Expression of the endocytic proteins dynamin and amphiphysin in rat gastric enterochromaffin-like cells.
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Dynamin and amphiphysin play crucial roles in a variety of endocytic processes. Previous investigations of expression and functions of these proteins were performed mostly on neurons. The aim of this study was to investigate the presence and interaction of dyn and amph in gastric enterochromaffin-like cells. These endocrine cells of the gastric mucosa play a pivotal role in the regulation of acid secretion. Exocytosis of histamine-containing secretory vesicles has been described in detail. However, the mechanisms of endocytosis are unknown in this neuroendocrine cell type. Using RT-PCR and western blotting, we detected dynamin-1, -2 and -3 in highly enriched isolated enterochromaffin-like cells. Dynamin-1 and -2 were expressed at similar high levels, whereas dynamin-3 was of low abundance. Immunofluorescence microscopy located dynamin-1 and -2 to the cytoplasm and cell surface, whereas dynamin-3 was distributed differently in the perinuclear area. The presence of amphiphysin-1 and -2 RNAs was revealed by RT-PCR and a new splice variant of amphiphysin-2 was detected. Amphiphysin-1 and -2 were also detected in enterochromaffin-like cells by immunohistochemistry in the same locations as dynamin-1 and -2. Amphiphysin-1 and dynamin-1 co-immunoprecipitated with amphiphysin-2. In addition, dynamin-1 and amphiphysin-2 partially colocalized at the plasma membrane. Our results confirm the interaction of dynamin and amphiphysin and imply a role in endocytosis in enterochromaffin-like cells. To our knowledge, this is the first demonstration of the co-expression of all three dynamin isoforms in a non-tumor cell. (+info)
Intra- and intermolecular domain interactions of the C-terminal GTPase effector domain of the multimeric dynamin-like GTPase Drp1.
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Mammalian Drp1 is a dynamin-like GTPase required for mitochondrial fission. Although it exists primarily as a cytosolic homo-tetramer in vivo, it can also self-assemble into higher order structures on the mitochondrial outer membrane, where it is required for proper mitochondrial division. Functional studies and sequence comparisons have revealed four different structural domains in Drp1, comprising N-terminal GTP-binding, middle, insert B, and C-terminal GTPase effector (GED) domains. Here we describe an intramolecular interaction within Drp1 between the GED and the N-terminal GTP-binding and middle domains. A point mutation (K679A) within the C-terminal GED domain inhibits this intramolecular association, without affecting the formation of Drp1 tetramers or the intermolecular associations among isolated C-terminal domains. Mutant Drp1 K679A exhibits impaired GTPase activity, and when overexpressed in mammalian cells it decreases mitochondrial division. Sedimentation experiments indicate that the K679A mutation either increases Drp1 complex formation or, more likely, decreases complex disassembly as compared with wild-type Drp1. Taken together, these data suggest that the C-terminal GED domain is important for stimulation of GTPase activity, formation and stability of higher order complexes, and efficient mitochondrial division. (+info)
Phospholipase C-gamma1 is a guanine nucleotide exchange factor for dynamin-1 and enhances dynamin-1-dependent epidermal growth factor receptor endocytosis.
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Phospholipase C-gamma1 (PLC-gamma1), which interacts with a variety of signaling molecules through its two Src homology (SH) 2 domains and a single SH3 domain has been implicated in the regulation of many cellular functions. We demonstrate that PLC-gamma1 acts as a guanine nucleotide exchange factor (GEF) of dynamin-1, a 100 kDa GTPase protein, which is involved in clathrin-mediated endocytosis of epidermal growth factor (EGF) receptor. Overexpression of PLC-gamma1 increases endocytosis of the EGF receptor by increasing guanine nucleotide exchange activity of dynamin-1. The GEF activity of PLC-gamma1 is mediated by the direct interaction of its SH3 domain with dynamin-1. EGF-dependent activation of ERK and serum response element (SRE) are both up-regulated in PC12 cells stably overexpressing PLC-gamma1, but knockdown of PLC-gamma1 by siRNA significantly reduces ERK activation. These results establish a new role for PLC-gamma1 in the regulation of endocytosis and suggest that endocytosis of activated EGF receptors may mediate PLC-gamma1-dependent proliferation. (+info)
Mitochondrial DNA maintenance in yeast requires a protein containing a region related to the GTP-binding domain of dynamin.
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Nuclear gene products replicate and partition mitochondrial DNA (mtDNA) molecules in the yeast Saccharomyces cerevisiae. However, few of the relevant genes have been identified. A mutation that results in temperature-sensitive loss of mtDNA identifies one of these genes, MGM1. Deletion of MGM1 shows that aside from its role in the mitochondrion, the gene has no essential cellular function. The MGM1 protein has a 200-amino-acid region that is highly related to a family of GTP-binding proteins of apparently diverse function that includes the microtubule-binding protein, dynamin D100. The temperature-sensitive strain partitions mtDNA molecules at the restrictive temperature, but a defect in mtDNA synthesis results in a reduction in the number of molecules per cell at each cell division. On the basis of the results of this study, we conclude that cells can partition single mitochondrial genomes, and that when a cell receives a single molecule at division it is able to restore the normal complement of multiple copies. (+info)
Mechanisms of dense core vesicle recapture following "kiss and run" ("cavicapture") exocytosis in insulin-secreting cells.
