Molecular Mechanisms of Exocytotic Prpces and Its Spatio-temporal Regulation

本课题旨在阐明胞吐过程的分子机制及其时空调控，主要研究结果如下：1）PKC在囊泡募集中的作用蛋白激酶C（PKC）促进囊泡从储备池向释放池的募集主要由PKCα介导。PKCβ也可增强分泌功能，但作用机制不同。PKC通过囊泡以0.03-0.06 μm/s的速率运动，增加囊泡募集，这种易化作用表现为持续反应中胞吐事件频率的增加。PKC促进囊泡募集的同时，质膜下囊泡数量也显著增加。2）运动蛋白在囊泡募集中的作用：肌动蛋白-肌球蛋白相互作用在嗜铬颗粒向囊泡释放位点的募集中起重要作用。在目前的研究中，有人建议，肌球蛋白-V是专门参与的小泡募集，而肌球蛋白-I参与的一般运动的小泡。此外，肌球蛋白-ATP酶被证明是必要的，在嗜铬细胞的小泡募集的调节。3）SNARE复合物的调节：钙调素与VAMP-2的C端结合，Syntaxin 1A与CaMkinase Ⅱ结合，调节SNARE复合物，是胞吐作用所必需的。

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.