Molecular Biology of the Cell

A Myosin IK-Abp1-PakB Circuit Acts as a Switch to Regulate Phagocytosis Efficiency.

Publication Date: 2010 Mar 3 PMID: 20200225
Authors: Dieckmann, R. - von Heyden, Y. - Kistler, C. - Gopaldass, N. - Hausherr, S. - Crawley, S. W. - Schwarz, E. C. - Diensthuber, R. P. - Cote, G. P. - Tsiavaliaris, G. - Soldati, T.
Journal: Mol Biol Cell

Monitoring Editor: Carole Parent Actin dynamics and myosin contractile forces are necessary for formation and closure of the phagocytic cup. In Dictyostelium, the actin-binding protein Abp1 and myosinIK are enriched in the closing cup and especially at an actin-dense constriction furrow formed around the neck of engulfed budded yeasts. This phagocytic furrow consists of concentric overlapping rings of MyoK, Abp1, Arp3, coronin and myosin II, following an order strikingly reminiscent of the overall organization of the lamellipodium of migrating cells. Mutation analyses of MyoK revealed that both a C-terminal farnesylation membrane anchor and a Gly-ProArg domain that interacts with profilin and Abp1, were necessary for proper localization in the furrow and efficient phagocytosis. Consequently, we measured the binding affinities of these interactions and unraveled further interactions with profilins, dynamin A and PakB. Due to the redundancy of the interaction network, we hypothesize that MyoK and Abp1 are restricted to regulatory roles and might affect the dynamic of cup progression. Indeed, phagocytic uptake was regulated antagonistically by MyoK and Abp1. MyoK is phosphorylated by PakB and positively regulates phagocytosis, whereas binding of Abp1 negatively regulates PakB and MyoK. We conclude that a MyoK-Abp1-PakB circuit acts as a switch regulating phagocytosis efficiency of large particles.

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Requirements for Transitional Endoplasmic Reticulum (tER) Site Structure and Function in Saccharomyces cerevisiae.

Publication Date: 2010 Mar 3 PMID: 20200224
Authors: Shindiapina, P. - Barlowe, C.
Journal: Mol Biol Cell

Monitoring Editor: Benjamin S. Glick Secretory proteins are exported from the ER at specialized regions known as the transitional ER (tER). COPII proteins are enriched at tER sites although the mechanisms underlying tER site assembly and maintenance are not understood. Here we investigated the dynamic properties of tER sites in Saccharomyces cerevisiae and probed protein and lipid requirements for tER site structure and function. Thermosensitive sec12 and sec16 mutations caused a collapse of tER sites in a manner that depended on nascent secretory cargo. Continual fatty acid synthesis was required for ER export and for normal tER site structure, whereas inhibition of sterol and ceramide synthesis produced minor effects. An in vitro assay to monitor assembly of Sec23p-GFP at tER sites was established to directly test requirements. tER sites remained active for approximately 10 min in vitro and depended on Sec12p function. Bulk phospholipids were also required for tER site structure and function in vitro whereas depletion of phophatidyl inositol selectively inhibited COPII budding but not assembly of tER site structures. These results indicate that tER sites persist through relatively stringent treatments in which COPII budding was strongly inhibited. We propose that tER site structures are stable elements that are assembled on an underlying protein and lipid scaffold.

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PKC{zeta} Regulates CDK5/P25 Signaling during Myogenesis.

Publication Date: 2010 Mar 3 PMID: 20200223
Authors: de Thonel, A. - Ferraris, S. E. - Pallari, H. M. - Imanishi, S. Y. - Kochin, V. - Hosokawa, T. - Hisanga, S. I. - Sahlgren, C. - Eriksson, J. E.
Journal: Mol Biol Cell

Monitoring Editor: M. Bishr Omary Atypical protein kinase Czeta (PKCzeta) is emerging as a mediator of differentiation. Here, we describe a novel role for PKCzeta in in myogenic differentiation, demonstrating that PKCzeta activity is indispensable for differentiation of both C2C12 and mouse primary myoblasts. PKCzeta was found to be associated with and to regulate the Cdk5/p35 signaling complex, an essential factor for both neuronal and myogenic differentiation. Inhibition of PKCzeta activity prevented both myotube formation and simultaneous reorganization of the nestin intermediate filament cytoskeleton, which is known to be regulated by Cdk5 during myogenesis. p35, the Cdk5 activator, was shown to be a specific phosphorylation target of PKCzeta. PKCzeta-mediated phosphorylation of Ser-33 on p35 promoted calpain-mediated cleavage of p35 to its more active and stable fragment, p25. Strikingly, both calpain activation and the calpain-mediated cleavage of p35 were shown to be PKCzeta-dependent in differentiating myoblasts. Overall, our results identify PKCzeta as a controller of myogenic differentiation by its regulation of the phosphorylation-dependent and calpain-mediated p35 cleavage, which is crucial for the amplification of the Cdk5 activity that is required during differentiation.

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LRPPRC and SLIRP Interact in a Ribonucleoprotein Complex that Regulates Posttranscriptional Gene Expression in Mitochondria.

