In FY15, my laboratory continued our investigations into molecular pathways important for primary cilium assembly and signaling. These studies involved the characterization of proteins important in regulating membrane trafficking, specifically proteins associated with the Rab family. Significant findings on ciliogenesis processes: We have made important new discovers into the cell signaling control of cilia assembly initiation via a previously uncharacterized preciliary Rab-dependent membrane trafficking pathway. This year we published our key findings in the journal Nature Cell Biology (Lu et al. 2015). In this work we demonstrated that 1) Rab8 is not required for pre-ciliary membrane trafficking steps leading to CV formation, but rather functions to grow this structure into the ciliary membrane at a time coincident with the assembly of the axoneme. 2) EHD1 and EHD3 have functional overlap in ciliogenesis in human cells and zebrafish embryos 3) using advanced imaging approaches (Correlative light and electron microscopy, CLEM, and super-resolution structured illumination microscopy, SIM) to demonstrate that EHD proteins have a unique localization to the ciliary pocket 4) using EM and SIM to show that EHD1/3 are needed for formation of the early ciliary vesicle (CV) structure from not previously described small distal appendage vesicles (DAV) 5) demonstrated that EHD proteins are need to reshape DAV to allow for SNARE mediated fusion to form the CV. In the process we identified a novel SNARE, SNAP29, required for ciliogenesis 6) showed that DAV to CV assembly is critical for mother centriole reorganization to become the basal body and for the recruitment of proteins needed to build the cilia, including Intraflagellar proteins and transition protein. We have also continued our work describing how cell signaling control of cilia assembly initiation via a previously uncharacterized preciliary Rab-dependent membrane trafficking pathway. Our major finding in FY15 have focused a Rab11-WDR44 effector switch dependent on phosphorylation. We anticipate submitting this work for publication by the end of FY15. Studies on regulation of Hedgehog ciliary signaling: We continue to investigates how these membrane trafficking pathways are involved in ciliary Hedgehog signaling regulation. We are using advanced imaging approaches including total internal fluorescence microscopy (TIRFM) to directly study Hedgehog regulator ciliary trafficking. Key equipment and methods: Key equipment and methods for our project include advanced microscope imaging systems and cell and zebrafish based genetics tools for characterizing protein functions. In FY11-12 we established live light microscopy systems to study ciliogenesis and ciliary signaling. The Marianas spinning disk confocal microscope acquired is unique at the NCI for its capacity to simultaneous image in two-color widefield, confocal or TIRF mode. Using this system we are able to image the processes of cilia assembly and disassembly, as well as to examine the transport of ciliary receptors important in signaling. This equipment/technology was also used for multiple collaborations with intramural and extramural researchers and lead to a recently published work in the Journal of Cell Biology (2013). We are upgrading the Marianas microscope to include a far red 640nm laser which will permit 4 color live imagine. In addition, we are collaborating with Dr. Jiji Chen (OML, Frederick National Labs) to develop STORM super resolution imaging to achieve spatial resolution of cilia structures at an even lower 20-50nm. We are also continuing to use Zebrafish is model system to investigate primary cilium function during development. Zebrafish encode 85% of the genes linked to ciliopathy in humans and is an established model for cancer studies. We have successful used genetic approaches (morpholinos) to investigate protein function in ciliogenesis. In FY15 we began developing CRISPR tools to generate genetic knock-in and knock-outs. In FY14 we received funding to establish a proper zebrafish facility. For this a aquatic tank system has been purchased and plans were finalized to construct an aquaculture facility in building to house the zebrafish and enable expansion of our fish capabilities to include generation of transgenic animals. CCR has since approved the renovation of a room in building 560. We anticipate this room will be ready in late 2015. This will greatly enhance our capabilities since we continue to perform our animal husbandry in numerous small domestic fish tanks. This facility will also house frogs from Dr. Ira Daar's laboratory. I have also been responsible for the purchase of a new 120kV electron microscope to be housed in a newly renovated room in building 560. We anticipate the arrival of this microscope in the fall of 2015 from FEI. We are collaborating with NIH researchers to carry out 3D electron microscopy studies. Meetings and Presentations: Abstracts on EHD-dependent regulation of ciliogenesis were accepted for oral presentation at the ASCB 2014 meeting (short talk, Dr. Christine Insinna presenter) and the FASEB Cilia meeting (talk, Dr. Chris Westlake presenter; Dr. Quanlong Lu poster presentation). An abstract on the Rab11-WDR44 effector switch were also presented at the ASCB 2014 meeting (e-poster, Dr. Vijay Walia). I gave invited talks on this work at the NCI-CCR Tenure Track Investigator Symposium, Department of Biology at Catholic University, and the ARVO 2015 meeting.
