Project Summary: In FY17, 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 process in normal ciliogenesis and in ciliopathy. Significant findings on laboratory projects: In 2017, we published a paper in Cell Reports (Shimada-Ishii et al. 2015) describing how ciliogenesis was affected by mutations in the CEP290 gene leading to the ciliopathy Leber's congenital amaurosis (LCA) and Jouberts Syndrome (JSRD). CEP290 is localized to the transition zone of the cilia and has been proposed to have a 'gate keeper' function in regulating the entry/exit of ciliary proteins. In this work, we demonstrated that JSRD CEP290 mutations causes ciliogenesis defects in patient fibroblast while LCA patient fibroblast ciliation was normal. Defects in ciliogenesis are associated with the ciliary vesicle stage (CV) previously shown by my laboratory to be regulated by EHD1. Interestingly, LCA ciliogenesis was observed the in optic cups, also blocked at the CV stage. Our findings for the tissue specific effects observed were consistent with phenotypes observed in patients. We demonstrated that JSRD, but not LCA, cilia had impaired trafficking of ciliary proteins. While JSRD patients fibroblast have reduced cilia number, the Hedgehog (Hh) signaling in these cilia was stronger than observed in parent (control) fibroblasts. Using live cell total internal reflection fluorescence (TIRF) microscopy, we demonstrated that the Hh receptor Smoothened had faster recover in the cilium in JSRD fibroblasts. From this work we concluded that elevated Hh signaling results from reduced 'gate keeping' function of CEP290 in JSRD patients. In FY17, we completed a project investigating F-Bar membrane reshaping protein function in ciliogenesis. Using advanced cell biology approaches including correlative light and electron microscopy (CLEM) and super-resolution SIM and STORM. In collaboration with Dr. Kedar Narayan in the CCR Center for Molecular Microscopy (CMM) we have used 3D-EM (FIBSEM) to investigating the mechanism of membrane assembly during intracellular ciliogenesis. Using FIB0-SEM we have made a number of new insights into the process of intracellular ciliogenesis. A manuscript describing F-Bar protein regulation of membrane tubulation important for ciliogenesis is currently being prepared for submission by the end of FY17. This year we also completed work for a manuscript describing the mechanism of cilium assembly initiation. 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. We have subsequently demonstrated that another Rab11 effector FIP3 is essential for the switch to pre-ciliary trafficking. Our mechanistic understanding of this Rab11 effector switch was greatly enhanced by an important collaboration Dr. Esben Lorentzen at Max Plank. We anticipate submitting a manuscript for this work by the end of August 2017. In FY17, we made significant progress on two other projects in the lab. The first project involves identification of a novel TRAPP complex protein, TRAPPC14, which regulates ciliogenesis via mediating interaction between Rab11-Rabin8 and the TRAPPII complex. We expect to submit a manuscript for this work early in 2018. In the second project, we have identified a novel membrane fusion SNARE complex important for ciliogenesis. Three SNAREs were identified using a SNARE siRNA library. We are continuing cell biology and biochemical studies to characterize the SNARE complex and anticipate submitting a manuscript in 2018 on this work. Meetings and Presentations: Abstracts on Akt regulation of a Rab11-effector switch important for ciliogenesis initiation were accepted for oral presentation at the ASCB 2016 meeting (short talk Dr. Chris Westlake presenter) and a poster at the 2017 FASEB Cilia Meeting. Abstracts for the F-Bar tubulation regulation in ciliogenesis and characterization of TRAPPC14 were also accepted for posters at the FASEB cilia meeting. I gave two invited talks at the CMM Workshop (ATRF) and have been invited to give a talk at the University of Nebraska (September 2017). My student Dr. Christine Insinna-Kettenhofen presented an invited talk at the NIH Developmental Biology workshop (Bethesda).