Function and assembly of toxin-stabilized domains

描述(申请人提供)：脂类和蛋白质如何在细胞中合作形成功能膜域仍然是细胞生物学中一个主要的悬而未决的问题。脂筏假说已经成为理解这一过程的一个广泛研究的模型。从膜转运到细胞信号转导，木筏参与了正常的细胞功能。它们还被认为与许多疾病有关，被认为是病原体进入细胞的主要部位。目前的模型认为，RAFT结构域通常以纳米级的成分波动形式存在于细胞中，但可以稳定下来形成功能筏。然而，稳定的木筏是如何组装和发挥作用的，目前还不清楚。在这个应用中，我们建议使用无毒的膜结合霍乱毒素B亚单位(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个与其糖脂受体神经节苷脂GM1结合的位点，神经节苷脂GM1本身是一种RAFT相关分子。CTxB具有同时聚集多个GM1分子的能力，被广泛认为是一种构建稳定的RAFT结构域的RAFT交联剂。此外，霍乱毒素进入细胞内被认为重要地依赖于它对RAFT依赖的、网状蛋白非依赖的内吞途径的靶向。然而，这些CTxB稳定结构域形成的机制，这一过程是如何在细胞中调节的，以及稳定的木筏是如何优先分类成网状蛋白非依赖性内吞载体的，仍然知之甚少。在这里，我们建议通过以下目的来研究这些问题：1)检验CTxB必须聚集RAFT相关糖脂以组装稳定的RAFT结构域的假设；2)确定毒素稳定的RAFT如何与肌动蛋白细胞骨架相互作用；以及3)确定稳定的RAFT是否在通过机械变形的膜摄取CTxB以使其被clathrin非依赖性内吞摄取方面发挥积极作用。这些目标的成功完成将提供对蛋白质和脂类合作形成稳定的筏、细胞骨架如何调节筏的组装以及稳定的筏在内吞作用中的作用的机械性见解。

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.