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The role of ciliary trafficking in Hedgehogreceptor signalingJynho Kim,1,2 Elaine Y. C. Hsia,3 Amira Brigui,4 Anne Plessis,4

Philip A. Beachy,1,2* Xiaoyan Zheng1,3*

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Defects in the biogenesis of or transport through primary cilia affect Hedgehog protein signaling, andmany Hedgehog pathway components traffic through or accumulate in cilia. The Hedgehog receptor Patchednegatively regulates the activity and ciliary accumulation of Smoothened, a seven-transmembrane proteinthat is essential for transducing the Hedgehog signal. We found that this negative regulation ofSmoothened required the ciliary localization of Patched, as specified either by its own cytoplasmic tailor by provision of heterologous ciliary localization signals. Surprisingly, given that Hedgehog binding pro-motes the exit of Patched from the cilium, we observed that an altered form of Patched that is retained inthe cilium nevertheless responded to Hedgehog, resulting in Smoothened activation. Our results indicatethat whereas ciliary localization of Patched is essential for suppression of Smoothened activation, theprimary event enabling Smoothened activation is binding of Hedgehog to Patched, and Patched ciliaryremoval is secondary.

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INTRODUCTION

Hedgehog (Hh) proteins function as cell-to-cell signals with embryonicroles in specification of tissue patterning and cell differentiation (1) andpostembryonic roles in organ homeostasis and regeneration (2, 3). Addi-tionally, pathway activity can stimulate or suppress the growth of variouscancers (4–7). During vertebrateHh signaling, the processed lipid-modifiedN-terminal signaling domain of Hh activates the pathway by binding toPatched1 (Ptch1) (8), a member of the resistance-nodulation-division(RND) family of proton-driven 12-transmembrane (12-TM) transporters,thus relieving Ptch1 suppression of the 7-TM protein Smoothened (Smo),which is structurally related to G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors. Activation of Smo, in turn, stimulatestranscriptionby inhibiting the constitutive proteolytic processingofGli familyproteins and switching them to their activated states.

The primary cilium plays a central role in transduction of vertebrate Hhsignals (9). ShhN, the signaling domain of mammalian Sonic Hh, binds tothe shaft of primary cilia, which contain Ptch1, and induces Ptch1 removalfrom the cilium, which is accompanied by ciliary accumulation of Smo(10, 11). Ciliary accumulation of activated Smo then triggers pathway ac-tivation through Gli proteins, which also accumulate in the primary ciliumbefore ciliary exit and nuclear entry (12, 13). Despite these findings, themechanisms by which Ptch1 suppresses Smo activity and by which Hh in-activates this inhibitory function of Ptch1 remain unclear. One model is thatPtch1 inhibits the pathway by excluding Smo from the cilium in resting cellsand thatHh binding triggers removal of Ptch1 from the cilium, thus enablingSmo to enter and activate signaling (11, 14). However, Smo accumulates inthe primary cilium without Hh stimulation in cells in which cytoplasmic

1Departments of Biochemistry and Developmental Biology, Institute for StemCell Biology and Regenerative Medicine, Stanford University School of Med-icine, Stanford, CA 94305, USA. 2Howard Hughes Medical Institute, StanfordUniversity School of Medicine, Stanford, CA 94305, USA. 3Department ofAnatomy and Regenerative Biology, George Washington University Schoolof Medicine and Health Sciences, Washington, DC 20037, USA. 4InstitutJacques Monod, CNRS, UMR 7592, Université Paris Diderot, Sorbonne ParisCité, F-75205 Paris, France.*Corresponding author. E-mail: pbeachy@stanford.edu (P.A.B.); xzheng@gwu.edu (X.Z.)

