MRCKα 1–478 cleavage product induces iEAAR assembly. (A) The image sequence of live confocal microscopy shows a MCF10A cell, part of a confluent epithelium, that expresses mCherry-MRCKα 1–478 (red channel) and simultaneously assembles the iEAAR. Polymerized actin was represented with the fire color scale, to better visualize the different phases of iEAAR assembly. Because during the movie the iEAAR was moving downward, we show two XY apical sections. (B) Chronological analysis of the phases of iEAAR assembly in six cells expressing mCherry-MRCKα 1–478. Discs represent the means, and horizontal bars SDs. (C) Quantification of mCherry-MRCKα 1–478 fluorescence intensity as a function of the time. To compare different cells, raw fluorescence intensity data were normalized by setting the minimum fluorescence at 0% and the maximum at 100%. Each colored line represents a different cell. Gray box indicates the time required for iEAAR assembly based on the quantification in B, and time 0 was set at the frame preceding cortical contraction. (D) Confluent MCF10A cells were transfected with mCherry-fused full-length MRCKα (1–1,719), two shorter fragments (1–965 and 1–478), or their relative kinase dead (KD) mutants and evaluated for the formation of iEAAR detected by blebbistatin staining (green). Bars, 10 µm. (E) Frequency analysis of cells that, upon transfection with these constructs, assemble the iEAAR.