Growth kinetics of filopodia adhesions after MLCK inhibition (A, B) or on soft polyacrylamide gels (C, D).

<p>The β3-integrin-EGFP (green) expressing REF52 fibroblasts on FN coated glass (A, B) or polyacrylamide gel (C, D) surfaces were time-lapse tracked with confocal microscopy. The cell membrane was stained by the fluorophore DiI (red). <b>A and B.</b> Inhibition of MLCK by ML-7 suppressed the cyclic protrusions and retractions of lamellipodium and the growth of filopodia adhesions. <b>A.</b> Time-lapse montage (b) of the filopodium containing cell edge region (white rectangle, a) showed the loss of the cyclic protrusions and retractions of the lamellipodium in the proximity of the filopodium in ML-7 treated cells (25 min–30 min after plating, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s009" target="_blank">Video S4</a>). <b>B.</b> Time-lapse montage (b) of the filopodia adhesion cell edge region (white rectangle, a) demonstrated the suppressed size growth of the filopodia β3-integrin adhesion in ML-7 treated cells. c. Comparison of the area (average of the tracked temporal states (0–600 s)) of the filopodia β3-integrin adhesions (7 adhesions, n = 17) in the ML-7 (30 or 40 µM) treated cells with that of the maturing filopodia β3-integrin adhesions (6 adhesions, n = 164) in the cells plated in normal culture media. Error bars correspond to the standard errors. <b>C and D.</b> The growth of filopodia adhesions in REF52-β3-integrin-EGFP cells on soft PAA substrates was associated with the restored cyclic protrusions and retractions of lamellipodia. The PAA substrate (7.4 kPa) was covalently coated with FN. <b>C.</b> Cell without stable surface adhesions (a, 103 min–113 min after plating, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s010" target="_blank">Video S5</a>) exhibited significant ruffling as shown by the time-lapse montage of the cell edge (b). <b>D.</b> The filopodia adhesion (white rectangle, b) at the edge of a cell (white square in a, 121 min–136 min after plating, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s011" target="_blank">Video S6</a>) matured in association with the restored cyclic protrusions and retractions of the lamellipodium (c). To better visualize lamellipodium activities, all montages were stretched vertically 2×. Scale bars: 5 µm (A, D–b), 10 µm (B, C, D–a). The width of a single image frame in the montage is indicated by the horizontal grey bar: A–b, 2.5 µm; B–b, 4.5 µm; C–b, 6.2 µm; D–c, 3 µm.