PubMed Central (PMC3 - NLM DTD)
(2,081,148 recursos)
Archive of life sciences journal literature at the U.S. National Institutes of Health (NIH), developed and managed by NIH's National Center for Biotechnology Information (NCBI) in the National Library of Medicine (NLM).
Mostrando recursos 101 - 120 de 9,978
101.
JRAB/MICAL-L2 Is a Junctional Rab13-binding Protein Mediating the Endocytic Recycling of Occludin - Terai, Tomoya; Nishimura, Noriyuki; Kanda, Ikuno; Yasui, Natsuo; Sasaki, Takuya
The dynamic turnover of tight junctions (TJs) is essential for epithelial-mesenchymal transitions and/or mesenchymal-epithelial transitions during epithelial morphogenesis. We previously demonstrated that Rab13 specifically mediates the endocytic recycling of occludin. Here, we identified MICAL-L2 (molecule interacting with CasL-like 2) as a novel Rab13-binding protein. Immunoprecipitation and immunofluorescence microscopy showed that MICAL-L2 specifically bound to the GTP-bound form of Rab13 via its C terminus, which contained a coiled-coil domain, and localized at TJs in epithelial MTD-1A cells. Recycling assay demonstrated that a MICAL-L2 mutant lacking the Rab13-binding domain (MICAL-L2-N) specifically inhibited the endocytic recycling of occludin but not transferrin receptor. Ca2+...
102.
Genome-wide Analysis of Re-replication Reveals Inhibitory Controls That Target Multiple Stages of Replication InitiationD? - Tanny, Robyn E.; MacAlpine, David M.; Blitzblau, Hannah G.; Bell, Stephen P.
DNA replication must be tightly controlled during each cell cycle to prevent unscheduled replication and ensure proper genome maintenance. The currently known controls that prevent re-replication act redundantly to inhibit pre-replicative complex (pre-RC) assembly outside of the G1-phase of the cell cycle. The yeast Saccharomyces cerevisiae has been a useful model organism to study how eukaryotic cells prevent replication origins from reinitiating during a single cell cycle. Using a re-replication-sensitive strain and DNA microarrays, we map sites across the S. cerevisiae genome that are re-replicated as well as sites of pre-RC formation during re-replication. Only a fraction of the genome...
103.
A Second SNARE Role for Exocytic SNAP25 in Endosome Fusion - Aikawa, Yoshikatsu; Lynch, Kara L.; Boswell, Kristin L.; Martin, Thomas F.J.
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25 kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca2+-dependent secretory vesicleplasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12 cells, which specifically cleaves SNAP25. BoNT E expression altered...
104.
Proteasome-mediated Degradation of Rac1-GTP during Epithelial Cell ScatteringD? - Lynch, Emma A.; Stall, Jennifer; Schmidt, Gudila; Chavrier, Philippe; D'Souza-Schorey, Crislyn
Epithelial cells disassemble their adherens junctions and scatter during processes such as tumor cell invasion as well as some stages of embryonic development. Control of actin polymerization is a powerful mechanism for regulating the strength of cellcell adhesion. In this regard, studies have shown that sustained activation of Rac1, a well-known regulator of actin dynamics, results in the accumulation of polymerized actin at cellcell contacts in epithelia and an increase in E-cadherinmediated adhesion. Here we show that active Rac1 is ubiquitinated and subject to proteasome-mediated degradation during the early stages of epithelial cell scattering. These findings delineate a mechanism for...
105.
The Vaccinia-related Kinases Phosphorylate the N? Terminus of BAF, Regulating Its Interaction with DNA and Its Retention in the Nucleus - Nichols, R. Jeremy; Wiebe, Matthew S.; Traktman, Paula
The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family whose members are characterized by homology to the vaccinia virus B1 kinase. The VRK orthologues encoded by Caenorhabditis elegans and Drosophila melanogaster play an essential role in cell division; however, substrates that mediate this role have yet to be elucidated. VRK1 can complement the temperature sensitivity of a vaccinia B1 mutant, implying that VRK1 and B1 have overlapping substrate specificity. Herein, we demonstrate that B1, VRK1, and VRK2 efficiently phosphorylate the extreme N? terminus of the BAF protein (Barrier to Autointegration Factor). BAF binds to both DNA and...
106.
Genome-wide Mapping of DNA Synthesis in Saccharomyces cerevisiae Reveals That Mechanisms Preventing Reinitiation of DNA Replication Are Not RedundantD? - Green, Brian M.; Morreale, Richard J.; Özaydin, Bilge; DeRisi, Joseph L.; Li, Joachim J.
