Hedgehog-stimulated chemotaxis is mediated by smoothened located outside the primary cilium

Abstract

Regulation of the Hedgehog (Hh) pathway relies on an interaction of two receptors. In the absence of Hh, Patched1 (Ptch1) inhibits the pathway. Binding of the ligand Hh to Ptch1 stimulates the localization of the activating receptor Smoothened (Smo) to the primary cilium, which is required for the transcriptional Hh response. Hh can also induce chemotaxis through a nontranscriptional pathway. We assessed the effects of defective ciliary localization of Smo on its subcellular trafficking and ability to mediate chemotactic signaling. Cells expressing mutants of Smo that could not localize to the primary cilium or cells lacking the primary cilium showed altered intracellular trafficking of Smo and, in response to Hh or Smo agonists, decreased transcriptional signaling and enhanced chemotactic responsiveness. Thus, the ciliary localization machinery appears to transport Smo to subcellular sites where it can mediate transcriptional signaling and away from locations where it can mediate chemotactic signaling. The subcellular localization of Smo is thus a crucial determinant of its signaling characteristics and implies the existence of a pool of Smo dedicated to chemotaxis.

Fig. 1

Reduced ciliary localization machinery correlates with altered intracellular itineraries of Smoothened. (A–D) Kif3A+/+ and Kif3A−/− MEFs (E–H) were transfected with SmoWT or SmoΔCLD and treated with 1 μg/ml cycloheximide and 100 μM chloroquine for 3 hours. Smo was visualized by immunofluorescence, and its localization divided in three classes; unperturbed localization to cytosol and membrane, indicated by grey arrow and grey fill in graphs; moderate perinuclear accumulation indicated in blue; widespread vesicular localization indicated in red. Fraction of cells with different Smo localization was quantified. Shown is mean of >60 cells in 3 independent experiments. Scale bar, 20 μm. (I–J) As for panels A–D using wild type MEFs, treatment for times indicated. (K–N) As for panels A–D. Immunofluorescence for indicated proteins was performed. Colocalization of Smo and organelle staining was analyzed and mean percentage of colocalizing pixels is indicated, ± SEM. (O) MEFs were transfected with SmoWT or SmoΔCLD and GFP, and surface biotinylation was performed. Shown is Western blot for surface labeled Smo, and Smo and GFP in total lysates. (P) As for panel O, lysates were treated wit Endo H to assess glycosylation status. Shown is Western blot for surface labeled Smo.

Fig. 2

Defective ciliary localization enhances chemotactic signaling by Smoothened. (A) MEFs were transfected with indicated forms of Smo and Gli-luciferase reporter. Luciferase activity was measured and corrected for co-transfected Renilla luciferase following 2 μM purmorphamine or control stimulation for 16 hours. Shown is mean fraction of vector, ± SEM, n=6. (B) MEFs were transfected, and transferred to a modified Boyden chamber, using 2 μM purmorphamine or 2 nM ShhN as attractant. Solvent control migration was subtracted to establish net chemotaxis. RFU, relative fluorescence unit; 1 cycle, 2 min. (C) Shown is average migration from 3 experiments as for panel B, using Smo−/− MEFs transfected with vector or indicated mutant forms of Smo, ± SEM, n≥3. For quantitative details, see Methods section. (D) As for panel C, using 2mM recombinant ShhN; (E) 500 nM SAG; (F) As for panel A, using Smo−/− MEFs transfected with mouse (M. musculus), zebrafish (D. rerio), or fruitfly (D. melanogaster) Smo. (G) As for panel C, using indicated constructs. (H) Smo−/− MEFs were transfected with SmoWT, pretreated with the indicated inhibitors for 10 min, and chemotaxis to purmorphamine was assessed. Concentrations used; cyclopamine, 5 μM; pertussis toxin (PTX), 1 μM; MK-571, 5 μM, PP1 and PP2, 10μM.

Fig. 3

Cells with defective ciliary function show enhanced chemotactic signaling by Smoothened. (A) Primary cilia on Kif3A+/+ and Kif3A−/− MEFs (B) were visualized by immunofluorescence for acetylated α-tubulin. Scale bar, 20 μm. (C) Kif3A MEFs were transfected with Gli-luciferase reporter and stimulated as for Fig. 2A. Shown is the mean ± SEM, n=4. P-value indicated is compared to Kif3A+/+ MEFs. (D) Chemotaxis to purmorphamine or FCS of Kif3A MEFs was assessed and shown as mean fluorescence ± SEM, n=4. Pretreatment with 500 ng/mL actinomycin D or 10 μM cyclopamine was for 10 min. P-value indicated by lines is compared to wild type MEFs. P-value indicated by grey symbols is compared to purmorphamine stimulated (no inhibitors). (E) As for panel D, using recombinant ShhN; (F) using SAG. (G, H) Primary cilia on wild type or Tg737^orpk MEFs were visualized (I) Tg737 MEFs were transfected with Gli-luciferase reporter, stimulated, and analyzed as for panel C. (J) As for panel E using Tg737^orpk or wild type MEFs. (K) As for panel J, using recombinant ShhN; (L) using SAG.

Fig. 4

Smoothened localized outside the primary cilium enhances neurite outgrowth. (A) Smo−/− ES cells were transfected with vector. After 1 days, cells were grown into embryoid bodies (EBs) and after 3 days replated without inducers, or neuralized and ventralized by addition of 1 μM retinoic acid (RA) and 200 nM SAG (B). After an additional 4 days in culture, class III β-tubulin-positive neurites were visualized. (C) Distance between crossing neurites was measured and frequency distribution analysis was performed. Over 100 measurements were made. (D) Induction of motor neurons was quantified by staining for Isl1/2. Number of Isl1/2-positive cells per EB was counted, and shown is mean ± SEM, n≥100 over 2 experiments. Scale bar, 100 μm. (E–H) As for panels A–D, using Smo+/− ES cells transfected with vector; (I–L) SmoWT; (M–P) SmoΔCLD. P-value indicated is compared to vector. (Q) Cells were transfected with SmoΔCLD and EBs were formed in the presence of RA and SAG, and the leukotriene inhibitor MK-886 (μM indicated). After 4d, EBs were stained for Isl1 and class III β-tubulin. Shown is mean ± SEM, n≥50.