Extended Data Fig. 6. MCs use integrin-dependent migration in tissues and 3D matrigel.
(a) Dermal MC shape and distribution in adult WT and Tln1ΔMC Ubow mice (Fig. 3a), stained with pan-MC marker avidin (purple) and anti-collagen IV (white). Representative images from one of n = 6 mice per genotype. (b) Workflow for the analysis of MC cluster formation with ClusterQuant2D software. (c) Average YFP+ and CFP+ cluster size in WT Ubow or Tln1ΔMC Ubow mice. A value of 1 indicates that no cell sits in close proximity to another cell (homogeneous distribution). A value > 1 indicates that fractions of cells sit in close proximity to another and form clusters of > 1 cell. Dots represent imaging fields of view (n (WT) = 10, n (Tln1ΔMC) = 8) from three WT and four Tln1ΔMC mice. Bars display the mean; ***P < 0.0001, two-sided t tests. (d) WT BMMCs lack mesenchymal-like morphology and migration in 3D collagen I matrices. Live imaging over 36 h and quantification of average cell speeds. Dots are individual cells (n = 30 randomly chosen cells). (e,f) Contribution of actin polymerization (e, cytochalasin D) and actomyosin contraction (f, Y-27632 or blebbistatin) for BMMC migration in 3D matrigel. Spinning-disk images of Lifeact–GFP expressing WT cells in 3D gels. Average cell speeds over 36 h from one experiment per genotype. Dots are individual cells (n = 20 randomly chosen cells per condition). Bars display the mean; ***P < 0.0001, two-sided t test (e). **P = 0.0019 (Y-27), ** P = 0.0046 (Bleb), two-sided t tests (f). (g) Example of a proliferating Tln1−/− BMMC cluster in 3D matrigel, live cell microscopy over 48 h. Rarely, individual cells move away from proliferating clusters, forming seeds for a new cluster (for example Cell 1 moves, then divides to form descendant cells 1.1 and 1.2). Tracks over 48 h are shown. Example sequence from two independent experiments. Scale bars: 25 µm (a), 10 µm (e,f), 30 µm (g).