Regulation of Ciliogenesis and Ciliary-related signaling

Abstract public domain (NIH RePORTER)

In FY20, my laboratory further investigated the molecular pathways important for primary cilium assembly and connections to human disease. Publications: We published a manuscript in the Journal of Biological Science where we examined the role of the TRAPP complex in ciliogenesis. Previously, we had showed that TRAPPII proteins are important for ciliogenesis by affecting Rabin8 MC accumulation (Westlake et al., 2011 PNAS), however it was unclear how this complex regulated this process. To address this question, we investigated novel Rabin8 interacting proteins. Interestingly, we identified an uncharacterized protein C7orf43 and showed that it is a direct interactor with Rabin8 and TRAPPC proteins. C7orf43 depletion impaired ciliogenesis in human cells and zebrafish embryos, and affected Rabin8 preciliary vesicle accumulation at the MC. Biochemical studies indicated that C7orf43 is a novel TRAPPII complex protein, referred to as TRAPPC14, that mediates Rabin8 interaction with this complex. Finally, we demonstrated that TRAPPC14 interacts with DAPs needed for preciliary vesicle accumulation at the MC, suggesting the TRAPPII complex functions as a molecular tether to help recruit GFP-Rabin8 preciliary vesicles to DAs. This study provides novel insight into the composition of the TRAPPII complex through identification of a new component that appears to specify complex function. This work also further shows the importance of DAPs in recruiting membrane vesicles to the MC. Cuenca, A., Insinna, C., Zhao, H., John, P., Weiss, M.A., Lu, Q., Walia, V., Specht, S., Manivannan, S., Stauffer, J., Peden, A.A., Westlake, C.J. The C7orf43/TRAPPC14 component links the TRAPPII complex to Rabin8 for preciliary vesicle tethering at the mother centriole during ciliogenesis. J Biol. Chem. 294:15418-15434, 2019. FY20 Projects: 1)Characterization of early ciliogenesis membrane structures. Previously, we showed that steps between DAV docking and CV assembly are critical for several key ciliogenesis processes (Lu et al., 2015 Nat Cell Biol; Insinna et al., 2019 Nat Commun). However, this work did not establish how the DAVs are formed into the CV. Thus, our objective is to understand the mechanism of CV assembly by examining the ultrastructure of upstream MC membrane structures. To answer questions about CV formation, we are using super resolution fluorescence microscopy and 3D electron microscopy (tomography and FIB-SEM) to characterize membrane structures associated with ciliogenesis 2) Investigation of membrane trafficking functions in MC uncapping. In a related project to 1) we are investigating the role of membrane structure and function in transition of the mother centriole into the basal body. This process requires the removal of a protein cap from the distal end of the mother centriole. Our previous work established that EHD1 is required MC uncapping (Lu et al., 2015 Nat Cell Biol). We are testing our hypothesize that EHD1 is directly involved in MC uncapping. Our investigations are utilizing the advanced imaging approaches described in Project #1, as well as biochemical studies of EHD1 interacting proteins. We are also utilizing EHD1 mutants with EHD1 RNAi knockdowns to investigate this proteins functioning in MC uncapping. 3) Characterization of novel membrane fusion SNARE complex important for ciliogenesis. We hypothesized that SNARES will play important roles in the assembly of the ciliary membrane. Membrane fusion requires four SNAREs forming a trans-complex between two membrane compartments. Our published work established that the Qbc SNARE SNAP29, functions in ciliogenesis (Lu et al., 2015 Nat Cell Biol) and therefore, we expect that at least two additional SNAREs will be required for ciliary fusion events. Interestingly, no SNAREs have been identified in published genome wide knockdown screens examining ciliogenesis.

Abstract public domain (NIH RePORTER)

Thus, to investigate other ciliogenic SNAREs, we performed an RNAi screen of the 38 human SNAREs in RPE-1 cells using 4 siRNA per gene. Through this work we identified 6 SNAREs needed for ciliogenesis. These candidate ciliogenic SNAREs are being characterized for specific functioning in ciliogenesis stage and RNAi rescue studies will also be performed to evaluate off-target effects. We are also examining SNARE function in zebrafish embryo ciliogenesis as we have previously reported (Lu et al., 2015, Nat Cell Biology; Insinna et al., 2019 Nat Commun; Cuenca et al., 2019 JBC). 4) Investigation of ciliogenesis dysfunction mechanisms linked to membrane trafficking regulation in cancer cells. Although ciliogenesis dysfunction has been reported in a number of cancer types, we know little about the molecular mechanisms affected. Interestingly, EM studies have showed cilia assembly dysfunction associated with at DAV- and CV-like stages in glioblastoma/astrocytoma lines, suggesting that the intracellular ciliogenesis pathway is affected in cancer. Moreover, our observation that LPA/PI3K/Akt regulates ciliogenesis initiation (Walia et al., 2019 Dev Cell) could have implications in cancer cells, as these signaling regulators are important in tumorigenesis. Indeed, PI3K inhibitors promoted ciliation in a mouse pancreatic cancer cell line expressing KRasG12D mutants in a serum-independent manner. Building on our new insights into ciliogenesis initiation, we analyzed ciliation in 56 cancer cell lines, most from the NCI-60 panel. From this work, we determined that 50% of the cell lines tested had less than 5% ciliation, the non-ciliator group, with a majority found without any cilia. Importantly, the PI3K inhibitor LY29004 significantly enhanced ciliation in two non-ciliator cell lines. We are investigating the connections to PTEN regulated PI3K and Akt signaling in these cells examining MC uncapping and connections to the cell cycle. We are also examining whether Akt phosphorylation of WDR44 and the Rab11-effector switch for Rabin8 preciliary trafficking is dysregulated in these cancer cells as we observed in normal RPE-1 cells as was reported in our recently published work (Walia et al., 2019 Dev Cell). Invited talks for FY20: Frontiers in Light Microscopy Symposium, CCR, NCI, NIH, Bethesda, MD (November 19th , 2019) Frontiers in Biology Seminar Series, Stanford University, Palo Alto, CA (March 19th, postponed due to public health concerns related to COVID-19) Cell and Experimental Biology 2020, Boston, MA. (April 22-24 2020, postponed to July 2020 due to public health concerns related to COVID-19). Department of Life Sciences Seminar Series, Brunel University, London, UK (June 11th, postponed due to public health concerns related to COVID-19). Department of Immunology and Cell Biology, Cambridge University, Cambridge, UK (June 15th, postponed due to public health concerns related to COVID-19). Symposium of the Collaborative Kidney Research Center SFB 1350. Regensburg, Germany (June 19-20 2020, postponed due to public health concerns related to COVID-19). Federation of American Societies for Experimental Biology (FASEB) - Small GTPases in Membrane Processes, Halifax, Canada (August 16-21 2020, postponed due to public health concerns related to COVID-19). Department of Radiology, The University of Ohio, Columbus, OH (September 2020, postponed due to public health concerns related to COVID-19). Meeting attendance and presentations by Westlake lab postdocs: ASCB, Washington DC, December 2019. - 3 poster presentations by Insinna et al.,, Cuenca et al., and Walia et al.. FASEB - Small GTPases in Membrane Processes, Halifax, Canada. (August 16-21 2020, postponed due to public health concerns related to COVID-19). - 3 abstracts were to be submitted by Lu et al., Zhao et al., Perera et al.