The tubulin code and its roles in regulation of ciliary transport

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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.