Antibodies and DNA constructs
The DNA constructs CHMP31–179
, and GFP-Vps4WT
have been described previously (Whitley et al., 2003
Dukes et al., 2008
), and the GFP construct used was pCS2-GFP (Chalmers et al., 2006
). Rabbit anti–claudin-1* (59–9000), mouse anti–claudin-2* (32–5600), mouse anti–claudin-4* (32–9400), rabbit anti-occludin (71–1500), mouse anti-occludin* (33–1500), mouse anti–ZO-1 (33–9100), mouse anti–E-cadherin (33–4000), mouse anti-TfR (13–6800), and rabbit anti-Rab11 (71–5300) were all purchased from Zymed (San Fransico, CA), and all used at a dilution of 1:25 for immunofluorescence and 1:1000 for Western blotting (anti–claudin-1, anti-occludin, and anti-TfR). Rabbit anti-PKCζ (C-20; sc-216; 1:75) and mouse anti-CD63 (sc5275; 1:1000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-ubiquitin (recognizes mono- and polyubiquitinylated conjugates; clone FK2; 1:50) and rabbit anti–furin convertase (canine; 1:300) were purchased from Enzo Life Sciences (Plymouth Meeting, PA). Mouse anti-Tsg101 (4A10; ab83; 1:1000), rabbit anti-NUMB* (ab14140; 1:50), mouse anti–desmoglein-2 (ab14415; 1:25), and mouse anti-M6PR (ab2733; 1:200) were purchased from AbCam (Cambridge, UK). Mouse anti-EEA1 was purchased from BD Biosciences (610457; San Jose, CA; 1:100). Mouse anti–β-tubulin (T4026; 1:5000) was purchased from Sigma-Aldrich (St. Louis, MO). Mouse anti-GP135 (Ojakian and Schwimmer, 1988
) was a kind gift from George Ojakian (SUNY Downstate Medical Center, Brooklyn, NY) and used at a dilution of 1:10. Mouse anti-LAMP1* (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) was a kind gift from Scott Lawrence (University College, London, UK) and used at a dilution of 1:500. Species-specific fluorophore-conjugated anti–immunoglobulin G secondary antibodies (Alexa Fluor 488, 546, and 633 nm) were all purchased from Molecular Probes, and each was used at a dilution of 1:200 (Invitrogen, Carlsbad, CA). Goat anti–mouse-HRP–conjugated secondary antibody was obtained from Sigma-Aldrich and goat anti–rabbit-HRP was purchased from Pierce (Rockford, IL) and used at a 1:5000 dilution.
Generation of adenovirus
The generation of high-titer, purified adenoviruses using the Iowa RapAd system has been described previously (Anderson et al., 2000
). Briefly, Ad-expressing CHMP31–179
GFP) was prepared by subcloning the DNA construct into pacAd5 CMV K-N pA shuttle vector (a kind gift from Beverly Davidson, University of Iowa, Iowa City, IA). This was then digested with PacI alongside pacAd5 9.2–100 sub360 backbone vector and mixed and transfected according to manufacturer's instructions, into low-passage HEK293 cells using Lipofectamine 2000 (Invitrogen). Cells were left for 10 d to allow recombination between digested shuttle and backbone vectors and for visible cytopathic effects to be observed. Lysates were then collected and used for further bulking of the virus. Ad vectors were then grown to high titer and purified using CsCl gradient methods, as previously described (Anderson et al., 2000
Caunt and McArdle, 2010
Cell culture, transfections, and transduction
MDCKII cells (Madin-Darby canine kidney cells; #00062107; European Collection of Cell Cultures, Salisbury, UK) and 16HBE14o- (human bronchial epithelial cells; a kind gift from Dieter Gruenert, University of California–San Francisco, San Francisco, CA;
Cozens et al., 1994
) were maintained at 37°C and 5% CO2
in DMEM supplemented with 10% (vol/vol) fetal bovine serum (FBS), 2 mM l
-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. CaCo-2 cells (#86010202; ECACC) were similarly maintained, except for the addition of 20% (vol/vol) FBS, 1x nonessential amino acids and 10 mM HEPES. All cell media and supplements were purchased from Lonza (Basel, Switzerland). Cells were tested for mycoplasma contamination using MycoAlert (Lonza). Cells were plated onto 13-mm coverslips in 24-well plates (Nunc, Roskilde, Denmark) and grown until ~80–90% confluent, at which time they were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
At 24 h posttransfection, cells were fixed with 4% (wt/vol) paraformaldehyde fixation buffer (PFA) in phosphate-buffered saline (PBS) for 20 min and permeabilized in methanol cooled to −20°C for 5 min. Cells stained with antibodies indicated by an asterisk (see Antibodies and DNA constructs) were fixed and permeabilized with methanol cooled to −20°C for 10 min. Cells were then blocked with 10% (vol/vol) FBS in PBS for 30 min. Primary and secondary antibodies were diluted in 2% FBS-PBS (2% FBS in PBS), and cells were incubated with primary antibodies for 2 h at 18°C and with secondary antibodies for 1 h. Cells were washed five times for 5 min (per wash) with 2% FBS-PBS following all antibody incubations. Stained cells were then mounted in Mowiol (Calbiochem, San Diego, CA) containing DAPI (Sigma-Aldrich) and examined on a Zeiss LSM510META laser-scanning confocal microscope with images taken.
