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

使用突变体和体外运动测定系统分析动力蛋白功能

基本信息

批准号：
07408034
负责人：
KAMIYA Ritsu
金额：
$ 3.33万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07408034/
关键词：
flagella dynein actin genes mutation Chlamydomonas cell motility force production 突然変異体

项目摘要

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.

真核生物纤毛和鞭毛跳动的基础是动力蛋白驱动的微管滑动运动，但波动运动是如何产生的还不清楚。为了解决这个问题，我们进行了两个系列的研究，旨在阐明微管和各种动力蛋白之间的相互作用。首先，我们试图分离和鉴定缺乏特定动力蛋白亚种的衣藻突变株，并通过分析突变株的运动性来评估缺失的动力蛋白的性质。其次，我们分离了特定的动力蛋白亚种，并利用体外动力系统分析了它们的性质。这些研究的主要发现如下：(1)缺失内臂动力蛋白一个子集的突变体ida5被发现在常规肌动蛋白的结构基因中存在突变，使得细胞中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臂内动力蛋白中的生理作用。(2)内臂动力蛋白和外臂动力蛋白的作用力特性有很大差异，尤其是在轴丝缓慢运动时，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ADP等核苷酸的调节。(4)外臂动力蛋白可以在各种条件下自激活产生力，而内臂动力蛋白则不能-它们需要中心对和径向辐条来生成力。简而言之，这项研究揭示了不同动力蛋白之间的巨大功能差异。这种多样性似乎对轴丝功能很重要。