Journal of Cell Biology

The cytoskeletal adapter protein 4.1G organizes the internodes in peripheral myelinated nerves.

Publication Date: 2012 Jan 30 PMID: 22291039
Authors: Ivanovic, A. - Horresh, I. - Golan, N. - Spiegel, I. - Sabanay, H. - Frechter, S. - Ohno, S. - Terada, N. - Mobius, W. - Rosenbluth, J. - Brose, N. - Peles, E.
Journal: J Cell Biol

Myelinating Schwann cells regulate the localization of ion channels on the surface of the axons they ensheath. This function depends on adhesion complexes that are positioned at specific membrane domains along the myelin unit. Here we show that the precise localization of internodal proteins depends on the expression of the cytoskeletal adapter protein 4.1G in Schwann cells. Deletion of 4.1G in mice resulted in aberrant distribution of both glial adhesion molecules and axonal proteins that were present along the internodes. In wild-type nerves, juxtaparanodal proteins (i.e., Kv1 channels, Caspr2, and TAG-1) were concentrated throughout the internodes in a double strand that flanked paranodal junction components (i.e., Caspr, contactin, and NF155), and apposes the inner mesaxon of the myelin sheath. In contrast, in 4.1G(-/-) mice, these proteins "piled up" at the juxtaparanodal region or aggregated along the internodes. These findings suggest that protein 4.1G contributes to the organization of the internodal axolemma by targeting and/or maintaining glial transmembrane proteins along the axoglial interface.

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Tension is required but not sufficient for focal adhesion maturation without a stress fiber template.

Publication Date: 2012 Jan 30 PMID: 22291038
Authors: Oakes, P. W. - Beckham, Y. - Stricker, J. - Gardel, M. L.
Journal: J Cell Biol

Focal adhesion composition and size are modulated in a myosin II-dependent maturation process that controls adhesion, migration, and matrix remodeling. As myosin II activity drives stress fiber assembly and enhanced tension at adhesions simultaneously, the extent to which adhesion maturation is driven by tension or altered actin architecture is unknown. We show that perturbations to formin and alpha-actinin 1 activity selectively inhibited stress fiber assembly at adhesions but retained a contractile lamella that generated large tension on adhesions. Despite relatively unperturbed adhesion dynamics and force transmission, impaired stress fiber assembly impeded focal adhesion compositional maturation and fibronectin remodeling. Finally, we show that compositional maturation of focal adhesions could occur even when myosin II-dependent cellular tension was reduced by 80%. We propose that stress fiber assembly at the adhesion site serves as a structural template that facilitates adhesion maturation over a wide range of tensions. This work identifies the essential role of lamellar actin architecture in adhesion maturation.

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The small G protein Arl1 directs the trans-Golgi-specific targeting of the Arf1 exchange factors BIG1 and BIG2.

Publication Date: 2012 Jan 30 PMID: 22291037
Authors: Christis, C. - Munro, S.
Journal: J Cell Biol

The small G protein Arf1 regulates Golgi traffic and is activated by two related types of guanine nucleotide exchange factor (GEF). GBF1 acts at the cis-Golgi, whereas BIG1 and its close paralog BIG2 act at the trans-Golgi. Peripheral membrane proteins such as these GEFs are often recruited to membranes by small G proteins, but the basis for specific recruitment of Arf GEFs, and hence Arfs, to Golgi membranes is not understood. In this paper, we report a liposome-based affinity purification method to identify effectors for small G proteins of the Arf family. We validate this with the Drosophila melanogaster Arf1 orthologue (Arf79F) and the related class II Arf (Arf102F), which showed a similar pattern of effector binding. Applying the method to the Arf-like G protein Arl1, we found that it binds directly to Sec71, the Drosophila ortholog of BIG1 and BIG2, via an N-terminal region. We show that in mammalian cells, Arl1 is necessary for Golgi recruitment of BIG1 and BIG2 but not GBF1. Thus, Arl1 acts to direct a trans-Golgi-specific Arf1 GEF, and hence active Arf1, to the trans side of the Golgi.

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Invasive matrix degradation at focal adhesions occurs via protease recruitment by a FAK-p130Cas complex.

