37 results on '"Sabanay H"'
Search Results
2. Genetic Deletion of Cadm4 Results in Myelin Abnormalities Resembling Charcot-Marie-Tooth Neuropathy
- Author
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Golan, N., primary, Kartvelishvily, E., additional, Spiegel, I., additional, Salomon, D., additional, Sabanay, H., additional, Rechav, K., additional, Vainshtein, A., additional, Frechter, S., additional, Maik-Rachline, G., additional, Eshed-Eisenbach, Y., additional, Momoi, T., additional, and Peles, E., additional
- Published
- 2013
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3. DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death
- Author
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Gozuacik, D, primary, Bialik, S, additional, Raveh, T, additional, Mitou, G, additional, Shohat, G, additional, Sabanay, H, additional, Mizushima, N, additional, Yoshimori, T, additional, and Kimchi, A, additional
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- 2008
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4. Focal adhesion formation by F9 embryonal carcinoma cells after vinculin gene disruption
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Volberg, T., primary, Geiger, B., additional, Kam, Z., additional, Pankov, R., additional, Simcha, I., additional, Sabanay, H., additional, Coll, J.L., additional, Adamson, E., additional, and Ben-Ze'ev, A., additional
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- 1995
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5. Selective interactions of cells with crystal surfaces. Implications for the mechanism of cell adhesion
- Author
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Hanein, D., primary, Sabanay, H., additional, Addadi, L., additional, and Geiger, B., additional
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- 1993
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6. The role of sphingolipids in the maintenance of fibroblast morphology. The inhibition of protrusional activity, cell spreading, and cytokinesis induced by fumonisin B1 can be reversed by ganglioside GM3.
- Author
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Meivar-Levy, I, Sabanay, H, Bershadsky, A D, and Futerman, A H
- Abstract
Previous studies demonstrated that inhibition of sphingolipid synthesis by the mycotoxin fumonisin B1 (FB1) disrupts axonal growth in cultured hippocampal neurons (Harel, R., and Futerman, A. H. (1993) J. Biol. Chem. 268, 14476-14481) by affecting the formation or stabilization of axonal branches (Schwarz, A., Rapaport, E., Hirschberg, K., and Futerman, A.H. (1995) J. Biol. Chem. 270, 10990-10998). We now demonstrate that long term incubation with FB1 affects fibroblast morphology and proliferation. Incubation of Swiss 3T3 cells with FB1 resulted in a decrease in synthesis of ganglioside GM3, the major glycosphingolipid in 3T3 fibroblasts and of sphingomyelin. The projected cell area of FB1-treated cells was approximately 45% less than control cells. FB1 had no affect on the organization of microtubules or intermediate filaments, but fewer actin-rich stress fibers were observed, and there was a loss of actin-rich lamellipodia at the leading edge. Three other processes involving the actin cytoskeleton, cytokinesis, microvilli formation, and the formation of long processes induced by protein kinase inhibitors, were all disrupted by FB1. All the effects of FB1 on cell morphology could be reversed by addition of ganglioside GM3 even in the presence of FB1, whereas the bioactive intermediates, sphinganine, sphingosine, and ceramide, were without effect. Finally, FB1 blocked cell proliferation and DNA synthesis in a reversible manner, although ganglioside GM3 could not reverse the effects of FB1 on cell proliferation. Together, these data suggest that ongoing sphingolipid synthesis is required for the assembly of both new membrane and of the underlying cytoskeleton.
- Published
- 1997
7. Cingulin: characterization and localization
- Author
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Citi, S., primary, Sabanay, H., additional, Kendrick-Jones, J., additional, and Geiger, B., additional
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- 1989
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8. Morphological switch to a resistant subpopulation in response to viral infection in the bloom-forming coccolithophore Emiliania huxleyi.
- Author
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Frada MJ, Rosenwasser S, Ben-Dor S, Shemi A, Sabanay H, and Vardi A
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- Eutrophication physiology, Gene Expression Profiling, Haptophyta genetics, Host-Pathogen Interactions genetics, Meiosis, Phytoplankton genetics, Phytoplankton growth & development, Phytoplankton virology, Ploidies, Haptophyta growth & development, Haptophyta virology, Phycodnaviridae pathogenicity
- Abstract
Recognizing the life cycle of an organism is key to understanding its biology and ecological impact. Emiliania huxleyi is a cosmopolitan marine microalga, which displays a poorly understood biphasic sexual life cycle comprised of a calcified diploid phase and a morphologically distinct biflagellate haploid phase. Diploid cells (2N) form large-scale blooms in the oceans, which are routinely terminated by specific lytic viruses (EhV). In contrast, haploid cells (1N) are resistant to EhV. Further evidence indicates that 1N cells may be produced during viral infection. A shift in morphology, driven by meiosis, could therefore constitute a mechanism for E. huxleyi cells to escape from EhV during blooms. This process has been metaphorically coined the 'Cheshire Cat' (CC) strategy. We tested this model in two E. huxleyi strains using a detailed assessment of morphological and ploidy-level variations as well as expression of gene markers for meiosis and the flagellate phenotype. We showed that following the CC model, production of resistant cells was triggered during infection. This led to the rise of a new subpopulation of cells in the two strains that morphologically resembled haploid cells and were resistant to EhV. However, ploidy-level analyses indicated that the new resistant cells were diploid or aneuploid. Thus, the CC strategy in E. huxleyi appears to be a life-phase switch mechanism involving morphological remodeling that is decoupled from meiosis. Our results highlight the adaptive significance of morphological plasticity mediating complex host-virus interactions in marine phytoplankton.
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- 2017
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9. Involvement of Rho GAP GRAF1 in maintenance of epithelial phenotype.
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Regev M, Sabanay H, Kartvelishvily E, Kam Z, and Bershadsky AD
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- Actin Cytoskeleton metabolism, Agar, Biomarkers metabolism, Cell Adhesion, Cell Line, Tumor, Cell Movement, Cell Shape, Epithelial Cells metabolism, Epithelial Cells ultrastructure, Epithelial-Mesenchymal Transition, Focal Adhesions metabolism, Gels, Gene Knockdown Techniques, Humans, Intercellular Junctions metabolism, Intercellular Junctions ultrastructure, Lentivirus metabolism, Mesoderm metabolism, Neoplasm Invasiveness, Phenotype, RNA, Small Interfering metabolism, rac1 GTP-Binding Protein metabolism, rho-Associated Kinases antagonists & inhibitors, rho-Associated Kinases metabolism, Epithelial Cells pathology, GTPase-Activating Proteins metabolism
- Abstract
Adhesion of epithelial cell to each other and to extracellular matrix, as well as cell migration ability and cytoskeleton organization undergo significant alterations in the course of neoplastic transformation, but regulatory mechanisms involved in these processes are not fully understood. Here, we studied the role of a Rho GAP protein GRAF1 (GTPase Regulator Associated with Focal adhesion kinase-1) in the regulation of the epithelial phenotype in cells of breast derived, non-malignant, MCF10A cell line. GRAF1 was shown to be localized to cell-cell junctions, and its depletion resulted in accelerated cell migration velocity, elongation of the cells and cell colonies, impaired monolayer integrity and significant disruption of desmosomes with a loss of associated keratin filaments. These processes were accompanied by formation of larger focal adhesions, an increased number of contractile actin stress fibers, reduction in epithelial markers and increase in mesenchymal markers such as epithelial-mesenchymal transition (EMT)-specific transcription factors Snail-1 and Snail-2, as well as N-cadherin, and vimentin. Moreover, unlike control cells, GRAF1 knocked-down cells demonstrated anchorage-independent growth in soft agar. GRAF1 expression in several highly invasive breast cancer cell lines was low, as compared to the non-malignant MCF10A cells, while overexpressing of GRAF1 in the malignant BT-549 cell line led to a decrease of mesenchymal markers, especially the Snail-1 and 2. Altogether, our analysis suggests that GRAF1 plays a role in the maintenance of normal epithelial phenotype and its depletion leads to an EMT-like process that might be involved in neoplastic transformation.
