Extended Data Fig. 2: Tracking and quantitative analysis of keratin filament movement during interphase and mitosis.

a, Time series of an embryo expressing K18-Emerald and RFP-Utrophin, with the corresponding major cellular events labelled in the left column. Separate K18-Emerald and RFP-Utrophin channels are shown. Right panels show 2D views through a single cell that assembles keratin filaments, for better visualization of keratin distribution within the cell, relative to the apical domain. Keratin filament assembly is initiated before the formation of the apical domain. When the apical domain forms, keratin filaments become enriched apically in close association with F-actin. During mitosis, the apical domain disassembles but keratin filaments remain apically localized, resulting in their asymmetric inheritance by the outer daughter cell. Data are from three independent experiments. b, Immunofluorescence of endogenous keratins in embryos fixed at different stages of apical domain formation recapitulates the pattern and localization of keratin filaments relative to the apical domain observed in live imaging experiments. Data are from three independent experiments. c, Computationally-rendered filaments obtained from live imaging data. In this example, five individual filaments were tracked over time with a 10-min interval between frames. Data are from three independent experiments. d, The log mean square displacement (MSD) versus log lag time graph indicates that the movement of keratin filaments is unconfined and diffusive (slope > 1). Pearson’s correlation. e, Volume of an individual keratin filament and total filament volume within a single tracked cell increase linearly over time. Pearson’s correlation. f, Quantification of filament speed, volume of filaments, and polarization index before apical domain formation, after apical domain formation, and during mitosis. After the formation of the apical domain, keratin filaments move more slowly, display a larger total volume, and become more apically polarized than before apical domain formation. During mitosis, keratin filaments move faster, but retain a large volume and high apical polarization. ***P = 0.0002; **P = 0.001; Kruskal–Wallis test for filament speed; **P = 0.003; ANOVA test for filament volume; **P = 0.003; Kruskal–Wallis test for polarization index. Scheme shows the parameters used for calculation of the polarization index. d1 is the distance between the volume-weighted centre of mass of the keratin filaments and the centre of mass of the cell. d2 is the length of the apical-basal axis of the cell. g, High-resolution immunofluorescence images show that keratin filaments align specifically along actin filaments extending from the apical domain. Green arrows indicate examples of keratin–actin colocalization. Data are from three independent experiments. h, Differences in F-actin accumulation at the apical domain of control embryos and embryos treated with cytochalasin D or a high concentration of SiR-Actin. Insets show zoomed views of individual 8-cell blastomeres, highlighting the loss of the apical domain in cytochalasin D-treated embryos, and a dense accumulation of apical F-actin in SiR-Actin-treated embryos. Data are from three independent experiments. Scale bars, 10 μm.