Publication Date: 2012 Jan 30 PMID: 22291036
Authors: Wang, Y. - McNiven, M. A.
Journal: J Cell Biol

Tumor cell migration and the concomitant degradation of extracellular matrix (ECM) are two essential steps in the metastatic process. It is well established that focal adhesions (FAs) play an important role in regulating migration; however, whether these structures contribute to matrix degradation is not clear. In this study, we report that multiple cancer cell lines display degradation of ECM at FA sites that requires the targeted action of MT1-MMP. Importantly, we have found that this MT1-MMP targeting is dependent on an association with a FAK-p130Cas complex situated at FAs and is regulated by Src-mediated phosphorylation of Tyr 573 at the cytoplasmic tail of MT1. Disrupting the FAK-p130Cas-MT1 complex significantly impairs FA-mediated degradation and tumor cell invasion yet does not appear to affect invadopodia formation or function. These findings demonstrate a novel function for FAs and also provide molecular insights into MT1-MMP targeting and function.

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Diacylglycerol kinase alpha controls RCP-dependent integrin trafficking to promote invasive migration.

Publication Date: 2012 Jan 23 PMID: 22270919
Authors: Rainero, E. - Caswell, P. T. - Muller, P. A. - Grindlay, J. - McCaffrey, M. W. - Zhang, Q. - Wakelam, M. J. - Vousden, K. H. - Graziani, A. - Norman, J. C.
Journal: J Cell Biol

Inhibition of alphavbeta3 integrin or expression of oncogenic mutants of p53 promote invasive cell migration by enhancing endosomal recycling of alpha5beta1 integrin under control of the Rab11 effector Rab-coupling protein (RCP). In this paper, we show that diacylglycerol kinase alpha (DGK-alpha), which phosphorylates diacylglycerol to phosphatidic acid (PA), was required for RCP to be mobilized to and tethered at the tips of invasive pseudopods and to allow RCP-dependent alpha5beta1 recycling and the resulting invasiveness of tumor cells. Expression of a constitutive-active mutant of DGK-alpha drove RCP-dependent invasion in the absence of mutant p53 expression or alphavbeta3 inhibition, and conversely, an RCP mutant lacking the PA-binding C2 domain was not capable of being tethered at pseudopod tips. These data demonstrate that generation of PA downstream of DGK-alpha is essential to connect expression of mutant p53s or inhibition of alphavbeta3 to RCP and for this Rab11 effector to drive the trafficking of alpha5beta1 that is required for tumor cell invasion through three-dimensional matrices.

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The synaptic vesicle SNARE neuronal Synaptobrevin promotes endolysosomal degradation and prevents neurodegeneration.

Publication Date: 2012 Jan 23 PMID: 22270918
Authors: Haberman, A. - Williamson, W. R. - Epstein, D. - Wang, D. - Rina, S. - Meinertzhagen, I. A. - Hiesinger, P. R.
Journal: J Cell Biol

Soluble NSF attachment protein receptors (SNAREs) are the core proteins in membrane fusion. The neuron-specific synaptic v-SNARE n-syb (neuronal Synaptobrevin) plays a key role during synaptic vesicle exocytosis. In this paper, we report that loss of n-syb caused slow neurodegeneration independent of its role in neurotransmitter release in adult Drosophila melanogaster photoreceptor neurons. In addition to synaptic vesicles, n-Syb localized to endosomal vesicles. Loss of n-syb lead to endosomal accumulations, transmembrane protein degradation defects, and a secondary increase in autophagy. Our evidence suggests a primary defect of impaired delivery of vesicles that contain degradation proteins, including the acidification-activated Cathepsin proteases and the neuron-specific proton pump and V0 adenosine triphosphatase component V100. Overexpressing V100 partially rescued n-syb-dependent degeneration through an acidification-independent endosomal sorting mechanism. Collectively, these findings reveal a role for n-Syb in a neuron-specific sort-and-degrade mechanism that protects neurons from degeneration. Our findings further shed light on which intraneuronal compartments exhibit increased or decreased neurotoxicity.

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FAK promotes recruitment of talin to nascent adhesions to control cell motility.

