REGULATION OF CYTOPLASMIC DYNEIN BASED VESICLE TRANSPORT

Dynactin是一种多蛋白复合物，是所有形式的细胞质动力蛋白驱动的细胞内运动所必需的。在动物细胞中，动力蛋白在分裂间期驱动许多类膜性细胞器的运动，并对有丝分裂中染色体的聚集和纺锤体的完整性起重要作用。动力蛋白-动力蛋白对微管在间期的组织和动员也是至关重要的。虽然对细胞质动力蛋白的功能有很多了解，但动力蛋白究竟如何促进运动仍不清楚。动力蛋白可以作为一种多功能的接头，使动力蛋白结合不同的细胞货物。我们的工作表明，dynactin可能会增强动力蛋白马达的持续合成能力。显然，动力蛋白具有多种复杂的功能，其复杂的结构就是证明。Dynactin可以被生物化学分解成三个不同的亚复合物，每个亚复合物在分子内都有不同的功能。这项工作导致了两个dynactin亚基的发现，p25和ArpX，一种新的肌动蛋白相关蛋白，不同于Arp 1，主要的dynactin亚基。所有11个dynactin亚基现已克隆，提供了一个全面的阿森纳的试剂为今后的研究dynactin的结构和功能。第一组目标集中在动力蛋白结构上。最近发现的dynactin亚基制备的分子生物学和免疫学试剂将用于定义dynactin ArpX复合物（一种假定的尖端封端复合物）的结构。ArpX复合物如何有助于Arp 1组装和传统的肌动蛋白聚合将得到解决。将对动力肌动蛋白亚基和亚复合物进行分析性超离心研究。这项工作将得到补充的表面等离子体共振研究。后来的目的是探索动力蛋白到达其两个主要亚细胞位置的机制。将使用去卷积显微镜和FEISEM（一种高分辨率SEM技术）对中心体动力蛋白进行可视化。将进行研究以确定dynactin募集到中心体和中心体结合结构域的机制。努力了解dynactin/膜结合的机制将集中在p62，ArpX复合物的组成部分，这是紧密相关的膜。将使用脂质标记、亲和色谱和化学交联来定义这种相互作用。

Dynactin is a multiprotein complex required for all forms of cytoplasmic dynein-driven intracellular motility. In animal cells, dynein drives the movement of many classes of membranous organelle in interphase and is important for chromosome congression and spindle integrity in mitosis. Dynein-dynactin also appears to be critical for microtubule organization and mobilization in interphase. Though a great deal is known about cytoplasmic dynein function, exactly how dynactin contributes to motility remains unclear. Dynactin may serve as a multifunctional adaptor that allows dynein to bind different cellular cargoes. Our work suggests that dynactin may enhance the processivity of the dynein motor. Apparently, dynactin is capable of a variety of complex functions, as evidenced by its intricate structure. Dynactin can be biochemically dissected into three different subcomplexes, each of which is expected to have a distinct function within the molecule. This work led to the discovery for two dynactin subunits, p25 and ArpX, a novel actin-related proteins that is distinct from Arp1, the major dynactin subunit. All eleven dynactin subunits are now cloned, providing a comprehensive arsenal of reagents for future studies of dynactin structure and function. The first set of Aims focus on dynactin structure. Molecular biological and immunological reagents prepared to recently discovered dynactin subunits will be used to define the structure of the dynactin ArpX complex (a putative pointed end capping complex). How ArpX complex contributes to Arp1 assembly and to conventional actin polymerization will be addressed. Analytical ultracentrifugation studies will be performed on dynactin subunits and subcomplexes. This work will be complemented by a surface plasmon resonance studies. Later Aims probe the mechanisms by which dynactin arrives at its two main subcellular locations. Centrosomal dynactin will be visualized using deconvolution microscopy and FEISEM, a high resolution SEM technique. Studies will be performed to define the mechanism of dynactin recruitment to centrosomes and the centrosome binding domain. Efforts to understand the mechanism of dynactin/membrane binding will focus on p62, a constituent of the ArpX complex, which is tightly associated with membranes. This interaction will be defined using lipid labeling, affinity chromatography and chemical cross-linking.