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。抑制这些细胞器运动的人类突变导致上呼吸道疾病和不孕症，而相关的运动形式对有丝分裂和许多细胞内颗粒的定向运输很重要。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运动酶。大多数鞭毛有两排不相同的动力蛋白附着在每个双微管上，它们都有助于微管的滑动和鞭毛的运动。外排动力蛋白与细胞质动力蛋白相似，含有2 - 3条约500 kD催化重链、2条约75 kD中间链和多条轻链。虽然动力蛋白作为纤毛和细胞质运动的微管相关马达的作用已得到充分认识，但对它们的调节或它们与其携带的负荷（细胞质中的囊泡或染色体，纤毛和鞭毛中的双重微管）的附着模式知之甚少。所提出的实验的目的是表征动力蛋白亚基和双态微管相关蛋白之间的相互作用，这对于鞭毛组装过程中动力蛋白的附着和外排动力蛋白马达的调节是重要的。以莱茵衣藻鞭毛外排动力蛋白为模型系统，对正常动力蛋白组装和功能所必需的基因进行了克隆、测序和遗传定位。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、鞭毛靶向和原位调控中的功能。本文提出的实验重点是通过已发表的和初步的组装突变研究发现的四个基因的产物，它们在动力蛋白组装中具有独特的作用：oda7， oda8， oda15和pfl3。所有的材料现在都可以用于快速确定这些基因的产物，这将为调节鞭毛动力蛋白马达的细胞内分布和活性的机制提供关键信息。

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.