siRNA oligonucleotides and reagents were purchased from Dharmacon (Thermo Fisher Scientific, Lafayette, CO). Tsg101 was depleted using ON-TARGETplus individual siRNA duplex (CCGUUUAGAUCAAGAAGUA; J-003549-06). As a control, ON-TARGETplus Non-targeting siRNA was used. CaCo-2 cells were plated at high density into either six-well plates (for Western blotting; Nunc), or onto 13-mm glass coverslips (for immunofluorescence), and incubated with complete CaCo-2 growth media (lacking antibiotics) at 37°C, 5% CO2 for ~4 h to adhere. Cells were transfected with 100 nM of siRNA using DharmaFECT 1 transfection reagent according to the manufacturer's instructions and incubated at 37°C, 5% CO2 for the desired time period, with media changed after 3 d.
Endocytosis and recycling biotin assays
The biotinylation assay to study endocytosis and recycling of tight junction proteins was modified from a method described previously (Nishimura and Sasaki, 2008
). Confluent cells plated on to 35-mm dishes were transferred to ice and washed with PBS supplemented with 0.9 mM CaCl2
and 0.33 mM MgCl2
(PBS-CM). Cells were then incubated with the cleavable non-membrane-permeable sulfo-NHS-SS-biotin (Pierce; in PBS-CM) at a concentration of 0.5 mg/ml for 30 min on ice. Free biotin was then quenched using 50 mM NH4
Cl (in PBS-CM) for 15 min (4°C). For the endocytosis assay, prewarmed complete growth medium was added, and cells were returned to 37°C for indicated times. Cells were then transferred to ice to stop endocytosis, and surface (nonendocytosed) biotin was stripped by reduction with 100 mM 2-mercaptoethanesulfonate (MESNA) in Tris-buffered saline supplemented with calcium and magnesium (TBS-CM) for three 10-min treatments (on ice). Internalized biotinylated cargo was protected from biotin stripping with MESNA by an intact membrane. Free –SH groups were then quenched by incubating cells with 5 mg/ml iodoacetamide (in PBS-CM) three times for 5 min each time.
For the recycling assay, this process was repeated with 20-min incubations at 37°C in complete growth medium. To control for potential loss of biotinylated cargo via degradation, a recycling condition was included that lacked the second MESNA treatment. Thus any loss in biotinylated cargo at this step, relative to the endocytosis step, would indicate a loss in signal due to degradation of cargo instead of recycling. Cells were lysed (1.25% [vol/vol] Triton X-100, 0.25% [wt/vol] SDS, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 5 mg/ml iodoacetamide, 10 μg/ml APMSF) on ice, pulse-sonicated, and centrifuged at 15,000 × g to remove large/nuclear debris. An equal volume of the postnuclear supernatant was taken from each sample for use as a loading control. Biotinylated proteins were collected by incubation with Neutravidin beads (Pierce), rotating overnight at 4°C. Beads were then washed by centrifuging at 1000 × g, five times with wash buffer (0.5% [vol/vol] Triton X-100, 0.1% [wt/vol] SDS, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA) and 3 times with PBS-CM. Reducing sample buffer was then added to each sample to release biotinylated proteins from the beads and, following boiling, samples were loaded onto a 15% Tris-glycine SDS–PAGE gel. Separated proteins were transferred to nitrocellulose and immunoblotted for the protein indicated. Signals were detected by enhanced chemiluminescence or Chemiglow West Chemiluminescence Substrate (Alpha Innotech, San Leandro, CA) and quantified using an Optichem detector with associated software (Ultra Violet Products, Cambridge, UK). For quantification of the biotinylated proteins, the amount of claudin-1 and occludin was normalized to their respective total protein bands, determined from nonisolated lysate samples. Where results were plotted graphically (Prism; GraphPad, La Jolla, CA), values were expressed as a percentage of the total claudin-1 or occludin biotinylated at the surface. For recycling assays using Ad-CHMP31–179GFP infections, MDCKII cells were allowed to grow past confluency on 35-mm dishes, and then infected with Ad-CHMP31–179GFP for 16 h and subsequently surface-biotinylated. The recycling assay was then performed as described previously.