dynein 2, the microtubule motor for retrograde ciliary trafficking, is genet-ically or pharmacologically impaired (13, 15–17), suggesting that Smomaytraffic into the cilium even in the presence of active Ptch1. Accumulation ofSmo in primary cilia in the absence of Hh-mediated Ptch1 inactivation alsooccurs in cells with functional impairment of other ciliary transport proteins(18–20), and Smo also accumulates in cilia of cells exposed to pharmaco-logical agents that directly bind and activate (or in some cases inactivate)Smo (11, 21). Thus, the relationship of the ciliary trafficking of Ptch1to activation and ciliary accumulation of Smo is unclear. To address theseissues, we used systematic deletions to show that sequences in the Ptch1cytoplasmic tail contributed to Ptch1 ciliary localization and that removalof these sequences disrupted the Smo inhibitory function of Ptch1. Fur-thermore, replacement of the Ptch1 cytoplasmic tailwith heterologous cil-iary localization signals (CLS) restored not only the ciliary localization butalso the Hh-responsive regulatory activity of Ptch1. We thus provide evi-dence that Ptch1 suppression of Hh pathway activity and response to Hhrequires localization to the primary cilium. We also found that modifica-tion of Ptch1 such that the protein remained in the primary cilium even inthe presence of ShhN nevertheless repressed downstream signaling activ-ity in the absence of ligand, and this inhibitory activity was relieved byexposure of the cells to ShhN. Therefore, although the removal of Ptch1from the primary cilium may fine-tune pathway activity, our evidence in-dicates that ciliary removal of Ptch1 is not essential for Hh-inducedpathway activation.

RESULTS

Ptch1 C-terminal cytoplasmic tail is necessary forciliary localizationThe CLS of Ptch1 has not been clearly defined, and such signals are in-sufficiently cataloged for reliable identification by sequence comparison.We constructed a series of truncations that progressively remove sequencesfrom an epitope-tagged form of Ptch1, expressed them in Ptch1−/− cells(Fig. 1A), and assessed ciliary localization by laser confocal microscopy(Fig. 1B). In addition,we tested for Smo suppression activity by cotransfect-ing these Ptch1 variants at low levels [1% (w/w)DNA]with aGli-controlled

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luciferase reporter. We used Ptch1−/− cells to avoid interference by endog-enous Ptch1, which is reported to homomultimerize (22). These cells do notexpress the Patched ortholog Ptch2; therefore, Ptch1 is the Hh receptor re-sponsible for Smo regulation and control of Hh pathway activity in thesecells (23, 24).

Removal of all ormost of the amino acid sequencesC-terminal to the finaltransmembrane segment (in constructs Ptch11162, Ptch11170, and Ptch11180)fully disrupted Ptch1 ciliary localization (Fig. 1, B and C) and inactivatedSmo suppression, leading to constitutively high reporter activity (Fig. 1C).The addition of amino acids from amino acid residue 1180 to the C terminusprogressively restored ciliary localization and Smo suppression (Fig. 1, B andC). Furthermore, Smo suppression correlated highly with the ability of thesesequences to target Ptch1 to the cilium (Fig. 1D). Restoration of Ptch1 cyto-plasmic tail sequences also restored the response to Hh stimulation (Fig. 1E).

Ptch1 cytoplasmic tail and Smo cytoplasmic tail mediateciliary localizationPtch1 with full cytoplasmic tail but lacking a segment of the TM7-TM8extracellular loop fully suppressed Smo activity but did not respondto Hh (Ptch1∆L2, Fig. 2A), consistent with loss of Hh binding (25, 26).We found that the Ptch1 C-terminal cytoplasmic tail (Ptch1CT) suf-ficed to specify ciliary localization when fused to peptides contain-ing tandem acylation signals from the kinase Lyn11 or the adaptorLAT (linker of activated T cells) (Fig. 2B), consistent with previousreports that acylated peptides targeted to membrane microdomains canreveal the activity of ciliary localization signals to which they are fused(27, 28).

As with Ptch1, truncation of the Smo cytoplasmic tail impaired Smoaccumulation in cilia and the ability of Smo to rescueHh pathway activity in

Fig. 1. ThePtch1 cy-toplasmic tail is nec-essary for ciliarylocalization.(A)Sche-matic diagram ofPtch1 cytoplasmic taildeletions. Ptch11434 isthe wild type (WT).Theinsetshowsabun-danceof the indicatedMyc-tagged Ptch1proteins expressed inHEK293 cells. aa,amino acids; b-Tub,b-tubulin. (B) Ptch1−/−

cells transfected withMyc-tagged Ptch1carrying various cyto-plasmic tail deletionswerefixedandstainedwith antibodies recog-nizingacetylatedtubu-lin (AcTub) (primarycilium, red) and Myc(Ptch1variants,green).Theinsetsshowshiftedoverlaysof theredandthe green channels.Scale bars, 5 mm. (C)The mean fluores-cence intensity ofPtch1 variants in ciliaof transfected Ptch1−/−

cellsand their ability tosuppressSmoactivityare plotted againstthe cytoplasmic tail

