Molecular Biology of the Cell

Systematic survey of deubiquitinase localisation identifies USP21 as a regulator of centrosome and microtubule associated functions.

Publication Date: 2012 Feb 1 PMID: 22298430
Authors: Urbe, S. - Liu, H. - Hayes, S. D. - Heride, C. - Rigden, D. J. - Clague, M. J.
Journal: Mol Biol Cell

Ubiquitination is a reversible modification that influences a broad range of physiological processes. There are approximately 90 DUBs encoded in the human genome, of which 79 are predicted to have catalytic activity. We have tagged 66 DUBs with GFP and systematically surveyed their sub-cellular distribution, identifying enzymes specific to the nucleus, plasma membrane, secretory and endocytic pathways. USP21 is unique in showing clear association with both centrosomes and microtubules. Using an in vitro assay, we show that microtubule binding is direct and identify a novel microtubule binding motif, encompassed within amino acids 59-75 of the N-terminus of USP21. Our functional studies indicate a key role for USP21 in the governance of microtubule and centrosome associated physiological processes: Depletion of USP21 in A549 cells compromises the re-establishment of a radial array of microtubules during recovery from cold-induced depolymerisation and also reduces the probability of primary cilium formation, whilst USP21 knockdown in PC12 cells inhibits NGF-induced neurite outgrowth.

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The coupling between sister kinetochore directional instability and oscillations in centromere stretch in metaphase PtK1 cells.

Publication Date: 2012 Feb 1 PMID: 22298429
Authors: Wan, X. - Cimini, D. - Cameron, L. A. - Salmon, E. D.
Journal: Mol Biol Cell

Kinetochores bound to kinetochore microtubules (kMTs) exhibit directional instability in mammalian and other mitotic vertebrate cells, oscillating between poleward (P) and away from the pole (AP) movements. These oscillations are coupled to changes in length of kMTs in a way that maintains a net stretch of the centromere. To understand how sister kinetochore directional instability and kMT plus-end dynamic instability are coupled to oscillations in centromere stretch, we tracked at high resolution the positions of fluorescent kinetochores and their poles for oscillating chromosomes within spindles of metaphase PtK1 cells. We found that the kinetics of P and AP movement are non-linear and different. By subtracting contributions from the poleward flux of kMTs, we found that maximum centromere stretch occurred when the leading kinetochore switched from depolymerization to polymerization, while minimum centromere stretch occurred on average 7 sec after the initially lagging kinetochore switched from polymerization to depolymerization. These differences produce oscillations in centromere stretch at about twice the frequency of kinetochore directional instability and at about twice the frequency of centromere oscillations back and forth across the spindle equator.

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The Ankrd 13 family of UIM-bearing proteins regulates EGF receptor endocytosis from the plasma membrane.

Publication Date: 2012 Feb 1 PMID: 22298428
Authors: Tanno, H. - Yamaguchi, T. - Goto, E. - Ishido, S. - Komada, M.
Journal: Mol Biol Cell

The mechanism of ubiquitin-dependent endocytosis of cell surface proteins is not completely understood. Here, we examined the role of the ankyrin repeat domain (Ankrd) 13A, 13B, and 13D proteins, which constitute a functionally unknown family of ubiquitin-interacting motif (UIM)-bearing proteins, in the process. Stimulation of human HeLa cells with epidermal growth factor (EGF) rapidly induced direct binding of Ankrd 13 proteins to ubiquitinated EGF receptor (EGFR) via the UIMs. The binding was inhibited when the Ankrd 13 proteins underwent UIM-dependent monoubiquitination, suggesting that their activity is regulated by ubiquitination of themselves. Ankrd 13 proteins bound specifically to Lys63-linked ubiquitin chains, which was consistent with a previous report that EGFR mainly undergoes Lys63-linked polyubiquitination. Ankrd 13 proteins were anchored, via the central region and UIMs, to the plasma membrane where they co-localized with EGFR. Finally, overexpression of wild-type as well as truncated-mutant Ankrd 13 proteins strongly inhibited rapid endocytosis of ubiquitinated EGFR from the surface in EGF-treated cells. We conclude that by binding to the Lys63-linked polyubiquitin moiety of EGFR at the plasma membrane, Ankrd 13 proteins regulate the rapid internalization of ligand-activated EGFR.

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Roles of putative Rho-GEF Gef2 in division-site positioning and contractile-ring function in fission yeast cytokinesis.

Publication Date: 2012 Feb 1 PMID: 22298427
Authors: Ye, Y. - Lee, I. J. - Runge, K. W. - Wu, J. Q.
Journal: Mol Biol Cell

Cytokinesis is crucial for integrating genome inheritance and cell functions. In multicellular organisms, Rho-GEFs and Rho GTPases are key regulators of division-plane specification and contractile-ring formation during cytokinesis, but how they regulate early steps of cytokinesis in fission yeast remains largely unknown. Here we show that putative Rho-GEF Gef2 and Polo kinase Plo1 coordinate to control the medial cortical localization and function of anillin-related protein Mid1. The division-site positioning defects of gef2 plo1-ts18 double mutant can be partially rescued by increasing Mid1 levels. We find that Gef2 physically interacts with the Mid1 N-terminus and modulates Mid1 cortical binding. Gef2 localization to cortical nodes and the contractile ring depends on its last 145 residues, and the DBL-homology (DH) domain is important for its function in cytokinesis. Our data suggest the interaction between Rho-GEFs and anillins is an important step of the signaling pathways during cytokinesis. In addition, Gef2 also regulates contractile-ring function late in cytokinesis and may negatively regulate the septation initiation network. Collectively, we propose that Gef2 facilitates and stabilizes Mid1 binding to the medial cortex, where the localized Mid1 specifies the division site and induces contractile-ring assembly.

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Phosphorylation of Stim1 at serine575 via netrin-2/Cdo-activated ERK1/2 is critical for the promyogenic function of Stim1.

