Identifying the Mechanism for Outer Arm Dynein Coordination in Ciliary Motion

确定睫状运动中外臂动力蛋白协调的机制

• 批准号: 10294939
• 负责人: Ruensern Tan
• 金额: $ 4.62万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10294939
• 关键词: Address; Affect; Behavior; Biophysics; Cell Surface Extensions; Characteristics; Cilia; Collaborations; Cryoelectron Microscopy; Cues; DNA; Defect; Development; Disease; Dynein ATPase; Engineering; Face; Feedback; Filament; Generations; Geometry; Goals; Gur; Hair; Head; In Vitro; Individual; Informal Social Control; Investigation; Label; Left; Length; Link; Liquid substance; Male Infertility; Mechanics; Mediating; Membrane; Methods; Microtubules; Modeling; Modification; Molecular; Molecular Conformation; Molecular Motors; Motion; Motor; Motor Activity; Movement; Mucous body substance; Mutate; Mutation; Organ; Organelles; Pattern; Physiological; Primary Ciliary Dyskinesias; Property; Protein Isoforms; Recombinants; Regulation; Research; Respiratory System; Side; Signal Transduction; Slide; Specialist; Sperm Motility; Stretching; Structure; Structure-Activity Relationship; Surface; Testing; Tetrahymena; Theoretical Studies; Therapeutic; Time; Tubular formation; Work; arm; biophysical properties; cell motility; ciliopathy; cilium motility; human disease; improved; in vitro Assay; in vitro activity; insight; molecular dynamics; molecular imaging; nanofabrication; optical traps; predictive modeling; programs; scaffold; simulation; single molecule; success

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.

项目摘要 纤毛是来自细胞表面的头发蛋白，在正弦波形中跳动以产生 移动。该运动是导致粘液通过呼吸道的左右右翼流动的 不对称和精子运动。每个纤毛由九个称为微管（MT）的刚性管状结构组成 双子围绕一对中央对MT的圆圈排列。微管电动机称为外臂动力蛋白 （OADS）将MT互相滑动，而双打之间的连接器将滑动转换为 弯曲。波动是在纤毛替代活动的相对侧面的oad产生的 纤毛。先前的研究表明，在没有外部线索的情况下，协调持续存在。型号 建议通过响应MT曲率和双重变化来实现OAD协调 跳动过程中的间距。另外，MT滑动可以通过产生自组织来调节运动活动 振荡。但是，OAD运动和力产生的机制在 与动力蛋白的细胞质同工型进行比较。因此，尚不清楚哪种模型适用于睫状 弯曲 四膜虫OAD重组表达的最新发展使我们能够研究其 机械师是第一次。这远，许多模型预测已经通过使用细胞质进行了测试 在结构上与OAD不同的动力蛋白。与细胞质动力蛋白（一种均二聚体）相反，OAD形成A （α，β和γ）重链的异三聚体，在物理ATP时没有遗传。理解 纤毛中动力蛋白自我调节的机制，我将表征运动和力的产生特征 单OAD电动机，并测试MT曲率和滑动如何在体外影响OAD活性。 这项研究的目的是直接测试模型通过 三个核心目的：首先，我们将测试OAD的分子特性，例如运动步进的协调 沿MT和力诱导的MT附着/脱离了MT的动力蛋白。其次，我们将在 体外测定模仿跳动纤毛中动力蛋白/MT相互作用的几何形状。第三，我们将使用已解决 轴突动力蛋白和细胞质动力蛋白的结构，使有向突变以识别结构 OAD的组成部分产生其非过程的运动，曲率感应和振荡行为。 这项工作将建立一个研究分子的实验和理论框架 OAD的机制，使我们能够确定自我协调振荡的最低要求 纤毛纤毛。