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lengths of Ptch1 variants. Ciliary intensity is themean ± SD of fluorescencefrom 10 to 20 cilia. Smo suppression activity is defined as RGFP/RPtch1, theratio of Gli reporter activity from Ptch1−/− cells cotransfected with green flu-orescent protein (GFP) or each individual Ptch1 variant: a higher value ofthis quotient indicates more effective suppression by a particular Ptch1variant. Error bars indicate SD; n = 3. (D) Smo suppression activity was

plotted as a function of ciliary intensity for each Ptch1 cytoplasmic tail de-letion. The Pearson correlation coefficient is 0.97 (r = 0.97). (E) Ptch1−/−

cells transfected for expression ofMyc-taggedPtch1 variants, Gli reporter,and control SV40-Renilla luciferase were grown to confluency, incubatedwith or without ShhN, and assayed for reporter activity. Error bars indicateSD; n = 3.

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Smo−/− cells (Fig. 2C). Furthermore, the cytoplasmic tail of Smo sufficed todeliver acylated LAT into primary cilia (Fig. 2D). Thus, although the exactmo-tif responsible for ciliary localization is not defined, both Ptch1 and Smo haveCLS that are necessary for the ability of these proteins to traffic into the cilium.

Signaling activity of truncated Ptch1 is restored byheterologous CLSWe tested the importance of ciliary localization for Ptch1 function by attach-ing heterologous CLS sequences to Ptch1 lacking the cytoplasmic tail(Ptch1N). We found that the fusion of the Smo cytoplasmic tail (SmoCT)

with Ptch1N (Ptch1N-SmoCT) rescued ciliary localization and the regulatoryfunction of Ptch1N and that Ptch1N-SmoCT localized to the primary cil-ium in 100% of transfected NIH/3T3 cells (Fig. 3, A and B). The Ptch1N-SmoCT fusion protein also suppressed Gli reporter activity when introducedinto Ptch1−/− cells, and this reporter activity was then induced in responseto ShhN (Fig. 3C). We also tested the effect of the third intracellular loop(icl3) of the 7-TM protein Sstr3 (Sstr3icl3), which contains a well-definedCLS responsible for Sstr3 ciliary localization (29, 30). Fusion of this CLS toPtch1N (Ptch1N-Sstr3icl3) also restored both ciliary localization and Hh-responsive regulatory activity (Fig. 3).

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Fig. 2. The cytoplasmic tails ofPtch1 and Smo specify ciliary

trafficking and Hh responsive-ness. (A) Summary of ciliary lo-calization and signaling activityof Ptch1 variants and a Ptch1cytoplasmic tail fusion protein.The constructs indicated in thefirst column were transfected athigh levels [50% (w/w) DNA] intoPtch1−/− cells to assess ciliary lo-calization by laser confocal mi-croscopy and cotransfected atlow levels [1% (w/w) DNA] withthe Gli reporter to test for signalingactivity. MEFs, mouse embryonicfibroblasts. (B) NIH/3T3 cells weretransfected with yellow fluores-cent protein (YFP)–tagged fusionproteins as schematically indi-cated at the top and fixed andstained with 4 ′,6-diamidino-2-phenylindole (DAPI) (nucleus,blue) and antibodies recogniz-ing AcTub (primary cilium, red)or GFP (fusion protein, green).The lower right insets show shiftedoverlays. Scale bars, 5 mm. (C)The cytoplasmic tail of Smo is re-quired for ciliary localization.Constructs with indicated cyto-plasmic tails of mouse Smo listedin the first column were trans-fected at high levels [50% (w/w)DNA] into Smo−/− cells to assessciliary localization by laser con-focal microscopy and cotrans-fected at low levels [1% (w/w)DNA] with the Gli reporter to testfor signaling activity. Smo793 isthe wild-type Smo. (D) The Smocytoplasmic tail is sufficient for cil-iary localization. NIH/3T3 cellstransfected with the indicatedconstructs were fixed and stainedas described in (B). Images anddata shown are representative ofmany cilia and signaling assaysfrom three experiments.

