REGULATION OF CYTOPLASMIC DYNEIN BASED VESICLE TRANSPORT

基于微管的运动为许多细胞内转运提供动力 功能包括许多膜的动态跳跃运动， 有丝分裂中的细胞器结合和细胞核及染色体运动。 细胞质动力蛋白，一种普遍存在的，可溶性的同种型， 酶轴丝动力蛋白，参与许多这些细胞内 动作 在体外，细胞质动力蛋白是一种微管刺激的 ATP酶可以驱动塑料珠和玻璃的滑动运动 相对于微管的盖玻片。 虽然细胞质动力蛋白 在体内运输微管上的膜囊泡，在体外， 需要其他可溶性因素的参与，例如20 S dynactin复合物，驱动囊泡运动。 A的进一步证据 细胞质动力蛋白驱动运动对动力蛋白复合体的需求 遗传学研究提供了这些事件。 Dynactin复合物含有十个不同的亚基，其中八个已经被发现。 通过肽测序、抗体交叉反应性和分子生物学鉴定 克隆技术 旋转阴影EM成像显示该分子是 由两个不同的结构域组成，一个37 nm的细丝， 类似于f-肌动蛋白和突出的肩膀和精细的侧臂。 抗体 装饰实验表明，37 nm的灯丝是由 肌动蛋白相关蛋白Arp 1、肌动蛋白加帽蛋白和p62亚基， 而精细侧臂含有p160/p150 Glued亚基。 在 在这里详细描述的实验中，Arp 1的生化特性和 p160/p150 Glued亚基将在体外进行研究。 复合物的途径 组装将在交联、破坏和重新组装中进行探索 实验 关于细胞质动力蛋白或动力蛋白之间的相互作用， 与膜复合，也不是动力蛋白刺激的机制 动力蛋白活性。 这两种大分子的相互作用 复合物与膜将被检查，特别注意 对动力蛋白结合的动力肌动蛋白（及其亚基）的影响。 的 动力蛋白复合体及其特异性亚基对动力蛋白ATP酶的影响 活动也将进行研究。 膜所需的膜蛋白 将通过重组到蛋白脂质体中分离运动性， 研究了它们与动力蛋白和动力肌动蛋白复合物的潜在相互作用 进一步.

Microtubule-based motility powers a number of intracellular transport functions including the dynamic saltatory movements of many membrane- bound organelles and nuclear and chromosome movement in mitosis. Cytoplasmic dynein, a ubiquitous, soluble isoform of the well-known enzyme axonemal dynein, participates in many of these intracellular movements. In vitro, cytoplasmic dynein is a microtubule-stimulated ATPase that can power the sliding movement of plastic beads and glass coverslips relative to microtubules. Although cytoplasmic dynein transports membrane vesicles on microtubules in vivo, in vitro the enzyme requires the participation of other soluble factors, such as the 20S dynactin complex, to drive vesicle movement. Further evidence of a requirement for dynactin complex in cytoplasmic dynein-driven motile events has been provided by genetic studies. Dynactin complex contains ten distinct subunits, eight of which have been identified by peptide sequencing, antibody cross-reactivity and molecular cloning techniques. Rotary shadow EM imaging reveals the molecule to be composed of two distinct structural domains, a 37 nm filament that resembles f-actin and a projecting shoulder and fine sidearm. Antibody decoration experiments show the 37 nm filament to be composed of the actin-related protein Arp1, actin-capping protein and the p62 subunit, while the fine sidearm contains the p160/p150Glued subunit. In experiments detailed here, the biochemical properties of the Arp1 and p160/p150Glued subunits will be studied in vitro. Pathways for complex assembly will be explored in crosslinking, disruption and reassembly experiments. Little is known about the interaction of cytoplasmic dynein or the dynein complex with membranes, nor is the mechanism by which dynactin stimulates dynein activity understood. The interaction of these two macromolecular complexes with membranes will be examined, paying particular attention to the effects of dynactin (and its subunits) on dynein binding. The effects of dynactin complex and its specific subunits on dynein ATPase activity will also be studied. Membrane proteins required for membrane motility will be isolated by reconstitution into proteoliposomes and their potential interactions with dynein and dynactin complex studied further.