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. 2009 Feb 24;106(8):2623-8.
doi: 10.1073/pnas.0812110106. Epub 2009 Feb 5.

Selective translocation of intracellular Smoothened to the primary cilium in response to Hedgehog pathway modulation

Yu Wang  1 , Zhe Zhou, Christopher T Walsh, Andrew P McMahon
Affiliations

Selective translocation of intracellular Smoothened to the primary cilium in response to Hedgehog pathway modulation

Yu Wang et al. Proc Natl Acad Sci U S A. .

Abstract

Smoothened (Smo), a 7-pass transmembrane protein, is essential for transduction of a Hedgehog (Hh) signal across the cell membrane. Smo is also the principle therapeutic target for several candidate drugs in the treatment of Hh-related diseases. Mammalian Smo translocates to the primary cilium in response to Sonic hedgehog (Shh) ligand-mediated signaling. A mechanistic understanding of Smo translocation and its interactions with drug candidates is pivotal to our understanding of Hh signaling and the design, development and application of successful drugs. We established a system in which Smo was dual-labeled with GFP and a 12-aa tag whose recognition by an enzymatic process enables the posttranslational labeling of Smo in the cell membrane within the living cell. These tools enable the simultaneous visualization of all cellular Smo, and more specifically, the cell membrane restricted subpopulation. Using this system, we demonstrate that cyclopamine, a widely used Hh antagonist, induces a cilial translocation of Smo similar to that reported for Shh ligand and several Hh agonists. In contrast, other antagonists abrogate the Shh-induced, cilial translocation of Smo. We present evidence that the majority of cilial-localized Smo originates from an intracellular source and may traffic to the primary cilium through an intraflagellar transport (IFT) pathway.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Generation of a dual labeled A1::Smo::GFP reporter construct. (A) A 12-aa A1 tag was added to the extracellular N terminus of human Smo, whereas GFP was fused to the intracellular C terminus. (B) AcpS enzyme transfers a fluorophore to the serine in the A1 tag (marked as red in A) through a Ppant group in the CoA-fluorophore substrate, thereby labeling A1::Smo::GFP with a specific fluorophore.
Fig. 2.
Fig. 2.
A1::Smo::GFP is functionally equivalent to endogenous Smo. (A–F) NIH/3T3 cells expressing GFP (A and B) or A1::Smo::GFP (C and D) were either untreated (A, C, and E) or treated with ShhN ligand (B, D, and F). GFP (green in A and B) and A1::Smo::GFP (green in C–F) were visualized by direct detection of GFP. Endogenous Smo (red in A–D) and A1::Smo::GFP (red in C and D) were detected with anti-Smo antibody. Cilial basal bodies were visualized with γ-tubulin antibody (blue in A–F). Arl13b was also identified with immunofluorescence (red in E and F). (Scale bars: 5 μm.) (G) Hh signaling was assessed in NIH/3T3 cells by activation of a Ptch1-promoter driven luciferase reporter in response to ShhN ligand. Cells either expressed GFP (blue bars) or A1::Smo::GFP (red bars) and in some samples they were transfected with short hairpin RNA constructs to knock down GFP (shGFP) and/or endogenous mouse Smo (shmSmo-1 and shmSmo-2; recognizing nonoverlapping regions specific to mouse and not human Smo). The result shown is the mean of 4 replicates. Error bars indicate SD.
Fig. 3.
Fig. 3.
Effects on Smo localization and Hh activity by various Hh agonists and antagonists. (A–G) Cells expressing A1::Smo::GFP were treated with DMSO (A); SAG (B), cyclopamine (CYC) (C); ShhN (D); or a combination of ShhN and either SANT-1 (E), SANT-2 (F), or GANT61 (G). A1::Smo::GFP (green in A–G) was visualized by direct detection of GFP. Primary cilia were marked with Arl13b antibody (red in A–G). (Scale bars: 5 μm.) (H) Hh signaling was activated by either ShhN ligand or RNAi knock down of suFU and assessed in Gli-luciferase assays. NIH/3T3 cells were either untreated or treated with one of the Hh antagonists, either CYC, SANT-1, SANT-2, or GANT61. The result shown is the mean of 4 replicates. Error bars depict SD.
Fig. 4.
Fig. 4.
AcpS labeling of A1::Smo::GFP. (A and B) After ShhN treatment, A1::Smo::GFP cells were incubated with both Texas Red (TxRed)-CoA and Alexa633-CoA either with (A) or without (B) AcpS for 20 min. (C) Wildtype NIH/3T3 cells were also treated with ShhN and incubated in a reaction with AcpS. (D) Experimental procedure for a “pulse–chase” labeling with 2 colors. (E and F) The images depict labeling at 1 h (E), before ShhN treatment, and at 3 h and 20 min (F), after the second labeling with Alexa633. (G) The background level of labeling was obtained following the same process but without ShhN treatment. A1::Smo::GFP was directly visualized by detecting GFP (green in A, B, and E–G). Endogenous Smo in wildtype NIH/3T3 cells was detected by Smo antibody (green in C). Labeling signal with TxRed and Alexa633 is shown in red and purple, respectively. Cilium basal bodies are marked with γ-tubulin antibody (blue in A–C and E–G). (Scale bars: 5 μm.)
Fig. 5.
Fig. 5.
A1::Smo::GFP colocalizes with IFT88. Cells expressing A1::Smo::GFP (green) were untreated (A) or treated with ShhN (B) and labeled at the cell surface with Alexa633 (purple), followed by staining with IFT88 (red) and γ-tubulin (blue). Arrows indicate localization to the basal bodies of primary cilia; arrowheads denote noncilial localization to intracellular puncta. (Scale bars: 5 μm.)
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