Regulation of protein transport in cilia

项目摘要 纤毛和鞭毛是保守的微管为基础的细胞延伸存在于大多数细胞中， 哺乳动物的身体除了它们在细胞运动和液体运输中的作用之外，纤毛还参与细胞的运动和液体运输。 传感和信号。在过去的二十年里，已经确定，许多发展中国家 异常和疾病是由纤毛功能障碍引起的。我们工作的目标是了解 细胞组装和维持纤毛，这两者都需要在细胞体和纤毛之间进行蛋白质转移。 细胞器决定纤毛蛋白质含量的一个关键机制是鞭毛内转运（IFT）， 大载体（“IFT列车”）的基于马达的运动性，其将蛋白质移入和移出纤毛。我们将使用 莱茵衣原体作为单细胞模型，以确定IFT如何识别蛋白质， 纤毛以及细胞如何调节纤毛蛋白运输的量和时间。在AIM 1中，我们将专注于 微管蛋白是纤毛和鞭毛的主要结构蛋白。微管蛋白和 当纤毛生长时，在IFT序列上进入纤毛的其它轴丝蛋白被上调。微管蛋白也进入 纤毛的扩散，我们将建立定量的贡献，每一个路线纤毛组装。我们将 确定IFT 54是否是先前表征的IFT 74-IFT 81微管蛋白结合模块的一部分，或者是否 形成独立的微管蛋白结合位点。所有三种蛋白质都通过它们的微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 与货物捆绑和纤毛长度有关。我们将尝试在孤立的IFT粒子上绘制TBD， 我们将研究IFT颗粒是否在纤毛内发生结构变化，从而可能解释 货物捆绑的差异。我们希望深入了解细胞如何调节微管蛋白的运输，这是 对于纤毛发生的时间和纤毛长度的调节至关重要。在目标2中，我们将重点关注 通过脂化作用将蛋白质转运至睫状体膜。这些蛋白质对于 纤毛的感觉和信号功能。通常，它们进入和退出纤毛以调节信号传导，但它们的作用 在这种交通中的IFT大多是未知的。在C. BBS蛋白或Arl 13 b中的莱茵氏突变体， 膜相关蛋白的模式受到严重影响。在人类中，BBS蛋白质的突变 和Ar 113 b分别导致Bardet-Biedl综合征（BBS）和Joubert综合征。两种突变体 显示纤毛中膜相关蛋白的丢失和异常积累，提出了一个问题， 是否有超过一条运输路线受到影响。我们将使用体内成像来确定 在这些突变体中错误定位的蛋白质在纤毛进入和出口中的IFT和扩散。我们将测试 假设由BBS和ARL 13 B突变直接引起的初始纤毛缺陷将诱导 随着时间的推移，额外的生化缺陷越来越多地损害纤毛。

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.