Chung JW, Yoon SR, Kim YS, Choi I
Hypoxia-induced IL-18 Increases Hypoxia-inducible Factor-1{alpha} Expression through a Rac1-dependent NF-{kappa}B Pathway.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: John Cleveland Interleukin-18 (IL-18) plays pivotal roles in linking inflammatory immune responses and tumor progression and metastasis, yet the manner in which this occurs remains to be sufficiently clarified. Here we report that hypoxia induces the transcription and secretion of IL-18, which subsequently induces the expression of hypoxia-inducible factor-1alpha (HIF-1alpha). Mechanistically, IL-18 induces HIF-1alpha through the activity of the GTPase Rac1, which inducibly associates with the IL-18 receptor beta (IL-18Rbeta) subunit, via a PI3K-AKT-NF-kappaB-dependent pathway. Importantly, the knockdown of the IL-18Rbeta subunit inhibited IL-18-driven tumor cell metastasis. Collectively, these findings demonstrate a feed-forward pathway in HIF-1alpha-mediated tumor progression, in which the induction of IL-18 by hypoxia or inflammatory cells augments the expression of both HIF-1alpha and tumor cell metastasis." [Abstract/Link to Full Text]

Manolea F, Claude A, Chun J, Rosas J, Melan�on P
Distinct Functions for Arf Nucleotide Exchange Factors at the Golgi Complex: GBF1 and BIGs Are Required for Assembly and Maintenance of the Golgi Stack and TGN, Respectively.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Benjamin Glick We examined the relative function of the two classes of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factors that regulate recruitment of coat proteins on the Golgi complex. Complementary overexpression and RNA-based knockdown approaches established that GBF1 regulates COPI recruitment on cis-Golgi compartments, while BIGs appear specialized for adaptor proteins on the trans-Golgi. Knockdown of GBF1 and/or COPI did not prevent export of VSVGtsO45 from the ER, but caused its accumulation into peripheral vesiculo-tubular clusters. In contrast, knockdown of BIG1 and BIG2 caused loss of clathrin adaptor proteins and redistribution of several TGN markers, but had no impact on COPI and several Golgi markers. Surprisingly, BIGs knockdown prevented neither traffic of VSVGtsO45 to the plasma membrane nor assembly of a polarized Golgi stack. Our observations indicate that COPII is the only coat required for sorting and export from the ER exit sites, while GBF1 but not BIGs, is required for COPI recruitment, Golgi subcompartmentalization and cargo progression to the cell surface. [Abstract/Link to Full Text]

Avezov E, Frenkel Z, Ehrlich M, Herscovics A, Lederkremer GZ
ER Mannosidase I Is Compartmentalized and Required for N-Glycan Trimming to Man5 6GlcNAc2 in Glycoprotein ER-associated Degradation.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Reid Gilmorez We had previously shown that ER-associated degradation (ERAD) of glycoproteins in mammalian cells involves trimming of 3-4 mannose residues from the N-linked oligosaccharide Man9GlcNAc2. A possible candidate for this activity, ER mannosidase I (ERManI), accelerates the degradation of ERAD substrates when overexpressed. Although in vitro, at low concentrations, ERManI removes only one specific mannose residue, at very high concentrations it can excise up to 4 alpha1,2-linked mannose residues. Using siRNA knock-down of ERManI, we show that this enzyme is required for trimming to Man5-6GlcNAc2 and for ERAD in cells in vivo, leading to the accumulation of Man9GlcNAc2 and Glc1Man9GlcNAc2 on a model substrate. Thus, trimming by ERManI to the smaller oligosaccharides would remove the glycoprotein from reglucosylation and calnexin binding cycles. ERManI appears strikingly concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC) that we had described previously. ERManI knock-down prevents substrate accumulation in the ERQC. We suggest that the ERQC provides a high local concentration of ERManI, and passage through this compartment would allow timing of ERAD, possibly through a cycling mechanism. When newly-made glycoproteins cannot fold properly, transport through the ERQC leads to trimming of a critical number of mannose residues, triggering a signal for degradation. [Abstract/Link to Full Text]