Publication Date: 2010 Mar 3 PMID: 20200222
Authors: Sasarman, F. - Brunel-Guitton, C. - Antonicka, H. - Wai, T. - Shoubridge, E. A. - Consortium, L.
Journal: Mol Biol Cell

Monitoring Editor: Thomas D. Fox Mutations in LRPPRC are responsible for the French Canadian variant of Leigh syndrome (LSFC), a neurodegenerative disorder caused by a tissue-specific deficiency in cytochrome c oxidase (COX). To investigate the pathogenic mechanism of disease we studied LRPPRC function in LSFC and control fibroblasts. The level of mutated LRPPRC is reduced in LSFC cells, and this results in decreased steady-state levels of most mitochondrial mRNAs, but not rRNAs or tRNAs, a phenotype that can be reproduced by siRNA-mediated knockdown of LRPPRC in control cells. Processing of the primary transcripts appears normal. The resultant defect in mitochondrial protein synthesis in LSFC cells disproportionately affects the COX subunits, leading to an isolated COX assembly defect. Further knockdown of LRPPRC produces a generalized assembly defect in all oxidative phosphorylation complexes containing mtDNA-encoded subunits, due to a severe decrease in all mitochondrial mRNAs. LRPPRC exists in a high molecular weight complex and it coimmunoprecipates with SLIRP, a stem-loop RNA binding protein. While this interaction does not depend on mitochondrial mRNA, both proteins show reduced stability in its absence. These results implicate LRPPRC in posttranscriptional mitochondrial gene expression as part of a ribonucleoprotein (RNP) complex that regulates the stability and handling of mature mRNAs.

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Retraction. Old yellow enzyme protects the actin cytoskeleton from oxidative stress.

Publication Date: 2010 Mar PMID: 20185771
Authors:
Journal: Mol Biol Cell

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Binding to the extracellular matrix and proteolytic processing: two key mechanisms regulating vascular endothelial growth factor action.

Publication Date: 2010 Mar PMID: 20185770
Authors: Ferrara, N.
Journal: Mol Biol Cell

Vascular endothelial growth factor (VEGF, VEGF-A) is a major regulator of physiological and pathological angiogenesis. One feature of VEGF is the existence of multiple isoforms arising from alternative exon splicing. Our initial biochemical and biological studies indicated that such isoforms are uniquely suited to generate angiogenic gradients by virtue of their differential ability to interact with the extracellular matrix (ECM). Although ECM-bound VEGF was bioactive, processing by physiologically relevant proteases such as plasmin was identified as a key mechanism to convert ECM-bound VEGF into freely diffusible forms. This retrospective article examines the early studies and also emphasizes the subsequent progress in our understanding of these processes in health and disease.

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h3/Acidic Calponin: An Actin-binding Protein that Controls ERK1/2 Activity in Non-muscle Cells.

Publication Date: 2010 Feb 24 PMID: 20181831
Authors: Appel, S. - Allen, P. G. - Vetterkind, S. - Jin, J. P. - Morgan, K. G.
Journal: Mol Biol Cell

Monitoring Editor: Josephine C. Adams Migration of fibroblasts is important in wound healing. Here, we demonstrate a role and a mechanism for h3/acidic calponin (aCaP, CNN3) in REF52.2 cell motility, a fibroblast line rich in actin filaments. We show that the actin binding protein, h3/acidic calponin, associates with stress fibers in the absence of stimulation but is targeted to the cell cortex and podosome-like structures after stimulation with a phorbol ester, PDBu. By coimmunoprecipitation and colocalization, we show that ERK1/2 and PKCalpha constitutively associate with h3/acidic calponin and are cotargeted with h3/acidic calponin in the presence of PDBu. This targeting can be blocked by a PKC inhibitor, but does not require phosphorylation of h3/acidic calponin at the PKC-sites S175 or T184. Knockdown of h3/acidic calponin results in a loss of PDBu-mediated ERK1/2 targeting, whereas PKCalpha targeting is unaffected. Caldesmon is an actin binding protein that regulates actomyosin interactions and is a known substrate of ERK1/2. Both ERK1/2 activity and nonmuscle l-caldesmon phosphorylation are blocked by h3/acidic calponin knockdown. Furthermore, h3/acidic calponin knockdown inhibits REF52.2 migration in an in vitro wound healing assay. Our findings are consistent with a model whereby h3/acidic calponin controls fibroblast migration by regulation of ERK1/2-mediated l-caldesmon phosphorylation.

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Mso1p Regulates Membrane Fusion through Interactions with the Putative N-Peptide Binding Area in Sec1p Domain 1.