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- 2017
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10. Altered lysosome distribution is an early neuropathological event in neurological forms of Gaucher disease.
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Zigdon H, Meshcheriakova A, Farfel-Becker T, Volpert G, Sabanay H, and Futerman AH
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- Actin Cytoskeleton metabolism, Actin Cytoskeleton ultrastructure, Animals, CD36 Antigens genetics, CD36 Antigens metabolism, Cell Death, Cerebral Cortex metabolism, Cerebral Cortex pathology, Disease Models, Animal, Gaucher Disease genetics, Gene Expression Profiling, Gene Expression Regulation, Glucosylceramidase deficiency, Glucosylceramidase genetics, Humans, Lysosomal Membrane Proteins genetics, Lysosomal Membrane Proteins metabolism, Lysosomes ultrastructure, Mice, Mice, Inbred C57BL, Mice, Transgenic, Microtubules metabolism, Microtubules ultrastructure, Neurons ultrastructure, Primary Cell Culture, Time Factors, Tubulin genetics, Tubulin metabolism, Gaucher Disease metabolism, Gaucher Disease pathology, Glucosylceramides metabolism, Lysosomes metabolism, Neurons metabolism
- Abstract
In the lysosomal storage disorder Gaucher disease (GD), glucosylceramide (GlcCer) accumulates due to the defective activity of glucocerebrosidase. A subset of GD patients develops neuropathology. We now show mislocalization of Limp2-positive puncta and a large reduction in the number of Lamp1-positive puncta, which are associated with impaired tubulin. These changes occur at an early stage in animal models of GD, prior to development of overt symptoms and considerably earlier than neuronal loss. Altered lysosomal localization and cytoskeleton disruption precede the neuroinflammatory pathways, axonal dystrophy and neuronal loss previously characterized in neuronal forms of GD., (© 2017 Federation of European Biochemical Societies.)
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- 2017
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11. Supramolecular Assembly and Coalescence of Ferritin Cages Driven by Designed Protein-Protein Interactions.
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Bellapadrona G, Sinkar S, Sabanay H, Liljeström V, Kostiainen M, and Elbaum M
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- Bacterial Proteins chemistry, Bacterial Proteins genetics, Ferritins chemistry, Ferritins genetics, HeLa Cells, Humans, Luminescent Proteins chemistry, Luminescent Proteins genetics, Macromolecular Substances chemistry, Macromolecular Substances metabolism, Models, Molecular, Protein Interaction Maps, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Scattering, Small Angle, X-Ray Diffraction, Bacterial Proteins metabolism, Ferritins metabolism, Luminescent Proteins metabolism, Recombinant Fusion Proteins chemistry
- Abstract
A genetically encoded system for expression of supramolecular protein assemblies (SMPAs) based on a fusion construct between ferritin and citrine (YFP) was transferred from a mammalian to a bacterial host. The assembly process is revealed to be independent of the expression host, while dimensions and level of order of the assembled structures were influenced by the host organism. An additional level of interactions, namely, coalescence between the preformed SMPAs, was observed during the purification process. SAXS investigation revealed that upon coalescence, the local order of the individual SMPAs was preserved. Finally, the chaotropic agent urea effectively disrupted both the macroscopic coalescence and the interactions at the nanoscale until the level of the single ferritin cage.
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- 2015
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12. Hijacking of an autophagy-like process is critical for the life cycle of a DNA virus infecting oceanic algal blooms.
- Author
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Schatz D, Shemi A, Rosenwasser S, Sabanay H, Wolf SG, Ben-Dor S, and Vardi A
- Subjects
- DNA Viruses ultrastructure, Gene Expression Regulation, Haptophyta ultrastructure, Microtubule-Associated Proteins genetics, Microtubule-Associated Proteins metabolism, Molecular Sequence Data, Seawater, Up-Regulation, Virion isolation & purification, Virion metabolism, Virus Replication, Autophagy, DNA Viruses pathogenicity, DNA Viruses physiology, Eutrophication physiology, Haptophyta virology, Host-Pathogen Interactions
- Abstract
Marine photosynthetic microorganisms are the basis of marine food webs and are responsible for nearly 50% of the global primary production. Emiliania huxleyi forms massive oceanic blooms that are routinely terminated by large double-stranded DNA coccolithoviruses. The cellular mechanisms that govern the replication cycle of these giant viruses are largely unknown. We used diverse techniques, including fluorescence microscopy, transmission electron microscopy, cryoelectron tomography, immunolabeling and biochemical methodologies to investigate the role of autophagy in host-virus interactions. Hallmarks of autophagy are induced during the lytic phase of E. huxleyi viral infection, concomitant with up-regulation of autophagy-related genes (ATG genes). Pretreatment of the infected cells with an autophagy inhibitor causes a major reduction in the production of extracellular viral particles, without reducing viral DNA replication within the cell. The host-encoded Atg8 protein was detected within purified virions, demonstrating the pivotal role of the autophagy-like process in viral assembly and egress. We show that autophagy, which is classically considered as a defense mechanism, is essential for viral propagation and for facilitating a high burst size. This cellular mechanism may have a major impact on the fate of the viral-infected blooms, and therefore on the cycling of nutrients within the marine ecosystem., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)
- Published
- 2014
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13. Zooplankton may serve as transmission vectors for viruses infecting algal blooms in the ocean.
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Frada MJ, Schatz D, Farstey V, Ossolinski JE, Sabanay H, Ben-Dor S, Koren I, and Vardi A
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- Animals, Copepoda virology, Models, Biological, Molecular Sequence Data, Plant Diseases virology, Plant Viruses isolation & purification, Disease Vectors, Eutrophication, Haptophyta virology, Host-Pathogen Interactions, Plant Viruses physiology, Zooplankton virology
- Abstract
Marine viruses are recognized as a major driving force regulating phytoplankton community composition and nutrient cycling in the oceans. Yet, little is known about mechanisms that influence viral dispersal in aquatic systems, other than physical processes, and that lead to the rapid demise of large-scale algal blooms in the oceans. Here, we show that copepods, abundant migrating crustaceans that graze on phytoplankton, as well as other zooplankton can accumulate and mediate the transmission of viruses infecting Emiliania huxleyi, a bloom-forming coccolithophore that plays an important role in the carbon cycle. We detected by PCR that >80% of copepods collected during a North Atlantic E. huxleyi bloom carried E. huxleyi virus (EhV) DNA. We demonstrated by isolating a new infectious EhV strain from a copepod microbiome that these viruses are infectious. We further showed that EhVs can accumulate in high titers within zooplankton guts during feeding or can be adsorbed to their surface. Subsequently, EhV can be dispersed by detachment or via viral-dense fecal pellets over a period of 1 day postfeeding on EhV-infected algal cells, readily infecting new host populations. Intriguingly, the passage through zooplankton guts prolonged EhV's half-life of infectivity by 35%, relative to free virions in seawater, potentially enhancing viral transmission. We propose that zooplankton, swimming through topographically adjacent phytoplankton micropatches and migrating daily over large areas across physically separated water masses, can serve as viral vectors, boosting host-virus contact rates and potentially accelerating the demise of large-scale phytoplankton blooms., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
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14. Accumulation of ordered ceramide-cholesterol domains in farber disease fibroblasts.
