Journal of Cell Biology

Eliminating the impact of the Impact Factor.

Publication Date: 2013 May 21 PMID: 23690180
Authors: Misteli, T.
Journal: J Cell Biol

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Live-cell imaging of exocyst links its spatiotemporal dynamics to various stages of vesicle fusion.

Publication Date: 2013 May 20 PMID: 23690179
Authors: Rivera-Molina, F. - Toomre, D.
Journal: J Cell Biol

Tethers play ubiquitous roles in membrane trafficking and influence the specificity of vesicle attachment. Unlike soluble N-ethyl-maleimide-sensitive fusion attachment protein receptors (SNAREs), the spatiotemporal dynamics of tethers relative to vesicle fusion are poorly characterized. The most extensively studied tethering complex is the exocyst, which spatially targets vesicles to sites on the plasma membrane. By using a mammalian genetic replacement strategy, we were able to assemble fluorescently tagged Sec8 into the exocyst complex, which was shown to be functional by biochemical, trafficking, and morphological criteria. Ultrasensitive live-cell imaging revealed that Sec8-TagRFP moved to the cell cortex on vesicles, which preferentially originated from the endocytic recycling compartment. Surprisingly, Sec8 remained with vesicles until full dilation of the fusion pore, supporting potential coupling with SNARE fusion machinery. Fluorescence recovery after photobleaching analysis of Sec8 at cell protrusions revealed that a significant fraction was immobile. Additionally, Sec8 dynamically repositioned to the site of membrane expansion, suggesting that it may respond to local cues during early cell polarization.

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Formation and dissociation of proteasome storage granules are regulated by cytosolic pH.

Publication Date: 2013 May 20 PMID: 23690178
Authors: Peters, L. Z. - Hazan, R. - Breker, M. - Schuldiner, M. - Ben-Aroya, S.
Journal: J Cell Biol

The 26S proteasome is the major protein degradation machinery of the cell and is regulated at many levels. One mode of regulation involves accumulation of proteasomes in proteasome storage granules (PSGs) upon glucose depletion. Using a systematic robotic screening approach in yeast, we identify trans-acting proteins that regulate the accumulation of proteasomes in PSGs. Our dataset was enriched for subunits of the vacuolar adenosine triphosphatase (V-ATPase) complex, a proton pump required for vacuole acidification. We show that the impaired ability of V-ATPase mutants to properly govern intracellular pH affects the kinetics of PSG formation. We further show that formation of other protein aggregates upon carbon depletion also is triggered in mutants with impaired activity of the plasma membrane proton pump and the V-ATPase complex. We thus identify cytosolic pH as a specific cellular signal involved both in the glucose sensing that mediates PSG formation and in a more general mechanism for signaling carbon source exhaustion.

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Diet controls Drosophila follicle stem cell proliferation via Hedgehog sequestration and release.

Publication Date: 2013 May 20 PMID: 23690177
Authors: Hartman, T. R. - Strochlic, T. I. - Ji, Y. - Zinshteyn, D. - O'Reilly, A. M.
Journal: J Cell Biol

A healthy diet improves adult stem cell function and delays diseases such as cancer, heart disease, and neurodegeneration. Defining molecular mechanisms by which nutrients dictate stem cell behavior is a key step toward understanding the role of diet in tissue homeostasis. In this paper, we elucidate the mechanism by which dietary cholesterol controls epithelial follicle stem cell (FSC) proliferation in the fly ovary. In nutrient-restricted flies, the transmembrane protein Boi sequesters Hedgehog (Hh) ligand at the surface of Hh-producing cells within the ovary, limiting FSC proliferation. Upon feeding, dietary cholesterol stimulates S6 kinase-mediated phosphorylation of the Boi cytoplasmic domain, triggering Hh release and FSC proliferation. This mechanism enables a rapid, tissue-specific response to nutritional changes, tailoring stem cell divisions and egg production to environmental conditions sufficient for progeny survival. If conserved in other systems, this mechanism will likely have important implications for studies on molecular control of stem cell function, in which the benefits of low calorie and low cholesterol diets are beginning to emerge.

