MDCK strain II cells were maintained in low-glucose DMEM (LG-DMEM) with 1.8 mM Ca2+
containing 1 g/l sodium bicarbonate and supplemented with 10% fetal bovine serum (FBS; Cell Generation, Fort Collins, CO), penicillin, streptomycin, and gentamicin (PSG; HCM) and grown at 37°C with 5% CO2
. shRalA and shRalB cells were generated by stable integration of shRNAs targeting canine RalA or RalB (RalA: sense, 5′-CGAGCTAATGTTGACAAGGTA-3′; RalB: sense, 5′-GAGTTTGTAGAAGACTATGAA-3′), respectively. shRNAs were delivered via transduction with recombinant lentiviral vectors that were pseudotyped with vesicular stomatitis virus G protein. Cells were selected and maintained in medium containing 5 μg/ml puromycin. Negative controls were generated by transducing MDCK II cells with lentiviral vectors encoding a nontargeting shRNA (sense, 5′-CCAGACCTTCAAGGAATCCAT-3′) and selecting them in puromycin. RalA rescue cell lines were generated by introducing three wobble-base point mutations into the first three codons of the target RalA hairpin sequence of simian RalA cDNA. This cDNA was ligated into pQCXIN and delivered into shRalA cells via retroviral transduction. Transduced cells were selected using 400 μg/ml G418 and assayed by immunofluorescence and immunoblotting for hairpin resistant RalA. The same process was performed to generate and screen RalB rescue cells. Inducible RalA mutant cell lines were generated using the MDCK T23 cell line, which expresses the tetracycline-repressible transactivator (Barth et al., 1997
). The 72L, 38R, and 47E point mutations were introduced into a RalA cDNA that was a generous gift from Larry Feig (Tufts University School of Medicine, Boston, MA), using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA). These cDNAs were introduced into MDCK T23 cells as described previously (Jou and Nelson, 1998
). Transient transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the suggested protocol. Tissue culture plates, 10 cm, were transfected with DNA or vehicle only 24 h prior to seeding in triplicate 12-mm, 0.4-μm Transwell polycarbonate filters following the protocol described for measurement of transepithelial resistance.
Antibodies and reagents
Mouse monoclonal antibodies (mAbs) against Sec6 (9H5) and Sec8 (2E12, 5C3, 10C2) were described previously (Hsu et al., 1996
; Kee et al., 1997
; Yeaman, 2003
). mAbs against claudin4, ZO-1, and occludin, as well as rabbit polyclonal antibodies against claudin1, claudin3, and claudin7, were obtained commercially (Zymed Laboratories, San Francisco, CA). RalA mAb (BD Transduction Laboratories, San Jose, CA), claudin2 mAb (Invitrogen), and myc mAb (Millipore, Billerica, MA) were also obtained commercially. A RalB mAb was a generous gift from Michael White (University of Texas Southwestern Medical Center, Dallas, TX). A rabbit polyclonal antibody against E-cadherin was a generous gift from W. James Nelson (Stanford University, Stanford, CA; Marrs et al., 1993
). Fluorescein isothiocyanate (FITC)–goat anti–mouse and Texas red–donkey anti–rabbit immunoglobulin G (IgG) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Horseradish peroxidase (HRP)–conjugated goat anti-mouse and goat anti-rabbit antibodies were purchased from Promega (Madison, WI). 125
I-Labeled goat anti-mouse IgG, 125
I-labeled goat anti–rabbit IgG, and 35
S-methionine were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA).
Measurement of transepithelial resistance
A well-described Ca2+
-switch method was used with cultures of MDCK II, shCtrl, shRalA, shRalB, RalA rescue, and RalB rescue cells (Gumbiner and Simons, 1986
; Gonzalez-Mariscal et al., 1990
). Each cell line was seeded on triplicate 0.4-μm Transwell polycarbonate filters at confluent density (8 × 105
cells/12-mm filter) in LG-DMEM with low Ca2+
(LCM; 5 μM Ca2+
), supplemented with 1 g/l sodium bicarbonate, 5% dialyzed FBS (Cell Generation), and PSG. Cells were cultured at 37°C for 3 h to allow for attachment to the filter. TER measurements were recorded both before and after replacing LCM with HCM. Two filters were left free of cells but otherwise treated identically, so that the intrinsic resistance of the membrane could be determined. For each filter, three independent readings were taken at each time point, using a Millicell (Millipore) electrical resistance device. Final values were obtained by subtracting the averaged blank value from the averaged sample reading and multiplying by the surface area of the filter. The final results are expressed as ohms · cm2
. Readings of samples in were recorded every hour after initial attachment in LCM. This technique resulted in frequent changes in media temperature, which caused higher TER values and decreased kinetics of TJ formation. In subsequent experiments, TER was not recorded until 6–10 h after HCM addition.
