Fig. 3.

PTCH1 diffusive movement in cilia is reduced in response to SHH and to cholesterol depletion. Single-molecule trajectories were divided into subtrajectories based on the likely mode of motion. (A–C) For each experimental condition, data from multiple cilia were combined by their position along the long axis of a normalized cilium. The relative fraction of each motion type (confined, diffusive, or directed motion) of the total time in each bin was calculated. (D) Pooled motion-type data from all recorded tracks of single PTCH1 molecules in cilia show that both SHH and MβCD treatment induce a significant increase in fraction of time spent in confinement, and a decrease in the fraction of time spent diffusing. No agonist, 85 trajectories from 41 cells; SHH treatment, 86 molecules from 35 cells; MβCD treatment, 17 molecules from 10 cells [not significant (NS), P > 0.05]. (E) Cholesterol depletion does not affect SHH-induced PTCH1 delocalization from cilia. PTCH1-ACP-YFP cells were treated for 4 h with SHH, MβCD, or both, fixed and immunostained, and imaged by confocal microscopy. Images were quantified for cilia localization of PTCH1 (mean ± SEM; **P < 0.01, ***P < 0.001). Treatment with SHH caused delocalization from cilia regardless of whether cells were treated with MβCD or not.