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Keratins control intercellular adhesion involving PKC-alpha-mediated desmoplakin phosphorylation.

Publication Date: 2013 May 20 PMID: 23690176
Authors: Kroger, C. - Loschke, F. - Schwarz, N. - Windoffer, R. - Leube, R. E. - Magin, T. M.
Journal: J Cell Biol

Maintenance of epithelial cell adhesion is crucial for epidermal morphogenesis and homeostasis and relies predominantly on the interaction of keratins with desmosomes. Although the importance of desmosomes to epidermal coherence and keratin organization is well established, the significance of keratins in desmosome organization has not been fully resolved. Here, we report that keratinocytes lacking all keratins show elevated, PKC-alpha-mediated desmoplakin phosphorylation and subsequent destabilization of desmosomes. We find that PKC-alpha activity is regulated by Rack1-keratin interaction. Without keratins, desmosomes assemble but are endocytosed at accelerated rates, rendering epithelial sheets highly susceptible to mechanical stress. Re-expression of the keratin pair K5/14, inhibition of PKC-alpha activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. Our findings identify a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth.

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Evolutionary comparisons reveal a positional switch for spindle pole oscillations in Caenorhabditis embryos.

Publication Date: 2013 May 20 PMID: 23690175
Authors: Riche, S. - Zouak, M. - Argoul, F. - Arneodo, A. - Pecreaux, J. - Delattre, M.
Journal: J Cell Biol

During the first embryonic division in Caenorhabditis elegans, the mitotic spindle is pulled toward the posterior pole of the cell and undergoes vigorous transverse oscillations. We identified variations in spindle trajectories by analyzing the outwardly similar one-cell stage embryo of its close relative Caenorhabditis briggsae. Compared with C. elegans, C. briggsae embryos exhibit an anterior shifting of nuclei in prophase and reduced anaphase spindle oscillations. By combining physical perturbations and mutant analysis in both species, we show that differences can be explained by interspecies changes in the regulation of the cortical Galpha-GPR-LIN-5 complex. However, we found that in both species (1) a conserved positional switch controls the onset of spindle oscillations, (2) GPR posterior localization may set this positional switch, and (3) the maximum amplitude of spindle oscillations is determined by the time spent in the oscillating phase. By investigating microevolution of a subcellular process, we identify new mechanisms that are instrumental to decipher spindle positioning.

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A light-triggered protein secretion system.

Publication Date: 2013 May 13 PMID: 23671313
Authors: Chen, D. - Gibson, E. S. - Kennedy, M. J.
Journal: J Cell Biol

Optical control of protein interactions has emerged as a powerful experimental paradigm for manipulating and studying various cellular processes. Tools are now available for controlling a number of cellular functions, but some fundamental processes, such as protein secretion, have been difficult to engineer using current optical tools. Here we use UVR8, a plant photoreceptor protein that forms photolabile homodimers, to engineer the first light-triggered protein secretion system. UVR8 fusion proteins were conditionally sequestered in the endoplasmic reticulum, and a brief pulse of light triggered robust forward trafficking through the secretory pathway to the plasma membrane. UVR8 was not responsive to excitation light used to image cyan, green, or red fluorescent protein variants, allowing multicolor visualization of cellular markers and secreted protein cargo as it traverses the cellular secretory pathway. We implemented this novel tool in neurons to demonstrate restricted, local trafficking of secretory cargo near dendritic branch points.

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Ligand-induced activation of a formin-NPF pair leads to collaborative actin nucleation.

