FN-PH deposition occurs preferentially under stable leading edge lamellipodia. (A) Gallery of frames from a live TIRFM video of a KO67 Halo-β1itg FN-PH fibroblast randomly migrating on FN-coated glass (Video 3). FN-PH is shown in green and paxillin-mCherry in magenta. Area 1 marks a stable lamellipodium at the leading edge, area 2 a retracting protrusion, and area 3 a short-lived protrusion at the leading edge that failed to stabilize. (B) Kymograph showing the bursts of FN-PH deposition (marked by stars) occurring along the white line over the course of the video. (C) Gallery of time frames at the leading edge (LE) and line profiles of FN-PH (green) and paxillin-mCherry (magenta) fluorescence along the direction of membrane protrusion (yellow line) at various time points labeled with FN deposition clusters 1–3. (D) Time profile of the total fluorescence intensity for paxillin-mCherry (solid colored lines) and FN-PH (dotted colored lines) within five clusters of stable leading edge focal adhesions, with mean signals shown in back lines. (E) Estimation plot of the time difference between the half-peaks of FN-PH and paxillin-mCherry fluorescent signals within 17 leading edge focal adhesions from four cells. The mean of differences is shown as 95% confidence interval. (F) Gallery of time frames at the rear end (RE) and line profiles of the FN-PH (green) and paxillin-mCherry (magenta) fluorescence along the direction of membrane retraction (yellow line) at various time points. (G) Gallery of time frames in a transient protrusion (TP) and line profiles of the FN-PH (green) and paxillin-mCherry (magenta) fluorescence along the direction of membrane protrusion (yellow line) at various time points. Scale bar = 10 μm.