REGULATION OF CYTOPLASMIC DYNEIN BASED VESICLE TRANSPORT

基于微管的运动为许多细胞内运输提供动力 功能包括许多膜的动态跳跃运动- 有丝分裂中细胞器的结合以及核和染色体的运动。 细胞质动力蛋白，一种普遍存在的、可溶的众所周知的 酶Axonemal dynein，参与这些细胞内的许多 动静。在体外，胞浆动力蛋白是一种微管刺激的 可以驱动塑料珠子和玻璃滑动的ATPase 相对于微管的盖片。尽管细胞质动力蛋白 体内转运微管上的膜小泡，体外酶 需要其他可溶因素的参与，如20s 动力蛋白复合体，驱动囊泡运动。更多的证据表明 胞质动力蛋白驱动的运动对动力蛋白复合体的要求 这些事件是由遗传学研究提供的。 动力蛋白复合体包含10个不同的亚基，其中8个亚基 通过多肽测序、抗体交叉反应和分子鉴定 克隆技术。旋转阴影电磁成像揭示了分子是 由两个不同的结构域组成，一个37 nm的灯丝 类似于肌动蛋白和突出的肩部和精致的手枪。抗体 装饰实验表明，37 nm的灯丝由 肌动蛋白相关蛋白Arp1、肌动蛋白封顶蛋白和p62亚单位， 而细小侧臂含有p160/p150Glued亚基。在……里面 这里详细的实验，Arp1和Arp1的生化性质 P160/p150Glued亚基将在体外进行研究。复合体的路径 将在交联、破坏和重新组装中探索组装 实验。 对细胞质动力蛋白或动力蛋白的相互作用知之甚少 与膜的复合体，也不是dynactin刺激的机制 了解动力蛋白的活动。这两种大分子的相互作用 将检查与膜的络合物，特别要注意 动力蛋白(及其亚基)对动力蛋白结合的影响。这个 动力蛋白复合体及其特异性亚基对动力蛋白ATPase的影响 活动也将被研究。膜所需的膜蛋白 运动性将通过重组到蛋白脂质体和 研究了它们与动力蛋白和动力蛋白复合体的潜在相互作用 再远一点。

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.