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: 8536861
• 负责人: XIN XIANG
• 金额: $ 28.05万
• 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/8536861
• 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）：减去定向的微管运动细胞质动力蛋白为膜货物（例如早期内体）的逆行运动提供了动力，但是运动碳相互作用的机制尚不清楚。该提案的目的是使用丝状曲霉尼古拉人作为模型系统剖析动力蛋白和早期内体之间的相互作用。在A. nidulans中，Dynein，dynactin和Nudf/Lis1积聚在动态微管和菌丝尖端附近的末端，在那里它们使早期内体与负端指导的转运相关。 Dynein，Dynactin或Nudf/Lis1的损失会损害减去端的转运，从而导致菌丝尖端的早期内体的异常积聚。 nuDF/lis1的丢失会影响动力蛋白结合的内体的运动而不是动力蛋白 - 粘体相互作用，会导致在菌丝尖端上明显的动力蛋白 - 二奈乳蛋白 - 二奈氏蛋白 - 二奈氏蛋白 - 内体共定位。值得注意的是，删除编码NUDF-NULL细胞中Dynactin复合物P25亚基的基因消除了这种定位。此外，p25在分析的dynactin成分中是独一无二的，因为它是早期内体运动所必需的，但对于动力蛋白介导的核分布或微管加末端的积累并不是动力蛋白和dynactin的积累。基于这些结果，我们假设p25介导了早期内体与dynactin-dynein超复合物之间的相互作用。具体目的1是确定结合dynactin-dynein与早期内体的P25机理。我们将提供直接的生化证据，表明P25对于早期 - 内体 - 二氧蛋白相互作用是必需的。我们将确定P25是否足以与早期内体相关，或者是否需要任何其他Dynactin成分与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，以确定专门针对P25-Dynactin相互作用的氨基酸残基，以及P25- Eartly-Endososom相互作用所需的氨基酸残基。具体目的2是鉴定桥接和/或调节早期内体和dynactin-dynein之间相互作用的蛋白质。我们将对早期 - 粘体 - 二氧蛋白相互作用（EEDI）所需的基因进行全基因组筛选。筛选标准可防止在动力蛋白介导的核分布途径中重新分离基因。迄今为止，我们收集了20个EEDI突变体，绝大多数代表非P25等位基因。我们将在互补组中组织突变体，并克隆EEDI基因。我们还将使用成像和生化方法进一步表征这些EEDI蛋白的特定作用。