Dissecting the interaction between dynein and early endosomes

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。本提案的目的是剖析动力蛋白和早期核内体之间的相互作用，以丝状真菌细粒曲霉为模型系统。在毛豆中，动力蛋白、动力蛋白和NUDF/LIS1聚集在菌丝尖端附近的动态微管正端，在那里它们参与早期核内体进行负端定向运输。动力蛋白、动力蛋白或NUDF/LIS1的缺失会损害负端定向转运，导致菌丝尖端早期核内体的异常积聚。NUDF/LIS1的缺失会影响动力蛋白结合的核内体的运动，而不是动力蛋白与核内体的相互作用，这导致了在菌丝尖端明显的动力蛋白-动力蛋白-早期核内体共定位。值得注意的是，在NUDF-null细胞中删除编码dynactin复合物p25亚基的基因可以消除这种定位。此外，p25在所分析的动力蛋白成分中是独特的，因为它是早期核内体运动所必需的，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端积累所必需的。基于这些结果，我们假设p25介导了早期内体和动力蛋白-动力蛋白超复合体之间的相互作用。特异性目的1是确定p25结合动力蛋白-动力蛋白与早期核内体的机制。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是否足以与早期核内体相互作用，或者是否需要任何其他动力蛋白成分与p25合作才能与早期核内体相互作用。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-early-endosome相互作用所需的氨基酸残基。特异性目的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早期内核-动力蛋白相互作用（eedi）所需的基因。筛选标准可防止动力蛋白介导的核分布途径中基因的重新分离。到目前为止，我们已经收集了20个eedi突变体，绝大多数是非p25等位基因。我们将把突变体组织成互补组，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蛋白的具体作用。

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.