Molecular Biology of the Cell

Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells.

Publication Date: 2010 Sep 1 PMID: 20739464
Authors: Chiu, T. T. - Patel, N. - Shaw, A. E. - Bamburg, J. R. - Klip, A.
Journal: Mol Biol Cell

Monitoring Editor: Laurent Blanchoin GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms had yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodelling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodelled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.

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Expression of the Salmonella Spp. Virulence Factor SifA in Yeast Alters RHO1 Activity on Peroxisomes.

Publication Date: 2010 Aug 25 PMID: 20739463
Authors: Vinh, D. B. - Ko, D. C. - Rachubinski, R. A. - Aitchison, J. D. - Miller, S. I.
Journal: Mol Biol Cell

Monitoring Editor: Suresh Subramani The Salmonella typhimurium effector protein SifA regulates the assembly and tubulation of the Salmonella phagosome. SifA localizes to the phagosome and interacts with the membrane via its prenylated tail. SifA is a structural homologue of another bacterial effector that acts as a GTP-exchange factor for Rho family GTPases and can bind GDP-RhoA. When coexpressed with a bacterial lipase that is activated by RhoA, SifA can induce tubulation of mammalian endosomes. In an effort to develop a genetic system to study SifA function, we expressed SifA and characterized its activity in yeast. GFP-SifA predominantly localized to yeast peroxisomal membranes. Under peroxisome-inducing condition, GFP-SifA reduced the number of free peroxisomes and promoted the formation of large peroxisomes with membrane invaginations. GFP-SifA activity depended on the recruitment to peroxisomes of wild-type Rho1p and Pex25p, a receptor for Rho1p. GFP-SifA could also rescue the actin organization defects in pex25Delta and rho1 mutants suggesting that SifA may recruit and potentiate Rho1p activity. We reexamined the distribution of GFP-SifA in mammalian cells and found the majority colocalizing with LAMP1-positive compartment and not with the peroxisomal marker PMP70. Together, these data suggest that SifA may use a similar mode of action via Rho proteins to alter yeast peroxisomal and mammalian endosomal membranes. Further definition of SifA activity on yeast peroxisomes could provide more insight into its role in regulating host membrane dynamics and small GTPases.

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Deficiency in the Multicopy Sycp3 like X-linked Genes Slx and Slxl1 Causes Major Defects in Spermatid Differentiation.

Publication Date: 2010 Aug 25 PMID: 20739462
Authors: Cocquet, J. - Ellis, P. J. - Yamauchi, Y. - Riel, J. M. - Karacs, T. P. - Rattigan, A. - Ojarikre, O. A. - Affara, N. A. - Ward, M. A. - Burgoyne, P. S.
Journal: Mol Biol Cell

Monitoring Editor: Julie Brill The human and mouse sex chromosomes are enriched in multicopy genes required for postmeiotic differentiation of round spermatids into sperm. The gene Sly is present in multiple copies on the mouse Y chromosome and encodes a protein that is required for the epigenetic regulation of postmeiotic sex chromosome expression. The X chromosome carries two multicopy genes related to Sly: Slx and Slxl1. Here we investigate the role of Slx/Slxl1 using transgenically-delivered small interfering RNAs to disrupt their function. We show that Slx and Slxl1 are important for normal sperm differentiation and male fertility. Slx/Slxl1 deficiency leads to delay in spermatid elongation and sperm release. A high proportion of delayed spermatids are eliminated via apoptosis, with a consequent reduced sperm count. The remaining spermatozoa are abnormal with impaired motility and fertilizing abilities. Microarray analyses reveal that Slx/Slxl1 deficiency affects the metabolic processes occurring in the spermatid cytoplasm but does not lead to a global perturbation of sex chromosome expression; this is in contrast with the effect of Sly-deficiency which leads to an upregulation of X and Y chromosome genes. This difference may be due to the fact that SLX/SLXL1 are exclusively cytoplasmic while SLY is found in the nucleus and cytoplasm of spermatids.

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{alpha}-arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling.

Publication Date: 2010 Aug 25 PMID: 20739461
Authors: O'Donnell, A. F. - Apffel, A. - Gardner, R. G. - Cyert, M. S.
Journal: Mol Biol Cell

Monitoring Editor: David G. Drubin Extracellular signals regulate trafficking events to reorganize proteins at the plasma membrane (PM), however few effectors of this regulation have been identified. beta-arrestins relay signaling cues to the trafficking machinery by controlling agonist-stimulated endocytosis of G-protein coupled receptors. In contrast, we show that yeast alpha-arrestins, Aly1 and Aly2, control intracellular sorting of Gap1, the general amino acid permease, in response to nutrients. These studies are the first to demonstrate association of alpha-arrestins with clathrin and clathrin adaptor proteins (AP), and show that Aly1 and Aly2 interact directly with the gamma subunit of AP-1, Apl4. Aly2-dependent trafficking of Gap1 requires AP-1, which mediates endosome-to-Golgi transport, and the nutrient-regulated kinase, Npr1, which phosphorylates Aly2. During nitrogen starvation, Npr1 phosphorylation of Aly2 may stimulate Gap1 incorporation into AP-1/clathrin-coated vesicles to promote Gap1 trafficking from endosomes to the trans-Golgi network (TGN). Ultimately, increased Aly1-/Aly2-mediated recycling of Gap1 from endosomes results in higher Gap1 levels within cells and at the PM by diverting Gap away from trafficking pathways that lead to vacuolar degradation. This work defines a new role for arrestins in membrane trafficking and offers insight into how alpha-arrestins coordinate signaling events with protein trafficking.

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SPG20 Protein Spartin is Recruited to Midbodies by ESCRT-III Protein Ist1 and Participates in Cytokinesis.

Publication Date: 2010 Aug 18 PMID: 20719964
Authors: Renvoise, B. - Parker, R. L. - Yang, D. - Bakowska, J. C. - Hurley, J. H. - Blackstone, C.
Journal: Mol Biol Cell

Monitoring Editor: Stephen Doxsey Hereditary spastic paraplegias (SPG1-46) are inherited neurological disorders characterized by lower extremity spastic weakness. Loss-of-function SPG20 gene mutations cause an autosomal recessive HSP known as Troyer syndrome. The SPG20 protein spartin localizes to lipid droplets and endosomes, and it interacts with the TIP47 as well as the ubiquitin E3 ligases AIP4 and AIP5. Spartin harbors an MIT domain at its N-terminus, and most proteins with MIT domains interact with specific ESCRT-III proteins. Using yeast two-hybrid and in vitro surface plasmon resonance assays, we demonstrate that the spartin MIT domain binds with micromolar affinity to the ESCRT-III protein Ist1, but not to ESCRT-III proteins CHMP1-7. Spartin colocalizes with Ist1 at the midbody, and depletion of Ist1 in cells by siRNA significantly decreases the number of cells where spartin is present at midbodies. Depletion of spartin does not affect Ist1 localization to midbodies but markedly impairs cytokinesis. A structure-based amino acid substitution in the spartin MIT domain (F24D) blocks the spartin-Ist1 interaction. Spartin F24D does not localize to the midbody and acts in a dominant-negative manner and impairs cytokinesis. These data suggest that Ist1 interaction is important for spartin recruitment to the midbody and that spartin participates in cytokinesis.

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The Sodium/Proton Exchanger NHE8 Regulates Late Endosomal Morphology and Function.

Publication Date: 2010 Aug 18 PMID: 20719963
Authors: Lawrence, S. P. - Bright, N. A. - Luzio, J. P. - Bowers, K.
Journal: Mol Biol Cell

Monitoring Editor: Jean E. Gruenberg The pH and lumenal environment of intracellular organelles is considered essential for protein sorting and trafficking through the cell. We provide the first evidence that a mammalian NHE sodium (potassium)/proton exchanger, NHE8, plays a key role in the control of protein trafficking and endosome morphology. At steady state, the majority of epitope-tagged NHE8 was found in the trans Golgi network of HeLa M cells, but a proportion was also localized to multivesicular bodies (MVB's). Depletion of NHE8 in HeLa M cells with siRNA resulted in the perturbation of MVB protein sorting, as shown by an increase in epidermal growth factor degradation. Additionally, NHE8-depleted cells displayed striking perinuclear clustering of endosomes and lysosomes and there was a ninefold increase in the cellular volume taken up by LAMP1/LBPA-positive, dense MVB's. Our data points to a role for the ion exchange activity of NHE8 being required to maintain endosome morphology, as overexpression of a nonfunctional point mutant protein (NHE8 E225Q) resulted in phenotypes similar to those seen after siRNA depletion of endogenous NHE8. Interestingly, we found that depletion of NHE8, despite its function as a sodium (potassium)/proton antiporter, did not affect the overall pH inside dense MVB's.

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Sprouty Proteins Inhibit Receptor-mediated Activation of Phosphatidylinositol-specific Phospholipase C.

