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

Abstract

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.

Keywords: Hedgehog signaling; Patched; Smoothened; primary cilia; single-molecule tracking.

Fig. 1.

PTCH1-ACP-YFP protein retains Hh-pathway functional activity. (A) Diagram showing PTCH1-ACP-YFP fusion protein. An ACP-tag was added to the extracellular loop1 of the PTCH1 protein and the YFP-tag is at the C terminus. (B) The PTCH1-ACP-YFP fusion protein was functional in inhibiting the Hh pathway. Levels of Gli1 RNA were assayed by quantitative RT-PCR in Ptch^−/− cells infected with an empty retrovirus (vector) only, or with PTCH1-ACP-YFP. (C) PTCH1-ACP-YFP is degraded in response to SHH. PTCH1-ACP-YFP–expressing cells were treated with SHH or with culture media only. Protein extracts from those cells were analyzed by standard immunoblots. PTCH1-ACP-YFP was detected with an anti-GFP antibody, and anti–γ-tubulin was used as a loading control. (D) PTCH1-ACP-YFP is responsive to SHH, as measured by Gli1 target gene expression in cells treated with SHH. Gli1 RNA was measured by quantitative RT-PCR. (E) PTCH1-ACP-YFP localized in cilia of Ptch^−/− cells. The protein was detected with an anti-GFP antibody, and cilia were marked with anti-acetylated tubulin antibody. (Scale bar: 1 μm.) (F) PTCH1-ACP-YFP delocalized from cilia in response to SHH. After 24 h of SHH treatment, levels of PTCH1-ACP-YFP were quantified by scanning confocal microscopy after immunostaining. Data are mean ± SEM (*P < 0.05, ***P < 0.001).

Fig. 2.

Characterization of PTCH1 motional dynamics in cilia. Representative kymograms and trajectories of PTCH1-ACP-YFP in cilia show three modes of motion: diffusion, confinement, and directional transport. Using two-color imaging, we observe the movement of both the bulk populations of PTCH1 molecules (YFP, in A and C) and single-molecule trajectories (DY647, in B and D). (A) Fluorescent image of YFP in the cilium. Cilia were identified based on the accumulation of YFP. (B) Image of an isolated DY647-labeled PTCH1-ACP-YFP molecule in the cilium shown in A. (C) Kymogram of the YFP channel for the cilium shown in A. (D) Kymogram of the red imaging channel of the same region shown in C. (E) Two-dimensional single-particle trajectory extracted from the period marked with a dashed line in D, color coded for time. (F and G) The same trajectory shown as the long axis (F) or short axis (G) position in time, color coded for the classified movement type. Boxed Insets show the 2D trajectory during an identified period of retrograde transport and confinement, respectively. (H) The signal photons maintain steady levels indicative of a single fluorophore followed by a single-photobleaching event. For clarity, the trace shows the average number of photons from a three-frame window. (Scale bar: 1 μm.)

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.

Fig. 4.

Cholesterol depletion affects SMO in cilia of Ptch1^−/− cells, and of SHH treated cells, but not SAG-treated cells. (A) Cells lacking PTCH1 accumulate SMO in cilia (image on the Left); however, 2-h treatment with MβCD reduces the levels (image on the Right). Fixed cells were stained with anti-SMO and anti-ARL13b to detect cilia and imaged by confocal microscopy. (B) Time-dependent delocalization of SMO from cilia in Ptch1^−/− cells after cholesterol depletion [mean ± SEM; not significant (NS), P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001]. (C) Reduction of Gli1 expression after MβCD treatment, quantified by RT-PCR (mean ± SEM). (D) SMO trajectory-motion types in Ptch1^−/− cells expressing SNAP-SMO and labeled with Alexa647 fluorescent substrate. Cells were imaged either at baseline, media-only condition, or after 30–90 min of 2-mM MβCD treatment. Trajectories were pooled and organized in bins along the long axis of the cilium. (E) MβCD treatment reduced the bulk SMO protein levels in cilia of Ptch1^−/− cells, but did not significantly change the SAG-induced accumulation of SNAP-SMO in cilia. SANT-1 blocked the accumulation of SNAP-SMO in cilia regardless of MβCD treatment. (F) Trajectories of SMO show no increase in binding at the base of cilia after cholesterol depletion in SAG-treated SNAP-SMO; PTCH-YFP cells. (G) When SAG is replaced with SHH, there is a significant increase in SMO binding at the base in cholesterol-depleted cells.

Fig. 5.

PTCH1 and SMO are dynamically segregated in the ciliary membrane. (A) Diffusion coefficients for PTCH1 and SMO in cilia are affected by SHH. The diffusion coefficients for PTCH1 and SMO in cilia were determined for clearly diffusing (DY/Alexa) 647-labeled molecules under different experimental conditions, as marked. Graphs show the distribution of measured diffusion coefficients from short subtrajectories determined by mean squared displacement analysis. Insets show the mean diffusion coefficients [not significant (NS), P > 0.05, *P < 0.05, **P < 0.01]. (B and C) Image reconstructions of all SMO-Alexa647 positions in trajectory data (red), superlocalized positions of PTCH1-YFP (green in B), and 5HT₆-YFP as a control (green in C). Dotted line outlines cilia. (Scale bar: 500 nm.) Arrows highlight anticorrelation of localizations.