Extended Data Fig. 2: Anaphase A and B and pH3-s10 dynamics in fixed and live zebrafish embryos.
From: Cytoplasmic flow is a cell size sensor that scales anaphase
a, Time-lapse of a cell (4-cell stage) in anaphase. b, Left, kymograph of the same cell as in (d). Chromosomes, blue. Microtubules, grey. Dashed lines indicated the approximate velocity of chromosomes and aster center. Right, kymograph of the same cell as in (d). Overlay of chromosomes (blue) and nuclear envelope (WGA, yellow). c, Aster and chromosome velocity during anaphase A (0–100 s, n=14aster/chromosome sets from 7 embryos) and B (100–200 s, n=11asters and 13 chromosome set from 7 embryos). Only 4-cell stage embryos were used. p-value = 1 × 10−7. During anaphase A, chromosomes move faster than the aster, while in anaphase B both velocities are comparable. Statistics, multiple analysis two-tailed Mann-Whitney test. Error bars, s.e.m. d, Staining of pH3-s10 and DAPI on fixed embryos. Bar, 50 µm. Insets, magnification of cell 1 and 2. Cells are in different stages of anaphase showing different levels of pH3-s10. Bars, 10 µm. e, Kymograph of a cell in anaphase. Right kymograph, H2B-mCherry. Left kymograph, pH3-s10-Cy5. Left images, snapshots at the time points highlighted in the kymograph. Note both in the kymograph and on the images the decay of pH3-s10 but not of H2B-mCherry. Bars, 20 µm. f, Semi-log plot of normalized fluorescent intensity as a function of time. Lines are exponential fits of pH3-s10 (yellow, n = 134 cells from 12 embryos, all developmental stages combined) and H2B-mCherry (blue, n=107cells from 12 embryos, all developmental stages combined) profiles. t = 0 s, anaphase onset. Note the decay of pH3-s10 but not of H2B-mcherry. g and h, Snapshots at t = 0 s (anaphase onset) and t = 100 s (begin of anaphase B) of an 8- and 16-cell stage cell. Astral microtubules are far away from the cell cortex. EMTB-3xGFP, microtubules. Bars, 50 µm. Insets chromosome region. Bars, 20 µm. Blue dashed box, magnification in (h). i, Magnification of a 4-, 8- and 16-cell stage, corresponding to cells in Extended Data Fig. 6 (b) and Extended Data Fig. 2 (g) and (h), respectively. Bars, 10 µm.