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Ferreira NS, Goldschmidt-Arzi M, Sabanay H, Storch J, Levade T, Ribeiro MG, Addadi L, and Futerman AH
- Abstract
Farber disease is an inherited metabolic disorder caused by mutations in the acid ceramidase gene, which leads to ceramide accumulation in lysosomes. Farber disease patients display a wide variety of symptoms with most patients eventually displaying signs of nervous system dysfunction. We now present a novel tool that could potentially be used to distinguish between the milder and more severe forms of the disease, namely, an antibody that recognizes a mixed monolayer or bilayer of cholesterol:C16-ceramide, but does not recognize either ceramide or cholesterol by themselves. This antibody has previously been used to detect cholesterol:C16-ceramide domains in a variety of cultured cells. We demonstrate that levels of cholesterol:C16-ceramide domains are significantly elevated in fibroblasts from types 4 and 7 Farber disease patients, and that levels of the domains can be modulated by either reducing ceramide or cholesterol levels. Moreover, these domains are located in membranes of the endomembrane system, and also in two unexpected locations, namely, the mitochondria and the plasma membrane. This study suggests that the ceramide that accumulates in severe forms of Farber disease cells is sequestered to distinct membrane subdomains, which may explain some of the cellular pathology observed in this devastating lysosomal storage disease.
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- 2014
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15. The expression of the beta cell-derived autoimmune ligand for the killer receptor nkp46 is attenuated in type 2 diabetes.
- Author
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Gur C, Enk J, Weitman E, Bachar E, Suissa Y, Cohen G, Schyr RB, Sabanay H, Horwitz E, Glaser B, Dor Y, Pribluda A, Hanna JH, Leibowitz G, and Mandelboim O
- Subjects
- Animals, Antigens, Ly genetics, Autoimmunity genetics, Diabetes Mellitus, Type 2 immunology, Gene Expression Regulation drug effects, Insulin metabolism, Insulin Secretion, Insulin-Secreting Cells immunology, Leptin administration & dosage, Ligands, Male, Mice, Natural Cytotoxicity Triggering Receptor 1 genetics, Protein Binding, Antigens, Ly metabolism, Diabetes Mellitus, Experimental, Diabetes Mellitus, Type 2 genetics, Diabetes Mellitus, Type 2 metabolism, Gene Expression, Insulin-Secreting Cells metabolism, Natural Cytotoxicity Triggering Receptor 1 metabolism
- Abstract
NK cells rapidly kill tumor cells, virus infected cells and even self cells. This is mediated via killer receptors, among which NKp46 (NCR1 in mice) is prominent. We have recently demonstrated that in type 1 diabetes (T1D) NK cells accumulate in the diseased pancreas and that they manifest a hyporesponsive phenotype. In addition, we found that NKp46 recognizes an unknown ligand expressed by beta cells derived from humans and mice and that blocking of NKp46 activity prevented diabetes development. Here we investigated the properties of the unknown NKp46 ligand. We show that the NKp46 ligand is mainly located in insulin granules and that it is constitutively secreted. Following glucose stimulation the NKp46 ligand translocates to the cell membrane and its secretion decreases. We further demonstrate by using several modalities that the unknown NKp46 ligand is not insulin. Finally, we studied the expression of the NKp46 ligand in type 2 diabetes (T2D) using 3 different in vivo models and 2 species; mice and gerbils. We demonstrate that the expression of the NKp46 ligand is decreased in all models of T2D studied, suggesting that NKp46 is not involved in T2D.
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- 2013
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16. Reconstructing adhesion structures in tissues by cryo-electron tomography of vitrified frozen sections.
- Author
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Bokstad M, Sabanay H, Dahan I, Geiger B, and Medalia O
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- Animals, Chickens, Cryoultramicrotomy methods, Frozen Sections, Vitrification, Cell Adhesion physiology, Cryoelectron Microscopy methods, Electron Microscope Tomography methods, Muscle, Smooth ultrastructure
- Abstract
Cryo-electron tomography enables three-dimensional insights into the macromolecular architecture of cells in a close-to-life state. However, it is limited to thin specimens, <1.0 μm in thickness, typically restricted to the peripheral areas of intact eukaryotic cells. Analysis of tissue ultrastructure, on the other hand, requires physical sectioning approaches, preferably cryo-sectioning, following which electron tomography (ET) may be performed. Nevertheless, cryo-electron microscopy of vitrified sections is a demanding technique and typically cannot be used to examine thick sections, >80-100 nm, due to surface crevasses. Here, we explore the potential use of cryo-ET of vitrified frozen sections (VFSs) for imaging cell adhesions in chicken smooth muscle and mouse epithelial tissues. By investigating 300-400 nm thick sections, which are collected on the EM grid and re-vitrified, we resolved fine 3D structural details of the membrane-associated dense plaques and flanking caveoli in smooth muscle tissue, and desmosomal adhesions in stratified epithelium. Technically, this method offers a simple approach for reconstructing thick volumes of hydrated frozen sections., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2012
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17. The cytoskeletal adapter protein 4.1G organizes the internodes in peripheral myelinated nerves.
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Ivanovic A, Horresh I, Golan N, Spiegel I, Sabanay H, Frechter S, Ohno S, Terada N, Möbius W, Rosenbluth J, Brose N, and Peles E
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- Animals, Axons metabolism, COS Cells, Cell Adhesion Molecules, Neuronal metabolism, Chlorocebus aethiops, Mice, Mice, Knockout, Microfilament Proteins genetics, Nerve Fibers, Myelinated metabolism, Microfilament Proteins metabolism, Myelin Sheath metabolism, Peripheral Nerves metabolism
- Abstract
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., (© 2012 Ivanovic et al.)
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- 2012
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18. Involvement of the Rho-mDia1 pathway in the regulation of Golgi complex architecture and dynamics.
