Figure 4(A-C) Confocal images of the apical cell surface of a βH-spectrin knock-down embryo showing the myosin-II marker Sqh::mCherry (green) and E-cadherin::mNeonGreen (magenta) during ventral furrow formation. (A) myosin-II accumulates normally at the apical surface and cells undergo an initial phase of constriction. The dashed rectangle indicates the region shown in (B and C) at higher magnification. Scale bar, 30 μm. Panels (B,C) show a sub-region within the ventral tissue at different time points. Initially a supracellular myosin-II network formed (0 s), before intercellular connections broke (13 s) resulting in membrane tears (dashed line) with tissue retractions (small arrow heads) along the anterior-posterior axis. The intercellular network re-established (39 s, 65 s) as new actomyosin fibers formed (big arrow heads) to reconnect neighboring cells. The intercellular network broke again causing tears (dashed lines) at different positions (104 s). Scale bars, 5 μm. (D,E) Confocal images showing the myosin-II marker Sqh::mCherry (green), E-cadherin::mNeonGreen (magenta) and the segmented membrane (red) of a single cell at different time points in a control embryo undergoing ratcheted constriction (D) and in a βH-spectrin knock-down embryo undergoing non-ratcheted pulsations (E). Scale bars, 2.5 μm. (F) Heatmaps showing the apical surface of individual cells over time of control embryos (left) and βH-spectrin knock-down embryos (right) normalized to the initial time point (nControl=233; nβH-spectrinKD=368; of 5 different embryos each). The color-coded scale bar is depicted on the left. Dark blue indicates a cell area bigger than the initial value (>1) and yellow indicates an area constricted to less than 1/5 of the initial area (<0.2). Three different cell behaviors in βH-spectrin knock-down embryos were identified: cells that constricted, cells that showed impaired constriction and cells that expanded. (G) Quantification of the percentage of cells that constricted (to an area < 50%, yellow), cells that showed impaired constriction (to an area >50 and <100%, green) and cells that expanded (to an area >100%, blue) after 7 min in control embryos and in βH-spectrin knock-down embryos. (H,I) Graph showing the average area over time of control cells (H, n=235 cells) and cells in βH-spectrin knock-down embryos (I) that constricted (yelow, n=274), of cells that showed impaired constriction (green, n=232) and of cells that expanded (blue, n=32). The lines indicate the mean value and the semi-transparent region the standard deviation. (J) Boxplots showing the distribution of apical cell area values 7.5 min after the onset of ventral furrow formation of ventral cells in control embryos, of ventral cells in βH-spectrin knock-down embryos that constricted, and ventral cells in βH-spectrin knock-down embryos that displayed impaired constriction and expanded. Sample numbers are equal to I. The values are presented as percentage of initial cell area. ANOVA result (all data points): F(3,717)=763.9, p=9.6e-223; Cohen's d compared to the control: dconstricted=0.1, dimpaired=1.5, dexpanded=3.7. Due to a big effect size (high sample number), statistical significance was assessed based on Cohen's d. A Cohen's d < 0.5 was considered not significant (n. s.). (K) Quantification of the peak constriction rate and expansion rate relative to control cells. Control cells and cells in βH-spectrin knock-down embryos that showed impaired constriction or expanded were compared. The mean value and standard deviation are shown. Please note the logarithmic scale. While in βH-spectrin knock-down cells the constriction rate was ~24% lower (mean value of 0.8) compared to control cells, the expansion rate increased to ~60% (mean value of 1.6). The absolute change of the expansion rate was 2.5 times higher than the change of the constriction rate. ANOVA result (all data points): F(3,717)=349.2, p=9.7e-140; pairwise comparison of βH-spectrin knock-down cells to control using Cohen's d>1.4.