In FY13, 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. 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. In addition, we have characterized roles for Rab11/8 and Rab-associated proteins in regulating the earliest steps in primary cilium assembly. With the addition of a new postdoctoral fellow to the lab in March FY14 we expanded our work with these cilia associated proteins to examine roles in cilia disassembly and association with cell cycle reentry/progression. Key equipment and methods for this 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 accepted co-author publication in the Journal of Cell Science (in press). 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. We continued to collaborate 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 super resolution imaging to achieve spatial resolution of cilia structures at an even lower 30-50nm. PALM was adapted for my labs Marianas microscope increasing the scope of use for this technology at NCI-Frederick. To further study the molecular mechanism of ciliogenesis and signaling pathways we generated cell lines expressing a new proteomics tool developed in FY12 that can be used for comparative and quantitative proteomics studies using mass spectrometry. The tandem affinity tag LAP sir4 system enables the expression of protein fusions that incorporates a unique trypsin sensitive peptide (sir4) not present in humans. We expect this technology will allow us to develop better quantitative mass spectrometry approaches to study disease-associated pathways. In addition, in collaboration with Dr. Jadrank Loncarek's lab we developed a cell fractionation protocol to isolate mother and daughter centrioles from cells for proteomics studies. The goal of this work is to characterize the differential protein composition of these structures during ciliogenesis. We hypothesize that novel proteins identified from these experiments will also reveal new insights into the cell cycle control of ciliogenesis and association with normal cell growth. 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 core 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. To improve whole animal fluorescence imaging capabilities in we purchased the mSAC technology for the Marianas microscope at the end of FY13. mSAC automatically corrects for spherical aberrations defects observed in thick section imaging. My laboratory is currently preparing manuscripts for this project. We anticipate submitting two publications in FY14. The first manuscript describes the role of Rabs and EHD proteins in assembly of the cilia membrane. This work utilized our developments in live microscopy imaging, CLEM, and super resolution microscopy and zebrafish morpholino studies. An abstract for this work was selected for an oral presentation at the FASEB Arf-Rab meeting in 2013 in Snowmass CO July 2013. Invited talks on this work were presented at Johns Hopkins University and the University of Lisbon in FY13 and a poster of this work was presented at the ASCB meeting in December 2012. A second manuscript for this project is will describe upstream growth factor signaling pathways important in cell growth that regulates preciliary vesicle transport and primary cilium formation
