Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol

Significance Primary cilia are antenna-like sensory organelles critical for many signal transduction pathways. One such pathway, called Hedgehog signaling, is essential for normal embryonic development and linked to tissue homeostasis and tumorigenesis. While the tightly regulated spatial and temporal localization of proteins in cilia controls activity, the mechanistic details are not fully understood. To characterize the movements of the Hedgehog receptor Patched1 in the cilium, we used single-molecule fluorescence microscopy, a tool that separates the behavior of individual proteins from the ensemble average, unveiling the underlying hidden physical behaviors. Our study reveals Hedgehog-induced changes in the motion of individual Patched1 molecules, which precede the exodus of Patched1 from cilia. These changes constitute one of the earliest measurable steps of Hedgehog-signal transduction. The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides.