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一个悬而未决的主要问题。脂质筏假说已经成为理解这一过程的一个被广泛研究的模型。筏涉及正常的细胞功能，从膜运输到细胞信号传导。它们还与许多疾病有关，被认为是病原体进入细胞的主要部位。目前的模型表明，筏结构域通常以纳米级成分波动的形式存在于细胞稳态，但可以稳定形成功能性筏。然而，稳定筏是如何组装和发挥作用的仍不清楚。在这个应用中，我们建议使用霍乱毒素的无毒膜结合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，其糖脂受体神经节苷脂GM1含有5个结合位点，其本身是一种筏相关分子。由于CTxB具有同时聚集多个GM1分子的能力，因此被广泛认为是一种raft交联剂，可以构建稳定的raft结构域。此外，霍乱毒素进入细胞被认为主要取决于其靶向筏依赖的、不依赖网格蛋白的内吞途径。然而，这些CTxB稳定结构域形成的机制，这一过程如何在细胞中被调节，以及稳定筏如何优先分类为不依赖网格蛋白的内吞载体，仍然知之甚少。在这里，我们提出通过以下目的来研究这些问题：1)验证CTxB必须聚集筏相关的糖脂来组装稳定的筏结构域的假设；2)确定毒素稳定筏如何与肌动蛋白细胞骨架相互作用；3)确定稳定筏是否通过机械变形膜靶向CTxB，使其被不依赖网格蛋白的内吞作用所吸收。这些目标的成功完成将提供蛋白质和脂质合作形成稳定筏的机制见解，筏的组装如何受到细胞骨架的调节，以及稳定筏在内吞作用中的作用。

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.