Publication Date: 2013 May 13 PMID: 23671312
Authors: Graziano, B. R. - Jonasson, E. M. - Pullen, J. G. - Gould, C. J. - Goode, B. L.
Journal: J Cell Biol

Formins associate with other nucleators and nucleation-promoting factors (NPFs) to stimulate collaborative actin assembly, but the mechanisms regulating these interactions have been unclear. Yeast Bud6 has an established role as an NPF for the formin Bni1, but whether it also directly regulates the formin Bnr1 has remained enigmatic. In this paper, we analyzed NPF-impaired alleles of bud6 in a bni1Delta background and found that Bud6 stimulated Bnr1 activity in vivo. Furthermore, Bud6 bound directly to Bnr1, but its NPF effects were masked by a short regulatory sequence, suggesting that additional factors may be required for activation. We isolated a novel in vivo binding partner of Bud6, Yor304c-a/Bil1, which colocalized with Bud6 and functioned in the Bnr1 pathway for actin assembly. Purified Bil1 bound to the regulatory sequence in Bud6 and triggered NPF effects on Bnr1. These observations define a new mode of formin regulation, which has important implications for understanding NPF-nucleator pairs in diverse systems.

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Dynamic bonds and polar ejection force distribution explain kinetochore oscillations in PtK1 cells.

Publication Date: 2013 May 13 PMID: 23671311
Authors: Civelekoglu-Scholey, G. - He, B. - Shen, M. - Wan, X. - Roscioli, E. - Bowden, B. - Cimini, D.
Journal: J Cell Biol

Duplicated mitotic chromosomes aligned at the metaphase plate maintain dynamic attachments to spindle microtubules via their kinetochores, and multiple motor and nonmotor proteins cooperate to regulate their behavior. Depending on the system, sister chromatids may display either of two distinct behaviors, namely (1) the presence or (2) the absence of oscillations about the metaphase plate. Significantly, in PtK1 cells, in which chromosome behavior appears to be dependent on the position along the metaphase plate, both types of behavior are observed within the same spindle, but how and why these distinct behaviors are manifested is unclear. Here, we developed a new quantitative model to describe metaphase chromosome dynamics via kinetochore-microtubule interactions mediated by nonmotor viscoelastic linkages. Our model reproduces all the key features of metaphase sister kinetochore dynamics in PtK1 cells and suggests that differences in the distribution of polar ejection forces at the periphery and in the middle of PtK1 cell spindles underlie the observed dichotomy of chromosome behavior.

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Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila.

Publication Date: 2013 May 13 PMID: 23671310
Authors: Takats, S. - Nagy, P. - Varga, A. - Pircs, K. - Karpati, M. - Varga, K. - Kovacs, A. L. - Hegedus, K. - Juhasz, G.
Journal: J Cell Biol

During autophagy, phagophores capture portions of cytoplasm and form double-membrane autophagosomes to deliver cargo for lysosomal degradation. How autophagosomes gain competence to fuse with late endosomes and lysosomes is not known. In this paper, we show that Syntaxin17 is recruited to the outer membrane of autophagosomes to mediate fusion through its interactions with ubisnap (SNAP-29) and VAMP7 in Drosophila melanogaster. Loss of these genes results in accumulation of autophagosomes and a block of autolysosomal degradation during basal, starvation-induced, and developmental autophagy. Viable Syntaxin17 mutant adults show large-scale accumulation of autophagosomes in neurons, severe locomotion defects, and premature death. These mutant phenotypes cannot be rescued by neuron-specific inhibition of caspases, suggesting that caspase activation and cell death do not play a major role in brain dysfunction. Our findings reveal the molecular mechanism underlying autophagosomal fusion events and show that lysosomal degradation and recycling of sequestered autophagosome content is crucial to maintain proper functioning of the nervous system.

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The cell biology of disease: Cellular and molecular mechanisms underlying muscular dystrophy.

