The tubulin code and its roles in regulation of ciliary transport

微管蛋白密码及其在纤毛运输调节中的作用

• 批准号: RGPIN-2022-04774
• 负责人: Bui, KhanhHuy
• 金额: $ 2.91万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=743444
• 关键词: tubulin; code; its; roles; regulation

BACKGROUND Cilia are complex organelles responsible for cell motility and sensory function and present in almost all human cell types. The primary structure of a cilium is the axoneme, comprised of radially interlinked doublet microtubules. Doublet microtubule serves as the skeleton for the cilium, giving support and acting as a track for ciliary transport. The function of the doublet microtubule is regulated through associated proteins and the "tubulin code", i.e., specific post-translational modifications (PTM) of tubulins. During ciliary assembly and maintenance, kinesin and dynein motors carry cargos and move along the doublet microtubule in anterograde (towards tips) and retrograde (towards base) directions, respectively. It has been shown that anterograde and retrograde transports run on different regions of the doublet microtubule, i.e., separate train tracks. Little is known about how the motors are regulated to ensure orderly traffic and proper assembly of the cilium. OBJECTIVES The objective of the proposed research is to understand the molecular mechanism for ciliary transport, particularly the regulation of anterograde and retrograde motors by the tubulin code. The specific aims are: Aim 1. Effect of PTM on doublet microtubule structure. We will obtain the ex vivo cryo-electron microscopy structures of the doublet microtubule from wild type and PTM deficient mutants; thereby providing fundamental insights into the structural alterations of the doublet microtubule with PTM deficiency. Aim 2. Structural and motility analysis of molecular motors on doublet microtubule. We will characterize the structure, interaction, and motility of kinesin-2 and dynein-2 binding to the doublet microtubule by cryo-electron microscopy and single-molecule fluorescence microscopy. Structures will provide the molecular interactions of motors with the doublet microtubule in different PTM states. At the same time, single-molecule fluorescence microscopy will characterize the motility of ciliary transport on wild type and PTM deficient mutants. Aim 3. Molecular mechanism of kinesin and dynein stepping on doublet microtubule. Here, we will investigate the details of how kinesins and dyneins move on the doublet microtubule under different tubulin codes by molecular dynamics. This computational approach allows us to understand how the specific post-translational modifications regulate the selectivity and motility in anterograde and retrograde transport. SIGNIFICANCE This proposed project presents an approach that bridges from atomic details of tubulins under different tubulin codes to the motility of molecular motors during ciliary transport. Understanding the regulation of the tubulin code will give principal insights into how biological systems can be modulated in a simple yet elegant way. Our expected outcome will provide us with a basic understanding of the regulation of ciliary functions and assembly and hence, insights into assembly-related aberrance.

背景纤毛是一种负责细胞运动和感觉功能的复杂细胞器，几乎存在于所有类型的人类细胞中。纤毛的初级结构是轴丝，由放射状相连的双微管组成。双微管是纤毛的骨架，起着支撑纤毛运输的作用。二联体微管的功能是通过相关蛋白和“微管蛋白密码”，即微管蛋白的翻译后修饰(PTM)来调节的。在纤毛组装和维持过程中，动蛋白和动力蛋白马达携带货物，并沿二重微管沿顺行(朝尖)和逆行(朝基)方向移动。已证明顺行和逆行运输在双线微管的不同区域上运行，即独立的列车轨道。关于如何调节马达以确保有序的交通和纤毛的正确组装，人们知之甚少。目的了解纤毛运输的分子机制，特别是微管蛋白编码对顺行和逆行运动的调控。其具体目的是：1.PTM对双微管结构的影响。我们将获得来自野生型和PTM缺陷突变体的二联体微管的体外冷冻电子显微镜结构，从而为研究PTM缺陷的二联体微管的结构变化提供基本的见解。目的2.双微管分子马达的结构和运动学分析。我们将通过冷冻电子显微镜和单分子荧光显微镜来表征与双线微管结合的动蛋白-2和动力蛋白-2的结构、相互作用和运动性。结构将提供发动机与处于不同PTM状态的双线微管的分子相互作用。同时，单分子荧光显微镜将表征野生型和PTM缺陷型突变体纤毛运输的运动性。目的3.激动素和动力蛋白踩在双线微管的分子机制。在这里，我们将用分子动力学的方法研究在不同的微管蛋白编码下，动蛋白和动力蛋白如何在微管上运动的细节。这种计算方法使我们能够理解特定的翻译后修饰如何调节顺行和逆行运输中的选择性和运动性。意义这个拟议的项目提出了一种方法，将不同微管蛋白编码下的微管蛋白的原子细节与纤毛运输过程中分子马达的运动性联系起来。了解微管蛋白密码的调节将使我们对生物系统如何以一种简单而优雅的方式进行调制提供基本的见解。我们的预期结果将使我们对纤毛功能和组装的调节有一个基本的了解，从而对组装相关的异常有深入的了解。