In FY12 we completed the assembly of two live optical microscopes to investigate the dynamics of membrane trafficking processes during ciliary membrane assembly and ciliary receptor/cargo transport. Both systems were reconfigured from microscopes that were either purchased in FY11 or existed prior to my arrival at NCI. One microscope was outfitted with an environmental chamber and high-resolution camera for live widefield imaging. The spinning disk confocal microscope was modified to enable simultaneous two-color widefield, confocal and TIRF imaging on two camera. This alteration enables better 3D fluorescent molecules tracking by combining the three different imaging platforms in a single experiment. In addition, we optimized the technique for correlative light and electron microscopy (CLEM). With this approach, we are now able to perform multicolor live cell imaging and subsequently examine the higher structure of primary cilium assembly using an electron microscope (EM). One of the key developments in this approach was to develop a methodology to track single cells/structures using live fluorescence optical microscopy and then be able to located the same cell/structure for EM imaging. Additionally we initiated a collaboration to use super resolution microscopy, a form of super resolution microscopy, to investigate primary cilium structure and function. To further study the molecular mechanism of these pathways we have developed a new proteomics tool that can be used for comparative and quantitative proteomics studies using mass spectrometry. A tandem affinity tag was generated for creating protein fusions that incorporates a unique trypsin sensitive peptide (sr4) not present in humans. This peptide can then be used to compare mass spectrometry analysis of different protein samples following addition of synthesized sr4 AQUA peptide which has stable amino acid isotope label which can be distinguished form the unlabeled sir4 peptide in a mass spectrometer. This tool is being used to further dissect the pathways important for primary cilium assembly and signaling and further to that we expect this will be a useful tool for other proteomics study. The lab carried out a screen of the NCI60 cell line panel to investigate primary cilium assembly and signaling-dependence. In this screen, we discovered more than 20 new cell lines that form cilia in culture. We further screened these cells with inhibitors linked signaling pathways important in ciliation to characterize how signaling affects ciliation in different cell lines. Finally, we are investigating the relationship between cancer-associated mutations in the NCI-60 cells and ciliation potential. .