Publication Date: 2010 Aug 18 PMID: 20719962
Authors: Akbulut, S. - Reddi, A. L. - Aggarwal, P. - Ambardekar, C. - Canciani, B. - Kim, M. K. - Hix, L. - Vilimas, T. - Mason, J. - Basson, M. A. - Lovatt, M. - Powell, J. - Collins, S. - Quatela, S. - Phillips, M. - Licht, J. D.
Journal: Mol Biol Cell

Monitoring Editor: Richard K. Assoian Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase (RTK) signaling, however their exact mechanism of action remains incompletely understood. We identified phosphatidylinositol-specific phospholipase (PLCgamma) as a partner of the Spry1 and Spry2 proteins. Spry/PLCgamma interaction was dependant on the SH2 domain of PLCgamma and a conserved N-terminal tyrosine residue in Spry1 and Spry2. Overexpression of Spry1 and Spry2 was associated with decreased PLCgamma phosphorylation, and decreased PLCgamma activity as measured by production of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), while cells deficient for Spry1 or Spry1, 2 and 4 showed increased production of IP3 at baseline and further increased in response to growth factor signals. Overexpression of Spry 1 or Spry2 or siRNA mediated knockdown of PLCgamma1 or PLCgamma2 abrogated the activity of a calcium-dependent reporter gene suggesting that Spry inhibited calcium-mediated signaling downstream of PLCgamma. Furthermore, Spry overexpression in T-cells, which are highly dependent on PLCgamma activity and calcium signaling, blocked T-cell receptor mediated calcium release. Accordingly, cultured T-cells from Spry1 gene knockout mice showed increased proliferation in response to T-cell receptor stimulation. These data highlight an important action of Spry, which may allow these proteins to influence signaling through multiple receptors.

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Activation of Akt is Essential for the Propagation of Mitochondrial Respiratory Stress Signaling and Activation of Transcriptional Coactivator hnRNPA2.

Publication Date: 2010 Aug 18 PMID: 20719961
Authors: Guha, M. - Fang, J. K. - Monks, R. - Birnbaum, M. J. - Avadhani, N. G.
Journal: Mol Biol Cell

Monitoring Editor: Thomas D. Fox Mitochondrial respiratory stress (also called mitochondrial retrograde signaling) activates a Ca(2+)/calcineurin-mediated signal that culminates in transcription activation/repression of a large number of nuclear genes. This signal is propagated through activation of the regulatory proteins NFkappaB cRel/p50, C/EBPdelta, CREB, and NFAT. Additionally, the heterogeneous ribonucleoprotein A2 (hnRNPA2) functions as a coactivator in upregulating the transcription of Cathepsin L, RyR1, and Glut-4, the target genes of stress signaling. Activation of IGF1R, which causes a metabolic switch to glycolysis, cell invasiveness, and resistance to apoptosis, is a phenotypic hallmark of C2C12 myoblasts subjected to mitochondrial stress. In this study, we report that mitochondrial stress leads to increased expression, activation, and nuclear localization of Akt1. Mitochondrial respiratory stress also activates Akt1-gene expression, which involves hnRNPA2 as a coactivator, indicating a complex interdependency of these two factors. Using Akt1(-/-) mouse embryonic fibroblasts and Akt1 mRNA-silenced C2C12 cells, we show that Akt1-mediated phosphorylation is crucial for the activation and recruitment of hnRNPA2 to the enhanceosome complex. Akt1 mRNA silencing in mtDNA-depleted cells resulted in reversal of the invasive phenotype, accompanied by sensitivity to apoptotic stimuli. These results show that Akt1 is an important regulator of the nuclear transcriptional response to mitochondrial stress.

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Integrin-mediated Cell Attachment Induces a PAK4-dependent Feedback Loop Regulating Cell Adhesion Through Modified Integrin {alpha}v{beta}5 Clustering and Turn-over.

Publication Date: 2010 Aug 18 PMID: 20719960
Authors: Li, Z. - Lock, J. G. - Olofsson, H. - Kowalewski, J. M. - Teller, S. - Liu, Y. - Zhang, H. - Stromblad, S.
Journal: Mol Biol Cell

Monitoring Editor: J. Silvio Gutkind Cell to extracellular matrix adhesion is regulated by a multitude of pathways initiated distally to the core cell-matrix adhesion machinery, such as via growth factor signaling. In contrast to these extrinsically sourced pathways, we now identify a regulatory pathway that is intrinsic to the core adhesion machinery, providing an internal regulatory feedback loop to fine tune adhesion levels. This auto-inhibitory negative feedback loop is initiated by cell adhesion to Vitronectin, leading to PAK4 activation, which in turn limits total cell-Vitronectin adhesion strength. Specifically, we show that PAK4 is activated by cell attachment to Vitronectin as mediated by PAK4 binding partner integrin alphavbeta5, and that active PAK4 induces accelerated integrin alphavbeta5 turn-over within adhesion complexes. Accelerated integrin turn-over is associated with additional PAK4-mediated effects, including inhibited integrin alphavbeta5 clustering, reduced integrin to F-actin connectivity and perturbed adhesion complex maturation. These specific outcomes are ultimately associated with reduced cell adhesion strength and increased cell motility. We thus demonstrate a novel mechanism deployed by cells to tune cell adhesion levels through the auto-inhibitory regulation of integrin adhesion.

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Par6{alpha} Interacts With the Dynactin Subunit p150Glued and Is a Critical Regulator of Centrosomal Protein Recruitment.

Publication Date: 2010 Aug 18 PMID: 20719959
Authors: Kodani, A. - Tonthat, V. - Wu, B. - Sutterlin, C.
Journal: Mol Biol Cell

Monitoring Editor: Stephen Doxsey The centrosome contains proteins that control the organization of the microtubule cytoskeleton in interphase and mitosis. Its protein composition is tightly regulated through selective and cell cycle-dependent recruitment, retention and removal of components. However, the mechanisms underlying protein delivery to the centrosome are not completely understood. We describe a novel function for the polarity protein Par6alpha in protein transport to the centrosome. We detected Par6alpha at the centrosome and centriolar satellites where it interacted with the centriolar satellite protein PCM-1 and the dynactin subunit p150(Glued). Depletion of Par6alpha caused the mislocalization of p150(Glued) and centrosomal components that are critical for microtubule anchoring at the centrosome. As a consequence, there were severe alterations in the organization of the microtubule cytoskeleton in the absence of Par6alpha and cell division was blocked. We propose a model in which Par6alpha controls centrosome organization through its association with the dynactin subunit p150(Glued).

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MAP1B Regulates Axonal Development by Modulating Rho-GTPase Rac1 Activity.

Publication Date: 2010 Aug 18 PMID: 20719958
Authors: Montenegro-Venegas, C. - Tortosa, E. - Rosso, S. - Peretti, D. - Bollati, F. - Bisbal, M. - Jausoro, I. - Avila, J. - Caceres, A. - Gonzalez-Billault, C.
Journal: Mol Biol Cell

Monitoring Editor: Erika Holzbaur Cultured neurons obtained from MAP1B-deficient mice have a delay in axon outgrowth and a reduced rate of axonal elongation as compared with neurons from wild-type mice. Here we show that MAP1B deficiency results in a significant decrease in Rac1 and cdc42 activity and a significant increase in Rho activity. We found that MAP1B interacted with Tiam1, a guanosine nucleotide exchange factor for Rac1. The decrease in Rac1/cdc42 activity was paralleled by decreases in the phosphorylation of the downstream effectors of these proteins, such as LIMK-1 and cofilin. The expression of a constitutively active form of Rac1, cdc42, or Tiam1 rescued the axon growth defect of MAP1B-deficient neurons. Taken together, these observations define a new and crucial function of MAP1B that we show to be required for efficient cross-talk between microtubules and the actin cytoskeleton during neuronal polarization.

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ZFP36L1 Negatively Regulates Erythroid Differentiation of CD34+ Hematopoietic Stem Cells by Interfering with Stat5b Pathway.

Publication Date: 2010 Aug 11 PMID: 20702587
Authors: Vignudelli, T. - Selmi, T. - Martello, A. - Parenti, S. - Grande, A. - Gemelli, C. - Zanocco-Marani, T. - Ferrari, S.
Journal: Mol Biol Cell

Monitoring Editor: Marianne Bronner-Fraser ZFP36L1 is a member of a family of CCCH tandem zinc finger proteins (TTP family) able to bind to AU-rich elements in the 3'-untranslated region of mRNAs, thereby triggering their degradation. The present study suggests that such mechanism is used during hematopoiesis to regulate differentiation by post-transcriptionally modulating the expression of specific target genes. In particular, it demonstrates that ZFP36L1 negatively regulates erythroid differentiation by directly binding the 3' untranslated region of Stat5b encoding mRNA. Stat5b down-regulation obtained by ZFP36L1 overexpression results, in human hematopoietic progenitors, in a drastic decrease of erythroid colonies formation. These observations have been confirmed by silencing experiments targeting Stat5b and by treating hematopoietic stem/progenitor cells with drugs able to induce ZFP36L1 expression. Moreover, this study shows that different members of ZFP36L1 family act redundantly, since cooverexpression of ZFP36L1 and family member ZFP36 determines a cumulative effect on Stat5b down-regulation. This work describes a mechanism underlying ZFP36L1 capability to regulate hematopoietic differentiation and suggests a new target for the therapy of hematopoietic diseases involving Stat5b/JAK2 pathway, such as chronic myeloproliferative disorders.

