SNARE complex formation in living cells

描述(申请人提供)：细胞能够通过高度调控的胞吐作用相互传递信息。胞吐作用允许细胞将激素、神经递质和其他化学物质释放到细胞外空间，方法是将小泡输送到细胞表面，与质膜融合并释放其内容物。SNARE蛋白对于膜融合的调节是必不可少的，它是通过在囊泡上的蛋白质和质膜上的蛋白质之间形成一个复合体来实现的，从而使两个膜紧密相连。这项工作旨在通过以下方式确定活体INS-1细胞中SNARE复合体形成的分子机制：(1)表征SNARE前复合体对突触素和突触素I浓度的依赖性，(2)确定哪些前复合体促进胞吐。提出的实验提供了一种直接测量SNARE复合体和胞吐作用的方法，该方法使用荧光标记的SNARs，将使用全内反射显微镜进行照明。最终，这些结果可能揭示，分泌的控制部分取决于细胞中存在的陷阱的相对数量。

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.