Monoclonal antibodies directed against β-catenin, GSK3β, and clathrin heavy chain were purchased from BD Pharmingen. The monoclonal anti-Wg (4D4) antibody was from the Developmental Studies Hybridoma Bank. Horseradish peroxidase (HRP)-conjugated secondary anti-mouse antibodies were obtained from Santa Cruz Biotechnology. MDC, sucrose, CPZ, and human holotransferrin were obtained from Sigma. Cy3 labeling reagent was obtained from Amersham Biosciences. Phosphate-buffered saline (PBS) was obtained from Invitrogen. Complete Protease Inhibitor cocktail tablets were obtained from Roche. Enhanced chemiluminescence (ECL) reagents were obtained from Perkin-Elmer. Polyfect was from Qiagen and Lipofectamine 2000 from Invitrogen.
Cell lines and transfection
Murine L cells were obtained from American Type Cell Culture and cultured in Dulbecco's modified Eagle's Medium supplemented with 10% fetal bovine serum, penicillin/streptomycin, and glutamine. Cells were cultured in a humidified incubator at 37°C with 7% CO2. Cell lines were transfected with the pEGFP vector (Clontech) alone or were co-transfected with either wild-type or K44E-mutated dynamin I. Transfections were performed in 6-well dishes with a total of 1.5 μg of DNA using Polyfect reagent according to manufacturer's instructions, using a 10-fold excess of dynamin DNA relative to pEGFP DNA.
Internalization of Wg into murine L cells was measured by confocal microscopy. L cells were plated on round coverslips and were pre-incubated with either vehicle or inhibitors at 24-hours post-plating. In cases where dynamin constructs were used, GFP was co-transfected in order to identify the transfected cell population. Cells were incubated with either control conditioned medium or Wg conditioned medium for 1 hour at 4°C, followed by transfer of cells to 37°C for an additional 1-hour incubation. In Figure 1, 4°C conditions represent cells maintained at this temperature for the second hour of the experiment. In cases where chemical inhibitors of endocytosis were used, cells were first pre-incubated with inhibitors at 37°C for 1 hour, followed by incubation with Wg at 4°C for 1 hour, followed by incubation at 37°C for an additional 1 hour. Cells were washed 2X with ice-cold PBS supplemented with CaCl2 and MgCl2 (1 mM each) ("PBS++"), followed by fixation at room temperature with 4% paraformaldehyde in PBS. Cells were washed 3X for 5 minutes with PBS++, followed by permeabilization in PBS++ supplemented with 0.1% Triton X-100 ("Washing/Permeabilization Buffer") for 15 minutes at room temperature. Subsequently, cells were blocked for 1 hour at room temperature with PBS++ supplemented with 0.1% Triton X-100 and 5% normal donkey serum ("Blocking Buffer"). Cells were incubated with anti-Wg (4D4) antibody (diluted 1:50 in Blocking Buffer) overnight at 4°C, followed by three 15-minute room-temperature washes with Washing/Permeabilization Buffer. Wg immunostaining was detected using either a Cy3- or Alexa Fluor 488-conjugated anti-mouse secondary antibodies in blocking buffer at room temperature for 1 hour, followed by three 15 minute room-temperature washes with Washing/Permeabilization Buffer. Coverslips were mounted using Fluoromount G and imaged on a confocal Zeiss Laser Scanning Microscope 5 using the 63× objective with oil immersion. In cases where endocytosis with transferrin was examined, holotransferrin was labeled with Cy3 according to manufacturer's instructions, followed by separation from unincorporated dye using Sephadex G-50 gel-filtration chromatography. The Cy3-transferrin was incubated with cells at ~10 μg/mL in the presence or absence of Wg.
Wnt-3A was purified from media conditioned from Wnt-3A-expressing L cells as previously described . β-catenin accumulation in L cells was measured both by immunoblot and immunocytochemistry. Briefly, murine L cells in 6-well tissue culture plates were pre-incubated with either Vehicle (DMSO at 0.33%), MDC (300 μM), hypertonic sucrose (0.45 M), or CPZ (30 μM) at 37°C for 1 hour, followed by stimulation with purified Wnt-3A (~100 ng/mL) for 2–3 hours at 37°C in the continued presence of vehicle or inhibitors. Following Wnt stimulation, the medium was aspirated and the cells were washed with ice-cold PBS. The cells were subsequently scraped into 10 mM Tris-HCl/100 mM NaCl/1% Triton X-100/1X protease inhibitor cocktail/pH 7.4 and solubilized by rotation at 4°C for 20 minutes. Lysates were clarified by refrigerated micro-centrifugation at 20000 × g for 20 minutes. In contrast to L cells, SW480 cells were swelled on ice in 10 mM Tris-HCl/0.2 mM MgCl2/0.25 M sucrose/1 mM EDTA/pH 7.4, followed by lysis by 20 strokes in a tight-fitting Dounce homogenizer. Lysates were first centrifuged at 1000 × g for 10 minutes at 4°C to pellet nuclei, debris, and unlysed cells. Supernatants from this spin were further clarified by refrigerated micro-centrifugation at 20000 × g for 1 hr. Supernatant from the micro-centrifugations was subsequently added to SDS-PAGE sample buffer and electrophoresed on a 10% gel. Transfer to nitrocellulose was performed at 12 V (constant current) for 40 minutes. Blots were blocked in PBS/0.1% Tween-20/5% non-fat dried milk ("Blotting Buffer") for 10 minutes, followed by incubation with a cocktail of primary anti-β-catenin and anti-GSK3β antibodies in Blotting Buffer overnight at 4°C with agitation. Blots were washed extensively with Blotting Buffer, followed by incubation with HRP-conjugated anti-mouse secondary antibodies in Blotting Buffer for 1 hour at room temperature. Following extensive washing in Blotting Buffer and PBS, immunoreactivity was assessed by ECL.