Takeshita N, Higashitsuji Y, Konzack S, Fischer R
Apical Sterol-rich Membranes Are Essential for Localizing Cell End Markers that Determine Growth Directionality in the Filamentous Fungus Aspergillus nidulans.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: David Drubin In filamentous fungi hyphal extension depends on the continuous delivery of vesicles to the growing tip. Here, we describe the identification of two cell-end marker proteins, TeaA and TeaR, in Aspergillus nidulans, corresponding to Tea1 and Mod5 in Schizosaccharomyces pombe. Deletion of teaA or teaR caused zig-zag-growing and meandering hyphae, respectively. The Kelch-repeat protein TeaA, the putatively prenylated TeaR protein, and the formin SepA were highly concentrated in the Spitzenk�rper, a vesicle transit station at the tip, and localized along the tip membrane. TeaA localization at tips depended on microtubules and TeaA was required for microtuble convergence in the hyphal apex. The CENP-E family kinesin KipA was necessary for proper localization of TeaA and TeaR, but not for their transportation. TeaA and TeaR localization were interdependent. TeaA interacted in vivo with TeaR, and TeaA colocalized with SepA. Sterol-rich membrane domains localized at the tip in teaA and teaR mutants like in wild type, and filipin treatment caused mislocalization of both proteins. This suggests that sterol-rich membrane domains determine cell-end factor destinations and thereby polarized growth. [Abstract/Link to Full Text]

Dickinson BL, Claypool SM, D'Angelo JA, Aiken ML, Venu N, Yen EH, Wagner JS, Borawski JA, Pierce AT, Hershberg R, Blumberg RS, Lencer WI
Ca2+-dependent Calmodulin-binding to FcRn Affects IgG Transport in the Transcytotic Pathway.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Keith Mostov The Fcgamma receptor FcRn transports IgG so as to avoid lysosomal degradation and to carry it bidirectionally across epithelial barriers to affect mucosal immunity. Here we identify a calmodulin-binding site within the FcRn cytoplasmic tail that affects FcRn trafficking. Calmodulin binding to the FcRn tail is direct, calcium-dependent, reversible, and specific to residues comprising a putative short amphipathic alpha-helix immediately adjacent to the membrane. FcRn mutants with single residue substitutions in this motif, or FcRn mutants lacking the cytoplasmic tail completely, exhibit a shorter half-life and attenuated transcytosis. Chemical inhibitors of calmodulin phenocopy the mutant FcRn defect in transcytosis. These results suggest a novel mechanism for regulation of IgG transport by calmodulin-dependent sorting of FcRn and its cargo away from a degradative pathway and into a bidirectional transcytotic route. [Abstract/Link to Full Text]

Chen D, Wilkinson CR, Watt S, Penkett CJ, Toone WM, Jones N, B�hler J
Multiple Pathways Differentially Regulate Global Oxidative Stress Responses in Fission Yeast.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Jonathan Weissman Cellular protection against oxidative damage is relevant to ageing and numerous diseases. We analyzed the diversity of genome-wide gene expression programs and their regulation in response to various types and doses of oxidants in Schizosaccharomyces pombe. A small core gene set, regulated by the AP-1-like factor Pap1p and the two-component regulator Prr1p, was universally induced irrespective of oxidant and dose. Strong oxidative stresses led to a much larger transcriptional response. The mitogen-activated protein kinase (MAPK) Sty1p and the bZIP factor Atf1p were critical for the response to hydrogen peroxide. A newly identified zinc-finger protein, Hsr1p, is uniquely regulated by all three major regulatory systems (Sty1p-Atf1p, Pap1p, and Prr1p) and in turn globally supports gene expression in response to hydrogen peroxide. Although the overall transcriptional responses to hydrogen peroxide and t-butylhydroperoxide were similar, to our surprise, Sty1p and Atf1p were less critical for the response to the latter. Instead, another MAPK, Pmk1p, was involved in surviving this stress, although Pmk1p played only a minor role in regulating the transcriptional response. These data reveal a considerable plasticity and differential control of regulatory pathways in distinct oxidative stress conditions, providing both specificity and backup for protection from oxidative damage. [Abstract/Link to Full Text]

