SNARE complex formation in living cells

描述（由申请人提供）：细胞能够通过高度调控的胞吐过程相互传递信息。胞吐作用使细胞释放激素、神经递质和其他化学物质到细胞外空间，通过运输囊泡到细胞表面，囊泡与质膜融合并释放其内容物。SNARE蛋白对膜融合的调节至关重要，通过在囊泡上的蛋白质和质膜上的蛋白质之间形成复合物，使两个膜靠近。本研究旨在通过以下方法确定活的INS-1细胞中SNARE复合物形成的分子机制：(1)表征SNARE预复合物对syntaxin和synaptotagmin I浓度的依赖性；(2)确定哪些预复合物促进胞外分泌。提出的实验提供了一种直接测量SNARE复合物和胞吸的方法，通过使用荧光标记的SNAREs，将使用全内反射显微镜照射。最终，这些结果可能揭示了分泌的控制部分取决于细胞中存在的SNAREs的相对数量。

DESCRIPTION (provided by applicant): Cells are able to transmit information to each other via the highly regulated process of exocytosis. Exocytosis allows cells to release hormones, neurotransmitters, and other chemicals into the extracellular space by transporting a vesicle to the cell surface that fuses with the plasma membrane and releases its contents. SNARE proteins are essential for the regulation of membrane fusion and do so by forming a complex between proteins on the vesicle and proteins on the plasma membrane that brings the two membranes in close proximity. The proposed work is designed to determine the molecular mechanism of SNARE complex formation in living INS-1 cells by: (1) characterizing the dependence of SNARE pre-complexes on syntaxin and synaptotagmin I concentration, (2) identifying which pre-complexes facilitate exocytosis. The proposed experiments offer a method of directly measuring SNARE complexes and exocytosis by using fluorescently labeled SNAREs that will be illuminated using total internal reflection microscopy. Ultimately, these results could reveal that control of secretion partially lies in the relative amounts of SNAREs present in a cell.