Journal of Cell Biology

Sweet recipe for cellular closeness.

Publication Date: 2010 Aug 30 PMID: 20805326
Authors: Leslie, M.
Journal: J Cell Biol

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A testis-specific regulator of complex and hybrid N-glycan synthesis.

Publication Date: 2010 Aug 30 PMID: 20805325
Authors: Huang, H. H. - Stanley, P.
Journal: J Cell Biol

Database analyses identified 4933434I20Rik as a glycosyltransferase-like gene expressed mainly in testicular germ cells and regulated during spermatogenesis. Expression of a membrane-bound form of the protein resulted in a marked and specific reduction in N-acetylglucosaminyltransferase I (GlcNAcT-I) activity and complex and hybrid N-glycan synthesis. Thus, the novel activity was termed GlcNAcT-I inhibitory protein (GnT1IP). Membrane-bound GnT1IP localizes to the ER, the ER-Golgi intermediate compartment (ERGIC), and the cis-Golgi. Coexpression of membrane-anchored GnT1IP with GlcNAcT-I causes association of the two proteins, inactivation of GlcNAcT-I, and mislocalization of GlcNAcT-I from the medial-Golgi to earlier compartments. Therefore, GnT1IP is a regulator of GlcNAcT-I and complex and hybrid N-glycan production. Importantly, the formation of high mannose N-glycans resulting from inhibition of GlcNAcT-I by GnT1IP markedly increases the adhesion of CHO cells to TM4 Sertoli cells. Testicular germ cells might use GnT1IP to induce the expression of high mannose N-glycans on glycoproteins, thereby facilitating Sertoli-germ cell attachment at a particular stage of spermatogenesis.

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The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage.

Publication Date: 2010 Aug 30 PMID: 20805324
Authors: Larsen, D. H. - Poinsignon, C. - Gudjonsson, T. - Dinant, C. - Payne, M. R. - Hari, F. J. - Rendtlew Danielsen, J. M. - Menard, P. - Sand, J. C. - Stucki, M. - Lukas, C. - Bartek, J. - Andersen, J. S. - Lukas, J.
Journal: J Cell Biol

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate-dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21(Cip1) accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.

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Two distinct mechanisms target GAD67 to vesicular pathways and presynaptic clusters.

Publication Date: 2010 Aug 30 PMID: 20805323
Authors: Kanaani, J. - Kolibachuk, J. - Martinez, H. - Baekkeskov, S.
Journal: J Cell Biol

The inhibitory neurotransmitter gamma-amino butyric acid (GABA) is synthesized by two isoforms of the enzyme glutamic acid decarboxylase (GAD): GAD65 and GAD67. Whereas GAD67 is constitutively active and produces >90% of GABA in the central nervous system, GAD65 is transiently activated and augments GABA levels for rapid modulation of inhibitory neurotransmission. Hydrophobic lipid modifications of the GAD65 protein target it to Golgi membranes and synaptic vesicles in neuroendocrine cells. In contrast, the GAD67 protein remains hydrophilic but has been shown to acquire membrane association by heterodimerization with GAD65. Here, we identify a second mechanism that mediates robust membrane anchoring, axonal targeting, and presynaptic clustering of GAD67 but that is independent of GAD65. This mechanism is abolished by a leucine-103 to proline mutation that changes the conformation of the N-terminal domain but does not affect the GAD65-dependent membrane anchoring of GAD67. Thus two distinct mechanisms target the constitutively active GAD67 to presynaptic clusters to facilitate accumulation of GABA for rapid delivery into synapses.

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The mitosis-to-interphase transition is coordinated by cross talk between the SIN and MOR pathways in Schizosaccharomyces pombe.

Publication Date: 2010 Aug 30 PMID: 20805322
Authors: Ray, S. - Kume, K. - Gupta, S. - Ge, W. - Balasubramanian, M. - Hirata, D. - McCollum, D.
Journal: J Cell Biol

The mechanisms that regulate cytoskeletal remodeling during the transition between mitosis and interphase are poorly understood. In fission yeast the MOR pathway promotes actin polarization to cell tips in interphase, whereas the SIN signaling pathway drives actomyosin ring assembly and cytokinesis. We show that the SIN inhibits MOR signaling in mitosis by interfering with Nak1 kinase-mediated activation of the most downstream MOR component, the NDR family kinase Orb6. Inactivation of the MOR may be a key function of the SIN because attenuation of MOR signaling rescued the cytokinetic defects of SIN mutants and allowed weak SIN signaling to trigger ectopic cytokinesis. Furthermore, failure to inhibit the MOR is toxic when the cell division apparatus is compromised. Together, our results reveal a mutually antagonistic relationship between the SIN and MOR pathways, which is important for completion of cytokinesis and coordination of cytoskeletal remodeling at the mitosis-to-interphase transition.

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Subgroup II PAK-mediated phosphorylation regulates Ran activity during mitosis.

