Project Summary Male infertility is caused by various defects during the complex mechanisms of spermatogenesis. Successful sperm production requires intracellular trafficking of proteins to the specific site in order to shape and build a sperm. During spermatogenesis, two main mechanisms of protein transport have been identified, the intra- manchette transport (IMT) and the intraflagellar transport (IFT). These transports utilize F-actin and microtubule tracks to deliver vesicular and non-vesicular cargos to specific domains. However, the protein complexes involved in these transports and the routes and mechanisms of transport are not well characterized. Recently, a mutation in the SPAG17 gene has been associated with male infertility in humans. This gene is a poorly understood gene that encodes for a protein believed to play a role in the motility of the sperm flagella. However, results from our laboratory have shown that the SPAG17 protein plays additional functions beyond flagellar motility. Our compelling data show that loss of SPAG17 results in deformities in the acrosome, nucleus, and the absence of mature sperm. Moreover, the delivery of some axonemal and IFT proteins is affected during spermiogenesis. It is not clear how SPAG17 plays this role and whether it is involved in the transport of proteins via microtubules and/or the actin cytoskeleton. In this application, we seek to investigate the mechanism of action for the SPAG17 protein and its interaction with the trafficking of proteins in two main structures that are essential for shaping the sperm head and the tail: the acrosome-acroplaxome and the manchette. We hypothesize that SPAG17 regulates male fertility by influencing spermatogenesis via intracellular trafficking of proteins. We will test our hypothesis in two independent aims: Aim 1 will characterize the association of SPAG17 with the acrosome-acroplaxome and the manchette structure. We will use state-of-the-art stochastic optical reconstruction microscopy (3D-STORM) to map the three-dimensional localization of SPAG17 in murine spermatids. We will resolve the subcellular distribution of SPAG17 integrated with the arrangement of microtubules and F-actin filaments present in both the acrosome-acroplaxome and the manchette structure. Aim 2 will determine the protein complexes associated with the trafficking of proteins regulated by SPAG17. We will perform immunoprecipitation studies to isolate the SPAG17 interactome and resolve the identity of the proteins by mass spectrometry. We will evaluate whether the transport of these proteins is affected in SPAG17 KO germ cells by using immunofluorescence and the state-of-the-art super-resolution structured illumination microscopy (SIM). We will confirm the association of these proteins in a complex by mapping their colocalization and co- immunoprecipitation studies after overexpressing them in CHO cells using pcDNA expressing vectors. This proposal will provide the basis for future studies...