Mechanisms of Intracellular Membrane Fusion

描述(申请人提供)：膜融合是许多内分泌和外分泌生理学领域的中心，这些过程中的不平衡会导致重要的疾病，如糖尿病。由于这笔赠款在当前资金周期中支持的工作，细胞膜融合的核心原则现在已经确立，包括最初驻留在相对膜中的同源SNARE蛋白的组装，以产生触发双层合并的稳定的桥接复合体。膜之间SNARE蛋白的组装如何驱动膜融合？对融合事件的机械理解，即多达四个独立的蛋白质折叠在一起以融合相对的双分子层，将需要各种生物物理方法的综合力量。由于SNARE融合生物化学和生物物理膜技术的不断进步，这项工作直到最近才成为可能。我们将使用不同的互补技术(细胞和分子生物学、电生理学、表面力/粘附力和光学成像)，实时跟踪圈套的动力学和功能。SNARE蛋白将在人工膜和生物膜中重组，从而允许在完全重组的膜环境中以及在较不灵活但更具生理性的细胞膜中进行测量。在这些系统中，我们将确定陷阱组装的能量学，膜组成和膜张力对融合的影响，以及融合孔本身的动力学。通过比较关键突变和其他扰动的影响，可以将从不同方法中获得的见解联系起来。通过遵循这个已经在病毒融合蛋白中证明其价值的计划，我们预计在未来五年内将出现关于细胞膜融合的新的重要信息。

DESCRIPTION (provided by applicant): Membrane fusion is central to many areas of endocrine and exocrine physiology, and imbalances in these processes give rise to important diseases, such as diabetes. As a result of the work supported by this grant during the current cycle of funding, the core principle of cellular membrane fusion is now well established, consisting of the assembly of cognate SNARE proteins initially residing in apposing membranes to yield a stable, bridging complex that triggers the bilayers to merge. How does the assembly of SNARE proteins between membranes drive membrane fusion? A mechanistic understanding of the fusion event, in which as many as four separate proteins fold together to fuse apposing bilayers, will require the combined power of a variety of biophysical approaches. Such an undertaking has become possible only recently, thanks to continuing advances in both SNARE fusion biochemistry and biophysical membrane technologies. We will use different complementary technologies (cellular and molecular biology, electrophysiology, surface force/adhesion, and optical imaging), to follow SNARE dynamics and function in real time. SNARE proteins will be reconstituted in both synthetic and biological membranes, thus allowing measurements in fully reconstituted membrane environments as well as in less flexible but more physiological cellular membranes. Within these systems, we will determine the energetics of SNARE-assembly, the consequences of membrane composition and membrane tension on fusion, and the dynamics of the fusion pore itself. Insights gained in the different approaches can be linked by comparing the effects of critical mutations and other perturbations. By following this program, whose value has been proven with viral fusion proteins, we expect new and important information concerning cellular membrane fusion to emerge during the next five years.