Publication Date: 2012 Feb 1 PMID: 22298426
Authors: Lee, H. J. - Bae, G. U. - Leem, Y. E. - Choi, H. K. - Kang, T. M. - Cho, H. - Kim, S. T. - Kang, J. S.
Journal: Mol Biol Cell

The promyogenic cell surface molecule, Cdo is required for activation of ERK and NFATc3 induced by netrin-2 in myogenic differentiation however the molecular mechanism leading to NFATc3 activation is still unknown. Stromal interaction molecule 1 (Stim1), an internal calcium sensor of the endoplasmic reticulum store, promotes myogenesis via activation of NFATc3. In this study we investigated the functional interaction between Cdo and Stim1 in myogenic differentiation. Overexpression and depletion of Stim1 enhanced or decreased myotube formation, respectively. Interestingly Stim1 protein levels were decreased in Cdo-deficient perinatal hindlimb muscles or primary myoblasts; this correlates with defective NFATc3 activation in Cdo(-/-) myoblasts upon differentiation. Forced activation of NFATc3 by overexpression of Calcineurin restored differentiation of Cdo-depleted C2C12 myoblasts. Furthermore, Cdo and Stim1 formed a complex in 293T cells or in differentiating C2C12 myoblasts. The netrin-2-mediated NFATc3 activation was coincident with robust interactions between Cdo and Stim1 in myoblasts and the ERK-mediated Stim1 phosphorylation at serine 575. The serine 575 phosphorylation was enhanced in C2C12 cells upon differentiation, and the alanine substitution of serine 575 failed to restore differentiation of Stim1-depleted myoblasts. Taken together, the cell adhesion signaling triggered by netrin-2/Cdo induces Stim1 phosphorylation at serine 575 by ERK which promotes myoblast differentiation.

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GPI-anchor Synthesis Is Indispensable for the Germline Development of the Nematode Caenorhabditis elegans.

Publication Date: 2012 Feb 1 PMID: 22298425
Authors: Murata, D. - Nomura, K. H. - Dejima, K. - Mizuguchi, S. - Kawasaki, N. - Matsuishi-Nakajima, Y. - Ito, S. - Gengyo-Ando, K. - Kage-Nakadai, E. - Mitani, S. - Nomura, K.
Journal: Mol Biol Cell

Glycosylphosphatidylinositol (GPI)-anchor attachment is one of the most common post-translational protein modifications. Using the nematode Caenorhabditis elegans, we determined that GPI-anchored proteins are present in germline cells and distal tip cells (DTCs), which are essential for the maintenance of the germline stem cell niche. We identified 24 C. elegans genes involved in GPI-anchor synthesis. Inhibition of various steps of GPI-anchor synthesis by RNAi or gene knockout resulted in abnormal development of oocytes and early embryos, and both lethal and sterile phenotypes were observed. The piga-1 gene (ortholog of human PIGA) codes for the catalytic subunit of the phosphatidylinositol N-acetylglucosaminyltransferase complex, which catalyzes the first step of GPI-anchor synthesis. We isolated piga-1 knockout worms and found that GPI-anchor synthesis is indispensable for the maintenance of mitotic germline cell number. The knockout worms displayed 100% lethality with decreased mitotic germline cells and abnormal eggshell formation. Using cell-specific rescue of the null allele, we showed that expression of piga-1 in somatic gonads and/or in germline is sufficient for normal embryonic development and the maintenance of the germline mitotic cells. These results clearly demonstrate that GPI-anchor synthesis is indispensable for germline formation and for normal development of oocytes and eggs.

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Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER- associated degradation.

Publication Date: 2012 Feb 1 PMID: 22298424
Authors: Izawa, T. - Nagai, H. - Endo, T. - Nishikawa, S. I.
Journal: Mol Biol Cell

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. While the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.

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Co-localization of Mec1 and Mrc1 is sufficient for Rad53 phosphorylation in vivo.

Publication Date: 2012 Feb 1 PMID: 22298423
Authors: Berens, T. J. - Toczyski, D. P.
Journal: Mol Biol Cell

When DNA is damaged, or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore, we constructed an in vivo replication-checkpoint mimic, which recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the endogenous replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires co-localization of Mrc1-LacI and Ddc2-LacI, and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the endogenous replication checkpoint, but that Ddc1 is absolutely required in the absence of Mrc1. We propose that co-localization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.

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Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis.

Publication Date: 2012 Jan 25 PMID: 22278744
Authors: Liang, X. - Da Paula, A. C. - Bozoky, Z. - Zhang, H. - Bertrand, C. A. - Peters, K. W. - Forman-Kay, J. D. - Frizzell, R. A.
Journal: Mol Biol Cell

CFTR is a cAMP/PKA-regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis post-translationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. 14-3-3beta, gamma and epsilon isoforms were expressed in airway cells and interacted with CFTR in co-immunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3beta and epsilon binding to immature and mature CFTR (bands B and C), and 14-3-3 over-expression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3beta increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3beta knockdown decreased CFTR B and C bands (70 and 55%), and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3beta interacted with the CFTR R region, and by NMR analysis, this interaction occurred at known PKA phosphorylated sites. In co-IP assays, forskolin stimulated the CFTR/14-3-3beta interaction while reducing CFTR's interaction with COPI. Thus, 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the ER. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.

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Midbody Assembly and its Regulation during Cytokinesis.

Publication Date: 2012 Jan 25 PMID: 22278743
Authors: Hu, C. K. - Coughlin, M. - Mitchison, T. J.
Journal: Mol Biol Cell

The midbody is a transient structure that connects two daughter cells at the end of cytokinesis, with the principle function to localize the site of abscission, which physically separates two daughter cells. Despite its importance, our understanding of midbody assembly and its regulation is still limited. Here we describe how the structural composition of the midbody changes during progression throughout cytokinesis, and explore the functional implications of these changes. Deriving from midzones, midbodies are organized by a set of microtubule interacting proteins that colocalize to a zone of microtubule overlap in the center. We found that these proteins split into three sub-groups that relocalize to different parts of the midbody: the bulge, the dark zone, and the flanking zone. We characterized these relocalizations, and defined domain requirements for three key proteins, MKLP1, KIF4 and PRC1. Two cortical proteins, Anillin and RhoA, localized to presumptive abscission sites in mature midbodies, where they may regulate the ESCRT machinery. Finally, we characterized the role of Plk1, a key regulator of cytokinesis, in midbody assembly. Our findings represent the most detailed description of midbody assembly and maturation to date, and may help elucidate how abscission sites are positioned and regulated.

