Extended Data Fig. 5: A dense F-actin meshwork within mitotic cells hinders the movement of keratin filaments away from the apical cortex.
From: Keratins are asymmetrically inherited fate determinants in the mammalian embryo
a, Immunofluorescence of embryos fixed specifically when a cell was undergoing mitosis or cytokinesis. Top, keratin filaments remain apically-localized throughout different mitotic stages, and become inherited by the prospective outer cell. Bottom, computational segmentation of the same cells highlighting the apical keratin distribution and asymmetric keratin inheritance. Quantification of proportion of endogenous keratin filaments present in the apical and basal regions of mitotic cells, and between prospective outer and inner daughter cells, showing a comparable asymmetry in endogenous keratin localization and inheritance as K18-Emerald dynamics in live embryos. ***P < 0.0001; unpaired, two-tailed Student’s t-test. b, Embryos microinjected with Pard6b siRNAs do not form an apical F-actin ring in the eight-cell embryo. Data are from three independent experiments. c, Mitotic cell within a fixed human embryo also displays an apical localization of keratins. d, A dense cytoplasmic F-actin meshwork is maintained throughout interphase and all stages of mitosis. Data are from three independent experiments. e, The F-actin meshwork is also present in cells across different stages of development. Representative images of a 3-cell, compacted 8-cell, and 16-cell embryo with all cells displaying a dense cytoplasmic F-actin meshwork. Data are from three independent experiments. f, Analysis of keratin filament movement during mitosis reveals that filament speed is inversely related to filament volume. n = 8 filaments; Pearson’s correlation. g, Acute cytochalasin D treatment for 15 min specifically during mitosis disrupts the F-actin meshwork, reduces the apical localization of keratins, and increases keratin filament speed. Cells treated with MG132 for 3 h retain an F-actin meshwork, but keratin apical localization is reduced and filament speed is unchanged. **P = 0.002 for CytoD; **P = 0.01 for MG132; Kruskal–Wallis test for polarization index; ***P = 0.0005; ANOVA test for filament speed. h, Scheme of a cell division producing an inner (green) and an outer (blue) cell. Keratin filaments localize close to the apical cortex of the forming outer daughter cell. The distance between the apical cortex and cytokinetic furrow, time between disassembly of the apical F-actin domain and cytokinesis, and the mean speed of keratin filament movement are indicated. Scale bars, 5 μm.
