Computational protocol: CLASP1 regulates endothelial cell branching morphology and directed migration

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[…] MT dynamics were analyzed from fluorescently-labeled EB3 (mApple-EB3) movies using plusTipTracker software, a MATLAB-based, open-source software package that combines automated detection, tracking, analysis, and visualization tools for movies of fluorescently labeled MT plus end binding proteins as previously described (; ; ; ; ). All MT growth parameter tracking and EC branching experiments were performed four times. [...] For analysis of cell branching, branches were defined as protrusions that extended from the cell >10 µm in length and were longer than they were wide. Branches were counted only if they met this criterion. ‘Branch origin’ and ‘branch length’ were measured as previously described (). Cell migration was performed using blinded analysis in duplicate, to ensure reproducibility, and was quantified by hand-tracking the nucleolus in successive images from a time-lapse phase-contrast image series using the MTrackJ tracking Plugin of ImageJ (NIH). Migratory displacement measurements were obtained through subtraction of each x and y coordinate from its predecessor. Individual displacement values were then binned into one of four categories, based on their overall directionality (Quadrant I, II, III, IV). Each displacement value was further qualified by distance (<250 µm or >250 µm). The total percentage of values that fell within each quadrant was binned and graphically represented in angular rose plot diagrams, where light gray represents values <250 µm and dark gray represents values >250 µm. Distance to origin was obtained using the ‘straight line tool’ in ImageJ. Instantaneous velocities were binned into 10 µm intervals and plotted into a histogram in Excel; a distribution curve was fitted to each histogram. Statistical analysis was performed using the Analyze-It plug-in for Microsoft Excel (Analyze-It Software, Ltd.; https://analyse-it.com/), and branching and migration data were compared using a two-tailed student t-test, with α set to 0.001 for statistical significance. […]

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