Publication Date: 2010 Feb 24 PMID: 20181830
Authors: Weber, M. - Chernov, K. - Turakainen, H. - Wohlfahrt, G. - Pajunen, M. - Savilahti, H. - Jantti, J.
Journal: Mol Biol Cell

Monitoring Editor: Thomas F.J. Martin Sec1p/Munc18 (SM) family proteins regulate SNARE complex function in membrane fusion through their interactions with syntaxins. In addition to syntaxins, only a few SM protein interacting proteins are known and typically, their binding modes with SM proteins are poorly characterized. We previously identified Mso1p as a Sec1p binding protein and showed that it is involved in membrane fusion regulation. Here we demonstrate that Mso1p and Sec1p interact at sites of exocytosis and that the Mso1p-Sec1p interaction site depends on a functional rab GTPase Sec4p and its GEF Sec2p. Random and targeted mutagenesis of Sec1p, followed by analysis of protein interactions, indicates that Mso1p interacts with Sec1p domain 1 and that this interaction is important for membrane fusion. In many SM family proteins, domain 1 binds to a N-terminal peptide of a syntaxin family protein. The Sec1p interacting syntaxins Sso1p and Sso2p lack the N-terminal peptide. We show that the putative N-peptide binding area in Sec1p domain 1 is important for Mso1p binding, and that Mso1p can interact with Sso1p and Sso2p. Our results suggest that Mso1p mimics N-peptide binding to facilitate membrane fusion.

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GLUT4 Is Sorted to Vesicles Whose Accumulation Beneath and Insertion into the Plasma Membrane Are Differentially Regulated by Insulin and Selectively Affected by Insulin Resistance.

Publication Date: 2010 Feb 24 PMID: 20181829
Authors: Xiong, W. - Jordens, I. - Gonzalez, E. - McGraw, T. E.
Journal: Mol Biol Cell

Monitoring Editor: Sandra Lemmon Insulin stimulates glucose transport by recruiting the GLUT4 glucose transporter to the plasma membrane. Here we use total internal reflection fluorescence microscopy to show that two trafficking motifs of GLUT4, a FQQI motif and a TELE-based motif, target GLUT4 to specialized vesicles that accumulate adjacent to the plasma membrane of unstimulated adipocytes. Mutations of these motifs redistributed GLUT4 to transferrin-containing recycling vesicles adjacent to the plasma membrane, and the degree of redistribution correlated with the increases of the GLUT4 mutants in the plasma membrane of basal adipocytes. These results establish that GLUT4 defaults to recycling endosomes when trafficking to specialized vesicles is disrupted, supporting the hypothesis that the specialized vesicles are derived from an endosomal compartment. Insulin stimulates both the accumulation of GLUT4 in the evanescent field and the fraction of this GLUT4 that is inserted into the plasma membrane. Unexpectedly, these two steps are differentially affected by the development of insulin resistance. We ascribe this selective insulin resistance to inherent differences in the sensitivities of GLUT4 vesicle accumulation and insertion into the plasma membrane to insulin. Differences in insulin sensitivities of various processes may be a general mechanism for the development of the physiologically important phenomenon of selective insulin resistance.

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Deficiency of the Transcriptional Regulator p8 Results in Increased Autophagy and Apoptosis, and Causes Impaired Heart Function.

Publication Date: 2010 Feb 24 PMID: 20181828
Authors: Kong, D. K. - Georgescu, S. P. - Cano, C. - Aronovitz, M. J. - Iovanna, J. L. - Patten, R. D. - Kyriakis, J. M. - Goruppi, S.
Journal: Mol Biol Cell

Monitoring Editor: Jonathan Chernoff Autophagy is a cytoprotective pathway used to degrade and recycle cytoplasmic content. Dysfunctional autophagy has been linked to both cancer and cardiomyopathies. Here we show a role for the transcriptional regulator p8 in autophagy. P8 RNAi increases basal autophagy markers in primary cardiomyocytes, in H9C2 and U2OS cells, and decreases cellular viability after autophagy induction. This autophagy is associated with caspase activation, and is blocked by atg5 silencing and by pharmacological inhibitors. FoxO3 transcription factor was reported to activate autophagy by enhancing the expression of autophagy-related genes. P8 expression represses FoxO3 transcriptional activity, and p8 knockdown affects FoxO3 nuclear localization. Thus, p8 RNAi increases FoxO3 association with bnip3 promoter, a known proautophagic FoxO3 target, resulting in higher bnip3 RNA and protein levels. Accordingly, bnip3 knockdown restores cell viability and blocks apoptosis of p8-deficient cells. In vivo, p8 (-/-) mice have higher autophagy and express higher cardiac bnip3 levels. These mice develop left ventricular wall thinning and chamber dilation, with consequent impaired cardiac function. Our studies provide evidence of a p8-dependent mechanism regulating autophagy by acting as FoxO3 corepressor, which may be relevant for diseases associated with dysregulated autophagy, as cardiovascular pathologies and cancer.

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Arginylation Regulates Intracellular Actin Polymer Level by Modulating Actin Properties and Binding of Capping and Severing Proteins.

