Secretion of FN-PH is cell autonomous and occurs at hotspots. (A) Time-lapse TIRFM images of a mixture of FN-PH-expressing (cell #1) or -nonexpressing (cells #2–4) KO67 Halo-β1itg fibroblasts (paxillin-mCherry in magenta and FN-PH in green) on FN-coated glass over the course of 8 h. The FN-PH expressing cell (#1) is depositing and assembling a clearly visible ventral matrix of FN-PH while the four nonexpressing surrounding cells (#2, 3, 4) are not. (B) FN-PH deposition before and after photobleaching: TIRFM images of a KO67 Halo-β1itg (paxillin-mCherry in magenta) depositing FN-PH (in green) on FN-coated glass (Video 1). Panels show the merge of paxillin-mCherry and FN-PH signals prior to photobleaching, right after photobleaching using the 488TIRF laser, and during recovery after photobleaching (1.5 and 3 h). Dotted circles show hotspots for FN-PH deposition, and white arrows indicate sites of fibrillogenesis. Graph represents the quantification of the mean green fluorescence over time in hot spots a, b, and c as well as in a control area d. (C–E) High-frequency imaging of FN-PH exocytosis following photobleaching of the cell shown in B. (C) Gallery of frames showing three representative FN-PH fusion events imaged in TIRFM (1.65 × 1.65 μm) in Video 2. Locations of cropped regions are indicated by arrows in E. (D) Time profile of the normalized fluorescence intensity (mean ±95% confidence interval) for 11 MatLab validated FN-PH fusion events detected during high-frequency acquisition. (E) Map of the validated FN-PH fusion events (white markers) overlaid on the cell picture from B taken after photobleaching or 1.5 h after. Scale bar = 20 μm.