权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular Analysis of Flagellar Dynein Function

鞭毛动力蛋白功能的分子分析

基本信息

批准号：
9914102
负责人：
Stephen M King
金额：
$ 40.72万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-05-01 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9914102
关键词：
Address Alleles Animal Model Binding Biochemical Biochemical Genetics Biology Biophysics Chlamydomonas Cilia Complex Coupling Cytoplasm Defect Development Docking Dynein ATPase Epithelial Epithelium Failure Feedback Female infertility Fertility Flagella Generations Genes Genetic Genetic Diseases Health Heart Abnormalities Homeostasis Human Human Development Human Genetics Hydrocephalus Infertility Lead Location Male Infertility Mechanics Methods Microtubules Molecular Molecular Analysis Molecular Chaperones Molecular Motors Monitor Motor Motor Activity Movement Organelles Organism Output Pattern Phenotype Play Point Mutation Polymers Primary Ciliary Dyskinesias Process Protein Engineering Proteins RNA Interference Role Sensory Site Situs Inversus Slide Structure Syndrome System Testing Theoretical model WD Repeat arm base biophysical properties biophysical techniques cell motility ciliopathy cilium motility design experience fluid flow insight leucine-rich repeat protein male mutant novel prefoldin sperm cell

项目摘要

Motile cilia and flagella play key roles in development, fertility, and organismal homeostasis; in humans, defects result in a broad array of phenotypes such as male/female infertility, hydrocephalus, severe bronchial problems and heart malformations. Cilia contain more than 700 distinct protein components and indeed, more than 5% of all human genes are involved in the assembly or function of these motile/sensory organelles. Ciliary motility is powered by the highly complex inner and outer dynein arm motors whose activity results in inter-doublet microtubule (MT) sliding and ciliary beating. However, the molecular mechanisms by which dyneins and other ciliary subsystems are pre-assembled in cytoplasm, docked at specific axonemal locations, and how their activity is controlled by the mechanical state or curvature of the axoneme to generate and propagate specific waveforms remain very unclear. In this proposal we will address key aspects of these fundamental problems in ciliary biology using two model organisms with very complementary attributes: Chlamydomonas will be used for genetic/biochemical and structural approaches, whereas RNAi methods in planaria will be employed to assess the function of novel factors in the context of a ciliated epithelium where thousands of motile cilia are synchronized through hydrodynamic coupling. We recently found that a WD-repeat protein (WDR92), which interacts with a prefoldin-like co-chaperone complex, is necessary to build fully functional motile cilia; lack of WDR92 results in axoneme assembly defects including missing dynein arms, incomplete outer doublet MTs and failure of the central pair complex to form. In Aim 1 we will use biochemical methods in Chlamydomonas to identify WDR92-interacting components in cytoplasm and then test their role in ciliary formation and function in planaria, as this will provide new paradigms for understanding how cytoplasmic factors influence the coordinate assembly of axonemal substructures. Once trafficked into the ciliary compartment, assembling outer arm dyneins at precise locations is a multi-factorial process that requires both specific docking proteins within the axonemal superstructure and soluble components in the ciliary matrix. In Aim 2, we will use biochemical/structural methods to define the mechanistic roles of two essential components in the precisely patterned assembly of the outer dynein arm that is absolutely critical for building a fully functional organelle. Axonemal dyneins must sense and respond to the curvature that they experience in order for regions of active sliding to oscillate across the structure and to propagate a wave of motor activity along the organelle generating a ciliary beat. We have predicted that the leucine-rich repeat protein LC1 which binds MTs and also associates with the MT-binding domain of one dynein heavy chain is key to this mechano-switching. In Aim 3, we will use a newly available LC1 null mutant to rigorously test these mechanistic hypotheses by expressing mutant versions of LC1 designed based on our biochemical/NMR structural studies. This will provide direct mechanistic insight into a conserved dynein regulatory system that is fundamental to the generation and propagation of ciliary beats.

能动纤毛和鞭毛在发育、生育和生物体内平衡中起着关键作用;在人类中，导致广泛的表型，如男性/女性不育、脑积水、严重的支气管问题和心脏畸形。纤毛含有超过700种不同的蛋白质成分，事实上，超过5%的所有人类基因都参与这些运动/感觉细胞器的组装或功能。纤毛运动是由高度复杂的内部和外部动力蛋白臂马达提供动力，其活动导致双联体间微管（MT）滑动和纤毛跳动。然而，动力蛋白和其他蛋白质的分子机制，纤毛子系统预先组装在细胞质中，停靠在特定的轴丝位置，以及它们的活动如何由轴丝的机械状态或曲率控制以产生和传播特定波形仍然非常不清楚。在本建议中，我们将解决这些睫状体疾病的基本问题的关键方面。生物学使用两种具有非常互补属性的模式生物：衣原体将用于遗传/生物化学和结构方法，而涡虫中的RNAi方法将用于评估新因子在纤毛上皮中的功能，其中数千根运动纤毛通过液力偶合器同步。我们最近发现，WD重复蛋白（WDR 92），与前折叠蛋白样辅助分子伴侣复合物相互作用，是建立功能齐全的运动纤毛所必需的;缺乏 WDR 92导致轴丝组装缺陷，包括动力蛋白臂缺失、不完整的外双联体MT和中心对复合物形成的失败。在目标1中，我们将使用衣原体的生化方法，鉴定细胞质中WDR 92相互作用组分，然后测试它们在纤毛形成和功能中的作用， planaria，因为这将为理解细胞质因子如何影响坐标提供新的范例轴丝亚结构的组装。一旦进入睫状室，组装外臂动力蛋白在精确位置的定位是一个多因素的过程，需要在细胞内的特定对接蛋白，纤毛基质中的轴丝超结构和可溶性成分。在目标2中，我们将使用生物化学/结构方法来定义两个基本组成部分的机械作用，外部动力蛋白臂的模式化组装，这对于构建功能齐全的细胞器至关重要。轴丝动力蛋白必须感知并响应它们所经历的弯曲，以使活跃的区域滑动以在结构上振荡并沿着细胞器传播运动活动波，纤毛的跳动我们已经预测，富含亮氨酸的重复蛋白LC 1结合MT，也与MT相关，与一个动力蛋白重链的MT结合结构域是这种机械转换的关键。在目标3中，我们将使用一种新的LC 1无效突变体，通过表达突变体版本来严格测试这些机制假设。的LC 1设计的基础上，我们的生化/NMR结构研究。这将提供直接的机械洞察力进入保守的动力蛋白调节系统，该系统是纤毛搏动的产生和传播的基础。