PubMed Central (PMC3 - NLM DTD)
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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 41 - 60 de 9,978
41.
Heterodimeric Capping Protein from Arabidopsis Is Regulated by Phosphatidic Acid - Huang, Shanjin; Gao, Lisa; Blanchoin, Laurent; Staiger, Christopher J.
The cytoskeleton is a key regulator of morphogenesis, sexual reproduction, and cellular responses to extracellular stimuli. Changes in the cellular architecture are often assumed to require actin-binding proteins as stimulus-response modulators, because many of these proteins are regulated directly by binding to intracellular second messengers or signaling phospholipids. Phosphatidic acid (PA) is gaining widespread acceptance as a major, abundant phospholipid in plants that is required for pollen tube tip growth and mediates responses to osmotic stress, wounding, and phytohormones; however, the number of identified effectors of PA is rather limited. Here we demonstrate that exogenous PA application leads to significant...
42.
A Holistic Kinesin Phylogeny Reveals New Kinesin Families and Predicts Protein FunctionsD? - Wickstead, Bill; Gull, Keith
Kinesin superfamily proteins are ubiquitous to all eukaryotes and essential for several key cellular processes. With the establishment of genome sequence data for a substantial number of eukaryotes, it is now possible for the first time to analyze the complete kinesin repertoires of a diversity of organisms from most eukaryotic kingdoms. Such a holistic approach using 486 kinesin-like sequences from 19 eukaryotes and analyzed by Bayesian techniques, identifies three new kinesin families, two new phylum-specific groups, and unites two previously identified families. The paralogue distribution suggests that the eukaryotic cenancestor possessed nearly all kinesin families. However, multiple losses in individual...
43.
Ran Is Required before Metaphase for Spindle Assembly and Chromosome Alignment and after Metaphase for Chromosome Segregation and Spindle Midbody OrganizationD?V? - Silverman-Gavrila, Rosalind V.; Wilde, Andrew
The Ran pathway has been shown to have a role in spindle assembly. However, the extent of the role of the Ran pathway in mitosis in vivo is unclear. We report that perturbation of the Ran pathway disrupted multiple steps of mitosis in syncytial Drosophila embryos and uncovered new mitotic processes that are regulated by Ran. During the onset of mitosis, the Ran pathway is required for the production, organization, and targeting of centrosomally nucleated microtubules to chromosomes. However, the role of Ran is not restricted to microtubule organization, because Ran is also required for the alignment of chromosomes at...
44.
CLIP-170 Homologue and NUDE Play Overlapping Roles in NUDF Localization in Aspergillus nidulansV? - Efimov, Vladimir P.; Zhang, Jun; Xiang, Xin
Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32°C, those that...
45.
The F-Box Protein Dia2 Regulates DNA ReplicationD? - Koepp, Deanna M.; Kile, Andrew C.; Swaminathan, Swarna; Rodriguez-Rivera, Veronica
Ubiquitin-mediated proteolysis plays a key role in many pathways inside the cell and is particularly important in regulating cell cycle transitions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases whose specificity is determined by a substrate-binding F-box protein. Dia2 is a Saccharomyces cerevisiae F-box protein previously described to play a role in invasive growth and pheromone response pathways. We find that deletion of DIA2 renders cells cold-sensitive and subject to defects in cell cycle progression, including premature S-phase entry. Consistent with a role in regulating DNA replication, the Dia2 protein binds replication origins. Furthermore, the dia2 mutant accumulates DNA damage...
46.
Yeast Nuclear Envelope Subdomains with Distinct Abilities to Resist Membrane Expansion - Campbell, Joseph L.; Lorenz, Alexander; Witkin, Keren L.; Hays, Thomas; Loidl, Josef; Cohen-Fix, Orna
Little is known about what dictates the round shape of the yeast Saccharomyces cerevisiae nucleus. In spo7? mutants, the nucleus is misshapen, exhibiting a single protrusion. The Spo7 protein is part of a phosphatase complex that represses phospholipid biosynthesis. Here, we report that the nuclear protrusion of spo7? mutants colocalizes with the nucleolus, whereas the nuclear compartment containing the bulk of the DNA is unaffected. Using strains in which the nucleolus is not intimately associated with the nuclear envelope, we show that the single nuclear protrusion of spo7? mutants is not a result of nucleolar expansion, but rather a property...
