Force-Mediated Membrane Fusion

 描述（由申请人提供）：膜融合在生物学中普遍存在，并且是细胞内囊泡运输、病毒进入和受精等多种过程所必需的。虽然负责细胞内囊泡融合和病毒-细胞融合的分子已被广泛研究，但对控制两个质膜之间融合的分子知之甚少。在过去的十年中，出现了一个新的观点，将肌动蛋白细胞骨架产生的力置于导致细胞-细胞融合的复杂事件序列的中心。然而，由于难以使用遗传学或生物化学控制机械力，力介导的膜融合的直接证据缺失，留下了关于力在细胞-细胞融合中的作用的未回答的基本问题：需要多大的力？清除蛋白质和形成接触部位是否需要外力？力是否引入膜缺陷和/或促进融合剂活性？在这个项目中，我们建议使用体外膜重建结合高分辨率光学和力显微镜来测试外力对膜融合的作用。我们将重点研究C. elegans，最近鉴定的细胞-细胞融合子，其融合活性与体内肌动蛋白组装相关。我们假设，融合蛋白和其他蛋白质，使质膜不稳定，需要降低融合的能量屏障，使肌动蛋白细胞骨架可以完成任务，潜在地提供了一种手段，以限制细胞-细胞融合的时间和地点的力量和融合蛋白一起行动。使用重组膜界面以及活细胞，我们将使用组合的荧光和力显微镜定量融合效率和动力学，我们将比较结果与Eff-1细胞-细胞融合剂更好地了解细胞内和病毒融合剂。这项研究的结果将提供一个新的生物物理学的角度对细胞-细胞融合，揭示融合启动过程中的融合因子和力之间的动态相互作用，并补充细胞-细胞融合研究的模式生物与机制的洞察力，这是唯一可能从体外重建。

 DESCRIPTION (provided by applicant): Membrane fusion is ubiquitous in biology and required for processes as diverse as intracellular vesicle trafficking, viral entry, and fertilizaton. While the molecules responsible for intracellular vesicle fusion and virus-cell fusion have been studied extensively, much less is known about what controls fusion between two plasma membranes. Over the past decade, a new perspective has emerged that places forces generated by the actin cytoskeleton at the center of the complex sequence of events that results in cell-cell fusion. However, direct evidence of force-mediated membrane fusion is missing due to the difficulty of controlling mechanical force using genetics or biochemistry, leaving unanswered basic questions about the role of force in cell-cell fusion: How much force is needed? Is force necessary to clear proteins and form contact sites? Does force introduce membrane defects and/or promote fusogen activity? In this project, we propose to use in vitro membrane reconstitution combined with high-resolution optical and force microscopy to test the role of external forces on membrane fusion. We will focus on Epithelial fusion failure (Eff-1) protein from C. elegans, a recently- identified cell-cell fusogen whose fusogenic activity is associated with actin assembly in vivo. We hypothesize that fusogens and other proteins that destabilize the plasma membrane are needed to lower the energy barrier to fusion such that the actin cytoskeleton can complete the task, potentially providing a means to restrict cell-cell fusio to times and locations where force and fusogens act in concert. Using reconstituted membrane interfaces as well as live cells, we will quantify fusion efficiency and dynamics using combined fluorescence and force microscopy, and we will compare results with Eff-1 cell-cell fusogen to better-known intracellular and viral fusogens. The results of this study will provide a new biophysical perspective on cell-cell fusion, reveal the dynamic interplay between fusogens and force during fusion initiation, and complement cell-cell fusion studies in model organisms with mechanistic insight that is only possible from in vitro reconstitution.