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GARP regulates the bioavailability and activation of TGFbeta

Publication Date: 2012 Jan 25 PMID: 22278742
Authors: Wang, R. - Zhu, J. - Dong, X. - Shi, M. - Lu, C. - Springer, T. A.
Journal: Mol Biol Cell

GARP associates with latent TGFbeta (proTGFbeta) on the surface of T regulatory cells and platelets; however, whether GARP functions in latent TGFbeta activation, and the structural basis of co-association remain unknown. We find that Cys192 and Cys331 of GARP disulfide link to the TGFbeta1 prodomain, and that GARP with C192A and C331A mutations can also noncovalently associate with proTGFbeta1. Noncovalent association is sufficiently strong for GARP to outcompete latent TGFbeta binding protein (LTBP) for binding to proTGFbeta1. Association between GARP and proTGFbeta1 prevents the secretion of TGFbeta1. Integrin alpha(V)beta(6) and to a lesser extent alpha(V)beta(8) are able to activate TGFbeta from the GARP-proTGFbeta1 complex. Activation requires the RGD motif of latent TGFbeta, disulfide linkage between GARP and latent TGFbeta, and membrane association of GARP. Our results show that GARP is a latent TGFbeta binding protein that functions in regulating the bioavailability and activation of TGFbeta.

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The first luminal loop confers insulin responsiveness to the glucose transporter 4.

Publication Date: 2012 Jan 19 PMID: 22262463
Authors: Kim, J. - Kandror, K. V.
Journal: Mol Biol Cell

GLUT4 is the major glucose transporter which is regulated by insulin and is alone responsible for the effect of insulin on post-prandial blood glucose clearance. However, the nature of the insulin sensitivity of GLUT4 remains unknown. Here, we have replaced the first luminal loop of cellugyrin, the four-transmembrane protein that does not respond to insulin, with that of GLUT4. The chimera protein is targeted to the intracellular insulin-responsive vesicles and is translocated to the plasma membrane upon insulin stimulation. The faithful targeting of the chimera depends on the expression of the sorting receptor sortilin, that interacts with the unique amino acid residues in the first luminal loop of GLUT4. Thus, the first luminal loop may confer insulin responsiveness to the GLUT4 molecule.

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The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2.

Publication Date: 2012 Jan 19 PMID: 22262462
Authors: Saitoh, N. - Sakamoto, C. - Hagiwara, M. - Agredano-Moreno, L. T. - Jimenez-Garcia, L. F. - Nakao, M.
Journal: Mol Biol Cell

The mammalian cell nucleus is functionally compartmentalized into various sub-structures. Nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Here, we report that nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus, and are instead localized at granular structures in the cytoplasm. To investigate how the nuclear architecture is formed, we performed a phenotypic screen of HeLa cells treated with a series of small interfering RNAs. Depletion of RANBP2 induced cytoplasmic intermediates of nuclear speckles in G1 phase. Detailed analyses of these structures suggested that a late step in the sequential nuclear entry of mitotic interchromatin granule components was disrupted, and that phosphorylated SR proteins were sequestered in an SR protein kinase-dependent manner. As a result, the cells had an imbalanced sub-cellular distribution of phosphorylated and hypophosphorylated SR proteins, which affected alternative splicing patterns. This study demonstrates that the speckled distribution of phosphorylated pre-mRNA processing factors is regulated by the nucleocytoplasmic transport system in mammalian cells, and that it is important for alternative splicing.

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YME1L controls the accumulation of respiratory chain subunits and is required for apoptotic resistance, cristae morphogenesis and cell proliferation.

Publication Date: 2012 Jan 19 PMID: 22262461
Authors: Stiburek, L. - Cesnekova, J. - Kostkova, O. - Fornuskova, D. - Vinsova, K. - Wenchich, L. - Houstek, J. - Zeman, J.
Journal: Mol Biol Cell

Mitochondrial AAA (ATPases associated with diverse cellular activities) proteases are involved in the quality control and processing of inner-membrane proteins. Here, we investigated the cellular activities of YME1L, the human ortholog of the Yme1 subunit of the yeast i-AAA complex, using stable shRNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600-1100 kDa. The stable knockdown of YME1L in HEK293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of non-assembled respiratory chain subunits (Ndufb6, ND1 and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.

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A Secreted MMP Is Required for Re-epithelialization during Wound Healing.

Publication Date: 2012 Jan 19 PMID: 22262460
Authors: Stevens, L. J. - Page-McCaw, A.
Journal: Mol Biol Cell

Matrix metalloproteinases (MMPs) are extracellular proteases highly expressed at wound sites. However, the precise function of MMPs during re-epithelialization in vivo has been elusive in mammalian models because of the high level of redundancy between the 24 mammalian MMPs. For this reason we used Drosophila melanogaster, whose genome encodes only two MMPs, one secreted type (Mmp1) and one membrane-anchored type (Mmp2), to study the function and regulation of the secreted class of MMPs in vivo. In the absence of redundancy, we found that the Drosophila secreted MMP, Mmp1, is required in the epidermis to facilitate re-epithelialization by remodeling the basement membrane, promoting cell elongation and actin cytoskeletal reorganization, and activating ERK signaling. Additionally, we report that the JNK pathway upregulates Mmp1 expression after wounding, but that Mmp1 is expressed independently of the JNK pathway in unwounded epidermis. When the JNK pathway is ectopically activated to overexpress Mmp1, the rate of healing is accelerated in an Mmp1-dependent manner. A primary function of Mmp1, under the control of the JNK pathway, is to promote basement membrane repair, which in turn may permit cell migration and the restoration of a continuous tissue.

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Chk1 and Wee1 kinases coordinate DNA replication, chromosome condensation and anaphase entry.

