MOLECULAR AND GENETIC ANALYSIS OF FLAGELLAR DYNEINS

这项工作的长期目标是了解产生和调节纤毛和鞭毛运动的机制。 抑制这些细胞器的运动性的人类突变导致上呼吸道疾病和不育，并且相关形式的运动性对于有丝分裂和许多细胞内颗粒的定向运输是重要的。 鞭毛动力蛋白ATP酶是为纤毛和鞭毛提供动力的大型多亚基马达酶。 大多数鞭毛有两排不同的动力蛋白连接到每个双微管，这两个都有助于微管滑动和鞭毛运动。 外排的动力蛋白更类似于细胞质动力蛋白，含有两个或三个Ca。500 kD催化重链，两个ca. 75 kD中间链和多个轻链。 虽然动力蛋白作为微管相关的纤毛和细胞质运动的马达的作用是公认的，很少有人知道他们的调节或他们的附着模式，他们携带的负载（囊泡或染色体在细胞质中，双微管纤毛和鞭毛）。所提出的实验的目标是表征动力蛋白亚基和双重微管相关蛋白之间的相互作用，这是重要的鞭毛组装过程中的动力蛋白附着和调节外排动力蛋白电机。 利用莱茵衣藻鞭毛的外排动力蛋白作为模型系统，对正常动力蛋白组装和功能所必需的基因进行了克隆、测序和遗传作图。 利用分子克隆、电子显微镜和生物化学方法研究了每个基因产物在细胞质组装、靶向鞭毛和原位调控中的功能。 这里提出的实验集中在四个基因的产物上，这四个基因是通过已发表的和初步的组装突变研究确定的，它们在动力蛋白组装中具有独特的作用：oda7，oda8，oda15和pfl3。 所有的材料，现在可用于快速的进展，在确定这些基因的产品，这应该提供关键信息的机制，调节细胞内的分布和活动的鞭毛动力蛋白电机。

The long-term goals of this work are to understand the mechanisms that generate and regulate motility in cilia and flagella. Human mutations that inhibit motility of these organelles cause upper respiratory illnesses and infertility, and related forms of motility are important for mitosis and directed transport of many intracellular particles. Flagellar dynein ATPases are large, multi-subunit motor enzymes that power cilia and flagella. Most flagella have two non-identical rows of dyneins attached to each doublet microtubule, both of which contribute to microtubule sliding and flagellar motility. Dyneins of the outer row more closely resemble cytoplasmic dyneins in containing two or three ca. 500 kD catalytic heavy chains, two ca. 75 kD intermediate chains and multiple light chains. While the role of dyneins as microtubule-associated motors for both ciliary and cytoplasmic motility is well recognized, little is known about their regulation or their mode of attachment to the loads they carry (vesicles or chromosomes in the cytoplasm, doublet microtubules in cilia and flagella). Goals of the proposed experiments are to characterize interactions between dynein subunits and doublet microtubule-associated proteins that are important for dynein attachment during flagellar assembly and for regulation of outer row dynein motors. Using the outer row dynein of Chlamydomonas reinhardtii flagella as a model system, genes essential for normal dynein assembly and function are being cloned, sequenced, and genetically mapped. The function of each gene product in cytoplasmic assembly, targeting to flagella, and in situ regulation is examined using molecular cloning, electron microscopic and biochemical approaches. Experiments proposed here focus on the products of four genes identified through published and preliminary studies of assembly mutations as having unique roles in dynein assembly: oda7, oda8, oda15 and pfl3. All materials are now available for rapid progress in determining the products of these genes, which should provide key information about mechanisms that regulate the intracellular distribution and activity of flagellar dynein motors.