Publication Date: 2012 Jan 23 PMID: 22270917
Authors: Lawson, C. - Lim, S. T. - Uryu, S. - Chen, X. L. - Calderwood, D. A. - Schlaepfer, D. D.
Journal: J Cell Biol

Cell migration is a dynamic process that involves the continuous formation, maturation, and turnover of matrix-cell adhesion sites. New (nascent) adhesions form at the protruding cell edge in a tension-independent manner and are comprised of integrin receptors, signaling, and cytoskeletal-associated proteins. Integrins recruit focal adhesion kinase (FAK) and the cytoskeletal protein talin to nascent adhesions. Canonical models support a role for talin in mediating FAK localization and activation at adhesions. Here, alternatively, we show that FAK promotes talin recruitment to nascent adhesions occurring independently of talin binding to beta1 integrins. The direct binding site for talin on FAK was identified, and a point mutation in FAK (E1015A) prevented talin association and talin localization to nascent adhesions but did not alter integrin-mediated FAK recruitment and activation at adhesions. Moreover, FAK E1015A inhibited cell motility and proteolytic talin cleavage needed for efficient adhesion dynamics. These results support an alternative linkage for FAK-talin interactions within nascent adhesions essential for the control of cell migration.

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Novel plant SUN-KASH bridges are involved in RanGAP anchoring and nuclear shape determination.

Publication Date: 2012 Jan 23 PMID: 22270916
Authors: Zhou, X. - Graumann, K. - Evans, D. E. - Meier, I.
Journal: J Cell Biol

Inner nuclear membrane Sad1/UNC-84 (SUN) proteins interact with outer nuclear membrane (ONM) Klarsicht/ANC-1/Syne homology (KASH) proteins, forming linkers of nucleoskeleton to cytoskeleton conserved from yeast to human and involved in positioning of nuclei and chromosomes. Defects in SUN-KASH bridges are linked to muscular dystrophy, progeria, and cancer. SUN proteins were recently identified in plants, but their ONM KASH partners are unknown. Arabidopsis WPP domain-interacting proteins (AtWIPs) are plant-specific ONM proteins that redundantly anchor Arabidopsis RanGTPase-activating protein 1 (AtRanGAP1) to the nuclear envelope (NE). In this paper, we report that AtWIPs are plant-specific KASH proteins interacting with Arabidopsis SUN proteins (AtSUNs). The interaction is required for both AtWIP1 and AtRanGAP1 NE localization. AtWIPs and AtSUNs are necessary for maintaining the elongated nuclear shape of Arabidopsis epidermal cells. Together, our data identify the first KASH members in the plant kingdom and provide a novel function of SUN-KASH complexes, suggesting that a functionally diverged SUN-KASH bridge is conserved beyond the opisthokonts.

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Evolution: The Ras protein superfamily: Evolutionary tree and role of conserved amino acids.

Publication Date: 2012 Jan 23 PMID: 22270915
Authors: Rojas, A. M. - Fuentes, G. - Rausell, A. - Valencia, A.
Journal: J Cell Biol

The Ras superfamily is a fascinating example of functional diversification in the context of a preserved structural framework and a prototypic GTP binding site. Thanks to the availability of complete genome sequences of species representing important evolutionary branch points, we have analyzed the composition and organization of this superfamily at a greater level than was previously possible. Phylogenetic analysis of gene families at the organism and sequence level revealed complex relationships between the evolution of this protein superfamily sequence and the acquisition of distinct cellular functions. Together with advances in computational methods and structural studies, the sequence information has helped to identify features important for the recognition of molecular partners and the functional specialization of different members of the Ras superfamily.

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FAK and talin: Who is taking whom to the integrin engagement party?

Publication Date: 2012 Jan 23 PMID: 22270914
Authors: Serrels, B. - Frame, M. C.
Journal: J Cell Biol

In this issue, Lawson et al. provide new insight into the relationship between FAK and talin during assembly of integrin adhesions on fibronectin. They show that FAK is upstream of talin, and that talin is not required for FAK recruitment or for integrin activation at nascent adhesions. However, FAK-talin binding is required for adhesion turnover and cell motility. The findings question the view that talin is always upstream of focal adhesion protein recruitment to clustered integrin sites.

