Identifying the Mechanism for Outer Arm Dynein Coordination in Ciliary Motion

项目摘要 活动的纤毛是细胞表面的毛发状突起，以正弦波形跳动产生 有动静。这种运动负责粘液流经呼吸道，器官从左到右。 不对称性和精子能动性。每个纤毛由9个坚硬的管状结构组成，称为微管。 双胞胎围绕一对中间的MT排列成一个圆圈。称为外臂动力蛋白的微管马达 (OAD)将MT二元组相对于彼此滑动，而二元组之间的连接器将滑动转换为 弯腰。这种波动是由于纤毛两侧的OAD沿长度交替活动而产生的 纤毛。以前的研究表明，在没有外部线索的情况下，协调仍然存在。模型 提出可以通过对MT曲率和二重线间的变化做出反应来实现OAD协调 打浆时的间距。或者，MT滑动可以通过产生自组织来调节运动活动 震荡。然而，OAD的运动和力量产生的机制仍然缺乏了解 与动力蛋白的胞质亚型进行比较。因此，目前还不清楚哪种模型(S)适用于睫状肌 弯曲 四膜虫OAD基因重组表达的最新进展为我们研究其 第一次是机械师。到目前为止，许多模型预测都是通过细胞质 在结构上与OAD不同的动力蛋白。与细胞质动力蛋白不同，同源二聚体OAD形成 (α，β，和γ)重链的异源三聚体，在生理上不是进行性的。要了解 纤毛中动力蛋白的自我调节机制，我将表征其运动和力的产生特征 并测试MT曲率和滑动对体外OAD活性的影响。 这项研究的目标是通过以下方式直接测试模型做出的特定生物物理预测 三个核心目标：首先，我们将测试OAD的分子特性，如电机步进的协调 沿MT和力诱导的MT附着/脱离MT。第二，我们将在 在一个跳动的纤毛中模拟动力蛋白/MT相互作用的几何形状的体外试验。第三，我们将使用已解决的 对轴丝动力蛋白和细胞质动力蛋白的结构进行定向突变以鉴定结构 OAD的组成部分，导致其非进行性运动、曲率感应和振荡行为。 这项工作将为分子的研究建立一个实验和理论框架 OAD的机制，并使我们能够确定自协调振荡的最低要求 活动的纤毛。

Project Summary Motile cilia are hair-like protrusions from the cell surface that beat in a sinusoidal waveform to produce movement. This movement is responsible for the flow of mucus through the respiratory tract, organ left-right asymmetry, and sperm motility. Each cilium is composed of nine rigid tubular structures called microtubule (MT) doublets arranged in a circle around a single central pair of MTs. Microtubule motors called outer arm dyneins (OADs) slide the MT doublets relative to one another while connectors between doublets convert the sliding into bending. The wave motion is generated as OADs on opposite sides of the cilia alternate activity down the length of the cilium. Previous studies have shown that coordination persists in the absence of external cues. Models propose that OAD coordination can be achieved by responding to changes in MT curvature and interdoublet spacing during beating. Alternatively, MT sliding can regulate motor activity by generating self-organized oscillations. However, the mechanism of OAD motility and force generation remains poorly understood in comparison to the cytoplasmic isoform of dynein. Therefore, it remains unclear which model(s) apply to ciliary bending The recent development of the recombinant expression of Tetrahymena OAD allows us to study its mechanics for the first time. Thus far, many of the model predictions have been tested by using cytoplasmic dynein which is structurally distinct from OAD. In contrast to cytoplasmic dynein, a homodimer, OAD forms a heterotrimer of (α, β, and γ) heavy chains and is not processive at physiological ATP. To understand the mechanism of dynein self-regulation in cilia, I will characterize the motility and force generation characteristics of single OAD motors, and test how MT curvature and sliding impact OAD activity in vitro. The goal of this study is to directly test the specific biophysical predictions made by the models through three core aims: First, we will test the molecular properties of OADs, such as coordination of motor stepping along MTs and force-induced MT attachment/detachment of dynein from the MT. Second, we will construct in vitro assays that mimic the geometries of dynein/MT interactions in a beating cilium. Third, we will use solved structures for axonemal dynein and cytoplasmic dynein to make directed mutations to identify the structural components of OAD that gives rise to its nonprocessive motility, curvature sensing, and oscillatory behavior. This work will establish an experimental and theoretical framework for the study of the molecular mechanism of OAD and enable us to determine the minimum requirements for self-coordinated oscillation of motile cilia.