Though the basic building blocks of the cytoskeleton were first identified more than fifty years ago, there remains much to learn about how cytoskeletal dynamics impact cellular function, and ultimately contribute to cellular homeostasis and human disease. In this project, we have sought to determine how a microtubule-associated protein, the dynein arm assembly factor Coiled-coiled domain-containing protein 103 (CCDC103) contributes to the normal maturation and effector function of human and zebrafish neutrophils by secondarily impacting α-tubulin lysine 40 (K40) acetylation, a critical post-translational modification affecting microtubule stability.
CCDC103 plays a critical role in the proper assembly of the axonemal dynein complex, and, which mutated, causes a disorder known in humans as Primary Ciliary Dyskinesia or PCD. Interestingly, studies have shown that myeloid cells from PCD patients have multiple functional and cytostructural defects, even though myeloid cells themselves do not possess motile cilia. We have previously shown that ccdc103 mutations are sufficient to disrupt neutrophil and macrophage migration and proliferation in zebrafish, mostly likely through its well-characterized role as a microtubule stabilizing factor.
Additionally, we identified multiple novel CCDC103 binding partners that may support its function, including the flagellar central-apparatus protein SPAG6 and the NAD-dependent deacetylase SIRT2, and showed that PCD-associated CCDC103 mutations abrogate interactions with both of these proteins. Zebrafish spag6 mutant embryos display multiple phenotypic hallmarks of motile cilia dysfunction, including low fertility and situs inversus, and recapitulate many of the same proliferation, morphology, and directed migration capacity defects also observed in ccdc103 mutants, suggesting that SPAG6 acts together with CCDC103 to stabilize cytoplasmic microtubules.
Conversely, SIRT2 is a key deacetylase of α-tubulin, a post-translational modification that increases microtubule resistance to mechanical stress, and so we set out to test the hypothesis that CCDC103 promotes neutrophil migration and effector functions by regulating SIRT2 activity. Here, we show that zebrafish ccdc103 mutant neutrophils have dysregulated levels of K40 acetylated TUBA1a, and that both the SIRT2-specific inhibitor AGK2 and CCDC103 overexpression are sufficient to restore normal α-tubulin acetylation.
Additionally, we show that increasing α-tubulin acetylation levels in differentiated CD34+ HSCs with AGK2 treatment increases the expression of neutrophil maturation and functional markers, and increases their directed migration capacity. Furthermore, in accordance with previous work showing that autophagy is critical for neutrophil differentiation, we show that increasing TUBA1A acetylation accelerates the fusion and formation of mature autolysosomes, thereby promoting neutrophil maturation.
Taken together, these results support the further exploration of the cilia-independent roles of motile cilia-associated proteins such as CCDC103, which may play hitherto un-appreciated roles in coordinating microtubule dynamics, cell metabolism, differentiation, and effector function.