Function and assembly of toxin-stabilized domains

毒素稳定结构域的功能和组装

• 批准号: 9925238
• 负责人: Anne K Kenworthy
• 金额: $ 36.99万
• 依托单位: UNIVERSITY OF VIRGINIA
• 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 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9925238
• 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各种复合物和结构域。底层 支配这些结构的组装和功能的原理仍然是谜。为了解决这个 知识的差距，我们专注于批判性地测试一个流行的模型的关键预测， 膜结构域组织：脂筏假说。目前的模型提出，筏域 通常以纳米级组成波动的形式在电池中稳定存在，但可以通过以下方式稳定 蛋白质形成功能性筏。霍乱毒素的无毒膜结合亚基（CTxB）是一种 可以将筏相关糖脂聚集成组装的稳定筏结构域的蛋白质的实例。 在上一个资助期，我们研究了毒素稳定域形成的机制， 并询问它们是否具有机械变形细胞膜的功能，以促进CTxB的摄取。 不依赖网格蛋白的内吞作用。我们使用一种新的CTxB变体进行了测试， 糖脂我们的研究结果导致了一个意外的发现，微管，动力蛋白和动力蛋白， 在CTxB的网格蛋白非依赖性摄取位点产生拉力。我们即将到来的目标 基金期间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- 以及CTxB如何选择性地分选成这些专门的网格蛋白- 独立的道路。使用细胞生物学和活细胞成像方法的组合，我们将 通过三个具体目标解决这些问题。首先，我们将确定是否稳定筏排序CTxB 不依赖网格蛋白的内吞途径。第二，我们将测试多网格蛋白的假设- 独立的途径利用微管和马达来使质膜成管状和断裂。 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， 不依赖网格蛋白的新生携带者。这项工作的顺利完成将提供基本的 深入了解稳定筏的功能，并揭示毒素操纵的新机制 并为了自己的目的劫持细胞机制。它也将促进我们对如何 微管和微管相关的马达在膜运输事件中起作用，并揭示了 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。

项目成果

Bigger Isn't Always Better: Bulking Up Impedes Receptor Internalization.

• DOI: 10.1016/j.bpj.2018.01.037
• 发表时间: 2018-03
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: A. Kenworthy

Analysis of diffusion in curved surfaces and its application to tubular membranes.

• DOI: 10.1091/mbc.e16-06-0445
• 发表时间: 2016-12-01
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Klaus CJ;Raghunathan K;DiBenedetto E;Kenworthy AK
• 通讯作者: Kenworthy AK

Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis.

• DOI: 10.1038/nature14064
• 发表时间: 2015-01-22
• 期刊: NATURE
• 影响因子: 64.8
• 作者: Renard, Henri-Francois;Simunovic, Mijo;Lemiere, Joel;Boucrot, Emmanuel;Garcia-Castillo, Maria Daniela;Arumugam, Senthil;Chambon, Valerie;Lamaze, Christophe;Wunder, Christian;Kenworthy, Anne K.;Schmidt, Anne A.;McMahon, Harvey T.;Sykes, Cecile;Bassereau, Patricia;Johannes, Ludger
• 通讯作者: Johannes, Ludger