Analysis of FN-PH deposition relative to EB3-RFP positive microtubules in a spreading cell protrusion. (A) Gallery of frames from a TIRFM video of a KO6 EB3-RFP FN-PH Halo-β1itg fibroblast labeled with 1 µM SiR-tubulin in the process of spreading on FN-coated glass for 20 min at a 5-s interval (Video 5). The change in FN signal, ΔFN-PH, is shown in green, EB3-RFP in magenta, and SiR-tubulin in cyan. (B) Kymograph showing the bursts of ΔFN-PH deposition (marked by green arrowheads) occurring inside the white dashed box in A over the course of the video. (C) Quantification of ΔFN-PH fusion events (green circles), also positive for EB3-RFP signal (magenta circles). Scale bar = 5 µm. Bar graph shows the mean ± SD of EB3-positive ΔFN-PH events in seven different cell protrusions.