Working Machinery of Dynein Molecules

动力蛋白是一种巨大而复杂的分子，是微管的负端定向马达。为了阐明其作为运动蛋白的机制，我们测量了单个动力蛋白分子与微管相互作用的分子行为。由于Dynein运动结构域的直径约为15 nm，是微管蛋白异源二聚体重复序列的两倍，因此目前还不清楚Dynein是只使用单个原丝还是使用多个原丝。为了回答这个问题，我们利用了锌诱导的微管蛋白薄片的特性，即马达结合位点只暴露在薄片的两边。以前的研究表明，玻璃表面的多个动力蛋白分子可以滑动锌板。然而，可能有多个动力蛋白分子与片状分子相互作用，滑动并不一定意味着单个动力蛋白分子可以连续地在单个原丝上行走。在本研究中，我们用tirf…技术研究了锌板上单分子运动的测定。我们用抗微管蛋白或MAP的抗体将锌片系在玻璃上，使动力蛋白结合部位暴露在溶液中。为了观察单一的动力蛋白分子，制备了绿色荧光蛋白标记的酵母胞质动力蛋白。我们成功地观察到GFP标记的dynein在锌板和微管上连续运动。然而，锌板上的速度约为微管上速度的一半。这些观察结果表明，双头动力蛋白分子能够在单根原丝上连续运动，但在多根原丝上运动更有效。接下来，我们用新开发的快速拉伸式实验测量了单头动力蛋白的解结力。结果表明，无核苷酸或AMPPNP的解结力较高，ADP-Vi或ADP的解结力较低，且与极性有关，正端的拉力较强，负端的拉力较弱。这些结果表明，动力蛋白双头结构中的后头比前头分离得更快，这使得动力蛋白分子能够进行前进运动。较少

Dynein is a huge and complicated molecule and minus-end directed motor of microtubules. To elucidate its machinery as a motor protein, we measured the molecular behavior of single dynein molecules focusing of the interaction with microtubules. Because dynein motor domain is as large as about 15nm in diameter, which is twice of the size of tubulin heterodimer repeat, it is not clear whether dynein uses only a single protofilament or multiple protofilaments. To answer this question, we exploited the property of zinc-induced tubulin sheets that the motor binding sites are only exposed at either edge of the sheet. Previous study has shown that multiple dynein molecules on glass surface can glide zinc-sheets. However, it is possible that multiple dynein molecules interact with the sheets and the gliding doesn't necessarily mean that single dynein molecule can walk on a single protofilament processively. In this study, we investigated the single molecule motility assay on zinc-sheets by TIRF … More M. We tethered the zinc-sheets on the glass using antibody against tubulin or MAPs, so that the dynein binding site is exposed in the solution. To observe single dynein molecules, GFP-tagged yeast Cytoplasmic dynein was prepared. We successfully observed that the GFP-tagged dynein moves processively on zinc-sheet as well as on microtubules. However, the velocity on zinc-sheet was about half of that on microtubules. These observations suggest that two-headed dynein molecule is able to move on single protofilament processively, but it can move more effectively on the multiple protofilaments. We next measured the unbinding force of single-headed dynein by newly developed quick stretch type experiment. The results suggested that the unbinding force was high in no nucleotide or AMPPNP and low in ADP-Vi or ADP, and also polarity dependent; the binding was strong against the pulling force from the plus-end and weak against that from the minus-end. These results showed that rear head within the double-headed structure of dynein is detach faster than the front head, which enable the processive movement of dynein molecules. Less