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First published online 16 September 2003
doi: 10.1242/jcs.00760

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The association of the tetraspanin D6.1A with the 6ß4 integrin supports cell motility and liver metastasis formation

Mikael Herlevsen^1,*, Dirk-Steffen Schmidt¹, Kaoru Miyazaki² and Margot Zöller^1,3,

¹ Department of Tumor Progression and Immune Defense, German Cancer Research Center, Heidelberg, Germany
² Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Yokahama, Japan
³ Department of Applied Genetics, University of Karlsruhe, Karlsruhe, Germany

View larger version (53K): [in a new window]   Fig. 1. Identification of rat 6ß4. (A) Lysate of Progressor cells was purified by immunoaffinity chromatography and separated by SDS-PAGE, which revealed two bands of 130 and 200 kDa. The trypsin digest of these 2 bands was subjected to mass spectrometry. (B) PCR analysis of the metastasizing lines BSp73ASML, Progressor and Regressor and of the non-metastasizing line BSp73AS with primers (see Materials and Methods) detecting the 7 AS deletion (ß4D) (left) and the +53 and/or +70 insertion (ß4B/ß4C) (right) of the ß4 integrin. The 3 metastasizing lines express the ß4A isoform. The non-metastasizing line does not express the ß4 integrin. (C) Staining of BSp73ASML, BSp73AS and BSp73AS-ß4 cells confirms that B5.5 recognizes 6ß4. Black line: negative control (secondary antibody), light gray area: staining with B5.5, dark gray area: staining with anti-ß1. Single parameter overlays are shown.

View larger version (39K): [in a new window]   Fig. 2. Comparison of the amino acid sequences of rat (R), mouse (M) and human (H) 6 integrin chain. Identical amino acids (aa) are indicated by a dash, conserved aa are in bold and are highlighted in gray and semi-conserved aa are in bold. Alignments and comparison of substituted aa were performed using the ClustalW from EMBL-EBI. The extracellular domain including the three beta propellar repeats, the transmembrane and cytoplasmic domains are indicated by a vertical line. Peptides identified by mass spectrometry are highlighted in gray.

View larger version (78K): [in a new window]   Fig. 3. Morphology, substrate adhesion and motility of BSp73AS-ß4 cells. (A) BSp73AS and BSp73AS-ß4 cells were grown on plastic culture dishes and were stained with Phalloidin-TRITC. As compared to the epitheloid BSp3AS cells, BSp73AS-ß4 cells resembled spindle shaped fibroblasts. Scale bar: 1.7 µm. (B) BSp73AS, BSp73AS-ß4, BSp73ASML and Progessor cells were labeled with [3H]thymidine and were seeded in triplicates on plates coated with BSA, recombinant Ln5, Col IV or Fn. After 20 minutes or 2 hours incubation, plates were vigorously washed. Adherent cells were detached and harvested. Radioactivity was determined in a ß-counter. The percentage (±s.d.) of adherent cells is shown. A significant increase in adhesion to the substrates as compared to BSA is indicated by an asterisk. Similar results have been obtained in repeated experiments using 804G supernatant as a source of Ln5. (C) BSp73AS, BSp73AS-ß4 and Progessor cells were seeded on recombinant Ln5-coated Petri dishes, where the central area was protected by a cover slide. After reaching subconfluency, the cover slide was removed. Thereafter medium was changed with the fresh medium contained only 1% FCS and either a control antibody, or B5.5 (10 µg/ml). After 72 hours, cultures were washed, cells were fixed and stained with Hematoxylin-Eosin. Pictures were taken at the boundary from where cells started to migrate (proximal) as well as from the center of the area initially protected by a cover slide (distant). Scale bar: 0.5 µm.