D'Silva PR, Schilke B, Hayashi M, Craig EA
Interaction of the J-Protein Heterodimer, Pam18/Pam16, of the Mitochondrial Import Motor with the Translocon of the Inner Membrane.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Jeffrey Brodsky Import of proteins across the inner mitochondrial membrane through the Tim23:Tim17 translocase requires the function of an essential import motor having mitochondrial Hsp70 (mtHsp70) at its core. The heterodimer composed of Pam18, the J-protein partner of mtHsp70, and the related protein Pam16 is a critical component of this motor. We report that three interactions contribute to association of the heterodimer with the translocon: the N-terminus of Pam16 with the matrix side of the translocon, the inner membrane space domain of Pam18 (Pam18IMS) with Tim17, and the direct interaction of Pam18's J-domain with Pam16's J-like domain. Pam16 plays a major role in translocon association, as alterations affecting the stability of the Pam18:Pam16 heterodimer dramatically affect association of Pam18, but not Pam16, with the translocon. Suppressors of the growth defects caused by alterations in Pam16's N-terminus were isolated and found to be due to mutations in a short segment of TIM44, the gene encoding the peripheral membrane protein that tethers mtHsp70 to the translocon. These data suggest a model in which Tim44 serves as a scaffold for precise positioning of mtHsp70 and its cochaperone Pam18 at the translocon. [Abstract/Link to Full Text]

Wiese C
Distinct Dgrip84 Isoforms Correlate with Distinct {gamma}-Tubulins in Drosophila.
Mol Biol Cell. 2007 Nov 15;
Monitoring Editor: Sandra Schmid gamma-Tubulin is an indispensable component of the animal centrosome and is required for proper microtubule organization. Within the cell, gamma-tubulin exists in a multi-protein complex containing between two (some yeasts) and six or more (metazoa) additional highly conserved proteins named gamma ring proteins (Grips) or gamma complex proteins (GCPs). gamma-Tubulin containing complexes isolated from Xenopus eggs or Drosophila embryos appear ring-shaped and have therefore been named the gamma-tubulin ring complex (gammaTuRC). Curiously, many organisms (including humans) have two distinct gamma-tubulin genes. In Drosophila, where the two gamma-tubulin isotypes have been studied most extensively, the gamma-tubulin genes are developmentally regulated: the 'maternal' gamma-tubulin isotype (named gammaTub37CD according to its location on the genetic map) is expressed in the ovary and is deposited in the egg, where it is thought to orchestrate the meiotic and early embryonic cleavages. The second gamma-tubulin isotype (gammaTub23C) is ubiquitously expressed and persists in most of the cells of the adult fly. In those rare cases where both gamma-tubulins coexist in the same cell, they show distinct subcellular distributions and cell-cycle-dependent changes (Raynaud-Messina et al., 2001. Eur. J. Cell Biol. 80, 643-649): gammaTub37CD mainly localizes to the centrosome, where its levels vary only slightly with the cell cycle. In contrast, the level of gammaTub23C at the centrosome increases at the beginning of mitosis, and gammaTub23C also associates with spindle pole microtubules. Here, we show that gammaTub23C forms discrete complexes that closely resemble the complexes formed by gammaTub37CD. Surprisingly, however, gammaTub23C associates with a distinct, longer splice variant of Dgrip84. This may reflect a role for Dgrip84 in regulating the activity and/or the location of the gamma-tubulin complexes formed with gammaTub37CD and gammaTub23C. [Abstract/Link to Full Text]

