Fig. 5: The actin star network ensures epithelial morphological stability.
From: A multicellular star-shaped actin network underpins epithelial organization and connectivity
A Confocal analysis and xz view of actin (green) distribution in DMSO-treated or blebbistatin-treated organoid-derived monolayers. Nuclei (grey) are shown. Green arrowheads point to AcSs. Scale bar, 10 μm. B Statistical analysis of mean cell height in control or blebbistatin-treated cells. Cell height in control = 10.43 µm (8.731–12.99) (median (IQR)), in blebbistatin-treated = 8.128 (6.392–9.742). Box plots represent the median (centre line), 25th and 75th percentiles (box bounds), and the minimum and maximum values (whiskers). N = 3 experiments, n (control) = 60 cells, n (blebbistatin-treated) = 65 cells. Two-sided Mann–Whitney, ****p = 1.64 × 10−⁸. Source data are provided as a Source Data file. C Statistical analysis of mean basal cell area in control or blebbistatin treated cells. Basal cell area in control = 63 µm2 (49.34–82.33) (median (IQR)), in blebbistatin treated = 73.14 (53.30–102). Box plots represent the median (centre line), 25th and 75th percentiles (box bounds), and the minimum and maximum values (whiskers). N = 3 experiments, n (control) = 675 cells, n (blebbistatin treated) = 585 cells. Two-sided Mann–Whitney, ****p = 2.92 × 10−⁸. Source data are provided as a Source Data file. D Confocal analysis illustrating cell shape changes in blebbistatin-treated organoid-derived monolayers compared to DMSO-treated. Top panel shows merged image with actin (green) and nuclei (grey), and bottom panel shows actin in inverted grey. Scale bar, 20 μm. E Images of membranes-tdTomato before (green) and 10 min after (magenta) photo-activation of azidoblebbistatin. Scale bar, 5 μm. F Statistical analysis of the basal cell area modification after photo-activation of azidoblebbistatin. Mean basal cell area fold change in non-activated condition = 1.015 ± 0.03 (mean ± SEM), in photo-activated condition = 2.353 ± 0.21. N = 3 experiments, n (non-activated) = 23 cells, n (photo-activated) = 10 cells. Two-sided unpaired t-test, ****p = 2.67 × 10−¹⁰. Source data are provided as a Source Data file. G Mechanical description of the AcS network contractility within a cell by AcS-cable stresses. In Supplementary Information, we show that a AcS-cable-tension model (model 2) maps into two contributions, an AcS-induced contractile stress α and an AcS-induced contribution to the junction tension χ, defining our model 1. H Theoretical analysis of the order/disorder rigidity transition in terms of the AcS-induced pulling stress α and tension χ. The black solid line represents the theoretically predicted critical rigidity transition line; see Eq. (S19) in Supplementary Information. The symbols and arrow denote the proposed path of cell differentiation. I Simulation of the local triangular order parameter upon AcS formation and subsequent optimal cell differentiation, or AcS inhibition under blebbistatin treatment and deficient cell differentiation. J Statistical quantification of the local triangular and hexatic order in silico. Local triangular order in AcS-positive cells = 0.959 (0.927–0.984) (median (IQR)), in AcS-negative cells = 0.402 (0.256–0.592). Local hexatic order in AcS-positive cells = 0.629 (0.321–0.800), in AcS-negative cells = 0.5130 (0.335–0.715). Box plots represent the median (centre line), 25th and 75th percentiles (box bounds), and the minimum and maximum values (whiskers). Triangular order, N = 1 simulation, n (AcS-positive cells) = 667, n (AcS-negative cells) = 74. Hexatic order, N = 1 simulation, n (AcS-positive cells) = 348, n (AcS-negative cells) = 50. Two-sided Mann–Whitney, ****p = 4.95 × 10−80, nsp = 0.156. Source data are provided as a Source Data file. K Statistical quantification of the local triangular and local hexatic order from experiments. Local triangular order in control cells = 0.847 (0.795−0.885) (median (IQR)), in blebbistatin-treated cells = 0.622 (0.448–0.746). Local hexatic order in control cells = 0.530 (0.505–0.555), in blebbistatin-treated cells = 0.436 (0.393–0.475). Two-sided unpaired t-test, *p (triangular) = 0.027, *p (hexatic) = 0.011. Source data are provided as a Source Data file.