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Tobias Walther: Floating ideas on lipids.

Publication Date: 2012 Jan 23 PMID: 22270913
Authors: Sedwick, C.
Journal: J Cell Biol

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A molecular switch on an arrestin-like protein relays glucose signaling to transporter endocytosis.

Publication Date: 2012 Jan 23 PMID: 22249293
Authors: Becuwe, M. - Vieira, N. - Lara, D. - Gomes-Rezende, J. - Soares-Cunha, C. - Casal, M. - Haguenauer-Tsapis, R. - Vincent, O. - Paiva, S. - Leon, S.
Journal: J Cell Biol

Endocytosis regulates the plasma membrane protein landscape in response to environmental cues. In yeast, the endocytosis of transporters depends on their ubiquitylation by the Nedd4-like ubiquitin ligase Rsp5, but how extracellular signals trigger this ubiquitylation is unknown. Various carbon source transporters are known to be ubiquitylated and endocytosed when glucose-starved cells are exposed to glucose. We show that this required the conserved arrestin-related protein Rod1/Art4, which was activated in response to glucose addition. Indeed, Rod1 was a direct target of the glucose signaling pathway composed of the AMPK homologue Snf1 and the PP1 phosphatase Glc7/Reg1. Glucose promoted Rod1 dephosphorylation and its subsequent release from a phospho-dependent interaction with 14-3-3 proteins. Consequently, this allowed Rod1 ubiquitylation by Rsp5, which was a prerequisite for transporter endocytosis. This paper therefore demonstrates that the arrestin-related protein Rod1 relays glucose signaling to transporter endocytosis and provides the first molecular insights into the nutrient-induced activation of an arrestin-related protein through a switch in post-translational modifications.

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Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane.

Publication Date: 2012 Jan 23 PMID: 22249292
Authors: Gerl, M. J. - Sampaio, J. L. - Urban, S. - Kalvodova, L. - Verbavatz, J. M. - Binnington, B. - Lindemann, D. - Lingwood, C. A. - Shevchenko, A. - Schroeder, C. - Simons, K.
Journal: J Cell Biol

The influenza virus (IFV) acquires its envelope by budding from host cell plasma membranes. Using quantitative shotgun mass spectrometry, we determined the lipidomes of the host Madin-Darby canine kidney cell, its apical membrane, and the IFV budding from it. We found the apical membrane to be enriched in sphingolipids (SPs) and cholesterol, whereas glycerophospholipids were reduced, and storage lipids were depleted compared with the whole-cell membranes. The virus membrane exhibited a further enrichment of SPs and cholesterol compared with the donor membrane at the expense of phosphatidylcholines. Our data are consistent with and extend existing models of membrane raft-based biogenesis of the apical membrane and IFV envelope.

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The dynamics of replication licensing in live Caenorhabditis elegans embryos.

Publication Date: 2012 Jan 23 PMID: 22249291
Authors: Sonneville, R. - Querenet, M. - Craig, A. - Gartner, A. - Blow, J. J.
Journal: J Cell Biol

Accurate DNA replication requires proper regulation of replication licensing, which entails loading MCM-2-7 onto replication origins. In this paper, we provide the first comprehensive view of replication licensing in vivo, using video microscopy of Caenorhabditis elegans embryos. As expected, MCM-2-7 loading in late M phase depended on the prereplicative complex (pre-RC) proteins: origin recognition complex (ORC), CDC-6, and CDT-1. However, many features we observed have not been described before: GFP-ORC-1 bound chromatin independently of ORC-2-5, and CDC-6 bound chromatin independently of ORC, whereas CDT-1 and MCM-2-7 DNA binding was interdependent. MCM-3 chromatin loading was irreversible, but CDC-6 and ORC turned over rapidly, consistent with ORC/CDC-6 loading multiple MCM-2-7 complexes. MCM-2-7 chromatin loading further reduced ORC and CDC-6 DNA binding. This dynamic behavior creates a feedback loop allowing ORC/CDC-6 to repeatedly load MCM-2-7 and distribute licensed origins along chromosomal DNA. During S phase, ORC and CDC-6 were excluded from nuclei, and DNA was overreplicated in export-defective cells. Thus, nucleocytoplasmic compartmentalization of licensing factors ensures that DNA replication occurs only once.

