Early stages of influenza virus entry into Mv-1 lung cells: involvement of dynamin.
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Viruses generally have one of two mechanisms for entry and uncoating. They can enter the cell either by endocytosis or by direct fusion at the plasma membrane. We have established a novel mink lung (Mv-1) cell line that expresses a dominant-interfering form of dynamin-1 (K44A) under the control of a tetracycline-responsive element and studied the early events in influenza infection using these cells. We found that influenza virus binds equally to both induced and uninduced cells, but in K44A-expressing cells, electron microscopy showed viruses trapped in deep coated pits and irregular-shaped tubular structures that contain discrete coated regions. We also show by immunofluorescence and confocal microscopy that entry of incoming virus into the nucleus is blocked in K44A-expressing cells. Virus replication was assayed by immunofluorescence microscopy and was strongly inhibited at both early and late times postinfection in K44A-expressing cells. Virus infectivity was inhibited by approximately 2 log units in cells expressing K44A dynamin when analyzed by influenza plaque assay. Overall these data show that dynamin is required for efficient influenza virus entry, presumably due to its function in release of vesicles from coated pits. (+info)
Ca(2+) influx inhibits dynamin and arrests synaptic vesicle endocytosis at the active zone.
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Ca(2+) entry into nerve terminals through clusters of voltage-dependent Ca(2+) channels (VDCCs) at active zones creates a microdomain of elevated intracellular free Ca(2+) concentration ([Ca(2+)](i)) that stimulates exocytosis. We show that this VDCC-mediated [Ca(2+)](i) elevation has no specific role in stimulating endocytosis but can inhibit endocytosis evoked by three different methods in isolated mammalian nerve terminals. The inhibition can be relieved by using either VDCC antagonists or fast, but not slow, binding intracellular Ca(2+) chelators. The Ca(2+)-dependent inhibition of endocytosis is mimicked in vitro by a low-affinity inhibition of dynamin I vesiculation of phospholipids. Increased [Ca(2+)](i) also inhibits dynamin II GTPase activity and receptor-mediated endocytosis in non-neuronal cells. VDCC-meditated Ca(2+) entry inhibits dynamin-mediated endocytosis at the active zone and provides neurons with a mechanism to clear recycling vesicles to nonactive zone regions during periods of high activity. (+info)
Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis.
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Endophilin/SH3p4 is a protein highly enriched in nerve terminals that binds the GTPase dynamin and the polyphosphoinositide phosphatase synaptojanin, two proteins implicated in synaptic vesicle endocytosis. We show here that antibody-mediated disruption of endophilin function in a tonically stimulated synapse leads to a block in the invagination of clathrin-coated pits adjacent to the active zone and therefore to a block of synaptic vesicle recycling. We also show that in a cell-free system, endophilin is not associated with clathrin coats and is a functional partner of dynamin. Our findings suggest that endophilin is part of a biochemical machinery that acts in trans to the clathrin coat from early stages to vesicle fission. (+info)
Role for dynamin in late endosome dynamics and trafficking of the cation-independent mannose 6-phosphate receptor.
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It is well established that dynamin is involved in clathrin-dependent endocytosis, but relatively little is known about possible intracellular functions of this GTPase. Using confocal imaging, we found that endogenous dynamin was associated with the plasma membrane, the trans-Golgi network, and a perinuclear cluster of cation-independent mannose 6-phosphate receptor (CI-MPR)-containing structures. By electron microscopy (EM), it was shown that these structures were late endosomes and that the endogenous dynamin was preferentially localized to tubulo-vesicular appendices on these late endosomes. Upon induction of the dominant-negative dynK44A mutant, confocal microscopy demonstrated a redistribution of the CI-MPR in mutant-expressing cells. Quantitative EM analysis of the ratio of CI-MPR to lysosome-associated membrane protein-1 in endosome profiles revealed a higher colocalization of the two markers in dynK44A-expressing cells than in control cells. Western blot analysis showed that dynK44A-expressing cells had an increased cellular procathepsin D content. Finally, EM revealed that in dynK44A-expressing cells, endosomal tubules containing CI-MPR were formed. These results are in contrast to recent reports that dynamin-2 is exclusively associated with endocytic structures at the plasma membrane. They suggest instead that endogenous dynamin also plays an important role in the molecular machinery behind the recycling of the CI-MPR from endosomes to the trans-Golgi network, and we propose that dynamin is required for the final scission of vesicles budding from endosome tubules. (+info)
Ligand endocytosis drives receptor dissociation and activation in the Notch pathway.
