Extended Data Fig. 6: Keratins promote actin stability and apical polarization.
From: Keratins are asymmetrically inherited fate determinants in the mammalian embryo
a, FRAP experiments for mRuby2-Actin performed at the apical domain of interphase cells with keratins, cells without keratins, and cells microinjected with desmosome siRNAs. Selected photobleached regions of interest (left) and kymographs of pre- and post-FRAP fluorescence intensities (right) are shown. Data are from three independent experiments. b, Analysis of FRAP experiments. Graphs show the fluorescence recovery of mRuby2-Actin over time for each condition. Thinner red lines indicate raw data after normalization, thicker red lines are fitted exponential curves, and thick black lines represent the mean fitted exponential curves. c, Cells lacking keratins and cells with reduced desmosome expression show a smaller immobile fraction of mRuby2-Actin compared to cells with keratins. **P = 0.0002 for without keratins; **P = 0.003 for desmosome KD; Kruskal–Wallis test. d, Immunofluorescence of 16-cell stage control embryos and embryos treated with cytochalasin D and SiR-Actin. Disruption of actin stability using cytochalasin D reduces accumulation of apical polarity markers PARD6B and PKCζ. By contrast, increasing actin stability using SiR-Actin increases apical polarity levels. *P = 0.03; ***P = 0.0009; ANOVA test for PARD6B; *P = 0.03; **P = 0.003; Kruskal–Wallis test for PKCζ. e, Desmosome knockdown in 16-cell stage embryos reduces levels of apical polarity markers PARD6B and PKCζ. ***P = 0.0002 for PARD6B; ***P = 0.001 for PKCζ; Unpaired, two-tailed Mann–Whitney U-test. f, Live imaging of K18-Emerald in an embryo displaying a cell division. After division, the daughter cell that did not inherit keratins (cyan) undergoes apical constriction to form the pluripotent inner cell mass35, whereas the outer daughter cell that inherited keratins (yellow) does not internalize. Data are from three independent experiments. g, Analysis of internalization events in cells that inherited (K+) or did not inherit (K−) keratin filaments after division. ***P < 0.0001; two-tailed Fisher’s exact test. h, Immunofluorescence of endogenous K8 and AMOT in a 16-cell stage embryo. Right panels indicate zoomed views of the apical region of cells with and without keratins, with separate K8 and AMOT channels for better visualization. Cells with keratins display higher levels of apical AMOT than cells lacking keratins and cells with K8 and K18 knockdown. *P = 0.04; ***P < 0.0001; Kruskal–Wallis test. Scale bars, 10 μm.
