ABSTRACT/PROJECT SUMMARY Primary ciliary dyskinesia (PCD) is a genetically heterogenous disorder resulting from dysfunctional motile cilia due to mutations in over 30 different genes discovered thus far. PCD is characterized by chronic pulmonary disease, infertility, laterality defects, and ultimately end-stage respiratory failure. The most common abnormality observed in motile cilia in cases of PCD are absent and/or defective inner and/or outer dynein arms, the protein structures essential for ciliary movement. However, the protein interactions and mechanisms that are necessary for axonemal dynein arm assembly are largely unknown. Previously, mutations in sperm- associated antigen 1 (SPAG1) were discovered to result in static cilia with missing and/or defective inner and outer dynein arms. Previous studies in primary human airway epithelial cell (hAEC) cultures demonstrates that SPAG1's expression is induced during ciliogenesis and SPAG1 localizes primarily in the cytoplasm and near basal bodies, but not in the ciliary axoneme. Immunoprecipitation (IP) studies for SPAG1 in primary hAEC lysates identified two known dynein axonemal assembly factors, DNAAF1 and DNAAF2, as well as a potential novel PCD gene, PIH1D2, to have co-precipitated with SPAG1.Therefore, the central hypothesis of this proposal is that SPAG1 plays a key role in the cytoplasmic assembly of axonemal dynein arms by interacting with other dynein axonemal assembly factors (DNAAFs). Aim 1 proposes to characterize the identified SPAG1 interactions with DNAAF1, DNAAF2, and PIH1D2 further. The expression and localization of these identified interactors will be examined by droplet digital PCR (ddPCR), westerns, and super-resolution microscopy. To determine the order that SPAG1 interactions occur during ciliated cell differentiation, time course co-IP studies will be performed. Proximity ligation assays (PLA) in differentiating hAEC will be performed to interrogate if DNAAF1, DNAAF2, or PIH1D2 are directly interacting with SPAG1. Aim 2 proposes to determine which isoforms and distinct subunit complexes of dynein arms require SPAG1 for their assembly. SPAG1 will be knocked out in hAEC cultures using CRISPR/Cas9 technology, and confirmation of this loss of SPAG1 will include measuring ciliary beat frequency and measuring the abundance of dynein arms. Mislocalization of known DNAAFs and dynein chains of different dynein arm isoforms in SPAG1-deficent cells will be studied using immunofluorescence techniques. The disassociation of known DNAAF interactions and known dynein chain complexes in the dynein arm assembly process will be studied using co-IP techniques in SPAG1- deficient cells. These studies will provide a greater understanding of the role SPAG1 has in the cytoplasmic pre-assembly of axonemal dynein arms. This expansion of knowledge will lead to improved diagnostics and the rationale for future therapeutic agents for primary ciliary dyskinesia.