Publication Date: 2010 Feb 24 PMID: 20181827
Authors: Saha, S. - Mundia, M. M. - Zhang, F. - Demers, R. W. - Korobova, F. - Svitkina, T. - Perieteanu, A. A. - Dawson, J. F. - Kashina, A.
Journal: Mol Biol Cell

Monitoring Editor: David G. Drubin Actin arginylation regulates lamella formation in motile fibroblasts, but the underlying molecular mechanisms are unknown. To understand how arginylation affects the actin cytoskeleton, we investigated the biochemical properties and the structural organization of actin filaments in wild type and arginyltransferase (Ate1) knockout cells. We found that Ate1 knockout results in a dramatic reduction of the actin polymer levels in vivo accompanied by a corresponding increase in the monomer level. Purified nonarginylated actin has altered polymerization properties, and actin filaments from Ate1 knockout cells show altered interactions with several associated proteins. Ate1 knockout cells have severe impairment of cytoskeletal organization throughout the cell. Thus, arginylation regulates the ability of actin to form filaments in the whole cell rather than preventing the collapse of pre-formed actin networks at the cell leading edge as proposed in our previous model. This regulation is achieved through interconnected mechanisms that involve actin polymerization per se, and binding of actin-associated proteins.

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Septin 14 Is Involved in Cortical Neuronal Migration via Interaction with Septin 4.

Publication Date: 2010 Feb 24 PMID: 20181826
Authors: Shinoda, T. - Ito, H. - Sudo, K. - Iwamoto, I. - Morishita, R. - Nagata, K. I.
Journal: Mol Biol Cell

Monitoring Editor: Josephine C. Adams Septins are a family of conserved GTP/GDP-binding proteins implicated in a variety of cellular functions such as cell cycle control and cytokinesis. Although several members of septin family, including Septin 14 (Sept14), are abundantly expressed in nervous tissues, little is known about their physiological functions, especially in neuronal development. Here we report that Sept14 is strongly expressed in the cortical plate of developing cerebral cortex. Knockdown experiments by using the method of in utero electroporation showed that reduction of Sept14 caused inhibition of cortical neuronal migration. Whereas cDNA encoding RNAi-resistant Sept14 rescued the migration defect, the C-terminal deletion mutant of Sept14 did not. Biochemical analyses revealed that C-terminal coiled-coil region of Sept14 interacts with Septin 4 (Sept4). Knockdown experiments showed that Sept4 is also involved in cortical neuronal migration in vivo. In addition, knockdown of Sept14 or Sept4 inhibited leading process formation in migrating cortical neurons. These results suggest that Sept14 is involved in neuronal migration in cerebral cortex via interaction with Sept4.

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The Protein Tyrosine Phosphatase PTP1B Is Required for Efficient Delivery of N-Cadherin to the Cell Surface.

Publication Date: 2010 Feb 24 PMID: 20181825
Authors: Hernandez, M. V. - Wehrendt, D. P. - Arregui, C. O.
Journal: Mol Biol Cell

Monitoring Editor: Vivek Malhotra PTP1B bound to mature N-cadherin promotes the association of beta-catenin into the complex, the stable expression of the complex at cell surface, and cadherin-mediated adhesion. Here we show that PTP1B is also required for N-cadherin precursor trafficking through early stages of the secretory pathway. This function does not require association of PTP1B with the precursor. In PTP1B null cells, the N-cadherin precursor showed higher sensitivity to endoglycosidase H than in cells reconstituted with the wild type enzyme. It also showed slower kinetics of ER to Golgi translocation and processing. Trafficking of the viral stomatitis vesicular glycoprotein, VSV-G, however, revealed no differences between PTP1B null and reconstituted cells. N-cadherin precursor complexes contained similar levels of alpha- and beta-catenin regardless of PTP1B expression. In contrast, the associated p120 catenin (p120) was significantly reduced in absence of PTP1B expression. An N-cadherin precursor construct defective in p120 binding, and expressed in PTP1B reconstituted cells, showed higher sensitivity to endoglycosidase H and slower kinetics of processing than the wild type precursor. Our results suggest that PTP1B promotes the association of p120 to the N-cadherin precursor, facilitating the trafficking of the complex from the ER to the Golgi complex.

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Tight Junction-associated MARVEL Proteins MarvelD3, Tricellulin, and Occludin Have Distinct but Overlapping Functions.

Publication Date: 2010 Feb 17 PMID: 20164257
Authors: Raleigh, D. R. - Marchiando, A. M. - Zhang, Y. - Shen, L. - Sasaki, H. - Wang, Y. - Long, M. - Turner, J. R.
Journal: Mol Biol Cell

Monitoring Editor: M. Bishr Omary In vitro studies have demonstrated that occludin and tricellulin are important for tight junction barrier function, but in vivo data suggest that loss of these proteins can be overcome. The presence of a heretofore unknown, yet related, protein could explain these observations. Here, we report marvelD3, a novel tight junction protein that, like occludin and tricellulin, contains a conserved four-transmembrane MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain. Phylogenetic tree reconstruction; analysis of RNA and protein tissue distribution; immunofluorescent and electron microscopic examination of subcellular localization; characterization of intracellular trafficking, protein interactions, dynamic behavior, and siRNA knockdown effects; and description of remodeling following in vivo immune activation show that marvelD3, occludin, and tricellulin have distinct but overlapping functions at the tight junction. Although marvelD3 is able to partially compensate for occludin or tricellulin loss, it cannot fully restore function. We conclude that marvelD3, occludin, and tricellulin define the tight junction-associated MARVEL protein (TAMP) family. The data further suggest that these proteins are best considered as a group with both redundant and unique contributions to epithelial function and tight junction regulation.