View larger version (101K): [in a new window]   Fig. 4. Co-clustering of 6ß4 with D6.1A. (A) Progressor cells were incubated for 15 minutes at 37°C with A2.6, C4.4, D6.1, D5.7 (all of which recognize metastasis-associated molecules), anti-CD9 (expressed on Progressor cells) and, as a negative control, anti-CD18 (not expressed on Progressor cells). Cells were washed with cold PBS and were incubated for 20 minutes at 37°C with an excess of FITC-labeled anti-mIgG. Thereafter cells were immediately transferred on ice. After washing and fixation, free binding sites of FITC-labeled anti-mIgG were blocked by incubation with PBS containing 10 µg/ml mouse IgG. Finally, cells were stained with B5.5-TxR (1 hour, 4°C), the staining solution also containing mIgG to avoid unspecific binding of B5.5-TxR to the FITC-labeled anti-mIgG. (B) Progressor cells were mixed with BSp73AS-D6.1A (indicated by one asterisk) that express the D6.1A tetraspanin, or BSp73AS-ß4 cells (indicated by two asterisks) that express 6ß4. As described in A, cells were stained with D6.1/anti-mIgG-FITC and B5.5-TxR or with B5.5/anti-mIgG-FITC and D6.1-TxR. Single stainings and overlays of green and red fluorescence are shown. The FITC-labeled anti-mIgG does not bind unspecifically (anti-CD18 staining) and B5.5-TxR or D6.1-TxR does not bind to FITC-labeled anti-mIgG that was blocked by mIgG (staining of the mixture of Progressor and BSp73AS-D6.1A or BSp73AS-ß4 cells). Although Progressor cells express CD44v6, C4.4A, D6.1A and D5.7A at a comparably high level, 6ß4 co-clustered only with CD44v6 (A2.6) and D6.1A. Scale bar: (A) 10 µm; (B) 5 µm.

View larger version (71K): [in a new window]   Fig. 4. Co-clustering of 6ß4 with D6.1A. (A) Progressor cells were incubated for 15 minutes at 37°C with A2.6, C4.4, D6.1, D5.7 (all of which recognize metastasis-associated molecules), anti-CD9 (expressed on Progressor cells) and, as a negative control, anti-CD18 (not expressed on Progressor cells). Cells were washed with cold PBS and were incubated for 20 minutes at 37°C with an excess of FITC-labeled anti-mIgG. Thereafter cells were immediately transferred on ice. After washing and fixation, free binding sites of FITC-labeled anti-mIgG were blocked by incubation with PBS containing 10 µg/ml mouse IgG. Finally, cells were stained with B5.5-TxR (1 hour, 4°C), the staining solution also containing mIgG to avoid unspecific binding of B5.5-TxR to the FITC-labeled anti-mIgG. (B) Progressor cells were mixed with BSp73AS-D6.1A (indicated by one asterisk) that express the D6.1A tetraspanin, or BSp73AS-ß4 cells (indicated by two asterisks) that express 6ß4. As described in A, cells were stained with D6.1/anti-mIgG-FITC and B5.5-TxR or with B5.5/anti-mIgG-FITC and D6.1-TxR. Single stainings and overlays of green and red fluorescence are shown. The FITC-labeled anti-mIgG does not bind unspecifically (anti-CD18 staining) and B5.5-TxR or D6.1-TxR does not bind to FITC-labeled anti-mIgG that was blocked by mIgG (staining of the mixture of Progressor and BSp73AS-D6.1A or BSp73AS-ß4 cells). Although Progressor cells express CD44v6, C4.4A, D6.1A and D5.7A at a comparably high level, 6ß4 co-clustered only with CD44v6 (A2.6) and D6.1A. Scale bar: (A) 10 µm; (B) 5 µm.

View larger version (93K): [in a new window]   Fig. 5. Co-localization of 6ß4 and D6.1A in BSp73AS-db cells. BSp73AS-db cells were seeded on recombinant Ln5. After 48 hours cells were stained with D6.1 or anti-CD9 plus FITC-labeled anti-mIgG and were counterstained with TxR-labeled B5.5 or were stained with B5.5 plus FITC-labeled anti-mIgG and were counterstained with TxR-labeled D6.1. As described in Fig. 4,Fig. 4, binding of the second, dye-labeled antibody to anti-mouse IgG was prevented by the addition of an excess of mouse IgG. Red and green fluorescence and the digital overlay are shown. Whereas in BSp73AS-db cells D6.1A and 6ß4 mostly co-localized irrespective of whether D6.1A or 6ß4 was cross-linked via the secondary antibody, CD9 hardly co-localized with 6ß4. Scale bar: 10 µm.