Abdi KM, Bennett V
Adducin Promotes Micron-Scale Organization of {beta}2-Spectrin in Lateral Membranes of Bronchial Epithelial Cells.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Ben Margolis Adducin promotes assembly of spectrin-actin complexes, and is a target for regulation by calmodulin, protein kinase C and rho kinase. We demonstrate here that adducin is required to stabilize preformed lateral membranes of human bronchial epithelial (HBE) cells through interaction with beta2-spectrin. We use a Tet-on regulated inducible siRNA system to deplete alpha-adducin from confluent HBE cells. Depletion of alpha-adducin resulted in increased detergent solubility of spectrin following normal membrane biogenesis during mitosis. Conversely, depletion of beta2-spectrin resulted in loss of adducin from the lateral membrane. siRNA-resistant alpha-adducin prevented loss of lateral membrane, but only if alpha-adducin retained the MARCKS domain that mediates spectrin-actin interactions. Phospho-mimetic versions of adducin with S/D substitutions at PKC phosphorylation sites in the MARCKS domain were not active in rescue. We find that adducin modulates long-range organization of the lateral membrane based on several criteria. First, the lateral membrane of adducin-depleted cells exhibited reduced height, increased curvature, expansion into the basal surface. Moreover, E-cadherin-GFP, which normally is restricted in lateral mobility, rapidly diffuses over distances up to 10 microns. We conclude that adducin acting through spectrin provides a novel mechanism to regulate global properties of the lateral membrane of bronchial epithelial cells. [Abstract/Link to Full Text]

Barkus RV, Klyachko O, Horiuchi D, Dickson BJ, Saxton WM
Identification of an Axonal Kinesin-3 Motor for Fast Anterograde Vesicle Transport that Facilitates Retrograde Transport of Neuropeptides.
Mol Biol Cell. 2007 Nov 7;
Monitoring Editor: Adam Linstedt A screen for genes required in Drosophila eye development identified a UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic anterograde axonal motor, kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of GFP-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to anterograde transport of mitochondria, which are likely transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism. [Abstract/Link to Full Text]

Liu P, Lu J, Cardoso WV, Vaziri C
The SPARC-related Factor SMOC-2 Promotes Growth Factor-induced Cyclin D1 Expression and DNA Synthesis via Integrin-linked Kinase (ILK).
Mol Biol Cell. 2007 Nov 7;
Monitoring Editor: Jean Schwarzbauer Secreted Modular Calcium-binding Protein-2 (SMOC-2) is a recently-identified SPARC-related protein of unknown function. In mRNA profiling experiments we found that SMOC-2 expression was elevated in quiescent (G0) mouse fibroblasts and repressed following mitogenic stimulation with serum. The G0-specific expression of SMOC-2 was similar to that of PDGFbetaR, a major mitogenic receptor. Therefore, we tested a possible role for SMOC-2 in growth factor-induced cell cycle progression. SMOC-2 overexpression augmented DNA synthesis induced by serum and fibroblast mitogens (including PDGF-BB and bFGF). Conversely, SMOC-2 ablation using siRNA attenuated DNA synthesis in response to PDGF-BB and other growth factors. Mitogen-induced expression of cyclin D1 was attenuated in SMOC-2-ablated cells and cyclin D1-overexpressing cells were resistant to inhibition of mitogenesis following SMOC-2 ablation. Therefore, cyclin D1 is limiting for G1 progression in SMOC-2-deficient cells. SMOC-2 ablation did not inhibit PDGF-induced PDGFbetaR autophosphorylation or PDGF-BB-dependent activation of MAPK and Akt kinases, suggesting that SMOC-2 is dispensable for growth factor receptor activation. However, Integrin-Linked Kinase (ILK) activity was reduced in SMOC-2-ablated cells. Ectopic expression of hyperactive ILK corrected the defective mitogenic response of SMOC-2-deficient cells. Therefore, SMOC-2 contributes to cell cycle progression by maintaining ILK activity during G1. These results identify a novel role for SMOC-2 in cell cycle control. [Abstract/Link to Full Text]

Jin R, Dobry CJ, McCown PJ, Kumar A
Large-Scale Analysis of Yeast Filamentous Growth by Systematic Gene Disruption and Overexpression.
Mol Biol Cell. 2007 Nov 7;
Monitoring Editor: Charles Boone Under certain conditions of nutrient stress, the budding yeast S. cerevisiae initiates a striking developmental transition to a filamentous form of growth, resembling developmental transitions required for virulence in closely related pathogenic fungi. In yeast, filamentous growth involves known MAPK and PKA signaling modules, but the full scope of this extensive filamentous response has not been delineated. Accordingly, we have undertaken the first systematic gene disruption and overexpression analysis of yeast filamentous growth. Standard laboratory strains of yeast are nonfilamentous; thus, we constructed a unique set of reagents in the filamentous Sigma1278b strain, encompassing 3627 integrated transposon insertion alleles and 2043 overexpression constructs. Collectively, we analyzed 4528 yeast genes with these reagents and identified 487 genes conferring mutant filamentous phenotypes upon transposon insertion and/or gene overexpression. Using a fluorescent protein reporter integrated at the MUC1 locus, we further assayed each filamentous growth mutant for aberrant protein levels of the key flocculence factor Muc1p. Our results indicate a variety of genes and pathways affecting filamentous growth. In total, this filamentous growth gene set represents a wealth of yeast biology, highlighting 84 genes of uncharacterized function and an underappreciated role for the mitochondrial retrograde signaling pathway as an inhibitor of filamentous growth. [Abstract/Link to Full Text]

