Regulation of two insulin granule populations within the reserve pool by distinct calcium sources

Insulin granule trafficking is a key step of glucose-stimulated insulin secretion from pancreatic cells. Using quantitative live cell imaging, we examined insulin granule movements within the reserve pool upon secretory stimulation in TC3 cells. For this study, we developed a custom image analysis program that permitted automatic tracking of the individual motions of over 20,000 granules. This analysis of a large sample size enabled us to study micro-populations of granules that were not quantifiable in previous studies. While over 90% of the granules depend on Ca²⁺ efflux from the endoplasmic reticulum for their mobilization, a small and fast-moving population of granules responds to extracellular Ca²⁺ influx after depolarization of the plasma membrane. We show that this differential regulation of the two granule populations is consistent with localized Ca²⁺ signals, and that the cytoskeletal network is involved in both types of granule movement. The fast-moving granules are correlated temporally and spatially to the replacement of the secreted insulin granules, which supports the hypothesis that these granules are responsible for replenishing the readily releasable pool. Our study provides a model by which glucose and other secretory stimuli can regulate the readily releasable pool through the same mechanisms that regulate insulin secretion.