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Zilberman Y, Alieva NO, Miserey-Lenkei S, Lichtenstein A, Kam Z, Sabanay H, and Bershadsky A
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- Actin Cytoskeleton metabolism, Adaptor Proteins, Signal Transducing genetics, Formins, Golgi Apparatus drug effects, Golgi Apparatus ultrastructure, HeLa Cells, Humans, Membrane Fusion, Myosin Type II metabolism, Paclitaxel pharmacology, Protein Multimerization, Protein Structure, Tertiary, Protein Transport, Signal Transduction, Time-Lapse Imaging, Tubulin Modulators pharmacology, rab GTP-Binding Proteins metabolism, Adaptor Proteins, Signal Transducing metabolism, Golgi Apparatus metabolism, rhoA GTP-Binding Protein metabolism
- Abstract
In mammalian cells, the Golgi apparatus is a ribbon-like, compact structure composed of multiple membrane stacks connected by tubular bridges. Microtubules are known to be important to Golgi integrity, but the role of the actin cytoskeleton in the maintenance of Golgi architecture remains unclear. Here we show that an increase in Rho activity, either by treatment of cells with lysophosphatidic acid or by expression of constitutively active mutants, resulted in pronounced fragmentation of the Golgi complex into ministacks. Golgi dispersion required the involvement of mDia1 formin, a downstream target of Rho and a potent activator of actin polymerization; moreover, constitutively active mDia1, in and of itself, was sufficient for Golgi dispersion. The dispersion process was accompanied by formation of dynamic F-actin patches in the Golgi area. Experiments with cytoskeletal inhibitors (e.g., latrunculin B, blebbistatin, and Taxol) revealed that actin polymerization, myosin-II-driven contractility, and microtubule-based intracellular movement were all involved in the process of Golgi dispersion induced by Rho-mDia1 activation. Live imaging of Golgi recovery revealed that fusion of the small Golgi stacks into larger compartments was repressed in cells with active mDia1. Furthermore, the formation of Rab6-positive transport vesicles derived from the Golgi complex was enhanced upon activation of the Rho-mDia1 pathway. Transient localization of mDia1 to Rab6-positive vesicles was detected in cells expressing active RhoA. Thus, the Rho-mDia1 pathway is involved in regulation of the Golgi structure, affecting remodeling of Golgi membranes.
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- 2011
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19. Intracellular localization of organized lipid domains of C16-ceramide/cholesterol.
- Author
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Goldschmidt-Arzi M, Shimoni E, Sabanay H, Futerman AH, and Addadi L
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- Cryoelectron Microscopy, Endoplasmic Reticulum metabolism, Endoplasmic Reticulum ultrastructure, Endosomes ultrastructure, HEK293 Cells, Humans, Lysosomes metabolism, Membrane Microdomains ultrastructure, Microscopy, Fluorescence, trans-Golgi Network ultrastructure, Ceramides metabolism, Cholesterol metabolism, Endosomes metabolism, Membrane Microdomains metabolism, trans-Golgi Network metabolism
- Abstract
Lipid microdomains, also called lipid rafts, consisting of sphingolipids and cholesterol, play important roles in membrane trafficking and in signaling. Despite years of study of the composition, size, half-life and dynamic organization of these domains, many open questions remain about their precise characteristics. To address some of these issues, we have developed a new experimental approach involving the use of specific monoclonal antibodies as recognition tools. One such antibody was raised against a homogeneous, mixed, ordered monolayer phase comprised of 60:40 mol% cholesterol:C16-ceramide, and has been used previously to demonstrate the existence of C16-ceramide/cholesterol domains in the membranes of cultured cells. We now use a combination of quantitative fluorescence microscopy, immuno-transmission electron microscopy and immuno-scanning cryo-electron microscopy, optimized for the study of intracellular lipid antigens. In a variety of cultured cells, C16-ceramide/cholesterol structural domains were found at high levels in late endosomes and in the trans-Golgi network, but were not found at statistically significant levels in early endosomes, lysosomes or the endoplasmic reticulum. We discuss the relevance of these results to understanding the role of lipid lateral organization in biological membranes., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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20. N-WASP is required for membrane wrapping and myelination by Schwann cells.
- Author
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Novak N, Bar V, Sabanay H, Frechter S, Jaegle M, Snapper SB, Meijer D, and Peles E
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- Animals, Cells, Cultured, Mice, Mice, Knockout, Wiskott-Aldrich Syndrome Protein, Neuronal deficiency, Cell Membrane metabolism, Myelin Sheath physiology, Schwann Cells cytology, Schwann Cells metabolism, Wiskott-Aldrich Syndrome Protein, Neuronal metabolism
- Abstract
During peripheral nerve myelination, Schwann cells sort larger axons, ensheath them, and eventually wrap their membrane to form the myelin sheath. These processes involve extensive changes in cell shape, but the exact mechanisms involved are still unknown. Neural Wiskott-Aldrich syndrome protein (N-WASP) integrates various extracellular signals to control actin dynamics and cytoskeletal reorganization through activation of the Arp2/3 complex. By generating mice lacking N-WASP in myelinating Schwann cells, we show that N-WASP is crucial for myelination. In N-WASP-deficient nerves, Schwann cells sort and ensheath axons, but most of them fail to myelinate and arrest at the promyelinating stage. Yet, a limited number of Schwann cells form unusually short internodes, containing thin myelin sheaths, with the occasional appearance of myelin misfoldings. These data suggest that regulation of actin filament nucleation in Schwann cells by N-WASP is crucial for membrane wrapping, longitudinal extension, and myelination.
- Published
- 2011
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21. Microtubule organization in the final stages of cytokinesis as revealed by cryo-electron tomography.
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Elad N, Abramovitch S, Sabanay H, and Medalia O
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- Animals, CHO Cells, Cell Line, Cells metabolism, Cells ultrastructure, Cricetinae, Cricetulus, Electron Microscope Tomography, Protein Transport, Cells cytology, Cytokinesis, Microtubules metabolism, Microtubules ultrastructure
- Abstract
The completion of cytokinesis is dominated by the midbody, a tightly-packed microtubule (MT)-based bridge that transiently connects the two daughter cells. Assembled from condensed, spindle-MTs and numerous associated proteins, the midbody gradually narrows down until daughter cell partitioning occurs at this site. Although described many years ago, detailed understanding of the abscission process remains lacking. Applying cryo-electron tomography to purified midbodies, in combination with fluorescence microscopy, we present here new insight into MT organization within the midbody. We find that the midbody is spatially divided into a core bundle of MTs that traverses the electron-dense overlap region (continuous MTs), surrounded by MTs that terminate within the overlap region (polar MTs). Residual continuous MTs remained intact up to the verge of abscission, whereas the residual polar MTs lost their organization and retreated from the overlap region at late cytokinesis stages. A detailed localization of the microtubule-bundling protein PRC1 supports the above notion. Our study thus provides a detailed account of the abscission process and suggests that the midbody, having acquired a distinct MT architecture as compared to the preceding central spindle, actively facilitates the final stage of cytokinesis.
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- 2011
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22. The neurexin superfamily of Caenorhabditis elegans.
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Haklai-Topper L, Soutschek J, Sabanay H, Scheel J, Hobert O, and Peles E
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- Animals, Gastrointestinal Tract metabolism, Gene Expression Regulation, Intercellular Junctions metabolism, Membrane Proteins metabolism, Nervous System metabolism, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Neural Cell Adhesion Molecules metabolism
- Abstract
The neurexin superfamily is a group of transmembrane molecules mediating cell-cell contacts and generating specialized membranous domains in polarized epithelial and nerves cells. We describe here the domain organization and expression of the entire, core neurexin superfamily in the nematode Caenorhabditis elegans, which is composed of three family members. One of the superfamily members, nrx-1, is an ortholog of vertebrate neurexin, the other two, itx-1 and nlr-1, are orthologs of the Caspr subfamily of neurexin-like genes. Based on reporter gene analysis, we find that nrx-1 is exclusively expressed in most if not all cells of the nervous system and localizes to presynaptic specializations. itx-1 and nrx-1 reporter genes are expressed in non-overlapping patterns within and outside the nervous system. ITX-1 protein co-localizes with β-G-spectrin to a subapical domain within intestinal cells. These studies provide a starting point for further functional analysis of this family of proteins., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2011
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23. A glial signal consisting of gliomedin and NrCAM clusters axonal Na+ channels during the formation of nodes of Ranvier.