Publication Date: 2013 May 13 PMID: 23671309
Authors: Rahimov, F. - Kunkel, L. M.
Journal: J Cell Biol

The muscular dystrophies are a group of heterogeneous genetic diseases characterized by progressive degeneration and weakness of skeletal muscle. Since the discovery of the first muscular dystrophy gene encoding dystrophin, a large number of genes have been identified that are involved in various muscle-wasting and neuromuscular disorders. Human genetic studies complemented by animal model systems have substantially contributed to our understanding of the molecular pathomechanisms underlying muscle degeneration. Moreover, these studies have revealed distinct molecular and cellular mechanisms that link genetic mutations to diverse muscle wasting phenotypes.

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Route to destruction: Autophagosomes SNARE lysosomes.

Publication Date: 2013 May 13 PMID: 23671308
Authors: Kramer, H.
Journal: J Cell Biol

Autophagy allows cells to encapsulate parts of their cytosol into unique double-membrane structures. These autophagosomes mature to fuse with lysosomes and deliver the enclosed contents for degradation. Three recent papers, including one by Takats et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201211160), have taken different routes to discover a role for Syntaxin 17 in the maturation of autophagosomes.

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Victor Ambros: The broad scope of microRNAs.

Publication Date: 2013 May 13 PMID: 23671307
Authors: Sedwick, C.
Journal: J Cell Biol

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p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes.

Publication Date: 2013 May 13 PMID: 23649808
Authors: Davalos, A. R. - Kawahara, M. - Malhotra, G. K. - Schaum, N. - Huang, J. - Ved, U. - Beausejour, C. M. - Coppe, J. P. - Rodier, F. - Campisi, J.
Journal: J Cell Biol

Cellular senescence irreversibly arrests proliferation in response to potentially oncogenic stress. Senescent cells also secrete inflammatory cytokines such as IL-6, which promote age-associated inflammation and pathology. HMGB1 (high mobility group box 1) modulates gene expression in the nucleus, but certain immune cells secrete HMGB1 as an extracellular Alarmin to signal tissue damage. We show that nuclear HMGB1 relocalized to the extracellular milieu in senescent human and mouse cells in culture and in vivo. In contrast to cytokine secretion, HMGB1 redistribution required the p53 tumor suppressor, but not its activator ATM. Moreover, altered HMGB1 expression induced a p53-dependent senescent growth arrest. Senescent fibroblasts secreted oxidized HMGB1, which stimulated cytokine secretion through TLR-4 signaling. HMGB1 depletion, HMGB1 blocking antibody, or TLR-4 inhibition attenuated senescence-associated IL-6 secretion, and exogenous HMGB1 stimulated NF-kappaB activity and restored IL-6 secretion to HMGB1-depleted cells. Our findings identify senescence as a novel biological setting in which HMGB1 functions and link HMGB1 redistribution to p53 activity and senescence-associated inflammation.

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The UBXN-2/p37/p47 adaptors of CDC-48/p97 regulate mitosis by limiting the centrosomal recruitment of Aurora A.

Publication Date: 2013 May 13 PMID: 23649807
Authors: Kress, E. - Schwager, F. - Holtackers, R. - Seiler, J. - Prodon, F. - Zanin, E. - Eiteneuer, A. - Toya, M. - Sugimoto, A. - Meyer, H. - Meraldi, P. - Gotta, M.
Journal: J Cell Biol

Coordination of cell cycle events in space and time is crucial to achieve a successful cell division. Here, we demonstrate that UBXN-2, a substrate adaptor of the AAA ATPase Cdc48/p97, is required to coordinate centrosome maturation timing with mitosis. In UBXN-2-depleted Caenorhabditis elegans embryos, centrosomes recruited more AIR-1 (Aurora A), matured precociously, and alignment of the mitotic spindle with the axis of polarity was impaired. UBXN-2 and CDC-48 coimmunoprecipitated with AIR-1 and the spindle alignment defect was partially rescued by co-depleting AIR-1, indicating that UBXN-2 controls these processes via AIR-1. Similarly, depletion in human cells of the UBXN-2 orthologues p37/p47 resulted in an accumulation of Aurora A at centrosomes and a delay in centrosome separation. The latter defect was also rescued by inhibiting Aurora A. We therefore postulate that the role of this adaptor in cell cycle regulation is conserved.

