Figure 1. Myosin II (MII) activity guides adhesion maturation in 3D.

(a) Adhesion and protrusion dynamics at the distal end of a pseudopod (images from Supplemental Movie 1). U2OS cells were transfected with GFP-paxillin (green) and cultured for 3 h in 2 mg/ml bovine collagen I matrices (magenta). A protrusion occurs after the first frame (indicated by dotted curves), and then new adhesions form (arrows), move rearward and grow. The vertical dashed lines provide fiduciary marks to judge adhesion movement and growth. Note the progressive alignment of the collagen fibers (magenta) highlighted by the rectangles, which is caused by force exerted through the rearward movement of the adhesions (arrows). Bar, 5 μm. (b) Box plots show the distribution of average (geometric mean) adhesion lengths per cell for each treatment group. Cells in 3D collagen gels were cultured for 3–5 h and, where indicated, treated with ~7 μM Y-27632 and ~3.5 μM ML-7. The “newly formed adhesions” are from cells in non-inhibited matrices (timelapse movies were used to identify and measure adhesions at the moment they were first detected). Bars show the 10th and 90th percentiles; the bottom and top box edges show the 25th and 75th percentiles; and the central line shows the median. Adhesions elongated significantly after initial formation, but length decreased upon ROCK/MLCK inhibition (Kruskal-Wallis test; from left to right, n = 20, 25, 22, and 14 cells). (c) Image of adhesions (green, GFP-paxillin) in a U2OS cell treated with Y-27632/ML-7 in a collagen matrix (magenta). Bar, 10 μm (insets, 3 μm).