In FY16, 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 laboratory projects: In 2015 we published our key findings in the journal Nature Cell Biology (Lu et al. 2015). In this work we demonstrated that a Rab-dependent membrane assembly process functions in ciliogenesis assembly. We identified new regulators of this process including membrane reorganizing proteins EHD1 and membrane fusion protein SNAP29. In FY16, my lab continues to investigate this process using advanced cell biology approaches inducing correlative light and electron microscopy (CLEM) and super-resolution structured illumination microscopy (SIM). In FY16, we have initiated a collaboration with Dr. Kedar Narayan and Dr. Sriram Subramaniam in the CCR Center for Molecular Microscopy (CMM) to use 3D-EM (FIBSEM) to further our work investigating the the mechanism of membrane assembly during intracellular ciliogenesis. Through this collaboration we have made made a number of insights into the process of intracellular ciliogenesis. We anticipate submitting a manuscript on this work in 2016. We have also made important new discovers into the cell signaling control of cilia assembly initiation via a previously uncharacterized preciliary Rab-dependent membrane trafficking pathway. We are preparing a manuscript describing how cell signaling controls cilia assembly initiation via a previously uncharacterized preciliary Rab-dependent membrane trafficking pathway. The key findings from this work include the discover of ta Rab11-effector switch regulated by Akt signaling. In non-ciliating cells Rab11 interacts with its effector WDR44 in an Akt-dependent phosphorylation manner. In the absence of Akt, Rab11 switches to a Rabin8 effector complex which is important for initiating early ciliogenesis events at the mother centriole. In FY16 we began an important collaboration for this work with Dr. Esben Lorentzen at Max Plank which has significantly enhanced our understanding of mechanism of the Rab11-effector switch. We will be submitting a manuscript on this work at the end of the summer in 2016. In FY15, we began an important collaboration with Dr. Anand Swaroop (NIH/NEI) to investigate the molecular mechanism of specific ciliopathy disorders. This work incorporates our expertise in ciliary imaging and ciliary signaling. We are using advanced imaging approaches to directly study ciliary transport, including molecules involved in Hedgehog signaling. This collaboration has has quickly progressed, and we are completing experiments for a manuscript that we expect to submit by the end of 2016. I will be the co-senior/corresponding author on this paper. 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. In FY16 we modified this system adding a sensitive smaller pixel imaging cMOS camera and upgraded the live cell heating/CO2 chamber. With these upgrades we will now be able to better 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) and Journal of Virology (2015). We are continuing to work with OMAL at NCO-Frederick to use 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 FY16 we successfully used CRISPR/Cas9 tools to generate genetic 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 approved the renovation of a room in building 560. In FY16, renovations were completed on the aquatic facility and we are now fully operational for our fish work which has greatly enhanced our capabilities. This facility also houses frogs from Dr. Ira Daar's laboratory. In FY14-15, I was responsible for the purchase of a new 120kV electron microscope to be housed in a newly renovated room in building 560. The microscope arrived at the end of FY15. However, because of planned renovations to building 560 it was decided not to set up the new EM microscope in 560. In FY16, I helped identify a new location and completed planning for renovated EM lab in building 426. Installation of the EM microscope began in July 2016 and should be complete sometime in August. We also continue to collaborate with NIH researchers to carry out 3D electron microscopy studies. Meetings and Presentations: Abstracts on Akt regulation of a Rab11-effector switch important for ciliogenesis initiation were accepted for oral presentation at the ASCB 2015 meeting (E-poster Dr. Vijay Walia presenter) and the 2016 Gordon Lysosome and Endosome meeting (talk, Dr. Chris Westlake presenter). An abstract has also been submitted for the FASEB small GTPase meeting in September 2016. I gave invited talks at the CMM Workshop (ATRF) and my student Dr. Christine Insinna presented an invited talk at the Frontiers in Light Microscopy symposium (Bethesda). Dr. Vijay Walia was awarded a FARE award in FY16.