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Gain-of-function mutations of PPM1D/Wip1 impair the p53-dependent G1 checkpoint.

Publication Date: 2013 May 13 PMID: 23649806
Authors: Kleiblova, P. - Shaltiel, I. A. - Benada, J. - Evcik, J. - Pechackova, S. - Pohlreich, P. - Voest, E. E. - Dundr, P. - Bartek, J. - Kleibl, Z. - Medema, R. H. - Macurek, L.
Journal: J Cell Biol

The DNA damage response (DDR) pathway and its core component tumor suppressor p53 block cell cycle progression after genotoxic stress and represent an intrinsic barrier preventing cancer development. The serine/threonine phosphatase PPM1D/Wip1 inactivates p53 and promotes termination of the DDR pathway. Wip1 has been suggested to act as an oncogene in a subset of tumors that retain wild-type p53. In this paper, we have identified novel gain-of-function mutations in exon 6 of PPM1D that result in expression of C-terminally truncated Wip1. Remarkably, mutations in PPM1D are present not only in the tumors but also in other tissues of breast and colorectal cancer patients, indicating that they arise early in development or affect the germline. We show that mutations in PPM1D affect the DDR pathway and propose that they could predispose to cancer.

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The asymmetric segregation of damaged proteins is stem cell-type dependent.

Publication Date: 2013 May 13 PMID: 23649805
Authors: Bufalino, M. R. - Deveale, B. - van der Kooy, D.
Journal: J Cell Biol

Asymmetric segregation of damaged proteins (DPs) during mitosis has been linked in yeast and bacteria to the protection of one cell from aging. Recent evidence suggests that stem cells may use a similar mechanism; however, to date there is no in vivo evidence demonstrating this effect in healthy adult stem cells. We report that stem cells in larval (neuroblast) and adult (female germline and intestinal stem cell) Drosophila melanogaster asymmetrically segregate DPs, such as proteins with the difficult-to-degrade and age-associated 2,4-hydroxynonenal (HNE) modification. Surprisingly, of the cells analyzed only the intestinal stem cell protects itself by segregating HNE to differentiating progeny, whereas the neuroblast and germline stem cells retain HNE during division. This led us to suggest that chronological life span, and not cell type, determines the amount of DPs a cell receives during division. Furthermore, we reveal a role for both niche-dependent and -independent mechanisms of asymmetric DP division.

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Defective nuclear import of Tpr in Progeria reflects the Ran sensitivity of large cargo transport.

Publication Date: 2013 May 13 PMID: 23649804
Authors: Snow, C. J. - Dar, A. - Dutta, A. - Kehlenbach, R. H. - Paschal, B. M.
Journal: J Cell Biol

The RanGTPase acts as a master regulator of nucleocytoplasmic transport by controlling assembly and disassembly of nuclear transport complexes. RanGTP is required in the nucleus to release nuclear localization signal (NLS)-containing cargo from import receptors, and, under steady-state conditions, Ran is highly concentrated in the nucleus. We previously showed the nuclear/cytoplasmic Ran distribution is disrupted in Hutchinson-Gilford Progeria syndrome (HGPS) fibroblasts that express the Progerin form of lamin A, causing a major defect in nuclear import of the protein, translocated promoter region (Tpr). In this paper, we show that Tpr import was mediated by the most abundant import receptor, KPNA2, which binds the bipartite NLS in Tpr with nanomolar affinity. Analyses including NLS swapping revealed Progerin did not cause global inhibition of nuclear import. Rather, Progerin inhibited Tpr import because transport of large protein cargoes was sensitive to changes in the Ran nuclear/cytoplasmic distribution that occurred in HGPS. We propose that defective import of large protein complexes with important roles in nuclear function may contribute to disease-associated phenotypes in Progeria.

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EphA2 cleavage by MT1-MMP triggers single cancer cell invasion via homotypic cell repulsion.

