In FY14, 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. Key findings include 1) discovery that 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) finding that EHD1 and EHD3 have functional overlap in ciliogenesis in human cells and zebrafish embryos 3) used 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) used 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. A manuscript is currently under revision at Nature Cell Biology. 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. Specifically we have begun to map how LPA/PI3K/Akt signaling pathway regulates Rabin8 pre-ciliary vesicle trafficking and ciliogenesis initiation. Our major finding in FY14 demonstrated: 1) LPA/LPAR1 regulates Rabin8 pre-ciliary vesicle trafficking and ciliogenesis via PI3K/Akt signaling 2) LPA/PI3K/Akt is a global regulator of ciliogenesis in other cells tested including a screen of the NCI60 panel. 3) regulation of Rabin8 pre-ciliary vesicle trafficking by Akt occurs by Rab11-effector switch involving WDR44 4) Akt-dependent phosphorylation on WDR44 Ser342 is important for Rab11 binding and impairment of this interaction promotes Rabin8 pre-ciliary trafficking 5) WDR44 ablation does not promote ciliogenesis suggesting that other Akt may have other substrates whose phospho-regulation is important for regulating ciliogenesis We anticipate submitting this work for publication early in FY15. Studies on regulation of Hedgehog ciliary signaling: We have also begun studies investigating how these membrane trafficking pathways are involved in ciliary Hedgehog signaling regulation. We have developed 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 Science (2013) and Mol. Cell. Biol. (2014). In FY12, we established a protocol for correlative light and electron microscopy (CLEM). This approach allows us to perform live cell imaging of ciliogenesis and subsequently examine the higher structure of primary cilium assembly using an electron microscope (EM). Using this technique has revealed new insight into the process of ciliogenesis at the structural and molecular level. In FY13/FY14 we collaborated with Dr. Jennifer Lippincott-Schwartz lab at NICHD to use structured illumination microscopy (SIM) super resolution microscopy to study cilia signaling and ciliogenesis pathways. SIM provides a two-fold increase in spatial imaging resolution (100nm) compared to conventional confocal microscopy. This approach has revealed new information on the localization of proteins to the developing cilia and cilia associated membranes. This work along with others interest in SIM technology at the NCI-Frederick was instrumental in the decision by CCR to purchase a SIM for the NCI. In addition, we have collaborated with Dr. Stephen Lockett (Frederick National Labs) to develop PALM and STORM super resolution imaging to achieve spatial resolution of cilia structures at an even lower 20-50nm. Zebrafish is a good model system for primary cilium function during development. Importantly zebrafish encode 85% of the genes linked to ciliopathy in humans and is an established model for cancer studies. To further our research into cilia disease associated pathways we established a zebrafish facility at NCI-Frederick in FY13. This facility contains wild-type and transgenic fish that are being used for whole embryo gene knockdown studies using modified DNA oligonucleotides called morpholinos. Transgenic animals expressing green fluorescent protein fusions were acquired through MTA agreements with extramural researchers to enable microscopy imaging in live animals. In FY14 we received funding to establish a proper zebrafish facility. For this a aquatic tank system has been purchased and plans are being 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. This facility will also house frogs from Dr. Ira Daar's laboratory. In addition, funds were acquired to purchase an upright light/fluorescence scope from Nikon. Finally, a research technologist was allocated by CCR to the operation and care of the fish facility. Meetings and Presentations: Abstracts on EHD-dependent regulation of ciliogenesis were accepted for oral presentation at the ASCB (e-poster, Dr. Quanlong Lu presenter) and Keystone Cilia meeting (Dr. Christine Insinna presenter) in FY14. Invited talks on this work were presented at the NCI Metabolism Branch seminar series.
