Fig. 3: Cell contractility triggers actin star formation.
From: A multicellular star-shaped actin network underpins epithelial organization and connectivity
A Confocal analysis of myosin-IIA-KI-GFP (magenta) localization in AcSs (green). Scale bar, 5 μm. B Statistical analyses of the signal intensity level of myosin-IIA-GFP in nodes and cables of AcSs. Myosin-IIA-GFP signal intensity in AcS nodes = 0.653 (0.525–0.812) (median (IQR)), in AcS cables = 0.029 (0.018–0.047). Box plots represent the median (centre line), 25th and 75th percentiles (box bounds), and the minimum and maximum values (whiskers). N = 3 experiments, n = 154 cells. Two sided Mann–Whitney test, ****p = 1.47 × 10−⁶⁰. Source data are provided as a Source Data file. C N-SIM analysis of P-MLC2 (phospho-myosin light chain 2, magenta) localization in AcSs (green). Scale bar, 5 μm. D Statistical analyses of the signal intensity level of P-MLC2 in nodes and cables of AcSs. P-MLC2 signal intensity in AcS nodes = 0.632 (0.496–0.909), in cables = 0.348 (0.216–0.558). Box plots represent the median (centre line), 25th and 75th percentiles (box bounds), and the minimum and maximum values (whiskers). N = 3 experiments, n = 98 cells. Two-sided Mann–Whitney, ****p = 3.93 × 10−¹³. Source data are provided as a Source Data file. E Confocal analysis of the apico-basal distribution of myosin-IIA-GFP (green) or P-MLC2 (magenta) in organoid-derived monolayers. Scale bar, 10 μm. F Statistical analyses of the signal intensity level of myosin-IIA-GFP and P-MLC2 in the apical and basal domain of differentiated cells. Mean apical myosin-IIA-GFP signal intensity in differentiated compartments = 725 ± 48 (mean ± SEM), basal myosin-IIA-GFP = 1159 ± 72, apical P-MLC2 = 1314 ± 63, basal P-MLC2 = 2078 ± 135. Box plots represent the median (centre line), 25th and 75th percentiles (box bounds), and the minimum and maximum values (whiskers). N = 3 experiments, n (cells in differentiated compartments) = 23 cells. Two-sided paired t-tests, ****p = 2.08 × 10−⁷. Source data are provided as a Source Data file. G Confocal analysis of myosin-IIA-GFP (green) and P-MLC2 (magenta) distribution in an organoid-derived monolayer. Nuclei (DNA) are stained with Hoechst33342 (blue). Crypt-like domains (c) are delimited with a dotted blue line. Scale bar, 20 μm. H Statistical analyses of the signal intensity level of myosin-IIA-GFP and P-MLC2 in differentiated and crypt-like compartments. Myosin-IIA-GFP signal intensity in differentiated compartments = 1987 (1548–2394) (median (IQR)), in crypt-like compartments = 1356 (1088–1791), P-MLC2 signal intensity in differentiated compartments = 3351 (2797–4369), in crypt-like compartments = 1360 (936.5–1701). Box plots represent the median (centre line), 25th and 75th percentiles (box bounds), and the minimum and maximum values (whiskers). N = 3 experiments, n (cells in differentiated compartments) = 23 cells, n (cells in crypt-like compartments) = 19 cells. Two-sided Mann–Whitney, **p = 0.0022, ****p = 4.14 × 10−⁸. Source data are provided as a Source Data file. I Confocal analysis and z-projection of actin distribution in the basal domain of control or blebbistatin-treated organoid-derived monolayers. Scale bar, left panel 20 μm, right panel 10 μm. J Time-lapse images of CellMask actin (green) in tdTomato (magenta) organoid-derived monolayer after 1 h blebbistatin treatment and then wash-out (t = 0 min). Crypt-like domains (c) are delimited with a dotted blue line. Scale bar, 10 μm. K Statistical analysis of the mean time of AcS re-formation after blebbistatin treatment and wash-out. Mean time (min) = 51.41 ± 16.81 (mean ± SD). N = 3 experiments, n = 101 cells. Source data are provided as a Source Data file.