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p23/Tmp21 Differentially Targets the Rac-GAP {beta}2-Chimaerin and Protein Kinase C via their C1 Domains.

Publication Date: 2010 Feb 17 PMID: 20164256
Authors: Wang, H. - Kazanietz, M. G.
Journal: Mol Biol Cell

Monitoring Editor: J. Silvio Gutkind The C1 domains in PKC isozymes and other signaling molecules are responsible for binding the lipid second messenger diacylglycerol and phorbol esters, and mediate translocation to membranes. Previous studies revealed that the C1 domain in alpha- and beta-chimaerins, diacylglycerol-regulated Rac-GAPs, interacts with the ER/Golgi protein p23/Tmp21. Here we found that p23/Tmp21 acts as a C1 domain-docking protein that mediates perinuclear translocation of beta2-chimaerin. Glu227 and Leu248 in the beta2-chimaerin C1 domain are crucial for binding p23/Tmp21 and perinuclear targeting. Interestingly, isolated C1 domains from individual PKC isozymes differentially interact with p23/Tmp21. In the case of PKCepsilon, it interacts with p23/Tmp21 specifically via its C1b domain, however this association is lost in response to phorbol esters. These results demonstrate that p23/Tmp21 acts as an anchor that distinctively modulates compartmentalization of C1 domain-containing proteins, and it plays an essential role in beta2-chimaerin relocalization. Our study also highlights the relevance of C1 domains in protein-protein interactions in addition to their well-established lipid-binding properties.

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Evolutionary gain of function for the ER membrane protein Sec62 from yeast to humans.

Publication Date: 2010 Mar PMID: 20071467
Authors: Muller, L. - de Escauriaza, M. D. - Lajoie, P. - Theis, M. - Jung, M. - Muller, A. - Burgard, C. - Greiner, M. - Snapp, E. L. - Dudek, J. - Zimmermann, R.
Journal: Mol Biol Cell

Because of similarity to their yeast orthologues, the two membrane proteins of the human endoplasmic reticulum (ER) Sec62 and Sec63 are expected to play a role in protein biogenesis in the ER. We characterized interactions between these two proteins as well as the putative interaction of Sec62 with ribosomes. These data provide further evidence for evolutionary conservation of Sec62/Sec63 interaction. In addition, they indicate that in the course of evolution Sec62 of vertebrates has gained an additional function, the ability to interact with the ribosomal tunnel exit and, therefore, to support cotranslational mechanisms such as protein transport into the ER. This view is supported by the observation that Sec62 is associated with ribosomes in human cells. Thus, the human Sec62/Sec63 complex and the human ER membrane protein ERj1 are similar in providing binding sites for BiP in the ER-lumen and binding sites for ribosomes in the cytosol. We propose that these two systems provide similar chaperone functions with respect to different precursor proteins.

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Glycolipid trafficking in Drosophila undergoes pathway switching in response to aberrant cholesterol levels.

Publication Date: 2010 Mar PMID: 20053687
Authors: Hortsch, R. - Lee, E. - Erathodiyil, N. - Hebbar, S. - Steinert, S. - Lee, J. Y. - Chua, D. S. - Kraut, R.
Journal: Mol Biol Cell

In lipid storage diseases, the intracellular trafficking of sphingolipids is altered by conditions of aberrant cholesterol accumulation. Drosophila has been used recently to model lipid storage diseases, but the effects of sterol accumulation on sphingolipid trafficking are not known in the fly, and the trafficking of sphingolipids in general has not been studied in this model organism. Here, we examined the uptake and intracellular distribution of a fluorescent glycolipid analog, BODIPY-lactosyl-ceramide, in Drosophila neurons. The uptake mechanism and intracellular trafficking route of this simple glycolipid are largely conserved. Our principle finding is that cholesterol steers trafficking of the glycolipid between Golgi, lysosome, and recycling compartments. Our analyses support the idea that cholesterol storage in Drosophila triggers a switch in glycolipid trafficking from the biosynthetic to the degradative endolysosomal pathway, whereas cholesterol depletion eliminates recycling of the glycolipid. Unexpectedly, we observe a novel phenomenon we term "hijacking," whereby lactosyl-ceramide diverts the trafficking pathway of an endocytic cargo, dextran, completely away from its lysosomal target. This work establishes that glycolipid trafficking in Drosophila undergoes changes similar to those seen in mammalian cells under conditions of cholesterol storage and therefore validates Drosophila as a suitable model organism in which to study lipid storage diseases.

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Fate specification and tissue-specific cell cycle control of the Caenorhabditis elegans intestine.

