Analysis of dynein function by use of mutants and in vitro motility assay systems

真核生物纤毛和鞭毛跳动的基础是由动力蛋白驱动的微管滑动运动，但波动运动是如何产生的尚不清楚。为了解决这一问题，我们开展了两条研究路线，旨在阐明微管与各种动力蛋白之间的相互作用。首先，我们试图分离和表征缺乏特定动力蛋白亚种的衣藻突变体，并通过分析突变体的运动性来评估缺失动力蛋白的性质。其次，我们分离了特定的动力蛋白亚种，并使用体外运动系统分析了它们的特性。这些研究的主要发现如下：(1)缺失内臂动力蛋白亚群的突变体ida5在常规肌动蛋白结构基因上发生突变，导致细胞内不产生常规肌动蛋白。该突变体的发现将有助于阐明肌动蛋白在内臂动力蛋白中的生理作用。(2)内臂和外臂动力机构的发力特性存在较大差异；特别是在轴索运动缓慢时，如弯曲起始过程中，内臂比外臂更重要。(3)动力蛋白的活性受核苷酸调控，如ADP。(4)外臂动力机构可以在各种条件下自激活产生力，而内臂动力机构则不能，它们需要中心副和径向辐条来产生力。总之，本研究揭示了不同动力蛋白之间的功能多样性。这种多样性似乎对轴突功能很重要。

The basis for the beating of eukaryotic cilia and flagella is the sliding movement of microtubules driven by dynein, but how undulating movements are produced is not understood. To approach this problem, we carried out two lines of studies aimed at elucidation of the interaction between microtubules and various kinds of dynein. First, we tried to isolate and characterize Chlamydomonas mutants that lack specific dynein subspecies, and assess the property of the missing dyneins by analyzing the motility of the mutants. Second, we isolated specific dynein subspecies, and assayd their properties using in vitro motility systems. Major findings from these studies are as follows : (1) ida5, a mutant missing a subset of inner arm dyneins, was found to have a mutation in the structural gene of conventional actin, such that no conventional actin is produced in the cell. The discovery of this mutant will help to clarify the physiological role of actin in inner-arm dyneins. (2) The force generating properties of dyneins were found to differ greatly between inner and outer arm dyneins ; in particular, the inner arm is more important than the outer arm when axoneme is moving slowly, such as in the process of bend initiation. (3) The activity of dynein is regulated by nucleotides, such as ADP.(4) Outer arm dynein can be self-activated to produce force under various conditions, while inner arm dyneins cannot-they need the central pair and radial spokes for force generation. In short, this study has revealed a great functional diversity among different dyneins. This diversity appears to be important for the axonemal function.