View larger version (61K): [in a new window]   Fig. 6. Co-immunoprecipitation of 6ß4 and D6.1A. (A) Lysates of Progressor cells were precipitated with B5.5, anti-ß4, anti-3, anti-ß1 and D6.1. After gel separation and transfer, membranes were stained with D6.1. (B) Lysates of biotinylated Progressor cells were immunoprecipitated with C4.4 (anti-C4.4A), D5.7 (anti-EpCAM), Ox50 (anti-panCD44), anti-CD9, anti-ß4, anti-6, B5.5, D6.1 and A8.10 (non-binding antibody). After gel separation and transfer, membranes were stained with streptavidin, D6.1 and anti-CD9. A large amount of D6.1A was recovered in D6.1-, anti-CD9-, anti-6 and anti-3 precipitates. D6.1A was also precipitated by B5.5, anti-ß4 and anti-ß1. Small amounts of D6.1A were recovered in precipitates with Ox50 and D5.7. No D6.1A was recovered in C4.4 and A8.10 precipitates. CD9 was only recovered in anti-CD9 and D6.1 precipitates.

View larger version (99K): [in a new window]   Fig. 7. Redistribution of D6.1A and 6ß4 during and after PMA treatment. (A,B) Progressor and BSp73AS-db cells were seeded on Ln5 (A and B2, recombinant Ln5; B1, 804G supernatant). After overnight adhesion, cells were treated for 1 to 2 hours with PMA (10–8 M). Untreated cells and PMA-treated cells were washed, fixed and permeabilized and were stained (immediately after fixation and permeabilization) with (A) phalloidin-TRITC, (B1) D6.1-FITC and B5.5-TxR or (B2) anti-CD9/anti-mIgG-FITC and B5.5-TxR. Single stainings and digital overlays are shown. (A) BSp73AS-db and Progressor cells were treated for 1 hour (BSp73AS-db) or 2 hours (Progressor) with PMA and were stained with phalloidin-TRITC at the indicated times after starting PMA treatment. In BSp73AS-db cells actin became organized in strong, growing bundles (open arrow). Later, the thin and partly very long filipodia (arrow; 4-5 hours) and the front of the leading lamella (arrowhead; 6 hours) were stained by phalloidin. After 10 hours adjacent cells were connected via thin filipodia (open arrowhead). Although less pronounced, actin bundles are still visible. Actin bundle formation was not seen in PMA-treated Progressor cells. However, actin was redistributed, being first enriched in filipodia (arrow) and thereafter in clusters in the body of the cells (open arrow). After 6 hours, actin increased at the base (arrowhead) and after 10 hours at the front of the leading lamella (open arrow head). Scale bar: 10 µm. (B1) During PMA treatment Progressor cells develop long filipodia, which were stained by B5.5 and D6.1 (closed arrow). Early in the recovery period, double staining was mainly seen intracellularly (open arrow); The leading lamella was hardly stained (arrowhead). After 10 hours the leading lamella was brightly stained by B5.5 and D6.1 (arrowhead). Scale bar: 10 µm. (B2) CD9 became very rapidly internalized during PMA treatment (arrow) and apparently co-localized with 6ß4 (open arrow). After 4 and 8 hours CD9 mostly was still found in large intracellular clusters (vesicles) (arrowhead). After 10 hours the leading lamella was brightly stained by B5.5 and faintly by anti-CD9 (open arrowhead). But, 6ß4 only rarely co-localized with CD9. Scale bar: 10 µm. (C) D6.1A-EYFP-transfected 804G cells were seeded on Ln5 (804G supernatant)-coated cover slides and were stained with B5.5 and TxR-labeled anti-mIgG. Top row: Resting 804G cells showing very little co-localization of D6.1A and 6ß4, and where present only in spikes. Scale bar: 20 µm. Middle rows: Co-localization of 6ß4 and D6.1A is readily seen after 30 minutes of PMA treatment (left). After 2 hours PMA treatment, cells developed long filipodia. Co-localization of D6.1A and 6ß4 is mainly seen intracellularly as depicted by the sagittal section through the filipodium of the above shown cell. Scale bars: 20 µm, sagittal section: 14 µm. Lower row: The migratory phenotype of PMA-treated 804G cells becomes most obvious when seeded at low density. Two hours after PMA treatment, cells were stained with B5.5 and TxR-labeled anti-mIgG. Two hours after PMA treatment, 6ß4 is enriched in the trail and the lagging edge of the cell, whereas the large lamelipodium is mostly devoid of the integrin. Left and right show the same cells, the brighter picture being included to demonstrate the formation of extended lamellae (arrow).

