Extended Data Fig. 4: Keratin filaments are stably retained during mitosis, and become asymmetrically inherited by outer daughter cells.
From: Keratins are asymmetrically inherited fate determinants in the mammalian embryo
a, Immunofluorescence shows the extensive remodelling of cortical F-actin and microtubules during different stages of mitosis. Data are from six independent experiments. b, Immunofluorescence for K8 shows endogenous keratin filaments retained within mitotic cells in embryos fixed at multiple stages of development. Data are from six independent experiments. c, FRAP experiments for K18-Emerald and mRuby2-Actin performed in whole live embryos. All cells selected for FRAP were at the eight-cell stage and in interphase, when the actin ring is visible. 3D views of entire pre-FRAP embryos (left), zoomed views of the photobleached regions of interest (middle), and kymographs of pre- and post-FRAP fluorescence intensities (right). Data are from three independent experiments. d, Analysis of FRAP experiments. Left graphs show fluorescence recovery of K18-Emerald (green) and mRuby2-Actin (red) over time. Thinner lines represent raw data after normalization, and thicker lines indicate fitted exponential curves. Right graph shows that K18-Emerald has a larger immobile fraction than mRuby2-Actin. ***P < 0.0001; unpaired, two-tailed Student’s t-test. e, Live imaging of embryos expressing K8-Emerald show a similar pattern of expression and inheritance as K18-Emerald. The outer daughter cell inherits most of the keratin filaments during an outer-inner division (top). Computational segmentation of the same cell at each stage of mitosis (bottom). Quantification of proportion of keratin filaments inherited by outer and inner cells in live embryos expressing K8-Emerald shows a comparable asymmetry in keratin inheritance as K18-Emerald. **P = 0.001; unpaired, two-tailed Student’s t-test. f, Time series of a cell expressing K18-Emerald undergoing a symmetric outer-outer division. Keratin filaments are uniformly inherited by both daughter cells during divisions producing two outer cells (top). Computational segmentation of the same cells at each time point (bottom), and whole embryo inset highlighting the outer location of both daughter cells (right). Data are from four independent experiments. Scale bars, 5 μm.
