Regulation of Ciliogenesis and Ciliary-related signaling

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

In FY23, my laboratory continued to investigate molecular pathways important for cilium assembly and connections to human ciliopathy disease and cancer cell signaling. For ciliogenesis studies we are examining both primary cilium and motile multiciliated cells. Our previously published work (Westlake et al., 2011, PNAS; Lu et al., Nat Cell Biol. 2015; Insinna et al., Nature Communication. 2019; Walia et a., Dev Cell, 2019; Cuenca et al., JBC, 2019) has helped establish the importance of membrane trafficking in the initiation and progression of primary ciliogenesis and this past year we published a review paper in this area (Zhao et al., Semin in Cell Dev Biol, 2023). Our past work has led to the understanding that the docking of small preciliary vesicles to the mother centriole is required to initiate ciliogenesis processes at the mother centriole and to assemble a larger ciliary vesicle (CV), upstream of ciliary axoneme growth. Moreover, we have shown that the CV stage is associated with ciliopathy (Shimada et al., Cell Rep. 2017). A key objective of the laboratory is to understand the molecular mechanism of intracellular ciliogenesis pathway and links to disease. The main approaches being used to study primary and motile ciliogenesis include advanced microscopy approaches (super resolution fluorescence [SRM] and volume electron microscopy), biochemistry/proteomics and animal models. Investigation of the architecture of ciliogenesis has led us to discover previously uncharacterized membrane structures associated with reorganization of the mother centriole in preparation for cilia growth. In collaboration with Dr. Kedar Narayan at the Center for Molecular Microscopy (CCR), we used FIB-SEM volume electron microscopy (vEM) to investigate membrane structure during ciliogenesis. This work extends from our previous studies investigating the role of membrane shaping and RAB trafficking regulators in early stages of ciliogenesis (Lu et al. Nature Cell Biology 2015, Insinna et al., Nature Communication 2019). We are using wild-type cells and cells with CRISPR-Cas9 knockout of ciliogenesis regulators to systematically identify new intermediate membrane structures important for cilium assembly. We plan to submit a manuscript for this work in the next 2 months which describes the role of membrane structure in building the CV and in uncapping of the mother centriole. This study also suggested an asymmetric ciliogenesis pathway involving vesicle trafficking to the mother centriole that we are continuing to investigate using advanced microscopy imaging. In FY23, we developed analysis tools (Python and MATLAB based) to investigate the centriole microenvironment to understand this mechanism which pointed to connections between asymmetric vesicle trafficking and different subcellular structures. For these and other ciliogenesis studies my group continues to pioneer the use of live cell imaging to investigate ciliogenesis, and we published a methods paper in FY23 about this imaging approach (Lu and Westlake, Methods Cell Biol 2023). We have extended this imaging application by using super resolution live cell microscopy with an Elyra7 microscope, purchased in FY2022, to perform ciliogenesis structure/function studies. Our work has also demonstrated a striking association of membrane tubulation in ciliogenesis, including in assembly of the CV (manuscript in preparation described above) and for connecting the intracellular cilia membrane to the plasma membrane (Insinna et al., Nature Communication 2019). Membrane tubulation is a poorly understood membrane transport process. In FY23 we undertook studies to examine the relationship between cellular membrane tubulation and ciliary membrane tubulation. We are preparing a manuscript we expect will be submitted in FY24 which examines the association of the. RAB11-RAB8 cascade (Westlake et al., PNAS 2011) in these membrane tubulation processes. Key approaches in this study include the use of FRAP and SRM (live cell and fixed cell imaging) to examine membrane trafficking dynamics in cilia and membrane tubules. We also are developing proximity labeling (BioID) and LC/MS approaches to identify protein function associated with these tubular membrane trafficking processes. We previously reported an Akt-dependent dependent signaling mechanism responsible for regulating ciliogenesis initiation (Walia et al, Developmental Cell 2019). In this work, we discovered WDR44, a RAB11 effector, is a negative regulator of ciliogenesis initiation. WDR44 phosphorylation by AKT blocks Rab11-dependent vesicle trafficking to the mother centriole. Following publication of this work we have identified families with variants in WDR44 displaying overlapping pleiotropic disease, many associated with ciliopathy. We have completed a study of 8 families with mutations in the WD40 repeat domain and are currently finishing revisions for a manuscript that was submitted to Nature Communications. This work has been undertaken in collaboration with Dr. Accogli (McGill University) and Dr. Park (Seoul National University) and I am the senior author of this manuscript. We are continuing to investigate disease mechanism associated with WDR44 by examining WDR44-RAB11 protein structure and have initiated a collaboration with Dr. Susan Lea (NCI). We are also examining patient variants outside of the WD40 repeats using cell biology and animal model (fish and mouse) systems. Finally, in a related study, my laboratory is investigating ciliogenesis initiation dysfunction in cancer associated with WDR44-RAB11 binding and regulated by PI3K/Akt signaling. Notably, the PI3K-Akt signaling pathway is frequently upregulated in cancer due to the absence of functional PTEN. In mid-FY23, I was able to hire a biologist to work on this project and we hope to complete studies aimed at linking PTEN and aberrant WDR44 activity in ciliogenesis dysfunction in cancer cells using cell culture models and xenograft studies this fiscal year. How cells develop hundreds of cilia in multiciliated cells (MCC) important for brain, lung and female reproductive function is poorly understood. We have established expertise and collaborations in MCC studies and I published a senior author paper in EMBO Reports in FY22 (Zhao et al. 2022). Mechanistically we are investigating the roles of RAB membrane trafficking regulators in MCC ciliogenesis. Currently, we are collaborating with the Aquatics Core and Dr. Ira Daar to use fish and frog embryos as a model for investigating MCC formation. Extramurally, we are collaborating with Dr. Jeremy Reiter (UCSF) to investigate the. role of membrane trafficking in MCC formation. These investigation into multiciliogenesis mechanism also employ vEM studies with Dr. Kedar Narayan (NCI). Our efforts in MCC investigation has led to a co-author contribution to a manuscript currently under revision from the Reiter lab.