Publication Date: 2010 Mar PMID: 20053685
Authors: Segref, A. - Cabello, J. - Clucas, C. - Schnabel, R. - Johnstone, I. L.
Journal: Mol Biol Cell

Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein beta-transducin repeat-containing protein (beta-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans beta-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant.

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Distinct and temporal roles of nucleosomal remodeling and histone deacetylation in the repression of the hTERT gene.

Publication Date: 2010 Mar PMID: 20053684
Authors: Wang, S. - Hu, C. - Zhu, J.
Journal: Mol Biol Cell

hTERT, the human telomerase reverse transcriptase, is highly expressed in stem cells and embryonic tissues but undetectable in most adult somatic cells. To understand its repression mechanisms in somatic cells, we investigated the endogenous hTERT gene regulation during differentiation of human leukemic HL60 cells. Our study revealed that silencing of the hTERT promoter was a biphasic process. Within 24 h after initiation of differentiation, hTERT mRNA expression decreased dramatically, accompanied by increased expression of Mad1 gene and disappearance of a nucleosome-free region at the hTERT core promoter. Subsequent to this early repression, nucleosomal remodeling continued at the promoter and downstream region for several days, as demonstrated by micrococcal nuclease and restriction enzyme accessibility assays. This later nucleosomal remodeling correlated with stable silencing of the hTERT promoter. Progressive changes of core histone modifications occurred throughout the entire differentiation process. Surprisingly, inhibition of histone deacetylation at the hTERT promoter did not prevent hTERT repression or nucleosomal deposition, indicating that nucleosomal deposition at the core promoter, but not histone deacetylation, was the cause of transcriptional repression. Our data also suggested that succeeding nucleosomal remodeling and histone deacetylation worked in parallel to establish the stable repressive status of hTERT gene in human somatic cells.

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Nuclear translocation of beta-actin is involved in transcriptional regulation during macrophage differentiation of HL-60 cells.

Publication Date: 2010 Mar PMID: 20053683
Authors: Xu, Y. Z. - Thuraisingam, T. - Morais, D. A. - Rola-Pleszczynski, M. - Radzioch, D.
Journal: Mol Biol Cell

Studies have shown that nuclear translocation of actin occurs under certain conditions of cellular stress; however, the functional significance of actin import remains unclear. Here, we demonstrate that during the phorbol 12-myristate 13-acetate (PMA)-induced differentiation of HL-60 cells toward macrophages, beta-actin translocates from the cytoplasm to the nucleus and that this process is dramatically inhibited by pretreatment with p38 mitogen-activated protein kinase inhibitors. Using chromatin immunoprecipitation-on-chip assays, the genome-wide maps of beta-actin binding to gene promoters in response to PMA treatment is analyzed in HL-60 cells. A gene ontology-based analysis shows that the identified genes belong to a broad spectrum of functional categories such as cell growth and differentiation, signal transduction, response to external stimulus, ion channel activity, and immune response. We also demonstrate a correlation between beta-actin occupancy and the recruitment of RNA polymerase II at six selected target genes, and beta-actin knockdown decreases the mRNA expression levels of these target genes induced by PMA. We further show that nuclear beta-actin is required for PMA-induced transactivation of one target gene, solute carrier family 11 member 1, which is important for macrophage activation. Our data provide novel evidence that nuclear accumulation of beta-actin is involved in transcriptional regulation during macrophage-like differentiation of HL-60 cells.

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Structural mutants of the spindle pole body cause distinct alteration of cytoplasmic microtubules and nuclear dynamics in multinucleated hyphae.

Publication Date: 2010 Mar PMID: 20053682
Authors: Lang, C. - Grava, S. - Finlayson, M. - Trimble, R. - Philippsen, P. - Jaspersen, S. L.
Journal: Mol Biol Cell

In the multinucleate fungus Ashbya gossypii, cytoplasmic microtubules (cMTs) emerge from the spindle pole body outer plaque (OP) in perpendicular and tangential directions. To elucidate the role of cMTs in forward/backward movements (oscillations) and bypassing of nuclei, we constructed mutants potentially affecting cMT nucleation or stability. Hyphae lacking the OP components AgSpc72, AgNud1, AgCnm67, or the microtubule-stabilizing factor AgStu2 grew like wild- type but showed substantial alterations in the number, length, and/or nucleation sites of cMTs. These mutants differently influenced nuclear oscillation and bypassing. In Agspc72Delta, only long cMTs were observed, which emanate tangentially from reduced OPs; nuclei mainly moved with the cytoplasmic stream but some performed rapid bypassing. Agnud1Delta and Agcnm67Delta lack OPs; short and long cMTs emerged from the spindle pole body bridge/half-bridge structures, explaining nuclear oscillation and bypassing in these mutants. In Agstu2Delta only very short cMTs emanated from structurally intact OPs; all nuclei moved with the cytoplasmic stream. Therefore, long tangential cMTs promote nuclear bypassing and short cMTs are important for nuclear oscillation. Our electron microscopy ultrastructural analysis also indicated that assembly of the OP occurs in a stepwise manner, starting with AgCnm67, followed by AgNud1 and lastly AgSpc72.

