Patched1 Regulates Hedgehog Signaling at the Primary Cilium
Rajat Rohatgi1,2*,
Ljiljana Milenkovic1* and
Matthew P. Scott1
1 Departments of Developmental Biology, Genetics, and Bioengineering and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
2 Department of Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA.
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Fig. 1. Rapid localization of Smo in primary cilia after activation of the Hh pathway and regulation by Ptc1. (A) Immunoblots with antibodies to Ptc1, Smo, and actin were used to assess amounts of endogenous proteins in extracts from NIH 3T3 cells treated with Shh or SAG (100 nM). (B) Enrichment of Smo in primary cilia of NIH 3T3 cells left untreated (control) or treated with Shh or SAG (100 nM) for 24 hours. (C) Mean intensity of Smo fluorescence in cilia of NIH 3T3 cells treated with Shh or SAG (100 nM). Each point shows the mean ± SEM of fluorescence from 10 to 20 cilia. (D and E) Constitutive presence of Smo in the cilia of unstimulated ptc1–/– MEFs and reversal by retrovirally transduced Ptc1-YFP. In (B) and (D), confocal images of the ciliary marker acetylated tubulin (red) and Smo (green) were detected by immunofluorescence; nuclei (blue) were stained with 4',6'-diamidino-2-phenylindole (DAPI).
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Fig. 2. Localization of Ptc1 in primary cilia. (A) Concentration of endogenous Ptc1 in cilia of NIH 3T3 cells stimulated with 100 nM SAG. (B) Localization of Ptc1-YFP in ptc1–/– MEFs infected with a retrovirus carrying an empty vector or the ptc1-YFP coding sequence. In (A) and (B), cilia (red) and Ptc1 (green) were visualized by immunofluorescence; nuclei (blue) were stained with DAPI. (C) Ptc1 staining in Shh-responsive cells of the neural tube (nt), notochord (nc), floor plate (fp), and paraxial mesoderm (m). Cross sections of wild-type (top row) or control ptc1–/– (bottom row) mouse embryos (E9.5) were imaged with a 40x objective. (D) Asymmetric, ciliary localization of Ptc1 in paraxial mesoderm cells. The cell boxed in white is magnified in the bottom panel; arrows indicate Ptc1 staining (red) around the base and in the shaft of cilia (green).
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Fig. 3. Interactions between Shh and Ptc1 at primary cilia. (A) Colocalization of Shh and Ptc1 at the cilium shown in a confocal image of a live ptc1–/– cell transfected with Ptc1-YFP (green) and incubated with ShhN-A594 (red, 300 ng/ml) for 45 min. Inversin-CFP (cyan) marks the cilium, the dotted line demarcates the cell border, and insets show magnified views of the cilia. (B) Mean Ptc1 fluorescence in cilia of NIH 3T3 cells treated with SAG (100 nM), Shh, or both. (C) Disappearance of Ptc1 from primary cilia after Shh treatment. NIH 3T3 cells preincubated with SAG for 24 hours were switched to control medium (untreated) or into Shh-containing medium. The red dashed baseline shows the amount of ciliary Ptc1 in cells treated with Shh for 4 hours without a 24-hour SAG pulse.
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Fig. 4. Accumulation of Smo and Ptc1 at cilia of NIH 3T3 cells exposed to 20 -hydroxycholesterol. (A and C) Localization of cilia (red) and Smo or Ptc1 (green) in cells treated with 10 µM 20-hydroxycholesterol or 7-hydroxycholesterol for 24 hours. (B) Time course of Smo accumulation at the primary cilium in NIH 3T3 cells treated with 20-hydroxycholesterol. (D) Increase in Ptc1 fluorescence in primary cilia after treatment with 20-hydroxycholesterol. In (B) and (D), each point shows the mean ± SEM of fluorescence from 10 to 20 cilia.
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