Function and assembly of toxin-stabilized domains

脂类和蛋白质在细胞膜上形成各种复合体和结构域。潜在的 管理这些结构的组装和功能的原则仍然是个谜。要解决这个问题 知识缺口，我们专注于批判性地测试流行模型的关键预测 膜域组织：脂筏假说。目前的模型认为，RAFT域 通常以纳米级的成分波动形式存在于细胞中的稳定状态，但可以通过 形成功能木筏的蛋白质。霍乱毒素的无毒膜结合亚基(CTxB)是一种 可以将RAFT相关糖脂聚集到组装的稳定RAFT结构域的蛋白质的例子。 在上一次资助期间，我们研究了毒素稳定结构域形成的机制。 并询问它们是否通过机械变形细胞膜来促进CTxB的吸收 胞苷非依赖性内吞作用。我们使用CTxB的一个新变种进行了测试，该变种不能集群 糖脂。我们的结果导致了一个意想不到的发现，微管、动力蛋白和动力蛋白 在网状蛋白独立摄取CTxB的位置产生拉力。我们即将到来的目标 资助期是为了更好地了解微管和动力蛋白/动力蛋白如何参与网状蛋白- 独立的内吞作用，以及CTxB是如何选择性地分类到这些特化的网状蛋白中的- 独立的小路。结合使用细胞生物学和活细胞成像方法，我们将 通过三个具体目标来解决这些问题。首先，我们将确定稳定的救生筏是否将CTxB 进入不依赖于笼状蛋白的内吞途径。第二，我们将检验多个笼状蛋白-- 独立的途径利用微管和马达来管状和断裂质膜。 最后，我们将确定负责连接微管和动力蛋白/动力蛋白的细胞机制。 新生的不依赖于笼状蛋白的载体。这项工作的成功完成将为 洞察稳定木筏的功能并揭示毒素操纵的新机制 为了自己的目的劫持蜂窝机器。它还将促进我们对如何 微管和微管相关马达在膜转运事件中的作用并揭示 微管、动力蛋白和动力蛋白在质膜上的新作用(S)。最后，它将定义新的 有助于组装不依赖于胞内蛋白的胞内载体的机制和机械。

Lipids and proteins form a variety of complexes and domains in cellular membranes. The underlying principles that govern the assembly and function of these structures remain enigmatic. To address this gap in knowledge, we have focused on critically testing key predictions of a prevalent model of membrane domain organization: the lipid raft hypothesis. Current models propose that raft domains normally exist as nanoscale compositional fluctuations at steady state in cells, but can be stabilized by proteins to form functional rafts. The non-toxic membrane binding subunit of cholera toxin (CTxB) is an example of a protein that can cluster raft-associated glycolipids to assembled stabilized raft domains. In the previous funding period, we examined the mechanisms by which toxin-stabilized domains form and asked whether they function to mechanically deform cell membranes to facilitate CTxB uptake by clathrin independent endocytosis. We tested this using a novel variant of CTxB that is unable to cluster glycolipids. Our results led to the unexpected discover that microtubules, dynein, and dynactin generate pulling forces at sites of clathrin independent uptake of CTxB. Our goal for the upcoming funding period is to better understand how microtubules and dynein/dynactin participate in clathrin- independent endocytosis and how CTxB is selectively sorted into these specialized clathrin- independent pathways. Using a combination of cell biological and live cell imaging approaches, we will tackle these questions through three specific aims. First, we will determine if stabilized rafts sort CTxB into clathrin independent endocytic pathways. Second, we will test the hypothesis that multiple clathrin- independent pathways exploit microtubules and motors to tubulate and scission the plasma membrane. Finally, we will identify cellular machinery responsible for linking microtubules and dynein/dynactin to nascent clathrin-independent carriers. Successful completion of this work will provide fundamental insights into the functions of stabilized rafts and uncover new mechanism by which toxins manipulate and hijack cellular machinery for their own purposes. It will also advance our understanding of how microtubules and microtubule-associated motors function in membrane trafficking events and reveal novel role(s) for microtubules, dynein, and dynactin at the plasma membrane. Finally, it will define new mechanisms and machinery that contribute to the assembly of clathrin independent endocytic carriers.