Publication Date: 2012 Jan 19 PMID: 22262459
Authors: Fasulo, B. - Koyama, C. - Yu, K. R. - Homola, E. M. - Hsieh, T. S. - Campbell, S. D. - Sullivan, W.
Journal: Mol Biol Cell

Defects in DNA replication and chromosome condensation are common phenotypes in cancer cells. A link between replication and condensation has been established, but little is known about the role of checkpoints in monitoring chromosome condensation. We investigate this function by live analysis, using the rapid division cycles in the early Drosophila embryo. We find S-phase and topoisomerase inhibitors delay both the initiation and the rate of chromosome condensation. These cell cycle delays are mediated by the cell cycle kinases chk1 and wee1. Inhibitors that cause severe defects in chromosome condensation and congression on the metaphase plate result in delayed anaphase entry. These delays are mediated by wee1 and are not the result of spindle assembly checkpoint activation. In addition, we provide the first detailed live analysis of the direct effect of widely used anti-cancer agents (aclarubicin, ICRF-193, VM26, doxorubicin, camptothecin, aphidicolin, hydroxyurea, cisplatin, mechlorethamine and X-rays) upon key nuclear and cytoplasmic cell cycle events.

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Actin Binding to WH2 Domains Regulates Nuclear Import of the Multifunctional Actin Regulator JMY.

Publication Date: 2012 Jan 19 PMID: 22262458
Authors: Bradley Zuchero, J. - Belin, B. - Dyche Mullins, R.
Journal: Mol Biol Cell

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. In response to DNA damage JMY accumulates in the nucleus and promotes p53-dependent apoptosis. JMY's actin regulatory activity relies on a cluster of three actin-binding WH2 domains that nucleate filaments directly and also promote nucleation activity of the Arp2/3 complex. In addition to these activities, we find that the WH2 cluster overlaps an atypical, bipartite nuclear localization sequence (NLS) and controls JMY's subcellular localization. Actin monomers bound to the WH2 domains block binding of Importins to the NLS and prevent nuclear import of JMY. Mutations that impair actin binding, or cellular perturbations that induce actin filament assembly and decrease the concentration of monomeric actin in the cytoplasm, cause JMY to accumulate in the nucleus. DNA damage induces both cytoplasmic actin polymerization and nuclear import of JMY, and we find that damage-induced nuclear localization of JMY requires both the WH2/NLS region and Importin beta. Based on our results we propose that actin assembly regulates nuclear import of JMY in response to DNA damage.

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Ezrin regulates microvillus morphogenesis by promoting distinct activities of Eps8 proteins.

Publication Date: 2012 Jan 19 PMID: 22262457
Authors: Zwaenepoel, I. - Naba, A. - da Cunha, M. M. - Del Maestro, L. - Formstecher, E. - Louvard, D. - Arpin, M.
Journal: Mol Biol Cell

The mechanisms that regulate actin filament polymerization resulting in the morphogenesis of the brush border microvilli in epithelial cells remain unknown. Eps8, the prototype of a family of proteins capable to cap and bundle actin filaments, has been shown to bundle the microvillar actin filaments. We report that Eps8L1a, a member of the Eps8 family, and a novel ezrin-interacting partner, controls microvillus length through its capping activity. Depletion of Eps8L1a leads to the formation of long microvilli whereas its overexpression has the opposite effect. We demonstrate that ezrin differentially modulates the actin capping and bundling activities of Eps8 and Eps8L1a during microvillus assembly. Coexpression of ezrin with Eps8 promotes the formation of membrane ruffles and tufts of microvilli while expression of ezrin and Eps8L1a induces the clustering of actin containing structures at the cell surface. These distinct morphological changes are neither observed when a mutant of ezrin defective in its binding to Eps8/Eps8L1a is coexpressed with Eps8 or Eps8L1a nor when ezrin is expressed with mutants of Eps8 or Eps8L1a defective in the actin bundling or capping activities respectively. Our data show a synergistic effect of ezrin and Eps8 proteins in the assembly and the organization of actin microvillar filaments.

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Essential diurnal Rac1 activation during retinal phagocytosis requires alphavbeta5 integrin but not tyrosine kinases FAK or MerTK.

Publication Date: 2012 Jan 19 PMID: 22262456
Authors: Mao, Y. - Finnemann, S. C.
Journal: Mol Biol Cell

Diurnal phagocytosis of shed photoreceptor outer segment particles by retinal pigment epithelial (RPE) cells belongs to a group of conserved clearance mechanisms employing alphav integrins upstream of tyrosine kinases and Rho GTPases. Here, we test the interdependence of tyrosine kinases FAK, MerTK and Rho GTPases during engulfment. RPE cells activated and redistributed Rac1 but not RhoA or Cdc42 during phagocytosis. Toxin B, overexpression of dominant-negative Rac1, or decreasing Rac1 expression prevented particle engulfment. Fluorescence microscopy showed that Rac1 inhibition had no obvious effect on F-actin arrangement in resting RPE but prevented recruitment of F-actin to surface-bound phagocytic particles. Quantification of active GTP-Rac1 in wild-type and mutant RPE in culture and in vivo revealed that Rac1 activation during phagocytosis requires alphavbeta5 integrin and its ligand MFG-E8 but not the receptor tyrosine kinase MerTK. Abolishing tyrosine kinase signaling downstream of alphavbeta5 towards MerTK by inhibiting focal adhesion kinase (FAK) specifically or tyrosine kinases generally neither prevented Rac1 activation nor F-actin recruitment during phagocytosis. Likewise, inhibiting Rac1 had no effect on FAK or MerTK activation. We conclude that MerTK activation via FAK and F-actin recruitment via Rac1 both require MFG-E8-ligated alphavbeta5 integrin. Both pathways are independently activated and required for clearance phagocytosis.

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Inhibition of fatty acid oxidation enhances oxidative protein folding and protects hepatocytes from endoplasmic reticulum stress.

Publication Date: 2012 Jan 19 PMID: 22262455
Authors: Tyra, H. M. - Spitz, D. R. - Rutkowski, D. T.
Journal: Mol Biol Cell

The unfolded protein response (UPR) signals protein misfolding in the endoplasmic reticulum (ER) to effect gene expression changes and restore ER homeostasis. While many UPR-regulated genes encode ER protein processing factors, others such as those encoding lipid catabolism enzymes seem unrelated to ER function. It is not known whether UPR-mediated inhibition of fatty acid oxidation influences ER function, and if so, by what mechanism. Here, we demonstrate that pharmacological or genetic inhibition of fatty acid oxidation renders liver cells partially resistant to ER stress-induced UPR activation both in vitro and in vivo. Reduced stress sensitivity appeared to be a consequence of increased cellular redox potential as judged by an elevated ratio of oxidized to reduced glutathione and enhanced oxidative folding in the ER. Accordingly, the ER folding benefit of inhibiting FA oxidation could be phenocopied by manipulating glutathione recycling during ER stress. Conversely, preventing cellular hyper-oxidation with N-acetyl cysteine partially negated the stress resistance provided by blocking FA oxidation. Our results suggest that ER stress can be ameliorated through alteration of the oxidizing environment within the ER lumen, and they provide a potential logic for the transient regulation of metabolic pathways by the UPR during stress.

