Simultaneous Imaging of Vesicle Trafficking and Calcium-Mediated Exocytosis in Pancreatic Beta-Cells

It has long been known that only a small fraction (<10%) of the insulin in pancreatic beta-cells can be released. Clinical treatments of type 2 diabetes focus on increasing insulin release, so an understanding of insulin vesicle trafficking and release may lead to novel therapeutic strategies. Towards this understanding, we have utilized a novel quantitative imaging assay, based on a double fusion to the integral vesicle protein phogrin. In this construct, a pH-dependent ecliptic pHluorinFP is inside the lumen of the granule and a mOrangeFP is outside the granule in the cytoplasm. The pHluorin fluorescence is quenched by the low pH inside the granule until exocytosis when the lumen of the granule mixes with the extracellular media. This increases the local pH and the green fluorescence can then be measured as a readout of exocytosis. Experiments were done on the multicolor TIRF system with a photoactivation module. We photoconverted the mOrange-phogrin to a deep red protein in the perinuclear region, and then watched where that population of granules went as we stimulated the cells with glucose. Having only a small subset of vesicles labeled greatly facilitates tracking. We used this approach with TIRF microscopy to measure secreted granules and determine if they came from previously docked granules or from regions beyond the TIRF imaging field. In beta-cells, we find that secreted vesicles do not come from a docked pool, with secretion happening within about 1 second of the vesicle's arrival at the membrane. This time is decreased even further with the addition of glucose. These data suggest that the releasable insulin pool in beta-cells may not be docked at the membrane as are synaptic vesicles, but rather are maintained deeper with in the cell.