To maintain genomic stability, reinitiation of eukaryotic DNA replication within a single cell cycle is blocked by multiple mechanisms that inactivate or remove replication proteins after G1 phase. Consistent with the prevailing notion that these mechanisms are redundant, we previously showed that simultaneous deregulation of three replication proteins, ORC, Cdc6, and Mcm2-7, was necessary to cause detectable bulk re-replication in G2/M phase in Saccharomyces cerevisiae. In this study, we used microarray comparative genomic hybridization (CGH) to provide a more comprehensive and detailed analysis of re-replication. This genome-wide analysis suggests that reinitiation in G2/M phase primarily occurs at a subset of...
107.
Coordinated Requirements of Human Topo II and Cohesin for Metaphase Centromere Alignment under Mad2-dependent Spindle Checkpoint SurveillanceD?V? - Toyoda, Yusuke; Yanagida, Mitsuhiro
Cohesin maintains sister chromatid cohesion until its Rad21/Scc1/Mcd1 is cleaved by separase during anaphase. DNA topoisomerase II (topo II) maintains the proper topology of chromatid DNAs and is essential for chromosome segregation. Here we report direct observations of mitotic progression in individual HeLa cells after functional disruptions of hRad21, NIPBL, a loading factor for hRad21, and topo II ?,? by RNAi and a topo II inhibitor, ICRF-193. Mitosis is delayed in a Mad2-dependent manner after disruption of either or both cohesin and topo II. In hRad21 depletion, interphase pericentric architecture becomes aberrant, and anaphase is virtually permanently delayed as preseparated...
108.
Rac1-null Mouse Embryonic Fibroblasts Are Motile and Respond to Platelet-derived Growth FactorD?V? - Vidali, Luis; Chen, Feng; Cicchetti, Gregor; Ohta, Yasutaka; Kwiatkowski, David J.
Previous studies of Rac1 in fibroblasts have used dominant negative constructs, which may have nonspecific effects. We used a conditional Rac1 allele to critically examine Rac1 function in mouse fibroblasts. Lack of Rac1 had dramatic effects on nonconfluent cells, which were elongated and had extensive blebbing, but no lamellipodia or ruffle formation. However, Rac1-null fibroblasts translocated using pseudopodia-like protrusions without lamellipodia, migrating toward a platelet-derived growth factor (PDGF) gradient as efficiently as their wild-type counterparts. Rac1-null fibroblasts closed wounds in vitro and spread on a fibronectin substrate, although at a slower rate than wild-type cells. However, Rac1-null cells were markedly...
109.
COG-7-deficient Human Fibroblasts Exhibit Altered Recycling of Golgi ProteinsD? - Steet, Richard; Kornfeld, Stuart
Recently, we reported that two siblings presenting with the clinical syndrome congenital disorders of glycosylation (CDG) have mutations in the gene encoding Cog7p, a member of the conserved oligomeric Golgi (COG) complex. In this study, we analyzed the localization and trafficking of multiple Golgi proteins in patient fibroblasts under a variety of conditions. Although the immunofluorescent staining pattern of several Golgi proteins was indistinguishable from normal, the staining of endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC)-53 and the vesicular-soluble N-ethylmaleimide-sensitive factor attachment protein receptors GS15 and GS28 was abnormal, and the steady-state level of GS15 was greatly decreased. Retrograde transport of...
110.
Caspase-2induced Apoptosis Requires Bid Cleavage: A Physiological Role for Bid in Heat Shockinduced DeathD? - Bonzon, Christine; Bouchier-Hayes, Lisa; Pagliari, Lisa J.; Green, Douglas R.; Newmeyer, Donald D.
The mechanisms through which Caspase-2 leads to cell death are controversial. Here we show, using a combination of cell-free and cell culture-based approaches, that cleavage of the Bcl-2-family protein Bid is required for the induction of apoptosis by Caspase-2. Caspase-2 promoted cytochrome c release from mitochondria in the presence of cytosol from wild-type, but not Bid-deficient, mouse embryonic fibroblasts (MEFs). Recombinant wild-type Bid, but not a noncleavable mutant (D59E), restored cytochrome c release. Similarly, Bid-null MEFs were relatively resistant to apoptosis triggered by active Caspase-2, and apoptosis was restored in Bid-null cells by the expression of wild-type, but not D59E,...
111.