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Enhanced H2AX phosphorylation, DNA replication fork arrest, and cell death in the absence of Chk1.

Publication Date: 2010 Mar PMID: 20053681
Authors: Gagou, M. E. - Zuazua-Villar, P. - Meuth, M.
Journal: Mol Biol Cell

H2AX phosphorylation at serine 139 (gammaH2AX) is a sensitive indicator of both DNA damage and DNA replication stress. Here we show that gammaH2AX formation is greatly enhanced in response to replication inhibitors but not ionizing radiation in HCT116 or SW480 cells depleted of Chk1. Although H2AX phosphorylation precedes the induction of apoptosis in such cells, our results suggest that cells containing gammaH2AX are not committed to death. gammaH2AX foci in these cells largely colocalize with RPA foci and their formation is dependent upon the essential replication helicase cofactor Cdc45, suggesting that H2AX phosphorylation occurs at sites of stalled forks. However Chk1-depleted cells released from replication inhibitors retain gammaH2AX foci and do not appear to resume replicative DNA synthesis. BrdU incorporation only occurs in a minority of Chk1-depleted cells containing gammaH2AX foci after release from thymidine arrest and, in cells incorporating BrdU, DNA synthesis does not occur at sites of gammaH2AX foci. Furthermore activated ATM and Chk2 persist in these cells. We propose that the gammaH2AX foci in Chk1-depleted cells may represent sites of persistent replication fork damage or abandonment that are unable to resume DNA synthesis but do not play a direct role in the Chk1 suppressed death pathway.

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Essential role of the p110beta subunit of phosphoinositide 3-OH kinase in male fertility.

Publication Date: 2010 Mar PMID: 20053680
Authors: Ciraolo, E. - Morello, F. - Hobbs, R. M. - Wolf, F. - Marone, R. - Iezzi, M. - Lu, X. - Mengozzi, G. - Altruda, F. - Sorba, G. - Guan, K. - Pandolfi, P. P. - Wymann, M. P. - Hirsch, E.
Journal: Mol Biol Cell

Phosphoinositide 3-kinases (PI3K) are key molecular players in male fertility. However, the specific roles of different p110 PI3K catalytic subunits within the spermatogenic lineage have not been characterized so far. Herein, we report that male mice expressing a catalytically inactive p110beta develop testicular hypotrophy and impaired spermatogenesis, leading to a phenotype of oligo-azoospermia and defective fertility. The examination of testes from p110beta-defective tubules demonstrates a widespread loss in spermatogenic cells, due to defective proliferation and survival of pre- and postmeiotic cells. In particular, p110beta is crucially needed in c-Kit-mediated spermatogonial expansion, as c-Kit-positive cells are lost in the adult testis and activation of Akt by SCF is blocked by a p110beta inhibitor. These data establish that activation of the p110beta PI3K isoform by c-Kit is required during spermatogenesis, thus opening the way to new treatments for c-Kit positive testicular cancers.

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The late endosome is essential for mTORC1 signaling.

Publication Date: 2010 Mar PMID: 20053679
Authors: Flinn, R. J. - Yan, Y. - Goswami, S. - Parker, P. J. - Backer, J. M.
Journal: Mol Biol Cell

The multisubunit mTORC1 complex integrates signals from growth factors and nutrients to regulate protein synthesis, cell growth, and autophagy. To examine how endocytic trafficking might be involved in nutrient regulation of mTORC1, we perturbed specific endocytic trafficking pathways and measured mTORC1 activity using S6K1 as a readout. When early/late endosomal conversion was blocked by either overexpression of constitutively active Rab5 (Rab5CA) or knockdown of the Rab7 GEF hVps39, insulin- and amino acid-stimulated mTORC1/S6K1 activation were inhibited, and mTOR localized to hybrid early/late endosomes. Inhibition of other stages of endocytic trafficking had no effect on mTORC1. Overexpression of Rheb, which activates mTOR independently of mTOR localization, rescued mTORC1 signaling in cells expressing Rab5CA, whereas hyperactivation of endogenous Rheb in TSC2-/- MEFs did not. These data suggest that integrity of late endosomes is essential for amino acid- and insulin-stimulated mTORC1 signaling and that blocking the early/late endosomal conversion prevents mTOR from interacting with Rheb in the late endosomal compartment.

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NPP-16/Nup50 function and CDK-1 inactivation are associated with anoxia-induced prophase arrest in Caenorhabditis elegans.