Publication Date: 2010 Aug 30 PMID: 20805321
Authors: Bompard, G. - Rabeharivelo, G. - Frank, M. - Cau, J. - Delsert, C. - Morin, N.
Journal: J Cell Biol

Ran is an essential GTPase that controls nucleocytoplasmic transport, mitosis, and nuclear envelope formation. These functions are regulated by interaction of Ran with different partners, and by formation of a Ran-GTP gradient emanating from chromatin. Here, we identify a novel level of Ran regulation. We show that Ran is a substrate for p21-activated kinase 4 (PAK4) and that its phosphorylation on serine-135 increases during mitosis. The endogenous phosphorylated Ran and active PAK4 dynamically associate with different components of the microtubule spindle during mitotic progression. A GDP-bound Ran phosphomimetic mutant cannot undergo RCC1-mediated GDP/GTP exchange and cannot induce microtubule asters in mitotic Xenopus egg extracts. Conversely, phosphorylation of GTP-bound Ran facilitates aster nucleation. Finally, phosphorylation of Ran on serine-135 impedes its binding to RCC1 and RanGAP1. Our study suggests that PAK4-mediated phosphorylation of GDP- or GTP-bound Ran regulates the assembly of Ran-dependent complexes on the mitotic spindle.

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The NuRD chromatin-remodeling complex regulates signaling and repair of DNA damage.

Publication Date: 2010 Aug 30 PMID: 20805320
Authors: Smeenk, G. - Wiegant, W. W. - Vrolijk, H. - Solari, A. P. - Pastink, A. - van Attikum, H.
Journal: J Cell Biol

Cells respond to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) by orchestrating events that coordinate cell cycle progression and DNA repair. How cells signal and repair DSBs is not yet fully understood. A genome-wide RNA interference screen in Caenorhabditis elegans identified egr-1 as a factor that protects worm cells against IR. The human homologue of egr-1, MTA2 (metastasis-associated protein 2), is a subunit of the nucleosome-remodeling and histone deacetylation (NuRD) chromatin-remodeling complex. We show that knockdown of MTA2 and CHD4 (chromodomain helicase DNA-binding protein 4), the catalytic subunit (adenosine triphosphatase [ATPase]) of NuRD, leads to accumulation of spontaneous DNA damage and increased IR sensitivity. MTA2 and CHD4 accumulate in DSB-containing chromatin tracks generated by laser microirradiation. Directly at DSBs, CHD4 stimulates RNF8/RNF168-dependent formation of ubiquitin conjugates to facilitate the accrual of RNF168 and BRCA1. Finally, we show that CHD4 promotes DSB repair and checkpoint activation in response to IR. Thus, the NuRD chromatin-remodeling complex is a novel regulator of DNA damage responses that orchestrates proper signaling and repair of DSBs.

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Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression.

Publication Date: 2010 Aug 23 PMID: 20733059
Authors: Liu, F. - Mih, J. D. - Shea, B. S. - Kho, A. T. - Sharif, A. S. - Tager, A. M. - Tschumperlin, D. J.
Journal: J Cell Biol

Tissue stiffening is a hallmark of fibrotic disorders but has traditionally been regarded as an outcome of fibrosis, not a contributing factor to pathogenesis. In this study, we show that fibrosis induced by bleomycin injury in the murine lung locally increases median tissue stiffness sixfold relative to normal lung parenchyma. Across this pathophysiological stiffness range, cultured lung fibroblasts transition from a surprisingly quiescent state to progressive increases in proliferation and matrix synthesis, accompanied by coordinated decreases in matrix proteolytic gene expression. Increasing matrix stiffness strongly suppresses fibroblast expression of COX-2 (cyclooxygenase-2) and synthesis of prostaglandin E(2) (PGE(2)), an autocrine inhibitor of fibrogenesis. Exogenous PGE(2) or an agonist of the prostanoid EP2 receptor completely counteracts the proliferative and matrix synthetic effects caused by increased stiffness. Together, these results demonstrate a dominant role for normal tissue compliance, acting in part through autocrine PGE(2), in maintaining fibroblast quiescence and reveal a feedback relationship between matrix stiffening, COX-2 suppression, and fibroblast activation that promotes and amplifies progressive fibrosis.

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Lipid activation of the signal recognition particle receptor provides spatial coordination of protein targeting.

Publication Date: 2010 Aug 23 PMID: 20733058
Authors: Lam, V. Q. - Akopian, D. - Rome, M. - Henningsen, D. - Shan, S. O.
Journal: J Cell Biol

The signal recognition particle (SRP) and SRP receptor comprise the major cellular machinery that mediates the cotranslational targeting of proteins to cellular membranes. It remains unclear how the delivery of cargos to the target membrane is spatially coordinated. We show here that phospholipid binding drives important conformational rearrangements that activate the bacterial SRP receptor FtsY and the SRP-FtsY complex. This leads to accelerated SRP-FtsY complex assembly, and allows the SRP-FtsY complex to more efficiently unload cargo proteins. Likewise, formation of an active SRP-FtsY GTPase complex exposes FtsY's lipid-binding helix and enables stable membrane association of the targeting complex. Thus, membrane binding, complex assembly with SRP, and cargo unloading are inextricably linked to each other via conformational changes in FtsY. These allosteric communications allow the membrane delivery of cargo proteins to be efficiently coupled to their subsequent unloading and translocation, thus providing spatial coordination during protein targeting.

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Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ.

Publication Date: 2010 Aug 23 PMID: 20733057
Authors: Ortiz, J. O. - Brandt, F. - Matias, V. R. - Sennels, L. - Rappsilber, J. - Scheres, S. H. - Eibauer, M. - Hartl, F. U. - Baumeister, W.
Journal: J Cell Biol

Ribosomes arranged in pairs (100S) have been related with nutritional stress response and are believed to represent a "hibernation state." Several proteins have been identified that are associated with 100S ribosomes but their spatial organization has hitherto not been characterized. We have used cryoelectron tomography to reveal the three-dimensional configuration of 100S ribosomes isolated from starved Escherichia coli cells and we have described their mode of interaction. In situ studies with intact E. coli cells allowed us to demonstrate that 100S ribosomes do exist in vivo and represent an easily reversible state of quiescence; they readily vanish when the growth medium is replenished.

