Regulation of Ciliogenesis and Ciliary-related signaling

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

In FY21, my laboratory continued to investigate the molecular pathways important for primary cilium assembly and connections to human disease (Project 1 and 2). We also initiated work investigating the molecular mechanism of motile cilia assembly (Project 3). Project 1) Membrane trafficking regulation of primary ciliogenesis: 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 lead us to conclude 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 primary objective of the laboratory in FY21 was to understand the mechanism of CV membrane assembly (Project 1a) and requirements for SNARE membrane fusion regulators (Project 1b) a) Dr. Lu and Dr. Huijie Zhao have investigated CV formation using super resolution fluorescence microscopy and 3D electron microscopy (FIB-SEM). We pioneered the use of CLEM confocal imaging and FIB-SEM in studying ciliogenesis structures as described in our 2019 paper by Insinna et al. (Nature Commun). In our current work, we have extended this approach to include super resolution structured illumination microscopy (SIM) to investigate the relationship between membrane structure and ciliogenesis initiation and progression. We have tested the function of EHD1 and Rab8a/b in regulating CV formation and have discovered new intermediate membrane structures between the preciliary vesicles and the CV stage requiring these membrane trafficking regulators. We have also demonstrated that EHD1 and membrane structure is directly involved in MC uncapping. Finally, we have determined that the transition zone, a region at the base of the cilium critical for regulating primary cilium signaling trafficking, is associated with assembly of early ciliary membrane structures. We also initiated live cell SIM imaging studies for this project to investigate ciliogenesis structure/function studies using a Zeiss Elyra7 microscope at NHLBI. A manuscript is being prepared for submission in 2021. Our FIB-SEM work is being done in collaboration with Dr. Kedar Narayan at the Center for Molecular Microscopy, Frederick National Laboratory. Based in part on the positive results achieved using the NHLBI microscope an RRS request was submitted and approved by CCR to purchase an Elyra7 to be housed at NCI-Frederick. b) Dr. Lu is also continuing to investigate the role of membrane fusion regulators in ciliogenesis. Based on our previous and current work we hypothesized that SNARES play important roles in the assembly of the ciliary membrane. Membrane fusion requires four SNAREs forming a trans-complex between two membrane compartments. We previously 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. From an RNAi screen of the 38 human SNAREs in RPE-1 cells using 4 siRNA per gene we have identified 6 SNAREs with ciliogenesis defects that we are characterizing using advanced microscopy imaging described above and in vivo using zebrafish embryos. Dr. Lu has been joined in this study by Dr. Ipsita Saha who will be investigating membrane trafficking networks associated with ciliogenesis. The Elyra7 microscope will be an important approach used in this work utilizing CRISPR knock-in cells lines generated with fluorescence tags inserted into different membrane trafficking regulators to enable examination of endogenous protein ciliogenic function. Project 2) Ciliogenesis initiation dysfunction in cancer Many ciliary signaling pathways are important drivers of cancer. Notably, there have been many reports describing ciliogenesis dysfunction in cancer cells. However, it is known why these tumors lack cilia and what the consequences of this dysfunction are on tumorigenesis. Interestingly, it has been reported that cilium assembly dysfunction is associated with CV-like stages in glioblastoma/astrocytoma lines. To investigate the molecular mechanism of ciliogenesis dysregulation in cancer cells we have carried out studies that extend from our recently published work describing PI3K/Akt signaling as a negative regulator of ciliogenesis initiation in normal cells (Walia et al., Dev Cell. 2019). Importantly, The PI3K-Akt signaling pathway is frequently upregulated in cancer. We reported that Akt directly negatively regulates Rab11-dependent preciliary trafficking by stabilizing binding between Rab11 and its effector, and Akt substrate, WDR44. Based on this work we hypothesized that PI3K/Akt signaling upregulation could cause ciliogenesis dysfunction in cancer cells. From our analysis of ciliation in 60 cancer cell lines, most from the NCI-60 panel, we determined that 50% of the cell lines had ciliogenesis dysfunction. Importantly, PI3K and Akt inhibitors could enhance ciliation in some of these cell lines. We next examined 9 cancer cells null for PTEN, a highly studied negative regulator of PI3K, and discovered that 7 of these cells lines failed to undergo uncapping of the mother centriole needed to initiate ciliogenesis. Remarkably, ciliogenesis initiation could be observed in all of the PTEN null cells following treatment with Akt inhibitors. Moreover, PTEN re-expression in the two lines we tested completely restored ciliogenesis. Currently, we are examining whether ablation of WDR44 by siRNA and CRISPR affects ciliogenesis in cancer cells with hyperactive PI3K/Akt signaling to determine if this protein could be a target for ciliation restoration in cancer cells. Project 3) Investigation of initiating processes in motile multiciliogenesis To investigate motile multiciliogenesis mechanisms we are also employing 3D FIB-SEM studies and our preliminary studies indicate that motile multiciliated cells (MCC) use a similar process as the primary cilium. Thus, we are investigating roles of membrane trafficking regulators in MCC ciliogenesis that have been linked to primary cilium assembly. In addition, Dr. Zhao has used bioinformatics approaches to identify potential uncharacterized multiciliogenesis factors. Using localization and knockdown studies he discovered that Ccdc108, a protein linked to sperm dysfunction and male infertility, has an evolutionarily conserved requirement in motile ciliation. Using frog embryos, Ccdc108 was found to be required for the migration and docking of basal bodies to the apical membrane in epidermis MCCs. His studies indicate that Ccdc108 and IFT-B complex components cooperate in centriole apical migration during multiciliogenesis. In FY21 a manuscript was submitted to EMBO Reports entitled Ccdc108 Regulates Multiciliogenesis via Interaction with the Intraflagellar Transport Machinery and is currently under revision. This work is a collaboration and co-corresponding authorship with Dr. Ira Daar (CCR, CDBL). FY21 Publications: A review article was published in Faculty Review (10:16, 2021) authored by Dr. Saurabh Shakya, and Dr. Christopher Westlake. Dr. Westlake and Dr. Quanlong Lu were co-authors on a publication in Developmental Cell (56:325-340, 2021). Dr. Lu and Dr. Westlake also have an article accepted for publication in Methods in Molecular Biology. Rab GTPases: Methods and Protocols entitled CLEM characterization of Rab8 and associated membrane trafficking regulators at primary cilium. Current Research: