# Molecular mechanism of CCDC32 in Cardiofacioneurodevelopmental syndrome

> **NIH NIH F31** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2024 · $37,346

## Abstract

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...

## Key facts

- **NIH application ID:** 10999030
- **Project number:** 1F31DE034311-01
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Dillon Sloan
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $37,346
- **Award type:** 1
- **Project period:** 2024-09-01 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10999030

## Citation

> US National Institutes of Health, RePORTER application 10999030, Molecular mechanism of CCDC32 in Cardiofacioneurodevelopmental syndrome (1F31DE034311-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10999030. Licensed CC0.

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