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Ptch1PY accumulates in the primary ciliumWe tested the model that Hh-dependent pathway activation depends on re-moval of Ptch1 from the cilium. Our analysis focused on two recognitionmotifs (PPXY) for binding byHECT (homologous to theE6APC terminus)E3 ubiquitin ligases (31), one in the cytoplasmic tail and one in the largecytoplasmic loop between TM6 and TM7 (Fig. 4A) (22). Ptch1 with muta-tions in both of these PPXYmotifs (Ptch1PY) persisted in the cilium even inthe presence of ShhN (Fig. 4, B andC). Using a biotin-labeling assay with anon–cell-permeable reagent to compare the amount of surface-localizedPtch1 and Ptch1PYafter stimulation with ShhN, we found that the amountof Ptch1 on the cell surface was reduced after 1 hour of exposure to ShhN,whereas Ptch1PY remained at the cell surface in the presence of ShhN(Fig. 4D).

This ciliary persistence was not due to disruption of ShhN binding be-cause structured illumination microscopy (SIM), which lowers the limit ofoptical resolution to less than 200 nm (32), showed that ShhN added to cellsexpressing Ptch1PY localized in a thin layer overlying regions of the ciliumwith higher concentrations of Ptch1PY protein (Fig. 4F). Consistent withthe hypothesis that Ptch1PY persistence on the ciliary membrane is due toloss of interaction with HECT E3 ubiquitin ligases, we found that, in con-trast to wild-type Ptch1, Ptch1PY did not coimmunoprecipitate with any ofthe eight tested HECT E3 ubiquitin ligases (Wwp1, Wwp2, Nedd4,Nedd4L, Itchy, HECW, Smurf1, and Smurf2) (Fig. 5), suggesting that theinteraction with and ubiquitylation by one or more of these E3 ubiquitinligases is necessary for trafficking of Ptch1 out of the cilium.

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Direct binding of ShhN to Ptch1, butnot ciliaryremoval of Ptch1, is required forShhN-inducedpathway activationThe persistence of Ptch1PY in the cilium pro-vides an opportunity to test the role of Ptch1ciliary removal upon Hh stimulation in activa-tion of the pathway. We found that Ptch1PY in-troduced into Ptch1−/− cells blocked the ciliaryaccumulation of Smo in a manner similar towild-type Ptch1 (Fig. 6A). Upon addition ofShhN, endogenous Smo accumulated not on-ly in the primary cilia of cells transfected forexpression of wild-type Ptch1 but also in theprimary cilia of cells transfected for expressionof Ptch1PY (Fig. 6A). Furthermore, we con-firmed simultaneous localization of Smo inthe primary cilium of 95% of ShhN-stimulatedcells expressing Ptch1PY (Fig. 6B). Imaging ofcilia containing both Ptch1PYand Smo by SIMrevealed that they localized in distinct nonover-lapping but intermingled regions of the primarycilium (Fig. 6C). The functional relevance ofthese nonoverlapping localizations is unclearbut may relate to the proclivity of Ptch1 andSmo to homomultimerize (22, 33).

We also examined the ability of Ptch1−/−

cells to respond to ShhN stimulation uponcotransfection with a Ptch1PY expressionconstruct and the Hh-sensitive Gli reporter.We found little difference in the ability ofwild-type Ptch1 and Ptch1PY to suppress tran-scriptional activity or in the responsiveness of

cells expressing either of these Ptch1 proteins to ShhN stimulation (Fig.6D). To extend this analysis to the single-cell level and to confirm that thetranscriptional response occurred in the very cells that show simultaneousPtch1PYandSmo ciliary localization,we examined the expression of nuclearb-galactosidase in ShhN-stimulated Ptch1−/− cells transfected to expressPtch1PY.Ptch1 inPtch1−/− cells is disruptedbyan in-frame insertionof lacZwithanuclear localization signal (34): the production of nuclear b-galactosidasethus provides a measure of transcriptional activation of the Ptch1 promoter,a Hh pathway target. As expected, nuclear b-galactosidase was abundant incontrol Ptch1−/− cells regardless of ShhN stimulation, whereas Ptch1−/−

