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The mechanism regulating the activity of oscillatory motor protein, dynein

调节振荡运动蛋白动力蛋白活性的机制

基本信息

批准号：
13440249
负责人：
SHINGYOJI Chikako
金额：
$ 8.32万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13440249/
关键词：
dynein sliding movement ATPase activity oscillation flagellar movement microtubule sea urchin sperm ダイニン力 1分子鞭毛

项目摘要

A prominent feature of flagellar motility is an oscillation. Flagellar oscillatory movement is based on the activity of dynein, which is one of the microtubule motor proteins. In this study we aimed to elucidate the mechanism regulating the dynein activity in the flagellar axonemes. We used sea urchin sperm flagella, which were demembranated, fragmented and then treated with elastase. An application of 1 mM ATP induced sliding of the elastase-treated axonemes into two groups of doublets, one of them contained the central pair. When several kinds of fluorescent ATP were used instead of ATP, we found that the dynein hydrolysed them without inducing the sliding. In the second experiment, we found that the velocity of sliding induced by ATP in the elastase-treated axonemes, which had the inner arms and 5% of the outer arms, was significantly increased by adding 350 kDa trypsin-fragments of the outer arms to the axonemes in the presence of ATP. Because the ATPase activity of the fragments was higher than that of the outer arms, the activity between dynein arms might be regulated through the doublet microtubules. In the third experiment, the effect of the central pair on dynein activity was studied by using singlet microtubules, which interacted with exposed dynein arms on doublets of the doublet groups and ultrastructural analysis of split axonemes. We found that calcium inhibits the activity of dynein arms on the doublets through the regulatory mechanism that involves the central pair and the radial spoke complex. This mechanism might control the switching of the dynein activity within the axoneme to induce the oscillatory bending movement of the flagellum.

鞭毛运动的一个显著特征是振荡。鞭毛的振荡运动是基于动力蛋白的活性，动力蛋白是微管马达蛋白之一。本研究旨在阐明鞭毛轴丝中动力蛋白活性的调控机制。我们使用海胆精子鞭毛，这是去膜，破碎，然后用弹性蛋白酶处理。应用1 mM ATP诱导弹性蛋白酶处理的轴丝滑动成两组的双峰，其中一个包含中央对。当用几种荧光ATP代替ATP时，我们发现动力蛋白水解它们而不诱导滑动。在第二个实验中，我们发现在弹性蛋白酶处理的轴丝（具有内臂和5%的外臂）中，通过添加外臂的350 kDa胰蛋白酶片段，ATP诱导的滑动速度显着增加在ATP存在下的轴丝。由于片段的ATP酶活性高于外臂，因此动力蛋白臂间的ATP酶活性可能是通过双微管调控的。在第三个实验中，中央对动力蛋白活性的影响进行了研究，通过使用单线态微管，与暴露的动力蛋白臂上的双峰的双峰组和分裂轴丝的超微结构分析相互作用。我们发现，钙离子通过涉及中央对和辐射辐条复合物的调节机制抑制了双联体上动力蛋白臂的活性。这一机制可能是通过控制轴丝内动力蛋白活性的转换而引起鞭毛的振荡弯曲运动。