REGULATION OF CYTOPLASMIC DYNEIN BASED VESICLE TRANSPORT

动力蛋白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多蛋白复合物。在动物细胞中，动力蛋白驱动间期许多类膜细胞器的运动，对有丝分裂中染色体的形成和纺锤体的完整性至关重要。动力蛋白-动力蛋白在间期的微管组织和动员中似乎也是至关重要的。虽然我们对细胞质动力蛋白的功能了解很多，但动力蛋白究竟是如何促进运动的仍不清楚。动力蛋白可以作为一个多功能适配器，允许动力蛋白结合不同的细胞货物。我们的研究表明，动力蛋白可以增强动力蛋白马达的加工能力。显然，dynactin具有多种复杂功能，其复杂的结构证明了这一点。Dynactin可以被生化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动态蛋白亚基的发现，p25和ArpX，一种新的肌动蛋白相关蛋白，不同于Arp1，主要的动态蛋白亚基。所有11个dynactin亚基现已克隆，为未来dynactin结构和功能的研究提供了全面的试剂库。第一组目标侧重于动态结构。根据最近发现的dynactin亚基制备的分子生物学和免疫学试剂将用于确定dynactin ArpX复合物（一种假定的尖端封盖复合物）的结构。将讨论ArpX复合物如何参与Arp1组装和常规肌动蛋白聚合。分析性超离心研究将在动力蛋白亚基和亚复合物上进行。这项工作将由表面等离子体共振研究补充。稍后的Aims探讨了动力学蛋白到达其两个主要亚细胞位置的机制。中心体动蛋白将使用反褶积显微镜和FEISEM（一种高分辨率扫描电镜技术）进行可视化。将进行研究，以确定动力蛋白募集到中心体和中心体结合域的机制。了解dynactin/膜结合机制的努力将集中在p62上，它是ArpX复合物的一个组成部分，与膜紧密相关。这种相互作用将通过脂质标记、亲和层析和化学交联来确定。

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.