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Concerted effort of centrosomal and Golgi-derived microtubules is required for proper Golgi complex assembly but not maintenance.

Publication Date: 2012 Jan 19 PMID: 22262454
Authors: Vinogradova, T. - Paul, R. - Grimaldi, A. D. - Loncarek, J. - Miller, P. M. - Yampolsky, D. - Magidson, V. - Khodjakov, A. - Mogilner, A. - Kaverina, I.
Journal: Mol Biol Cell

Assembly of an integral Golgi complex is driven by microtubule-dependent transport. Conversely, the Golgi itself functions as an unconventional microtubule-organizing center (MTOC). This raises a question of whether Golgi assembly requires centrosomal microtubules, or the Golgi can be self-organized relying on it's own MTOC activity. The computational model presented here predicts that each microtubule population is capable of gathering Golgi stacks, but not of establishing Golgi complex integrity or polarity. In contrast, the concerted effort of two microtubule populations would assemble an integral polarized Golgi complex. Indeed, while laser ablation of the centrosome did not alter already-formed Golgi complexes, acentrosomal cells fail to re-assemble an integral complex upon nocodazole washout. Moreover, polarity of post-Golgi trafficking was compromised under these conditions, leading to strong deficiency in polarized cell migration. Our data indicate that centrosomal microtubules complement Golgi self-organization for proper Golgi assembly and motile cell polarization.

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Derlin-1 and UBXD8 are engaged in dislocation and degradation of lipidated ApoB-100 at lipid droplets.

Publication Date: 2012 Jan 11 PMID: 22238364
Authors: Suzuki, M. - Otsuka, T. - Ohsaki, Y. - Cheng, J. - Taniguchi, T. - Hashimoto, H. - Taniguchi, H. - Fujimoto, T.
Journal: Mol Biol Cell

Apolipoprotein B-100 (ApoB) is the principal component of very low-density lipoprotein. Poorly lipidated nascent ApoB is extracted from the Sec61 translocon and degraded by proteasomes. ApoB lipidated in the ER lumen is also subjected to proteasomal degradation, but where and how it dislocates to the cytoplasm remains unknown. In the present study, we demonstrated that ApoB after lipidation was dislocated to the cytoplasmic surface of lipid droplets (LDs) and accumulated as ubiquitinated ApoB in Huh7 cells. Depletion of UBXD8, which was almost confined to LDs in this cell type, decreased recruitment of p97 to LDs and caused an increase of both ubiquitinated ApoB on the LD surface and lipidated ApoB in the ER lumen. In contrast, abrogation of Derlin-1 function induced an accumulation of lipidated ApoB in the ER lumen, but did not increase ubiquitinated ApoB on the LD surface. UBXD8 and Derlin-1 bound with each other and with lipidated ApoB, and showed co-localization around LDs. These results indicate that ApoB after lipidation is dislocated from the ER lumen to the LD surface for proteasomal degradation and that Derlin-1 and UBXD8 are engaged in the pre- and post-dislocation step, respectively.

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Caveolin Targeting to Late Endosome/Lysosomal Membranes is Induced by Perturbations of Lysosomal pH and Cholesterol Content.

Publication Date: 2012 Jan 11 PMID: 22238363
Authors: Mundy, D. I. - Li, W. P. - Luby-Phelps, K. - Anderson, R. G.
Journal: Mol Biol Cell

Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intra-lysosomal pH since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions however, live cell imaging shows cavicles actively docking with lysosomes, suggesting these structures may be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally and the degradation rate of caveolin-1 is not altered by any of these conditions indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking.

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Desmoplakin Controls Microvilli Length but not Cell Adhesion or Keratin Organization in the Intestinal Epithelium.

Publication Date: 2012 Jan 11 PMID: 22238362
Authors: Sumigray, K. D. - Lechler, T.
Journal: Mol Biol Cell

Maintaining proper cell-cell adhesion in the intestine is essential for tissue homeostasis and barrier function. This is thought to be mediated by cell adhesion structures, including tight junctions, adherens junctions, and desmosomes, that concentrate in the apical junctional region. While clear roles for adherens and tight junctions have been established in simple epithelia, the function of desmosomes has not been addressed. In stratified epithelia, desmosomes impart mechanical strength to tissues by organizing and anchoring the keratin filament network. Here, we report that the essential desmosomal protein desmoplakin is not essential for cell adhesion in the intestinal epithelium. Surprisingly, keratin filament localization is also unperturbed, although they no longer anchor at desmosomes. Unexpectedly however, desmoplakin is important for proper microvillus structure. Our study highlights the tissue-specific functions of desmosomes and reveals that the canonical functions for these structures are not conserved in a simple epithelium.

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Matrix rigidity regulates a switch between TGF-beta1-induced apoptosis and epithelial-mesenchymal transition.

Publication Date: 2012 Jan 11 PMID: 22238361
Authors: Leight, J. L. - Wozniak, M. A. - Chen, S. - Lynch, M. L. - Chen, C. S.
Journal: Mol Biol Cell

The transforming growth factor-beta (TGF-beta) signaling pathway is often misregulated during cancer progression. In early stages of tumorigenesis, TGF-beta acts as a tumor suppressor by inhibiting proliferation and inducing apoptosis. However, as the disease progresses, TGF-beta switches to promote tumorigenic cell functions, such as epithelial-mesenchymal transition (EMT) and increased cell motility. Dramatic changes in the cellular microenvironment are also correlated with tumor progression, including an increase in tissue stiffness. However, it is unknown if these changes in tissue stiffness can regulate the effects of TGF-beta. To this end, we examined NMuMG and MDCK epithelial cells cultured on polyacrylamide gels with varying rigidity and treated with TGF-beta1. Interestingly, changing matrix rigidity switched the functional response to TGF-beta1. Decreasing rigidity increased TGF-beta1-induced apoptosis, while increasing rigidity resulted in EMT. Matrix rigidity did not change Smad signaling, but instead regulated the PI3K/Akt signaling pathway. Direct genetic and pharmacologic manipulations further demonstrated a role for PI3K/Akt signaling in the apoptotic and EMT responses. These findings demonstrate that matrix rigidity regulates a previously undescribed switch in TGF-beta-induced cell functions and provide insight into how changes in tissue mechanics during disease may contribute to the cellular response to TGF-beta.