cells transfected with either wild-type Ptch1 or Ptch1PY showed very lownuclear b-galactosidase that increased upon ShhN stimulation (Fig. 6, Eand F). Moreover, Ptch1−/− cells transfected with Ptch1PY displayed simulta-neous presence of ciliary Ptch1PY and nuclear b-galactosidase upon ShhNstimulation (Fig. 6, E and G).

DISCUSSION

Our results indicate that Ptch1 function is modular: N-terminal se-quences through the 12th TM domain confer Smo suppression, as well asbinding and response to Hh, and ciliary localization is conferred by the Ptch1cytoplasmic tail. Although the ciliary localization specified by the Ptch1 cyto-plasmic tail was required for Hh-sensitive Smo regulation, the Ptch1 cyto-plasmic tail itself was dispensable if a heterologous CLS was providedinstead.

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Fig. 3. Exogenous ciliary localization signalsrescue the impaired signaling activity of Ptch1N.(A) Schematic diagrams of YFP-tagged Ptch1fusion proteins. (B) NIH/3T3 cells transfectedwith the indicated YFP-tagged Ptch1 constructswere fixed and stained with DAPI (nucleus, blue)and antibodies recognizingAcTub (primary cilium,red) orGFP (fusionprotein, green). The insets showshifted overlays. Scale bars, 5 mm. (C) Ptch1−/−

cells were transiently transfected for expressionof YFP-tagged Ptch1 variants, Gli reporter, andcontrol SV40-Renilla luciferase. After transfection,cells were grown to confluency, incubated with orwithout ShhN, and assayed for reporter activity.Error bars indicate SD; n = 3.

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Fig. 4. Ptch1PY fails to exit the primary cilium. (A)Schematic diagram of Ptch1 shows conserved PPXYmotifs within the large TM6-TM7cytoplasmic loop andthe cytoplasmic tail. (B) Ptch1−/− cells were trans-fected with Myc-tagged Ptch1WT (wild-type Ptch1) orPtch1PY. After incubation with or without ShhN for4 hours, cells were fixed and stained for acetylatedtubulin (primary cilium, red) andMyc (Ptch1 variants,green). The insets show shifted overlays. Scale bars,5 mm. (C) Mean fluorescence intensity in cilia ofPtch1WT or Ptch1PY in transfected Ptch1−/− cells withorwithout ShhN treatment for 4 hours. Eachbar showsthemean±SDof fluorescence from10 to 20 cilia, andrepresentative images are shown in (B). Unpairedt test was used for statistical analysis. ****P < 0.0001.n.s., not significant (P>0.05). (D) Cell surfaceproteinsfrom HEK293 cells transfected with Myc-taggedPtch1WT or Ptch1PY were biotinylated, isolated usingstreptavidin beads, and examined for the presenceof Ptch1 by immunoblotting for Myc. IP, immuno-precipitation; WB, Western blot. (E) NIH/3T3 cellswere transfectedwithMyc-taggedPtch1PY. After incu-bation with or without mCherry-tagged ShhN for4 hours, cells were fixed and stained with antibodiesrecognizing dsRed (ShhN, red), acetylated tubulin(primary cilium, blue), or Myc (Ptch1 variants, green)and imaged by confocal microscopy. Scale bars,5 mm. (F) Cells transfected and stained as in (E) wereimaged by SIM. Scale bars, 2.5 mm. Images shown in(E) and (F) are representative of many cilia.

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We also found that although ciliary localization of Ptch1 and directbinding of Hh to Ptch1 were required for Hh-sensitive Smo regulation,Hh-induced ciliary removal of Ptch1 was not necessary for ciliary accumu-lation of Smo and for downstream pathway activation. This finding is anal-ogous to the finding inDrosophila (where primary cilia are not required forHh signaling) that the trafficking of Patched from the plasma membrane,

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which is normally triggered by Hh stimula-tion, is required neither for Hh-dependentactivation of Smo nor for initiation of down-stream signaling events (35).

