Regulation of Ciliogenesis and Ciliary-related signaling

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

In FY22, my laboratory investigated molecular pathways important for cilium assembly and connections to human disease. This work comprised work on 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 ciliogenesis. This 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). It is not known if all cells use this intracellular pathway and as such we have expanded out studies to include cells thought to use alternative mechanisms (extracellular pathway) that are also important in disease. A continued primary objective of the laboratory in FY21 has been to investigate the intracellular ciliogenesis pathway associated with CV assembly. Using super resolution fluorescence microscopy and 3D electron microscopy (FIB-SEM) we are examining the ultrastructure of ciliogenesis. We continue to use CLEM approaches with FIB-SEM that we first described in our 2019 paper ( Insinna et al. Nature Commun) and in FY22 we published a methods paper on this technique (Lu and Westlake, 2021). FIB-SEM work is being performed in collaboration with Dr. Kedar Narayan at the Center for Molecular Microscopy, Frederick National Laboratory. In our current work we investigated the role of membrane shaping (EHD1) and RAB trafficking (Rab8a/b) regulators in early stages of ciliogenesis. Through this work we have discovered new intermediate membrane structures important for cilium assembly. We have demonstrated that EHD1 and membrane structure is directly involved in preparing the centriole to build cilia. In FY22, we acquired a live cell super resolution imaging microscope (Elyra7) to perform ciliogenesis structure/function studies to further investigate this membrane assembly mechanism. This approach has allowed us to uncover an asymmetric ciliogenesis pathway not previously appreciated. We have expanded our analysis of the centriole microenvironment to understand this pathway and have developed analysis tools to analyze other subcellular structures involvement in this mechanism. A manuscript is expected to be submitted early in FY22 on this work. In addition to this work we investigated the role of RAB and membrane fusion regulators in ciliogenesis. We previously established that RAB11, RAB8 and the SNARE SNAP29, functions in ciliogenesis (Lu et al., 2015 Nat Cell Biol). We are examining the membrane assembly pathways associated with this RAB11-RAB8 cascade and have completed siRNA screens for additional SNAREs. For the SNARE project, we have attempted to generate CRISPR knockout cells where possible and we are also investigating ciliogenesis requirements for these proteins in zebrafish embryos. To investigate membrane trafficking networks associated with ciliogenesis a key approach is the application of the Elyra7 microscope. We are also developing molecular biology tools to generate CRISPR knock-in cells lines where we can tag membrane trafficking regulators with fluorescent proteins to enable examination of endogenous protein ciliogenic function. We previously published a paper in Developmental Cell (Walia et al, 2019) describing the membrane trafficking protein WDR44 as a negative regulator of ciliogenesis. WDR44 blocks Rab11-dependent vesicle trafficking to the mother centriole needed for initiating ciliogenesis. This past year we completed a study examining seven patients with X-linked mutations in WDR44 which have various ciliopathy phenotypes. Fibroblasts isolated from severely affected patients have aberrant ciliogenesis initiation and reduced Hedgehog signaling implicating dysfunctional cilia as causative in disease. We also demonstrated that zebrafish embryos expressing human WDR44 variants develop ciliopathy. Through biochemical studies we discovered a surprising gain-of-function mechanism whereby WDR44 variants bind with higher affinity to RAB11 than the wild-type protein to block ciliogenesis initiation. Our work reveals that interdomain interactions in WDR44 affect ciliogenesis initiation to cause a previously uncharacterized ciliopathy-related disorder. A manuscript is being prepared for submission with our collaborators Dr. Accogli (McGill University) and Dr. Park (Seoul National University). In a related study, my laboratory investigated ciliogenesis initiation dysfunction in cancer. WDR44-RAB11 binding and ciliogenesis initiation is regulated by PI3K/Akt signaling. Notably, the PI3K-Akt signaling pathway is frequently upregulated in cancer due to the absence of functional PTEN. Thus we hypothesized that PI3K/Akt signaling upregulation could cause ciliogenesis dysfunction in cancer cells. We plan to complete studies aimed at linking PTEN and aberrant WDR44 activity in ciliogenesis dysfunction in cancer cells using cell culture models and xenograft studies. In FY22, our group published our first study examining multiciliogenesis mechanisms in EMBO Reports (Zhao et al. 2022). Multiciliated cells (MCC) have to replicate hundreds of centrioles and assembly cilia by a poorly understood mechanism. Using a bioinformatics approach potential uncharacterized multiciliogenesis factors were identified. By localization and knockdown studies it was discovered that Ccdc108, a protein linked to sperm dysfunction and male infertility, has an evolutionarily conserved requirement in motile ciliation. In collaboration with Dr. Ira Daar's laboratory in CCR we used frog, fish and mammalian systems to demonstrate that Ccdc108 is required for the migration and docking of basal bodies to the apical membrane in MCCs. Remarkably, these studies demonstrated that Ccdc108 and the intraflagellar transport (IFT)-B complex cooperate in centriole apical migration during multiciliogenesis. This is the first study to link the IFT-B complex to ciliogenesis mechanism upstream of axoneme assembly. We also continued our investigation into multiciliogenesis mechanism using 3D FIB-SEM ultrastructure studies. Our preliminary studies indicate that motile multiciliated cells (MCC) use a similar process as the primary cilium. Thus, we are investigating roles of RAB membrane trafficking regulators in MCC ciliogenesis. My group also published several other papers in FY22. Dr. Quanlong Lu and Dr. Westlake published a paper in Methods in Molecular Biology. Rab GTPases: Methods and Protocols entitled "CLEM characterization of Rab8 and associated membrane trafficking regulators at primary cilium". This work described methods for coupling fluorescence and electron microscopy to study protein localization to ultrastructure during ciliogenesis. My group also published a review paper in Semin Cell Dev Biol.(22: S1084-9521. 2022) authored by Dr. Huijie Zhao, Ziam Kahn and Dr. Westlake. Finally, Dr. Westlake and members of the laboratory were co-authors on five publications published in MBoC, JCell Biol (2 papers), AJHG, and JBC.