Recent Articles in Molecular and Cellular Biology

Chan MC, Nguyen PH, Davis BN, Ohoka N, Hayashi H, Du K, Lagna G, Hata A
A novel regulatory mechanism of the bone morphogenetic protein (BMP) signaling pathway involving the carboxyl-terminal tail domain of BMP type II receptor.
Mol Cell Biol. 2007 Aug;27(16):5776-89.
Bone morphogenetic protein (BMP) signaling regulates many different biological processes, including cell growth, differentiation, and embryogenesis. BMPs bind to heterogeneous complexes of transmembrane serine/threonine (Ser/Thr) kinase receptors known as the BMP type I and II receptors (BMPRI and BMPRII). BMPRII phosphorylates and activates the BMPRI kinase, which in turn activates the Smad proteins. The cytoplasmic region of BMPRII contains a "tail" domain (BMPRII-TD) with no enzymatic activity or known regulatory function. The discovery of mutations associated with idiopathic pulmonary artery hypertension mapping to BMPRII-TD underscores its importance. Here, we report that Tribbles-like protein 3 (Trb3) is a novel BMPRII-TD-interacting protein. Upon BMP stimulation, Trb3 dissociates from BMPRII-TD and triggers degradation of Smad ubiquitin regulatory factor 1 (Smurf1), which results in the stabilization of BMP receptor-regulated Smads and potentiation of the Smad pathway. Downregulation of Trb3 inhibits BMP-mediated cellular responses, including osteoblast differentiation of C2C12 cells and maintenance of the smooth muscle phenotype of pulmonary artery smooth muscle cells. Thus, Trb3 is a critical component of a novel mechanism for regulation of the BMP pathway by BMPRII. [Abstract/Link to Full Text]

Pfaff KL, Straub CT, Chiang K, Bear DM, Zhou Y, Zon LI
The zebra fish cassiopeia mutant reveals that SIL is required for mitotic spindle organization.
Mol Cell Biol. 2007 Aug;27(16):5887-97.
A critical step in cell division is formation of the mitotic spindle, which is a bipolar array of microtubules that mediates chromosome separation. Here, we report that the SCL-interrupting locus (SIL), a vertebrate-specific cytosolic protein, is necessary for proper mitotic spindle organization in zebrafish and human cells. A homozygous lethal zebrafish mutant, cassiopeia (csp), was identified by a genetic screen for mitotic mutant. csp mutant embryos have an increased mitotic index, have highly disorganized mitotic spindles, and often lack one or both centrosomes. These phenotypes are caused by a loss-of-function mutation in zebrafish sil. To determine if the requirement for SIL in mitotic spindle organization is conserved in mammals, we generated an antibody against human SIL, which revealed that SIL localizes to the poles of the mitotic spindle during metaphase. Furthermore, short hairpin RNA knockdown of SIL in human cells recapitulates the zebrafish csp mitotic spindle defects. These data, taken together, identify SIL as a novel, vertebrate-specific regulator of mitotic spindle assembly. [Abstract/Link to Full Text]

