U373 MG (human glioblastoma) and HeLa (human epithelioid carcinoma) cells were obtained from the American Type Culture Collection (Rockville, MD). U373 MG were grown in Eagles media (GIBCO BRL, Gaithersburg, MD) containing 10% FCS. HeLa cells were grown in DME media (Bio-Whittaker, Walkersville, MD) containing 10% FCS and supplemented with 2 mM glutamine. Cells were maintained at 37°C in a humidified atmosphere with 10% CO2.
PBS: 10 mM sodium phosphate buffer (pH 7.4), 150 mM NaCl. HBS buffer: 10 mM Hepes (pH 7.4), 150 mM NaCl. SDS sample buffer: 0.125 M Tris (pH 6.8), 10% SDS, 25% glycerol, 0.01% bromophenol blue. Detachment buffer: 2 mM EDTA, PBS, 0.05% BSA. Wash media: serum-free DME media supplemented with 0.05% heat-inactivated BSA and 10 mM Hepes (pH 7.4). BSA was heat-inactivated at 80°C for 20 min.
Anti-LAR Monoclonal Antibodies
The anti-LAR monoclonal antibodies 75.3A, 11.1A, and 71.2E have been previously described (34
). The anti-LAR monoclonal antibody 75.11.16 was raised by a similar approach using a GST-LAR Fn5-protein as antigen. The epitopes in the LAR extracellular domain recognized by these antibodies are as follows: mAb 75.3A, LAR Ig domain 1, 2, or 3; mAb 11.1A, between FnIII domain 8 and the transmembrane segment; mAb 71.2E, FnIII domain 6 or 7; mAb 75.11.16, LAR FnIII domain 5.
The antilaminin mAbs used were: antihuman laminin β1 chain (clone I) and antihuman laminin γ1 chain (clone II) from GIBCO BRL; and antihuman laminin (LAM-89) from Sigma Chemical Co. (St. Louis, MO). Affinity-purified rabbit polyclonal anti-Engelbreth-Holm-Swarm (EHS)-laminin was also from Sigma Chemical Co., and rabbit polyclonal anti-mouse laminin B1/B2 chain was from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-human β2 microglobulin mAb MIG-B5 was from BioSource International (Camarillo, CA). Affini-pure rabbit anti–mouse IgG was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Monoclonal anti-human integrin β1, anti-human talin, and anti-human vinculin antibodies were from Upstate Biotechnology Inc. HRP-conjugated rabbit anti–mouse IgG antibody and TRITC-conjugated goat anti– mouse IgG1 were from Southern Biotechnology Associates Inc. (Birmingham, AL).
Laminin and ECM Proteins
The following preparations of ECM proteins were used: Matrigel™ (basement membrane matrix from the mouse EHS sarcoma; Becton Dickinson, Bedford, MA); purified laminin–nidogen complex from the EHS sarcoma, and human fibronectin (Boehringer Mannheim, Indianapolis, IN).
GST–LAR–FnIII Fusion Proteins
Fusion proteins of glutathione S-transferase (GST) with various LAR FnIII domains were constructed using the E. coli expression vector pGEX-2T (Pharmacia Biotech. Inc., Piscataway, NJ; 32). The GST-fusion proteins used in this study are shown schematically in Fig. . Various segments of LAR cDNA with appropriate restriction sites for subcloning were generated by PCR and inserted in-frame into the pGEX-2T vector downstream of the GST coding sequence. Their nucleotide sequences were confirmed by DNA sequencing. Fusion protein synthesis was induced in exponentially growing Escherichia coli cultures by adding 0.1 mM isopropylthiogalactoside, and continued for 2 h at 25°C. Fusion proteins were extracted by solubilizing the bacterial pellet with 1% Triton X-100/PBS, containing 1.0 mM PMSF and 1.0 mM benzamidine for 30 min at room temperature, followed by sonication for 30 s at 4°C. Fusion proteins were affinity-purified using glutathione Sepharose beads. Beads were washed five times with PBS. The purity and sizes of the fusion proteins were confirmed by SDS-PAGE followed by Coomassie blue staining.