- Author
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Feinberg K, Eshed-Eisenbach Y, Frechter S, Amor V, Salomon D, Sabanay H, Dupree JL, Grumet M, Brophy PJ, Shrager P, and Peles E
- Subjects
- Action Potentials physiology, Analysis of Variance, Animals, Blotting, Western, Cell Adhesion Molecules, Neuronal genetics, Cells, Cultured, Electrophysiology, Fluorescent Antibody Technique, Mice, Mice, Knockout, Microscopy, Electron, Myelin Sheath metabolism, Nerve Fibers, Myelinated metabolism, Nerve Growth Factors metabolism, Neural Conduction, RNA, Messenger genetics, RNA, Messenger metabolism, Reverse Transcriptase Polymerase Chain Reaction, Axons metabolism, Cell Adhesion Molecules metabolism, Cell Adhesion Molecules, Neuronal metabolism, Ranvier's Nodes metabolism, Schwann Cells metabolism, Sodium Channels metabolism
- Abstract
Saltatory conduction requires high-density accumulation of Na(+) channels at the nodes of Ranvier. Nodal Na(+) channel clustering in the peripheral nervous system is regulated by myelinating Schwann cells through unknown mechanisms. During development, Na(+) channels are first clustered at heminodes that border each myelin segment, and later in the mature nodes that are formed by the fusion of two heminodes. Here, we show that initial clustering of Na(+) channels at heminodes requires glial NrCAM and gliomedin, as well as their axonal receptor neurofascin 186 (NF186). We further demonstrate that heminodal clustering coincides with a second, paranodal junction (PNJ)-dependent mechanism that allows Na(+) channels to accumulate at mature nodes by restricting their distribution between two growing myelin internodes. We propose that Schwann cells assemble the nodes of Ranvier by capturing Na(+) channels at heminodes and by constraining their distribution to the nodal gap. Together, these two cooperating mechanisms ensure fast and efficient conduction in myelinated nerves.
- Published
- 2010
- Full Text
- View/download PDF
24. DAP-kinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-XL and induction of autophagy.
- Author
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Zalckvar E, Berissi H, Mizrachy L, Idelchuk Y, Koren I, Eisenstein M, Sabanay H, Pinkas-Kramarski R, and Kimchi A
- Subjects
- Animals, Apoptosis Regulatory Proteins chemistry, Apoptosis Regulatory Proteins genetics, Beclin-1, Calcium-Calmodulin-Dependent Protein Kinases genetics, Cell Line, Death-Associated Protein Kinases, Humans, Membrane Proteins chemistry, Membrane Proteins genetics, Models, Molecular, Phosphorylation, Protein Conformation, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, bcl-X Protein chemistry, bcl-X Protein genetics, Apoptosis Regulatory Proteins metabolism, Autophagy physiology, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Membrane Proteins metabolism, bcl-X Protein metabolism
- Abstract
Autophagy, an evolutionarily conserved process, has functions both in cytoprotective and programmed cell death mechanisms. Beclin 1, an essential autophagic protein, was recently identified as a BH3-domain-only protein that binds to Bcl-2 anti-apoptotic family members. The dissociation of beclin 1 from its Bcl-2 inhibitors is essential for its autophagic activity, and therefore should be tightly controlled. Here, we show that death-associated protein kinase (DAPK) regulates this process. The activated form of DAPK triggers autophagy in a beclin-1-dependent manner. DAPK phosphorylates beclin 1 on Thr 119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl-XL and the induction of autophagy. These results reveal a substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and they provide a new phosphorylation-based mechanism that reduces the interaction of beclin 1 with its inhibitors to activate the autophagic machinery.
- Published
- 2009
- Full Text
- View/download PDF
25. Anomalous features of EMT during keratinocyte transformation.
- Author
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Geiger T, Sabanay H, Kravchenko-Balasha N, Geiger B, and Levitzki A
- Subjects
- Cell Adhesion, Cell Line, Transformed, Cell Line, Tumor, Cell Movement, Female, Human papillomavirus 16 pathogenicity, Humans, Keratinocytes chemistry, Mesenchymal Stem Cells chemistry, Papillomavirus Infections, Uterine Cervical Neoplasms chemistry, Uterine Cervical Neoplasms pathology, rac1 GTP-Binding Protein physiology, Cell Transformation, Neoplastic, Cell Transformation, Viral, Epithelial Cells pathology, Keratinocytes virology, Mesenchymal Stem Cells pathology
- Abstract
During the evolution of epithelial cancers, cells often lose their characteristic features and acquire a mesenchymal phenotype, in a process known as epithelial-mesenchymal transition (EMT). In the present study we followed early stages of keratinocyte transformation by HPV16, and observed diverse cellular changes, associated with EMT. We compared primary keratinocytes with early and late passages of HF1 cells, a cell line of HPV16-transformed keratinocytes. We have previously shown that during the progression from the normal cells to early HF1 cells, immortalization is acquired, while in the progression to late HF1, cells become anchorage independent. We show here that during the transition from the normal state to late HF1 cells, there is a progressive reduction in cytokeratin expression, desmosome formation, adherens junctions and focal adhesions, ultimately leading to poorly adhesive phenotype, which is associated with anchorage-independence. Surprisingly, unlike "conventional EMT", these changes are associated with reduced Rac1-dependent cell migration. We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa. Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated.
- Published
- 2008
- Full Text
- View/download PDF
26. A central role for Necl4 (SynCAM4) in Schwann cell-axon interaction and myelination.
- Author
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Spiegel I, Adamsky K, Eshed Y, Milo R, Sabanay H, Sarig-Nadir O, Horresh I, Scherer SS, Rasband MN, and Peles E
- Subjects
- Animals, COS Cells, Cell Adhesion Molecules, Chlorocebus aethiops, Fluorescent Antibody Technique, Immunoglobulins, Male, Microscopy, Electron, Peripheral Nervous System physiology, RNA biosynthesis, RNA genetics, RNA, Small Interfering genetics, RNA, Small Interfering physiology, Rats, Reverse Transcriptase Polymerase Chain Reaction, Axons physiology, Cell Adhesion Molecules, Neuronal physiology, Myelin Sheath physiology, Schwann Cells physiology, Tumor Suppressor Proteins physiology
- Abstract
Myelination in the peripheral nervous system requires close contact between Schwann cells and the axon, but the underlying molecular basis remains largely unknown. Here we show that cell adhesion molecules (CAMs) of the nectin-like (Necl, also known as SynCAM or Cadm) family mediate Schwann cell-axon interaction during myelination. Necl4 is the main Necl expressed by myelinating Schwann cells and is located along the internodes in direct apposition to Necl1, which is localized on axons. Necl4 serves as the glial binding partner for axonal Necl1, and the interaction between these two CAMs mediates Schwann cell adhesion. The disruption of the interaction between Necl1 and Necl4 by their soluble extracellular domains, or the expression of a dominant-negative Necl4 in Schwann cells, inhibits myelination. These results suggest that Necl proteins are important for mediating axon-glia contact during myelination in peripheral nerves.