Jones NP, Katan M
Role of phospholipase Cgamma1 in cell spreading requires association with a beta-Pix/GIT1-containing complex, leading to activation of Cdc42 and Rac1.
Mol Cell Biol. 2007 Aug;27(16):5790-805.
The significance of multiprotein signaling complexes in cell motility is becoming increasingly important. We have previously shown that phospholipase Cgamma1 (PLCgamma1) is critical for integrin-mediated cell spreading and motility (N. Jones et al., J. Cell Sci. 118:2695-2706, 2005). In the current study we show that, on a basement membrane-type matrix, PLCgamma1 associates with the adaptor protein GIT1 and the Rac1/Cdc42 guanine exchange factor beta-Pix; GIT1 and beta-Pix form tight complexes independently of PLCgamma1. The association of PLCgamma1 with the complex requires both GIT1 and beta-Pix and the specific array region (gammaSA) of PLCgamma1. Mutations of PLCgamma1 within the gammaSA region reveal that association with this complex is essential for the phosphorylation of PLCgamma1 and the progression to an elongated morphology after integrin engagement. Short interfering RNA (siRNA) depletion of either beta-Pix or GIT1 inhibited cell spreading in a fashion similar to that seen with siRNA against PLCgamma1. Furthermore, siRNA depletion of PLCgamma1, beta-Pix, or GIT1 inhibited Cdc42 and Rac1 activation, while constitutively active forms of Cdc42 or Rac1, but not RhoA, were able to rescue the elongation of these cells. Signaling of the PLCgamma1/GIT1/beta-Pix complex to Cdc42/Rac1 was found to involve the activation of calpains, calcium-dependent proteases. Therefore, we propose that the association of PLCgamma1 with complexes containing GIT1 and beta-Pix is essential for its role in integrin-mediated cell spreading and motility. As a component of this complex, PLCgamma1 is also involved in the activation of Cdc42 and Rac1. [Abstract/Link to Full Text]

Chen LY, Liu D, Songyang Z
Telomere maintenance through spatial control of telomeric proteins.
Mol Cell Biol. 2007 Aug;27(16):5898-909.
The six human telomeric proteins TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 can form a complex called the telosome/shelterin, which is required for telomere protection and length control. TPP1 has been shown to regulate both POT1 telomere localization and telosome assembly through its binding to TIN2. It remains to be determined where such interactions take place and whether cellular compartmentalization of telomeric proteins is important for telomere maintenance. We systematically investigated here the cellular localization and interactions of human telomeric proteins. Interestingly, we found TIN2, TPP1, and POT1 to localize and interact with each other in both the cytoplasm and the nucleus. Unexpectedly, TPP1 contains a functional nuclear export signal that directly controls the amount of TPP1 and POT1 in the nucleus. Furthermore, binding of TIN2 to TPP1 promotes the nuclear localization of TPP1 and POT1. We also found that disrupting TPP1 nuclear export could result in telomeric DNA damage response and telomere length disregulation. Our findings highlight how the coordinated interactions between TIN2, TPP1, and POT1 in the cytoplasm regulate the assembly and function of the telosome in the nucleus and indicate for the first time the importance of nuclear export and spatial control of telomeric proteins in telomere maintenance. [Abstract/Link to Full Text]

Scaglione KM, Bansal PK, Deffenbaugh AE, Kiss A, Moore JM, Korolev S, Cocklin R, Goebl M, Kitagawa K, Skowyra D
SCF E3-mediated autoubiquitination negatively regulates activity of Cdc34 E2 but plays a nonessential role in the catalytic cycle in vitro and in vivo.
Mol Cell Biol. 2007 Aug;27(16):5860-70.
One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless (K0)Cdc34(DeltaC) is indistinguishable from Cdc34(DeltaC) in ubiquitination of the prototype SCF(Cdc4) substrate Sic1 in vitro, and replacement of the CDC34 gene with either the (K0)cdc34(DeltaC) or the cdc34(DeltaC) allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF. [Abstract/Link to Full Text]

