Functional Compartmentalization of Hedgehog Signal Transduction in Primary Cilia Abstract Primary cilia are small, antenna-like organelles critical for vertebrate development and physiology. Defects in cilia result in human diseases called ciliopathies, characterized by a wide spectrum of phenotypes, highlighting their important role in multiple cell types and organs. Although not completely surrounded by a membrane, cilia form a distinct compartment that receives and transmits extracellular signals. Their function critically depends on the dynamic changes in protein composition and localization. The Hedgehog (Hh) signaling transduction, essential for embryonic development, adult tissue regeneration and cancer, largely takes place at primary cilia. The Hh-transduction proteins Smoothened (Smo), Patched1 (Ptch1), Suppressor of Fused (SuFu) and Gli2 shuttle into and out of cilia when cells receive the Hh signal, and changes in localization correlate with pathway activation. The molecular basis for most of these steps is not clear. In this proposal, we will build upon our previous work in Hh signal transduction and in the cell biology of cilia and centrosomes, to answer unresolved questions about protein compartmentalization in cilia and Hh signal transduction. We will apply high-resolution optical microscopy, molecular genetics and novel biochemical methods to determine how specific interactions with ciliary protein complexes control Hh-transducer dynamics in cilia, and, more broadly, how ciliary responsiveness is established; investigate how Ptch1 interactions within the cilium regulate Smo activation; finally, we will elucidate how removal of Hh-transducers from cilia is controlled, extending our recent discovery of Rilp-like proteins as regulators of this process. In summary, we propose to establish a high-resolution, spatially and temporally resolved picture of Hedgehog signaling in the primary cilium. We believe that only by having such an in- depth analysis will it be possible to understand the molecular basis of Hedgehog signaling. !
Primary cilia are complex cellular organelles, present in most cell types, that function in reception and transduction of extracellular signals; defects in cilia cause a spectrum of human diseases called ciliopathies. The dynamic localization of specific proteins required for signal transduction processes is critical for cilium function as a signaling center. We propose to use state-of-the-art imaging and biochemical methods to understand the regulated trafficking and compartmentalization of signaling proteins within cilia, with a focus on the Hedgehog signal transduction pathway; the signaling components in this pathway have complex behavior with respect to cilium localization, and defects in this pathway cause birth defects and cancer.