POLKADOTS Are Foci of Functional Interactions in T-Cell Receptormediated Signaling to NF-?BD?V? - Rossman, Jeremy S.; Stoicheva, Natalia G.; Langel, Felicia D.; Patterson, George H.; Lippincott-Schwartz, Jennifer; Schaefer, Brian C.
Stimulation of the T-cell receptor (TCR) results in the activation of several transcription factors, including NF-?B, that are crucial for T-cell proliferation and gain of effector functions. On TCR engagement, several proteins within the TCR-directed NF-?B signaling pathway undergo dynamic spatial redistribution, but the significance of these redistribution events is largely unknown. We have previously described TCR-induced cytoplasmic structures called POLKADOTS (punctate and oligomeric killing or activating domains transducing signals) that are enriched in the NF-?B signaling intermediate, Bcl10. We now show that these structures are formed only under conditions that promote efficient NF-?B activation. Furthermore, POLKADOTS formation is dependent...
112.
Factors Controlling Fibroblast Growth Factor Receptor-1's Cytoplasmic Trafficking and Its Regulation as Revealed by FRAP Analysis - Dunham-Ems, Star M.; Pudavar, Haridas E.; Myers, Jason M.; Maher, Pamela A.; Prasad, Paras N.; Stachowiak, Michal K.
Biochemical and microscopic studies have indicated that FGFR1 is a transmembrane and soluble protein present in the cytosol and nucleus. How FGFR1 enters the cytosol and subsequently the nucleus to control cell development and associated gene activities has become a compelling question. Analyses of protein synthesis, cytoplasmic subcompartmental distribution and movement of FGFR1-EGFP and FGFR1 mutants showed that FGFR1 exists as three separate populations (a) a newly synthesized, highly mobile, nonglycosylated, cytosolic receptor that is depleted by brefeldin A and resides outside the ER-Golgi lumen, (b) a slowly diffusing membrane receptor population, and (c) an immobile membrane pool increased by...
113.
Disulfide Transfer between Two Conserved Cysteine Pairs Imparts Selectivity to Protein Oxidation by Ero1 - Sevier, Carolyn S.; Kaiser, Chris A.
The membrane-associated flavoprotein Ero1p promotes disulfide bond formation in the endoplasmic reticulum (ER) by selectively oxidizing the soluble oxidoreductase protein disulfide isomerase (Pdi1p), which in turn can directly oxidize secretory proteins. Two redox-active disulfide bonds are essential for Ero1p oxidase activity: Cys100-Cys105 and Cys352-Cys355. Genetic and structural data indicate a disulfide bond is transferred from Cys100-Cys105 directly to Pdi1p, whereas a Cys352-Cys355 disulfide bond is used to reoxidize the reduced Cys100-Cys105 pair through an internal thiol-transfer reaction. Electron transfer from Cys352-Cys355 to molecular oxygen, by way of a flavin cofactor, maintains Cys352-Cys355 in an oxidized form. Herein, we identify a...
114.
Type I Collagen in Hsp47-null Cells Is Aggregated in Endoplasmic Reticulum and Deficient in N-Propeptide Processing and FibrillogenesisD? - Ishida, Yoshihito; Kubota, Hiroshi; Yamamoto, Akitsugu; Kitamura, Akira; Bächinger, Hans Peter; Nagata, Kazuhiro
Heat-shock protein of 47 kDa (Hsp47) is a molecular chaperone that recognizes collagen triple helices in the endoplasmic reticulum (ER). Hsp47-knockout mouse embryos are deficient in the maturation of collagen types I and IV, and collagen triple helices formed in the absence of Hsp47 show increased susceptibility to protease digestion. We show here that the fibrils of type I collagen produced by Hsp47-/- cells are abnormally thin and frequently branched. Type I collagen was highly accumulated in the ER of Hsp47-/- cells, and its secretion rate was much slower than that of Hsp47+/+ cells, leading to accumulation of the insoluble...
115.
The Slp4-a Linker Domain Controls Exocytosis through Interaction with Munc18-1·Syntaxin-1a ComplexD? - Tsuboi, Takashi; Fukuda, Mitsunori
Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a is specifically localized on dense-core vesicles in certain neuroendocrine cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A. However, the precise molecular mechanism of its inhibitory effect on exocytosis has never been elucidated and is still a matter of controversy. Here we show by deletion and chimeric analyses that the linker domain of Slp4-a interacts with the Munc18-1·syntaxin-1a complex by directly binding to Munc18-1 and that this interaction promotes docking of dense-core vesicles to the plasma membrane in PC12 cells. Despite increasing the number of plasma membrane docked vesicles, expression of Slp4-a strongly...
