Dynein arm-function studied by mutants and in vitro motility

动力蛋白在鞭毛跳动机制中起着最重要的作用。鞭毛动力蛋白由内臂和外臂上多达10条不同的重链组成，但每条重链的具体功能尚不清楚。为了探讨各种重链的功能，我们进行了两个系列的实验：分离缺乏特定重链的衣藻突变体，以及分离的重链的运动活性测定。在第一种方法中，我们分离了一个缺少外臂部分β重链的突变体和两种缺乏不同组的内臂重链的突变体。对这些突变体的研究表明：(1)在外臂动力蛋白的功能中，β重链比α重链更重要，以及(2)已知的8个内臂重链中的5个对于鞭毛搏动的产生是不必要的。在第二条研究中，我们首先开发了一个系统，在该系统中，分离的外臂动力蛋白，一个α/β亚粒子和一个伽马亚粒子，可以重组并连接到一个外臂无臂突变体oda1上。这项研究再次证明了β重链在外臂功能中的重要作用。最后，我们开发了一个使用分离的内臂动力蛋白亚种的体外运动测试系统。7个亚种中的6个(包含8个重链)被发现在体外具有移位微管的活性。不同的亚种在最大速度和引起微管旋转的能力上有所不同。这些研究表明，鞭毛含有不同功能的动力蛋白。这些不同的动力蛋白如何合作产生规则的鞭毛跳动是一个有趣的未来问题。

Dynein plays a most important function in the flagellar beating mechanism. Flagellar dynein comprises as many as ten different heavy chains in the inner and outer arms, but the specific function of each heavy chain is unknown. To approach the function of various heavy chains, we carried out two lines of experiments : isolation Chlamydomonas mutants that lack particular heavy chains, and assay of the motile activity of isolated heavy chains.In the first approach, we isolated a mutant that lacks part of the beta heavy chain of the outer arm and two kinds of mutants that lack different sets of inner-arm heavy chains. Studies on these mutants have indicated that (1) the beta heavy chain is more important than the alpha heavy chain in the function of outer arm dynein, and (2) five out of the eight known inner-arm heavy chains are unnecessary for the generation of flagellar beating.In the second line of study, we first developed a system in which dissociated outer-arm dynein, an alpha/beta subparticle and a gamma subparticle, can be recombined and attached to an outer arm-less mutant, oda1. This study again demonstrated that the beta heavy chain is important in the outer arm function. Finally, we developed an in vitro motility assay system using isolated inner arm dynein subspecies. Six out of the seven subspecies (containing eight heavy chains) were found to have activities to translocate microtubules in vitro. Different subspecies differ in maximal velocity and in the ability to cause microtubule rotation. These studies indicate that the flagella contain various dyneins that differ functionally. How these different dyneins cooperate in producing regular flagellar beating is a fascinating future problem.