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Cdk Phosphorylation of a Nucleoporin Controls Localization of Active Genes Through the Cell Cycle.

Publication Date: 2010 Aug 11 PMID: 20702586
Authors: Brickner, D. G. - Brickner, J. H.
Journal: Mol Biol Cell

Monitoring Editor: Martin Hetzer Many inducible genes in yeast are targeted to the nuclear pore complex when active. We find that the peripheral localization of the INO1 and GAL1 genes is regulated through the cell cycle. Active INO1 and GAL1 localized at the nuclear periphery during G1, became nucleoplasmic during S-phase and then returned to the nuclear periphery during G2/M. Loss of peripheral targeting followed the initiation of DNA replication and was lost in cells lacking a cyclin dependent kinase (Cdk) inhibitor. Furthermore, the Cdk1 kinase and two Cdk phosphorylation sites in the nucleoporin Nup1 were required for peripheral targeting of INO1 and GAL1. Introduction of aspartic acid residues in place of either of these two sites in Nup1 bypassed the requirement for Cdk1 and resulted in targeting of INO1 and GAL1 to the nuclear periphery during S-phase. Thus, phosphorylation of a nuclear pore component by cyclin dependent kinase controls the localization of active genes to the nuclear periphery through the cell cycle.

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Keeping the Vimentin Network Under Control: Cell-matrix Adhesion-associated Plectin 1f Affects Cell Shape and Polarity of Fibroblasts.

Publication Date: 2010 Aug 11 PMID: 20702585
Authors: Burgstaller, G. - Gregor, M. - Winter, L. - Wiche, G.
Journal: Mol Biol Cell

Monitoring Editor: M. Bishr Omary Focal adhesions (FAs) located at the ends of actin/myosin-containing contractile stress fibers form tight connections between fibroblasts and their underlying extracellular matrix. We show here that mature FAs and their derivative fibronectin fibril-aligned fibrillar adhesions (FbAs) serve as docking sites for vimentin intermediate filaments (IFs) in a plectin isoform 1f (P1f)-dependent manner. Time-lapse video microscopy revealed that FA-associated P1f captures mobile vimentin filament precursors which then serve as seeds for de novo IF network formation via end-to-end fusion with other mobile precursors. As a consequence of IF association, the turnover of FAs is reduced. P1f-mediated IF network formation at FbAs creates a resilient cage-like core structure that encases and positions the nucleus while being stably connected to the exterior of the cell. We show that the formation of this structure affects cell shape with consequences for cell polarization.

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The Rapamycin-sensitive Phosphoproteome Reveals That TOR Controls PKA Toward Some but Not All Substrates.

Publication Date: 2010 Aug 11 PMID: 20702584
Authors: Soulard, A. - Cremonesi, A. - Moes, S. - Schutz, F. - Jeno, P. - Hall, M. N.
Journal: Mol Biol Cell

Monitoring Editor: Daniel J. Lew Regulation of cell growth requires extensive coordination of several processes including transcription, ribosome biogenesis, translation, nutrient metabolism, and autophagy. In yeast, the protein kinases Target Of Rapamycin (TOR) and Protein Kinase A (PKA) regulate these processes and are thereby the main activators of cell growth in response to nutrients. How TOR, PKA and their corresponding signaling pathways are coordinated to control the same cellular processes is not understood. Quantitative analysis of the rapamycin-sensitive phosphoproteome combined with targeted analysis of PKA substrates suggests that TOR complex 1 (TORC1) activates PKA but only toward a subset of substrates. Furthermore, we show that TORC1 signaling impinges on BCY1, the negative regulatory subunit of PKA. Inhibition of TORC1 with rapamycin leads to BCY1 phosphorylation on several sites including T129. Phosphorylation of BCY1 T129 results in BCY1 activation and inhibition of PKA. TORC1 inhibits BCY1 T129 phosphorylation by phosphorylating and activating the S6K homolog SCH9 that in turn inhibits the MAP kinase MPK1. MPK1 phosphorylates BCY1 T129 directly. Thus, TORC1 activates PKA toward some substrates by preventing MPK1-mediated activation of BCY1.

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V-ATPase-mediated granular acidification is regulated by the V-ATPase accessory subunit Ac45 in POMC-producing cells.

Publication Date: 2010 Aug 11 PMID: 20702583
Authors: Jansen, E. J. - Hafmans, T. G. - Martens, G. J.
Journal: Mol Biol Cell

Monitoring Editor: Thomas F.J. Martin The vacuolar (H(+))-ATPase (V-ATPase) is an important proton pump and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive, but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral-pH-dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing.

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Small Molecule Inhibition of HIV-1-Induced MHC-I Downregulation Identifies a Temporally Regulated Switch in Nef Action.

Publication Date: 2010 Aug 11 PMID: 20702582
Authors: Dikeakos, J. D. - Atkins, K. M. - Thomas, L. - Emert-Sedlak, L. - Byeon, I. J. - Jung, J. - Ahn, J. - Wortman, M. D. - Kukull, B. - Saito, M. - Koizumi, H. - Williamson, D. M. - Hiyoshi, M. - Barklis, E. - Takiguchi, M. - Suzu, S. - Gronenborn, A. M. - Smithgall, T. E. - Thomas, G.
Journal: Mol Biol Cell

Monitoring Editor: Howard Riezman HIV-1 Nef triggers down-regulation of cell-surface MHC-I by assembling a Src family kinase (SFK)-ZAP-70/Syk-PI3K cascade (Hung et al., Cell Host & Mic, 1:121-133 (2007)). Here, we report that chemical disruption of the Nef-SFK interaction with the small molecule inhibitor 2c blocks assembly of the multi-kinase complex and represses HIV-1 mediated MHC-I down-regulation in primary CD4(+) T-cells. 2c did not interfere with the PACS-2 dependent trafficking of Nef required for the Nef-SFK interaction or the AP-1 and PACS-1 dependent sequestering of internalized MHC-I, suggesting the inhibitor specifically interfered with the Nef-SFK interaction required for triggering MHC-I down-regulation. Transport studies revealed Nef directs a highly regulated program to down-regulate MHC-I in primary CD4(+) T-cells. During the first two days following infection, Nef assembles the 2c-sensitive multi-kinase complex to trigger down-regulation of cell-surface MHC-I. By three days post-infection Nef switches to a stoichiometric mode that prevents surface delivery of newly synthesized MHC-I. Pharmacologic inhibition of the multi-kinase cascade prevents the Nef-dependent block in MHC-I transport, suggesting the signaling and stoichiometric modes are causally linked. Together, these studies resolve the seemingly controversial models that describe Nef-induced MHC-I down-regulation and provide new insights into the mechanism of Nef action.

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Coordination of Substrate Binding and ATP Hydrolysis in Vps4-mediated ESCRT-III Disassembly.

Publication Date: 2010 Aug 11 PMID: 20702581
Authors: Davies, B. A. - Azmi, I. F. - Payne, J. - Shestakova, A. - Horazdovsky, B. F. - Babst, M. - Katzmann, D. J.
Journal: Mol Biol Cell

Monitoring Editor: Judith Klumperman ESCRT-III undergoes dynamic assembly and disassembly to facilitate membrane exvagination processes including multivesicular body (MVB) formation, enveloped virus budding, and membrane abscission during cytokinesis. The AAA-ATPase Vps4 is required for ESCRT-III disassembly, however the coordination of Vps4 ATP hydrolysis with ESCRT-III binding and disassembly is not understood. Vps4 ATP hydrolysis has been proposed to execute ESCRT-III disassembly as either a stable oligomer or an unstable oligomer whose dissociation drives ESCRT-III disassembly. An in vitro ESCRT-III disassembly assay was developed to analyze Vps4 function during this process. The studies presented here support a model in which Vps4 acts as a stable oligomer during ATP hydrolysis and ESCRT-III disassembly. Moreover, Vps4 oligomer binding to ESCRT-III induces coordination of ATP hydrolysis at the level of individual Vps4 subunits. These results suggest that Vps4 functions as a stable oligomer that acts upon individual ESCRT-III subunits to facilitate ESCRT-III disassembly.

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Different Localizations and Cellular Behaviors of Leiomodin and Tropomodulin in Mature Cardiomyocyte Sarcomeres.

Publication Date: 2010 Aug 4 PMID: 20685966
Authors: Skwarek-Maruszewska, A. - Boczkowska, M. - Zajac, A. L. - Kremneva, E. - Svitkina, T. - Dominguez, R. - Lappalainen, P.
Journal: Mol Biol Cell

Monitoring Editor: Laurent Blanchoin Leiomodin (Lmod) is a muscle-specific F-actin nucleating protein that is related to the F-actin pointed-end capping protein tropomodulin (Tmod). However, Lmod contains a unique approximately 150-residue C-terminal extension that is required for its strong nucleating activity. Overexpression or depletion of Lmod compromises sarcomere organization, but the mechanism by which Lmod contributes to myofibril assembly is not well understood. We show that Tmod and Lmod localize through fundamentally different mechanisms to the pointed ends of two distinct subsets of actin filaments in myofibrils. Tmod localizes to two narrow bands immediately adjacent to M-lines, whereas Lmod displays dynamic localization to two broader bands, which are generally more separated from M-lines. Lmod's localization and F-actin nucleation activity are enhanced by interaction with tropomyosin. Unlike Tmod, the myofibril localization of Lmod depends on sustained muscle contraction and actin polymerization. We further show that Lmod expression correlates with the maturation of myofibrils in cultured cardiomyocytes, and that it associates with sarcomeres only in differentiated myofibrils. Collectively, the data suggest that Lmod contributes to the final organization and maintenance of sarcomere architecture by promoting tropomyosin-dependent actin filament nucleation.