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Dissection of the NUP107 nuclear pore subcomplex reveals a novel interaction with spindle assembly checkpoint protein MAD1 in C. elegans.

Publication Date: 2012 Jan 11 PMID: 22238360
Authors: Rodenas, E. - Gonzalez-Aguilera, C. - Ayuso, C. - Askjaer, P.
Journal: Mol Biol Cell

Nuclear pore complexes consist of several subcomplexes. The NUP107 complex is important for nucleocytoplasmic transport, nuclear envelope assembly and kinetochore function. However, the underlying molecular mechanisms and the roles of individual complex members remain elusive. We report the first description of a genetic disruption of NUP107 in metazoan. Caenorhabditis elegans NUP107/npp-5 mutants display temperature-dependent lethality. Surprisingly, NPP-5 is dispensable for incorporation of most nucleoporins into nuclear pores and for nuclear protein import. In contrast, NPP-5 is essential for proper kinetochore localization of NUP133/NPP-15, another NUP107 complex member, whereas recruitment of NUP96/NPP-10C and ELYS/MEL-28 is NPP-5-independent. We found that kinetochore protein NUF2/HIM-10 and Aurora B/AIR-2 kinase are less abundant on mitotic chromatin upon NPP-5 depletion. npp-5 mutants are hypersensitive to anoxia, suggesting that the spindle assembly checkpoint (SAC) is compromised. Indeed, NPP-5 interacts genetically and physically with SAC protein MAD1/MDF-1, whose nuclear envelope accumulation requires NPP-5. Thus, our results strengthen the emerging connection between nuclear pore proteins and chromosome segregation.

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Cell-free Reconstitution of Vacuole Membrane Fragmentation Reveals Regulation of Vacuole Size and Number by TORC1.

Publication Date: 2012 Jan 11 PMID: 22238359
Authors: Michaillat, L. - Baars, T. L. - Mayer, A.
Journal: Mol Biol Cell

Size and copy number of organelles are influenced by an equilibrium of membrane fusion and fission. We studied this equilibrium on vacuoles, the lysosomes of yeast. Vacuole fusion can readily be reconstituted and quantified in vitro but fission of the organelle could not be studied in a similar way so far. Here, we present a cell-free system that reconstitutes fragmentation of purified yeast vacuoles (lysosomes) into smaller vesicles. Fragmentation in vitro reproduces physiological aspects. It requires the dynamin-like GTPase Vps1p, V-ATPase pump activity, cytosolic proteins, and ATP and GTP hydrolysis. We used the in vitro system to uncover that the vacuole-associated TOR complex 1 (TORC1) stimulates vacuole fragmentation but not the opposing reaction of vacuole fusion. Under nutrient restriction, TORC1 is inactivated and the continuing fusion activity then dominates the fusion/fission equilibrium, decreasing the copy number and increasing the volume of the vacuolar compartment. This result can explain why nutrient restriction not only induces autophagy and a massive buildup of vacuolar/lysosomal hydrolases but leads to a concomitant increase in volume of the vacuolar compartment by coalescence of the organelles into a single large compartment.

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Fer kinase regulates cell migration through alpha-dystroglycan glycosylation.

Publication Date: 2012 Jan 19 PMID: 22238358
Authors: Yoneyama, T. - Angata, K. - Bao, X. - Courtneidge, S. - Chanda, S. K. - Fukuda, M.
Journal: Mol Biol Cell

Glycans of alpha-dystroglycan (alpha-DG), which is expressed at the epithelial cell-basement membrane (BM) interface, play an essential role in epithelium development and tissue organization. Laminin-binding glycans on alpha-DG expressed on cancer cells suppress tumor progression by attenuating tumor cell migration from the BM. However, mechanisms controlling laminin-binding glycan expression are not known yet. Here, we used siRNA library screening and identified Fer kinase, a non-receptor type tyrosine kinase, as a key regulator of laminin-binding glycan expression. Fer overexpression decreased laminin-binding glycan expression, while siRNA-mediated down-regulation of Fer kinase increased glycan expression on breast and prostate cancer cell lines. Loss of Fer kinase function via siRNA or mutagenesis increased transcription levels of glycosyltransferases, including POMT1, beta3GnT1, and LARGE, which are required to synthesize laminin-binding glycans. Consistently, inhibition of Fer expression decreased cell migration in the presence of laminin fragment. Fer kinase regulates STAT3 phosphorylation and consequent activation while knockdown of STAT3 increased laminin-binding glycan expression on cancer cells. These results indicate that the Fer pathway negatively controls expression of genes required to synthesize laminin-binding glycans, thus impairing BM attachment and increasing tumor cell migration.

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Zonula Occludens (ZO)-1 and -2 Regulate Apical Cell Structure and the Zonula Adherens Cytoskeleton in Polarized Epithelia.