47.
Actin-depolymerizing Protein Adf1 Is Required for Formation and Maintenance of the Contractile Ring during Cytokinesis in Fission YeastD?V? - Nakano, Kentaro; Mabuchi, Issei
The role of the actin-depolymerizing factor (ADF)/cofilin-family protein Adf1 in cytokinesis of fission yeast cells was studied. Adf1 was required for accumulation of actin at the division site by depolymerizing actin at the cell ends, assembly of the contractile ring through severing actin filaments, and maintenance of the contractile ring once formed. Genetic and cytological analyses suggested that it collaborates with profilin and capping protein in the mitotic reorganization of the actin cytoskeleton. Furthermore, it was unexpectedly found that Adf1 and myosin-II also collaborate in assembling the contractile ring. Tropomyosin was shown to antagonize the function of Adf1 in the...
48.
EDEM Is Involved in Retrotranslocation of Ricin from the Endoplasmic Reticulum to the Cytosol - Slominska-Wojewodzka, Monika; Gregers, Tone F.; Wälchli, Sébastien; Sandvig, Kirsten
The plant toxin ricin is transported retrogradely from the cell surface to the endoplasmic reticulum (ER) from where the enzymatically active part is retrotranslocated to the cytosol, presumably by the same mechanism as used by misfolded proteins. The ER degradation enhancing ?-mannosidase I-like protein, EDEM, is responsible for directing aberrant proteins for ER-associated protein degradation. In this study, we have investigated whether EDEM is involved in ricin retrotranslocation. Overexpression of EDEM strongly protects against ricin. However, when the interaction between EDEM and misfolded proteins is inhibited by kifunensin, EDEM promotes retrotranslocation of ricin from the ER to the cytosol. Furthermore,...
49.
The Transitional Junction: A New Functional Subcellular Domain at the Intercalated Disc - Bennett, Pauline M.; Maggs, Alison M.; Baines, Anthony J.; Pinder, Jennifer C.
We define here a previously unrecognized structural element close to the heart muscle plasma membrane at the intercalated disc where the myofibrils lead into the adherens junction. At this location, the plasma membrane is extensively folded. Immunofluorescence and immunogold electron microscopy reveal a spectrin-rich domain at the apex of the folds. These domains occur at the axial level of what would be the final Z-disc of the terminal sarcomere in the myofibril, although there is no Z-disc-like structure there. However, a sharp transitional boundary lies between the myofibrillar I-band and intercalated disc thin filaments, identifiable by the presence of Z-disc...
50.
Domains within the GARP Subunit Vps54 Confer Separate Functions in Complex Assembly and Early Endosome Recognition - Quenneville, Nicole R.; Chao, Tzu-Yuan; McCaffery, J. Michael; Conibear, Elizabeth
Tethering complexes contribute to the specificity of membrane fusion by recognizing organelle features on both donor and acceptor membranes. The Golgi-associated retrograde protein (GARP) complex is required for retrograde traffic from both early and late endosomes to the trans-Golgi network (TGN), presenting a paradox as to how a single complex can interact specifically with vesicles from multiple upstream compartments. We have found that a subunit of the GARP complex, Vps54, can be separated into N- and C-terminal regions that have different functions. Whereas the N-terminus of Vps54 is important for GARP complex assembly and stability, a conserved C-terminal domain mediates...
51.
Differential Requirements for Actin Polymerization, Calmodulin, and Ca2+ Define Distinct Stages of Lysosome/Phagosome Targeting - Stockinger, Walter; Zhang, Shao C.; Trivedi, Vishal; Jarzylo, Larissa A.; Shieh, Eugenie C.; Lane, William S.; Castoreno, Adam B.; Nohturfft, Axel
Fusion of phagosomes with late endocytic organelles is essential for cellular digestion of microbial pathogens, senescent cells, apoptotic bodies, and retinal outer segment fragments. To further elucidate the biochemistry of the targeting process, we developed a scintillation proximity assay to study the stepwise association of lysosomes and phagosomes in vitro. Incubation of tritium-labeled lysosomes with phagosomes containing scintillant latex beads led to light emission in a reaction requiring cytosol, ATP, and low Ca2+ concentrations. The nascent complex was sensitive to disruption by alkaline carbonate, indicating that the organelles had docked but not fused. Through inhibitor studies and fluorescence microscopy we...
