Regulation of Ciliogenesis and Ciliary-related signaling

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

The Westlake Laboratory is dedicated to understanding the molecular mechanisms underlying primary and motile ciliogenesis, which are essential for normal development and tissue homeostasis. Cilia are critical signaling hubs that regulate pathways such as Hedgehog, Notch, and PDGF. Disruption of these and other ciliary signaling processes is strongly linked to genetic disorders and cancer. To investigate ciliogenesis, we employ a multidisciplinary approach combining advanced microscopy, including super-resolution fluorescence (SRM) and volume electron microscopy (vEM), biochemistry, proteomics, and animal models. Our previous work (Westlake et al., 2011, PNAS; Lu et al., 2015, Nat Cell Biol; Insinna et al., 2019, Nat Commun; Walia et al., Dev Cell 2019; Cuenca et al., JBC 2019; Accogli et al., Nat Commun 2024; Saha et al., Cell Rep 2024) has elucidated key requirements for membrane trafficking during the initiation and progression of primary ciliogenesis. We demonstrated that the small GTPase Rab11-Rab8 cascade plays a central role in regulating ciliogenesis and that the docking of small preciliary vesicles to the mother centriole is essential for the formation of a larger ciliary vesicle (CV), which acts upstream of axoneme assembly. A major discovery from our group revealed that PI3K-Akt signaling directly regulates ciliogenesis by controlling Rab11 binding to its effector WDR44, thereby suppressing ciliogenesis initiation (Walia et al., Dev Cell 2019). We have also shown that defects in these membrane trafficking pathways are associated with ciliopathies (Shimada et al., Cell Rep 2017; Accogli et al., Nat Commun 2024). In our recent Nature Communications paper (Accogli et al., 2024), we reported families carrying pathogenic variants in WDR44 that exhibit a pleiotropic spectrum of ciliopathy-related disease. We are now investigating the structural dynamics of the WDR44-Rab11 complex and assessing additional disease-associated WDR44 variants using both cell line and animal models (zebrafish and mouse). In FY25, we continued our focus on ciliogenesis mechanisms, publishing a senior-author paper in Cell Reports and contributing to two collaborative publications. In our Cell Reports (Saha et al., 2024) paper, we provided new mechanistic insights into the Rab11-Rab8 cascade, a pathway central to ciliogenesis, neurite outgrowth, and apical lumen formation. Through biophysical approaches and advanced fluorescence imaging, we demonstrated how Rab11 and the Rab8 GEF Rabin8 jointly recruit Rab8 to membranes, including the ciliary membrane and dynamic membrane tubules. Using SRM, we were able to visualize the real-time dynamics of Rab11, Rab8, and Rabin8 association with membranes, an achievement reported for the first time. Additionally, we are developing new analysis tools for studying membrane trafficking in ciliogenesis. In a collaborative project with Dr. Kedar Narayan (Center for Molecular Microscopy), we utilized FIB-SEM vEM to characterize membrane structures during ciliogenesis. Comparing wild-type cells with CRISPR-Cas9 knockouts of ciliogenesis regulators, we identified novel intermediate membrane structures crucial for cilium assembly. This manuscript is under revision and will also be submitted to bioRxiv this summer. Our laboratory is also investigating the dysregulation of ciliogenesis initiation in cancer, focusing on how the Rab11-Rab8 cascade and associated signaling pathways are altered. Furthermore, we have expanded into studying multiciliogenesis, a less understood process essential for brain, lung, and female reproductive health. We use fluorescence and vEM imaging to investigate multiciliated cell (MCC) assembly and have proposed that MCCs utilize similar membrane trafficking mechanisms as primary cilia. Our expertise in MCC biology is supported by a series of recent publications (Zhao et al., EMBO Rep 2022; Choksi et al., Nature 2024; Lee et al., Commun Biol; 2024; Lee et al., JCB, 2022; Kumar et al., JCB 2021).