- Published
- 2007
- Full Text
- View/download PDF
27. A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death.
- Author
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Reef S, Zalckvar E, Shifman O, Bialik S, Sabanay H, Oren M, and Kimchi A
- Subjects
- Amino Acid Sequence, Animals, Caspases metabolism, Cell Death physiology, Cell Line, Cyclin-Dependent Kinase Inhibitor p16, HeLa Cells, Humans, Membrane Potentials, Mice, Microscopy, Electron, Mitochondria metabolism, Molecular Sequence Data, NIH 3T3 Cells, Proteasome Endopeptidase Complex metabolism, Protein Biosynthesis, Proto-Oncogene Proteins c-bcl-2 metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Tumor Suppressor Protein p14ARF chemistry, Tumor Suppressor Protein p14ARF genetics, Tumor Suppressor Protein p53 metabolism, Autophagy physiology, Tumor Suppressor Protein p14ARF metabolism
- Abstract
The tumor suppressor functions of p19(ARF) have been attributed to its ability to induce cell cycle arrest or apoptosis by activating p53 and regulating ribosome biogenesis. Here we describe another cellular function of p19(ARF), involving a short isoform (smARF, short mitochondrial ARF) that localizes to a Proteinase K-resistant compartment of the mitochondria. smARF is a product of internal initiation of translation at Met45, which lacks the nucleolar functional domains. The human p14(ARF) mRNA likewise produces a shorter isoform. smARF is maintained at low levels via proteasome-mediated degradation, but it increases in response to viral and cellular oncogenes. Ectopic expression of smARF reduces mitochondrial membrane potential (DeltaPsim) without causing cytochrome c release or caspase activation. The dissipation of DeltaPsim does not depend on p53 or Bcl-2 family members. smARF induces massive autophagy and caspase-independent cell death that can be partially rescued by knocking down ATG5 or Beclin-1, suggesting a different prodeath function for this short isoform.
- Published
- 2006
- Full Text
- View/download PDF
28. Ermin, a myelinating oligodendrocyte-specific protein that regulates cell morphology.
- Author
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Brockschnieder D, Sabanay H, Riethmacher D, and Peles E
- Subjects
- Animals, Cells, Cultured, Cytoskeletal Proteins biosynthesis, Cytoskeletal Proteins genetics, Mice, Mice, Neurologic Mutants, Mice, Transgenic, Myelin Proteins biosynthesis, Myelin Proteins genetics, Rats, Cytoskeletal Proteins physiology, Myelin Proteins physiology, Oligodendroglia cytology, Oligodendroglia metabolism
- Abstract
Oligodendrocytes form an insulating multilamellar structure of compact myelin around axons, thereby allowing rapid propagation of action potentials. Despite the considerable clinical importance of myelination, little is known about the molecular mechanisms that enable oligodendrocytes to generate their specialized membrane wrapping. Here, we used microarray expression profiling of oligodendrocyte-ablated mutant mice to identify new glial molecules that are involved in CNS myelination. This effort resulted in the identification of Ermin, a novel cytoskeletal molecule that is exclusively expressed by oligodendrocytes. Ermin appears at a late stage during myelination, and in the mature nerves, it is localized to the outer cytoplasmic lip of the myelin sheath and the paranodal loops. In cultured oligodendrocytes, Ermin becomes visible in well differentiated MBP-positive cells, where it is concentrated at the tip of F-actin-rich processes (termed "Ermin spikes"). Ectopic expression of Ermin, but not of a mutant protein lacking its actin-binding domain, induced the formation of numerous cell protrusions and a pronounced change in cell morphology. Our results demonstrate that Ermin is a novel marker of myelinating oligodendroglia and suggest that it plays a role in cytoskeletal rearrangements during the late wrapping and/or compaction phases of myelinogenesis.
- Published
- 2006
- Full Text
- View/download PDF
29. Gliomedin mediates Schwann cell-axon interaction and the molecular assembly of the nodes of Ranvier.
- Author
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Eshed Y, Feinberg K, Poliak S, Sabanay H, Sarig-Nadir O, Spiegel I, Bermingham JR Jr, and Peles E
- Subjects
- Age Factors, Amino Acid Sequence, Animals, Ankyrins metabolism, Blotting, Northern methods, Blotting, Western methods, Cell Adhesion Molecules immunology, Cell Adhesion Molecules, Neuronal metabolism, Cell Compartmentation, Cells, Cultured, Chlorocebus aethiops, Claudins, Cloning, Molecular methods, Cytoskeletal Proteins, Fluorescent Antibody Technique methods, Ganglia, Spinal metabolism, Gene Expression Regulation, Developmental, Humans, Macromolecular Substances immunology, Membrane Proteins metabolism, Microfilament Proteins metabolism, Microscopy, Immunoelectron methods, Myelin Basic Protein metabolism, Myelin-Associated Glycoprotein metabolism, Neurofilament Proteins metabolism, Phosphoproteins metabolism, Protein Binding physiology, Protein Structure, Tertiary, Ranvier's Nodes ultrastructure, Rats, Receptors, Peptide metabolism, S100 Proteins metabolism, Schwann Cells ultrastructure, Sciatic Nerve growth & development, Sciatic Nerve metabolism, Sodium Channels metabolism, Spectrin metabolism, Transfection methods, Axons metabolism, Cell Adhesion Molecules metabolism, Macromolecular Substances metabolism, Ranvier's Nodes metabolism, Schwann Cells metabolism
- Abstract
Accumulation of Na(+) channels at the nodes of Ranvier is a prerequisite for saltatory conduction. In peripheral nerves, clustering of these channels along the axolemma is regulated by myelinating Schwann cells through a yet unknown mechanism. We report the identification of gliomedin, a glial ligand for neurofascin and NrCAM, two axonal immunoglobulin cell adhesion molecules that are associated with Na+ channels at the nodes of Ranvier. Gliomedin is expressed by myelinating Schwann cells and accumulates at the edges of each myelin segment during development, where it aligns with the forming nodes. Eliminating the expression of gliomedin by RNAi, or the addition of a soluble extracellular domain of neurofascin to myelinating cultures, which caused the redistribution of gliomedin along the internodes, abolished node formation. Furthermore, a soluble gliomedin induced nodal-like clusters of Na+ channels in the absence of Schwann cells. We propose that gliomedin provides a glial cue for the formation of peripheral nodes of Ranvier.
- Published
- 2005
- Full Text
- View/download PDF
30. Maintenance of the diacylglycerol level in the Golgi apparatus by the Nir2 protein is critical for Golgi secretory function.
- Author
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Litvak V, Dahan N, Ramachandran S, Sabanay H, and Lev S
- Subjects
- Calcium-Binding Proteins metabolism, Cell Membrane metabolism, Choline, Chromatography, Thin Layer, Cytosine chemistry, Diphosphates chemistry, Eye Proteins metabolism, Glycosaminoglycans chemistry, HeLa Cells, Humans, Lipid Metabolism, Lipids chemistry, Membrane Glycoproteins metabolism, Membrane Proteins metabolism, Microscopy, Fluorescence, Models, Biological, Phosphatidylcholines chemistry, Phospholipid Transfer Proteins metabolism, Protein Structure, Tertiary, RNA Interference, Saccharomyces cerevisiae Proteins metabolism, Saccharomycetales, Time Factors, Viral Envelope Proteins metabolism, trans-Golgi Network metabolism, Calcium-Binding Proteins physiology, Diglycerides metabolism, Eye Proteins physiology, Golgi Apparatus metabolism, Golgi Apparatus physiology, Membrane Proteins physiology
- Abstract
The level of diacylglycerol (DAG) in the Golgi apparatus is crucial for protein transport to the plasma membrane. Studies in budding yeast indicate that Sec14p, a phosphatidylinositol (PI)-transfer protein, is involved in regulating DAG homeostasis in the Golgi complex. Here, we show that Nir2, a peripheral Golgi protein containing a PI-transfer domain, is essential for maintaining the structural and functional integrity of the Golgi apparatus in mammalian cells. Depletion of Nir2 by RNAi leads to substantial inhibition of protein transport from the trans-Golgi network to the plasma membrane, and causes a reduction in the DAG level in the Golgi apparatus. Remarkably, inactivation of cytidine [corrected] 5'-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis restores both effects. These results indicate that Nir2 is involved in maintaining a critical DAG pool in the Golgi apparatus by regulating its consumption via the CDP-choline pathway, demonstrating the interface between secretion from the Golgi and lipid homeostasis.
