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

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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.