Publication Date: 2012 Jan 25 PMID: 22190737
Authors: Fanning, A. S. - Van Itallie, C. - Anderson, J. M.
Journal: Mol Biol Cell

The structure and function of both adherens (AJ) and tight junctions (TJ) are dependent on the cortical actin cytoskeleton. The Zonula Occludens (ZO)-1 and -2 proteins have context-dependent interactions with both junction types and bind directly to F-actin and other cytoskeletal proteins, suggesting they might regulate cytoskeletal activity at cell junctions. To address this hypothesis, we generated stable MDCK cell lines depleted of both ZO-1 and ZO-2. Both paracellular permeability and the localization of TJ proteins are disrupted in ZO-1/-2 depleted cells. In addition, immunocytochemistry and electron microscopy revealed a significant expansion of the perijunctional actomyosin ring associated with the AJ. These structural changes are accompanied by a recruitment of pMLC and ROCK1, contraction of the actomyosin ring and expansion of the apical domain. Despite these changes in the apical cytoskeleton, there are no detectable changes in cell polarity, localization of adherens junction proteins or the organization of the basal and lateral actin cytoskeleton. We conclude that ZO proteins are not just required for tight junction assembly, but that they also regulate the organization and functional activity of the apical cytoskeleton, particularly the perijunctional actomyosin ring, and speculate these activities are relevant both to cellular organization and epithelial morphogenesis.

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The phosphoinositide-associated protein Rush hour regulates endosomal trafficking in Drosophila.

Publication Date: 2012 Feb PMID: 22160599
Authors: Gailite, I. - Egger-Adam, D. - Wodarz, A.
Journal: Mol Biol Cell

Endocytosis regulates multiple cellular processes, including the protein composition of the plasma membrane, intercellular signaling, and cell polarity. We have identified the highly conserved protein Rush hour (Rush) and show that it participates in the regulation of endocytosis. Rush localizes to endosomes via direct binding of its FYVE (Fab1p, YOTB, Vac1p, EEA1) domain to phosphatidylinositol 3-phosphate. Rush also directly binds to Rab GDP dissociation inhibitor (Gdi), which is involved in the activation of Rab proteins. Homozygous rush mutant flies are viable but show genetic interactions with mutations in Gdi, Rab5, hrs, and carnation, the fly homologue of Vps33. Overexpression of Rush disrupts progression of endocytosed cargo and increases late endosome size. Lysosomal marker staining is decreased in Rush-overexpressing cells, pointing to a defect in the transition between late endosomes and lysosomes. Rush also causes formation of endosome clusters, possibly by affecting fusion of endosomes via an interaction with the class C Vps/homotypic fusion and vacuole protein-sorting (HOPS) complex. These results indicate that Rush controls trafficking from early to late endosomes and from late endosomes to lysosomes by modulating the activity of Rab proteins.

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Yeast formin Bni1p has multiple localization regions that function in polarized growth and spindle orientation.

Publication Date: 2012 Feb PMID: 22160598
Authors: Liu, W. - Santiago-Tirado, F. H. - Bretscher, A.
Journal: Mol Biol Cell

Formins are conserved proteins that assemble unbranched actin filaments in a regulated, localized manner. Budding yeast's two formins, Bni1p and Bnr1p, assemble actin cables necessary for polarized cell growth and organelle segregation. Here we define four regions in Bni1p that contribute to its localization to the bud and at the bud neck. The first (residues 1-333) requires dimerization for its localization and encompasses the Rho-binding domain. The second (residues 334-821) covers the Diaphanous inhibitory-dimerization-coiled coil domains, and the third is the Spa2p-binding domain. The fourth region encompasses the formin homology 1-formin homology 2-COOH region of the protein. These four regions can each localize to the bud cortex and bud neck at the right stage of the cell cycle independent of both F-actin and endogenous Bni1p. The first three regions contribute cumulatively to the proper localization of Bni1p, as revealed by the effects of progressive loss of these regions on the actin cytoskeleton and fidelity of spindle orientation. The fourth region contributes to the localization of Bni1p in tiny budded cells. Expression of mislocalized Bni1p constructs has a dominant-negative effect on both growth and nuclear segregation due to mislocalized actin assembly. These results define an unexpected complexity in the mechanism of formin localization and function.

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Three-dimensional ultrastructure of the septin filament network in Saccharomyces cerevisiae.

Publication Date: 2012 Feb PMID: 22160597
Authors: Bertin, A. - McMurray, M. A. - Pierson, J. - Thai, L. - McDonald, K. L. - Zehr, E. A. - Garcia, G. 3rd - Peters, P. - Thorner, J. - Nogales, E.
Journal: Mol Biol Cell

Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P(2)-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo-electron tomography. We found networks of filaments both perpendicular and parallel to the mother-bud axis that resemble septin arrays on lipid monolayers, displaying a repeat pattern that mirrors the molecular dimensions of the corresponding septin preparations in vitro. Thus these in situ structures most likely represent septin filaments. In viable mutants lacking a single septin, in situ filaments are still present, although more disordered, consistent with other evidence that the in vivo function of septins requires filament formation.

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Altered nuclear tRNA metabolism in La-deleted Schizosaccharomyces pombe is accompanied by a nutritional stress response involving Atf1p and Pcr1p that is suppressible by Xpo-t/Los1p.

Publication Date: 2012 Feb PMID: 22160596
Authors: Cherkasova, V. - Lopez Maury, L. - Bacikova, D. - Pridham, K. - Bahler, J. - Maraia, R. J.
Journal: Mol Biol Cell

Deletion of the sla1(+) gene, which encodes a homologue of the human RNA-binding protein La in Schizosaccharomyces pombe, causes irregularities in tRNA processing, with altered distribution of pre-tRNA intermediates. We show, using mRNA profiling, that cells lacking sla1(+) have increased mRNAs from amino acid metabolism (AAM) genes and, furthermore, exhibit slow growth in Edinburgh minimal medium. A subset of these AAM genes is under control of the AP-1-like, stress-responsive transcription factors Atf1p and Pcr1p. Although S. pombe growth is resistant to rapamycin, sla1-Delta cells are sensitive, consistent with deficiency of leucine uptake, hypersensitivity to NH4, and genetic links to the target of rapamycin (TOR) pathway. Considering that perturbed intranuclear pre-tRNA metabolism and apparent deficiency in tRNA nuclear export in sla1-Delta cells may trigger the AAM response, we show that modest overexpression of S. pombe los1(+) (also known as Xpo-t), encoding the nuclear exportin for tRNA, suppresses the reduction in pre-tRNA levels, AAM gene up-regulation, and slow growth of sla1-Delta cells. The conclusion that emerges is that sla1(+) regulates AAM mRNA production in S. pombe through its effects on nuclear tRNA processing and probably nuclear export. Finally, the results are discussed in the context of stress response programs in Saccharomyces cerevisiae.