52.
Loss of P4 ATPases Drs2p and Dnf3p Disrupts Aminophospholipid Transport and Asymmetry in Yeast Post-Golgi Secretory VesiclesD? - Alder-Baerens, Nele; Lisman, Quirine; Luong, Lambert; Pomorski, Thomas; Holthuis, Joost C.M.
Eukaryotic plasma membranes generally display asymmetric lipid distributions with the aminophospholipids concentrated in the cytosolic leaflet. This arrangement is maintained by aminophospholipid translocases (APLTs) that use ATP hydrolysis to flip phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external to the cytosolic leaflet. The identity of APLTs has not been established, but prime candidates are members of the P4 subfamily of P-type ATPases. Removal of P4 ATPases Dnf1p and Dnf2p from budding yeast abolishes inward translocation of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS, PE, and phosphatidylcholine (PC) across the plasma membrane and causes cell surface exposure of endogenous PE. Here, we show that yeast...
53.
UNC-83 Is a KASH Protein Required for Nuclear Migration and Is Recruited to the Outer Nuclear Membrane by a Physical Interaction with the SUN Protein UNC-84 - McGee, Matthew D.; Rillo, Regina; Anderson, Amy S.; Starr, Daniel A.
UNC-84 is required to localize UNC-83 to the nuclear envelope where it functions during nuclear migration. A KASH domain in UNC-83 was identified. KASH domains are conserved in the nuclear envelope proteins Syne/nesprins, Klarsicht, MSP-300, and ANC-1. Caenorhabditis elegans UNC-83 was shown to localize to the outer nuclear membrane and UNC-84 to the inner nuclear membrane in transfected mammalian cells, suggesting the KASH and SUN protein targeting mechanisms are conserved. Deletion of the KASH domain of UNC-83 blocked nuclear migration and localization to the C. elegans nuclear envelope. Some point mutations in the UNC-83 KASH domain disrupted nuclear migration, even...
54.
A PDZ-binding Motif as a Critical Determinant of Rho Guanine Exchange Factor Function and Cell Phenotype - Liu, Miaoliang; Horowitz, Arie
We identified a Rho guanine exchange factor (GEF) expressed as two splice variants, which differ only in either having or lacking a Postsynaptic density 95, Disk large, Zona occludens-1 (PDZ) motif. The PDZ adaptor protein synectin bound the longer splice variant, Syx1, which was targeted to the plasma membrane in a synectin-dependent manner. The shorter variant, Syx2, was diffusely distributed in the cytoplasm. Fluorescence resonance energy transfer (FRET) imaging revealed similar differences between the spatial patterns of active RhoA in Syx1 versus Syx2-expressing cells. Expression of Syx1 augmented endothelial cell (EC) migration and tube formation, whereas Syx2 expression did not....
55.
Regulated Phosphorylation of Budding Yeast's Essential Myosin V Heavy Chain, Myo2pD? - Legesse-Miller, Aster; Zhang, Sheng; Santiago-Tirado, Felipe H.; Van Pelt, Colleen K.; Bretscher, Anthony
The tail of the yeast myosin V encoded by Myo2p is known to bind several receptors for cargo delivery along polarized actin cables. However, it is not known how Myo2p activity is regulated or how it selects between cargos. Here we show that Myo2p is reversibly phosphorylated in vivo. A short peptide at the N-terminal end of the cargo-binding domain contains three residues contributing to single or doubly phosphorylated species. We confirm that the tail consists of two proteolytically resistant subdomains and identify a functionally important region N-terminal to sudomain 1 that includes the phosphorylation sites. Mutagenesis of the phosphorylation...
56.
Kinesin-1 and Dynein Are the Primary Motors for Fast Transport of Mitochondria in Drosophila Motor AxonsD?V? - Pilling, Aaron D.; Horiuchi, Dai; Lively, Curtis M.; Saxton, William M.
To address questions about mechanisms of filament-based organelle transport, a system was developed to image and track mitochondria in an intact Drosophila nervous system. Mutant analyses suggest that the primary motors for mitochondrial movement in larval motor axons are kinesin-1 (anterograde) and cytoplasmic dynein (retrograde), and interestingly that kinesin-1 is critical for retrograde transport by dynein. During transport, there was little evidence that force production by the two opposing motors was competitive, suggesting a mechanism for alternate coordination. Tests of the possible coordination factor P150Glued suggested that it indeed influenced both motors on axonal mitochondria, but there was no evidence...
