Requirement for deformation of the nucleus during MMP-independent migration at rate-limiting pore conditions. (A) Time-lapse sequence (left) and population speed (right) of leading edge and main cell body in bovine dermal and rat tail collagen (1.7 mg/ml) in the presence of GM6001. Tracking of the leading extension (white dotted lines) and body (green dotted lines) from starting (*) to end (triangle) position after 5 h. Speed is depicted as median with box blot and whiskers (5th/95th percentile; 45 cells, 3 independent experiments). ***, P < 0.0001; n.s., nonsignificant. (B) Nuclear deformation in moving or migration-arrested nuclei in HT1080 cells expressing H2B/EGFP and cytoplasmic DsRed2 in 6.6 mg/ml bovine dermal collagen in the presence of protease inhibitor cocktail (PI). Overview image from movie sequence (top; Video 5) highlighting moving (green arrowheads) and immobilized nuclei (red arrowheads). Middle: time-lapse example sequences of moving and immobilized nuclei from cells marked in the top image (numbers, time in min). Bottom: diameters of deformed nuclei from independent cells during migration or immobilization. 10 cells from one representative experiment out of n = 2; *, P < 0.05. (C) Morphology of cell body and nucleus during migration in 3D bovine dermal or rat tail collagen matrix (numbers, collagen concentration in mg/ml) in the absence or presence of GM6001. Cultures were fixed 10–16 h after polymerization and stained as indicated, including collagen cleavage neoepitope detection (cyan signal). Nuclear deformation imposed by collagen structures in moving cells in the presence of GM6001 (diameters 4–5 µm; empty arrowheads); conditions of immobilization induce nuclear rounding with occasional thin prolapse (white arrowheads). Arrows, direction of migration and protrusion, respectively. (D) Classification of nuclear morphologies under conditions of cell migration (middle) and immobilization (right). Left: representative xz cross section of a nondeformed HT/MT1 cell (50 µm²). Arrows, nuclear diameters used for quantification in B and E. Numbers show arbitrary ranges for diameters and corresponding cross sections for each phenotype. Red symbols (top-left corner) represent population phenotypes marked in E. (E) Nuclear diameters from cells cultured in collagen matrices of varying pore sizes in the absence or presence of GM6001. After 16 h, samples were fixed, stained with DAPI and phalloidin, and cells with polarized morphologies were analyzed by confocal microscopy for nuclear diameters (compare C). Red dashed ovals, subset of deformed nuclei with hourglass shape associated with movement (middle gray zone) or local prolapse in largely arrested cells (dark gray zone). Black dashed ovals highlight round nuclei of arrested phenotypes (light gray zone). Horizontal lines represent the medians (of each 21 nuclei per group; 2–3 independent experiments). Tables indicate the frequency of hourglass and prolapse phenotypes for each condition. Bars: (A) 50 µm; (B–D) 10 µm.