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The Osmoprotective Transcription Factor NFAT5/TonEBP Modulates NF-{kappa}B Activity.

Publication Date: 2010 Aug 4 PMID: 20685965
Authors: Roth, I. - Leroy, V. - Kwon, H. M. - Martin, P. Y. - Feraille, E. - Hasler, U.
Journal: Mol Biol Cell

Monitoring Editor: William P. Tansey Tonicity responsive binding protein (TonEBP or NFAT5) is a widely expressed transcription factor whose activity is regulated by extracellular tonicity. TonEBP plays a key role in osmoprotection by binding to ORE/TonE elements of genes that counteract the deleterious effects of cell shrinkage. Here, we show that in addition to this 'classical' stimulation, TonEBP protects cells against hypertonicity by enhancing NF-kappaB activity. We show that hypertonicity enhances NF-kappaB stimulation by LPS but not TNF-alpha, and demonstrate overlapping Akt-dependent signal transduction pathways elicited by hypertonicity and TGFalpha. Activation of p38 kinase by hypertonicity and downstream activation of Akt play key roles in TonEBP activity, IkappaBalpha degradation and p65 nuclear translocation. TonEBP affects neither of these latter events and is itself insensitive to NF-kappaB signaling. Rather, we reveal a tonicity-dependent interaction between TonEBP and p65 and show that NF-kappaB activity is considerably enhanced following binding of NF-kappaB-TonEBP complexes to kappaB elements of NF-kappaB responsive genes. We demonstrate the key roles of TonEBP and Akt in renal collecting duct epithelial cells and in macrophages. These findings reveal a novel role for TonEBP and Akt in NF-kappaB activation on the onset of hypertonic challenge.

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A Novel Pzg-NURF Complex Regulates Notch Target Gene Activity.

Publication Date: 2010 Aug 4 PMID: 20685964
Authors: Kugler, S. J. - Nagel, A. C.
Journal: Mol Biol Cell

Monitoring Editor: Marcos Gonzalez-Gaitan Drosophila putzig was identified as a member of the TRF2/DREF complex that is involved in core promoter selection. Additionally, putzig regulates Notch signaling, however independently of DREF. Here, we show that Putzig associates with the NURF complex. Loss of any NURF component including the NURF-specific subunit Nurf 301 impedes binding of Putzig to Notch target genes, suggesting that NURF recruits Putzig to these sites. Accordingly, Putzig can be copurified with any NURF-member. Moreover, Nurf 301 mutants show reduced Notch target gene activity and enhance Notch mutant phenotypes. These data suggest a novel Putzig-NURF chromatin complex required for epigenetic activation of Notch targets.

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The Farnesylated Nuclear Proteins Kugelkern and Lamin Dm0 Affect Nuclear Morphology by Directly Interacting with the Nuclear Membrane.

Publication Date: 2010 Aug 4 PMID: 20685963
Authors: Polychronidou, M. - Hellwig, A. - Grosshans, J.
Journal: Mol Biol Cell

Monitoring Editor: Martin Hetzer Nuclear shape changes are observed during a variety of developmental processes, pathological conditions and ageing. The mechanisms underlying nuclear shape changes in the above mentioned situations have mostly remained unclear. To address the molecular mechanism behind nuclear shape changes, we analyzed how the farnesylated nuclear envelope proteins Kugelkern and lamin Dm0 affect the structure of the nuclear membrane. We found that Kugelkern and lamin Dm0 affect nuclear shape without requiring filament formation or the presence of a classical nuclear lamina. We could also show that the two proteins do not depend on a group of selected inner nuclear membrane proteins for their localization to the nuclear envelope. Surprisingly, we found that farnesylated Kugelkern and lamin Dm0 protein constructs change the morphology of protein free liposomes. Based on these findings we propose that farnesylated proteins of the nuclear membrane induce nuclear shape changes by being asymmetrically inserted into the phospholipid bilayer via their farnesylated C-terminal part.

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Identification of a Nuclear Export Signal in the Catalytic Subunit of AMP-activated Protein Kinase.

Publication Date: 2010 Aug 4 PMID: 20685962
Authors: Kazgan, N. - Williams, T. - Forsberg, L. J. - Brenman, J. E.
Journal: Mol Biol Cell

Monitoring Editor: Ramanujan S. Hegde The metabolic regulator AMP-activated protein kinase (AMPK) maintains cellular homeostasis through regulation of proteins involved in energy-producing and energy-consuming pathways. While AMPK phosphorylation targets include cytoplasmic and nuclear proteins, the precise mechanisms that regulate AMPK localization, and thus its access to these substrates, are unclear. We identify highly conserved carboxy-terminal hydrophobic amino acids that function as a Leptomycin-B sensitive, CRM1-dependent nuclear export sequence (NES) in the AMPK catalytic subunit (AMPKalpha). When this sequence is modified AMPKalpha shows increased nuclear localization via a Ran-dependent import pathway. Cytoplasmic localization can be restored by substituting well-defined snurportin-1 or protein kinase A inhibitor (PKIA) CRM1-binding NESs into AMPKalpha. We demonstrate a functional requirement in vivo for the AMPKalpha carboxy-terminal NES, as transgenic Drosophila expressing AMPKalpha lacking this NES fail to rescue lethality of AMPKalpha null mutant flies and show decreased activation loop phosphorylation under heat shock stress. Sequestered to the nucleus, this truncated protein shows highly reduced phosphorylation at the key Thr172 activation residue, suggesting that AMPK activation predominantly occurs in the cytoplasm under unstressed conditions. Thus, modulation of CRM1-mediated export of AMPKalpha via its C-terminal NES provides an additional mechanism for cells to use in the regulation of AMPK activity and localization.

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Vestigial Is Required during Late-Stage Muscle Differentiation in Drosophila melanogaster Embryos.

Publication Date: 2010 Aug 4 PMID: 20685961
Authors: Deng, H. - Bell, J. B. - Simmonds, A. J.
Journal: Mol Biol Cell

Monitoring Editor: Marcos Gonzalez-Gaitan The somatic muscles of Drosophila develop in a complex pattern that is repeated in each embryonic hemi-segment. During early development, progenitor cells fuse to form a syncytial muscle, which further differentiates via expression of muscle-specific factors which induce specific responses to external signals to regulate late-stage processes like migration and attachment. Initial communication between somatic muscles and the epidermal tendon cells is critical for both of these processes. However, later establishment of attachments between longitudinal muscles at the segmental borders is largely independent of the muscle-epidermal attachment signals and relatively little is known about how this event is regulated. Using a combination of null mutations and a truncated version of SD that binds Vg but not DNA we show that e Vestigial (Vg) is required in ventral longitudinal muscles to induce formation of stable intermuscular attachments. In several muscles, this activity may be independent of SD. Further, the cell-specific differentiation events induced by Vg in two cells fated to form attachments are coordinated by Drosophila Epidermal Growth Factor (DER) signaling. Thus, Vg is a key factor to induce specific changes in VL1-4 identity and is required for these cells to be competent to form stable intermuscular attachments with each other.

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Ang2/Fat-free Is a Conserved Subunit of the Golgi-associated Retrograde Protein (GARP) Complex.

Publication Date: 2010 Aug 4 PMID: 20685960
Authors: Perez-Victoria, F. J. - Schindler, C. - Magadan, J. G. - Mardones, G. A. - Delevoye, C. - Romao, M. - Raposo, G. - Bonifacino, J. S.
Journal: Mol Biol Cell

Monitoring Editor: Adam Linstedt The Golgi-associated Retrograde Protein (GARP) complex mediates tethering and fusion of endosome-derived transport carriers to the trans-Golgi network (TGN). In the yeast S. cerevisiae, GARP comprises four subunits named Vps51p, Vps52p, Vps53p and Vps54p. Orthologues of the GARP subunits, except for Vps51p, have been identified in all other eukaryotes. A yeast two-hybrid screen of a human cDNA library yielded a phylogenetically conserved protein, Ang2/Fat-free, which interacts with human Vps52, Vps53 and Vps54. Human Ang2 is larger than yeast Vps51p, but exhibits significant homology in an N-terminal coiled-coil region that mediates assembly with other GARP subunits. Biochemical analyses show that human Ang2, Vps52, Vps53 and Vps54 form an obligatory 1:1:1:1 complex that strongly interacts with the regulatory Habc domain of the TGN SNARE, Syntaxin 6. Depletion of Ang2 or the GARP subunits similarly impairs protein retrieval to the TGN, lysosomal enzyme sorting, endosomal cholesterol traffic and autophagy. These findings indicate that Ang2 is the missing component of the GARP complex in most eukaryotes.

