ABSTRACT Craniofacial malformations represent the largest category of birth defects and a major expense to the healthcare system and affected families. As craniofacial morphogenesis is complex and requires the coordinated interplay of several cellular pathways, mutations in many genes in these pathways can give rise to craniofacial defects. Coiled coil domain containing protein 32 (CCDC32) is an understudied gene recently found to underlie a craniofacial syndrome known as Cardiofacioneurodevelopmental Syndrome (CFNDS). Patients with defective CCDC32 exhibit a number of morphological abnormalities including bilateral cleft lip/palate, hypo- or hypertelorism, and microcephaly among others. Preliminary cellular data have shown CCDC32’s involvement in endocytosis and ciliogenesis, although molecular data to explain these functions is lacking. I have found that CCDC32 interacts with the adaptor protein complex 2 (AP2), a key factor in regulating endocytosis, via two well characterized motifs and exhibits the novel function of disassembling this complex. Moreover, I discovered CCDC32 binds the related AP3 complex, which is involved in trafficking throughout the Golgi and endolysosomal compartments. My preliminary data shows that knocking down CCDC32 in cells results in defects in intracellular trafficking including abnormal and reversible accumulation of ciliary components, and previously unobserved ciliary assembly dynamics. I hypothesize that CCDC32 promotes ciliary maintenance via AP3, and also regulates ciliary function via AP2. My approach will use cellular, biochemical, and structural methods to address the molecular mechanism of CCDC32’s role in endocytosis and ciliary assembly. In Aim 1, I will characterize the membrane trafficking interactions of CCDC32 that regulate ciliary maintenance. To determine a role for AP3 in intracellular ciliary trafficking, I will knock down AP3 in cells and observe cilia dynamics. To determine the molecular mechanism of CCDC32 interaction, I will determine a high-resolution CryoEM structure of the AP3- CCDC32 complex. To confirm the cellular importance of this interaction, I will use targeted mutagenesis to disrupt complex assembly in vivo. In Aim 2, I will determine how AP2-mediated endocytosis supports CCDC32’s role in ciliary function. CCDC32 and AP2 both promote endocytosis and bind one another, although the necessity and nature of this interaction has not been established. To assess an AP2-dependent function for CCDC32 in endocytosis, I will use targeted mutagenesis in vivo. I have also shown that CCDC32 disassembles AP2. To elucidate this novel mechanism, I will obtain CryoEM structures of CCDC32 with AP2 alpha/sigma. I will validate that AP2 disassembly occurs at membranes using biochemical reconstitution. To understand the effect of CCDC32 on endocytosis at the cilium, I will use TIRF endocytosis assays. Via these experiments, I will build the first molecular model of CCDC32 in cilia biology. This study wi...