Project Summary / Abstract The primary cilium is critical to vertebrate development and the prevention of disease. Severe defects in this organelle lead to prenatal lethality in extreme cases and a variety of structural birth defects and degenerative diseases in less extreme cases. The primary cilium serves as a cellular antenna to monitor the extracellular environment and feed information back to the cell to coordinate its action with that of the surrounding cells. Several signaling pathways are directly or indirectly regulated by cilia but of these, Hedgehog is particularly important in vertebrate development. Understanding how the cilium is assembled and how the signaling environment is created and maintained is critical to understanding how this organelle functions in the etiology of human diseases. The cilium is assembled by the process of intraflagellar transport (IFT). During IFT, large protein complexes called IFT particles (composed of IFT-A, IFT-B, and BBSome sub particles) are carried along the ciliary microtubules by kinesin and dynein motors. IFT transports proteins made in the cell body into the cilium to build and maintain the structure. In addition to building cilia, IFT is an integral part of the Hedgehog signaling. Hedgehog signaling is initiated by Hedgehog ligand binding to ciliary-localized Ptch1. Activated Ptch1 leaves the cilium and allows Smo to activate. Activated Smo accumulates in cilia, and this drives a third receptor, Gpr161, out of cilia. The Gli transcription factors then accumulate at the ciliary tip and become activated before moving into the nucleus to regulate gene expression. In past funding cycles, we discovered that the Ift25/Ift27 subcomplex of IFT-B is not needed for ciliary assembly but is critical for Hedgehog signaling by removing Ptch1 from cilia upon pathway activation and keeping Smo levels low at the basal state. Our work showed that Ift25/Ift27 couple the BBSome to IFT-B through an adaptor protein Lztfl1. The BBSome is likely to be the receptor recognition module of the IFT particle. Understanding the mechanism regulating the interaction of Smo and Ptch1 with the IFT particle is critical to understanding Hedgehog signaling. In the last cycle, we found that ubiquitination is likely to be the key regulatory event. At the basal state, Smo is ubiquitinated on lysines K444 and K448, which marks the receptor for removal from cilia by IFT. Mutation of these lysines or pharmacologically blocking ubiquitination causes Smo to inappropriately accumulate in cilia at the basal state. The E2 Ube2l3 and the E3 Wwp1 localize to cilia and appear to be responsible for the regulatory ubiquitination of Smo. Wwp1 binds to Ptch1. Activation of signaling removes both Ptch1 and Wwp1 from cilia, thus providing an elegant mechanism for Ptch1 to regulate ciliary Smo levels. We propose to extend this work by identifying adaptors that link ubiquitinated Smo to the IFT particle and the determine the mechanism controlling Ptch1’...