In FY18, my laboratory further investigated the 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. Laboratory projects overview: 1) We completed work on the F-BAR membrane reshaping protein PACSIN1 and -2 role in ciliogenesis. We used advanced cell biology approaches, in particular genetics and imaging to resolve the role of PACSIN proteins. Imaging studies included correlative light and electron microscopy (CLEM) and super-resolution SIM and STORM and focus ion beam scanning electron microscopy (FIB-SEM). Using genetic approaches we demonstrated that the PACSINs function at early stages of ciliogenesis, in particular the assembly of the ciliary vesicle. We also detected PACSIN1/2-positive membrane tubules associated with early stages of ciliogenesis. In collaboration with Dr. Kedar Narayan in the CCR Center for Molecular Microscopy (CMM) we used CELM FIBSEM to investigate the mechanism of membrane assembly during intracellular ciliogenesis. The findings from FIB-SEM have provided new insights into the process of intracellular ciliogenesis. First, we showed that membrane tubules are generated from the ciliary vesicle and intracellular cilia membrane. Second, we discovered that these membrane tubules locate and fuse with the plasma membrane, providing a mechanism for how the intracellular cilium emerges from the cell surface. We also discovered membrane tubule formation on the ciliary pocket membrane and showed that these are associated with the trafficking of ciliary cargo. A manuscript describing this work was prepared and submitted for review in FY18. We are continuing this project, following up on novel observations made by FIB-SEM related to how membrane structure regulates early stages of ciliogenesis. In particular, we are interested in the relationship between the developing ciliary membrane and uncapping of the mother centriole. We have also extended this work to investigate ciliogenesis defects observed in cancer cells, which fail to recruit membranes to the mother centriole and uncap this structure for axonemal growth. 2) We submitted a second manuscript for review in FY18 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 unciliated 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. Revision experiments are underway. 3) We are completing work examining the role of the TRAPP complex in ciliogenesis. We have discovered a novel TRAPPC protein, TRAPPC14, which regulates Rabin8 preciliary trafficking and which mediates interaction between Rabin8 and the larger TRAPPII complex. We expect to submit a manuscript for this work in 2018. 4) In another project, we are characterizing a novel membrane fusion SNARE complex important for ciliogenesis. Cell biology and biochemical studies are ongoing to characterize the SNARE complex. Meetings and Presentations: ASCB Philadelphia December 2017 - Abstract title - 3D FIB-SEM analysis of PACSIN membrane tubulation regulation of intracellular ciliogenesis (poster) FASEB Small GTPase meeting September 2018 - Abstract title Primary cilia assembly initiation is regulated by a Rab effector switch (tbd) I gave invited talks on this labs work for the Department of Molecular Genetics at the University of Toronto and for the Department of Cell and Developmental Biology at Vanderbilt University.