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A novel function for Cyclin A2: Control of cell invasion via RhoA signaling.

Publication Date: 2012 Jan 9 PMID: 22232705
Authors: Arsic, N. - Bendris, N. - Peter, M. - Begon-Pescia, C. - Rebouissou, C. - Gadea, G. - Bouquier, N. - Bibeau, F. - Lemmers, B. - Blanchard, J. M.
Journal: J Cell Biol

Cyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2-depleted cells display a cortical distribution of actin filaments and increased migration. These defects are rescued by restoration of wild-type Cyclin A2, which directly interacts with RhoA, or by a Cyclin A2 mutant unable to associate with Cdk. In vitro, Cyclin A2 potentiates the exchange activity of a RhoA-specific guanine nucleotide exchange factor. Consistent with this, Cyclin A2 depletion enhances migration of fibroblasts and invasiveness of transformed cells via down-regulation of RhoA activity. Moreover, Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma in matched human tumors. All together, these data show that Cyclin A2 negatively controls cell motility by promoting RhoA activation, thus demonstrating a novel Cyclin A2 function in cytoskeletal rearrangements and cell migration.

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Structural specializations of alpha4beta7, an integrin that mediates rolling adhesion.

Publication Date: 2012 Jan 9 PMID: 22232704
Authors: Yu, Y. - Zhu, J. - Mi, L. Z. - Walz, T. - Sun, H. - Chen, J. - Springer, T. A.
Journal: J Cell Biol

The lymphocyte homing receptor integrin alpha(4)beta(7) is unusual for its ability to mediate both rolling and firm adhesion. alpha(4)beta(1) and alpha(4)beta(7) are targeted by therapeutics approved for multiple sclerosis and Crohn's disease. Here, we show by electron microscopy and crystallography how two therapeutic Fabs, a small molecule (RO0505376), and mucosal adhesion molecule-1 (MAdCAM-1) bind alpha(4)beta(7). A long binding groove at the alpha(4)-beta(7) interface for immunoglobulin superfamily domains differs in shape from integrin pockets that bind Arg-Gly-Asp motifs. RO0505376 mimics an Ile/Leu-Asp motif in alpha(4) ligands, and orients differently from Arg-Gly-Asp mimics. A novel auxiliary residue at the metal ion-dependent adhesion site in alpha(4)beta(7) is essential for binding to MAdCAM-1 in Mg(2+) yet swings away when RO0505376 binds. A novel intermediate conformation of the alpha(4)beta(7) headpiece binds MAdCAM-1 and supports rolling adhesion. Lack of induction of the open headpiece conformation by ligand binding enables rolling adhesion to persist until integrin activation is signaled.

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alpha-Actinin-4/FSGS1 is required for Arp2/3-dependent actin assembly at the adherens junction.

Publication Date: 2012 Jan 9 PMID: 22232703
Authors: Tang, V. W. - Brieher, W. M.
Journal: J Cell Biol

We have developed an in vitro assay to study actin assembly at cadherin-enriched cell junctions. Using this assay, we demonstrate that cadherin-enriched junctions can polymerize new actin filaments but cannot capture preexisting filaments, suggesting a mechanism involving de novo synthesis. In agreement with this hypothesis, inhibition of Arp2/3-dependent nucleation abolished actin assembly at cell-cell junctions. Reconstitution biochemistry using the in vitro actin assembly assay identified alpha-actinin-4/focal segmental glomerulosclerosis 1 (FSGS1) as an essential factor. alpha-Actinin-4 specifically localized to sites of actin incorporation on purified membranes and at apical junctions in Madin-Darby canine kidney cells. Knockdown of alpha-actinin-4 decreased total junctional actin and inhibited actin assembly at the apical junction. Furthermore, a point mutation of alpha-actinin-4 (K255E) associated with FSGS failed to support actin assembly and acted as a dominant negative to disrupt actin dynamics at junctional complexes. These findings demonstrate that alpha-actinin-4 plays an important role in coupling actin nucleation to assembly at cadherin-based cell-cell adhesive contacts.

