Understanding the cellular roles of EB1 proteins

了解 EB1 蛋白的细胞作用

• 批准号: 6445318
• 负责人: STEPHEN L ROGERS
• 金额: $ 4.42万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6445318
• 关键词: Drosophilidae; affinity chromatography; atomic force microscopy; cell cycle; confocal scanning microscopy; fluorescence microscopy; immunoprecipitation; larva; microtubule associated protein; microtubules; mitotic spindle apparatus; postdoctoral investigator; protein binding; protein protein interaction; protein structure function; receptor binding; tissue /cell culture; tubulin; yeast two hybrid system

Microtubules (Mts) are polymers of the cytoskeleton that serve to organize the cytoplasm and to act as tracks for the delivery of cargo by motor proteins. In order to perform these functions, the ends of Mts sometimes need to form stable linkages with targets, such as organelles or cortical domains. These linkages are thought to form between MT plus end-associated proteins and target-bound 'receptors.' The EB1 family of proteins is believed to be one such MT-bound protein. We propose to study the cellular functions of EB1 in Drosophila and to understand the molecular mechanisms that target it to MT plus ends by pursuing three specific aims. In the first, we will inhibit the function of EB1 in cultured cells using RNAi and use immunofluorescence- microscopy to assess the role EB1 plays in MT organization during interphase and mitosis. We will also employ real-time confocal microscopy to observe the role of inhibitory antibodies on the mitotic spindles of living fly embryos. In aim two, we will reconstitute EB1 targeting to the plus ends of MTs using in vitro assays and study the interactions between tubulin and EB1 using biochemical techniques, as well as atomic force microscopy. In the third, we will identify EB1- interacting proteins using biochemical approaches, such as affinity chromatography and immunoprecipitation, and by using two-hybrid interaction to identify EB1Q effector proteins and candidate EB1 receptors. Collectively, these studies will allow us to assign cellular functions to EB1 and to determine how this protein interacts with the MT cytoskeleton.

微管 (Mts) 是细胞骨架的聚合物，用于组织细胞质并充当运动蛋白运送货物的轨道。为了执行这些功能，Mts 末端有时需要与目标形成稳定的连接，例如细胞器或皮质域。这些连接被认为是在 MT 加末端相关蛋白和靶标结合“受体”之间形成的。 EB1 蛋白家族被认为是这样一种 MT 结合蛋白。我们建议研究果蝇中 EB1 的细胞功能，并通过追求三个特定目标来了解将其靶向 MT 加末端的分子机制。首先，我们将使用 RNAi 抑制培养细胞中 EB1 的功能，并使用免疫荧光显微镜评估 EB1 在间期和有丝分裂期间在 MT 组织中发挥的作用。我们还将采用实时共聚焦显微镜来观察抑制性抗体对活果蝇胚胎有丝分裂纺锤体的作用。在目标二中，我们将使用体外测定重建针对 MT 正端的 EB1 靶向，并使用生化技术以及原子力显微镜研究微管蛋白和 EB1 之间的相互作用。在第三部分中，我们将使用生化方法（例如亲和层析和免疫沉淀）来鉴定 EB1 相互作用蛋白，并使用两种杂交相互作用来鉴定 EB1Q 效应蛋白和候选 EB1 受体。总的来说，这些研究将使我们能够将细胞功能分配给 EB1，并确定该蛋白质如何与 MT 细胞骨架相互作用。