35S-Labeling of Conditioned Media
U373 MG cells were grown to 80% confluency in 100-mm tissue culture dishes in Eagle's media containing 10% FCS. Cells were then washed twice with serum-free media and metabolically labeled with [35S]methionine/cysteine (Dupont-NEN, Boston, MA). In brief, cells were incubated for 20 h in methionine-free media supplemented with [35S]methionine/cysteine (80 μCi/ml), 5% dialyzed FCS (1,000 D cut-off), and 10 mM Hepes buffer (pH 7.4). Labeled media was collected at 4°C, the protease inhibitors (1.0 mM PMSF and 1.0 mM benzamidine) were added, and the media was centrifuged at 1,500 rpm in a Beckman J-6M/E centrifuge at 4°C.
The different GST-LAR-fusion proteins (20 μg) bound to glutathione Sepharose beads were incubated with [35S]methionine-labeled media (5 ml) collected from U373 MG cells. Incubation was carried out for 2 h at 4°C with rotation. The incubation was terminated by centrifuging the beads at 1,000 rpm in a Beckman J-6M/E centrifuge for 5 min at 4°C. The beads were washed four times with 5 ml of ice-cold HBS buffer. After the fourth wash, the beads were resuspended in SDS sample buffer, reduced with β-mercaptoethanol, boiled, and subjected to SDS-PAGE on a 5% gel. A sample of the media without incubation with the fusion proteins was run on the gel to assess the number and relative concentration of the labeled bands in the media. Gels were processed for autoradiography with Enhance™ (Dupont-NEN) according to the manufacturer's instructions. Dried gels were exposed to x-ray film in the presence of an enhancer screen.
Binding of GST-LAR Fn5 (with or without the exon 13 sequence) to Matrigel™ was assayed as follows. Glutathione bead–bound GST-LAR Fn5+ or Fn5− (20-μl beads coated with 40 μg protein) was diluted into 10 ml HBS containing 2 mM EDTA, 1.0 mM PMSF, and 1.0 mM benzamidine, and mixed with various amount of Matrigel (150–750 μg) for 2 h at 4°C with rotation. The beads were then precipitated by centrifugation and washed four times with 5 ml of ice-cold HBS buffer each. After the final centrifugation, the beads were resuspended in SDS sample buffer, reduced with β-mercaptoethanol, boiled, and subjected to SDS-PAGE on a 6.5% gel and immunoblotting. Blots were probed with the rabbit polyclonal anti-mouse laminin B1/B2 chain antibody, followed by HRP-conjugated goat anti–rabbit antibody. Bound antibodies were visualized by the enhanced chemiluminescence reagent (Amersham Life Science Inc., Arlington Heights, IL).
Binding of GST-LAR Fn5 proteins to the purified laminin-nidogen complex was assayed in a similar manner, except that the concentration of laminin-nidogen was kept constant (150 μg in 10 ml), while the amount of GST-LAR Fn5 protein bound to glutathione beads varied (50–150 μg of protein per 40 μl beads), and the mixtures were incubated for 3.5 h at 4°C with rotation. Samples were analyzed on a 5% SDS gel, and blots were probed with an affinity-purified polyclonal anti-laminin antibody.
Immunoblotting with Antilaminin Antibodies
Conditioned media collected from U373 MG cells was incubated with the GST-LAR fusion proteins as described in the binding assay above. Electrophoresis was carried out on a 3–12% gradient SDS-polyacrylamide gel under nonreducing conditions, or on a 5% SDS-polyacrylamide gel under reducing conditions. After electrophoresis, the gels were blotted to nitrocellulose and probed with monoclonal antibodies specific to the laminin β1 chain, laminin γ1 chain, or laminin (LAM-89). Bound anti-laminin antibodies were detected with HRP-conjugated rabbit anti–mouse IgG antibodies and the enhanced chemiluminescence reagent. Purified laminin-1 was run in parallel in all the gels as a positive control.
