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Invasive behaviour of glioblastoma cell lines is associated with altered organisation of the cadherin-catenin adhesion system

¹ Department of Pharmacology, Center of Excellence of Neurodegenerative Disease, University of Milan, IN CNR, Cellular and Molecular Pharmacology Section, Italy
² Department of Pharmacology, Center of Excellence of Neurodegenerative Disease, University of Milan, Italy

View larger version (65K): [in a new window]   Fig. 1. Characterisation of the cadherin-mediated junctional complex in primary cultured cortical astrocytes. (A) Western blot analysis of the total expression of the components of the cadherin-based junctional system in brain, astrocytes and MDCK cells. 20 µg aliquots of homogenates were loaded onto 11% SDS-polyacrylamide gel and immunoblotted with the indicated antibodies. The MDCK cell homogenate was probed with the E-cadherin antibody, and the brain and astrocyte homogenate with the pan-cadherin antibody. (B) Affinity chromatography. The rat cortical astrocyte lysate was incubated with immobilised GST-LIN-7A fusion protein. The bound material (Bound) was resolved on 10% SDS-PAGE and immunostained for ß-catenin. 10% of the total astrocyte lysate (Lys) used in the experiment was probed with the same antibody. (C) Co-immunoprecipitation of ß-catenin with the LIN-7 antibody. The rat cortical astrocytes were extracted in lysis buffer and immunoprecipitated with the LIN-7 antibody (IP: LIN-7) or the pre-immune serum (IP: Pre). The immunoprecipitates were loaded onto 11% SDS-PAGE and immunoblotted with the ß-catenin and LIN-7 antibodies. Molecular weight standards expressed in kDa are indicated on the left (A-C). (D) Confocal analysis of double immunofluorescence staining for LIN-7 and ß-catenin in cultured primary astrocytes. The astrocytes were plated onto poly-L-lysine-coated glass coverslips, cultured until they reached confluence (recently confluent) or had been confluent for a longer period (long confluent) before fixation in 4% paraformaldehyde. Bar, 10 µm.

View larger version (20K): [in a new window]   Fig. 2. Biochemical characterisation of the cadherin-mediated junctional complex in the T98G and U373MG glioblastoma cell lines. (A) In vitro cell migration assay of T98G and U373MG cells. Serum-free medium containing 0.1% BSA (basal) or conditioned media from the same cells (stimulated) were used as chemoattractants. The data are expressed as the mean number of migrating cells per field±s.d. for triplicate samples from a representative experiment. (B) Western blot analysis of total cadherin, ß-catenin and LIN-7 expression in glioblastoma cell lines grown to confluence. 50 µg aliquots of total glioblastoma cell homogenates were loaded onto 11% SDS-PAGE and immunoblotted with the indicated antibodies. (C) Surface biotinylation assay. After being grown to confluence, the glioblastoma cell lines were surface biotinylated with NHS-ss-biotin. After cell lysis, the surface-biotinylated proteins were recovered using streptavidin beads, loaded onto 10% SDS-PAGE and immunoblotted with the indicated antibodies. The results were densitometrically analysed using the NIH Image 1.61 programme. The corresponding ß-catenin/N-cadherin ratio (expressed as relative units) is shown on the right. (D) Affinity chromatography. Confluent cell lysates were incubated with immobilised GST-LIN-7A fusion protein: the bound material (Bound) was resolved by 10% SDS-PAGE and immunostained for ß-catenin. (E) Co-immunoprecipitation of ß-catenin with the LIN-7 antibody. Lysates of confluent cells were immunoprecipitated with the LIN-7 antibody (IP), and the immunocomplexes were resolved by 11% SDS-PAGE and immunoprobed for ß-catenin. 10% of the total T98G or U373MG lysate (Lys) used in the experiments was probed with the indicated antibody (D-E). Molecular weight standards expressed in kDa are indicated on the left (B-E).

