Direct observation of dynein-microtubule binding

鞭毛轴突外对微管中，动力蛋白外臂的排列间隔为24 nm，7种内臂的排列间隔均为96 nm。这样的常规安排是如何产生的，几乎完全是未知的。在这项研究中，我们旨在通过直接观察动力蛋白-微管结合过程来探讨这一机制。利用全反射光学和荧光标记的蛋白质，我们观察到动力蛋白与微管的结合是一个合作过程，并利用昆虫培养细胞中表达的外动力蛋白臂对接复合体(oda-DC)对合作结合进行了定量。在这些研究的同时，我们搜索了负责结合内部动力蛋白手臂的蛋白质，并发现tektin蛋白参与了衣藻轴丝中动力蛋白e的附着。这是第一个显示tektin参与动力蛋白排列的研究；tektin最先在海胆精子鞭毛中发现，一直被认为是决定轴突纵向周期性的标尺。在另一种不同的实验中，我们使用一种名为光漂白后荧光恢复的技术来检测动力蛋白-微管结合的动态性质。利用电穿孔介导的荧光标记的dynein亚基掺入，我们发现衣藻的内臂dynein在鞭毛内不断地被翻转。这些发现应该会为动力蛋白臂如何附着在轴丝微管上的机制提供重要的见解。

In thee outer doublet microtubule in the flagellar axoneme, the outer dynein arms are arranged with a 24 nm interval, while each of the seven kinds of inner arms are arranged with a 96 nm interval. How such a regular arrangement is produced is almost entirely unknown. In this study, we aimed to approach this mechanism by directly observing the dynein-microtubule binding process. Using total reflection optics and fluorescently labeled proteins, we observed that the association of dynein to the microtubule is a cooperative process, quantified the cooperative binding using outer-dynein-arm-docking complex (ODA-DC) expressed in insect culture cells. Along with these studies, we searched for proteins responsible for the binding of inner dynein arms and discovered that the protein tektin is involved in the attachment of dynein e in Chlamydomonas axonemes. This is the first study to show tektin involvement in the dynein arrangement ; tektin, first discovered in sea urchin sperm flagella, has been thought to function as a ruler that determines the longitudinal periodicity in the axoneme. In a still different line of experiment, we examined the dynamic nature of dynein-microtubule association using a technique called fluorescence recovery after photobleaching. Using electroporation-mediated incorporation of fluorescently labeled dynein subunit, we found that inner arm dynein of Chlamydomonas is constantly being turned over within the flagella. These findings should provide important insights into the mechanism as to how dynein arms are attached to axonemal microtubules.