- Published
- 2005
- Full Text
- View/download PDF
31. Differential regulation of endoplasmic reticulum structure through VAP-Nir protein interaction.
- Author
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Amarilio R, Ramachandran S, Sabanay H, and Lev S
- Subjects
- Amino Acid Motifs, Calcium-Binding Proteins chemistry, Calcium-Binding Proteins genetics, Cell Line, Conserved Sequence, Endoplasmic Reticulum ultrastructure, Eye Proteins chemistry, Eye Proteins genetics, Eye Proteins metabolism, HeLa Cells, Humans, Kv Channel-Interacting Proteins, Membrane Proteins chemistry, Membrane Proteins genetics, Membrane Proteins metabolism, Membrane Transport Proteins, Microtubules chemistry, Microtubules metabolism, Protein Binding, Vesicular Transport Proteins, Calcium-Binding Proteins metabolism, Endoplasmic Reticulum chemistry, Endoplasmic Reticulum metabolism
- Abstract
The endoplasmic reticulum (ER) exhibits a characteristic tubular structure that is dynamically rearranged in response to specific physiological demands. However, the mechanisms by which the ER maintains its characteristic structure are largely unknown. Here we show that the integral ER-membrane protein VAP-B causes a striking rearrangement of the ER through interaction with the Nir2 and Nir3 proteins. We provide evidence that Nir (Nir1, Nir2, and Nir3)-VAP-B interactions are mediated through the conserved FFAT (two phenylalanines (FF) in acidic tract) motif present in Nir proteins. However, each interaction affects the structural integrity of the ER differently. Whereas the Nir2-VAP-B interaction induces the formation of stacked ER membrane arrays, the Nir3-VAP-B interaction leads to a gross remodeling of the ER and the bundling of thick microtubules along the altered ER membranes. In contrast, the Nir1-VAP-B interaction has no apparent effect on ER structure. We also show that the Nir2-VAP-B interaction attenuates protein export from the ER. These results demonstrate new mechanisms for the regulation of ER structure, all of which are mediated through interaction with an identical integral ER-membrane protein.
- Published
- 2005
- Full Text
- View/download PDF
32. Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1.
- Author
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Poliak S, Salomon D, Elhanany H, Sabanay H, Kiernan B, Pevny L, Stewart CL, Xu X, Chiu SY, Shrager P, Furley AJ, and Peles E
- Subjects
- Animals, Axons ultrastructure, Cell Communication genetics, Contactin 2, Gene Targeting, Mice, Mice, Knockout, Microscopy, Electron, Mutation genetics, Nerve Fibers, Myelinated ultrastructure, Nerve Tissue Proteins genetics, Neural Conduction genetics, Neuroglia metabolism, Neuroglia ultrastructure, Potassium Channels genetics, Ranvier's Nodes ultrastructure, Shaker Superfamily of Potassium Channels, Axons metabolism, Cell Adhesion Molecules, Neuronal metabolism, Membrane Proteins, Nerve Fibers, Myelinated metabolism, Nerve Tissue Proteins deficiency, Potassium Channels metabolism, Ranvier's Nodes metabolism
- Abstract
In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily. Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location. Furthermore, we show that the localization of Caspr2 and clustering of K+ channels at the juxtaparanodal region depends on the presence of TAG-1, an immunoglobulin-like cell adhesion molecule that binds Caspr2. These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon-glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane.
- Published
- 2003
- Full Text
- View/download PDF
33. Junctional protein MAGI-3 interacts with receptor tyrosine phosphatase beta (RPTP beta) and tyrosine-phosphorylated proteins.
- Author
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Adamsky K, Arnold K, Sabanay H, and Peles E
- Subjects
- Adherens Junctions metabolism, Adherens Junctions ultrastructure, Amino Acid Sequence physiology, Animals, Cadherins metabolism, Cadherins ultrastructure, Cell Membrane ultrastructure, Cells, Cultured, Epithelial Cells metabolism, Epithelial Cells ultrastructure, Guanylate Kinases, Humans, Immunohistochemistry, Intercellular Junctions ultrastructure, Membrane Proteins metabolism, Membrane Proteins ultrastructure, Microfilament Proteins, Microscopy, Electron, Molecular Sequence Data, Nerve Tissue Proteins ultrastructure, Phosphoproteins metabolism, Phosphoproteins ultrastructure, Phosphorylation, Protein Structure, Tertiary physiology, Protein Tyrosine Phosphatases ultrastructure, Rats, Receptor-Like Protein Tyrosine Phosphatases, Class 5, Tight Junctions metabolism, Tight Junctions ultrastructure, Tyrosine metabolism, Zonula Occludens-1 Protein, Cell Communication physiology, Cell Membrane metabolism, Intercellular Junctions metabolism, Nerve Tissue Proteins metabolism, Nucleoside-Phosphate Kinase metabolism, Protein Tyrosine Phosphatases metabolism
- Abstract
Receptor protein tyrosine phosphatase beta (RPTP beta) mediates cell-cell and cell-matrix interactions. By searching for intracellular proteins that interact with the cytoplasmic region of this phosphatase using the two-hybrid method, we identified several proteins containing PDZ domains. One of these proteins, MAGI-3, contains a guanylate-kinase-like region, six PDZ and two WW domains. The interaction between RPTP beta and MAGI-3 was confirmed by co-immunoprecipitation and pulldown experiments in transfected cells. Immunofluorescence and immunoelectron microscopy revealed that MAGI-3 is concentrated in specific sites at the plasma membrane and in the nucleus. In epithelial cells, MAGI-3 was localized with ZO-1 and cingulin at tight junctions, whereas in primary cultured astrocytes it was found in E-cadherin-based cell-cell contacts and in focal adhesion sites. Although MAGI-3 itself was not phosphorylated on tyrosine residues, it became associated with tyrosine-phosphorylated proteins following a short treatment of the cells with vanadate. In glioblastoma SF763T cells MAGI-3 was associated with a tyrosine-phosphorylated protein with the apparent molecular weight of 130 kDa, whereas in Caco2 cells it was associated with a 90 kDa protein. Finally, we show that p130 served as a substrate for RPTP beta and that its dephosphorylation required the C-terminal sequence of the phosphatase, which mediated the interaction with MAGI-3. These findings suggest a possible role for MAGI-3 as a scaffolding molecule that links receptor tyrosine phosphatase with its substrates at the plasma membrane.
