Fig. 1 | Nature Communications

Fig. 1

From: LINC complex-Lis1 interplay controls MT1-MMP matrix digest-on-demand response for confined tumor cell migration

Fig. 1

MT1-MMP-dependent pericellular collagenolysis is an adaptive response to matrix porosity. a MDA-MB-231 cells were embedded in 2.0 mg/ml fluorescent type I collagen (cyan) and polymerization was induced at neutral pH at 37 °C. Cells were fixed after 2.5 hrs and stained for cleaved collagen neoepitope (Col1-3⁄4C antibody, red), α-tubulin (green) and DAPI (blue). Dashed box indicates the region used for line-scan analysis in panel b with yellow, orange and pink dots indicating nucleus center, posterior and anterior limits of regions used for line-scan analysis. Dashed lines indicate initial cell position (thick) and cell rear (thin), respectively. Inset shows nucleus and cleaved collagen signal and arrowheads point to nucleo-anterior collagenolysis. b Averaged maximal fluorescence intensity profiles of cleaved collagen in 37 °C (orange curve) or 20 °C (purple curve) polymerized collagen±SD (left Y-axis) and DAPI (right Y-axis) along cell axis. n, number of cells used to calculate averaged intensity profiles from three independent experiments; “0” on X-axis corresponds to nucleus center. c MDA-MB-231 cells embedded in 20 °C polymerized gel analyzed as in a. d Morphological analysis of DAPI-stained nuclei (see Supplementary Fig. 1a for nucleus shape scoring criteria) in MDA-MB-231 cells in 3D collagen matrix under indicated experimental conditions. Data are mean % ± SEM from three independent experiments (except Ctrl at [37 °C], N = 2 and siNT, N = 6); (n), number of cells analyzed. P-values of Kruskal–Wallis test as compared to control condition in each dataset. e MDA-MB-231 cells expressing H2BGFP were embedded in 3D 37 °C or 20 °C polymerized gels. Cells were treated with ethanol (Ctrl) or GM as indicated. Nuclei were automatically tracked from time-lapse sequences obtained from three independent experiments and plot shows the distribution of nuclei speed. n, number of cells analyzed from three independent experiments. Data were transformed using the log transformation y=log(y) to make data conform to normality and analyzed using one-way ANOVA test. f, g Pericellular collagenolysis by MDA-MB-231 cells treated with GM (f) or silenced for MT1-MMP (g) in 37 °C or 20 °C polymerized gels measured as mean intensity of Col1-3⁄4C signal per cell (see Supplementary Fig. 1b for representative images). Values for vehicle-treated (panel f) or siNT-treated cells in 37 °C polymerized gel (g) were set to 100%. n, number of cells analyzed from three to five independent experiments (except experiments in 20 °C polymerized gel, N = 2); Kruskal–Wallis (f) and Mann-Whitney (g) tests. h MDA-MB-231 cells expressing H2BGFP or GFPLMNA were embedded in 3D collagen gel polymerized at 20 °C and invasion speed was analyzed as in e. n number of cells analyzed from three independent experiments. Unpaired t-test. i Pericellular collagenolysis by MDA-MB-231 cells expressing GFPLMNA in 37 °C (set to 100%) or 20 °C polymerized gels (see Supplementary Fig. 2g for representative images). n, number of cells analyzed from three independent experiments; Mann-Whitney test. j Analysis of nuclear deformation in MDA-MB-231 cells knocked down for LMNA in 37 °C polymerized gel as in d. Data are mean % ± SEM from three independent experiments (except siNT, N = 6); (n), number of cells analyzed. P-values of One-way ANOVA test as compared to control condition. k Pericellular collagenolysis by MDA-MB-231 cells knocked down for LMNA in 37 °C polymerized gel normalized to mean intensity of siNT-treated cells ± SEM (see Supplementary Fig. 3c for representative images); n number of cells analyzed from three independent experiments; Kruskal–Wallis test. l Relative invasion of cells penetrating 3D collagen to depths ≥ 30 μm (see Supplementary Fig. 3d for representative images). Data represent mean ± SEM normalized to invasion of control cells from three independent experiments. n number of cells analyzed from three independent experiments; Kruskal-Wallis test. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Scale bar=10 μm

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