The Role of SPAG1 in the Cytoplasmic Assembly of Axonemal Dynein Arms in Human Airway Epithelia

Motile cilia are microtubule-based hair-like organelles that extend from the apical surface of cells and are essential for the propulsion of fluid or cells across the respiratory, ependymal, oviduct, and nodal epithelium. Dysfunctional motile cilia, due to genetic variants in proteins associated with the structure, regulation, and biogenesis of motile cilia, result in primary ciliary dyskinesia (PCD), a genetically heterogeneous, rare disorder characterized by chronic oto-sino-pulmonary disease, infertility, and laterality defects. The most common ciliary defect seen in PCD is absence of the multiprotein motor complexes known as axonemal dynein arms. While the composition and regulation of dynein arms have been extensively studied, how these large, complex structures are assembled remains poorly understood. Mutations in the gene sperm-associated antigen 1 (SPAG1) cause PCD with defects in both the inner and outer dynein arms, resulting in completely immotile cilia. Due to the observed dynein defects and the cytoplasmic location of SPAG1, it has been proposed to be a dynein axonemal assembly factor (DNAAF) that facilitates the assembly of dynein arms in the cytoplasm before their transport into the cilium. To further elucidate SPAG1’s role in dynein assembly, we examined its expression, interactions, and ciliary defects caused by its absence in human airway epithelia. A previously uncharacterized 60-kDa isoform of SPAG1 was discovered. The full-length and 94-kDa isoforms of SPAG1 expression corresponded with ciliogenesis, while the 60-kDa isoform was constitutively expressed. Immunoprecipitations showed that SPAG1 interacts with multiple DNAAFs, dynein chains, and canonical components of the R2TP complex, and we identified protein interaction domains between SPAG1 and another DNAAF, DNAAF2. Examination of PCD subjects with an atypical ultrastructural defect for SPAG1 variants demonstrated that the 60-kDa SPAG1 can partially compensate for the absence of full-length SPAG1 to assemble a reduced number of outer dynein arms. In SPAG1 mutants, dynein heavy chain (DHC) protein levels and their interaction with dynein intermediate chains (DICs) were reduced. In summary, our data demonstrate that SPAG1 is necessary for axonemal dynein arm assembly by scaffolding R2TP-like complexes composed of several DNAAFs that facilitate the folding of the DHCs and/or binding of the DHCs to the DIC complex.Doctor of Philosoph