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Cajal body surveillance of U snRNA export complex assembly.

Publication Date: 2010 Aug 23 PMID: 20733056
Authors: Suzuki, T. - Izumi, H. - Ohno, M.
Journal: J Cell Biol

Phosphorylated adaptor for RNA export (PHAX) is the key export mediator for spliceosomal U small nuclear RNA (snRNA) precursors in metazoa. PHAX is enriched in Cajal bodies (CBs), nuclear subdomains involved in the biogenesis of small ribonucleoproteins. However, CBs' role in U snRNA export has not been demonstrated. In this study, we show that U snRNA precursors microinjected into Xenopus laevis oocyte nuclei temporarily concentrate in CBs but gradually decrease as RNA export proceeds. Inhibition of PHAX activity by the coinjection of a specific anti-PHAX antibody or a dominant-negative PHAX mutant inhibits U snRNA export and simultaneously enhances accumulation of U snRNA precursors in CBs, indicating that U snRNAs transit through CBs before export and that binding to PHAX is required for efficient exit of U snRNAs from CBs. Similar results were obtained with U snRNAs transcribed from microinjected genes. These results reveal a novel function for CBs, which ensure that U snRNA precursors are properly bound by PHAX.

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The APC/C recruits cyclin B1-Cdk1-Cks in prometaphase before D box recognition to control mitotic exit.

Publication Date: 2010 Aug 23 PMID: 20733055
Authors: van Zon, W. - Ogink, J. - ter Riet, B. - Medema, R. H. - te Riele, H. - Wolthuis, R. M.
Journal: J Cell Biol

The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) is activated at prometaphase by mitotic phosphorylation and binding of its activator, Cdc20. This initiates cyclin A degradation, whereas cyclin B1 is stabilized by the spindle checkpoint. Upon checkpoint release, the RXXL destruction box (D box) was proposed to direct cyclin B1 to core APC/C or Cdc20. In this study, we report that endogenous cyclin B1-Cdk1 is recruited to checkpoint-inhibited, phosphorylated APC/C in prometaphase independently of Cdc20 or the cyclin B1 D box. Like cyclin A, cyclin B1 binds the APC/C by the Cdk cofactor Cks and the APC3 subunit. Prior binding to APC/C(Cdc20) makes cyclin B1 a better APC/C substrate in metaphase, driving mitotic exit and cytokinesis. We conclude that in prometaphase, the phosphorylated APC/C can recruit both cyclin A and cyclin B1 in a Cks-dependent manner. This suggests that the spindle checkpoint blocks D box recognition of APC/C-bound cyclin B1, whereas distinctive complexes between the N terminus of cyclin A and Cdc20 evade checkpoint control.

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A chromatin-bound kinase, ERK8, protects genomic integrity by inhibiting HDM2-mediated degradation of the DNA clamp PCNA.

Publication Date: 2010 Aug 23 PMID: 20733054
Authors: Groehler, A. L. - Lannigan, D. A.
Journal: J Cell Biol

Proliferating cell nuclear antigen (PCNA) acts as a scaffold, coordinator, and stimulator of numerous processes required for faithful transmission of genetic information. Maintaining PCNA levels above a critical threshold is essential, but little is known about PCNA protein turnover. We now show that ERK8 (extracellular signal-regulated kinase 8) is required for PCNA protein stability. ERK8 contains a conserved PCNA-interacting protein (PIP) box. Chromatin-bound ERK8 (ERK8(CHROMATIN)) interacts via this motif with PCNA(CHROMATIN), which acts as a platform for numerous proteins involved in DNA metabolism. Silencing ERK8 decreases PCNA levels and increases DNA damage. Ectopic expression of PCNA blocks DNA damage induced by ERK8 loss. ERK8 prevents HDM2-mediated PCNA destruction by inhibiting the association of PCNA with HDM2. This regulation is physiologically relevant as ERK8 activity is inhibited in transformed mammary cells. Our results reveal an unanticipated mechanism to control PCNA levels in normal cycling mammary epithelial cells and implicate ERK8 in the regulation of genomic stability.

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SNX27 mediates PDZ-directed sorting from endosomes to the plasma membrane.

Publication Date: 2010 Aug 23 PMID: 20733053
Authors: Lauffer, B. E. - Melero, C. - Temkin, P. - Lei, C. - Hong, W. - Kortemme, T. - von Zastrow, M.
Journal: J Cell Biol

Postsynaptic density 95/discs large/zonus occludens-1 (PDZ) domain-interacting motifs, in addition to their well-established roles in protein scaffolding at the cell surface, are proposed to act as cis-acting determinants directing the molecular sorting of transmembrane cargo from endosomes to the plasma membrane. This hypothesis requires the existence of a specific trans-acting PDZ protein that mediates the proposed sorting operation in the endosome membrane. Here, we show that sorting nexin 27 (SNX27) is required for efficient PDZ-directed recycling of the beta(2)-adrenoreceptor (beta(2)AR) from early endosomes. SNX27 mediates this sorting function when expressed at endogenous levels, and its recycling activity requires both PDZ domain-dependent recognition of the beta(2)AR cytoplasmic tail and Phox homology (PX) domain-dependent association with the endosome membrane. These results identify a discrete role of SNX27 in PDZ-directed recycling of a physiologically important signaling receptor, and extend the concept of cargo-specific molecular sorting in the recycling pathway.

