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 运动纤毛是细胞表面的毛发状突起，以正弦波形跳动， 运动这种运动是负责流动的粘液通过呼吸道，器官左-右 不对称性和精子活力。每根纤毛由9个刚性管状结构组成，称为微管（MT） 在单个中心对MT周围的圆中排列的双峰。称为外臂动力蛋白的微管马达 （OAD）使MT双合件相对于彼此滑动，而双合件之间的连接器将滑动转换成 弯曲。波动是由纤毛两侧的OAD沿着长度交替活动产生的 的纤毛。先前的研究表明，协调在没有外部线索的情况下仍然存在。模型 提出OAD协调可以通过响应MT曲率和互偶极子的变化来实现 在打浆过程中的间隔。或者，MT滑动可以通过产生自组织的运动来调节运动活动。 振荡然而，OAD运动性和力产生的机制仍然知之甚少， 与动力蛋白的细胞质同种型比较。因此，仍不清楚哪种模型适用于睫状体 弯曲 四膜虫OAD重组表达的最新进展使我们能够研究其在细胞中的表达。 机械师第一次到目前为止，许多模型预测已经通过使用细胞质来测试。 动力蛋白在结构上与OAD不同。与细胞质动力蛋白（一种同源二聚体）相比，OAD形成了一种 在一些实施方案中，该蛋白是（α、β和γ）重链的异源三聚体，并且在生理ATP下不进行性。了解 动力蛋白在纤毛自我调节的机制，我将描述运动和力量产生的特点 的单个OAD电机，并测试MT曲率和滑动如何影响体外OAD活动。 本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，如运动步进的协调性， 沿着MT和力诱导的MT附着/动力蛋白从MT脱离。第二，我们将在 体外试验，模拟的几何动力蛋白/MT的相互作用，在跳动的纤毛。第三，我们将使用解决 轴丝动力蛋白和细胞质动力蛋白的结构进行定向突变，以确定结构 OAD的组成部分，引起其非进行性运动，弯曲感测和振荡行为。 本工作为分子生物学的研究建立了实验和理论框架 OAD的机制，使我们能够确定自协调振荡的最低要求， 活动纤毛