Force-Mediated Membrane Fusion

 描述(申请人提供)：膜融合在生物学中普遍存在，在细胞内囊泡运输、病毒进入和受精等各种过程中都需要膜融合。虽然负责细胞内囊泡融合和病毒-细胞融合的分子已经被广泛研究，但关于是什么控制两个质膜之间的融合还知之甚少。在过去的十年里，出现了一种新的视角，将肌动蛋白细胞骨架产生的力量置于导致细胞-细胞融合的复杂事件序列的中心。然而，由于难以利用遗传学或生物化学来控制机械力，力介导的膜融合的直接证据缺失，留下了力在细胞-细胞融合中的作用的基本问题：需要多少力？清除蛋白质和形成接触部位是否需要力量？外力是否会导致膜缺陷和/或促进FusoGen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显微镜和力显微镜来测试外力对膜融合的作用。我们将关注线虫上皮融合失败(EFF-1)蛋白，这是最近发现的一种细胞-细胞FusoGen，其融合活性与体内肌动蛋白组装相关。我们假设，融合原和其他破坏质膜稳定的蛋白质是降低融合能量障碍所必需的，这样肌动蛋白细胞骨架就可以完成这一任务，这可能提供了一种手段，将细胞融合限制在力和融合原协同作用的时间和位置。使用重组膜界面以及活细胞，我们将使用联合荧光和力显微镜来量化融合效率和动力学，并将结果与EFF-1细胞-细胞FusoGen进行比较，以更好地了解细胞内和病毒融合原。这项研究的结果将为细胞-细胞融合提供一个新的生物物理视角，揭示融合起始过程中融合子和力之间的动态相互作用，并以只有在体外重建才可能的机制洞察力来补充模型生物中的细胞-细胞融合研究。

 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.