View larger version (97K): [in a new window]   Fig. 7. Redistribution of D6.1A and 6ß4 during and after PMA treatment. (A,B) Progressor and BSp73AS-db cells were seeded on Ln5 (A and B2, recombinant Ln5; B1, 804G supernatant). After overnight adhesion, cells were treated for 1 to 2 hours with PMA (10–8 M). Untreated cells and PMA-treated cells were washed, fixed and permeabilized and were stained (immediately after fixation and permeabilization) with (A) phalloidin-TRITC, (B1) D6.1-FITC and B5.5-TxR or (B2) anti-CD9/anti-mIgG-FITC and B5.5-TxR. Single stainings and digital overlays are shown. (A) BSp73AS-db and Progressor cells were treated for 1 hour (BSp73AS-db) or 2 hours (Progressor) with PMA and were stained with phalloidin-TRITC at the indicated times after starting PMA treatment. In BSp73AS-db cells actin became organized in strong, growing bundles (open arrow). Later, the thin and partly very long filipodia (arrow; 4-5 hours) and the front of the leading lamella (arrowhead; 6 hours) were stained by phalloidin. After 10 hours adjacent cells were connected via thin filipodia (open arrowhead). Although less pronounced, actin bundles are still visible. Actin bundle formation was not seen in PMA-treated Progressor cells. However, actin was redistributed, being first enriched in filipodia (arrow) and thereafter in clusters in the body of the cells (open arrow). After 6 hours, actin increased at the base (arrowhead) and after 10 hours at the front of the leading lamella (open arrow head). Scale bar: 10 µm. (B1) During PMA treatment Progressor cells develop long filipodia, which were stained by B5.5 and D6.1 (closed arrow). Early in the recovery period, double staining was mainly seen intracellularly (open arrow); The leading lamella was hardly stained (arrowhead). After 10 hours the leading lamella was brightly stained by B5.5 and D6.1 (arrowhead). Scale bar: 10 µm. (B2) CD9 became very rapidly internalized during PMA treatment (arrow) and apparently co-localized with 6ß4 (open arrow). After 4 and 8 hours CD9 mostly was still found in large intracellular clusters (vesicles) (arrowhead). After 10 hours the leading lamella was brightly stained by B5.5 and faintly by anti-CD9 (open arrowhead). But, 6ß4 only rarely co-localized with CD9. Scale bar: 10 µm. (C) D6.1A-EYFP-transfected 804G cells were seeded on Ln5 (804G supernatant)-coated cover slides and were stained with B5.5 and TxR-labeled anti-mIgG. Top row: Resting 804G cells showing very little co-localization of D6.1A and 6ß4, and where present only in spikes. Scale bar: 20 µm. Middle rows: Co-localization of 6ß4 and D6.1A is readily seen after 30 minutes of PMA treatment (left). After 2 hours PMA treatment, cells developed long filipodia. Co-localization of D6.1A and 6ß4 is mainly seen intracellularly as depicted by the sagittal section through the filipodium of the above shown cell. Scale bars: 20 µm, sagittal section: 14 µm. Lower row: The migratory phenotype of PMA-treated 804G cells becomes most obvious when seeded at low density. Two hours after PMA treatment, cells were stained with B5.5 and TxR-labeled anti-mIgG. Two hours after PMA treatment, 6ß4 is enriched in the trail and the lagging edge of the cell, whereas the large lamelipodium is mostly devoid of the integrin. Left and right show the same cells, the brighter picture being included to demonstrate the formation of extended lamellae (arrow).