Samples were fixed on ice with 4% paraformaldehyde for 20 min and quenched with Ringer's saline (154 mM NaCl, 1.8 mM Ca2+, 7.2 mM KCl, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.4) containing 50 mM NH4Cl. Samples were then permeabilized with CSK buffer (1% Triton X-100, 10 mM 1,4-piperazinediethanesulfonic acid, pH 6.8, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2) for 10 min or with Ringer's saline containing 0.1% SDS for 5 min; both buffers were supplemented with protease inhibitors (1 mM Pefabloc [Sigma-Aldrich, St. Louis, MO] and 10 μg/ml each of aprotinin, antipain, leupeptin, and pepstatin A). After samples were blocked with 0.2% fish-skin gelatin in Ringer's saline (blocking buffer) for 1 h, primary antibodies were diluted in blocking buffer and applied for 1 h at room temperature. After five washes with blocking buffer, FITC- or Texas red–conjugated secondary antibodies and 4′,6-diamidino-2-phenylindole were applied for 30 min at room temperature. Samples were washed five times with blocking buffer, and filters and coverslips were mounted onto slides using Elvanol (Dupont, Wilmington, DE)–paraphenylene diamine. Images were obtained using either a Zeiss (Thornwood, NY) 510 scanning confocal microscope (63× objective) equipped with a krypton/argon laser (FITC excitation using 488-nm laser line, Texas red excitation using 543-laser line) or with a Leica (Wetzlar, Germany) DMI 6000B microscope equipped with a BD CARV II (BD Biosciences, San Diego, CA) spinning disk confocal imager. Stacks of confocal images were collected from several different fields using a 63× objective and a Photometrics (Tucson, AZ) QuantEM 512SC EM-CCD high-speed camera. For studies examining immunofluorescence intensity at TJs, confluent monolayers of cells were subjected to a Ca2+ switch and processed as described at 10 h following Ca2+ addition. Acquired z-series optical sections were merged, and the immunofluorescence intensity along the TJ was traced and quantified using the wand tool with ImageJ software (National Institutes of Health, Bethesda, MD). One-way ANOVA with Tukey's posttest statistical analyses was performed for each protein and cell type, and differences are considered significant when p < 0.05.
A Ca2+ switch was performed on MDCK II, shCtrl, shRalA, and shRalB cells seeded on triplicate 12-mm, 0.4-μm Transwell polycarbonate filters. At 9 h following Ca2+ readdition, 1 × 106 dpm of 3H-inulin was added in 0.5 ml of warm media to the apical chambers of all cell types, including bank filters. Filters were placed at 37°C, except for removal of 100 μl from basal chambers immediately after inulin addition and every 30 min thereafter, for a total of 4 h. After removal of basal samples, 100 μl of nonlabeled fresh media was applied to basal chambers at each time point. The basal samples were placed in 3 ml of scintillation fluid, and each was counted with a Beckman LS 6500 multiplier purpose scintillation counter (Beckman Coulter, Brea, CA) for 0.3 min in triplicate.
Biotin assay for endocytosis
Totals of 3 × 106
shCtrl, shRalA, and shRalB cells were seeded on triplicate 24-mm, 0.4-μm–pore size Transwell polycarbonate filters, and a Ca2+
switch was performed as described. At 9 h following Ca2+
readdition, filters were placed on ice and washed twice with Ringer's saline. Apical and basal chambers were biotinylated with 0.5 mg/ml NHS-SS-biotin (Thermo Scientific, Waltham, MA). All filters were washed three times with DMEM/0.2% BSA, and three filters of each cell type were lysed immediately after the last wash. Three filters of each cell type were placed into warm media and incubated at 37°C for 30 min, whereas the other three remained on ice. Endocytosis was stopped by placing warm filters on ice and washing with ice-cold Ringer's saline. Filters were reduced and quenched following a method described previously (Graeve et al., 1989
). Filters were lysed with radioimmunoprecipitation assay buffer (RIPA; 50 mM Tris-HCl, pH 7.5, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) containing protease inhibitors. Following centrifugation for 10 min at 14,000 rpm, 10% of the total extract was removed for analysis, whereas the remainder was incubated with streptavidin agarose resin (Thermo Scientific) overnight at 4°C. Beads were pelleted by gentle centrifugation and washed as described for immunoprecipitation. Immunoprecipitates were eluted from beads with SDS–PAGE sample buffer and processed for SDS–PAGE as described. Immunoblots were performed using antibodies specific to E-cadherin, and band intensities were quantified, as described.
