Sunday, May 25, 2025
5/25/2025
Structure and Function of Complexes that Regulate Ciliary Motility - PROJECT SUMMARY The overall goal of the proposed research is to provide a better understanding of how cilia move by dissecting the structure and function of ciliary complexes that regulate dynein activity and ciliary motility. Cilia and flagella are conserved and ubiquitous microtubule-based organelles with important roles in cell locomotion, fluid transport, sensation, cell signaling, and development, which are critical processes for the survival and proper function of many eukaryotic cells and tissues. In humans, defects in the motility and assembly of cilia are responsible for numerous congenital diseases, such as primary ciliary dyskinesia, chronic respiratory disease, infertility, brain developmental defects, congenital heart disease, and randomization of the left-right body axis. Our previous studies of both inhibited and actively beating wild type and mutant cilia have opened a new window into the functional organization of motile cilia. However, long-standing fundamental questions remain, for example, about how regulatory signals change dynein’s activity on a molecular level, what are the roles of the different regulatory complexes during ciliary motility, and how dyneins are spatially and temporally coordinated to generate the oscillatory beating typical for cilia. Building on a strong premise of both published and preliminary data, this proposal directly addresses these critical open questions through three specific aims that are directed at (Aim 1) revealing the proteome and near-atomic resolution structure of the full-length radial spoke RS3 in mouse respiratory cilia, (Aim 2) understanding the functional roles of the regulatory ATPase domain of DRC11 – a nexin-dynein regulatory complex subunit – for proper regulation of ciliary motility, and (Aim 3) characterizing ciliary components that assemble only on specific doublets to ascertain if their inherently asymmetric distribution contributes to generating ciliary beating and/or different waveforms. We use a powerful and innovative combination of modern approaches, including a multi-scale imaging approach that combines near-atomic resolution cryo-EM single particle analysis, cryo-electron tomography to image mutant cilia and tagged proteins with molecular resolution, and expansion light microscopy to determine the doublet-specific distribution of specific ciliary proteins. We expect that our combined studies will provide important new conceptual and mechanistic insights into ciliary motility and regulation, which will also impact our understanding of ciliary diseases.
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