View larger version (65K): [in a new window]   Fig. 3. Confocal analysis of ß-catenin and N-cadherin distribution in subconfluent and confluent T98G and U373MG cells. The cells were plated onto poly-L-lysine-coated glass coverslips, fixed in 4% paraformaldehyde and immunostained. The arrows indicate ß-catenin- and N-cadherin-enriched spots in the T98G cells and lamellae and lamellipodia-like structures connecting adjacent cells in the U373MG cells. The arrowheads indicate filopodia-connecting neighbouring cells. Bar, 10 µm.

View larger version (148K): [in a new window]   Fig. 4. Ultrastructural analysis of cell-cell contacts. (a,b) Scanning electron microscope analysis of subconfluent T98G (a) and U373MG cells (b). The arrows indicate thin parallel cellular processes contacting neighbouring T98G cells (a) or lamellipodia-like structures (b). The arrowheads indicate sparse, branched and thin cellular processes making contact with the substratum (b) (Bars, 5 µm). (c-f) Transmission electron microscope analysis of confluent T98G (c,e) and U373MG (d,f) cells, showing perpendicular (c,d) and parallel sections (e,f). The arrows in (e) indicate well defined adherens junctions. (c,d, bars, 1 µm) (e,f, bars, 0.3 µm).

View larger version (90K): [in a new window]   Fig. 5. Morphological analysis of the degree of maturation of cell-cell contacts in T98G and U373MG cell lines. Confocal analysis of immunofluorescence staining. The glioblastoma cell lines were plated onto poly-L-lysine-coated glass coverslips, fixed in 4% paraformaldehyde and double-stained for ß-catenin and the indicated antibodies or the FITC-conjugated phalloidin to stain actin. The arrowheads indicate mature cell-cell contacts, and the arrows indicate immature cell-cell contacts. Bar, 10 µm.

View larger version (43K): [in a new window]   Fig. 6. Biochemical analysis of the degree of maturation of cell-cell contacts in T98G and U373MG cell lines. (A) Western blot analysis of the amount of LIN-7 and ß-catenin recovered in the TX-100-soluble (S) and TX-100-insoluble (I) fractions. T98G or U373MG glioblastoma cells grown to confluence were extracted in TX-100. Equivalent volumes of the soluble or insoluble fractions were separated by 10% SDS-PAGE and immunostained using the indicated antibodies. (B) Total P-tyr content in T98G and U373MG cells. 5x105 cells were plated, cultured for 72 hours and extracted in lysis buffer containing 0.02% SDS and 100 µM pervanadate. Five percent of the cell lysate used in the immunoprecipitation experiments was probed for P-tyr. (C,D) Tyrosine phosphorylation of - and ß-catenin. The cell lysates were immunoprecipitated with anti--(C) or anti-ß-catenin (D) antibodies and probed for P-tyr. To confirm similar protein loading, 10% of the immunoprecipitates were probed for - (C) or ß-catenin (D). Molecular weight standards expressed in kDa are indicated on the left.

View larger version (96K): [in a new window]   Fig. 7. Analysis of cell-cell adhesion in invasive T98G cells. (A) Matrigel invasion assay using as chemoattractants either serum-free medium containing 0.1% BSA (basal) or conditioned media from the same cells cultured in Matrigel-coated Petri dish (stimulated). The data are the mean number of cells per field±s.d. of triplicate samples from a representative experiment. (B) Western blot analysis of cadherin, ß-catenin and LIN-7 total expression in T98G cells cultured on poly-L-lysine (lane 1) or Matrigel (lane 2). The same amounts of total cell homogenates were loaded onto 11% SDS-PAGE and immunoblotted with the indicated antibodies. Molecular weight standards expressed in kDa are indicated on the left. (C) Brightfield and (D) confocal laser microscopy of T98G cells cultured in Matrigel. The cells (2x105 cells/35 mm diameter dish) were seeded onto Matrigel-coated glass coverslips and cultured for three days. After fixation in 4% paraformaldehyde, the cells were double-stained for ß-catenin and the indicated antibodies. The arrows indicate ß-catenin-enriched cell-cell contacts with disorganised filaments of F-actin, virtually no LIN-7 and the accumulation of phosphorylated proteins. Bar, 10 µm.