Growth kinetics of β3-integrin-EGFP clusters of filopodia tip or base adhesions (A–D.).

<p>Each curve represented one filopodia adhesion as REF52-β3-integrin-EGFP cells spread on FN coated glass (cells after plating, 44–56 min (Tip a), 12–24 min (Tip b), 17–29 min (Tip c), 25–37 min (Tip c), 5–17 min (Base a), 7–19 min (Base b)). The length (A), width (B), area (C) and average fluorescence intensity (D, average = sum of grey value/number of pixels) were plotted for β3-integrin-EGFP clusters for filopodia tip adhesions (thin curves, representing 4 maturing filopodia adhesions in 3 cells) and filopodia base adhesions (thick opaque curves, representing 2 maturing filopodia adhesions in 1 cell). Curves Tip-a and Base-a corresponded to the filopodia adhesions shown in Fig. 1 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s001" target="_blank">Fig. S1</a> respectively. The traces were color coded (similar to the colored zones in Fig. 1B) to indicate the different local movements (advancing or pausing) of the lamellipodium in relation to the growth of the filopodia adhesion (<i>black</i>: before being reached by lamellipodium; yellow: being passed by an advancing lamellipodium; <i>pink</i>: with the lamellipodium persisted at the distal end of the filopodia adhesion without net advancement). <b>E.</b> The spatial distribution of β3-integrins in filopodia adhesions. For the filopodia adhesions analyzed in A–D, the fluorescence intensity profiles were generated along the long axes of filopodia β3-integrin-EGFP clusters in the last frame of their tracked sequences, as indicated by the dashed arrow in the <i>Insert</i> (same as analyzed in Fig. 1; green: β3-integrin-EGFP; red: membrane). The range of the x axis for the intensity profile curves corresponded to the full lengths of filopodia adhesions between their distal and proximal ends. <b>F.</b> Vinculin recruitment to filopodia adhesions. The white squared cell edge region of a HFF cell (a, 20 min plated on FN coated glass) was magnified (b–f) in respective signals and their overlays. <b>G.</b> Scatter plot of average fluorescence intensities of vinculin clusters within filopodia adhesions with respect to their areas. The grey and pink data points corresponded to the filopodia adhesions before reached by lamellipodia or in contact with lamellipodia respectively. Open symbols correspond to the filopodia adhesions indicated by the arrowheads (in respective colors) in e and f. Scale bars: 2 µm (F–b, E insert), 10 µm (F–a).