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The septin cytoskeleton facilitates membrane retraction during motility and blebbing.

Publication Date: 2012 Jan 9 PMID: 22232702
Authors: Gilden, J. K. - Peck, S. - M Chen, Y. C. - Krummel, M. F.
Journal: J Cell Biol

Increasing evidence supports a critical role for the septin cytoskeleton at the plasma membrane during physiological processes including motility, formation of dendritic spines or cilia, and phagocytosis. We sought to determine how septins regulate the plasma membrane, focusing on this cytoskeletal element's role during effective amoeboid motility. Surprisingly, septins play a reactive rather than proactive role, as demonstrated during the response to increasing hydrostatic pressure and subsequent regulatory volume decrease. In these settings, septins were required for rapid cortical contraction, and SEPT6-GFP was recruited into filaments and circular patches during global cortical contraction and also specifically during actin filament depletion. Recruitment of septins was also evident during excessive blebbing initiated by blocking membrane trafficking with a dynamin inhibitor, providing further evidence that septins are recruited to facilitate retraction of membranes during dynamic shape change. This function of septins in assembling on an unstable cortex and retracting aberrantly protruding membranes explains the excessive blebbing and protrusion observed in septin-deficient T cells.

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Dynein-dependent processive chromosome motions promote homologous pairing in C. elegans meiosis.

Publication Date: 2012 Jan 9 PMID: 22232701
Authors: Wynne, D. J. - Rog, O. - Carlton, P. M. - Dernburg, A. F.
Journal: J Cell Biol

Meiotic chromosome segregation requires homologue pairing, synapsis, and crossover recombination, which occur during meiotic prophase. Telomere-led chromosome motion has been observed or inferred to occur during this stage in diverse species, but its mechanism and function remain enigmatic. In Caenorhabditis elegans, special chromosome regions known as pairing centers (PCs), rather than telomeres, associate with the nuclear envelope (NE) and the microtubule cytoskeleton. In this paper, we investigate chromosome dynamics in living animals through high-resolution four-dimensional fluorescence imaging and quantitative motion analysis. We find that chromosome movement is constrained before meiosis. Upon prophase onset, constraints are relaxed, and PCs initiate saltatory, processive, dynein-dependent motions along the NE. These dramatic motions are dispensable for homologous pairing and continue until synapsis is completed. These observations are consistent with the idea that motions facilitate pairing by enhancing the search rate but that their primary function is to trigger synapsis. This quantitative analysis of chromosome dynamics in a living animal extends our understanding of the mechanisms governing faithful genome inheritance.

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Axon degeneration: Molecular mechanisms of a self-destruction pathway.

Publication Date: 2012 Jan 9 PMID: 22232700
Authors: Wang, J. T. - Medress, Z. A. - Barres, B. A.
Journal: J Cell Biol

Axon degeneration is a characteristic event in many neurodegenerative conditions including stroke, glaucoma, and motor neuropathies. However, the molecular pathways that regulate this process remain unclear. Axon loss in chronic neurodegenerative diseases share many morphological features with those in acute injuries, and expression of the Wallerian degeneration slow (WldS) transgene delays nerve degeneration in both events, indicating a common mechanism of axonal self-destruction in traumatic injuries and degenerative diseases. A proposed model of axon degeneration is that nerve insults lead to impaired delivery or expression of a local axonal survival factor, which results in increased intra-axonal calcium levels and calcium-dependent cytoskeletal breakdown.

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Keith Burridge: Cultivating knowledge on Rho.

Publication Date: 2012 Jan 9 PMID: 22232699
Authors: Sedwick, C.
Journal: J Cell Biol

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dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions.