Although our data showed that Ptch1 cil-iary removal is not essential for pathway ac-tivation, Hh-induced ciliary removal ofPtch1 may have an important role in tuningthe response to Hh signaling during devel-opment. Yue et al. (36) demonstrated thatHh-dependent proliferation of granular neu-ronal precursors is reduced by deletion ofthe Ptch1 PPXY sequences or by in-activation of the HECT E3 ubiquitin ligasesthat interact with them. Given our findingthat Ptch1 suppression of Smo activity re-quired ciliary localization, clearance ofPtch1 from the cilium and its subsequentdegradation may result in a higher degreeof Hh responsiveness. Hh-induced ciliaryclearance of Ptch1, thus, may be critical infine-tuning pathway activity and hence thedifferentiation program induced by theamount ofHhpresent at a particular positionin a field of Hh-responsive cells. Never-theless, our data indicate that Hh bindingto Ptch1 is the critical event for suppressionof Ptch1 activity and that ciliary removal, al-though potentially important, is a secondaryevent. The mechanism of Ptch1 inactivationby Hh binding awaits molecular elucidationbut may involve stabilization of a particularconformation among several alterna-tives typically associated with transporterfunction.

MATERIALS AND METHODS

Cell cultureHuman embryonic kidney (HEK) 293 cells(Life Technologies), Ptch1−/− MEFs, andstably transfected HEK293-ShhN cells(37) were maintained in Dulbecco’s modi-fied Eagle’s medium (DMEM) supplemen-ted with 10% fetal bovine serum (OmegaScientific) and 1% penicillin/streptomycin/glutamine. NIH/3T3 cells (American TypeCulture Collection) were maintained inDMEM with 1% penicillin/streptomycin/glutamine and 10% bovine calf serum(HyClone) as previously described (13).

Constructs and reagentsMouse full-length Ptch1 and Ptch1 with cytoplasmic tail deletions werefused with either 3× Myc tag or a single YFP tag at the C terminus.Ptch1N-SmoCT-YFP was constructed by directly fusing the N terminus ofPtch1 (amino acids 1 to 1169) with the Smo cytoplasmic tail (amino acids550 to 793) followed by a YFP tag. Ptch1N-Sstr3icl3-YFP was constructedby directly fusing Ptch1 (amino acids 1 to 1169) with the icl3 of Sstr3 (30)

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Fig. 6. Ciliary removal of Ptch1 is not required for Shh-induced pathway activation. (A) Ptch1−/− cells wereWT PY

transfected with Myc-tagged Ptch1 or Ptch1 . After incubation with or without ShhN for 4 hours, cells

were fixed andstainedwithanti-Smo(red)andanti-Myc (Ptch1, green). The insets showshifted overlays. Scalebars, 5 mm. (B) Quantification of percentage of transfected cells with simultaneous ciliary presence ofSmo and Ptch1WT or Ptch1PY. Note that in the absence of ShhN, there are no cilia that contain both Ptch1andSmo (black bars are not visible). (C) Cells transfected and stained as in (A) were imagedbySIM. Scalebars, 2.5 mm. (D) Ptch1−/− cells transiently transfected for expression of Myc-tagged Ptch1 variants, Gliluciferase reporter, and control SV40-Renilla luciferase were grown to confluency, incubated with or with-out ShhN, and assayed for reporter activity. Error bars indicate SD; n=3. (E)Ptch1−/− cells transfectedwithGFP-tagged Ptch1WT or Ptch1PY were incubated with or without ShhN overnight, fixed, and stained withb-galactosidase (b-gal) (pathway activity, red), GFP (Ptch1, green), AcTub (primary cilium,white) antibodies,and DAPI (nucleus, blue). The insets show a separate green channel view of ciliary Ptch1. The presence ofnuclear b-galactosidase indicates pathway activation. Scale bars, 5 mm. (F) Mean fluorescence intensity ofb-galactosidase in the nuclei of Ptch1−/− cells and Ptch1−/− cells transfectedwith Ptch1WT or Ptch1PY. Eachbar shows themean ± SD of fluorescence from 10 to 20 cells. Images are representative of two experiments.(G) Quantification of percentage of transfected ciliated cells that contain both nuclear b-galactosidase andciliary Ptch1.