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PKR Differentially Regulates IRS1 and IRS2 in HepG2 Cells.

Publication Date: 2010 Aug 4 PMID: 20685959
Authors: Yang, X. - Nath, A. - Opperman, M. J. - Chan, C.
Journal: Mol Biol Cell

Monitoring Editor: Carole Parent Initially identified to be activated upon virus infection, the double-stranded RNA-dependent protein kinase (PKR) is best known for triggering cell defense responses by phosphorylating eIF-2alpha, thus suppressing RNA translation. We as well as others showed that the phosphorylation of PKR is down-regulated by insulin. In the present study, we further uncovered a novel function of PKR in regulating the IRS proteins. We found that PKR up-regulates the inhibitory phosphorylation of IRS1 at Ser312, which suppresses the tyrosine phosphorylation of IRS1. This effect of PKR on the phosphorylation of IRS1 is mediated by 2 other protein kinases, JNK and IKK. In contrast, PKR regulates IRS2, another major IRS family protein in the liver, at the transcriptional rather than the post-translational level, and this effect is mediated by the transcription factor, FoxO1, which has been previously shown to be regulated by insulin and plays a significant role in glucose homeostasis and energy metabolism. In summary, we found for the first time that initially known as a virus infection response gene, PKR regulates the upstream central transmitters of insulin signaling, IRS1 and IRS2, through different mechanisms.

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A PI3K/PKB-Independent Activation of mTOR Signaling Is Sufficient to Induce Skeletal Muscle Hypertrophy.

Publication Date: 2010 Aug 4 PMID: 20668162
Authors: Goodman, C. A. - Miu, M. H. - Frey, J. W. - Mabrey, D. M. - Lincoln, H. C. - Ge, Y. - Chen, J. - Hornberger, T. A.
Journal: Mol Biol Cell

Monitoring Editor: Carl-Henrik Heldin It has been widely proposed that signaling by mTOR is both necessary and sufficient for the induction of skeletal muscle hypertrophy. Evidence for this hypothesis is largely based on studies which used stimuli that activate mTOR via a PI3K/PKB-dependent mechanism. However, the stimulation of signaling by PI3K/PKB can also activate several mTOR-independent growth promoting events, and thus, it is not clear if signaling by mTOR is permissive, or sufficient, for the induction of hypertrophy. Furthermore, the presumed role of mTOR in hypertrophy is derived from studies which utilized rapamycin to inhibit mTOR, yet, there is very little direct evidence that mTOR is the rapamycin-sensitive element that confers the hypertrophic response. In this study we determined that, in skeletal muscle, overexpression of Rheb stimulates a PI3K/PKB-independent activation of mTOR signaling and this is sufficient for the induction of a rapamycin-sensitive hypertrophic response. Transgenic mice with muscle specific expression of various mTOR mutants were also used to demonstrate that mTOR is the rapamycin-sensitive element that conferred the hypertrophic response and that the kinase activity of mTOR is necessary for this event. Combined, these results provide direct genetic evidence that a PI3K/PKB-independent activation of mTOR signaling is sufficient to induce hypertrophy.

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Selective Association of Peroxiredoxin 1 with Genomic DNA and COX-2 Upstream Promoter Elements in Estrogen Receptor Negative Breast Cancer Cells.

Publication Date: 2010 Sep PMID: 20631257
Authors: Wang, X. - He, S. - Sun, J. M. - Delcuve, G. P. - Davie, J. R.
Journal: Mol Biol Cell

In a search for proteins differentially cross-linked to DNA by cisplatin or formaldehyde in normal breast epithelial and breast cancer cell lines, we identified peroxiredoxin 1 (PRDX1) as a protein preferentially cross-linked to DNA in estrogen receptor negative (ER-) MDA-MB-231 but not in estrogen receptor positive (ER+) MCF7 breast cancer cells. Indirect immunofluorescence microscopic analyses showed that PRDX1 was located in the cytoplasm and nucleus of normal and breast cancer cells, with nuclear PRDX1 associated with promyelocytic leukemia protein bodies. We demonstrated that PRDX1 association with the transcription factor nuclear factor-kappaB (NF-kappaB) in MDA-MB-231 but not in MCF7 cells contributed to PRDX1-selective recruitment to MDA-MB-231 genomic DNA. Furthermore, PRDX1 was associated with the cyclooxygenase (COX)-2 upstream promoter region at sites occupied by NF-kappaB in ER- but not in ER+ breast cancer cells. PRDX1 knockdown attenuated COX-2 expression by reducing NF-kappaB occupancy at its upstream promoter element in MDA-MB-231 but not in MCF7 cells. A phosphorylated form of PRDX1 was only present in ER- breast cancer cells. Because PRDX1 phosphorylation is known to inhibit its peroxidase activity and to promote PRDX1 oligomerization, we propose that PRDX1 acts as a chaperone to enhance the transactivation potential of NF-kappaB in ER- breast cancer cells.

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The QKI-6 RNA Binding Protein Regulates Actin-interacting Protein-1 mRNA Stability during Oligodendrocyte Differentiation.

Publication Date: 2010 Sep PMID: 20631256
Authors: Doukhanine, E. - Gavino, C. - Haines, J. D. - Almazan, G. - Richard, S.
Journal: Mol Biol Cell

The quaking viable (qk(v)) mice represent an animal model of dysmyelination. The absence of expression of the QKI-6 and QKI-7 cytoplasmic isoforms in oligodendrocytes (OLs) during CNS myelination causes the qk(v) mouse phenotype. The QKI RNA-binding proteins are known to regulate RNA metabolism of cell cycle proteins and myelin components in OLs; however, little is known of their role in reorganizing the cytoskeleton or process outgrowth during OL maturation and differentiation. Here, we identify the actin-interacting protein (AIP)-1 mRNA as a target of QKI-6 by using two-dimensional differential gel electrophoresis. The AIP-1 mRNA contains a consensus QKI response element within its 3'-untranslated region that, when bound by QKI-6, decreases the half-life of the AIP-1 mRNA. Although the expression of QKI-6 is known to increase during OL differentiation and CNS myelination, we show that this increase is paralleled with a corresponding decrease in AIP-1 expression in rat brains. Furthermore, qk(v)/qk(v) mice that lack QKI-6 and QKI-7 within its OLs had an increased level of AIP-1 in OLs. Moreover, primary rat OL precursors harboring an AIP-1 small interfering RNA display defects in OL process outgrowth. Our findings suggest that the QKI RNA-binding proteins regulate OL differentiation by modulating the expression of AIP-1.

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Cdc42 Regulates Apical Junction Formation in Human Bronchial Epithelial Cells through PAK4 and Par6B.

Publication Date: 2010 Sep PMID: 20631255
Authors: Wallace, S. W. - Durgan, J. - Jin, D. - Hall, A.
Journal: Mol Biol Cell

Cdc42 has been implicated in numerous biochemical pathways during epithelial morphogenesis, including the control of spindle orientation during mitosis, the establishment of apical-basal polarity, the formation of apical cell-cell junctions, and polarized secretion. To investigate the signaling pathways through which Cdc42 mediates these diverse effects, we have screened an siRNA library corresponding to the 36 known Cdc42 target proteins, in a human bronchial epithelial cell line. Two targets, PAK4 and Par6B, were identified as necessary for the formation of apical junctions. PAK4 is recruited to nascent cell-cell contacts in a Cdc42-dependent manner, where it is required for the maturation of primordial junctions into apical junctions. PAK4 kinase activity is essential for junction maturation, but overexpression of an activated PAK4 mutant disrupts this process. Par6B, together with its binding partner aPKC, is necessary both for junction maturation and for the retention of PAK4 at sites of cell-cell contact. This study demonstrates that controlled regulation of PAK4 is required for apical junction formation in lung epithelial cells and highlights potential cross-talk between two Cdc42 targets, PAK4 and Par6B.

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Induction of Liver Steatosis and Lipid Droplet Formation in ATF6{alpha}-Knockout Mice Burdened with Pharmacological Endoplasmic Reticulum Stress.

Publication Date: 2010 Sep PMID: 20631254
Authors: Yamamoto, K. - Takahara, K. - Oyadomari, S. - Okada, T. - Sato, T. - Harada, A. - Mori, K.
Journal: Mol Biol Cell

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates homeostatic responses collectively termed the unfolded protein response. Among the three principal signaling pathways operating in mammals, activating transcription factor (ATF)6alpha plays a pivotal role in transcriptional induction of ER-localized molecular chaperones and folding enzymes as well as components of ER-associated degradation, and thereby mouse embryonic fibroblasts deficient in ATF6alpha are sensitive to ER stress. However, ATF6alpha-knockout mice show no apparent phenotype under normal growing conditions. In this report, we burdened mice with intraperitoneal injection of the ER stress-inducing reagent tunicamycin and found that wild-type mice were able to recover from the insult, whereas ATF6alpha-knockout mice exhibited liver dysfunction and steatosis. Thus, ATF6alpha-knockout mice accumulated neutral lipids in the liver such as triacylglycerol and cholesterol, which was ascribable to blockage of beta-oxidation of fatty acids caused by decreased mRNA levels of the enzymes involved in the process, suppression of very-low-density lipoprotein formation due to destabilized apolipoprotein B-100, and stimulation of lipid droplet formation resulting from transcriptional induction of adipose differentiation-related protein. Accordingly, the hepatocytes of tunicamycin-injected knockout mice were filled with many lipid droplets. These results establish links among ER stress, lipid metabolism, and steatosis.

