Assembly of one apical F-actin ring occurs within the extruding epithelial cell. (A) The figure represents an epithelial MCF10A cell undergoing cell extrusion. The sequence of images are derived from XY and XZ optical sections of confluent epithelial cells MCF10A stably transduced with LifeAct-EGFP or LifeAct-Ruby and mixed at a 1:1 ratio. The extruding cell is stained with LifeAct-EGFP and shows the assembly of cell-autonomous aEAAR. (B) Cartoon representing the sequential events of epithelial extrusion according to our experimental observations. Blue, plasma membrane; green, F-actin of the extruding cell; red, F-actin of neighboring cells. (C) Grayscale image sequence of XY optical section of a representative aEAAR, showing four identifiable phases of its assembly: (1) cortical contraction, (2) formation of F-actin patches, (3) connection among patches, and (4) coalescence of patches in a unique ring-like structure. (D) Statistical analysis of time distribution of aEAAR assembly phases measured starting from the frame preceding cortical contraction. Extruding-cell autonomous events are represented in green, and events of neighboring cells in red. Discs represent the means, and horizontal bars SDs. (E) Left: Optical section of a cell with a fully assembled aEAAR detected by LifeAct-EGFP live staining (grayscale). Right, enlargement of the aEAAR. (F) F-actin density is measured as the fluorescence intensity of the aEAAR as a function of the distance from aEAAR center. The radial profile was calculated by taking the mean of the rescaled radial profile intensity of several aEAARs (n = 44). Insets: Distributions of distances from aEAAR center of maximum F-actin density and half decay length respectively. Blue dots, samples; black dot, mean.