- Published
- 2003
- Full Text
- View/download PDF
34. Retention of a cell adhesion complex at the paranodal junction requires the cytoplasmic region of Caspr.
- Author
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Gollan L, Sabanay H, Poliak S, Berglund EO, Ranscht B, and Peles E
- Subjects
- Amino Acid Sequence, Animals, Brain Chemistry, Cell Adhesion Molecules, Neuronal deficiency, Cell Adhesion Molecules, Neuronal metabolism, Cell Line, Cells, Cultured, Contactins, Cytoplasm metabolism, Cytoskeletal Proteins chemistry, Cytoskeletal Proteins metabolism, Humans, Membrane Proteins metabolism, Mice, Mice, Knockout, Models, Biological, Neurons cytology, Neurons metabolism, Optic Nerve metabolism, Optic Nerve ultrastructure, Receptors, Cell Surface genetics, Sciatic Nerve metabolism, Sequence Deletion, Transgenes genetics, Cell Adhesion Molecules metabolism, Intercellular Junctions metabolism, Neuropeptides, Ranvier's Nodes metabolism, Receptors, Cell Surface metabolism
- Abstract
An axonal complex of cell adhesion molecules consisting of Caspr and contactin has been found to be essential for the generation of the paranodal axo-glial junctions flanking the nodes of Ranvier. Here we report that although the extracellular region of Caspr was sufficient for directing it to the paranodes in transgenic mice, retention of the Caspr-contactin complex at the junction depended on the presence of an intact cytoplasmic domain of Caspr. Using immunoelectron microscopy, we found that a Caspr mutant lacking its intracellular domain was often found within the axon instead of the junctional axolemma. We further show that a short sequence in the cytoplasmic domain of Caspr mediated its binding to the cytoskeleton-associated protein 4.1B. Clustering of contactin on the cell surface induced coclustering of Caspr and immobilized protein 4.1B at the plasma membrane. Furthermore, deletion of the protein 4.1B binding site accelerated the internalization of a Caspr-contactin chimera from the cell surface. These results suggest that Caspr serves as a "transmembrane scaffold" that stabilizes the Caspr/contactin adhesion complex at the paranodal junction by connecting it to cytoskeletal components within the axon.
- Published
- 2002
- Full Text
- View/download PDF
35. Cell-adhesion to crystal surfaces. Adhesion-induced physiological cell death.
- Author
-
Hanein D, Yarden A, Sabanay H, Addadi L, and Geiger B
- Subjects
- Animals, Cells, Cultured, Epithelium ultrastructure, Extracellular Matrix, Kidney ultrastructure, Oligopeptides, Receptors, Cell Surface, Signal Transduction, Surface Properties, Xenopus laevis, Apoptosis physiology, Cell Adhesion physiology, Crystallography, Tartrates pharmacology
- Abstract
Cultured epithelial cells interact massively, rapidly and stereospecifically with the ¿011¿ faces of calcium (R,R)-tartrate tetrahydrate crystals. It was suggested that the massive rapid adhesion represents an exaggerated and isolated form of the first initial events in the attachment of cultured cells to conventional tissue culture surfaces (Hanein, et al., Cells and Materials, 5, 197-210; 1995). Attachment is however not followed by normal cell spreading and development of focal adhesions, but results in massive cell death. In this study, the fate of the crystal-bound cells was characterized by electron microscopy, flow cytometry and microscopic morphometry and was found to display the characteristics of physiological cell death. We show that the direct interaction with the highly homogenous and repetitive ¿011¿ faces per se does not trigger the transduction of lethal transmembrane signals. We suggest that the excessive direct interactions between the cell membrane and the crystal, by impairing cell motion, prevent the evolution of RGD-dependent cell adhesion. This implies that the deprivation of proper extracellular matrix (ECM)-receptor contacts of substrate-attached epithelial cells eventually triggers physiological cell death.
- Published
- 1996
- Full Text
- View/download PDF
36. Spatial and temporal relationships between vinculin and talin in the developing chicken gizzard smooth muscle.
- Author
-
Volberg T, Sabanay H, and Geiger B
- Subjects
- Animals, Cell Membrane analysis, Chick Embryo, Cytoplasm analysis, Fluorescent Antibody Technique, Gizzard, Avian, Immunologic Techniques, Microscopy, Electron, Muscle Development, Muscle, Smooth embryology, Muscle, Smooth growth & development, Muscle, Smooth ultrastructure, Talin, Vinculin, Cytoskeletal Proteins analysis, Muscle Proteins analysis, Muscle, Smooth analysis
- Abstract
The spatiotemporal relationships between vinculin and talin in developing chicken gizzard smooth muscle were investigated. Immunofluorescence and immunoelectron-microscopic labeling revealed that both proteins are associated with membrane-bound dense plaques in muscle cells; however, the most intense labeling for vinculin was located rather closer to the membrane than that for talin. The localization of vinculin and talin in embryonic chicken gizzards indicated that both are primarily cytoplasmic during the first 2 embryonic weeks. Only around days 16-18 does talin apparently become associated with the plasma membrane, this being concomitant with the appearance of distinct myofilament-bound dense plaques. Vinculin, on the other hand, remains primarily cytoplasmic and appears in the plaques only 1-3 days after hatching. It is thus proposed that the interactions of the dense plaque with myofilaments or with the membrane do not depend on the presence of vinculin in the plaque. Electrophoretic analyses indicated that, during development, there is no major change in the differential expression of specific vinculin isoforms. Quantitative immunoblotting analysis indicated that the vinculin content (relative to total extracted protein) is virtually constant during the last week of embryonic life. However, within 3 days of hatching, the vinculin concentration increases remarkably to over twice the embryonic level, and then slowly increases until it reaches the adult levels, which are three to four times higher than the embryonic level. The concentration of metavinculin (a 160-Kd vinculin-related protein) showed only a limited increase after hatching. We discuss the possible roles of vinculin and talin in the assembly of membrane-bound dense plaques during the different phases of smooth-muscle development.
- Published
- 1986
- Full Text
- View/download PDF
37. Cingulin, a new peripheral component of tight junctions.
- Author
-
Citi S, Sabanay H, Jakes R, Geiger B, and Kendrick-Jones J
- Subjects
- Amino Acids analysis, Animals, Antibodies, Antibodies, Monoclonal, Cell Membrane ultrastructure, Chickens, Epithelium ultrastructure, Fluorescent Antibody Technique, Intestines ultrastructure, Microscopy, Electron, Microvilli ultrastructure, Molecular Weight, Intercellular Junctions ultrastructure, Membrane Proteins analysis
- Abstract
The tight junction (Zonula occludens), a belt-like region of contact between cells of polarized epithelia, serves as a selective barrier to small molecules and as a total barrier to large molecules, and is involved in the separation between lumenal and basolateral compartments of the epithelium. In the electron microscope, tight junctions show focal regions of apparent fusion between the adjoining cell membranes, and freeze-fractured membranes display an elaborate network of branching and anastomosing strands. Very little is known about the molecular composition and architecture of tight junctions. The first specific zonula occludens-associated protein, designated ZO-1, has recently been identified in mammalian epithelial and endothelial cells. Here we describe the identification and purification of a new component of this junctional complex in avian brush-border cells, which we name cingulin. Cingulin is an acidic, heat-stable protein, with a highly elongated shape. Immunofluorescence and immunoelectron microscopy of brush-border cells with anti-cingulin antibodies show that cingulin is localized in the apical zone of the terminal web, at the endofacial surfaces of the zonula occludens.
- Published
- 1988
- Full Text
- View/download PDF
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