The localizations of three members of the neuronal calcium sensor (NCS) family were studied in HeLa cells. Using hippocalcin-EYFP and NCS-1-ECFP, it was found that their localization differed dramatically in resting cells. NCS-1 had a distinct predominantly perinuclear localization (similar to trans-Golgi markers), whereas hippocalcin was present diffusely throughout the cell. Upon the elevation of intracellular Ca 2؉ , hippocalcin rapidly translocated to the same perinuclear compartment as NCS-1. Another member of the family, neurocalcin ␦, also translocated to this region after a rise in Ca

No abstract

Accumulating evidence suggests that the kinetics of release from single secretory vesicles can be regulated and that quantal size can be modified during fast kiss-and-run fusion. Multiple pathways for vesicle retrieval have been identified involving clathrin and dynamin. It has been unclear whether dynamin could participate in a fast kiss-and-run process to reclose a transient fusion pore and thereby limit vesicle release. We have disrupted dynamin function in adrenal chromaffin cells by expression of the amphiphysin Src-homology domain 3 (SH3) or by application of guanosine 5-[␥-thio]triphosphate (GTP␥S), and have monitored single vesicle release events, evoked by digitonin and Ca 2؉ , by using carbon-fiber amperometry. Under both conditions, there was an increase in mean quantal size accompanying an increase in the half-width of amperometric spikes and a slowing of the fall time. These data suggest the existence of a dynamin-dependent process that can terminate vesicle release under basal conditions. Protein kinase C activation changed release kinetics and decreased quantal size by shortening the release period. The effects of phorbol ester treatment were not prevented by expression of the amphiphysin SH3 domain or by GTP␥S suggesting the existence of alternative dynamin-independent process underlying fast kiss-and-run exocytosis. I t has been debated over many years whether regulated exocytosis always occurs through a pathway involving full fusion of the secretory vesicle or whether it can occur through a transient kiss-and-run (1) mechanism as first proposed to occur at the neuromuscular junction (2). In the classical pathway (3), full fusion of the vesicle with the plasma membrane allows complete emptying of the vesicle contents, and vesicle recycling then occurs through a dynamin-dependent pathway (4) involving clathrin-coated vesicles (5). Exocytosis involves the formation of a transient fusion pore (6 -8).Recycling of the vesicle in kiss-and-run exocytosis could occur by rapid reclosure of such a pore. This mechanism would allow for very rapid recycling and reuse of synaptic vesicles that could be important to maintain neurotransmission (9). Rapid endocytosis on a time scale of seconds has been directly observed in neurons (10, 11) and neuroendocrine cells (12-15), and a dynamin-dependent but clathrin-independent form of endocytosis has been described as potentially underlying kiss-and-run events (15,16). It is clear, however, that a much faster type of kiss-and-run event can occur on a time scale of a few milliseconds (17). This third type of exo͞endocytic event, which we will term ''fast kiss-and-run,'' could be important, if sufficiently rapid, as it could limit the amount released per vesicle and thereby modify quantal size.Recent work has established the existence of kiss-and-run exocytosis of dense-core granules in adrenal chromaffin cells (17,18), and various experimental data are consistent with regulation of quantal size in these cells by partial release of vesicle contents because of fast kiss-...