Caco-2 TER measurements
CaCo-2 cells were plated into six-well Nunc plates and transfected with ON-TARGETplus Non-targeting siRNA or Tsg101 siRNA, as detailed previously. Cells were incubated at 37°C, 5% CO2 for 3 d. Cells were washed in PBS, trypsinized, and resuspended in CaCo-2 growth media. Cells were plated in triplicate on Transwell (#3470; Corning Life Sciences, Corning, NY) permeable polyester filters (0.4-μm pore size, 0.33-cm2 surface area) at 2.5 × 105 cells/filter and incubated at 37°C, 5% CO2. TER was measured every 24 h for 4 d using a EVOM TER machine with an Endohm-6 chamber (World Precision Instruments, Sarasota, FL), with media changed after every reading.
CaCo-2 three-dimensional cyst culture and immunofluorescence
CaCo-2 cells were plated into six-well Nunc plates and transfected with ON-TARGETplus Non-targeting siRNA or Tsg101 siRNA, as detailed previously. Cells were incubated at 37°C, 5% CO2 for 3 d. Cells were washed in PBS, trypsinized and resuspended in antibiotic-free CaCo-2 growth media. A cell:matrix mix was prepared containing 5.8 × 104 CaCo-2 cells/ml, 0.02 M HEPES (pH 7.4), 1 mg/ml Collagen I (Inamed Biomaterials, Fremont, CA), and 40% Growth Factor Reduced BD Matrigel Matrix (BD Biosciences). The cell:matrix mix was plated into an eight-chamber slide, incubated at 37°C, 5% CO2 for 1 h to solidify, and overlaid with antibiotic-free CaCo-2 growth medium. Cysts were allowed to develop for 10 d at 37°C, 5% CO2, with media changed every 3–4 d. After a 10-d culture, cysts were washed in PBS, treated with 5U/ml Collagenase VII (Sigma-Aldrich) for 15 min, and fixed in 4% (wt/vol) PFA for 30 min at room temperature. Cysts were washed three times for 20 min with 1X wash buffer (10X stock, pH 7.4: 1.3 M sodium chloride, 70 mM dibasic heptahydrate sodium phosphate, 30 mM monobasic monohydrate sodium phosphate, 77 mM sodium azide, 1% [wt/vol] BSA, 2% [vol/vol] Triton-X 100, 0.4% [vol/vol] Tween-20; all Sigma-Aldrich). Cysts were incubated with blocking buffer (10% [vol/vol] FBS in 1X wash buffer) for 1 h at room temperature, and the incubated with primary antibodies diluted in blocking buffer overnight at 4°C. Cysts were washed three times for 20 min with 1X wash buffer and incubated with secondary antibodies diluted in blocking buffer for 1 h at room temperature. After a further three 20-min washes with 1X wash buffer, cysts were rinsed twice in PBS and postfixed in 4% (wt/vol) PFA for 30 min at room temperature. Cysts were washed three times for 5 min with PBS and incubated with DAPI (4 μg/ml) for 30 min at room temperature to stain the nuclei. After a final rinse in PBS, cysts were mounted in Mowiol (Calbiochem) and examined on a Zeiss LSM510 META laser-scanning confocal microscope (Welwyn Garden City, UK).