Function and assembly of toxin-stabilized domains

毒素稳定结构域的功能和组装

• 批准号: 9403684
• 负责人: Anne K Kenworthy
• 金额: $ 37.01万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9403684
• 关键词: Address; Bacterial Toxins; Binding; Biological; Cell Surface Receptors; Cell membrane; Cells; Cellular Membrane; Cholera; Cholera Toxin; Cholera Toxin Protomer B; Clathrin; Complex; Couples; Cytoplasm; Disease; Dynein ATPase; Endocytosis; Endocytosis Pathway; Endoplasmic Reticulum; Ensure; Event; Funding; Genetic Screening; Glycolipids; Goals; Knowledge; Link; Lipid Binding; Lipids; Mammalian Cell; Mechanics; Mediating; Membrane; Membrane Microdomains; Microtubules; Modeling; Motor; Nature; Pathway interactions; Process; Property; Proteins; Proteomics; Role; Shiga Toxin; Site; Structure; Testing; Toxin; Variant; Work; base; coated pit; dynactin; imaging approach; insight; live cell imaging; membrane model; nanoscale; novel; pathogen; protein complex; trafficking; uptake

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.

脂质和蛋白质在细胞膜中形成多种复合物和结构域。底层的 控制这些结构的组装和功能的原则仍然是个谜。为了解决这个问题 知识差距，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。当前模型提出筏域 通常在细胞中以纳米级成分波动的形式存在，但可以通过以下方式稳定 蛋白质形成功能筏。霍乱毒素 (CTxB) 的无毒膜结合亚基是一种 可以将筏相关糖脂聚集成组装稳定筏结构域的蛋白质的示例。 在上一个资助期间，我们研究了毒素稳定结构域形成的机制 并询问它们是否具有使细胞膜机械变形以促进 CTxB 摄取的功能 网格蛋白独立的内吞作用。我们使用 CTxB 的一种新变体进行了测试，该变体无法聚类 糖脂。我们的结果意外地发现微管、动力蛋白和动力蛋白 在网格蛋白独立摄取 CTxB 的位点产生拉力。我们未来的目标 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格蛋白- 独立的内吞作用以及 CTxB 如何选择性地分类到这些专门的网格蛋白中 独立的途径。结合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，我们将确定稳定筏是否分类 CTxB 进入网格蛋白独立的内吞途径。其次，我们将检验多种网格蛋白的假设 独立途径利用微管和马达来管状和分裂质膜。 最后，我们将确定负责将微管和动力蛋白/动力蛋白连接到 新生的网格蛋白独立携带者。这项工作的顺利完成将为 深入了解稳定筏的功能并揭示毒素操纵的新机制 并劫持细胞机器以达到自己的目的。它还将加深我们对如何 微管和微管相关运动在膜运输事件中发挥作用并揭示 微管、动力蛋白和动力蛋白在质膜上的新作用。最后，它将定义新的 有助于组装网格蛋白独立内吞载体的机制和机器。