Two-Photon Excitation Imaging of Insulin Exocytosis

Abstract

To elucidate the final steps of secretion, we have established twophoton extracellular polar tracer (TEP) imaging, with which we can quantify all exocytic events in the plane of focus within intact pancreatic islets. We can also estimate the precise diameters of vesicles independently of the spatial resolution of the optical microscope and measure the fusion pore dynamics at nanometer resolution using TEP-imaging based quantification (TEPIQ). During insulin exocytosis, it took 2 s for the fusion pore to dilate from 1.4 nm in diameter to 6 nm in diameter. Such unusual stability of the pore may be due to the crystallization of the intragranular contents. Opening of the pore was preceded by unrestricted lateral diffusion of lipids along the inner wall of the pore, supporting the idea that this structure was mainly composed of membrane lipids. TEP imaging has been also applied to other representative secretory glands, and has revealed hitherto unexpected diversity in spatial organization of exocytosis and endocytosis, which are relevant for physiology and pathology of secretory tissues. In the pancreatic islet, compound exocytosis was characteristically inhibited (<5%), partly due to the rarity of SNAP25 redistribution into the exocytosed vesicle membrane. Such mechanisms necessitate transport of insulin granules to the cell surface for fusion, possibly rendering exocytosis more sensitive to the metabolic state. TEP imaging and TEPIQ analysis will be powerful tools for elucidating molecular and cellular mechanisms of exocytosis and related disease, and to develop new therapeutic agencies as well as diagnostic tools.