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).