Dissecting the interaction between dynein and early endosomes

剖析动力蛋白和早期内体之间的相互作用

• 批准号: 8321957
• 负责人: XIN XIANG
• 金额: $ 29.07万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321957
• 关键词: Affect; Alleles; Amino Acids; Amyotrophic Lateral Sclerosis; Aspergillus nidulans; Binding; Biochemical; Biological Models; Brain Diseases; Cell Nucleus; Complex; Cytoskeleton; Defect; Dynein ATPase; Early Endosome; Endosomes; Eukaryota; Eukaryotic Cell; Genes; Genetic; Goals; Human; Hyphae; Image; Intracellular Transport; LIS1 protein; Lead; Link; Mediating; Microtubules; Molds; Motor; Movement; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear; Null Lymphocytes; Organelles; Pathway interactions; Plus End of the Microtubule; Positioning Attribute; Process; Proteins; Role; Signaling Molecule; Structure; Syndrome; System; Testing; base; cell type; dynactin; gene interaction; genetic analysis; genome wide association study; mutant; neuronal survival; prevent

DESCRIPTION (provided by applicant): The minus-end-directed microtubule motor cytoplasmic dynein powers the retrograde movement of membranous cargoes such as early endosomes, but the mechanism of motor-cargo interaction is unclear. The objective of this proposal is to dissect the interaction between dynein and early endosomes using the filamentous fungus Aspergillus nidulans as a model system. In A. nidulans, dynein, dynactin and NUDF/LIS1 accumulate at the dynamic microtubule plus ends near the hyphal tip, where they engage early endosomes for their minus-end-directed transport. Loss of dynein, dynactin or NUDF/LIS1 impairs minus-end-directed transport, causing an abnormal buildup of early endosomes at the hyphal tip. Loss of NUDF/LIS1, which affects the movement of dynein-bound endosomes rather than dynein-endosome interaction, causes an obvious dynein-dynactin-early- endosome co-localization at the hyphal tip. Remarkably, deleting the gene encoding the p25 subunit of the dynactin complex in NUDF-null cells abolishes this localization. Moreover, p25 is unique among the analyzed dynactin components in that it is required for early endosome movement but not for dynein-mediated nuclear distribution, or the microtubule plus-end accumulation of dynein and dynactin. Based on these results, we hypothesize that p25 mediates the interaction between early endosomes and the dynactin-dynein supercomplex. Specific Aim 1 is to determine the mechanism of p25 in binding dynactin-dynein to early endosomes. We will provide direct biochemical evidence that p25 is necessary for early-endosome-dynein interaction. We will determine whether p25 is sufficient for associating with early endosomes or if any other dynactin components are required to cooperate with p25 for its interaction with early endosomes. We will also perform a structure-function analysis on p25 to determine the amino acid residues required specifically for p25-dynactin interaction as well as those required for p25-early-endosome interaction. Specific Aim 2 is to identify proteins that bridge and/or regulate the interaction between early endosomes and dynactin-dynein. We will perform a genome-wide screen for genes that are required for early-endosome-dynein interaction (eedi). The screen criteria prevent the re-isolation of genes in the dynein-mediated nuclear distribution pathway. To date, we have collected 20 eedi mutants, and the vast majority represents non-p25 alleles. We will organize the mutants in complementation groups and clone the eedi genes. We will also use imaging and biochemical approaches to further characterize the specific roles of these EEDI proteins.

描述（由申请人提供）：负端定向微管运动细胞质动力蛋白为膜质货物（如早期内体）的逆行运动提供动力，但运动-货物相互作用的机制尚不清楚。本研究的目的是以丝状真菌构巢曲霉为模型系统，研究动力蛋白与早期内体之间的相互作用。以. nidulans、动力蛋白、动力肌动蛋白和NUDF/LIS 1聚集在靠近菌丝顶端的动态微管正末端，在那里它们与早期内体接合以进行负末端定向运输。动力蛋白、dynactin或NUDF/LIS 1的缺失会损害负末端定向运输，导致菌丝顶端早期内体的异常积累。NUDF/LIS 1的缺失影响了动力蛋白结合的内体的运动，而不是动力蛋白-内体相互作用，导致在菌丝顶端明显的动力蛋白-动力肌动蛋白-早期内体共定位。值得注意的是，在NUDF无效细胞中删除编码动力肌动蛋白复合物的p25亚基的基因消除了这种定位。此外，p25是唯一的分析动力蛋白的成分，因为它是所需的早期内体运动，但不是动力蛋白介导的核分布，或微管加端积累的动力蛋白和动力蛋白。基于这些结果，我们假设p25介导早期内体和动力蛋白-动力蛋白超复合物之间的相互作用。具体目标1是确定p25在结合动力蛋白-动力蛋白到早期内体中的机制。我们将提供直接的生化证据，p25是必要的早期内体动力蛋白的相互作用。我们将确定p25是否足以与早期内体相关联，或者是否需要任何其他动力蛋白组分与p25合作以与早期内体相互作用。我们还将对p25进行结构-功能分析，以确定p25-dynactin相互作用所需的特定氨基酸残基以及p25-早期内体相互作用所需的氨基酸残基。具体目标2是鉴定桥接和/或调节早期内体和动力蛋白-动力蛋白之间相互作用的蛋白质。我们将进行全基因组筛选，寻找早期内体动力蛋白相互作用（eedi）所需的基因。筛选标准防止了动力蛋白介导的核分布途径中基因的重新分离。迄今为止，我们已经收集了20个eedi突变体，其中绝大多数代表非p25等位基因。我们将把突变体组织成互补群，并克隆eedi基因。我们还将使用成像和生物化学方法来进一步表征这些EEDI蛋白的特定作用。