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: 8087258
• 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/8087258
• 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. PUBLIC HEALTH RELEVANCE: Dynein-mediated retrograde movement of early endosomes that carry signaling molecules towards the nucleus is crucial for neuronal survival. Deficiencies in the dynein complex and the dynactin complex that interacts with dynein cause devastating neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Perry syndrome.

描述(申请人提供)：负端导向的微管马达细胞质动力蛋白推动膜性货物的逆行运动，如早期的内小体，但马达-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Nidulans Aspergillusnidulans为模型系统，剖析动力蛋白与早期内体之间的相互作用。在根结线虫中，动力蛋白、动力蛋白和NUDF/Lis1聚集在菌丝顶端附近的动态微管正端，在那里它们与早期的内体接触，进行负端定向运输。动力蛋白、动力蛋白或NUDF/Lis1的缺失会损害负端定向运输，导致菌丝顶端早期内吞体内的异常堆积。NUDF/Lis1的缺失影响了dynein结合的内小体的运动，而不是dynein与内小体的相互作用，导致了明显的dynein-dynactin-早期-内吞体共定位在菌丝顶端。值得注意的是，在NUDF缺失的细胞中，删除编码dynactin复合体p25亚单位的基因就会取消这种定位。此外，在所分析的动力蛋白成分中，p25是独一无二的，因为它是早期内小体运动所必需的，而不是动力蛋白介导的核分布或动力蛋白和动力蛋白的微管正端积累所必需的。基于这些结果，我们假设p25介导了早期内小体和动力蛋白-动力蛋白超复合体之间的相互作用。具体目的1是确定p25结合动力蛋白-动力蛋白与早期内小体的机制。我们将提供直接的生化证据，证明p25是早期内体-动力蛋白相互作用所必需的。我们将确定p25是否足以与早期内体结合，或者是否需要任何其他动力蛋白成分与p25协同作用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相互作用所需的氨基酸残基以及p25-早期-内小体相互作用所需的氨基酸残基。具体目标2是识别桥接和/或调节早期内体和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。筛选标准防止动力蛋白介导的核分配途径中的基因重新分离。到目前为止，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组，并克隆EEDI基因。我们还将使用成像和生化方法来进一步表征这些EEDI蛋白的具体作用。 公共卫生相关性：动力蛋白介导的携带信号分子朝向细胞核的早期内小体的逆行运动对神经元存活至关重要。动力蛋白复合体和与动力蛋白相互作用的动力蛋白复合体的缺陷会导致毁灭性的神经退行性疾病，如肌萎缩侧索硬化症(ALS)和佩里综合征。