Project Summary: Microtubules (MTs) and the structures they form play essential roles in eukaryotic cells. Best known as dynamic polymers assembled from a/b-tubulin heterodimers, MTs are absolutely required in numerous cellular processes, including mitosis. Many of these activities depend on dynamic MT behavior, but there are critical cellular functions that require stable microtubules. Stable singlet MTs in neurons act as tracks for axonal transport, stable doublet MTs in axonemes generate force in cilia, and stable triplet MTs are found in centrioles and basal bodies that organize centrosomes and cilia, respectively. Despite their importance, we know little of how stable MT-based structures are assembled, maintained, and disassembled. Because the same tubulin dimers assemble dynamic and stable MTs in most organisms, including the ciliate Tetrahymena thermophila, the different MT behaviors are attributed to associated proteins and protein modifications. In doublet and triplet MTs, some associated proteins are found inside the hollow MT; these microtubule inner proteins (MIPs) are the focus of our work. Originally discovered using various forms of electron microscopy, MIPs appeared as structures of unknown composition inside axonemal doublet microtubules. MIPs are proposed to mitigate the deformation and stress on doublet MTs caused by ciliary beating. Ciliary beating moves extracellular fluid in a single direction, which is necessary for many essential processes, such as clearing mucus from airways, facilitating the movement of eggs in the fallopian tube, and generating cerebrospinal fluid flow in the brain. Structurally analogous to the motile cilium, the flagellum is required for sperm motility. Defects disrupting motile cilia cause a wide range of human pathologies, including primary ciliary dyskinesia (PCD), hydrocephalus, and infertility in both sexes. Understanding of how ciliary defects lead to motility problems and disease is limited. Previously, we identified Rib72A and Rib72B in Tetrahymena cilia as MIPs required for normal cilia beating. Comparative proteomic analyses of axonemes isolated from wild type and rib72A-, rib72B- null cells identified additional MIPs, such as Fap115 and Calciphosin-like protein, whose assembly is defective in the mutants. We further characterized Fap115 and showed it to be essential for normal cell motility and axoneme stability. Meanwhile, by comparing the doublet MT structures of Tetrahymena, Chlamydomonas reinhardtii, and Bos taurus, we find both conservation and diversity of MIPs in these evolutionarily distant organisms, revealing essential and divergent functions. The long-term goal of this project is to use biophysical, genetic, and advanced microscopy tools to better understand the function and assembly mechanisms of motile cilia. To do this, we plan to identify Tetrahymena MIPs in both axonemal doublet and basal body triplet MTs, to map protein interactions that drive MIP localization and assembly,...