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Endocytosis of the ligand delta; is required for activation of the receptor Notch during Drosophila development. The Notch extracellular domain (NotchECD) dissociates from the Notch intracellular domain (NotchICD) and is trans-endocytosed into delta;-expressing cells in wild-type imaginal discs. Reduction of dynamin-mediated endocytosis in developing eye and wing imaginal discs reduces Notch dissociation and Notch signalling. Furthermore, dynamin-mediated delta endocytosis is required for Notch trans-endocytosis in Drosophila cultured cell lines. Endocytosis-defective delta proteins fail to mediate trans-endocytosis of Notch in cultured cells, and exhibit aberrant subcellular trafficking and reduced signalling capacity in Drosophila. We suggest that endocytosis into delta-expressing cells of NotchECD bound to delta plays a critical role during activation of the Notch receptor and is required to achieve processing and dissociation of the Notch protein. (+info)
Syndapin isoforms participate in receptor-mediated endocytosis and actin organization.
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Syndapin I (SdpI) interacts with proteins involved in endocytosis and actin dynamics and was therefore proposed to be a molecular link between the machineries for synaptic vesicle recycling and cytoskeletal organization. We here report the identification and characterization of SdpII, a ubiquitously expressed isoform of the brain-specific SdpI. Certain splice variants of rat SdpII in other species were named FAP52 and PACSIN 2. SdpII binds dynamin I, synaptojanin, synapsin I, and the neural Wiskott-Aldrich syndrome protein (N-WASP), a stimulator of Arp2/3 induced actin filament nucleation. In neuroendocrine cells, SdpII colocalizes with dynamin, consistent with a role for syndapin in dynamin-mediated endocytic processes. The src homology 3 (SH3) domain of SdpI and -II inhibited receptor-mediated internalization of transferrin, demonstrating syndapin involvement in endocytosis in vivo. Overexpression of full-length syndapins, but not the NH(2)-terminal part or the SH3 domains alone, had a strong effect on cortical actin organization and induced filopodia. This syndapin overexpression phenotype appears to be mediated by the Arp2/3 complex at the cell periphery because it was completely suppressed by coexpression of a cytosolic COOH-terminal fragment of N-WASP. Consistent with a role in actin dynamics, syndapins localized to sites of high actin turnover, such as filopodia tips and lamellipodia. Our results strongly suggest that syndapins link endocytosis and actin dynamics. (+info)
Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state.
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MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits. (+info)
Phragmoplastin polymerizes into spiral coiled structures via intermolecular interaction of two self-assembly domains.
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Phragmoplastin, a high molecular weight GTPase belonging to the dynamin superfamily of proteins, becomes associated with the cell plate during cytokinesis in plants. Growth of the cell plate requires continuous fusion of vesicles, and phragmoplastin appears to play a role in the formation of vesicle-tubule-vesicle structures at the cell plate. In this study, we have demonstrated that two self-assembly domains (SA1 and SA2) are involved in polymerization of phragmoplastin. SA1 is about 42 amino acids long and is located near the N terminus overlapping with the GTP-binding region. SA2, containing at least 24 amino acids, is located in the middle of the molecule outside the GTP-binding domain. Peptides containing either SA1 or SA2 interact efficiently with the full-length phragmoplastin. The SA1 domain of one phragmoplastin molecule also binds to SA2 of another as confirmed in vitro by using radiolabeled peptides. This interaction leads to the formation of polymers with a staggered contoured spiral structure. Electron microscopy studies revealed that helical arrays of phragmoplastin can be induced by reducing salt concentration. Our results suggest that phragmoplastin may assemble into helical arrays that wrap around and squeeze vesicles into vesicle-tubule-vesicle structures observed on the forming cell plate. (+info)