Regulation of protein transport in cilia

纤毛中蛋白质运输的调节

基本信息

批准号：
10356914
负责人：
Karl Lechtreck
金额：
$ 31.5万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-10 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10356914
关键词：
Affect Age Antibodies Bardet-Biedl Syndrome Binding Binding Sites Biochemical Blindness Carrier Proteins Cell membrane Cells Chlamydomonas Chlamydomonas reinhardtii Chronic Cilia Complex Data Defect Deterioration Development Diffusion Disease Ensure Flagella Fluorescence Resonance Energy Transfer Frequencies Goals Harvest Heterogeneity Human Impairment In Vitro Joubert syndrome Length Lipids Liquid substance Maintenance Male Infertility Maps Masks Membrane Membrane Lipids Membrane Proteins Microtubules Modeling Modification Monitor Motor Mutation Obesity Organelles Pathway interactions Pattern Performance Phospholipase D Positioning Attribute Protein Export Pathway Protein Import Protein Precursors Proteins Regulation Research Respiratory Tract Infections Ribosomes Role Route Sensory Signal Transduction Structural Protein Testing Time Training Travel Tubulin Work base carbonate dehydratase cell motility ciliopathy cilium biogenesis in vivo in vivo imaging insight interest mutant particle protein transport tool

项目摘要

Project Summary Cilia and flagella are conserved microtubule-based cell extensions present on most cells in the mammalian body. In addition to their role in cell locomotion and fluid transport, cilia participate in cellular sensing and signaling. Over the past two decades, it has been established that numerous developmental anomalies and diseases are caused by dysfunctional cilia. The goal of our work is to understand how cells assemble and maintain cilia, which both require protein transfer between the cell body and the organelle. A key mechanism that determines the protein content of cilia is intraflagellar transport (IFT), a motor-based motility of large carriers (“IFT trains”) that move proteins in and out of cilia. We will use Chlamydomonas reinhardtii as a unicellular model to determine how IFT identifies proteins destined for the cilium and how the cells regulate the volume and timing of ciliary protein traffic. In Aim1, we will focus on the transport of tubulin, the main structural protein of cilia and flagella. The amount of tubulin and other axonemal proteins entering cilia on IFT trains is upregulated while cilia grow. Tubulin also enters cilia by diffusion and we will establish the quantitative contribution of each route in cilia assembly. We will determine if IFT54 is part of the previously characterized IFT74-IFT81 tubulin-binding module or if it forms an independent tubulin-binding site. All three proteins interact with tubulin via their tubulin-binding domains (TBDs). Isolated IFT complexes will be used to study if the TBDs undergo biochemical changes related to cargo binding and cilia length. We will attempt to map the TBDs on isolated IFT particles and we will study whether IFT particles undergo structural changes inside cilia potentially explaining the differences in cargo binding. We expect to gain insights into how cells regulate tubulin transport, which is critical for the timing of ciliogenesis and the regulation of ciliary length. In Aim 2, we will focus on the transport of proteins associated to the ciliary membrane by lipidation. Such proteins are critical for the sensory and signaling functions of cilia. Often, they enter and exit cilia to modulate signaling but the role of IFT in this traffic is mostly unknown. In cilia of C. reinhardtii mutants in BBS proteins or Arl13b, the patterns of membrane-associated proteins are severely affected. In humans, mutations in BBS proteins and Arl13b result in Bardet-Biedl syndrome (BBS) and Joubert syndrome, respectively. Both mutants show loss and abnormal accumulation of membrane-associated proteins in cilia, raising the question whether more than one route of transport is affected. We will use in vivo imaging to determine the role of IFT and diffusion in ciliary entry and export of proteins mislocalized in these mutants. We will test a hypothesis that initial ciliary defects caused directly by the bbs and arl13b mutations will induce additional biochemical defects increasingly impairing cilia over time.

项目摘要纤毛和鞭毛是在大多数细胞上都存在的基于微管的细胞扩展哺乳动物的身体。除了它们在细胞运动和液体转运中的作用外，纤毛还参与细胞传感和信号传导。在过去的二十年中，已经确定了许多发展异常和疾病是由功能失调的纤毛引起的。我们工作的目标是了解如何细胞组装并维持纤毛，这两者都需要蛋白质在细胞体之间转移细胞器。确定纤毛蛋白质含量的关键机制是flagellar transpers（ift），a 大型载体（“ IFT列车”）的基于运动的运动，将蛋白质移入和退出纤毛。我们将使用衣原体reinhardtii是一个单细胞模型，以确定IFT如何识别注定的蛋白质纤毛以及细胞如何调节纤毛蛋白质流量的体积和时机。在AIM1中，我们将集中精力关于小管蛋白的运输，纤毛和鞭毛的主要结构蛋白。微管蛋白和在纤毛生长的同时，对进入IFT火车上纤毛的其他轴突蛋白进行了更新。微管蛋白也进入纤毛通过扩散，我们将建立纤毛组装中每条路线的定量贡献。我们将确定IFT54是否是先前表征的IFT74-IFT81微管蛋白结合模块的一部分，还是它是否是形成一个独立的小管蛋白结合位点。所有三种蛋白质都通过小管蛋白结合与微管蛋白相互作用域（TBD）。分离的IFT复合物将用于研究TBD是否发生生化变化与货物结合和纤毛长度有关。我们将尝试在孤立的IFT颗粒上绘制TBD，然后我们将研究IFT颗粒是否经历纤毛内部的结构变化可能解释货物结合的差异。我们希望能够了解细胞如何调节小管蛋白的转运，即纤毛生成的时间和纤毛长度的调节至关重要。在AIM 2中，我们将专注于通过脂质化与睫状膜相关的蛋白质的转运。这种蛋白质对纤毛的感觉和信号传导功能。通常，他们进入并退出纤毛以调节信号，但角色此流量中的IFT大多未知。在BBS蛋白或ARL13B中的Reinhardtii突变体的纤毛中膜相关蛋白的模式受到严重影响。在人类中，BBS蛋白中的突变和ARL13B分别导致Bardet-Biedl综合征（BBS）和Joubert综合征。两个突变体显示纤毛中膜相关蛋白的损失和异常积累，提出了一个问题是否影响多种运输途径。我们将使用体内成像来确定 IFT和扩散在这些突变体中被错误定位的纤毛进入和输出。我们将测试假设直接由BB和ARL13B突变引起的初始睫状缺陷将引起随着时间的推移，纤毛障碍损害的其他生化缺陷会增加。