Regulation of protein transport in cilia

项目摘要 纤毛和鞭毛是一种保守的以微管为基础的细胞延伸，存在于 哺乳动物的身体。纤毛除了在细胞运动和液体运输中发挥作用外，还参与细胞 感知和发送信号。在过去的二十年里，人们已经确定，许多发展 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，这两者都需要细胞体和细胞之间的蛋白质转移 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)，一种 以运动为基础的大型载体(“IFT列车”)的运动，将蛋白质运进运出纤毛。我们将使用 莱茵衣藻作为单细胞模型来确定IFT如何识别目的蛋白 纤毛以及细胞如何调节纤毛蛋白的流量和时间。在Aim1，我们将专注于 纤毛和鞭毛的主要结构蛋白微管蛋白的转运。微管蛋白和 在IFT列车上进入纤毛的其他轴丝蛋白在纤毛生长时上调。微管蛋白也进入 纤毛通过扩散，我们将建立每条路径在纤毛组装中的定量贡献。我们会 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 形成一个独立的微管蛋白结合位点。这三种蛋白质都通过微管蛋白结合与微管蛋白相互作用。 域(待定)。分离的IFT复合体将用于研究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粒子和 我们将研究IFT颗粒是否在纤毛内经历结构变化，这可能解释 货物捆绑方面的差异。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，我们将重点介绍 通过脂化作用运输与睫状膜相关的蛋白质。这类蛋白质对 纤毛的感觉和信号功能。通常，它们进出纤毛是为了调节信号，但作用是 IFT在这一流量中的比例几乎是未知的。在BBS蛋白或Arl13b中突变的莱茵梭菌纤毛中， 膜相关蛋白的模式受到严重影响。在人类中，BBS蛋白的突变 Ar13b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丢失和异常积累，提出了这个问题 是否有多条运输路线受到影响。我们将使用体内成像来确定 这些突变体中错位的蛋白质在纤毛进出中的IFT和扩散。我们将测试一个 假设由Bbs和Ar13b突变直接导致的初始睫状体缺陷将导致 随着时间的推移，额外的生化缺陷会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.