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followedby aYFP tag. ShhN-mCherrywas constructed by fusingmCherry atthe C terminus of ShhN. Ptch1PY, with two point mutations, Y631F andY1320F, was constructed by the QuikChange method (Agilent). Full-lengthNedd4, Neddd4L, Itchy, Smurf1, and Smurf2 were obtained from Addgeneand fused with the HA (hemagglutinin) or FLAG tag at the N terminus.Full-length WWP1 and WWP2 were obtained from Open Biosystemsand fused with the FLAG tag at the N terminus. Full-length HECW wasobtained from DNASU Plasmid Repository and fused with the FLAG tagat the N terminus. ShhN-conditioned medium was prepared as previouslydescribed (37).

AntibodiesAntibodies were used at the following concentrations: rabbit antibody rec-ognizing Smo (13), 1:1000 dilution of 0.7 mg/ml; mouse monoclonal anti-body recognizing acetylated tubulin (Sigma-Aldrich), 1:2000; rabbitantibody recognizingMyc (Santa Cruz Biotechnology), 1:1000; mouse anti-body recognizing Myc (9E10, Santa Cruz Biotechnology), 1:200; rabbitantibody recognizing GFP (Invitrogen), 1:200; chicken antibody recogniz-ing GFP (Abcam), 1:5000; rabbit antibody recognizing HA (Invitrogen),1:1000; rabbit antibody recognizing dsRed (Clontech), 1:1000; mouse an-tibody recognizing FLAG (M2, Sigma-Aldrich), 1:2000; rabbit antibodyrecognizing b-galactosidase (MP Biomedicals), 1:2000; horseradish perox-idase (HRP)–conjugated goat anti-mouse (W4021, Promega), 1:10,000;and HRP-conjugated goat anti-rabbit (111-035-144, Jackson Immuno-Research Laboratories), 1:10,000. Fluorophore-conjugated secondary anti-bodies were from Jackson ImmunoResearch Laboratories and used at1:500.

Protein immunoprecipitationHEK293 cells were transfected using FuGENE 6 transfection reagent(Promega). Cells were lysed at 48 hours after transfection in a buffer con-taining 1% NP-40, 50 mM tris-HCl (pH 7.5), 150 mM NaCl, and proteaseinhibitors for 30 min at 4 °C. The lysate was clarified by centrifugation at12,000g for 20 min at 4 °C and incubated with antibody-coupled proteinA–coated magnetic beads (Invitrogen) for 2 hours at 4 °C. Beads werewashed and proteins were recovered directly in SDS–polyacrylamide gelelectrophoresis (SDS-PAGE) sample buffer.

Cell surface biotinylationHEK293 cells were washed at 48 hours after transfection with phosphate-buffered saline (PBS) (pH 7.4) and incubated for 60 min in PBS containingSulfo-NHS-LC-Biotin (1 mg/ml) (Pierce) at 4 °C. The reaction wasquenched by washing the cells with 100 mM glycine in PBS three times.Cellswere then lysed in a buffer containing 1%NP-40, 50mM tris-HCl (pH7.5), 150mMNaCl, and protease inhibitors for 30min. The lysatewas clar-ified by centrifugation at 12,000g for 20 min at 4 °C, and biotinylated pro-teins were recovered by binding to streptavidin-Sepharose beads (GEhealthcare) for 2 hours at 4 °C. Beads were washed and proteins wererecovered in SDS-PAGE sample buffer.

ImmunoblottingProteins separated on Criterion TGX SDS-PAGE gels (Bio-Rad) weretransferred onto polyvinylidene difluoride membranes, blocked in 5%milkin PBS/0.1% Tween 20, and detected with the relevant primary andsecondary antibodies.

Gli reporter assaysNIH/3T3 cells plated at 5 × 104 to 9 × 104 cells per well in 24-well plateswere transfected the next day using FuGENEHD (Promega) with Gli lucif-erase reporter, control pRL-SV40-Renilla luciferase, and other DNA

constructs as indicated. After reaching confluence (~2 days), cells wereshifted to 0.5% serummedium and incubated for 24 hourswith conditionedmedium containing ShhN.