Yoshida H, Ichikawa H, Tagata Y, Katsumoto T, Ohnishi K, Akao Y, Naoe T, Pandolfi PP, Kitabayashi I
PML-retinoic acid receptor alpha inhibits PML IV enhancement of PU.1-induced C/EBPepsilon expression in myeloid differentiation.
Mol Cell Biol. 2007 Aug;27(16):5819-34.
PML and PU.1 play important roles in myeloid differentiation. PML-deficient mice have an impaired capacity for terminal maturation of their myeloid precursor cells. This finding has been explained, at least in part, by the lack of PML action to modulate retinoic acid-differentiating activities. In this study, we found that C/EBPepsilon expression is reduced in PML-deficient mice. We showed that PU.1 directly activates the transcription of the C/EBPepsilon gene that is essential for granulocytic differentiation. The type IV isoform of PML interacted with PU.1, promoted its association with p300, and then enhanced PU.1-induced transcription and granulocytic differentiation. In contrast to PML IV, the leukemia-associated PML-retinoic acid receptor alpha fusion protein dissociated the PU.1/PML IV/p300 complex and inhibited PU.1-induced transcription. These results suggest a novel pathogenic mechanism of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia. [Abstract/Link to Full Text]

Bilanges B, Argonza-Barrett R, Kolesnichenko M, Skinner C, Nair M, Chen M, Stokoe D
Tuberous sclerosis complex proteins 1 and 2 control serum-dependent translation in a TOP-dependent and -independent manner.
Mol Cell Biol. 2007 Aug;27(16):5746-64.
The tuberous sclerosis complex (TSC) proteins TSC1 and TSC2 regulate protein translation by inhibiting the serine/threonine kinase mTORC1 (for mammalian target of rapamycin complex 1). However, how TSC1 and TSC2 control overall protein synthesis and the translation of specific mRNAs in response to different mitogenic and nutritional stimuli is largely unknown. We show here that serum withdrawal inhibits mTORC1 signaling, causes disassembly of translation initiation complexes, and causes mRNA redistribution from polysomes to subpolysomes in wild-type mouse embryo fibroblasts (MEFs). In contrast, these responses are defective in Tsc1(-/-) or Tsc2(-/-) MEFs. Microarray analysis of polysome- and subpolysome-associated mRNAs uncovered specific mRNAs that are translationally regulated by serum, 90% of which are TSC1 and TSC2 dependent. Surprisingly, the mTORC1 inhibitor, rapamycin, abolished mTORC1 activity but only affected approximately 40% of the serum-regulated mRNAs. Serum-dependent signaling through mTORC1 and polysome redistribution of global and individual mRNAs were restored upon re-expression of TSC1 and TSC2. Serum-responsive mRNAs that are sensitive to inhibition by rapamycin are highly enriched for terminal oligopyrimidine and for very short 5' and 3' untranslated regions. These data demonstrate that the TSC1/TSC2 complex regulates protein translation through mainly mTORC1-dependent mechanisms and implicates a discrete profile of deregulated mRNA translation in tuberous sclerosis pathology. [Abstract/Link to Full Text]

Bell EL, Klimova TA, Eisenbart J, Schumacker PT, Chandel NS
Mitochondrial reactive oxygen species trigger hypoxia-inducible factor-dependent extension of the replicative life span during hypoxia.
Mol Cell Biol. 2007 Aug;27(16):5737-45.
Physiological hypoxia extends the replicative life span of human cells in culture. Here, we report that hypoxic extension of replicative life span is associated with an increase in mitochondrial reactive oxygen species (ROS) in primary human lung fibroblasts. The generation of mitochondrial ROS is necessary for hypoxic activation of the transcription factor hypoxia-inducible factor (HIF). The hypoxic extension of replicative life span is ablated by a dominant negative HIF. HIF is sufficient to induce telomerase reverse transcriptase mRNA and telomerase activity and to extend replicative life span. Furthermore, the down-regulation of the von Hippel-Lindau tumor suppressor protein by RNA interference increases HIF activity and extends replicative life span under normoxia. These findings provide genetic evidence that hypoxia utilizes mitochondrial ROS as signaling molecules to activate HIF-dependent extension of replicative life span. [Abstract/Link to Full Text]

