Antibodies and Reagents
Primary antibodies (see also Table S1) targeting β1-integrin were purchased from Thermo Fischer Scientific (TS2/16; #MA2910). Anti-β4-integrin antibodies were purchased from Santa Cruz (sc-6628) and anti-α6-integrin (555734-GoH3) and anti-E-cadherin (610181) antibodies were from BD Biosciences. Anti-LN-γ1 (L9393), anti-β-actin (A5441), anti-β-tubulin (T4026) and anti-talin (T3287) antibodies were from Sigma-Aldrich. Anti-Calnexin (ab10286) and anti-EEA1 (ab2900) antibodies were from Abcam. Peroxidase-conjugated secondary antibodies for western immunoblotting and fluorophore-conjugated secondary antibodies for immunofluorescence stainings were purchased from Jackson Immunoresearch. Goat and rat total IgGs, used as negative controls in immunoprecipitations, were also from Jackson Immunoresearch. Alexa Fluor 488 phalloidin from Invitrogen was used for staining of F-actin and DAPI from Sigma-Aldrich was used for staining of nuclei. Surface-biotinylated and immunoprecipitated proteins were detected from western immunoblots using peroxidase streptavidin (Jackson Immunoresearch).
Cell culture and Treatments
MDCK-II cells (ATCC:CCL-34) were routinely cultured in 5% fetal bovine serum (Invitrogen) containing minimal essential medium (MEM, Invitrogen) with 1% penicillin and streptomycin. For analysis of matrix adhesions in confluent cells, cells were seeded onto coverslips or glass bottom dishes (for TIRF microscopy) and cultured in serum containing medium for 6 days. When indicated, cells were cultured in the presence of GM6001 (Sigma-Aldrich) at different concentrations for 24 h.
KO of integrins via lentivirus-mediated expression sgRNAs and Cas9
Gene editing with lentivirus-mediated co-expression of Cas9 and sgRNA-constructs was done with some modifications as previously described. Second exon of canine ITGA6 and fourth exon of canine ITGB4 were used as a template for gRNA-design. Target sequences with no off-target sites with less than 3 mismatches in the canine genome database (European Nucleotide Archive), were selected based on FASTA similarity search tool (EMBL-EBI; Table S2). Two targeting sequences for each gene were selected and the corresponding gRNA oligos with BsmBI overhangs were subcloned into lentiCRISPRv1 (β4- and α6-KOs; Addgene #49535) or lentiCRISPRv2 (β1-KOs; Addgene #52961,).
For generation of the lentiviruses, 70–80% confluent 293T cells on CellBind 10 cm dishes (Corning) were co-transfected with lentiCRISPRv1 (20 µg), pPAX2 (15 µg) and VSVg (5 µg) by Lipofectamine 2000 reagent (Invitrogen) in OptiMEM (Invitrogen). Medium was collected over a period of 24 h to 96 h post-transfection in 12 h patches. Virus-containing medium was pooled and filtered through a 0.44 µm filter, followed by pelleting of the virus by ultracentrifugation at 100,000 × g for 2 h at 4°C. 1/10th dilution of the 100X virus concentrate was used for infection of MDCK cells seeded at 6×104/24-well, 24 h prior. 24 h post-infection, virus-containing media was exchanged with normal media to allow cells to recover. 48 h post-infection, cells were trypsinized and re-seeded in the presence of 6 µg/ml puromycin, followed by 24 h of selection. Puromycin-resistant cells were analyzed for KO frequency using immunofluorescence staining (Fig. S1B). Clonal cell populations were generated and analyzed for loss of protein expression as shown in figure S1C and S1D. Three clonal cell lines for each gRNA construct were generated and used in replicate experiments.
Cell surface biotinylation
Cell surface biotinylation was adapted from. Cells were seeded at a density of 4.5×104/cm2 onto 10 cm tissue culture dishes 24 h prior. Cells were washed thrice with biotinylation buffer (20 mM HEPES, 130 mM NaCl, 5 mM KCl, 0.8 mM MgCl2 and 1 mM CaCl2), followed by biotinylation with 0.5 mg/ml Sulfo-NHS-LC-Biotin (Pierce) in biotinylation buffer 30 min on ice with gentle rotation. Cells were washed three times with 10 mM Tris-HCl, 0.15 M NaCl, pH 7.45, followed by lysis with RIPA buffer (10 mM Tris-HCl, 0.15 M NaCl, 0.5% SDS, 1% IGEPAL, 1% sodium deoxycholate) supplemented with protease inhibitors (Roche).
Immunoprecipitation and Avidin-precipitation
RIPA-lysates were rotated 30 min +4°C with Benzonase nuclease (Novagen) and filtered through a 0.45 µm Spin-X column (Corning). Protein concentration was determined with the bicinchoninic acid assay (Pierce). For analysis of surface biotinylated integrins, lysates with 150 µg total protein were incubated o/n at +4°C with 3 µg of primary antibodies or control IgGs, and immunoprecipitated with 1.5 mg of Protein-G Dynabeads (Invitrogen) or 30 µl of protein-G agarose beads (Pierce) for 2–3 h +4°C . Beads were washes thrice with RIPA buffer and once with 10 mM Tris-HCl, pH 6.8. For analysis of metabolically labelled integrins, 200–300 µg of total protein was incubated with 5–7.5 µg of primary antibodies or control IgGs and processed as above. Beads were cooked with 2X Laemmli sample buffer with 2% β-mercaptoethanol 4 min at +97°C. Cell surface biotinylated cell lysates were avidin-precipitated with avidin agarose beads (Pierce).