Publication Date: 2012 Jan 9 PMID: 22213802
Authors: Giagtzoglou, N. - Yamamoto, S. - Zitserman, D. - Graves, H. K. - Schulze, K. L. - Wang, H. - Klein, H. - Roegiers, F. - Bellen, H. J.
Journal: J Cell Biol

Notch signaling governs binary cell fate determination in asymmetrically dividing cells. Through a forward genetic screen we identified the fly homologue of Eps15 homology domain containing protein-binding protein 1 (dEHBP1) as a novel regulator of Notch signaling in asymmetrically dividing cells. dEHBP1 is enriched basally and at the actin-rich interface of pII cells of the external mechanosensory organs, where Notch signaling occurs. Loss of function of dEHBP1 leads to up-regulation of Sanpodo, a regulator of Notch signaling, and aberrant trafficking of the Notch ligand, Delta. Furthermore, Sec15 and Rab11, which have been previously shown to regulate the localization of Delta, physically interact with dEHBP1. We propose that dEHBP1 functions as an adaptor molecule for the exocytosis and recycling of Delta, thereby affecting cell fate decisions in asymmetrically dividing cells.

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Ndfip1 regulates nuclear Pten import in vivo to promote neuronal survival following cerebral ischemia.

Publication Date: 2012 Jan 9 PMID: 22213801
Authors: Howitt, J. - Lackovic, J. - Low, L. H. - Naguib, A. - Macintyre, A. - Goh, C. P. - Callaway, J. K. - Hammond, V. - Thomas, T. - Dixon, M. - Putz, U. - Silke, J. - Bartlett, P. - Yang, B. - Kumar, S. - Trotman, L. C. - Tan, S. S.
Journal: J Cell Biol

PTEN (phosphatase and tensin homologue deleted on chromosome TEN) is the major negative regulator of phosphatidylinositol 3-kinase signaling and has cell-specific functions including tumor suppression. Nuclear localization of PTEN is vital for tumor suppression; however, outside of cancer, the molecular and physiological events driving PTEN nuclear entry are unknown. In this paper, we demonstrate that cytoplasmic Pten was translocated into the nuclei of neurons after cerebral ischemia in mice. Critically, this transport event was dependent on a surge in the Nedd4 family-interacting protein 1 (Ndfip1), as neurons in Ndfip1-deficient mice failed to import Pten. Ndfip1 binds to Pten, resulting in enhanced ubiquitination by Nedd4 E3 ubiquitin ligases. In vitro, Ndfip1 overexpression increased the rate of Pten nuclear import detected by photobleaching experiments, whereas Ndfip1(-/-) fibroblasts showed negligible transport rates. In vivo, Ndfip1 mutant mice suffered larger infarct sizes associated with suppressed phosphorylated Akt activation. Our findings provide the first physiological example of when and why transient shuttling of nuclear Pten occurs and how this process is critical for neuron survival.

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PAI-1-regulated miR-21 defines a novel age-associated fibrogenic pathway in muscular dystrophy.

Publication Date: 2012 Jan 9 PMID: 22213800
Authors: Ardite, E. - Perdiguero, E. - Vidal, B. - Gutarra, S. - Serrano, A. L. - Munoz-Canoves, P.
Journal: J Cell Biol

Disruption of skeletal muscle homeostasis by substitution with fibrotic tissue constitutes the principal cause of death in Duchenne muscular dystrophy (DMD) patients, yet the implicated fibrogenic mechanisms remain poorly understood. This study identifies the extracellular PAI-1/urokinase-type plasminogen activator (uPA) balance as an important regulator of microribonucleic acid (miR)-21 biogenesis, controlling age-associated muscle fibrosis and dystrophy progression. Genetic loss of PAI-1 in mdx dystrophic mice anticipated muscle fibrosis through these sequential mechanisms: the alteration of collagen metabolism by uPA-mediated proteolytic processing of transforming growth factor (TGF)-beta in muscle fibroblasts and the activation of miR-21 expression, which inhibited phosphatase and tensin homologue and enhanced AKT signaling, thus endowing TGF-beta with a remarkable cell proliferation-promoting potential. Age-associated fibrogenesis and muscle deterioration in mdx mice, as well as exacerbated dystrophy in young PAI-1(-/-) mdx mice, could be reversed by miR-21 or uPA-selective interference, whereas forced miR-21 overexpression aggravated disease severity. The PAI-1-miR-21 fibrogenic axis also appeared dysregulated in muscle of DMD patients, providing a basis for effectively targeting fibrosis and muscular dystrophies in currently untreatable individuals.