Publication Date: 2013 Apr 29 PMID: 23629968
Authors: Sugiyama, N. - Gucciardo, E. - Tatti, O. - Varjosalo, M. - Hyytiainen, M. - Gstaiger, M. - Lehti, K.
Journal: J Cell Biol

Changes in EphA2 signaling can affect cancer cell-cell communication and motility through effects on actomyosin contractility. However, the underlying cell-surface interactions and molecular mechanisms of how EphA2 mediates these effects have remained unclear. We demonstrate here that EphA2 and membrane-anchored membrane type-1 matrix metalloproteinase (MT1-MMP) were selectively up-regulated and coexpressed in invasive breast carcinoma cells, where, upon physical interaction in same cell-surface complexes, MT1-MMP cleaved EphA2 at its Fibronectin type-III domain 1. This cleavage, coupled with EphA2-dependent Src activation, triggered intracellular EphA2 translocation, as well as an increase in RhoA activity and cell junction disassembly, which suggests an overall repulsive effect between cells. Consistent with this, cleavage-prone EphA2-D359I mutant shifted breast carcinoma cell invasion from collective to rounded single-cell invasion within collagen and in vivo. Up-regulated MT1-MMP also codistributed with intracellular EphA2 in invasive cells within human breast carcinomas. These results reveal a new proteolytic regulatory mechanism of cell-cell signaling in cancer invasion.

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Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds.

Publication Date: 2013 Apr 29 PMID: 23629967
Authors: Martinelli, R. - Kamei, M. - Sage, P. T. - Massol, R. - Varghese, L. - Sciuto, T. - Toporsian, M. - Dvorak, A. M. - Kirchhausen, T. - Springer, T. A. - Carman, C. V.
Journal: J Cell Biol

Basic mechanisms by which cellular barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust barrier function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique ventral lamellipodia that propagate via integrins toward and across these "micro-wounds" to close them. This novel actin remodeling activity progressively healed multiple micro-wounds in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that ventral lamellipodia formed as a response to force imbalance and specifically loss of isometric tension. Ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that barrier disruptions are detected as local release of isometric tension/force unloading, which is directly coupled to reactive oxygen species-dependent self-restorative actin remodeling dynamics.

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Deacetylation of p53 induces autophagy by suppressing Bmf expression.

Publication Date: 2013 Apr 29 PMID: 23629966
Authors: Contreras, A. U. - Mebratu, Y. - Delgado, M. - Montano, G. - Hu, C. A. - Ryter, S. W. - Choi, A. M. - Lin, Y. - Xiang, J. - Chand, H. - Tesfaigzi, Y.
Journal: J Cell Biol

Interferon gamma (IFN-gamma)-induced cell death is mediated by the BH3-only domain protein, Bik, in a p53-independent manner. However, the effect of IFN-gamma on p53 and how this affects autophagy have not been reported. The present study demonstrates that IFN-gamma down-regulated expression of the BH3 domain-only protein, Bmf, in human and mouse airway epithelial cells in a p53-dependent manner. p53 also suppressed Bmf expression in response to other cell death-stimulating agents, including ultraviolet radiation and histone deacetylase inhibitors. IFN-gamma did not affect Bmf messenger RNA half-life but increased nuclear p53 levels and the interaction of p53 with the Bmf promoter. IFN-gamma-induced interaction of HDAC1 and p53 resulted in the deacetylation of p53 and suppression of Bmf expression independent of p53's proline-rich domain. Suppression of Bmf facilitated IFN-gamma-induced autophagy by reducing the interaction of Beclin-1 and Bcl-2. Furthermore, autophagy was prominent in cultured bmf(-/-) but not in bmf(+/+) cells. Collectively, these observations show that deacetylation of p53 suppresses Bmf expression and facilitates autophagy.

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Heterochromatin protein 1 promotes self-renewal and triggers regenerative proliferation in adult stem cells.