HeLa cells at 30–50% confluency were detached by incubation in detachment buffer for 20 min at 37°C. Cells were washed three times at 4°C in wash media. Cells were resuspended in wash media at a concentration of 0.4 × 106 cells/ml, and 0.12 × 106 cells were plated per well on laminin– nidogen-coated 24-well tissue culture plates. Laminin–nidogen (Boehringer Mannheim) was coated at a concentration of 20 μg/ml PBS overnight at 4°C. Before plating the cells, the laminin–nidogen-coated wells were blocked for 1 h at room temperature by incubation with 0.5% heat-inactivated BSA, and were then washed once with PBS. Cells were incubated for 1 h at 37°C to allow the cells to attach and begin to spread. Unattached cells were removed by washing with wash media at room temperature. The cells were then incubated with the anti-LAR mAb 75.3A, 75.11.16, or a control anti-human β2 microglobulin mAb, at a concentration of 20 μg/ml in wash media for 10 min at 37°C. A further control was the omission of a first antibody. The media containing the unbound antibodies was removed, and the bound antibodies were cross-linked with 60 μg/ml Affini-pure™ rabbit anti–mouse IgG. Incubation at 37°C was continued for a further 35 min. The cells were washed three times with PBS and fixed in 3.7% paraformaldehyde/PBS for 10 min at room temperature. The cells were washed four times with PBS and photographed by phase contrast microscopy on a Axiovert 135 microscope (Carl Zeiss Inc., Thornwood, NY) using Tri-X400 film (Eastman Kodak Co., Rochester, NY). Fractions of spread cells were quantified by counting at least four random fields for each of four separate experiments.
The laminin–nidogen complex (20 μg/ml) was coated overnight at 4°C onto sterile, acid-washed 12-mm glass coverslips (Fisher Scientific Co.
, Pittsburgh, PA) in 24-well dishes (Costar Corp., Cambridge, MA). HeLa cells were plated on the laminin–nidogen-coated coverslips, and were treated as described for the cross-linking assay above. Cell surface LAR was stained by a modification of the method of Serra-Pagès et al.
). In brief, before LAR staining cells were fixed in 3.7% paraformaldehyde (Fisher Scientific Co.
) in PBS for 10 min. Fixation was required before LAR staining in order to preserve the laminin–nidogen-induced cell morphology. For staining internalized LAR and other intracellular proteins (talin, vinculin, and actin), the cells were permeabilized with 0.4% Triton X-100 in PBS for 5 min after fixation. After four washes with PBS, nonspecific binding sites were blocked with 10% goat serum in PBS (for LAR, talin, and vinculin stainings) or 1% BSA in PBS (for actin staining) for 30 min. LAR, talin, and vinculin were stained for 1 h with monoclonal anti-LAR (11.1A), anti-talin, and anti-vinculin antibodies (10 μg/ml), respectively, in PBS with 2% goat serum. After four washes with PBS, the bound antibodies were detected by incubation for 1 h with TRITC-conjugated goat anti–mouse IgG1
(at 1:100 dilution) in PBS with 2% goat serum. Actin was visualized by incubation of the cells with 2U rhodamine-conjugated phalloidin (Molecular Probes Inc., Eugene, OR) in blocking solution for 30 min according to the manufacturer's instructions. For all stainings, the specificity of staining was determined in control experiments in which either the primary antibody or both the primary and secondary antibodies were omitted.
After labeling, all slides were washed three times with PBS and mounted in Fluorsave (Calbiochem-Novabiochem Corp., La Jolla, CA). Slides were viewed on an Axiophot™ microscope (Carl Zeiss Inc.) equipped for epifluorescence. Photographs were taken on Tri-X400 black and white film (Eastman Kodak Co.).