Elucidating the physical mechanisms of membrane fission

项目摘要。通过以下过程将膜分离成离散的隔室： 膜分裂对于从细胞分裂到病毒进入的各种细胞过程是必不可少的。而 众所周知，专门的裂变机器发动蛋白通过膜管的收缩来诱导裂变， 最近的证据表明，其他蛋白质通过以前未知的机制驱动裂变。特别是 胰蛋白酶1 N-末端同源（ENTH）结构域是膜弯曲的有效驱动力（福特等人，性质 2002），并且最近显示在膜裂变中起作用。特别是，最近的一份报告提出， 插入一个楔形的两亲性螺旋的ENTH曲线和不稳定的膜，证明了 在具有降低的螺旋疏水性的ENTH突变体中降低膜分裂能力（Boucrot等人， Cell 2012）。然而，我们的研究小组最近表明，致密的、膜结合的ENTH之间的碰撞， 蛋白质产生空间压力，在没有螺旋插入的情况下驱动膜弯曲 （Stachowiak等人，Nature Cell Biology 2012）。这些结果促使我们问：空间位压是否也 负责ENTH的膜分裂？在我的初步研究中，我发现ENTH突变体 当结合时，降低的螺旋疏水性能够驱动分裂至与野生型ENTH相似的程度 以相当的密度附着在膜上。有趣的是，我还发现，全长epsin，其中包含一个庞大的， 本质上无序的C-末端结构域，比单独的ENTH结构域更有力地驱动裂变。这些 结果表明，虽然螺旋插入对于将蛋白质紧密结合到膜表面是重要的， 不是裂变所必需的。然而，一旦以足够的密度结合到膜表面， 任意结构的分子可以产生空间压力，增加膜的曲率，直到分裂 发生。总之，我的发现揭示了膜分裂的一种新机制。的目的 拟议的研究是定量比较这种新机制与其他关键机制的膜 裂变第一个具体的目标将描绘楔状螺旋插入和蛋白质拥挤的具体作用 来驱动膜分裂。第二个具体目标将研究发动蛋白如何与螺旋协同工作 插入和蛋白质拥挤来驱动强大的裂变。第三个具体目标将利用定量成像， 活细胞来研究螺旋插入和蛋白质拥挤如何在生理学上调节裂变动力学。 上下文这项工作将创造创新的生物物理工具，同时研究膜裂变和 在体外和活细胞中的蛋白质-脂质相互作用。这项研究的总体成果将是一个更深层次的 了解膜分裂的物理机制，包括膜分裂的新机制 通过蛋白质聚集进行裂变。这里描述的大胆假设断言，任何膜结合蛋白质都可以 有助于裂变，这一想法将影响对不同膜区室化的理解 过程，包括内吞作用、细胞分裂和病毒进入。

Project Summary. The separation of membranes into discrete compartments through the process of membrane fission is essential for diverse cellular processes ranging from cell division to viral entry. While the specialized fission machine dynamin is well-known to induce fission through constriction of membrane tubes, recent evidence shows that other proteins drive fission by previously unknown mechanisms. In particular, the epsin 1 N-terminal homology (ENTH) domain is a potent driver of membrane curvature (Ford et al., Nature 2002), and has recently been shown to play a role in membrane fission. Specifically, a recent report proposed that insertion of a wedge-like amphipathic helix by ENTH curves and destabilizes membranes, as evidenced by decreasing membrane fission ability among ENTH mutants with decreasing helix hydrophobicity (Boucrot et al., Cell 2012). However, our group recently showed that collisions among dense, membrane-bound ENTH proteins generate steric pressure, which drives membrane bending in the absence of helix insertions (Stachowiak et al., Nature Cell Biology 2012). These results prompted us to ask: is steric pressure also responsible for membrane fission by ENTH? In my preliminary studies, I found that ENTH mutants with reduced helix hydrophobicity are capable of driving fission to a similar degree as wild-type ENTH when bound to the membrane at comparable density. Interestingly, I also found that full-length epsin, which contains a bulky, intrinsically-disordered C-terminal domain, drives fission more potently than the ENTH domain alone. These results imply that, while helix insertions are important for binding proteins tightly to membrane surfaces, helices are not required for fission. However, once bound to the membrane surface at sufficient density, bulky molecules of arbitrary structure can create steric pressure that increases membrane curvature until fission occurs. Taken together, my findings reveal a novel mechanism for membrane fission. The objective of the proposed research is to quantitatively compare this new mechanism with other key mechanisms of membrane fission. The first specific aim will delineate the specific roles of wedge-like helix insertion and protein crowding in driving membrane fission. The second specific aim will examine how dynamin works cooperatively with helix insertion and protein crowding to drive robust fission. The third specific aim will utilize quantitative imaging of live cells to examine how helix insertion and protein crowding modulate fission dynamics in a physiological context. This work will create innovative biophysical tools for the simultaneous study of membrane fission and protein-lipid interactions both in vitro and in live cells. The overall outcome of this research will be a deeper understanding of the physical mechanisms of membrane fission, including the novel mechanism of membrane fission by protein crowding. The bold hypothesis described here asserts that any membrane-bound protein can contribute to fission, an idea that will influence understanding of diverse membrane compartmentalizing processes, including endocytosis, cell division, and viral entry.