57.
HIV-1 Tat Regulates Endothelial Cell Cycle Progression via Activation of the Ras/ERK MAPK Signaling PathwayD? - Toschi, Elena; Bacigalupo, Ilaria; Strippoli, Raffaele; Chiozzini, Chiara; Cereseto, Anna; Falchi, Mario; Nappi, Filomena; Sgadari, Cecilia; Barillari, Giovanni; Mainiero, Fabrizio; Ensoli, Barbara
Tat, the transactivator of HIV-1 gene expression, is released by acutely HIV-1-infected T-cells and promotes adhesion, migration, and growth of inflammatory cytokine-activated endothelial and Kaposi's sarcoma cells. It has been previously demonstrated that these effects of Tat are due to its ability to bind through its arginine-glycine-aspartic (RGD) region to the ?5?1 and ?v?3 integrins. However, the signaling pathways linking Tat to the regulation of cellular functions are incompletely understood. Here, we report that Tat ligation on human endothelial cells results in the activation of the small GTPases Ras and Rac and the mitogen-activated protein kinase ERK, specifically through its...
58.
Cholesterol Is Required for Efficient Endoplasmic Reticulum-to-Golgi Transport of Secretory Membrane ProteinsV? - Ridsdale, Andrew; Denis, Maxime; Gougeon, Pierre-Yves; Ngsee, Johnny K.; Presley, John F.; Zha, Xiaohui
Although cholesterol is synthesized in the endoplasmic reticulum (ER), compared with other cellular membranes, ER membrane has low cholesterol (36%). Most of the molecular machinery that regulates cellular cholesterol homeostasis also resides in the ER. Little is known about how cholesterol itself affects the ER membrane. Here, we demonstrate that acute cholesterol depletion in ER membranes impairs ER-to-Golgi transport of secretory membrane proteins. Cholesterol depletion is achieved by a brief inhibition of cholesterol synthesis with statins in cells grown in cholesterol-depleted medium. We provide evidence that secretory membrane proteins vesicular stomatitis virus glycoprotein and scavenger receptor A failed to be...
59.
The Gcs1 Arf-GAP Mediates Snc1,2 v-SNARE Retrieval to the Golgi in Yeast - Robinson, Micah; Poon, Pak Phi; Schindler, Christina; Murray, Lois E.; Kama, Rachel; Gabriely, Galina; Singer, Richard A.; Spang, Anne; Johnston, Gerald C.; Gerst, Jeffrey E.
Gcs1 is an Arf GTPase-activating protein (Arf-GAP) that mediates Golgi-ER and post-Golgi vesicle transport in yeast. Here we show that the Snc1,2 v-SNAREs, which mediate endocytosis and exocytosis, interact physically and genetically with Gcs1. Moreover, Gcs1 and the Snc v-SNAREs colocalize to subcellular structures that correspond to the trans-Golgi and endosomal compartments. Studies performed in vitro demonstrate that the Snc-Gcs1 interaction results in the efficient binding of recombinant Arf1?17N-Q71L to the v-SNARE and the recruitment of purified coatomer. In contrast, the presence of Snc had no effect on Gcs1 Arf-GAP activity in vitro, suggesting that v-SNARE binding does not attenuate...
60.
Splicing Speckles Are Not Reservoirs of RNA Polymerase II, but Contain an Inactive Form, Phosphorylated on Serine2 Residues of the C-Terminal DomainD? - Xie, Sheila Q.; Martin, Sonya; Guillot, Pascale V.; Bentley, David L.; Pombo, Ana
Splicing speckles are major nuclear domains rich in components of the splicing machinery and polyA+ RNA. Although speckles contain little detectable transcriptional activity, they are found preferentially associated with specific mRNA-coding genes and gene-rich R bands, and they accumulate some unspliced pre-mRNAs. RNA polymerase II transcribes mRNAs and is required for splicing, with some reports suggesting that the inactive complexes are stored in splicing speckles. Using ultrathin cryosections to improve optical resolution and preserve nuclear structure, we find that all forms of polymerase II are present, but not enriched, within speckles. Inhibition of polymerase activity shows that speckles do not...
41.
