Fig. 5

Ectopic activation of PKC expands the apical domain and induces premature actomyosin polarisation. a 8-cell stage embryos treated with DMSO or OAG and immunostained for F-actin and Pard6. Yellow arrows indicate the borders of the cell-contact-free domain and white arrows indicate the borders of the Pard6 apical domain. b Quantification of the Pard6 surface coverage rate in embryos from (a). The surface coverage rate is calculated as the ratio between the length of Pard6 positive domain and the length of the cell-contact-free surface. Data are shown as individual data points with Box and Whiskers graph. ****p < 0.0001, two-tailed unpaired Student’s t test. N = 8 embryos for DMSO and N = 6 embryos for OAG, four independent experiments. c 4-cell stage embryos treated with DMSO or OAG and immunostained for F-actin, ppMRLC and Pard6. Arrowheads indicate apically polarised F-actin or ppMRLC. d Cell-contact-free enrichment of F-actin and Pard6 as a function of the IEA in embryos from (c). Dots represent individual measurements. e Quantification of cell-contact-free cortical enrichment of ppMRLC in embryos from (c). Data are shown as individual data points with a Box and Whiskers graph (bottom: 25%; top: 75%; line: median; whiskers: min to max). ****p < 0.0001, Mann–Whitney test. N = 18 embryos for DMSO and N = 10 embryos for OAG, six independent experiments. f 4-cell stage embryos expressing GFP-MRLC or GFP-MRLC + PKCɑ-A25E in two blastomeres were immunostained for GFP, F-actin and Pard6. Arrowheads indicate the injected blastomeres. g, h Quantification of cell-contact-free enrichment of GFP-MRLC (g) or F-actin (h) in embryos from (f). Data are shown as individual data points with Box and Whiskers graph (bottom: 25%; top: 75%; line: median; whiskers: min to max). ***p < 0.001, ****p < 0.0001, Mann–Whitney test. N = 6 embryos, three independent experiments. i Scheme of the PKC-localised activation using the CRY2-CIB1 photoactivatable system. j Time-lapse snapshots of the localisation of CRY2-PKC-KD under localised blue-light illumination. k Blastomeres expressing CIB1-Zsgreen-CAAX and CRY2-PKC-KD were regionally illuminated using a 458 nm wavelength and immunostained for F-actin, ppMRLC and Pard6. Arrowheads or dotted circles indicate the illuminated region. Squares indicate the magnified regions. (N = 24 embryos, 11 independent experiments). All scale bars, 15 μm