Dephosphorylation of Dynamin1 is a Ca2+ Sensor that Triggers Clathrin- Independent Vesicle Recycling Processes in Pancreatic Beta Cells

Abstract

By tagging fluorphores to different endocytic proteins such as clathrin and dynamin, clathrin-mediated receptor internalization process has been visualized in a number of non-excitable cells in vivo using total internal reflection fluorescence microscopy. In contrast, systematic examination of the tempo-spatial relationship between different exocytotic and endocytic proteins has not been done in excitable cells. The clathrin-dependent receptor endocytosis in non-excitable cells has a long life time. In contrast, the clathrin-dependent vesicle recycling process is faster and subjected to further acceleration by increase in cytoplasmic Ca2+ concentration ([Ca2+]i), as proved by our previous data in pancreatic beta cells and also by recently papers on Calyx neurons. Moreover, elevated [Ca2+]i triggers and accelerates a type of clathrin-independent but dynamin-dependent endocytosis in beta cells. As dynamin1 is a neuronal specific subtype of dynamin that can be dephorsphorylated at S774 and S778 positions upon stimulation-induced Ca2+ influx, we test whether it acts as a signal molecule to sensor increase in [Ca2+]i and act to trigger vesicle recycling processes in our insulin-secreting cells. By mutating the S774 and S778 into A and E, we can mimic dephorsphorylated and phorsphorylated status of dynamin1. We show that dephorsphorylated dynamin1 has a statistically shortened life time as compared to the life times of dynamin1 in cells expressed wide-type dynamin1 or dynamin1 S774ES778E mutant. Moreover, dynamin1 S774AS778A is more likely to be recruited to the vesicle fusion sites, which represent clathrin-independent but dynamin-dependent endoytic events in beta cells. In the end, the fast capacitance decay evoked by homogenous elevation in [Ca2+]i induced by flash-photolysis is selective inhibited by expressing either dynamin1 mutants in INS-1 cells, further reinforce the important role of dynamin1 phorsphorylation-dephorphosphorylation cycle in clathrin-independent vesicle retrieval process.