View larger version (85K): [in a new window]   Fig. 7. Redistribution of D6.1A and 6ß4 during and after PMA treatment. (A,B) Progressor and BSp73AS-db cells were seeded on Ln5 (A and B2, recombinant Ln5; B1, 804G supernatant). After overnight adhesion, cells were treated for 1 to 2 hours with PMA (10–8 M). Untreated cells and PMA-treated cells were washed, fixed and permeabilized and were stained (immediately after fixation and permeabilization) with (A) phalloidin-TRITC, (B1) D6.1-FITC and B5.5-TxR or (B2) anti-CD9/anti-mIgG-FITC and B5.5-TxR. Single stainings and digital overlays are shown. (A) BSp73AS-db and Progressor cells were treated for 1 hour (BSp73AS-db) or 2 hours (Progressor) with PMA and were stained with phalloidin-TRITC at the indicated times after starting PMA treatment. In BSp73AS-db cells actin became organized in strong, growing bundles (open arrow). Later, the thin and partly very long filipodia (arrow; 4-5 hours) and the front of the leading lamella (arrowhead; 6 hours) were stained by phalloidin. After 10 hours adjacent cells were connected via thin filipodia (open arrowhead). Although less pronounced, actin bundles are still visible. Actin bundle formation was not seen in PMA-treated Progressor cells. However, actin was redistributed, being first enriched in filipodia (arrow) and thereafter in clusters in the body of the cells (open arrow). After 6 hours, actin increased at the base (arrowhead) and after 10 hours at the front of the leading lamella (open arrow head). Scale bar: 10 µm. (B1) During PMA treatment Progressor cells develop long filipodia, which were stained by B5.5 and D6.1 (closed arrow). Early in the recovery period, double staining was mainly seen intracellularly (open arrow); The leading lamella was hardly stained (arrowhead). After 10 hours the leading lamella was brightly stained by B5.5 and D6.1 (arrowhead). Scale bar: 10 µm. (B2) CD9 became very rapidly internalized during PMA treatment (arrow) and apparently co-localized with 6ß4 (open arrow). After 4 and 8 hours CD9 mostly was still found in large intracellular clusters (vesicles) (arrowhead). After 10 hours the leading lamella was brightly stained by B5.5 and faintly by anti-CD9 (open arrowhead). But, 6ß4 only rarely co-localized with CD9. Scale bar: 10 µm. (C) D6.1A-EYFP-transfected 804G cells were seeded on Ln5 (804G supernatant)-coated cover slides and were stained with B5.5 and TxR-labeled anti-mIgG. Top row: Resting 804G cells showing very little co-localization of D6.1A and 6ß4, and where present only in spikes. Scale bar: 20 µm. Middle rows: Co-localization of 6ß4 and D6.1A is readily seen after 30 minutes of PMA treatment (left). After 2 hours PMA treatment, cells developed long filipodia. Co-localization of D6.1A and 6ß4 is mainly seen intracellularly as depicted by the sagittal section through the filipodium of the above shown cell. Scale bars: 20 µm, sagittal section: 14 µm. Lower row: The migratory phenotype of PMA-treated 804G cells becomes most obvious when seeded at low density. Two hours after PMA treatment, cells were stained with B5.5 and TxR-labeled anti-mIgG. Two hours after PMA treatment, 6ß4 is enriched in the trail and the lagging edge of the cell, whereas the large lamelipodium is mostly devoid of the integrin. Left and right show the same cells, the brighter picture being included to demonstrate the formation of extended lamellae (arrow).

View larger version (96K): [in a new window]   Fig. 8. Influence of co-expession of 6ß4 and D6.1A on cell adhesion and migration. (A) BSp73AS-ß4, BSp73AS-D6.1A, BSp73AS-db, BSp73ASML and Progressor cells were labeled with [3H]thymidine and seeded on BSA- or recombinant Ln5-coated 96 well plates in the absence or presence of anti-3, B5.5 or D6.1 (10 µg/ml). After 2 hours, unbound cells were washed off, adherent cells were detached and were counted in a ß-counter. Mean cpm ± s.d. of triplicates are shown. Significant inhibition of binding by the antibodies is indicated by an asterisk. Binding of BSp73AS-ß4, BSp73AS-D6.1A, BSp73AS-db and BSp73ASML cells to Ln5 was slightly inhibited by anti-3. B5.5 and D6.1 only inhibited Ln5 binding of BSp73AS-db, BSp73ASML and Progressor cells. (B) BSp73AS, BSp73AS-ß4, BSp73AS-D6.1A, BSp73AS-db and Progressor cells were seeded on Petri dishes coated with recombinant Ln5, where the central area was protected by a cover slide. After reaching subconfluency, the cover slide was removed. Thereafter medium was changed, fresh medium containing only 1% FCS and, where indicated, PMA (10–8 M). After 72 hours, cultures were washed, cells were fixed and stained with Hematoxylin-Eosin. Pictures were taken at the boundary from where cells started to migrate (proximal) as well as from the center of the area initially protected by a cover slide (distant). Scale bar: 0.5 µm.

