Function and assembly of toxin-stabilized domains

描述（由申请人提供）：脂质和蛋白质如何在细胞中合作形成功能性膜结构域仍然是细胞生物学中未回答的主要问题。脂筏假说已成为一个广泛研究的模型，以了解这一过程。筏已经涉及从膜运输到细胞信号传导的正常细胞功能。它们也与许多疾病有关，并被认为是病原体进入细胞的主要场所。目前的模型提出，筏域通常存在的纳米级组成的波动在稳定状态下的细胞，但可以稳定，形成功能筏。然而，稳定筏如何组装和发挥作用仍不清楚。在本申请中，我们建议使用无毒的霍乱毒素膜结合B亚基（CTxB）作为模型来解决这些基本问题。CTxB是一种同型五聚体，含有5个糖脂受体神经节苷脂GM 1的结合位点，神经节苷脂GM 1本身是一种筏相关分子。由于CTxB能够同时聚集多个GM 1分子，因此被广泛认为是构建稳定筏结构域的筏交联剂。此外，霍乱毒素进入细胞被认为是重要的依赖于其靶向筏依赖性，网格蛋白非依赖性内吞途径。然而，这些CTxB稳定的结构域形成的机制，该过程如何在细胞中调节，以及稳定的筏如何优先分选成网格蛋白非依赖性内吞载体仍然知之甚少。在这里，我们建议调查这些问题，通过以下目的：1）测试的假设，CTxB必须集群筏相关的糖脂组装稳定筏域; 2）以确定如何毒素稳定筏相互作用与肌动蛋白细胞骨架;和3）以确定是否稳定筏发挥积极作用，靶向CTxB通过机械变形膜的网格蛋白非依赖性内吞作用的摄取。这些目标的成功完成将提供机制的见解，蛋白质和脂质合作，形成稳定的筏，筏组装是如何调节的细胞骨架，以及稳定的筏在胞吞作用中的作用。

DESCRIPTION (provided by applicant): How lipids and proteins cooperate to form functional membrane domains in cells remains a major unanswered question in cell biology. The lipid raft hypothesis has become a widely studied model for understanding this process. Rafts have been implicated in normal cellular functions ranging from membrane trafficking to cell signaling. They have also have been linked to a number of diseases and are thought to represent a major site for pathogen entry into cells. Current models propose that raft domains normally exist as nanoscale compositional fluctuations at steady state in cells, but can be stabilized to form functional rafts. However, how stabilized rafts assemble and function remains unclear. In this application, we propose to address these fundamental questions using the non-toxic membrane binding B-subunit of cholera toxin (CTxB) as a model. CTxB is a homopentamer that contains 5 binding sites for its glycolipid receptor, ganglioside GM1, itself a raft-associated molecule. Give its ability to cluster multiple GM1 molecules simultaneously, CTxB is widely regarded to serve as a raft crosslinker that builds stabilized raft domains. Moreover, the entry of cholera toxin int cells is thought to depend importantly on its targeting to raft-dependent, clathrin-independent endocytic pathways. However, the mechanisms by which these CTxB- stabilized domains form, how this process is regulated in cells, and how stabilized rafts are preferentially sorted into clathrin-independent endocytic carriers remain poorly understood. Here, we propose to investigate these questions though the following aims: 1) to test the hypothesis that CTxB must cluster raft-associated glycolipids to assemble stabilized raft domains; 2) to determine how toxin-stabilized rafts interact with the actin cytoskeleton; and 3) to determine if stabilized raft play an active role in targeting CTxB for uptake by clathrin independent endocytosis by mechanically deforming membranes. Successful completion of these aims will provide mechanistic insights into proteins and lipids cooperate to form stabilized rafts, how raft assembly is regulated by the cytoskeleton, and the role of stabilized rafts in endocytosis.