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Calpains promote alpha2beta1 integrin turnover in nonrecycling integrin pathway.

Publication Date: 2012 Feb PMID: 22160595
Authors: Rintanen, N. - Karjalainen, M. - Alanko, J. - Paavolainen, L. - Maki, A. - Nissinen, L. - Lehkonen, M. - Kallio, K. - Cheng, R. H. - Upla, P. - Ivaska, J. - Marjomaki, V.
Journal: Mol Biol Cell

Collagen receptor integrins recycle between the plasma membrane and endosomes and facilitate formation and turnover of focal adhesions. In contrast, clustering of alpha2beta1 integrin with antibodies or the human pathogen echovirus 1 (EV1) causes redistribution of alpha2 integrin to perinuclear multivesicular bodies, alpha2-MVBs. We show here that the internalized clustered alpha2 integrin remains in alpha2-MVBs and is not recycled back to the plasma membrane. Instead, receptor clustering and internalization lead to an accelerated down-regulation of alpha2beta1 integrin compared to the slow turnover of unclustered alpha2 integrin. EV1 infection or integrin degradation is not associated with proteasomal or autophagosomal processes and shows no significant association with lysosomal pathway. In contrast, degradation is dependent on calpains, such that it is blocked by calpain inhibitors. We show that active calpain is present in alpha2-MVBs, internalized clustered alpha2beta1 integrin coprecipitates with calpain-1, and calpain enzymes can degrade alpha2beta1 integrin. In conclusion, we identified a novel virus- and clustering-specific pathway that diverts alpha2beta1 integrin from its normal endo/exocytic traffic to a nonrecycling, calpain-dependent degradative endosomal route.

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Epidermal growth factor induction of front-rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2.

Publication Date: 2012 Feb PMID: 22160594
Authors: Ho, E. - Dagnino, L.
Journal: Mol Biol Cell

Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front-rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front-rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin beta1, and Rac1. Furthermore, EGF induction of front-rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front-rear polarity and forward movement.

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Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the Escherichia coli signal recognition particle.

Publication Date: 2012 Feb PMID: 22160593
Authors: Welte, T. - Kudva, R. - Kuhn, P. - Sturm, L. - Braig, D. - Muller, M. - Warscheid, B. - Drepper, F. - Koch, H. G.
Journal: Mol Biol Cell

Protein insertion into the bacterial inner membrane is facilitated by SecYEG or YidC. Although SecYEG most likely constitutes the major integration site, small membrane proteins have been shown to integrate via YidC. We show that YidC can also integrate multispanning membrane proteins such as mannitol permease or TatC, which had been considered to be exclusively integrated by SecYEG. Only SecA-dependent multispanning membrane proteins strictly require SecYEG for integration, which suggests that SecA can only interact with the SecYEG translocon, but not with the YidC insertase. Targeting of multispanning membrane proteins to YidC is mediated by signal recognition particle (SRP), and we show by site-directed cross-linking that the C-terminus of YidC is in contact with SRP, the SRP receptor, and ribosomal proteins. These findings indicate that SRP recognizes membrane proteins independent of the downstream integration site and that many membrane proteins can probably use either SecYEG or YidC for integration. Because protein synthesis is much slower than protein transport, the use of YidC as an additional integration site for multispanning membrane proteins may prevent a situation in which the majority of SecYEG complexes are occupied by translating ribosomes during cotranslational insertion, impeding the translocation of secretory proteins.

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Timing of centrosome separation is important for accurate chromosome segregation.

Publication Date: 2012 Feb PMID: 22130796
Authors: Silkworth, W. T. - Nardi, I. K. - Paul, R. - Mogilner, A. - Cimini, D.
Journal: Mol Biol Cell

Spindle assembly, establishment of kinetochore attachment, and sister chromatid separation must occur during mitosis in a highly coordinated fashion to ensure accurate chromosome segregation. In most vertebrate cells, the nuclear envelope must break down to allow interaction between microtubules of the mitotic spindle and the kinetochores. It was previously shown that nuclear envelope breakdown (NEB) is not coordinated with centrosome separation and that centrosome separation can be either complete at the time of NEB or can be completed after NEB. In this study, we investigated whether the timing of centrosome separation affects subsequent mitotic events such as establishment of kinetochore attachment or chromosome segregation. We used a combination of experimental and computational approaches to investigate kinetochore attachment and chromosome segregation in cells with complete versus incomplete spindle pole separation at NEB. We found that cells with incomplete spindle pole separation exhibit higher rates of kinetochore misattachments and chromosome missegregation than cells that complete centrosome separation before NEB. Moreover, our mathematical model showed that two spindle poles in close proximity do not "search" the entire cellular space, leading to formation of large numbers of syntelic attachments, which can be an intermediate stage in the formation of merotelic kinetochores.

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Localization and retention of p90 ribosomal S6 kinase 1 in the nucleus: implications for its function.

Publication Date: 2012 Feb PMID: 22130794
Authors: Gao, X. - Chaturvedi, D. - Patel, T. B.
Journal: Mol Biol Cell

Ribosomal S6 kinase 1 (RSK1) belongs to a family of proteins with two kinase domains. Following activation in the cytoplasm by extracellular signal-regulated kinases (ERK1/2), it mediates the cell-proliferative, cell-growth, and survival-promoting actions of a number of growth factors and other agonists. These diverse biological actions of RSK1 involve regulation of both cytoplasmic and nuclear events. However, the mechanisms that permit nuclear accumulation of RSK1 remain unknown. Here, we show that phosphorylation of RSK1 on S221 is important for its dissociation from the type Ialpha regulatory subunit of protein kinase A (PKA) in the cytoplasm and that RSK1 contains a bipartite nuclear localization sequence that is necessary for its nuclear entry. Once inside, the active RSK1 is retained in the nucleus via its interactions with PKA catalytic subunit and AKAP95. Mutations of RSK1 that do not affect its activity but disrupt its entry into the nucleus or expression of AKAP95 forms that do not enter the nucleus inhibit the ability of active RSK1 to stimulate DNA synthesis. Our findings identify novel mechanisms by which active RSK1 accumulates in the nucleus and also provide new insights into how AKAP95 orchestrates cell cycle progression.

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