Publication Date: 2013 Apr 29 PMID: 23629965
Authors: Zeng, A. - Li, Y. Q. - Wang, C. - Han, X. S. - Li, G. - Wang, J. Y. - Li, D. S. - Qin, Y. W. - Shi, Y. - Brewer, G. - Jing, Q.
Journal: J Cell Biol

Adult stem cells (ASCs) capable of self-renewal and differentiation confer the potential of tissues to regenerate damaged parts. Epigenetic regulation is essential for driving cell fate decisions by rapidly and reversibly modulating gene expression programs. However, it remains unclear how epigenetic factors elicit ASC-driven regeneration. In this paper, we report that an RNA interference screen against 205 chromatin regulators identified 12 proteins essential for ASC function and regeneration in planarians. Surprisingly, the HP1-like protein SMED-HP1-1 (HP1-1) specifically marked self-renewing, pluripotent ASCs, and HP1-1 depletion abrogated self-renewal and promoted differentiation. Upon injury, HP1-1 expression increased and elicited increased ASC expression of Mcm5 through functional association with the FACT (facilitates chromatin transcription) complex, which consequently triggered proliferation of ASCs and initiated blastema formation. Our observations uncover an epigenetic network underlying ASC regulation in planarians and reveal that an HP1 protein is a key chromatin factor controlling stem cell function. These results provide important insights into how epigenetic mechanisms orchestrate stem cell responses during tissue regeneration.

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The level of origin firing inversely affects the rate of replication fork progression.

Publication Date: 2013 Apr 29 PMID: 23629964
Authors: Zhong, Y. - Nellimoottil, T. - Peace, J. M. - Knott, S. R. - Villwock, S. K. - Yee, J. M. - Jancuska, J. M. - Rege, S. - Tecklenburg, M. - Sclafani, R. A. - Tavare, S. - Aparicio, O. M.
Journal: J Cell Biol

DNA damage slows DNA synthesis at replication forks; however, the mechanisms remain unclear. Cdc7 kinase is required for replication origin activation, is a target of the intra-S checkpoint, and is implicated in the response to replication fork stress. Remarkably, we found that replication forks proceed more rapidly in cells lacking Cdc7 function than in wild-type cells. We traced this effect to reduced origin firing, which results in fewer replication forks and a consequent decrease in Rad53 checkpoint signaling. Depletion of Orc1, which acts in origin firing differently than Cdc7, had similar effects as Cdc7 depletion, consistent with decreased origin firing being the source of these defects. In contrast, mec1-100 cells, which initiate excess origins and also are deficient in checkpoint activation, showed slower fork progression, suggesting the number of active forks influences their rate, perhaps as a result of competition for limiting factors.

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Stress granules as crucibles of ALS pathogenesis.

Publication Date: 2013 Apr 29 PMID: 23629963
Authors: Li, Y. R. - King, O. D. - Shorter, J. - Gitler, A. D.
Journal: J Cell Biol

Amyotrophic lateral sclerosis (ALS) is a fatal human neurodegenerative disease affecting primarily motor neurons. Two RNA-binding proteins, TDP-43 and FUS, aggregate in the degenerating motor neurons of ALS patients, and mutations in the genes encoding these proteins cause some forms of ALS. TDP-43 and FUS and several related RNA-binding proteins harbor aggregation-promoting prion-like domains that allow them to rapidly self-associate. This property is critical for the formation and dynamics of cellular ribonucleoprotein granules, the crucibles of RNA metabolism and homeostasis. Recent work connecting TDP-43 and FUS to stress granules has suggested how this cellular pathway, which involves protein aggregation as part of its normal function, might be coopted during disease pathogenesis.

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Carla Koehler: Small TIMs are a big deal.

Publication Date: 2013 Apr 29 PMID: 23629962
Authors: Sedwick, C.
Journal: J Cell Biol

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A tumor suppressor role of the Bub3 spindle checkpoint protein after apoptosis inhibition.

