(671) Adenylate cyclase 6 interactome regulates mucociliary clearance

Background: Mucociliary clearance (MCC) is a critical multilayered defense system that eliminates microbes, pathogens and environmental pollutants from the airways. Impaired MCC is a key pathogenic defect in inherited disorders such as cystic fibrosis (CF) and primary ciliary dyskinesia (PCD), resulting in recurrent infection and inflammation that ultimately leads to lung function decline. Adenylate cyclase (AC) is a membrane-bound glycoprotein that catalyzes the conversion of ATP to cAMP. cAMP is a critical regulator of ciliary beating, MCC, and organization of compartmental signaling in motile cilia^[1]. We previously identified AC6 as a predominant AC isoform in mammalian airway epithelium, and established that AC6 physically and functionally associates with CFTR at the epithelial cell surface^[2]. Additionally, we demonstrated that AC6 primarily regulates cAMP-induced CFTR-dependent Cl^- current in airway epithelial cells^[2].Furthermore, we showed that AC6 regulates ciliary length and cilia-generated flow^[3]. In this study, we will investigate the critical role of AC6-mediated regulation of mucociliary clearance via ciliary beat frequency (CBF).

Methods: CBF measurements were performed on tracheal rings generated from AC6 KO and control WT mice using a 3-D printed micro-guillotine system. CBF was assessed in the presence and absence of isoproterenol, a cAMP agonist. To understand the mechanism of AC6 in regulating MCC, a medium-throughput protein array screen was performed to identify putative AC6 interacting partners.

Results: CBF was significantly decreased in AC6 KO mice tracheal epithelial cells (TEC) as compared to control cells. Isoproterenol treatment significantly increased CBF in WT-TEC but failed to do so in AC6 KO-TEC. We addressed the possibility of AC6 interacting partner CCDC65 (identified in ProtoArray® screen) in the regulation of CBF through AC6. CCDC65 is a crucial regulator of beating patterns and microtubule sliding in ciliary axonemes. Moreover, Horani and colleagues reported that a frameshift mutation in CCDC65 resulted in PCD, further supporting a potential role for CCDC65 in MCC^[4]. We demonstrated that CCDC65 did indeed interact with AC6 when transiently expressed in HEK293 cells and that this interaction was responsive to stimulated phosphorylation.

Conclusions: We identified CBF stimulation as a mechanism by which AC6 regulates MCC. Furthermore, isoproterenol mediated regulation of CBF requires AC6, supporting our hypothesis that AC6 plays a role in regulating ciliary function via maintaining the cAMP gradient. We further propose that the AC6 interaction with CCDC65 is crucial to maintain CBF in airway epithelial cells.

Acknowledgements: This work is supported by Cystic Fibrosis Foundation (Award number: 003361H221)

References: 1.Schmid, A., et al., Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells. Journal of cell science, 2006. 119(20): p. 4176-4186.

2.Thomas, A., et al., AC6 is the major adenylate cyclase forming a diarrheagenic protein complex with cystic fibrosis transmembrane conductance regulator in cholera. Journal of Biological Chemistry, 2018. 293(33): p. 12949-12959.

3.Arora, K., et al., AC6 regulates the microtubule-depolymerizing kinesin KIF19A to control ciliary length in mammals. Journal of Biological Chemistry, 2020. 295(42): p. 14250-14259.

4.Horani, A., et al., CCDC65 mutation causes primary ciliary dyskinesia with normal ultrastructure and hyperkinetic cilia. PloS one, 2013. 8(8): p. e72299.