View larger version (86K): [in a new window]   Fig. 7. Redistribution of D6.1A and 6ß4 during and after PMA treatment. (A,B) Progressor and BSp73AS-db cells were seeded on Ln5 (A and B2, recombinant Ln5; B1, 804G supernatant). After overnight adhesion, cells were treated for 1 to 2 hours with PMA (10–8 M). Untreated cells and PMA-treated cells were washed, fixed and permeabilized and were stained (immediately after fixation and permeabilization) with (A) phalloidin-TRITC, (B1) D6.1-FITC and B5.5-TxR or (B2) anti-CD9/anti-mIgG-FITC and B5.5-TxR. Single stainings and digital overlays are shown. (A) BSp73AS-db and Progressor cells were treated for 1 hour (BSp73AS-db) or 2 hours (Progressor) with PMA and were stained with phalloidin-TRITC at the indicated times after starting PMA treatment. In BSp73AS-db cells actin became organized in strong, growing bundles (open arrow). Later, the thin and partly very long filipodia (arrow; 4-5 hours) and the front of the leading lamella (arrowhead; 6 hours) were stained by phalloidin. After 10 hours adjacent cells were connected via thin filipodia (open arrowhead). Although less pronounced, actin bundles are still visible. Actin bundle formation was not seen in PMA-treated Progressor cells. However, actin was redistributed, being first enriched in filipodia (arrow) and thereafter in clusters in the body of the cells (open arrow). After 6 hours, actin increased at the base (arrowhead) and after 10 hours at the front of the leading lamella (open arrow head). Scale bar: 10 µm. (B1) During PMA treatment Progressor cells develop long filipodia, which were stained by B5.5 and D6.1 (closed arrow). Early in the recovery period, double staining was mainly seen intracellularly (open arrow); The leading lamella was hardly stained (arrowhead). After 10 hours the leading lamella was brightly stained by B5.5 and D6.1 (arrowhead). Scale bar: 10 µm. (B2) CD9 became very rapidly internalized during PMA treatment (arrow) and apparently co-localized with 6ß4 (open arrow). After 4 and 8 hours CD9 mostly was still found in large intracellular clusters (vesicles) (arrowhead). After 10 hours the leading lamella was brightly stained by B5.5 and faintly by anti-CD9 (open arrowhead). But, 6ß4 only rarely co-localized with CD9. Scale bar: 10 µm. (C) D6.1A-EYFP-transfected 804G cells were seeded on Ln5 (804G supernatant)-coated cover slides and were stained with B5.5 and TxR-labeled anti-mIgG. Top row: Resting 804G cells showing very little co-localization of D6.1A and 6ß4, and where present only in spikes. Scale bar: 20 µm. Middle rows: Co-localization of 6ß4 and D6.1A is readily seen after 30 minutes of PMA treatment (left). After 2 hours PMA treatment, cells developed long filipodia. Co-localization of D6.1A and 6ß4 is mainly seen intracellularly as depicted by the sagittal section through the filipodium of the above shown cell. Scale bars: 20 µm, sagittal section: 14 µm. Lower row: The migratory phenotype of PMA-treated 804G cells becomes most obvious when seeded at low density. Two hours after PMA treatment, cells were stained with B5.5 and TxR-labeled anti-mIgG. Two hours after PMA treatment, 6ß4 is enriched in the trail and the lagging edge of the cell, whereas the large lamelipodium is mostly devoid of the integrin. Left and right show the same cells, the brighter picture being included to demonstrate the formation of extended lamellae (arrow).