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Differential Requirements for Clathrin-dependent Endocytosis at Sites of Cell-Substrate Adhesion.

Publication Date: 2010 Sep PMID: 20631253
Authors: Batchelder, E. M. - Yarar, D.
Journal: Mol Biol Cell

Clathrin-dependent endocytosis is a major route for the cellular import of macromolecules and occurs at the interface between the cell and its surroundings. However, little is known about the influences of cell-substrate attachment in clathrin-coated vesicle formation. Using biochemical and imaging-based methods, we find that cell-substrate adhesion reduces the rate of endocytosis. Clathrin-coated pits (CCPs) in proximity to substrate contacts exhibit slower dynamics in comparison to CCPs found more distant from adhesions. Direct manipulation of the extracellular matrix (ECM) to modulate adhesion demonstrates that tight adhesion dramatically reduces clathrin-dependent endocytosis and extends the lifetimes of clathrin structures. This reduction is in part mediated by integrin-matrix engagement. In addition, we demonstrate that actin cytoskeletal dynamics are differentially required for efficient endocytosis, with a stronger requirement for actin polymerization in areas of adhesion. Together, these results reveal that cell-substrate adhesion regulates clathrin-dependent endocytosis and suggests that actin assembly facilitates vesicle formation at sites of adhesion.

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Chloride Intracellular Channel 4 Is Critical for the Epithelial Morphogenesis of RPE Cells and Retinal Attachment.

Publication Date: 2010 Sep PMID: 20610659
Authors: Chuang, J. Z. - Chou, S. Y. - Sung, C. H.
Journal: Mol Biol Cell

Retinal detachment is a sight-threatening condition. The molecular mechanism underlying the adhesion between the RPE and photoreceptors is poorly understood because the intimate interactions between these two cell types are impossible to model and study in vitro. In this article, we show that chloride intracellular channel 4 (CLIC4) is enriched at apical RPE microvilli, which are interdigitated with the photoreceptor outer segment. We used a novel plasmid-based transfection method to cell-autonomously suppress CLIC4 in RPE in situ. CLIC4 silenced RPE cells exhibited a significant loss of apical microvilli and basal infoldings, reduced retinal adhesion, and epithelial-mesenchymal transition. Ectopically expressing ezrin failed to rescue the morphological changes exerted by CLIC4 silencing. Neural retinas adjacent to the CLIC4-suppressed RPE cells display severe dysplasia. Finally, a high level of aquaporin 1 unexpectedly appeared at the apical surfaces of CLIC4-suppressed RPE cells, together with a concomitant loss of basal surface expression of monocarboxylate transporter MCT3. Our results suggested that CLIC4 plays an important role in RPE-photoreceptor adhesion, perhaps by modulating the activity of cell surface channels/transporters. We propose that these changes may be attributable to subretinal fluid accumulation in our novel retinal detachment animal model.

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Dissecting BAR Domain Function in the Yeast Amphiphysins Rvs161 and Rvs167 during Endocytosis.

Publication Date: 2010 Sep PMID: 20610658
Authors: Youn, J. Y. - Friesen, H. - Kishimoto, T. - Henne, W. M. - Kurat, C. F. - Ye, W. - Ceccarelli, D. F. - Sicheri, F. - Kohlwein, S. D. - McMahon, H. T. - Andrews, B. J.
Journal: Mol Biol Cell

BAR domains are protein modules that bind to membranes and promote membrane curvature. One type of BAR domain, the N-BAR domain, contains an additional N-terminal amphipathic helix, which contributes to membrane-binding and bending activities. The only known N-BAR-domain proteins in the budding yeast Saccharomyces cerevisiae, Rvs161 and Rvs167, are required for endocytosis. We have explored the mechanism of N-BAR-domain function in the endocytosis process using a combined biochemical and genetic approach. We show that the purified Rvs161-Rvs167 complex binds to liposomes in a curvature-independent manner and promotes tubule formation in vitro. Consistent with the known role of BAR domain polymerization in membrane bending, we found that Rvs167 BAR domains interact with each other at cortical actin patches in vivo. To characterize N-BAR-domain function in endocytosis, we constructed yeast strains harboring changes in conserved residues in the Rvs161 and Rvs167 N-BAR domains. In vivo analysis of the rvs endocytosis mutants suggests that Rvs proteins are initially recruited to sites of endocytosis through their membrane-binding ability. We show that inappropriate regulation of complex sphingolipid and phosphoinositide levels in the membrane can impinge on Rvs function, highlighting the relationship between membrane components and N-BAR-domain proteins in vivo.

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FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network.

Publication Date: 2010 Sep PMID: 20610657
Authors: Jing, J. - Junutula, J. R. - Wu, C. - Burden, J. - Matern, H. - Peden, A. A. - Prekeris, R.
Journal: Mol Biol Cell

Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through several retrograde transport pathways to maintain the composition and function of the TGN. However, the molecular mechanisms involved in these distinct retrograde pathways remain to be fully understood. Here we have used fluorescence and electron microscopy as well as various functional transport assays to show that Rab11a/b and its binding protein FIP1/RCP are both required for the retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN, but not for the retrieval of mannose-6-phosphate receptor from late endosomes. Furthermore, by proteomic analysis we identified Golgin-97 as a FIP1/RCP-binding protein. The FIP1/RCP-binding domain maps to the C-terminus of Golgin-97, adjacent to its GRIP domain. Binding of FIP1/RCP to Golgin-97 does not affect Golgin-97 recruitment to the TGN, but appears to regulate the targeting of retrograde transport vesicles to the TGN. Thus, we propose that FIP1/RCP binding to Golgin-97 is required for tethering and fusion of recycling endosome-derived retrograde transport vesicles to the TGN.

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The HOG Pathway Dictates the Short-Term Translational Response after Hyperosmotic Shock.

Publication Date: 2010 Sep PMID: 20587780
Authors: Warringer, J. - Hult, M. - Regot, S. - Posas, F. - Sunnerhagen, P.
Journal: Mol Biol Cell

Cellular responses to environmental changes occur on different levels. We investigated the translational response of yeast cells after mild hyperosmotic shock by isolating mRNA associated with multiple ribosomes (polysomes) followed by array analysis. Globally, recruitment of preexisting mRNAs to ribosomes (translational response) is faster than the transcriptional response. Specific functional groups of mRNAs are recruited to ribosomes without any corresponding increase in total mRNA. Among mRNAs under strong translational up-regulation upon shock, transcripts encoding membrane-bound proteins including hexose transporters were enriched. Similarly, numerous mRNAs encoding cytoplasmic ribosomal proteins run counter to the overall trend of down-regulation and are instead translationally mobilized late in the response. Surprisingly, certain transcriptionally induced mRNAs were excluded from ribosomal association after shock. Importantly, we verify, using constructs with intact 5' and 3' untranslated regions, that the observed changes in polysomal mRNA are reflected in protein levels, including cases with only translational up-regulation. Interestingly, the translational regulation of the most highly osmostress-regulated mRNAs was more strongly dependent on the stress-activated protein kinases Hog1 and Rck2 than the transcriptional regulation. Our results show the importance of translational control for fine tuning of the adaptive responses.

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Molecular characterization of EG-VEGF-mediated angiogenesis: differential effects on microvascular and macrovascular endothelial cells.

Publication Date: 2010 Aug 15 PMID: 20587779
Authors: Brouillet, S. - Hoffmann, P. - Benharouga, M. - Salomon, A. - Schaal, J. P. - Feige, J. J. - Alfaidy, N.
Journal: Mol Biol Cell

Endocrine gland derived vascular endothelial growth factor (EG-VEGF) also called prokineticin (PK1), has been identified and linked to several biological processes including angiogenesis. EG-VEGF is abundantly expressed in the highest vascularized organ, the human placenta. Here we characterized its angiogenic effect using different experimental procedures. Immunohistochemistry was used to localize EG-VEGF receptors (PROKR1 and PROKR2) in placental and umbilical cord tissue. Primary microvascular placental endothelial cell (HPEC) and umbilical vein-derived macrovascular EC (HUVEC) were used to assess its effects on proliferation, migration, cell survival, pseudovascular organization, spheroid sprouting, permeability and paracellular transport. siRNA and neutralizing antibody strategies were used to differentiate PROKR1- from PROKR2-mediated effects. Our results show that 1) HPEC and HUVEC express both types of receptors 2) EG-VEGF stimulates HPEC's proliferation, migration and survival, but increases only survival in HUVECs. and 3) EG-VEGF was more potent than VEGF in stimulating HPEC sprout formation, pseudovascular organization, and it significantly increases HPEC permeability and paracellular transport. More importantly, we demonstrated that PROKR1 mediates EG-VEGF angiogenic effects, whereas PROKR2 mediates cellular permeability. Altogether, these data characterized angiogenic processes mediated by EG-VEGF, depicted a new angiogenic factor in the placenta, and suggest a novel view of the regulation of angiogenesis in placental pathologies.

