Mechanoregulation of Ciliary Motility

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Project Summary Mechanoregulation is a fundamental mechanism for the control of dynamic and multiscale biological systems. A mechanoregulatory network is responsible for the motility of cilia by converting the action of thousands of individual dynein motors bound to doublet microtubules in the cilium into a single waveform. This waveform has evolved to efficiently displace fluid, allowing either cell self-propulsion or the transport of extracellular liquid over epithelial surfaces. Ciliary motility in humans is therefore essential for the movement of sperm cells, the removal of bacteria and viruses from the respiratory tract, and the circulation of cerebrospinal fluid in the brain. Cilia are also used by protozoan pathogens for movement, contributing to their pathogenicity. The biflagellate alga, Chlamydomonas reinhardtii, has become the model system for studying the relationship between cilia ultrastructure and ciliary motilty. Using this organism, we have determined single-particle electron cryomicroscopy (cryo-EM) structures of the bases of dyneins and mechanoregulatory complexes natively bound to doublet microtubules. These structures map the interconnected network of microtubules, mechanoregulators, and dynein motors in unparalleled atomic detail. The structures reveal the mechanisms that dock mechanoregulators to doublet microtubules and generate new hypotheses for how they control dynein behavior. These preliminary structural studies provide a unique opportunity to better understand the structure, function and assembly pathway of the largest mechanoregulator, the radial spoke. In aim 1, I propose to elucidate the complete structure of a native radial spoke using cryo-EM and cross-linking mass spectrometry. Structural information will resolve how its 20+ unique subunits interact and function together to respond to both mechanical and chemical signals. Due to the high conservation of radial-spoke subunits among organisms, our structure will provide insights into the etiology of ciliopathy-causing mutations in humans. In aim 2, I propose to test hypotheses that have arisen from our “on-doublet” structures using an interdisciplinary combination of structure-guided mutagenesis, waveform analysis by high-speed microcinematography, and structural characterization using electron cryotomography. This work will provide experimental evidence for the fundamental molecular mechanisms that control ciliary motility. In aim 3, I propose to use a proteomic and structural approach to determine the mechanisms of radial-spoke assembly. This work will test our current structure-based model of assembly and has the potential to identify the first radial-spoke biogenesis factors. Collectively, these studies will provide unprecedented mechanistic insight into the mechanoregulatory pathways that control ciliary motility and promises to open new avenues for the treatment of ciliopathies.