116.
Adenomatous Polyposis Coli on Microtubule Plus Ends in Cell Extensions Can Promote Microtubule Net Growth with or without EB1D?V? - Kita, Katsuhiro; Wittmann, Torsten; Näthke, Inke S.; Waterman-Storer, Clare M.
In interphase cells, the adenomatous polyposis coli (APC) protein accumulates on a small subset of microtubules (MTs) in cell protrusions, suggesting that APC may regulate the dynamics of these MTs. We comicroinjected a nonperturbing fluorescently labeled monoclonal antibody and labeled tubulin to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and had a decreased catastrophe frequency compared with non-APC-decorated MTs. Endogenous APC associated briefly with shortening MTs. To determine the relationship between APC and its binding partner EB1, we monitored EB1-green fluorescent protein and endogenous APC concomitantly in living...
117.
Luteinizing Hormone Receptor Ectodomain Splice Variant Misroutes the Full-Length Receptor into a Subcompartment of the Endoplasmic ReticulumD? - Apaja, Pirjo M.; Tuusa, Jussi T.; Pietilä, E. Maritta; Rajaniemi, Hannu J.; Petäjä-Repo, Ulla E.
The luteinizing hormone receptor (LHR) is a G proteincoupled receptor that is expressed in multiple RNA messenger forms. The common rat ectodomain splice variant is expressed concomitantly with the full-length LHR in tissues and is a truncated transcript corresponding to the partial ectodomain with a unique C-terminal end. Here we demonstrate that the variant alters the behavior of the full-length receptor by misrouting it away from the normal secretory pathway in human embryonic kidney 293 cells. The variant was expressed as two soluble forms of Mr 52,000 and Mr 54,000, but although the protein contains a cleavable signal sequence, no...
118.
The Cadherin-11 Cytoplasmic Juxtamembrane Domain Promotes ?-Catenin Turnover at Adherens Junctions and Intercellular MotilityD?V? - Kiener, Hans P.; Stipp, Christopher S.; Allen, Philip G.; Higgins, Jonathan M.G.; Brenner, Michael B.
Cadherins mediate homophilic cell adhesion and contribute to tissue morphogenesis and architecture. Cadherin cell adhesion contacts are actively remodeled and impact cell movement and migration over other cells. We found that expression of a mutant cadherin-11 lacking the cytoplasmic juxtamembrane domain (JMD) diminished the turnover of ?-catenin at adherens junctions as measured by fluorescence recovery after photobleaching. This resulted in markedly diminished cell intercalation into monolayers reflecting reduced cadherin-11-dependent cell motility on other cells. Furthermore, the actin cytoskeleton in cadherin-11 ?JMD cells revealed a more extensive cortical F-actin ring that correlated with significantly higher levels of activated Rac1. Together, these...
119.
Motion Matters: Secretory Granule Motion Adjacent to the Plasma Membrane and Exocytosis - Allersma, Miriam W.; Bittner, Mary A.; Axelrod, Daniel; Holz, Ronald W.
Total internal reflection fluorescence microscopy was used to monitor changes in individual granule motions related to the secretory response in chromaffin cells. Because the motions of granules are very small (tens of nanometers), instrumental noise in the quantitation of granule motion was taken into account. ATP and Ca2+, both of which prime secretion before fusion, also affect granule motion. Removal of ATP in permeabilized cells causes average granule motion to decrease. Nicotinic stimulation causes a calcium-dependent increase in average granule motion. This effect is more pronounced for granules that undergo exocytosis than for those that do not. Fusion is not...
120.
FUS1 Regulates the Opening and Expansion of Fusion Pores between Mating YeastD?V? - Nolan, Scott; Cowan, Ann E.; Koppel, Dennis E.; Jin, Hui; Grote, Eric
Mating yeast cells provide a genetically accessible system for the study of cell fusion. The dynamics of fusion pores between yeast cells were analyzed by following the exchange of fluorescent markers between fusion partners. Upon plasma membrane fusion, cytoplasmic GFP and DsRed diffuse between cells at rates proportional to the size of the fusion pore. GFP permeance measurements reveal that a typical fusion pore opens with a burst and then gradually expands. In some mating pairs, a sudden increase in GFP permeance was found, consistent with the opening of a second pore. In contrast, other fusion pores closed after permitting...
101.