SDS-PAGE and Western immunoblotting
RIPA-lysates were prepared into 1X Laemmli sample buffer with 1% β-mercaptoethanol and cooked 4 min at +97°C. Proteins were separated in 6% SDS-PAGE and blotted o/n at +4°C at 20V in with 20% ethanol in 0.025 M Tris 0.192 M Glycine onto a nitrocellulose membrane (Perkin-Elmer). Membranes were reacted with primary antibodies o/n at +4°C, followed by incubation with peroxidase-conjugated secondary antibodies (Jackson Immunoresearch). Antibody bands were detected with the Lumi-Light chemiluminescence kit (Roche) with the LAS-3000 imager. Protein bands were quantified with the Quantity One software (Biorad) and relative levels of KOs were determined either by directly dividing from control or from a standard curve prepared from serially diluted control.
Metabolic labeling and Autoradiography
Metabolic labeling protocol was adapted from. Briefly, cells were seeded as 4×105/cm onto 6 cm Ø tissue culture dishes and cultured for 3 days followed by 18 h of labeling at +37°C with 100 µCi of EasyTag™ EXPRE35S35S Protein Labeling Mix (PerkinElmer) in medium containing 1/10th normal concentration of methionine and cysteine. Labelled cells were washed three times with cold PBS and lysed in 1 ml of RIPA buffer on ice. For analysis of metabolically labelled integrins, 200–300 µg of total protein was incubated with 5–7.5 µg of primary antibodies or unspecific IgGs. Immunoprecipitation and SDS-PAGE were performed as described above. Gels were de-stained for 10 min at room temperature (RT) with 45% methanol and 10% acetic acid) and impregnated with Kodak™ ENLIGHTNING™ Rapid Autoradiography Enhancer (PerkinElmer) for 30 min at RT in the dark. Gels were dried and exposed onto BioMax® XAR films (Carestream).
Cells were fixed with 4% PFA in PBS+/+ (PBS with 0.5 mM MgCl2 and 0.9 mM CaCl2) for 15 min at RT. After quenching for 20 min with 0.2 M glycine in PBS, cells were permeabilized with 0.1% TX-100 in PBS for 10 min. All subsequent steps on saponin-permeabilized cells contained 0.02% saponin. Permeabilized cells were blocked with 0.5% BSA in PBS for minimum of 30 min and primary antibodies were prepared in blocking buffer and incubated with cells o/n at +4°C. After 1 h incubation with secondary antibodies at RT, cells were mounted with Immu-Mount (company). When used, DAPI and phalloidin dyes were prepared with secondary antibodies. For staining with PAN-cytokeratin antibody, cells were fixed with 1:1 mixed methanol and acetone at -20°C and quenching and permeabilization steps omitted.
Microscopy and Image analysis
Cells were seeded onto glass coverslips (for confocal microscopy) or glass-bottom dishes (for TIRF) and cultured to confluency. For immunofluorescence staining, cells were fixed with 4% PFA in PBS+/+ (PBS with 0.5 mM MgCl2 and 0.9 mM CaCl2) for 15 min at RT. After quenching for 20 min with 0.2 M glycine in PBS, cells were permeabilized with 0.1% TX-100 in PBS for 10 min. Permeabilized cells were blocked with 0.5% BSA in PBS for minimum of 30 min and primary antibodies, were prepared in blocking buffer and incubated with cells overnight at +4°C. After 1 h incubation with secondary antibodies at RT, cells were mounted with Immu-MountTM (Thermo Fisher Scientific). When used, DAPI and phalloidin dyes were prepared with secondary antibodies. Confocal images were acquired with the Zeiss LSM-780 laser scanning confocal microscope using 40X Plan-Apochromat objective (N.A = 1.4) and TIRF images with the Zeiss Cell Observer spinning disc confocal microscope using the alpha Plan-Apochromat 63X oil objective with an N.A of 1.46. For TIRF image acquisition, immunofluorescence stained samples in glass bottom dishes were left unmounted and were kept in PBS. Co-localization in TIRF images was assessed with the Pearson's correlation coefficient measured with the Co-localization Threshold plugin in FIJI using Costes method auto threshold determination and excluding zero intensity pixels. Unthresholded PCC values were used in the analysis due to the high labelling density of the matrix staining, which interferes with the thresholding algorithm. One channel was rotated 90° degrees relative to the other channel and the misaligned images analyzed to demonstrate the absence of random correlation.
Absolute values were tested for significant differences with one-way analysis of variance (ANOVA) using Tukey's post-hoc test. Fold changes were tested for significant differences with two-tailed one-sample t-test. All statistical analyses were carried out with SPSSv20.