Abstract: FR-PO627
Defining the Interaction Network of the CPLANE Complex Reveals Cilia and Actin Regulatory Components
Session Information
- Cystic Kidney Diseases: Mechanisms and Models
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1201 Genetic Diseases of the Kidneys: Cystic
Authors
- Kalot, Rita Kassem, McGill University Health Centre, Montreal, Quebec, Canada
- Babayeva, Sima, McGill University Health Centre, Montreal, Quebec, Canada
- Torban, Elena, McGill University Health Centre, Montreal, Quebec, Canada
Background
Almost all vertebrate cells have a single primary cilium that protrudes to the extracellular space and acts as a signalling nexus. Defects in primary cilia cause a set of diseases termed ciliopathies that affect multiple organs including the kidneys. Mutations in ciliogenesis and planar cell polarity (CPLANE) genes FUZZY, INTURNED and WDPCP lead to human ciliopathies. Although the homologs of CPLANE proteins in Drosophila Melanogaster regulate the planar cell polarity and actin polymerization, their functions in ciliation and the etiology of ciliopathies remain elusive. We hypothesize that CPLANE proteins regulate ciliary and actin-regulating proteins to control primary cilia formation.
Methods
Co-immunoprecipitation assay was used to validate the interactions between IQGAP1 and CPLANE proteins. IQGAP1 knockdown was achieved by siRNA transfection in hTERT-RPE-1 cells. Ciliation and YAP localization were assessed by immunofluorescence using anti-ARL13b and anti-YAP antibody, respectively. BioID baits were genrated by gateway cloning and transfected in FlpIn TREX293 cells. Pulldown coupled to mass spectrometry was used to identify CPLANE interactors. Protein identification and analysis of interactions were done in Prohits, and SAINT.
Results
Using a preliminary Bio-ID we identified IQGAP-1(an actin-regulating protein) as a novel interactor of Fuzzy. We established that IQGAP1 interacts with all CPLANE proteins and showed that the knockdown of IQGAP1 reduces ciliation and cilia length in htERT-RPE-1 cells. Since IQGAP1 does not localize to the ciliary basal body, we propose an indirect link where IQGAP1 regulates cilia length by modulating YAP1 downstream ciliary transcriptional targets.To better understand the roles of CPLANE in ciliogenesis, we generated CPLANE BioID baits and conducted an interaction study. Coupling BioID with MS and bioinformatics we identify multiple CPLANE interactors that further link actin regulation to primary cilia functions.
Conclusion
This study unravels a link between the CPLANE complex and actin regulators. Identifying CPLANE-specific proximity interactors helps discover novel molecular components regulating ciliation, particularly through the regulation of actin dynamics. This bridges the gap between actin regulatory pathways and ciliation and proposes potenitial actin-targeting therapeutics as treatment for ciliopathies.
Funding
- Government Support – Non-U.S.