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RAB-6.2 and the retromer regulate glutamate receptor recycling through a retrograde pathway.

Publication Date: 2012 Jan 9 PMID: 22213799
Authors: Zhang, D. - Isack, N. R. - Glodowski, D. R. - Liu, J. - Chen, C. C. - Xu, X. Z. - Grant, B. D. - Rongo, C.
Journal: J Cell Biol

Regulated membrane trafficking of AMPA-type glutamate receptors (AMPARs) is a key mechanism underlying synaptic plasticity, yet the pathways used by AMPARs are not well understood. In this paper, we show that the AMPAR subunit GLR-1 in Caenorhabditis elegans utilizes the retrograde transport pathway to regulate AMPAR synaptic abundance. Mutants for rab-6.2, the retromer genes vps-35 and snx-1, and rme-8 failed to recycle GLR-1 receptors, resulting in GLR-1 turnover and behavioral defects indicative of diminished GLR-1 function. In contrast, expression of constitutively active RAB-6.2 drove the retrograde transport of GLR-1 from dendrites back to cell body Golgi. We also find that activated RAB-6.2 bound to and colocalized with the PDZ/phosphotyrosine binding domain protein LIN-10. RAB-6.2 recruited LIN-10. Moreover, the regulation of GLR-1 transport by RAB-6.2 required LIN-10 activity. Our results demonstrate a novel role for RAB-6.2, its effector LIN-10, and the retromer complex in maintaining synaptic strength by recycling AMPARs along the retrograde transport pathway.

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Ubiquitylation of the nuclear pore complex controls nuclear migration during mitosis in S. cerevisiae.

Publication Date: 2012 Jan 9 PMID: 22213798
Authors: Hayakawa, A. - Babour, A. - Sengmanivong, L. - Dargemont, C.
Journal: J Cell Biol

Nuclear pore complexes (NPCs) correspond to large protein transport complexes responsible for selective nucleocytoplasmic exchange. Although research has revealed much about the molecular architecture and roles of the NPC subcomplexes, little is known about the regulation of NPC functions by posttranslational modifications. We used a systematic approach to show that more than half of NPC proteins were conjugated to ubiquitin. In particular, Nup159, a nucleoporin exclusively located on the cytoplasmic side of the NPC, was monoubiquitylated by the Cdc34/SCF (Skp1-Cdc53-F-box E3 ligase) enzymes. Preventing this modification had no consequences on nuclear transport or NPC organization but strongly affected the ability of Nup159 to target the dynein light chain to the NPC. This led to defects in nuclear segregation at the onset of mitosis. Thus, defining ubiquitylation of the yeast NPC highlights yet-unexplored functions of this essential organelle in cell division.

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The vesicular SNARE Synaptobrevin is required for Semaphorin 3A axonal repulsion.

Publication Date: 2012 Jan 9 PMID: 22213797
Authors: Zylbersztejn, K. - Petkovic, M. - Burgo, A. - Deck, M. - Garel, S. - Marcos, S. - Bloch-Gallego, E. - Nothias, F. - Serini, G. - Bagnard, D. - Binz, T. - Galli, T.
Journal: J Cell Biol

Attractive and repulsive molecules such as Semaphorins (Sema) trigger rapid responses that control the navigation of axonal growth cones. The role of vesicular traffic in axonal guidance is still largely unknown. The exocytic vesicular soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) Synaptobrevin 2 (Syb2) is known for mediating neurotransmitter release in mature neurons, but its potential role in axonal guidance remains elusive. Here we show that Syb2 is required for Sema3A-dependent repulsion but not Sema3C-dependent attraction in cultured neurons and in the mouse brain. Syb2 associated with Neuropilin 1 and Plexin A1, two essential components of the Sema3A receptor, via its juxtatransmembrane domain. Sema3A receptor and Syb2 colocalize in endosomal membranes. Moreover, upon Sema3A treatment, Syb2-deficient neurons failed to collapse and transport Plexin A1 to cell bodies. Reconstitution of Sema3A receptor in nonneuronal cells revealed that Sema3A further inhibited the exocytosis of Syb2. Therefore, Sema3A-mediated signaling and axonal repulsion require Syb2-dependent vesicular traffic.

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