Mechanisms of Intracellular Membrane Fusion

描述（由申请人提供）：膜融合是内分泌和外分泌生理学许多领域的核心，这些过程的不平衡会引起重要疾病，如糖尿病。在当前的资助周期内，这项资助支持的工作的结果是，细胞膜融合的核心原理现在已经很好地建立起来，包括同源SNARE蛋白的组装，最初驻留在相对的膜上，产生稳定的桥接复合物，从而触发双分子层合并。SNARE蛋白在膜间的组装是如何驱动膜融合的？对融合事件的机制理解，其中多达四个单独的蛋白质折叠在一起融合对立的双层，将需要多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，这样的任务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/附着力和光学成像）来实时跟踪SNARE的动态和功能。SNARE蛋白将在合成膜和生物膜中重建，从而允许在完全重建的膜环境中以及在不太灵活但更生理的细胞膜中进行测量。在这些系统中，我们将确定snare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，可以将不同方法中获得的见解联系起来。通过这个项目，其价值已被病毒融合蛋白证明，我们期待在未来五年内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.