Modulation of migration efficiency and nuclear deformation in dense collagen lattices by integrin- and actomyosin-mediated force generation. (A) Dose-dependent reduction of pMLC^T18S19 content in the presence of ROCK inhibitor Y-27632 in HT/MT1 cells. Signal intensity was calculated by densitometry and normalized to the total MLC signal. (B) Dose-dependent inhibition of collagen contraction by anti–β1 integrin mAb 4B4 (left) or Y-27632 (right). Cell-free and cell-containing collagen lattices treated with GM6001 (20 µM), and mAb 4B4, control IgG1 (3 µg/ml), or Y-27632 at indicated concentration. Matrix contraction was measured from gel areas (top images) after 48 h (left) or 24 h (right) as triplicates (means and SD from one representative experiment). Dashed horizontal lines mark 0, 50, and 100% gel contraction. (C) Mean population speed from single-cell analysis after 19–24 h of cell tracking in bovine collagen (1.7 mg/ml) in the absence or presence of MMP inhibitor GM6001 (20 µM) and mAb 4B4 or Y-27632. Medians and boxes and whiskers from 60–150 cells (n = 2–3 for Y-27632; n = 3–5 for 4B4). Dashed lines indicate 100% reference for MMP-independent movement and 90% inhibition. ***, P < 0.0001; **, P < 0.01; n.s., not significant. (D and H) Median elongation (cell body length divided by width) after 10 h of MMP-independent migration (50–90 cells from n = 2; ***, P < 0.0001; n.s., not significant). Horizontal line, median. (E) xy and xz image cross sections of confocal reflectance from bovine collagen fibrils (top) and quantification of pore cross sections (1.7 vs. 0.8 mg/ml concentration). (F and I) Population speed from single-cell analysis in bovine dermal collagen in the presence of 1 µg/ml mAb 4B4 (F) or 2 µM Y-27632 (I) and, where indicated, 20 µM GM6001. 60–108 cells from 2–3 independent experiments; ***, P < 0.0001; **, P < 0.01; *, P < 0.05; n.s., nonsignificant. (G and J) Nuclear diameter analysis as described in the legend of Fig. 3 E. Red dashed ovals, subset of deformed nuclei with hourglass (“h”) shape associated with movement (middle gray zone) or local prolapse (“p”) in largely arrested cells (dark gray zone). r/e, round or ellipsoid nuclei. Arrowheads, deformation of nuclei; arrows, migration direction. Cells migrated in bovine collagen in the presence of either 1 µg/ml 4B4 antibody or 2 µM Y-27632 and, when indicated, 20 µM GM6001. 11–27 cells each; n = 2. Bars: (B) 5 mm; (D) 25 µm; (E, G, and J) 10 µm.