The tubulin code and its roles in regulation of ciliary transport

纤毛是负责细胞运动和感觉功能的复杂细胞器，存在于几乎所有的人类细胞类型中。纤毛的初级结构是轴丝，由径向互连的双线微管组成。双微管作为纤毛的骨架，为纤毛运输提供支持和通道。双微管的功能通过相关蛋白和“微管蛋白密码”调节，即，微管蛋白的特异性翻译后修饰（PTM）。在纤毛的组装和维持过程中，驱动蛋白和动力蛋白马达分别携带货物并沿双微管沿着顺行（朝向尖端）和逆行（朝向基部）方向移动。已经表明，顺行和逆行运输在双重微管的不同区域运行，即，分开的铁轨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。该研究的目的是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马达的调节。具体目标是：目标1。PTM对双联体微管结构的影响。我们将获得野生型和PTM缺陷突变体的双联体微管的离体冷冻电子显微镜结构;从而为PTM缺陷的双联体微管的结构改变提供基本见解。目标二。双态微管分子马达的结构与运动分析。我们将通过冷冻电子显微镜和单分子荧光显微镜来表征驱动蛋白-2和动力蛋白-2与双态微管结合的结构、相互作用和运动性。结构将提供马达与处于不同PTM状态的双态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缺陷突变体上纤毛运输的运动性。目标3。驱动蛋白和动力蛋白对双态微管作用的分子机制。在此，我们将通过分子动力学的方法来研究不同微管蛋白编码下驱动蛋白和动力蛋白在双态微管上的运动。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转运的选择性和运动性。意义这个项目提出了一个方法，桥梁从不同的微管蛋白编码下的微管蛋白的原子细节的纤毛运输过程中的分子马达的运动。了解微管蛋白编码的调节将使我们对生物系统如何以简单而优雅的方式进行调节有了深入的了解。我们预期的结果将为我们提供一个基本的了解纤毛功能和组装的调节，因此，装配相关的畸变的见解。

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.