Chauvin C, Salhi S, Jean-Jean O
Human eukaryotic release factor 3a depletion causes cell cycle arrest at G1 phase through inhibition of the mTOR pathway.
Mol Cell Biol. 2007 Aug;27(16):5619-29.
Eukaryotic release factor 3 (eRF3) is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. Studies have related eRF3 with cell cycle regulation, cytoskeleton organization, and tumorigenesis. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. eRF3a is the major factor acting in translation termination, and its expression level controls termination complex formation. Here, we investigate the role of eRF3a in cell cycle progression using short interfering RNAs and flow cytometry. We show that eRF3a depletion induces a G1 arrest and that eRF3a GTP-binding activity, but not the eRF3a N-terminal domain, is required to restore G1-to-S-phase progression. We also show that eRF3a depletion decreases the global translation rate and reduces the polysome charge of mRNA. Finally, we show that two substrates of the mammalian TOR (mTOR) kinase, 4E-BP1 and protein kinase S6K1, are hypophosphorylated in eRF3a-depleted cells. These results strongly suggest that the G1 arrest and the decrease in translation induced by eRF3a depletion are due to the inhibition of mTOR activity and hence that eRF3a belongs to the regulatory pathway of mTOR activity. [Abstract/Link to Full Text]

Ai D, Wang J, Amen M, Lu MF, Amendt BA, Martin JF
Nuclear factor 1 and T-cell factor/LEF recognition elements regulate Pitx2 transcription in pituitary development.
Mol Cell Biol. 2007 Aug;27(16):5765-75.
Pitx2, a paired-related homeobox gene that is mutated in Rieger syndrome I, is the earliest known marker of oral ectoderm. Pitx2 was previously shown to be required for tooth, palate, and pituitary development in mice; however, the mechanisms regulating Pitx2 transcription in the oral ectoderm are poorly understood. Here we used an in vivo transgenic approach to investigate the mechanisms regulating Pitx2 transcription. We identified a 7-kb fragment that directs LacZ expression in oral ectoderm and in many of its derivatives. Deletion analysis of transgenic embryos reduced this fragment to a 520-bp region that directed LacZ activity to Rathke's pouch. A comparison of the mouse and human sequences revealed a conserved nuclear factor 1 (NF-1) recognition element near a consensus T-cell factor (TCF)/LEF binding site. The mutation of either site individually abolished LacZ activity in transgenic embryos, identifying Pitx2 as a direct target of Wnt signaling in pituitary development. These findings uncover a requirement for NF-1 and TCF factors in Pitx2 transcriptional regulation in the pituitary and provide insight into the mechanisms controlling region-specific transcription in the oral ectoderm and its derivatives. [Abstract/Link to Full Text]

Huang FT, Yu K, Balter BB, Selsing E, Oruc Z, Khamlichi AA, Hsieh CL, Lieber MR
Sequence dependence of chromosomal R-loops at the immunoglobulin heavy-chain Smu class switch region.
Mol Cell Biol. 2007 Aug;27(16):5921-32.
The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the Smu locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The Smu R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core Smu repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core Smu repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core Smu repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence. [Abstract/Link to Full Text]

Yu EY, Steinberg-Neifach O, Dandjinou AT, Kang F, Morrison AJ, Shen X, Lue NF
Regulation of telomere structure and functions by subunits of the INO80 chromatin remodeling complex.
Mol Cell Biol. 2007 Aug;27(16):5639-49.
ATP-dependent chromatin remodeling complexes have been implicated in the regulation of transcription, replication, and more recently DNA double-strand break repair. Here we report that the Ies3p subunit of the Saccharomyces cerevisiae INO80 chromatin remodeling complex interacts with a conserved tetratricopeptide repeat domain of the telomerase protein Est1p. Deletion of IES3 and some other subunits of the complex induced telomere elongation and altered telomere position effect. In telomerase-negative mutants, loss of Ies3p delayed the emergence of recombinational survivors and stimulated the formation of extrachromosomal telomeric circles in survivors. Deletion of IES3 also resulted in heightened levels of telomere-telomere fusions in telomerase-deficient strains. In addition, a delay in survivor formation was observed in an Arp8p-deficient mutant. Because Arp8p is required for the chromatin remodeling activity of the INO80 complex, the complex may promote recombinational telomere maintenance by altering chromatin structure. Consistent with this notion, we observed preferential localization of multiple subunits of the INO80 complex to telomeres. Our results reveal novel functions for a subunit of the telomerase complex and the INO80 chromatin remodeling complex. [Abstract/Link to Full Text]

Tateno H, Li H, Schur MJ, Bovin N, Crocker PR, Wakarchuk WW, Paulson JC
Distinct endocytic me