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Coordinated RhoA signaling at the leading edge and uropod is required for T cell transendothelial migration.

Publication Date: 2010 Aug 23 PMID: 20733052
Authors: Heasman, S. J. - Carlin, L. M. - Cox, S. - Ng, T. - Ridley, A. J.
Journal: J Cell Biol

Transendothelial migration (TEM) is a tightly regulated process whereby leukocytes migrate from the vasculature into tissues. Rho guanosine triphosphatases (GTPases) are implicated in TEM, but the contributions of individual Rho family members are not known. In this study, we use an RNA interference screen to identify which Rho GTPases affect T cell TEM and demonstrate that RhoA is critical for this process. RhoA depletion leads to loss of migratory polarity; cells lack both leading edge and uropod structures and, instead, have stable narrow protrusions with delocalized protrusions and contractions. By imaging a RhoA activity biosensor in transmigrating T cells, we find that RhoA is locally and dynamically activated at the leading edge, where its activation precedes both extension and retraction events, and in the uropod, where it is associated with ROCK-mediated contraction. The Rho guanine nucleotide exchange factor (GEF) GEF-H1 contributes to uropod contraction but does not affect the leading edge. Our data indicate that RhoA activity is dynamically regulated at the front and back of T cells to coordinate TEM.

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How cyclin A destruction escapes the spindle assembly checkpoint.

Publication Date: 2010 Aug 23 PMID: 20733051
Authors: Di Fiore, B. - Pines, J.
Journal: J Cell Biol

The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase essential to mitosis, which ensures that specific proteins are degraded at specific times to control the order of mitotic events. The APC/C coactivator, Cdc20, is targeted by the spindle assembly checkpoint (SAC) to restrict APC/C activity until metaphase, yet early substrates, such as cyclin A, are degraded in the presence of the active checkpoint. Cdc20 and the cyclin-dependent kinase cofactor, Cks, are required for cyclin A destruction, but how they enable checkpoint-resistant destruction has not been elucidated. In this study, we answer this problem: we show that the N terminus of cyclin A binds directly to Cdc20 and with sufficient affinity that it can outcompete the SAC proteins. Subsequently, the Cks protein is necessary and sufficient to promote cyclin A degradation in the presence of an active checkpoint by binding cyclin A-Cdc20 to the APC/C.

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Mass spectrometry-based proteomics in cell biology.

Publication Date: 2010 Aug 23 PMID: 20733050
Authors: Walther, T. C. - Mann, M.
Journal: J Cell Biol

The global analysis of protein composition, modifications, and dynamics are important goals in cell biology. Mass spectrometry (MS)-based proteomics has matured into an attractive technology for this purpose. Particularly, high resolution MS methods have been extremely successful for quantitative analysis of cellular and organellar proteomes. Rapid advances in all areas of the proteomic workflow, including sample preparation, MS, and computational analysis, should make the technology more easily available to a broad community and turn it into a staple methodology for cell biologists.

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HDM2 ERKs PCNA.

Publication Date: 2010 Aug 23 PMID: 20733049
Authors: Nguyen, H. D. - Bielinsky, A. K.
Journal: J Cell Biol

In this issue, a study by Groehler and Lannigan (2010. J. Cell Biol. doi:10.1083/jcb.201002124) sheds light on the regulation of proliferating cell nuclear antigen (PCNA) turnover and how it is counteracted by the small chromatin-bound kinase ERK8 (extracellular signal-regulated kinase 8). Importantly, inactivation of ERK8 results in genome instability and is associated with cell transformation.

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Martin Hetzer: taking the nuclear membrane beyond the barrier.

Publication Date: 2010 Aug 23 PMID: 20733048
Authors: Hetzer, M.
Journal: J Cell Biol

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Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells.

Publication Date: 2010 Aug 23 PMID: 20713605
Authors: Howes, M. T. - Kirkham, M. - Riches, J. - Cortese, K. - Walser, P. J. - Simpson, F. - Hill, M. M. - Jones, A. - Lundmark, R. - Lindsay, M. R. - Hernandez-Deviez, D. J. - Hadzic, G. - McCluskey, A. - Bashir, R. - Liu, L. - Pilch, P. - McMahon, H. - Robinson, P. J. - Hancock, J. F. - Mayor, S. - Parton, R. G.
Journal: J Cell Biol

Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.

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Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis.

Publication Date: 2010 Aug 23 PMID: 20713604
Authors: Nezis, I. P. - Shravage, B. V. - Sagona, A. P. - Lamark, T. - Bjorkoy, G. - Johansen, T. - Rusten, T. E. - Brech, A. - Baehrecke, E. H. - Stenmark, H.
Journal: J Cell Biol

Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. In this study, we show that autophagy controls DNA fragmentation during late oogenesis in Drosophila melanogaster. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. Our results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death.

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Identification of novel filament-forming proteins in Saccharomyces cerevisiae and Drosophila melanogaster.

