MP1 endosomes target mature FAs, but neither FCs nor newly formed FAs. (A) HeLa cells expressing GFP-MP1 (green) and mCherry-Paxillin (red) are used for detailed quantification of FA targeting. Top: images from the video sequence showing overlay of FAs (red) and endosomal (green) masks. Bottom: center of each FA was identified as a round sphere (purple) and used for further FA tracking. FA tracks (black arrows) during time lapse are illustrated in a line of different colors, indicating temporal changes of FA positions (as shown on the color bar below). (B) Quantification of the FA targeting by p14–MP1-carrying endosomes (mean in percentage ± SEM) and of FA targeting (per frame) during the time lapse. Green arrows indicate parameter combination of highly dynamic FAs, blue arrows of mature stable FAs, and red arrows of sliding FAs. Experiments were repeated three times, n = 10 (n = the number of cells used for quantification). (C) Schematic segmentation of FAs dissected with respect to the change of its fluorescence signal over time (left image). Right graph depictures mean calculation of fluorescence MP1–Paxillin colocalization in the FA segments. Experiments were repeated three times, n = 10 (n = the number of cells used for quantification).