Publication Date: 2010 Mar PMID: 20053678
Authors: Hajeri, V. A. - Little, B. A. - Ladage, M. L. - Padilla, P. A.
Journal: Mol Biol Cell

Oxygen, an essential nutrient, is sensed by a multiple of cellular pathways that facilitate the responses to and survival of oxygen deprivation. The Caenorhabditis elegans embryo exposed to severe oxygen deprivation (anoxia) enters a state of suspended animation in which cell cycle progression reversibly arrests at specific stages. The mechanisms regulating interphase, prophase, or metaphase arrest in response to anoxia are not completely understood. Characteristics of arrested prophase blastomeres and oocytes are the alignment of condensed chromosomes at the nuclear periphery and an arrest of nuclear envelope breakdown. Notably, anoxia-induced prophase arrest is suppressed in mutant embryos lacking nucleoporin NPP-16/NUP50 function, indicating that this nucleoporin plays an important role in prophase arrest in wild-type embryos. Although the inactive form of cyclin-dependent kinase (CDK-1) is detected in wild-type-arrested prophase blastomeres, the inactive state is not detected in the anoxia exposed npp-16 mutant. Furthermore, we found that CDK-1 localizes near chromosomes in anoxia-exposed embryos. These data support the notion that NPP-16 and CDK-1 function to arrest prophase blastomeres in C. elegans embryos. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.

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Numb independently antagonizes Sanpodo membrane targeting and Notch signaling in Drosophila sensory organ precursor cells.

Publication Date: 2010 Mar PMID: 20053677
Authors: Tong, X. - Zitserman, D. - Serebriiskii, I. - Andrake, M. - Dunbrack, R. - Roegiers, F.
Journal: Mol Biol Cell

In Drosophila, mitotic neural progenitor cells asymmetrically segregate the cell fate determinant Numb in order to block Notch signaling in only one of the two daughter cells. Sanpodo, a membrane protein required for Notch signaling in asymmetrically dividing cells, is sequestered from the plasma membrane to intracellular vesicles in a Numb-dependent way after neural progenitor cell mitosis. However, the significance of Numb-dependent Sanpodo regulation is unclear. In this study, we conducted a structure-function analysis to identify the determinants of Sanpodo targeting in vivo. We identified an NPAF motif in the amino-terminal cytoplasmic tail of Sanpodo, which is conserved among insect Sanpodo homologues. The Sanpodo NPAF motif is predicted to bind directly to the Numb phosphotyrosine-binding domain and is critical for Numb binding in vitro. Deletion or mutation of the NPAF motif results in accumulation of Sanpodo at the plasma membrane in Numb-positive cells in vivo. Genetic analysis of Sanpodo NPAF mutants shows that Numb-dependent Sanpodo endocytic targeting can be uncoupled from Notch signaling regulation. Our findings demonstrate that Sanpodo contains an evolutionarily conserved motif that has been linked to Numb-dependent regulation in vertebrates and further support the model that Numb regulates Notch signaling independently of Sanpodo membrane trafficking in neural progenitor cells.

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Global up-regulation of microtubule dynamics and polarity reversal during regeneration of an axon from a dendrite.

Publication Date: 2010 Mar PMID: 20053676
Authors: Stone, M. C. - Nguyen, M. M. - Tao, J. - Allender, D. L. - Rolls, M. M.
Journal: Mol Biol Cell

Axon regeneration is crucial for recovery after trauma to the nervous system. For neurons to recover from complete axon removal they must respecify a dendrite as an axon: a complete reversal of polarity. We show that Drosophila neurons in vivo can convert a dendrite to a regenerating axon and that this process involves rebuilding the entire neuronal microtubule cytoskeleton. Two major microtubule rearrangements are specifically induced by axon and not dendrite removal: 1) 10-fold up-regulation of the number of growing microtubules and 2) microtubule polarity reversal. After one dendrite reverses its microtubules, it initiates tip growth and takes on morphological and molecular characteristics of an axon. Only neurons with a single dendrite that reverses polarity are able to initiate tip growth, and normal microtubule plus-end dynamics are required to initiate this growth. In addition, we find that JNK signaling is required for both the up-regulation of microtubule dynamics and microtubule polarity reversal initiated by axon injury. We conclude that regulation of microtubule dynamics and polarity in response to JNK signaling is key to initiating regeneration of an axon from a dendrite.

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Intracellular targeting signals and lipid specificity determinants of the ALA/ALIS P4-ATPase complex reside in the catalytic ALA alpha-subunit.

Publication Date: 2010 Mar PMID: 20053675
Authors: Lopez-Marques, R. L. - Poulsen, L. R. - Hanisch, S. - Meffert, K. - Buch-Pedersen, M. J. - Jakobsen, M. K. - Pomorski, T. G. - Palmgren, M. G.
Journal: Mol Biol Cell

Members of the P(4) subfamily of P-type ATPases are believed to catalyze flipping of phospholipids across cellular membranes, in this way contributing to vesicle biogenesis in the secretory and endocytic pathways. P(4)-ATPases form heteromeric complexes with Cdc50-like proteins, and it has been suggested that these act as beta-subunits in the P(4)-ATPase transport machinery. In this work, we investigated the role of Cdc50-like beta-subunits of P(4)-ATPases for targeting and function of P(4)-ATPase catalytic alpha-subunits. We show that the Arabidopsis P(4)-ATPases ALA2 and ALA3 gain functionality when coexpressed with any of three different ALIS Cdc50-like beta-subunits. However, the final cellular destination of P(4)-ATPases as well as their lipid substrate specificity are independent of the nature of the ALIS beta-subunit they were allowed to interact with.

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