Immunofluorescence and quantification ofmicroscopy imagesCells were fixed in 4% formaldehyde for 10 min and then washed threetimes with PBS. Fixed cells were placed in blocking solution (PBS with1% normal goat serum and 0.1% Triton X-100) for 30 min. Primary anti-bodies were diluted in blocking solution and used to stain cells for 1 hour atroom temperature. After three washes in PBS, secondary antibodies andDAPI (Invitrogen) were added in blocking solution at a dilution of 1:500 for1 hour at room temperature. The samples were mounted in VECTASHIELDmounting medium (Vector Laboratories) for microscopy. Microscopy wasperformed with a Leica SD6000 spinning disc confocal microscope, a ZeissLSM710 laser scanning confocal microscope (with a 63× objective), or anOMX structured illumination microscope. Quantitative analyses were per-formed using ImageJ, as described previously (21). To assess protein amountsin primary cilia or nuclei, images used for comparison within an experimentwere obtainedwith identical settings on themicroscope and then used for quan-tification without any manipulation. A mask was constructed by manually out-lining the cilium or nucleus in the image taken in the acetylated tubulin or DAPIchannel, respectively. This mask was then applied to the image taken in thechannel for the protein of interest, and the fluorescence at the cilium or in thenucleus was measured. Local background correction was performed by movingthemask tomeasure fluorescence at a representative nearby region and thensubtracting this value from that of ciliary or nuclear fluorescence. All pointsrepresent mean fluorescence ± SD from 10 to 20 individual cilia or nuclei.Statistical analysis was performed using GraphPad Prism software.

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Acknowledgments:We thank Applied Precision for OMX imaging; Addgene and DNASUfor the plasmids expressing Nedd4, Nedd4L, Itchy, HECW, Smurf1, and Smurf2; andmembers of the Beachy laboratory for comments on the manuscript. Funding: This re-search was supported in part by funding from the NIH to X.Z. and P.A.B. and from theARC to A.B. and A.P. P.A.B. is an investigator of the Howard Hughes Medical Institute.Author contributions: J.K., X.Z., and P.A.B. designed experiments and analyzed data;J.K., E.Y.C.H., and X.Z. performed experiments; A.B. and A.P. provided data forming thebasis for the Ptch1PY mutation; and J.K., X.Z., and P.A.B. wrote the paper. Competinginterests: The authors declare that they have no competing interests. Data and materialsavailability: All materials are available upon request.

Submitted 23 December 2014Accepted 13 May 2015Final Publication 2 June 201510.1126/scisignal.aaa5622Citation: J. Kim, E. Y. C. Hsia, A. Brigui, A. Plessis, P. A. Beachy, X. Zheng, The role ofciliary trafficking in Hedgehog receptor signaling. Sci. Signal. 8, ra55 (2015).

Ja

www.SCIENCESIGNALING.org 2 June 2015 Vol 8 Issue 379 ra55 8

nuary 4, 2021

The role of ciliary trafficking in Hedgehog receptor signalingJynho Kim, Elaine Y. C. Hsia, Amira Brigui, Anne Plessis, Philip A. Beachy and Xiaoyan Zheng

DOI: 10.1126/scisignal.aaa5622 (379), ra55.8Sci. Signal. 

required for cells to respond to Hedgehog.Patched must reach the cilium to inhibit Smoothened in the absence of Hedgehog, but exit from the primary cilium is notrepression and stimulate Smoothened activity despite Patched not exiting the cilia in the presence of Hedgehog. Thus, were unable to exit the cilia not only repressed Smoothened activity but also enabled Hedgehog to alleviate thisnecessary and sufficient to mediate localization of proteins to the primary cilium. Unexpectedly, Patched mutants that

. determined that the C-terminal domain of Patched contained the ciliary localization sequence and waset alcilium. Kim alleviates repression of the transmembrane protein Smoothened but also stimulates Patched to exit from the primaryoccurs in a cellular sensory process called the primary cilium. Binding of Hedgehog to the receptor Patched not only

In mammals, regulation of the Hedgehog pathway, which is involved in development and tissue homeostasis,No need to leave

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