CFAP65 is essential for C2a projection integrity in axonemes: implications for organ-specific ciliary dysfunction and infertility.

Defects in motile cilia and flagella lead to motile ciliopathies, including primary ciliary dyskinesia (PCD), which manifests as multi-organ dysfunction such as hydrocephalus, infertility, and respiratory issues. CFAP65 variants are a common cause of male infertility, but its localization and function have remained unclear. In this study, we systematically evaluated CFAP65's role using Cfap65 knockout mice and human patients with CFAP65 variants. The knockout mice displayed severe sperm flagellar defects (MMAF), high hydrocephalus incidence, but no significant impact on respiratory cilia. Similarly, the patients exhibited MMAF and infertility without respiratory symptoms. CFAP65 was found to anchor at the base of the C2a projection of the axoneme, interacting with proteins such as CFAP70 and MYCBPAP. Loss of CFAP65 caused disorganization of the sperm head-shaping microtubule structure and impaired protamine precursor removal, leading to nuclear condensation defects and poor assisted reproductive outcomes. Importantly, the assembly of CFAP65 was unaffected in mice with defects in the radial spokes (RSs) and nexin-dynein regulatory complex (N-DRC), indicating that CFAP65 assembly is independent of these components. However, CFAP65 deficiency led to the disintegration of the C2a projection, compromising ciliary and flagellar integrity. These findings establish CFAP65 as an essential component of the C2a projection, critical for the structure and function of sperm flagella and ependymal cilia, but not respiratory cilia, underscoring the organ-specific consequences of C2a projection defects in PCD.