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，存在于几乎所有人类细胞类型中。纤毛的主要结构是轴突，由径向相连的双线微管组成。双微管作为纤毛的骨架，为纤毛的运输提供支持和轨道。双态微管的功能是通过相关蛋白和“微管蛋白编码”，即微管蛋白的特异性翻译后修饰（PTM）来调节的。在纤毛装配和维护过程中，驱动蛋白和动力蛋白电机分别沿双态微管顺行（向尖端）和逆行（向基部）方向携带货物和移动。研究表明，顺行和逆行运输运行在双态微管的不同区域，即分开的列车轨道上。人们对这些马达是如何调节以确保有序的交通和纤毛的适当组装知之甚少。本研究的目的是了解纤毛转运的分子机制，特别是微管蛋白代码对顺行和逆行马达的调节。具体目标是：目标1。PTM对双态微管结构的影响。我们将获得野生型和PTM缺陷突变体双态微管的离体低温电镜结构；从而为PTM缺乏的双态微管的结构改变提供了基本的见解。目标2。双态微管分子马达的结构与运动特性分析。我们将通过低温电子显微镜和单分子荧光显微镜来表征动力蛋白-2和动力蛋白-2结合到双态微管的结构、相互作用和运动性。结构将提供马达与双态微管在不同PTM状态下的分子相互作用。同时，单分子荧光显微镜将表征野生型和PTM缺陷突变体纤毛运输的运动性。目标3。动力蛋白和动力蛋白作用于双态微管的分子机制。在这里，我们将通过分子动力学研究在不同微管蛋白编码下，动力蛋白和动力蛋白如何在双态微管上移动的细节。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。本项目提出了一种从不同微管蛋白编码下的微管的原子细节到纤毛运输过程中分子马达运动的桥梁方法。了解微管蛋白编码的调控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节有更深入的了解。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，从而深入了解与组装相关的异常。

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.