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The molecular mechanisms underlying "kiss and run" or "cavicapture" exocytosis of dense core secretory vesicles are presently unclear. Although dynamin-1 has previously been implicated in the recapture process in neurons, the recruitment of this fission protein to a single exocytosing vesicle has not been examined in real time during peptide release from pancreatic beta-cells. Imaged simultaneously in clonal insulin-secreting cells by dual color total internal reflection fluorescence microscopy, monomeric red fluorescent protein (mRFP)-tagged neuropeptide Y and green fluorescent protein (GFP)-tagged synaptotagmin-1 or synaptobrevin-2 rapidly diffused from sites of exocytosis, whereas the vesicle membrane protein phogrin and tissue plasminogen activator (tPA) were retained, consistent with fusion pore closure. Vesicle recovery frequently involved the recruitment of enhanced GFP-tagged dynamin-1, and GTPase-defective dynamin-1(K44E) increased the dwell time of tPA-mRFP at the plasma membrane. By contrast, recruitment of GFP chimeras of clathrin, epsin, and amphiphysin was not observed. Expression of dynamin-1(K535A), mutated in the pleckstrin homology domain, caused the apparent full fusion of vesicles, as reported by the additional release of tPA-mRFP (15-nm diameter) and enhanced GFP-tagged phogrin. We conclude that re-uptake of vesicles after peptide release by cavicapture corresponds to a novel form of endocytosis in which dynamin-1 stabilizes and eventually closes the fusion pore, with no requirement for "classical" endocytosis for retreat from the plasma membrane. (+info)
Fzo1, a protein involved in mitochondrial fusion, inhibits apoptosis.
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Mitochondrial morphology and physiology are regulated by the processes of fusion and fission. Some forms of apoptosis are reported to be associated with mitochondrial fragmentation. We showed that overexpression of Fzo1A/B (rat) proteins involved in mitochondrial fusion, or silencing of Dnm1 (rat)/Drp1 (human) (a mitochondrial fission protein), increased elongated mitochondria in healthy cells. After apoptotic stimulation, these interventions inhibited mitochondrial fragmentation and cell death, suggesting that a process involved in mitochondrial fusion/fission might play a role in the regulation of apoptosis. Consistently, silencing of Fzo1A/B or Mfn1/2 (a human homolog of Fzo1A/B) led to an increase of shorter mitochondria and enhanced apoptotic death. Overexpression of Fzo1 inhibited cytochrome c release and activation of Bax/Bak, as assessed from conformational changes and oligomerization. Silencing of Mfn or Drp1 caused an increase or decrease of mitochondrial sensitivity to apoptotic stimulation, respectively. These results indicate that some of the proteins involved in mitochondrial fusion/fission modulate apoptotic cell death at the mitochondrial level. (+info)
Vesicle endocytosis requires dynamin-dependent GTP hydrolysis at a fast CNS synapse.
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Molecular dependence of vesicular endocytosis was investigated with capacitance measurements at the calyx of Held terminal in brainstem slices. Intraterminal loading of botulinum toxin E revealed that the rapid capacitance transient implicated as "kiss-and-run" was unrelated to transmitter release. The release-related capacitance change decayed with an endocytotic time constant of 10 to 25 seconds, depending on the magnitude of exocytosis. Presynaptic loading of the nonhydrolyzable guanosine 5'-triphosphate (GTP) analog GTPgS or dynamin-1 proline-rich domain peptide abolished endocytosis. These compounds had no immediate effect on exocytosis, but caused a use-dependent rundown of exocytosis. Thus, the guanosine triphosphatase dynamin-1 is indispensable for vesicle endocytosis at this fast central nervous system (CNS) synapse. (+info)
Dynamin I phosphorylation and the control of synaptic vesicle endocytosis.
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The GTPase dynamin I is essential for synaptic vesicle endocytosis in nerve terminals. It is a nerve terminal phosphoprotein that is dephosphorylated on nerve terminal stimulation by the calcium-dependent protein phosphatase calcineurin and then rephosphorylated by cyclin-dependent kinase 5 on termination of the stimulus. Because of its unusual phosphorylation profile, the phosphorylation status of dynamin I was assumed to be inexorably linked to synaptic vesicle endocytosis; however, direct proof of this link has been elusive until very recently. This review will describe current knowledge regarding dynamin I phosphorylation in nerve terminals and how this regulates its biological function with respect to synaptic vesicle endocytosis. (+info)