Dynamic imaging of secretion from pancreatic beta-cells by confocal fluorescence microscopy.

摘要

Insulin is stored in secretory vesicles of pancreatic beta-cells and secreted by exocytosis in response to external stimuli, most notably glucose. A better understanding of stimulus-secretion coupling is of great interest due to the possible role of defective insulin secretion in type 2 diabetes. Studies of stimulus-secretion coupling require analytical techniques that allow the monitoring of secretion with high temporal and spatial resolution.; In this work, a fluorescence imaging approach has been developed for spatially and temporally resolved measurements of secretion from beta-cells using extracellular fluorogenic reactions and confocal laser-scanning fluorescence microscopy. In this method, Zn2+ efflux from beta-cells is monitored as a marker of insulin secretion because of the co-release of Zn 2+ and insulin from beta-cells by exocytosis. To detect Zn 2+ release, cells are incubated in buffer containing the fluorogenic Zn2+ indicator Zinquin, while fluorescence signals are monitored by confocal fluorescence microscopy. When secretion is evoked by stimulation, the released Zn2+ reacts with Zinquin in the extracellular space to form a fluorescent complex, resulting in an increase in fluorescence at the site of release. Spatial organization of beta-cell secretion has been investigated using this spatially resolved technique. It was revealed that secretion from single beta-cells was localized and the active areas of release spatially co-localized with L-type Ca2+ channels on the plasma membrane of cells.; The sensitivity and temporal resolution of this approach was further improved with the use of a novel Zn2+ indicator FluoZin-3. Temporally resolved exocytotic release from individual secretory vesicles has been observed for the first time by a fluorescence imaging technique. Unlike other single-cell techniques that limit their applications to single cells, this approach allows the monitoring of secretion from clusters of cells and single islets with sub-second temporal resolution. For the first time, oscillatory secretion from cell clusters and single islets was observed with high temporal resolution. Complex patterns of oscillatory secretion, spatial heterogeneity, and spatial synchrony of secretion from cell clusters and islets have been revealed. The high sensitivity, high temporal and spatial resolution of this technique would make it a valuable tool for physiological studies of insulin secretion and exocytosis.