Wild-type mouse Smo was tagged at the N terminus (with the insertion C terminal to the signal sequence) with YFP or the SNAP tag in the pCS2+ vector. For retroviral infections, SNAP-Smo and YFP-Smo were subcloned into pMSCV-pac. To produce retroviral supernatants, Bosc23 packaging cells were transfected using Fugene-6, supernatants containing viral particles were collected 48 h after the transfection, polybrene was added at 4 µg/ml, and supernatants were filtered through a 0.45-µm filter and used to infect fibroblasts (Pear et al., 1993
; Bailey et al., 2002
). The constructs for the catalytic subunits of PKA (both α and β; Uhler and McKnight, 1987
) were obtained from Addgene, and the cAMP response element–driven luciferase reporter (pGL4.29) was obtained from Promega. Constructs encoding GFP-tagged wild-type dynamin or the two dominant-negative dynamin proteins were provided by S. Schmid (The Scripps Research Institute, La Jolla, CA).
Small molecules and recombinant proteins
SAG, Fsk, H-89, and KT5720 were obtained from Enzo Life Sciences, Inc.; dynamin inhibitory peptide (DIP) and a control scrambled peptide (CIP) were obtained from Tocris Bioscience; dideoxy-Fsk was obtained from EMD; the SNAP substrates were obtained from Covalys and New England Biolabs, Inc.; and the puromycin and cycloheximide were obtained from Sigma-Aldrich. The 293 EcR Shh cells used to make Shh-conditioned media are available from American Type Culture Collection (ATCC; CRL-2782). They carry a stably integrated construct for full-length mouse Shh under an ecdysone-inducible promoter. The Shh produced by these cells is expected to be processed via an autocatalytic reaction, undergoing internal cleavage and lipidation. To produce conditioned media, cells were grown in high glucose Dulbecco's minimum essential medium, 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, 2 mM GlutaMAX, 1 mM sodium pyruvate, and 0.1 mM MEM nonessential amino acid supplement containing 10% FBS (Hyclone, defined grade; Thermo Fisher Scientific). At the time of induction with 1.5 µM muristerone A, cells were switched to media containing 2% FBS. Conditioned media was collected after ~72 h of induction, filtered through a 0.22-µM filter, and snap frozen in liquid nitrogen. Conditioned media was used at a dilution of 1:4 or 1:5 unless otherwise noted.
NIH3T3 cells were obtained from ATCC. The smo
−/−:YFP-Smo cells (Rohatgi et al., 2009
) and smo
−/−:SNAP-Smo cell lines were generated by infection of smo
−/− cells (Sinha and Chen, 2006
) with a retrovirus carrying YFP-Smo or SNAP-Smo cloned into pMSCVpac, followed by selection in 2 µg/ml puromycin and isolation of single clones. The 293 EcR Shh cells used to make Shh conditioned media are available from ATCC (Taipale et al., 2000
For assays of ciliary Smo accumulation, cells were grown to confluence in medium (high-glucose Dulbecco's minimum essential medium, 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, 2 mM GlutaMAX, 1 mM sodium pyruvate, and 0.1 mM MEM nonessential amino acid supplement) containing 10% FBS (Hyclone, defined grade), then switched to medium containing 0.5% FBS for 24 h. All cells were transfected using Fugene6 (Roche).
Polyclonal rabbit antisera against mouse Smo (anti-SmoC) were produced and purified as described previously (Rohatgi et al., 2007
). The anti-SmoN polyclonal antibody was produced (Josman Laboratories) against amino acids 36–234 of the mouse Smo protein, and affinity-purified before use. The mouse anti-acetylated tubulin antibody was obtained from Sigma-Aldrich, the anti-SNAP antibody was obtained from Thermo Fisher Scientific, the rabbit anti-YFP antibody was obtained from Abcam (ab290), and the goat anti–rabbit or goat anti–mouse secondary antibodies coupled to Alexa Fluor 594, Alexa Fluor 488, or Alexa Fluor 647 were obtained from Invitrogen.
Immunofluorescence and microscopy
Cultured cells were fixed with 4% PFA in PBS for 10 min at 4°C and washed three times with PBS. Fixed cells were placed in blocking solution (PBS with 1% vol/vol normal goat serum and 0.1% vol/vol Triton X-100) for 30 min. Primary antibodies (1:1,000 for anti-Smo or anti-acetylated tubulin) were diluted in blocking solution and used to stain cells for 1 h at room temperature. After washing three times in PBS, Alexa Fluor–coupled secondary antibodies were added in blocking solution at 1:500 for 1 h at room temperature. DAPI was included in the final washes before the samples were mounted in Fluoromount G (SouthernBiotech) for microscopy. Microscopy was performed on an inverted laser scanning confocal microscope (DMIRE2; Leica). Images were taken with a 63× objective lens and 4× zoom (Leica). When possible, images were depicted with the color channels slightly shifted relative to each other (“shifted overlay”) to more clearly show colocalization of different probes in the cilia. In all figures, the scale bar is 5 µm.