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Quantitative analysis of the mechanism of endocytic actin patch assembly and disassembly in fission yeast.

Publication Date: 2010 Aug 15 PMID: 20587778
Authors: Sirotkin, V. - Berro, J. - Macmillan, K. - Zhao, L. - Pollard, T. D.
Journal: Mol Biol Cell

We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30-50% between individual patches. The pathway begins with accumulation of 30-40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (approximately 120 End4p and approximately 230 Pan1p), activators of Arp2/3 complex (approximately 200 Wsp1p and approximately 340 Myo1p) and approximately 300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (approximately 7000 actins, approximately 200 capping proteins, and approximately 900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over approximately 10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803-2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100-200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.

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The Rsr1/Bud1 GTPase Interacts with Itself and the Cdc42 GTPase during Bud-Site Selection and Polarity Establishment in Budding Yeast.

Publication Date: 2010 Sep PMID: 20587777
Authors: Kang, P. J. - Beven, L. - Hariharan, S. - Park, H. O.
Journal: Mol Biol Cell

Cell polarization occurs along a single axis that is generally determined in response to spatial cues. In budding yeast, the Rsr1 GTPase and its regulators direct the establishment of cell polarity at the proper cortical location in response to cell type-specific cues. Here we use a combination of in vivo and in vitro approaches to understand how Rsr1 polarization is established. We find that Rsr1 associates with itself in a spatially and temporally controlled manner. The homotypic interaction and localization of Rsr1 to the mother-bud neck and to the subsequent division site are dependent on its GDP-GTP exchange factor Bud5. Analyses of rsr1 mutants suggest that Bud5 recruits Rsr1 to these sites and promotes the homodimer formation. Rsr1 also exhibits heterotypic interaction with the Cdc42 GTPase in vivo. We show that the polybasic region of Rsr1 is necessary for the efficient homotypic and heterotypic interactions, selection of a proper growth site, and polarity establishment. Our findings thus suggest that dimerization of GTPases may be an efficient mechanism to set up cellular asymmetry.

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Mathematical modeling of endocytic actin patch kinetics in fission yeast: disassembly requires release of actin filament fragments.

Publication Date: 2010 Aug 15 PMID: 20587776
Authors: Berro, J. - Sirotkin, V. - Pollard, T. D.
Journal: Mol Biol Cell

We used the dendritic nucleation hypothesis to formulate a mathematical model of the assembly and disassembly of actin filaments at sites of clathrin-mediated endocytosis in fission yeast. We used the wave of active WASp recruitment at the site of the patch formation to drive assembly reactions after activation of Arp2/3 complex. Capping terminated actin filament elongation. Aging of the filaments by ATP hydrolysis and gamma-phosphate dissociation allowed actin filament severing by cofilin. The model could simulate the assembly and disassembly of actin and other actin patch proteins using measured cytoplasmic concentrations of the proteins. However, to account quantitatively for the numbers of proteins measured over time in the accompanying article (Sirotkin et al., 2010, MBoC 21: 2792-2802), two reactions must be faster in cells than in vitro. Conditions inside the cell allow capping protein to bind to the barbed ends of actin filaments and Arp2/3 complex to bind to the sides of filaments faster than the purified proteins in vitro. Simulations also show that depolymerization from pointed ends cannot account for rapid loss of actin filaments from patches in 10 s. An alternative mechanism consistent with the data is that severing produces short fragments that diffuse away from the patch.

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The closely related RNA helicases, UAP56 and URH49, preferentially form distinct mRNA export machineries and coordinately regulate mitotic progression.

Publication Date: 2010 Aug 15 PMID: 20573985
Authors: Yamazaki, T. - Fujiwara, N. - Yukinaga, H. - Ebisuya, M. - Shiki, T. - Kurihara, T. - Kioka, N. - Kambe, T. - Nagao, M. - Nishida, E. - Masuda, S.
Journal: Mol Biol Cell

Nuclear export of mRNA is an essential process for eukaryotic gene expression. The TREX complex couples gene expression from transcription and splicing to mRNA export. Sub2, a core component of the TREX complex in yeast, has diversified in humans to two closely related RNA helicases, UAP56 and URH49. Here, we show that URH49 forms a novel URH49-CIP29 complex, termed the AREX (alternative mRNA export) complex, whereas UAP56 forms the human TREX complex. The mRNAs regulated by these helicases are different at the genome-wide level. The two sets of target mRNAs contain distinct subsets of key mitotic regulators. Consistent with their target mRNAs, depletion of UAP56 causes mitotic delay and sister chromatid cohesion defects, whereas depletion of URH49 causes chromosome arm resolution defects and failure of cytokinesis. In addition, depletion of the other human TREX components or CIP29 causes mitotic defects similar to those observed in UAP56- or URH49-depleted cells, respectively. Taken together, the two closely related RNA helicases have evolved to form distinct mRNA export machineries, which regulate mitosis at different steps.

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Regulation of genotoxic stress response by homeodomain-interacting protein kinase 2 through phosphorylation of cyclic AMP response element-binding protein at serine 271.

Publication Date: 2010 Aug 15 PMID: 20573984
Authors: Sakamoto, K. - Huang, B. W. - Iwasaki, K. - Hailemariam, K. - Ninomiya-Tsuji, J. - Tsuji, Y.
Journal: Mol Biol Cell

CREB (cyclic AMP response element-binding protein) is a stimulus-induced transcription factor that plays pivotal roles in cell survival and proliferation. The transactivation function of CREB is primarily regulated through Ser-133 phosphorylation by cAMP-dependent protein kinase A (PKA) and related kinases. Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression. Ser-271 to Glu-271 substitution potentiated the CREB transactivation function. ChIP assays in SH-SY5Y neuroblastoma cells demonstrated that CREB Ser-271 phosphorylation by HIPK2 increased recruitment of a transcriptional coactivator CBP (CREB binding protein) without modulation of CREB binding to the BDNF CRE sequence. HIPK2-/- MEF cells were more susceptible to apoptosis induced by etoposide, a DNA-damaging agent, than HIPK2+/+ cells. Etoposide activated CRE-dependent transcription in HIPK2+/+ MEF cells but not in HIPK2-/- cells. HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression. These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress.

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EHBP-1 functions with RAB-10 during endocytic recycling in Caenorhabditis elegans.

Publication Date: 2010 Aug 15 PMID: 20573983
Authors: Shi, A. - Chen, C. C. - Banerjee, R. - Glodowski, D. - Audhya, A. - Rongo, C. - Grant, B. D.
Journal: Mol Biol Cell

Caenorhabditis elegans RAB-10 functions in endocytic recycling in polarized cells, regulating basolateral cargo transport in the intestinal epithelia and postsynaptic cargo transport in interneurons. A similar role was found for mammalian Rab10 in MDCK cells, suggesting that a conserved mechanism regulates these related pathways in metazoans. In a yeast two-hybrid screen for binding partners of RAB-10 we identified EHBP-1, a calponin homology domain (CH) protein, whose mammalian homolog Ehbp1 was previously shown to function during endocytic transport of GLUT4 in adipocytes. In vivo we find that EHBP-1-GFP colocalizes with RFP-RAB-10 on endosomal structures of the intestine and interneurons and that ehbp-1 loss-of-function mutants share with rab-10 mutants specific endosome morphology and cargo localization defects. We also show that loss of EHBP-1 disrupts transport of membrane proteins to the plasma membrane of the nonpolarized germline cells, a defect that can be phenocopied by codepletion of RAB-10 and its closest paralog RAB-8. These results indicate that RAB-10 and EHBP-1 function together in many cell types and suggests that there are differences in the level of redundancy among Rab family members in polarized versus nonpolarized cells.

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Sequential counteracting kinases restrict an asymmetric gene expression program to early G1.

Publication Date: 2010 Aug 15 PMID: 20573982
Authors: Mazanka, E. - Weiss, E. L.
Journal: Mol Biol Cell

Gene expression is restricted to specific times in cell division and differentiation through close control of both activation and inactivation of transcription. In budding yeast, strict spatiotemporal regulation of the transcription factor Ace2 ensures that it acts only once in a cell's lifetime: at the M-to-G1 transition in newborn daughter cells. The Ndr/LATS family kinase Cbk1, functioning in a system similar to metazoan hippo signaling pathways, activates Ace2 and drives its accumulation in daughter cell nuclei, but the mechanism of this transcription factor's inactivation is unknown. We found that Ace2's nuclear localization is maintained by continuous Cbk1 activity and that inhibition of the kinase leads to immediate loss of phosphorylation and export to the cytoplasm. Once exported, Ace2 cannot re-enter nuclei for the remainder of the cell cycle. Two separate mechanisms enforce Ace2's cytoplasmic sequestration: 1) phosphorylation of CDK consensus sites in Ace2 by the G1 CDKs Pho85 and Cdc28/CDK1 and 2) an unknown mechanism mediated by Pho85 that is independent of its kinase activity. Direct phosphorylation of CDK consensus sites is not necessary for Ace2's cytoplasmic retention, indicating that these mechanisms function redundantly. Overall, these findings show how sequential opposing kinases limit a daughter cell specific transcriptional program to a brief period during the cell cycle and suggest that CDKs may function as cytoplasmic sequestration factors.