Publication Date: 2010 Aug 23 PMID: 20713603
Authors: Noree, C. - Sato, B. K. - Broyer, R. M. - Wilhelm, J. E.
Journal: J Cell Biol

The discovery of large supramolecular complexes such as the purinosome suggests that subcellular organization is central to enzyme regulation. A screen of the yeast GFP strain collection to identify proteins that assemble into visible structures identified four novel filament systems comprised of glutamate synthase, guanosine diphosphate-mannose pyrophosphorylase, cytidine triphosphate (CTP) synthase, or subunits of the eIF2/2B translation factor complex. Recruitment of CTP synthase to filaments and foci can be modulated by mutations and regulatory ligands that alter enzyme activity, arguing that the assembly of these structures is related to control of CTP synthase activity. CTP synthase filaments are evolutionarily conserved and are restricted to axons in neurons. This spatial regulation suggests that these filaments have additional functions separate from the regulation of enzyme activity. The identification of four novel filaments greatly expands the number of known intracellular filament networks and has broad implications for our understanding of how cells organize biochemical activities in the cytoplasm.

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Oxidative status of muscle is determined by p107 regulation of PGC-1alpha.

Publication Date: 2010 Aug 23 PMID: 20713602
Authors: Scime, A. - Soleimani, V. D. - Bentzinger, C. F. - Gillespie, M. A. - Le Grand, F. - Grenier, G. - Bevilacqua, L. - Harper, M. E. - Rudnicki, M. A.
Journal: J Cell Biol

Mice lacking p107 exhibit a white adipose deficiency yet do not manifest the metabolic changes typical for lipodystrophy, and instead exhibit low levels of serum triglycerides and a normal liver phenotype. When fed a high fat diet, p107-null mice still did not accumulate fat in the liver, and display markedly elevated energy expenditures together with an increased energy preference for lipids. Skeletal muscle was therefore examined, as this is normally the major tissue involved in whole body lipid metabolism. Notably, p107-deficient muscle express increased levels of peroxisome proliferator-activated receptor gamma co-activator-1alpha (PGC-1alpha) and contained increased numbers of the pro-oxidative type I and type IIa myofibers. Chromatin immunoprecipitation revealed binding of p107 and E2F4 to the PGC-1alpha proximal promoter, and this binding repressed promoter activity in transient transcription assays. Ectopic expression of p107 in muscle tissue in vivo results in a pronounced 20% decrease in the numbers of oxidative type IIa myofibers. Lastly, isolated p107-deficient muscle tissue display a threefold increase in lipid metabolism. Therefore, p107 determines the oxidative state of multiple tissues involved in whole body fat metabolism, including skeletal muscle.

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BAG-6 is essential for selective elimination of defective proteasomal substrates.

Publication Date: 2010 Aug 23 PMID: 20713601
Authors: Minami, R. - Hayakawa, A. - Kagawa, H. - Yanagi, Y. - Yokosawa, H. - Kawahara, H.
Journal: J Cell Biol

BAG-6/Scythe/BAT3 is a ubiquitin-like protein that was originally reported to be the product of a novel gene located within the human major histocompatibility complex, although the mechanisms of its function remain largely obscure. Here, we demonstrate the involvement of BAG-6 in the degradation of a CL1 model defective protein substrate in mammalian cells. We show that BAG-6 is essential for not only model substrate degradation but also the ubiquitin-mediated metabolism of newly synthesized defective polypeptides. Furthermore, our in vivo and in vitro analysis shows that BAG-6 interacts physically with puromycin-labeled nascent chain polypeptides and regulates their proteasome-mediated degradation. Finally, we show that knockdown of BAG-6 results in the suppressed presentation of MHC class I on the cell surface, a procedure known to be affected by the efficiency of metabolism of defective ribosomal products. Therefore, we propose that BAG-6 is necessary for ubiquitin-mediated degradation of newly synthesized defective polypeptides.

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Regulation of the autophagy protein LC3 by phosphorylation.

Publication Date: 2010 Aug 23 PMID: 20713600
Authors: Cherra, S. J. 3rd - Kulich, S. M. - Uechi, G. - Balasubramani, M. - Mountzouris, J. - Day, B. W. - Chu, C. T.
Journal: J Cell Biol

Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP(+)) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease-associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl-cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity.

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Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors: a possible link to Rho family GTPases.

Publication Date: 2010 Aug 23 PMID: 20713598
Authors: Lee, C. S. - Choi, C. K. - Shin, E. Y. - Schwartz, M. A. - Kim, E. G.
Journal: J Cell Biol

Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology-pleckstrin homology module, the catalytic site of these GEFs, with dissociation constants of approximately 0.3 microM. Binding to the GEFs required assembly of the MII into filaments and actin-stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly, inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of betaPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells.

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Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L.

Publication Date: 2010 Aug 23 PMID: 20713597
Authors: Matsunaga, K. - Morita, E. - Saitoh, T. - Akira, S. - Ktistakis, N. T. - Izumi, T. - Noda, T. - Yoshimori, T.
Journal: J Cell Biol

Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.

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KIF17 stabilizes microtubules and contributes to epithelial morphogenesis by acting at MT plus ends with EB1 and APC.