Inhibition of endocytosis
MDCK II cells stably expressing the human transferrin receptor were seeded at confluent density on 12-mm Transwell polycarbonate filters, and a Ca2 -switch was performed as described. HCM readdition was supplemented with DMSO or 80 μM dynasore (Sigma-Aldrich). TER of triplicate filters was recorded in triplicate beginning 8 h after HCM readdition. For immunofluorescence, samples were treated with DMSO or 80 μM dynasore for 2 h before addition of fluorescent human transferrin for 25 min. Samples were fixed for immunofluorescence as described but not permeabilized.
Human transferrin receptor was expressed in shCtrl, shRalA, and shRalB cells by transduction with a replication-deficient adenoviral vector. Cells were transduced 1 d after seeding at confluent density on 12-mm Transwell filters in HCM. Three days after transduction, cells were incubated in serum-free DMEM at 37°C for 30 min. Filters were then placed on ice and washed with PBS2+
(phosphate-buffered saline [PBS] with 0.9 mM CaCl2
and 0.5 mM MgCl2
) before labeling with 125
I-transferrin on ice for 45 min. All filters were washed five times with cold PBS2+
. Triplicate filters of each cell type were then warmed to 37°C for indicated times prior to immediate transfer to cold PBS2+
. All filters were then washed twice, alternating between DMEM, pH 3.5, and PBS2+
. Media and PBS2+
from these washes were saved in 5-ml glass tubes. Following the final wash, filters were excised from collars and transferred to 5-ml glass tubes. Radioactivity associated with filters and media samples were quantified using a Titertek Plus Series Automatic Gamma Counter (Huntsville, AL). The model used, in which clearance from the cell surface into early endosomes assumes a first-order rate constant, is represented by the equation
, where St
is the percentage of total transferrin at the cell surface at time t, S0
is at time zero, EE0 is the percentage of total transferrin in early endosomes at time zero, k1 is the internalization rate, k–1 is the rate of transfer from early endosomes to the cell surface. t is time in minutes, and e is the natural logarithm base. The internalization rate of transferrin was determined using a model described previously (Sheff et al., 1999
Ral GTP pulldown
Cells expressing myc-tagged RalA or RalB were seeded at confluent density (14 × 106
cells) in LCM lacking serum on six 10-cm tissue culture plates. They were subjected to the described Ca2+
-switch protocol using HCM without serum. Plates were cultured in HCM at 37°C for the indicated length of time before being placed on ice and washed twice with Ringer's saline. Each plate was lysed with 1 ml of GTP lysis buffer (1% NP40, 50 mM Tris, pH 7.5, 200 mM NaCl, 10 mM MgCl2
, and 10 mM dithiothreitol) containing protease inhibitors (1 mM Pefabloc and 10 μg/ml each of aprotinin, antipain, leupeptin, and pepstatin A). Lysates were cleared at 20,000 × g
, and 10% of the supernatant from each sample was set aside for assessment of total protein levels; the remaining supernatants were transferred to glutathione–agarose beads (Pierce; 25-μl bead volume/sample) prebound to GST-Exo84 PH RBD (RalA wild-type samples) or GST-Sec5 RBD (RalB wild-type samples). Samples were rotated for 45 min at 4°C and then washed twice with GTP lysis buffer and prepared for SDS–PAGE as described previously (Pasdar and Nelson, 1988
Fluorescence labeling of the lipid membrane
BODIPY-FL-C5 sphingomyelin (Molecular Probes, Invitrogen) and defatted BSA were used to prepare sphingomyelin/BSA complexes (5 nmol/ml) in P buffer (10 mM HEPES, pH 7.4, 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl2, and 145 mM NaCl). A Ca2+ switch was performed on shCtrl, shRalA, and shRalB cells seeded at confluent density on 12-mm Transwell filters, as described previously. Five filters of each cell type were placed on ice 10 h after the switch to HCM, and apical and basal chambers were washed twice with cold P buffer. For labeling, 0.5 ml of 5 μM sphingomyelin/BSA complex was added to apical chambers, and 1 ml of P buffer was added to basal chambers. Filters were incubated on ice for 10 min and then washed extensively with P buffer. For each cell type, one filter was immediately processed for confocal microscopy, as described previously, and one additional filter was incubated in P buffer on ice for 1 h and then processed for confocal microscopy. The remaining three filters of each cell type were incubated on ice for 1 h in P buffer containing 1 mg/ml defatted BSA (0.5 ml apical, 1 ml basal). The solutions from apical and basal compartments were collected at the end of this incubation, and fluorescence intensities were quantified using a Wallac Victor2 1420 Multi Label Counter (PerkinElmer).