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Transgene rescue identifies an essential function for Drosophila beta spectrin in the nervous system and a selective requirement for ankyrin-2-binding activity.

Publication Date: 2010 Aug 15 PMID: 20573981
Authors: Mazock, G. H. - Das, A. - Base, C. - Dubreuil, R. R.
Journal: Mol Biol Cell

The protein spectrin is ubiquitous in animal cells and is believed to play important roles in cell shape and membrane stability, cell polarity, and endomembrane traffic. Experiments here were undertaken to identify sites of essential beta spectrin function in Drosophila and to determine whether spectrin and ankyrin function are strictly linked to one another. The Gal4-UAS system was used to drive tissue-specific overexpression of a beta spectrin transgene or to knock down beta spectrin expression with dsRNA. The results show that 1) overexpression of beta spectrin in most of the cell types studied was lethal; 2) knockdown of beta spectrin in most tissues had no detectable effect on growth or viability of the organism; and 3) nervous system-specific expression of a UAS-beta spectrin transgene was sufficient to overcome the lethality of a loss-of-function beta spectrin mutation. Thus beta spectrin expression in other cells was not required for development of fertile adult males, although females lacking nonneuronal spectrin were sterile. Previous data indicated that binding of the DAnk1 isoform of ankyrin to spectrin was partially dispensable for viability. Domain swap experiments here uncovered a different requirement for neuronal DAnk2 binding to spectrin and establish that DAnk2-binding is critical for beta spectrin function in vivo.

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Overlapping roles of Drosophila Drak and Rok kinases in epithelial tissue morphogenesis.

Publication Date: 2010 Aug 15 PMID: 20573980
Authors: Neubueser, D. - Hipfner, D. R.
Journal: Mol Biol Cell

Dynamic regulation of cytoskeletal contractility through phosphorylation of the nonmuscle Myosin-II regulatory light chain (MRLC) provides an essential source of tension for shaping epithelial tissues. Rho GTPase and its effector kinase ROCK have been implicated in regulating MRLC phosphorylation in vivo, but evidence suggests that other mechanisms must be involved. Here, we report the identification of a single Drosophila homologue of the Death-associated protein kinase (DAPK) family, called Drak, as a regulator of MRLC phosphorylation. Based on analysis of null mutants, we find that Drak broadly promotes proper morphogenesis of epithelial tissues during development. Drak activity is largely redundant with that of the Drosophila ROCK orthologue, Rok, such that it is essential only when Rok levels are reduced. We demonstrate that these two kinases synergistically promote phosphorylation of Spaghetti squash (Sqh), the Drosophila MRLC orthologue, in vivo. The lethality of drak/rok mutants can be rescued by restoring Sqh activity, indicating that Sqh is the critical common effector of these two kinases. These results provide the first evidence that DAPK family kinases regulate actin dynamics in vivo and identify Drak as a novel component of the signaling networks that shape epithelial tissues.

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WASP family proteins: their evolution and its physiological implications.

Publication Date: 2010 Aug 15 PMID: 20573979
Authors: Veltman, D. M. - Insall, R. H.
Journal: Mol Biol Cell

WASP family proteins control actin polymerization by activating the Arp2/3 complex. Several subfamilies exist, but their regulation and physiological roles are not well understood, nor is it even known if all subfamilies have been identified. Our extensive search reveals few novel WASP family proteins. The WASP, WASH, and SCAR/WAVE subfamilies are evolutionarily ancient, with WASH the most universally present, whereas WHAMM/JMY first appears in invertebrates. An unusual Dictyostelium WASP homologue that has lost the WH1 domain has retained its function in clathrin-mediated endocytosis, demonstrating that WASPs can function with a remarkably diverse domain topology. The WASH and SCAR/WAVE regulatory complexes are much more rigidly maintained; their domain topology is highly conserved, and all subunits are present or lost together, showing that the complexes are ancient and functionally interdependent. Finally, each subfamily has a distinctive C motif, indicating that this motif plays a specific role in each subfamily's function, unlike the generic V and A motifs. Our analysis identifies which features are universally conserved, and thus essential, and which are branch-specific modifications. It also shows the WASP family is more widespread and diverse than currently appreciated and unexpectedly biases the physiological role of the Arp2/3 complex toward vesicle traffic.

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Phosphatidic acid plays a regulatory role in clathrin-mediated endocytosis.

Publication Date: 2010 Aug 15 PMID: 20573978
Authors: Antonescu, C. N. - Danuser, G. - Schmid, S. L.
Journal: Mol Biol Cell

Clathrin-mediated endocytosis (CME) is the main route of internalization of receptor-ligand complexes. Relatively little is known about the role of specific lipids in CME, in particular that of phosphatidic acid (PA). We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production. DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically. This corresponded to a decreased rate of clathrin-coated pit (CCP) initiation and increased lifetimes of productive CCPs, as determined by quantitative live-cell total internal reflection fluorescence microscopy. Unexpectedly, PLD inhibition caused an increase in cellular PA, suggesting that PLD activity negatively regulates PA synthesis by other more productive pathways. Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition. Importantly, the constitutive internalization of transferrin receptors was unaffected by either treatment. These findings demonstrate that PA plays a regulatory rather than obligatory role in CME and differentially regulates ligand-stimulated CME of EGFR.

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Regulation of exocytosis and fusion pores by synaptotagmin-effector interactions.

Publication Date: 2010 Aug 15 PMID: 20573977
Authors: Zhang, Z. - Hui, E. - Chapman, E. R. - Jackson, M. B.
Journal: Mol Biol Cell

Synaptotagmin (syt) serves as a Ca(2+) sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca(2+), but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca(2+) triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.

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Plakophilin 2 couples actomyosin remodeling to desmosomal plaque assembly via RhoA.

Publication Date: 2010 Aug 15 PMID: 20554761
Authors: Godsel, L. M. - Dubash, A. D. - Bass-Zubek, A. E. - Amargo, E. V. - Klessner, J. L. - Hobbs, R. P. - Chen, X. - Green, K. J.
Journal: Mol Biol Cell

Plakophilin 2 (PKP2), an armadillo family member closely related to p120 catenin (p120ctn), is a constituent of the intercellular adhesive junction, the desmosome. We previously showed that PKP2 loss prevents the incorporation of desmosome precursors enriched in the plaque protein desmoplakin (DP) into newly forming desmosomes, in part by disrupting PKC-dependent regulation of DP assembly competence. On the basis of the observation that DP incorporation into junctions is cytochalasin D-sensitive, here we ask whether PKP2 may also contribute to actin-dependent regulation of desmosome assembly. We demonstrate that PKP2 knockdown impairs cortical actin remodeling after cadherin ligation, without affecting p120ctn expression or localization. Our data suggest that these defects result from the failure of activated RhoA to localize at intercellular interfaces after cell-cell contact and an elevation of cellular RhoA, stress fibers, and other indicators of contractile signaling in squamous cell lines and atrial cardiomyocytes. Consistent with these observations, RhoA activation accelerated DP redistribution to desmosomes during the first hour of junction assembly, whereas sustained RhoA activity compromised desmosome plaque maturation. Together with our previous findings, these data suggest that PKP2 may functionally link RhoA- and PKC-dependent pathways to drive actin reorganization and regulate DP-IF interactions required for normal desmosome assembly.

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Phosphatidic acid induces ligand-independent epidermal growth factor receptor endocytic traffic through PDE4 activation.

Publication Date: 2010 Aug 15 PMID: 20554760
Authors: Norambuena, A. - Metz, C. - Jung, J. E. - Silva, A. - Otero, C. - Cancino, J. - Retamal, C. - Valenzuela, J. C. - Soza, A. - Gonzalez, A.
Journal: Mol Biol Cell

Endocytosis modulates EGFR function by compartmentalizing and attenuating or enhancing its ligand-induced signaling. Here we show that it can also control the cell surface versus intracellular distribution of empty/inactive EGFR. Our previous observation that PKA inhibitors induce EGFR internalization prompted us to test phosphatidic acid (PA) generated by phospholipase D (PLD) as an endogenous down-regulator of PKA activity, which activates rolipram-sensitive type 4 phosphodiesterases (PDE4) that degrade cAMP. We found that inhibition of PA hydrolysis by propranolol, in the absence of ligand, provokes internalization of inactive (neither tyrosine-phosphorylated nor ubiquitinated) EGFR, accompanied by a transient increase in PA levels and PDE4s activity. This EGFR internalization is mimicked by PA micelles and is strongly counteracted by PLD2 silencing, rolipram or forskolin treatment, and PKA overexpression. Accelerated EGFR endocytosis seems to be mediated by clathrin-dependent and -independent pathways, leading to receptor accumulation in juxtanuclear recycling endosomes, also due to a decreased recycling. Internalized EGFR can remain intracellular without degradation for several hours or return rapidly to the cell surface upon discontinuation of the stimulus. This novel regulatory mechanism of EGFR, also novel function of signaling PA, can transmodulate receptor accessibility in response to heterologous stimuli.

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