Compliance mechanosensing promotes fast MT assembly in both 2D and 3D, but 3D ECM engagement makes MT growth persistence insensitive to compliance. (A) Color-coded MT growth track subpopulation overlays from 2-min time-lapse movies of GFP-EB3 (frame rate = 2s) on representative cells cultured on 8.7 kPa or 0.7 kPa 2D ECMs, for comparison with 8.7 kPa or 0.7 kPa 3D ECMs or with cells cultured in 8.7 kPa 3D ECMs and treated with 20 µM blebbistatin (3D 8.7 kPa + Blebb). A color code key for MT growth dynamics classifications is shown below. Bars, 10 µm. (B and C) Comparison of percentages of the population of MTs whose growth dynamics were categorized in the four subpopulations described in A in cells under the conditions described in A or on 55 kPa 3D ECMs. Percentage values shown below. (D and E) Comparison of mean MT growth speeds and growth excursion lifetimes in cells under the conditions described in A or on 55 kPa 3D ECMs. *, P < 0.001. Error bars indicate standard error of the mean.