Project Summary/Abstract New genetic and molecular methods have shed increasing light on the pathobiology of ciliopathies, including Primary ciliary dyskinesia (PCD). However, the mechanisms underlying surprising migration defects observed in non-ciliated neutrophils isolated from PCD patients have not been investigated. Mutations in the coiled-coiled domain containing protein 103 (CCDC103), which is a conserved dynein arm assembly factor that can promote microtubule stability, are associated with multiple independent families. We identified ccdc103 in a screen for novel genes expressed in the anteriolateral plate mesoderm of the zebrafish, and subsequent to that screen, it was reported that mutations in CCDC103 could cause PCD in humans. We have shown that ccdc103 expressed in embryonic zebrafish myeloid cell populations, and by utilizing the zebrafish ccdc103 mutant (schmalhans (smh)), an established model for PCD, we found that smh mutants have fewer myeloid cells at 24 hours post- fertilization (hpf). Additionally, both embryonic neutrophils and macrophages were unable to migrate efficiently towards wound sites, findings consistent with reports that Ccdc103 stabilizes microtubules. Using a yeast-two hybrid screen, we identified Spag6 as a potential interacting partner of Ccdc103. SPAG6 has been shown to be critical for cell proliferation and migration, is overexpressed in several myeloid malignancies and myelodysplastic syndromes, and, most interestingly, has been shown to promote microtubule acetylation. Acetylation of the K40 residue of alpha-tubulin is known to be critical for microtubule stability and longevity. We engineered spag6 mutant zebrafish embryos and found they have a decreased number of myeloid cells. Therefore, our results suggest that Ccdc103 may interact with a protein complex that coordinates myeloid cell proliferation and function through affecting microtubule stability independent of its role in motile cilia, which may explain largely overlooked functional defects observed in PCD patient neutrophils. In our first aim, we will determine whether there is an evolutionarily conserved requirement for CCDC103 in human myeloid cells, and if CCDC103 is required for human myeloid proliferation and migration. We will use CRISPR-mediated genome editing of human HL-60 cells and CD34+ HSCs to determine how loss of CCDC103 affects the ability of these cells to grow, migrate, and phagocytose foreign material. Our second aim seeks to establish the molecular mechanism by which CCDC103 stabilizes microtubules by investigating the role of CCDC103 in SPAG6-mediated tubulin acetylation. We will use myeloid-specific overexpression of CCDC103 in embryonic zebrafish myeloid cells in addition to pharmacological inhibitors of tubulin deacetylases to elucidate both the role of CCDC103 in myeloid microtubule acetylation and the role of microtubule acetylation in normal myeloid function. Ultimately, these studies will not only shed light on the unexp...