For measurement of β-catenin by immunofluorescence, L cells were plated on round coverslips in 12-well dishes and allowed to adhere overnight. 24-hours post-plating, L cells were pre-incubated and stimulated in the same manner as previously described for the immunoblot analysis. In cases where the dynamin constructs were used, cells were co-transfected with GFP to mark the transfected cell population. Wnt stimulations were performed 48 hours post-transfection. Following stimulation, the medium was aspirated and cells were gently rinsed twice with PBS++. Cells were subsequently fixed in 4% PFA in PBS for 15 minutes at room temperature, followed by three 5-minute washes in PBS++. Cells were permeabilized in Washing/Permeabilization Buffer for 15 minutes at room temperature. Permeabilization of the nuclear envelope was accomplished by a 10-second incubation with room-temperature methanol, followed by three 5-minute washes in PBS++. Cells were blocked for 1 hour at room temperature with Blocking Buffer. Incubation of primary anti-β-catenin antibody (diluted 1:100 in Blocking Buffer) was overnight at 4°C. Primary antibody was aspirated and cells were given three 15-minute washes with Washing/Permeabilization Buffer, followed by incubation with Cy3-conjugated secondary anti-mouse antibody in Blocking Buffer for 1 hour at room temperature. Secondary antibody was aspirated and cells were given three 15-minute washes with Washing/Permeabilization Buffer before mounting the coverslips in Fluoromount G. Immunostaining experiments were observed with a Zeiss Axioplan 2 microscope using the 40× objective.
Measurement of TCF/LEF activity
L cells stably harboring the TOPFLASH and LacZ constructs ("LSL cells") were plated in 96-well microtiter dishes to approximately 80–90% confluency. 24 hours post-plating, LSL cells were pre-incubated for 1 hour in the presence of either Vehicle (DMSO at 0.33%), MDC (300 μM), sucrose (0.45 M), or CPZ (30 μM). Subsequently, cells were stimulated with purified Wnt-3A (~100 ng/mL) for 5 hours at 37°C in the continued presence of vehicle or inhibitors. Expression of luciferase was measured in a Lumat LB 9507 luminometer (Berthold) using the TROPIX kit according to manufacturer's instructions. All assays were performed in triplicate, and the relative luciferase units were normalized to LacZ readings.
siRNA targeting of endocytic proteins
siRNAs directed against clathrin heavy chain were synthesized in the Stanford Protein and Nucleic Acid Facility. Sequences for clathrin heavy chain (sense: 5'-GCAAUGAGCUGUUUGAAGATT-3'; anti-sense: 5'UCUUCAAACAGCUCAUUGCTT-3') which were identical in human and murine isoforms of the proteins were selected from siRNAs described previously.(Huang et al., 2004) Using Lipofectamine 2000, siRNAs (~2 μg) were transfected along with empty pcDNA3 or pEGFP vector (2 μg) into LSL cells in 6-well plates, followed by a second round of transfections 24 hours later. Approximately 12 hours after the second transfection, the LSL cells were split into fresh media in preparation for reporter assays (either 24 well or 96 well plates) and harvesting for immunoblots. In reporter experiments, cells were incubated with purified Wnt-3A (~100 ng/mL) for approximately 8 hours, followed by measurement of TOPFLASH and LacZ activities.
JB conceived of and executed the experiments in this study. RN provided advice with design of experiments and interpretation of results, as well as funding for this work.
We thank Derk ten Berge and Michael Povelones for helpful discussions about experiments and interpretation of data. We also greatfully acknowledge Amanda Mikels for generation of the LSL cell line and Joeke van der Velde for purification of the Wnt-3A protein. Roel Nusse is an Investigator with the Howard Hughes Medical Institute. Jeremy T. Blitzer is a member of the Medical Scientist Training Program at Stanford University School of Medicine. This work was in part supported by a grants from the National Institutes of Health (DK067834) and the JDRF.