PROJECT SUMMARY Motile cilia generate fluid flow in organisms ranging from microbes to humans. Defective ciliary function can produce severe pathologies, including infertility, severe respiratory infection, and hydrocephaly. Motile cilia beat in a coordinated fashion to effectively move fluids across cell surfaces. While the role for the microtubule cytoskeleton and dynein activity is well characterized for undulating cilia, the mechanics for how hundreds of cilia in multiciliary arrays beat in a coordinated and connected array remains a significant gap in the field. One critical feature of coordinated ciliary motility is the spatial organization of cilia. The position of cilia is precisely patterned at the cortex by basal bodies (BBs), which are microtubule-based structures that nucleate cilia. BBs anchor to the cell cortex through three BB-appendage structures that asymmetrically extend from BBs. BB-appendages function in maintaining BB organization, as these structures physically bridge neighboring BBs to each other and to the cell cortex. The coupling of BBs together and to the cortex creates a structured matrix to dissipate the forces generated by ciliary beating and to translate such forces to the cell. This is most evident by the finding that loss of cortical actin leads to beating cilia being ripped from the cell cortex. However, how cortical actin anchors BBs to the cell cortex in multiciliated cells has yet to be determined. The ciliate model organism Tetrahymena thermophila is an excellent system to address these gaps in knowledge. These cells have well-characterized BB-appendage connections between neighboring BBs, and between BBs and the cell cortex allowing us to elucidate the role for actin in BB anchoring and BB organization in promoting coordinated ciliary motility. Furthermore, Tetrahymena actin localizes to BBs and cilia, where roles for actin in ciliary beating or coordinated waveforms have yet to be established. Thus, the role of ciliary and cortical localized actin will be elucidated using Tetrahymena. An important aspect of this proposal is to uncover the basic components of these actin networks to begin to understand their differential regulation and contributions to ciliary motility. In addition, the existing ultrastructural understanding of BBs, BB-appendages, cortical contacts and ciliary axonemes provides a foundation for examining actin's requirement for structural integrity and localization within these structures. In this proposal, the role for actin that localizes to cilia and the cell cortex will be determined in promoting coordinated and efficient ciliary motility by utilizing molecular-genetic, cell biological and ultrastructural approaches. These goals will be accomplished according to my detailed research plan, by the exceptional qualifications of Dr. Pearson, my postdoctoral advisory committee and utilizing the facilities and training resources available at the University of Colorado, Anschutz Medical Cam...