Molecular Mechanisms of Exocytotic Prpces and Its Spatio-temporal Regulation

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The aim of this research project is to elucidate the molecular mechanism of exocytotic process and its spatio-temporal regulation, and we have obtained the following results.1) Roles of PKC in vesicle recruitment.The facilitation of the vesicle recruitment from the reserve pool to the releasable pool by protein kinase C (PKC) is mediated predominantly by PKCα. PKCβ also increases the secretory function, but by a distinct mechanism. Vesicle recruitment was increased by PKC through the vesicle movement at the rate of 0.03-0.06 μm/s, and the facilitation was detected as an increase in the frequency of exocytotic events in the sustained response. The facilitation of the vesicle recruitment by PKC is accompanied with a substantial increase in the vesicle numbers beneathe the plasma membrane. The receptor molecule for the activated PKC, RACK1, seemed to have roles as anchoring and signaling molecules in the regulation.2) Roles of motor proteins in vesicle recruitment.The important role of actin-myosin interaction in the recruitment of chromaffin granules to the release sites has been suggested. In the present research, it was suggested that myosin-V is specifically involved in the vesicle recruitment, while myosin-I is involved in the general movement of the vesicles. In addition, myosin-ATPase was shown to be essential for the regulation of the vesicle recruitment in chromaffin cells. The inhibition of myosin-ATPase resulted a decrease in the vesicle numbers beneathe the plasma membrane.3) Regulation of SNARE complex.Binding of calmodulin to C-teminus of VAMP-2, and binding between SyntaxinlA and CaMkinase II regulate SNARE complex and are essential for exocytosis.