Publication Date: 2010 Aug 9 PMID: 20696710
Authors: Jaulin, F. - Kreitzer, G.
Journal: J Cell Biol

Epithelial polarization is associated with selective stabilization and reorganization of microtubule (MT) arrays. However, upstream events and downstream consequences of MT stabilization during epithelial morphogenesis are still unclear. We show that the anterograde kinesin KIF17 localizes to MT plus ends, stabilizes MTs, and affects epithelial architecture. Targeting of KIF17 to plus ends of growing MTs requires kinesin motor activity and interaction with EB1. In turn, KIF17 participates in localizing adenomatous polyposis coli (APC) to the plus ends of a subset of MTs. We found that KIF17 affects MT dynamics, polymerization rates, and MT plus end stabilization to generate posttranslationally acetylated MTs. Depletion of KIF17 from cells growing in three-dimensional matrices results in aberrant epithelial cysts that fail to generate a single central lumen and to polarize apical markers. These findings implicate KIF17 in MT stabilization events that contribute to epithelial polarization and morphogenesis.

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Syndecan-3 and Notch cooperate in regulating adult myogenesis.

Publication Date: 2010 Aug 9 PMID: 20696709
Authors: Pisconti, A. - Cornelison, D. D. - Olguin, H. C. - Antwine, T. L. - Olwin, B. B.
Journal: J Cell Biol

Skeletal muscle postnatal growth and repair depend on satellite cells and are regulated by molecular signals within the satellite cell niche. We investigated the molecular and cellular events that lead to altered myogenesis upon genetic ablation of Syndecan-3, a component of the satellite cell niche. In the absence of Syndecan-3, satellite cells stall in S phase, leading to reduced proliferation, increased cell death, delayed onset of differentiation, and markedly reduced numbers of Pax7(+) satellite cells accompanied by myofiber hypertrophy and an increased number of centrally nucleated myofibers. We show that the aberrant cell cycle and impaired self-renewal of explanted Syndecan-3-null satellite cells are rescued by ectopic expression of the constitutively active Notch intracellular domain. Furthermore, we show that Syndecan-3 interacts with Notch and is required for Notch processing by ADAM17/tumor necrosis factor-alpha-converting enzyme (TACE) and signal transduction. Together, our data support the conclusion that Syndecan-3 and Notch cooperate in regulating homeostasis of the satellite cell population and myofiber size.

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Drosophila Mtm and class II PI3K coregulate a PI(3)P pool with cortical and endolysosomal functions.

Publication Date: 2010 Aug 9 PMID: 20696708
Authors: Velichkova, M. - Juan, J. - Kadandale, P. - Jean, S. - Ribeiro, I. - Raman, V. - Stefan, C. - Kiger, A. A.
Journal: J Cell Biol

Reversible phosphoinositide phosphorylation provides a dynamic membrane code that balances opposing cell functions. However, in vivo regulatory relationships between specific kinases, phosphatases, and phosphoinositide subpools are not clear. We identified myotubularin (mtm), a Drosophila melanogaster MTM1/MTMR2 phosphoinositide phosphatase, as necessary and sufficient for immune cell protrusion formation and recruitment to wounds. Mtm-mediated turnover of endosomal phosphatidylinositol 3-phosphate (PI(3)P) pools generated by both class II and III phosphatidylinositol 3-kinases (Pi3K68D and Vps34, respectively) is needed to down-regulate membrane influx, promote efflux, and maintain endolysosomal homeostasis. Endocytosis, but not endolysosomal size, contributes to cortical remodeling by mtm function. We propose that Mtm-dependent regulation of an endosomal PI(3)P pool has separable consequences for endolysosomal homeostasis and cortical remodeling. Pi3K68D depletion (but not Vps34) rescues protrusion and distribution defects in mtm-deficient immune cells and restores functions in other tissues essential for viability. The broad interactions between mtm and class II Pi3K68D suggest a novel strategy for rebalancing PI(3)P-mediated cell functions in MTM-related human disease.

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Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL.

Publication Date: 2010 Aug 9 PMID: 20696707
Authors: Fricker, N. - Beaudouin, J. - Richter, P. - Eils, R. - Krammer, P. H. - Lavrik, I. N.
Journal: J Cell Biol

Cellular FADD-like interleukin-1beta-converting enzyme inhibitory proteins (c-FLIPs; isoforms c-FLIP long [c-FLIP(L)], c-FLIP short [c-FLIP(S)], and c-FLIP Raji [c-FLIP(R)]) regulate caspase-8 activation and death receptor (DR)-induced apoptosis. In this study, using a combination of mathematical modeling, imaging, and quantitative Western blots, we present a new mathematical model describing caspase-8 activation in quantitative terms, which highlights the influence of c-FLIP proteins on this process directly at the CD95 death-inducing signaling complex. We quantitatively define how the stoichiometry of c-FLIP proteins determines sensitivity toward CD95-induced apoptosis. We show that c-FLIP(L) has a proapoptotic role only upon moderate expression in combination with strong receptor stimulation or in the presence of high amounts of one of the short c-FLIP isoforms, c-FLIP(S) or c-FLIP(R). Our findings resolve the present controversial discussion on the function of c-FLIP(L) as a pro- or antiapoptotic protein in DR-mediated apoptosis and are important for understanding the regulation of CD95-induced apoptosis, where subtle differences in c-FLIP concentrations determine life or death of the cells.

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ER sliding dynamics and ER-mitochondrial contacts occur on acetylated microtubules.

