Multiple forms of "kiss-and-run" exocytosis revealed by evanescent wave microscopy.
ABSTRACT Exocytotic release of neuropeptides and hormones is generally believed to involve the complete merger of the secretory vesicle with the plasma membrane. However, recent data have suggested that "kiss-and-run" mechanisms may also play a role. Here, we have examined the dynamics of exocytosis in pancreatic MIN6 beta cells by imaging lumen- (neuropeptide Y/pH-insensitive yellow fluorescent protein; NPY.Venus) or vesicle membrane-targeted fluorescent probes (synaptobrevin-2/enhanced green fluorescent protein; synapto.pHluorin, or phosphatase on the granule of insulinoma-enhanced green fluorescent protein, phogrin.EGFP) by evanescent wave microscopy. Unexpectedly, NPY.Venus release events occurred much less frequently (13%-40% maximal rate) than those of synapto.pHluorin, even though the latter molecule, but not phogrin.EGFP, usually diffused away from the site of fusion. Thus, the majority of exocytosis occurs in these cells by kiss-and-run events that involve either the release of small molecules only, small molecules and selected membrane proteins, or all soluble cargoes ("pure," "mixed," and "full" kiss-and-run, respectively). Changes in the activity of synaptotagmin IV, achieved here by overexpression of the wild-type protein, may allow different stimuli to alter the ratio of these events, and thus the release of selected vesicle cargoes.
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ABSTRACT: Sorting of proinsulin to the regulated secretory pathway of pancreatic beta cells and retention of insulin in dense-core granules of this pathway is remarkably efficient. To monitor the specificity of these events, the secretion of two exogenous secretory proteins not known to carry information for sorting or retention in the regulated pathway was investigated in INS-1 cells. SEGFP, a fusion protein consisting of a signal peptide N-terminal to EGFP (mutant green fluorescent protein with enhanced fluorescence) and secreted alkaline phosphatase (SEAP) were expressed in INS-1 cells by transfection and by infection with recombinant adenovirus, respectively. Secretion of SEGFP was monitored by quantitative western blotting and that of SEAP by its activity. Secreted alkaline phosphatase showed high basal secretion (6.6% total) but only modest (3.6-fold) stimulation of secretion by secretagogues, in keeping with secretion largely through the constitutive pathway. By contrast SEGFP had a secretory pattern similar to insulin, with low basal secretion (0.8% total) and 16-fold stimulation by secretagogues. Granular localization of SEGFP was confirmed by high resolution electron microscopy immunocytochemistry. Pulse-chase experiments indicated retention of SEGFP in granules at least 24 h after synthesis. The secretory SEGFP, but not cytosolic EGFP, formed disulphide-linked oligomers. This could be implicated in its regulated secretion. These data indicate that in INS-1 cells SEGFP, but not SEAP, is unexpectedly handled as a regulated secretory protein and stored along with insulin in granules. This raises questions about the specificity and mechanism of the sorting of proteins to granules in INS-1 cells or their retention therein or both.Diabetologia 10/2000; 43(9):1157-64. · 6.49 Impact Factor
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ABSTRACT: In the exocytosis of neurotransmitter, fusion pore opening represents the first instant of fluid contact between the vesicle lumen and extracellular space. The existence of the fusion pore has been established by electrical measurements, but its molecular composition is unknown. The possibility that synaptotagmin regulates fusion pores was investigated with amperometry to monitor exocytosis of single dense-core vesicles. Overexpression of synaptotagmin I prolonged the time from fusion pore opening to dilation, whereas synaptotagmin IV shortened this time. Both synaptotagmin isoforms reduced norepinephrine flux through open fusion pores. Thus, synaptotagmin interacts with fusion pores, possibly by associating with a core complex of membrane proteins and/or lipid.Science 12/2001; 294(5544):1111-5. · 31.03 Impact Factor
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ABSTRACT: Perhaps synaptic vesicles can recycle so rapidly because they avoid complete exocytosis, and release transmitter through a fusion pore that opens transiently. This view emerges from imaging whole terminals where the fluorescent lipid FM1-43 seems unable to leave vesicles during transmitter release. Here we imaged single, FM1-43-stained synaptic vesicles by evanescent field fluorescence microscopy, and tracked the escape of dye from single vesicles by watching the increase in fluorescence after exocytosis. Dye left rapidly and completely during most or all exocytic events. We conclude that vesicles at this terminal allow lipid exchange soon after exocytosis, and lose their dye even if they connected with the plasma membrane only briefly. At the level of single vesicles, therefore, observations with FM1-43 provide no evidence that exocytosis of synaptic vesicles is incomplete.Neuron 10/2002; 35(6):1085-97. · 15.77 Impact Factor