Gel electrophoresis and immunoblotting
Protein samples were incubated in SDS–PAGE sample buffer for 10 min at 65°C before separation on 10, 12.5, or 15% SDS polyacrylamide gels. Proteins were electrophoretically transferred onto Immobilon polyvinylidene fluoride membrane (Millipore) and blocked with BLOTTO (5% nonfat dry milk, 0.5% normal goat serum, and 0.1% sodium azide in Tris-buffered saline [TBS]) overnight at 4°C. Immunoblots were incubated with primary antibodies overnight at 4°C and then washed five times with TST (TBS containing 0.1% Tween-20), 10 min each. Finally, immunoblots were incubated with either 125I-labeled goat anti-mouse or goat anti-rabbit (Amersham, GE Healthcare, Piscataway, NJ) or HRP-labeled goat anti-mouse or goat anti-rabbit antibodies (GE Healthcare) for 45 min at room temperature. Immunoblots were washed as described, then twice with TBS. The immunoblots were exposed to either phosphorimager screens (Molecular Dynamics, Sunnyvale, CA) and signal was quantified using a phosphorimager (Typhoon, Molecular Dynamics) and ImageQuant software, version 5.0 (GE Healthcare), or incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and visualized with a ChemiDoc-It Imaging System and quantified with Visionworks software (UVP, Upland, CA).
Detergent solubility assay
MDCK II, shRalA, and shRalB cells were seeded on 0.4-μm Transwell polycarbonate filters at confluent densities (3 × 106 cells/24-mm filter) and subjected to a Ca2+. Samples were lysed in CSK containing 0.5% Triton-X 100 plus protease inhibitors (as described earlier) 0, 10, or 65 h after the switch to HCM. After a 10-min incubation on ice, lysates were centrifuged at 20,000 × g for 10 min. The supernatant was removed, and the pellet was resuspended in CSK containing 1% SDS and processed by SDS–PAGE, as described.
Protein stability assay
Confluent plates of cells were left nontreated or were treated with 4 μg/ml cyclohexamide for 3, 6, or 9 h at 37°C. Following treatment, samples were placed on ice and washed twice with Ringer's buffer. Samples were then lysed for 20 min on ice with CSK plus protease inhibitors. Lysates were centrifuged for 10 min at 20,000 × g, and supernatants were analyzed by SDS–PAGE.
Ca2+ depletion assay
TER of confluent cell monolayers on Transwell filters was measured before washing twice with Hank's buffered salt solution (HBSS; 5.33 mM KCl, 0.441 mM KH2PO4, 4.17 mM NaHCO3, 137.93 mM NaCl, 0.338 mM Na2HPO4, 5.56 mM dextrose, 0.0266 mM phenol red). Samples were incubated in HBSS plus 2.5 mM ethylene glycol tetraacetic acid (EGTA) for 10 min at 37°C, and the TER of all filters was measured. All filters were then washed twice with HBSS before HCM was added. Cells were cultured at 37°C for the times indicated in , at which points TER was measured again. One filter of each cell type was processed for immunofluorescence (as described) immediately following the calcium chelation, to determine the subcellular localization of occludin.
Confluent monolayers of MDCK II, shRalA, and shRalB cells on Transwell polycarbonate filters were washed twice with LG-DMEM without cystine or methionine and then incubated in the same medium for 1 h at 37°C. Samples were then labeled with 170 μCi of 35S-methionine per Transwell filter for 1 h at 37°C. Samples were washed three times on ice with Ringer's buffer and lysed with RIPA. Lysates were precleared with Pansorbin (Calbiochem, San Diego, CA), and 10% of the starting extract was removed for analysis, whereas the remainder was incubated with anti-Sec8 primary antibodies (mAbs 2E12, 5C3, 10C2) prebound to protein A–Sepharose (GE Healthcare) for 2 h at 4°C. Beads were pelleted by gentle centrifugation and washed under stringent conditions; twice with HS- buffer (0.1% SDS, 0.5% deoxycholate, 0.5% Triton X-100, 20 mM Tris-HCl, pH 7.5, 120 mM NaCl, 25 mM KCl, 5 mM EDTA, and 5 mM EGTA), twice with HS- buffer containing 1 M NaCl, and once with low-salt washing buffer (10 mM Tris-HCl, pH 7.5, and 2 mM EDTA). Immunoprecipitates were eluted from beads with SDS–PAGE sample buffer and processed for SDS–PAGE as described. Intensities of the Sec8, Sec6, and Sec15 bands were quantified using ImageQuant software, and the ratios of Sec8:Sec6 and Sec8:Sec15 were compared. Sec6 and Sec15 were identified by molecular weight, and their identities were verified by immunoprecipitation with specific antibodies to each protein.