Publication Date: 2010 Aug 9 PMID: 20696706
Authors: Friedman, J. R. - Webster, B. M. - Mastronarde, D. N. - Verhey, K. J. - Voeltz, G. K.
Journal: J Cell Biol

The endoplasmic reticulum (ER) network is extremely dynamic in animal cells, yet little is known about the mechanism and function of its movements. The most common ER dynamic, termed ER sliding, involves ER tubule extension along stable microtubules (MTs). In this study, we show that ER sliding occurs on nocodazole-resistant MTs that are posttranslationally modified by acetylation. We demonstrate that high MT curvature is a good indicator of MT acetylation and show in live cells that ER sliding occurs predominantly on these curved, acetylated MTs. Furthermore, increasing MT acetylation by drug treatment increases the frequency of ER sliding. One purpose of the ER sliding on modified MT tracts could be to regulate its interorganelle contacts. We find that all mitochondria and many endosomes maintain contact with the ER despite the movements of each. However, mitochondria, but not endosomes, preferentially localize to acetylated MTs. Thus, different ER dynamics may occur on distinct MT populations to establish or maintain contacts with different organelles.

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Structure of the Sec13-Sec16 edge element, a template for assembly of the COPII vesicle coat.

Publication Date: 2010 Aug 9 PMID: 20696705
Authors: Whittle, J. R. - Schwartz, T. U.
Journal: J Cell Biol

Ancestral coatomer element 1 (ACE1) proteins assemble latticework coats for COPII vesicles and the nuclear pore complex. The ACE1 protein Sec31 and Sec13 make a 2:2 tetramer that forms the edge element of the COPII outer coat. In this study, we report that the COPII accessory protein Sec16 also contains an ACE1. The 165-kD crystal structure of the central domain of Sec16 in complex with Sec13 was solved at 2.7-A resolution. Sec16 and Sec13 also make a 2:2 tetramer, another edge element for the COPII system. Domain swapping at the ACE1-ACE1 interface is observed both in the prior structure of Sec13-Sec31 and in Sec13-Sec16. A Sec31 mutant in which domain swapping is prevented adopts an unprecedented laminated structure, solved at 2.8-A resolution. Our in vivo data suggest that the ACE1 element of Sec31 can functionally replace the ACE1 element of Sec16. Our data support Sec16 as a scaffold for the COPII system and a template for the Sec13-Sec31 coat.

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The cell biology of taste.

Publication Date: 2010 Aug 9 PMID: 20696704
Authors: Chaudhari, N. - Roper, S. D.
Journal: J Cell Biol

Taste buds are aggregates of 50-100 polarized neuroepithelial cells that detect nutrients and other compounds. Combined analyses of gene expression and cellular function reveal an elegant cellular organization within the taste bud. This review discusses the functional classes of taste cells, their cell biology, and current thinking on how taste information is transmitted to the brain.

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Yukiko Goda: memories are made of this.

Publication Date: 2010 Aug 9 PMID: 20696703
Authors: Short, B.
Journal: J Cell Biol

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Ephexin4 and EphA2 mediate cell migration through a RhoG-dependent mechanism.

Publication Date: 2010 Aug 9 PMID: 20679435
Authors: Hiramoto-Yamaki, N. - Takeuchi, S. - Ueda, S. - Harada, K. - Fujimoto, S. - Negishi, M. - Katoh, H.
Journal: J Cell Biol

EphA2, a member of the Eph receptor family, is frequently overexpressed in a variety of human cancers, including breast cancers, and promotes cancer cell motility and invasion independently of its ligand ephrin stimulation. In this study, we identify Ephexin4 as a guanine nucleotide exchange factor (GEF) for RhoG that interacts with EphA2 in breast cancer cells, and knockdown and rescue experiments show that Ephexin4 acts downstream of EphA2 to promote ligand-independent breast cancer cell migration and invasion toward epidermal growth factor through activation of RhoG. The activation of RhoG recruits its effector ELMO2 and a Rac GEF Dock4 to form a complex with EphA2 at the tips of cortactin-rich protrusions in migrating breast cancer cells. In addition, the Dock4-mediated Rac activation is required for breast cancer cell migration. Our findings reveal a novel link between EphA2 and Rac activation that contributes to the cell motility and invasiveness of breast cancer cells.

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Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2.

Publication Date: 2010 Aug 9 PMID: 20679434
Authors: Xu, N. - Hegarat, N. - Black, E. J. - Scott, M. T. - Hochegger, H. - Gillespie, D. A.
Journal: J Cell Biol

Using chemical genetics to reversibly inhibit Cdk1, we find that cells arrested in late G2 are unable to delay mitotic entry after irradiation. Late G2 cells detect DNA damage lesions and form gamma-H2AX foci but fail to activate Chk1. This reflects a lack of DNA double-strand break processing because late G2 cells fail to recruit RPA (replication protein A), ATR (ataxia telangiectasia and Rad3 related), Rad51, or CtIP (C-terminal interacting protein) to sites of radiation-induced damage, events essential for both checkpoint activation and initiation of DNA repair by homologous recombination. Remarkably, inhibition of Akt/PKB (protein kinase B) restores DNA damage processing and Chk1 activation after irradiation in late G2. These data demonstrate a previously unrecognized role for Akt in cell cycle regulation of DNA repair and checkpoint activation. Because Akt/PKB is frequently activated in many tumor types, these findings have important implications for the evolution and therapy of such cancers.

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p125A exists as part of the mammalian Sec13/Sec31 COPII subcomplex to facilitate ER-Golgi transport.