Publication Date: 2013 Apr 29 PMID: 23609535
Authors: Morais da Silva, S. - Moutinho-Santos, T. - Sunkel, C. E.
Journal: J Cell Biol

Most solid tumors contain aneuploid cells, indicating that the mitotic checkpoint is permissive to the proliferation of chromosomally aberrant cells. However, mutated or altered expression of mitotic checkpoint genes accounts for a minor proportion of human tumors. We describe a Drosophila melanogaster tumorigenesis model derived from knocking down spindle assembly checkpoint (SAC) genes and preventing apoptosis in wing imaginal discs. Bub3-deficient tumors that were also deficient in apoptosis displayed neoplastic growth, chromosomal aneuploidy, and high proliferative potential after transplantation into adult flies. Inducing aneuploidy by knocking down CENP-E and preventing apoptosis does not induce tumorigenesis, indicating that aneuploidy is not sufficient for hyperplasia. In this system, the aneuploidy caused by a deficient SAC is not driving tumorigenesis because preventing Bub3 from binding to the kinetochore does not cause hyperproliferation. Our data suggest that Bub3 has a nonkinetochore-dependent function that is consistent with its role as a tumor suppressor.

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Sanpodo controls sensory organ precursor fate by directing Notch trafficking and binding gamma-secretase.

Publication Date: 2013 Apr 29 PMID: 23609534
Authors: Upadhyay, A. - Kandachar, V. - Zitserman, D. - Tong, X. - Roegiers, F.
Journal: J Cell Biol

In Drosophila peripheral neurogenesis, Notch controls cell fates in sensory organ precursor (SOP) cells. SOPs undergo asymmetric cell division by segregating Numb, which inhibits Notch signaling, into the pIIb daughter cell after cytokinesis. In contrast, in the pIIa daughter cell, Notch is activated and requires Sanpodo, but its mechanism of action has not been elucidated. As Sanpodo is present in both pIIa and pIIb cells, a second role for Sanpodo in regulating Notch signaling in the low-Notch pIIb cell has been proposed. Here we demonstrate that Sanpodo regulates Notch signaling levels in both pIIa and pIIb cells via distinct mechanisms. The interaction of Sanpodo with Presenilin, a component of the gamma-secretase complex, was required for Notch activation and pIIa cell fate. In contrast, Sanpodo suppresses Notch signaling in the pIIb cell by driving Notch receptor internalization. Together, these results demonstrate that a single protein can regulate Notch signaling through distinct mechanisms to either promote or suppress signaling depending on the local cellular context.

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DNA synthesis by Pol eta promotes fragile site stability by preventing under-replicated DNA in mitosis.

Publication Date: 2013 Apr 29 PMID: 23609533
Authors: Bergoglio, V. - Boyer, A. S. - Walsh, E. - Naim, V. - Legube, G. - Lee, M. Y. - Rey, L. - Rosselli, F. - Cazaux, C. - Eckert, K. A. - Hoffmann, J. S.
Journal: J Cell Biol

Human DNA polymerase eta (Pol eta) is best known for its role in responding to UV irradiation-induced genome damage. We have recently observed that Pol eta is also required for the stability of common fragile sites (CFSs), whose rearrangements are considered a driving force of oncogenesis. Here, we explored the molecular mechanisms underlying this newly identified role. We demonstrated that Pol eta accumulated at CFSs upon partial replication stress and could efficiently replicate non-B DNA sequences within CFSs. Pol eta deficiency led to persistence of checkpoint-blind under-replicated CFS regions in mitosis, detectable as FANCD2-associated chromosomal sites that were transmitted to daughter cells in 53BP1-shielded nuclear bodies. Expression of a catalytically inactive mutant of Pol eta increased replication fork stalling and activated the replication checkpoint. These data are consistent with the requirement of Pol eta-dependent DNA synthesis during S phase at replication forks stalled in CFS regions to suppress CFS instability by preventing checkpoint-blind under-replicated DNA in mitosis.

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