Abstract
Glucose evokes the biphasic insulin release, though, the mechanism for docking and fusion of insulin granules during glucose-stimulation is well not characterized. Total internal reflection fluorescent (TIRF) microscopy imaging analysis is a powerful tool to determine the molecular mechanism for insulin exocytosis in the primary pancreatic β cells. We imaged and analyzed the motion of single insulin granules near the plasma membrane (<100 nm) using TIRF with green fluorescent protein (GFP)-tagged insulin granules. In normal primary β cells, the granules that were fusing during the first phase originate from previously docked granules, and those during the second phase originate from ‘newcomers’. In diabetic GK rat β cells, the number of fusion events from previously docked granules was markedly reduced, and, in contrast, the fusion from newcomers was still preserved. We have then tried to determine the site of insulin exocytosis using dual color-TIRF imaging. Because the cytomatrix at the active zone (CAZ) is known to be implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitter, we examined the expression and function of ELKS, a protein structurally related to the CAZ protein CAST in pancreatic β cells. We found that most of ELKS clusters were on the docking site of insulin granules that were colocalized with syntaxin clusters. Dual color-TIRF images of single granule motion showed that the fusion events of insulin granules were mostly occurred on the ELKS clusters. In this symposium, we will present the newest data concerning about the insulin exocytotic site. [Jpn J Physiol 55 Suppl:S33 (2005)]