Publication Date: 2010 Aug 9 PMID: 20679433
Authors: Ong, Y. S. - Tang, B. L. - Loo, L. S. - Hong, W.
Journal: J Cell Biol

Coat protein II (COPII)-mediated export from the endoplasmic reticulum (ER) involves sequential recruitment of COPII complex components, including the Sar1 GTPase, the Sec23/Sec24 subcomplex, and the Sec13/Sec31 subcomplex. p125A was originally identified as a Sec23A-interacting protein. Here we demonstrate that p125A also interacts with the C-terminal region of Sec31A. The Sec31A-interacting domain of p125A is between residues 260-600, and is therefore a distinct domain from that required for interaction with Sec23A. Gel filtration and immunodepletion studies suggest that the majority of cytosolic p125A exists as a ternary complex with the Sec13/Sec31A subcomplex, suggesting that Sec 13, Sec31A, and p125A exist in the cytosol primarily as preassembled Sec13/Sec31A/p125A heterohexamers. Golgi morphology and protein export from the ER were affected in p125A-silenced cells. Our results suggest that p125A is part of the Sec13/Sec31A subcomplex and facilitates ER export in mammalian cells.

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S-glutathionylation activates STIM1 and alters mitochondrial homeostasis.

Publication Date: 2010 Aug 9 PMID: 20679432
Authors: Hawkins, B. J. - Irrinki, K. M. - Mallilankaraman, K. - Lien, Y. C. - Wang, Y. - Bhanumathy, C. D. - Subbiah, R. - Ritchie, M. F. - Soboloff, J. - Baba, Y. - Kurosaki, T. - Joseph, S. K. - Gill, D. L. - Madesh, M.
Journal: J Cell Biol

Oxidant stress influences many cellular processes, including cell growth, differentiation, and cell death. A well-recognized link between these processes and oxidant stress is via alterations in Ca(2+) signaling. However, precisely how oxidants influence Ca(2+) signaling remains unclear. Oxidant stress led to a phenotypic shift in Ca(2+) mobilization from an oscillatory to a sustained elevated pattern via calcium release-activated calcium (CRAC)-mediated capacitive Ca(2+) entry, and stromal interaction molecule 1 (STIM1)- and Orai1-deficient cells are resistant to oxidant stress. Functionally, oxidant-induced Ca(2+) entry alters mitochondrial Ca(2+) handling and bioenergetics and triggers cell death. STIM1 is S-glutathionylated at cysteine 56 in response to oxidant stress and evokes constitutive Ca(2+) entry independent of intracellular Ca(2+) stores. These experiments reveal that cysteine 56 is a sensor for oxidant-dependent activation of STIM1 and demonstrate a molecular link between oxidant stress and Ca(2+) signaling via the CRAC channel.

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The inositol 5-phosphatase SHIP2 regulates endocytic clathrin-coated pit dynamics.

Publication Date: 2010 Aug 9 PMID: 20679431
Authors: Nakatsu, F. - Perera, R. M. - Lucast, L. - Zoncu, R. - Domin, J. - Gertler, F. B. - Toomre, D. - De Camilli, P.
Journal: J Cell Biol

Phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P(2)) and its phosphorylated product PI 3,4,5-triphosphate (PI(3,4,5)P(3)) are two major phosphoinositides concentrated at the plasma membrane. Their levels, which are tightly controlled by kinases, phospholipases, and phosphatases, regulate a variety of cellular functions, including clathrin-mediated endocytosis and receptor signaling. In this study, we show that the inositol 5-phosphatase SHIP2, a negative regulator of PI(3,4,5)P(3)-dependent signaling, also negatively regulates PI(4,5)P(2) levels and is concentrated at endocytic clathrin-coated pits (CCPs) via interactions with the scaffold protein intersectin. SHIP2 is recruited early at the pits and dissociates before fission. Both knockdown of SHIP2 expression and acute production of PI(3,4,5)P(3) shorten CCP lifetime by enhancing the rate of pit maturation, which is consistent with a positive role of both SHIP2 substrates, PI(4,5)P(2) and PI(3,4,5)P(3), on coat assembly. Because SHIP2 is a negative regulator of insulin signaling, our findings suggest the importance of the phosphoinositide metabolism at CCPs in the regulation of insulin signal output.

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Gamma-tubulin regulates the anaphase-promoting complex/cyclosome during interphase.

Publication Date: 2010 Aug 9 PMID: 20679430
Authors: Nayak, T. - Edgerton-Morgan, H. - Horio, T. - Xiong, Y. - De Souza, C. P. - Osmani, S. A. - Oakley, B. R.
Journal: J Cell Biol

A cold-sensitive gamma-tubulin allele of Aspergillus nidulans, mipAD159, causes defects in mitotic and cell cycle regulation at restrictive temperatures that are apparently independent of microtubule nucleation defects. Time-lapse microscopy of fluorescently tagged mitotic regulatory proteins reveals that cyclin B, cyclin-dependent kinase 1, and the Ancdc14 phosphatase fail to accumulate in a subset of nuclei at restrictive temperatures. These nuclei are permanently removed from the cell cycle, whereas other nuclei, in the same multinucleate cell, cycle normally, accumulating and degrading these proteins. After each mitosis, additional daughter nuclei fail to accumulate these proteins, resulting in an increase in noncycling nuclei over time and consequent inhibition of growth. Extensive analyses reveal that these noncycling nuclei result from a nuclear autonomous, microtubule-independent failure of inactivation of the anaphase-promoting complex/cyclosome. Thus, gamma-tubulin functions to regulate this key mitotic and cell cycle regulatory complex.

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