Antibody labeling experiments
In all panels, cilia and total Smo protein were detected with anti-acetylated tubulin or anti-SmoC, respectively, after cell permeabilization. For live cell antibody labeling, cells were incubated (30 min, 37°C) in media containing 0.5–1 µg/ml anti-SmoC, 1:1,000 anti-YFP, or 1–2 µg/ml anti-SmoN. After washing and fixation, the antibodies were detected with a secondary antibody before cell permeabilization. Cilia were labeled after a second washing and permeabilization step. For antibody-chase experiments, cells were washed three times with warm media after antibody feeding and then chased for an additional 2 h in the presence or absence of Shh.
SNAP labeling and pulse-chase
SNAP fluorescent substrates were used at 5 µM, non–cell-permeable C8 propanoic acid benzylguanine (CBG) block were used at 20 µM, and cell-permeable benzylguanine (BG) block were used at 10 or 20 µM. Live cells expressing SNAP-tagged proteins were stained for 15 min at 37°C. In control experiments to test the cell permeability of fluorescent substrates, the labeling period was extended to 30 or 60 min to make sure that substrates did not leak into the cell even in this prolonged period. For pulse-chase experiments, nonfluorescent blocking substrates were added during the chase period. For tracing surface Smo in pulse-chase experiments, cells were labeled with BG-547, washed, treated with Shh or other agonists, and fixed at different time points after induction. Two different approaches were taken to trace intracellular Smo. First, cell-surface Smo was blocked with CBG block (a non–cell-permeable molecule), and intracellular Smo was labeled with BG-505, a cell-permeable SNAP substrate. After washing off free BG-505, cells were exposed to an Hh agonist for varying periods of time before fixation. In an alternative approach, surface Smo was blocked with CBG block before cells were exposed to Shh. At different time points after Shh addition, cells were stained with BG-547 immediately before fixation to selectively reveal intracellular Smo that had moved to the cilium. The first approach detects only those Smo molecules that were present in the cell at the beginning of the experiment before Shh, whereas the second approach detects all Smo that is on the surface at the time of fixation, whether it existed at the beginning of the experiment or was newly delivered during the experiment. In all of the experiments, cells were stained after fixation and permeabilization with anti-SmoC to detect total Smo.
Cells treated with Shh for 1 h were cooled to 4°C and biotinylated for 15 min with the cleavable cross-linker sulfo-NHS-S-S-biotin (Thermo Fisher Scientific). After quenching any remaining reagent, cells were lysed, and biotinylated proteins were isolated on streptavidin-linked magnetic beads (Invitrogen). Proteins were eluted with sample buffer containing DTT (100 mM) and analyzed by immunoblotting.
Inhibition of endocytosis
Two different approaches were taken to block endocytosis: incubation of cells with a soluble, cell-permeable DIP (Marks and McMahon, 1998
); or transfection of cells with GFP-tagged dominant-negative mutants of dynamin (K44A and I690K), previously shown to block endocytosis (Song et al., 2004
). For experiments with DIP, NIH3T3 cells were incubated with DIP or a scrambled control peptide for 30 min before the addition of Shh or SAG for 2 h. Transferrin uptake was used to assess the efficiency of DIP and dynamin mutants in blocking endocytosis.
Alexa Fluor 594–conjugated transferrin (50 µM) from Invitrogen was added to cells in serum-free media and allowed to bind at room temperature for 2 min. After the binding, cells were washed with media and incubated for 15 min at 37°C to allow internalization. Cells were rapidly cooled by adding chilled media to stop further endocytosis, acid-washed (0.1 M glycine, pH 2.5, and 150 mM NaCl) to strip off surface-bound (but not internalized) transferrin, and fixed for analysis.
Image and data analysis
All analysis was performed by importing images as TIFF files into ImageJ. For the quantitative analysis of Smo levels in primary cilia, all images used for comparisons within an experiment were taken with identical gain, offset, and laser power settings on the microscope and used for quantitation without any manipulation. A mask was constructed by manually outlining cilia in the image taken in the acetylated-tubulin channel. This mask was applied to the image taken in the Smo channel and the fluorescence at cilia measured. Local background correction was performed by moving the mask to measure fluorescence at a representative nearby region, and this value was subtracted from that of ciliary fluorescence. All points represent mean (±SEM) fluorescence from ~10–30 individual cilia.
The data shown in were quantified in two ways. has two parallel but separate experiments, one in which surface Smo is followed and a second in which internal Smo is followed (). In both cases, the level of total Smo was measured by staining with anti-SmoC. The anti-SmoC staining data from both experiments () were combined, and the mean total Smo at each time point was plotted. In the experiments following surface or internal Smo, BG-547 staining at each time point was averaged and plotted. For , instead of averaging the signals separately, the ratio of the BG-547 signal to the anti-SmoC signal (this is roughly proportional to the fraction of total Smo that is labeled with BG-547) for each cilium was individually calculated. These ratios were averaged for the two separate experiments at each time point, yielding the two curves.
Online supplemental material
Fig. S1 shows controls for , , and . Fig. S2 shows characterization of SNAP-Smo cells and SNAP substrates. Fig. S3 show that Smo protein undergoes constant turnover. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200907126/DC1