Down-regulation of myosin II by compliance mechanosensing promotes fast, short-lived MT growth excursions. (A) Workflow used in the plusTipTracker software package for detecting fluorescent EB3 comets, tracking their movement, and classifying MT growth dynamics based on growth speed and growth lifetime. (B) Color scheme for the four subpopulations of MT growth tracks derived by plusTipTracker software and depicted in C and D. (C) Color-coded MT growth track subpopulation overlays from 2 min time-lapse movies of GFP-EB3 (frame rate = 2 s) on representative cells for comparison between a cell plated on glass with (Glass + Blebb) or without (Glass) 20 µM blebbistatin treatment, or on a more compliant ECM (0.7 kPa). Bars, 10 µm. (D) Percentage of the population of MTs whose dynamics were categorized in the four subpopulations described in B in cells under the conditions described in C. (E) Comparison of percentages of mean MT growth speeds and growth excursion lifetimes in cells under the conditions described in C. *, P < 0.001. Error bars indicate standard error of the mean.