Myosin 2-Induced Mitotic Rounding Enables Columnar Epithelial Cells to Interpret Cortical Spindle Positioning Cues  Soline Chanet, Rishabh Sharan, Zia Khan, Adam C. Martin  Current Biology  Volume 27, Issue 21, Pages 3350-3358.e3 (November 2017) DOI: 10.1016/j.cub.2017.09.039 Copyright © 2017 Terms and Conditions

Current Biology 2017 27, 3350-3358.e3DOI: (10.1016/j.cub.2017.09.039) Copyright © 2017 Terms and Conditions

Figure 1 Myosin Activity Is Required for Planar Cell Division (A) Left: schematic illustrating the viewpoint and a cell dividing parallel to the plane of the tissue (top) or perpendicular to the plane of the tissue (bottom). Right: still images of dividing cells labeled for myosin (green) and the plasma membrane (magenta) in mitotic domain 1 of WT (top) and ROCK-inhibitor-injected (bottom) embryos. In cells dividing within the plane of the epithelium, the cytokinetic ring appeared as a ribbon from this viewpoint, whereas it appeared as a ring in cells dividing perpendicular to the plane of the epithelium; see schematic (left). (B) Quantification of division angles for WT and ROCK-inhibitor-injected embryos. Division angles are defined as the angle between the axis of division and the plane of the epithelium. Cells in late metaphase and anaphase were considered. Blue bars indicate the relative frequency of the measured values, and red lines indicate the median. n = 53 divisions, 7 WT embryos; and n = 73 divisions, 4 ROCK-inhibitor-injected embryos. (C) Examples of dividing cells in domain 1 in fixed embryos of the indicated genotype. Mud (red) stained the centrosomes and is also slightly enriched in cortical domains closest to the centrosomes. In en face views, detection of the two centrosomes in the same plane indicated planar division. Bottom panels are cross-sectional views. Microtubules were labeled with α-tubulin (αtub). (D) Quantification of division angles for the indicated genotypes. n = 29 divisions, 5 Zip-RNAi embryos; n = 33 divisions, 4 sqh1; sqh-AE embryos; and n = 34 divisions, 4 sqh1; sqh-TA embryos. ∗∗∗p < 0.01, two-sample Kolmogorov-Smirnov test, compared to the WT distribution. Scale bars, 10 μm. See also Movies S1, S2, S3, S4, and S5. Current Biology 2017 27, 3350-3358.e3DOI: (10.1016/j.cub.2017.09.039) Copyright © 2017 Terms and Conditions

Figure 2 Reduced Myosin Activity Does Not Affect Pins Cortical Localization (A) Example of domain 1 dividing cells in WT, pinsp62 mutant, and Dlg-RNAi embryos showing the requirement of cortical Pins for correct division within the plane of the epithelium. αtub, α-tubulin. (B) Quantification of division angles in pinsp62 mutant and Dlg-RNAi embryos. n = 70 divisions, 9 pinsp62 embryos; and n = 76 divisions, 4 Dlg-RNAi embryos. ∗∗∗p < 0.01, two-sample Kolmogorov-Smirnov test (compared to WT; see Figure 1C). (C) Fixed images of dividing cells in mitotic domain 1 of the indicated genotype. Pins (red) is enriched in a cortical belt around dividing cells in embryos with reduced myosin activity, similar to WT embryos. Top panels show en face views, and bottom panels show cross-section views. Yellow arrowheads point to spindles that were not aligned toward Pins cortical accumulation. Scale bars, 10 μm. See also Figures S1 and S2 and Movie S6. Current Biology 2017 27, 3350-3358.e3DOI: (10.1016/j.cub.2017.09.039) Copyright © 2017 Terms and Conditions

Figure 3 Reduced Myosin Activity Disrupts Mitotic Rounding (A) Still images of live embryos showing dividing cells in domain 1. WT myosin (sqh-TS::GFP) accumulated around the cell cortex as the cells rounded up upon mitotic entry. Mutant myosin (sqh-TA::GFP and sqh-AE::GFP) in contrast did not accumulate around the cortex. Cortical myosin was also depleted in ROCK-inhibitor-injected embryos. Scale bar, 10 μm. (B) Cross-section views of dividing cells in metaphase, immediately before cytokinetic ring assembly. In WT embryos, cells rounded up, shortening their apical-basal (a-b) axis. In contrast, cells stayed elongated along the apical-basal axis in sqh1; sqh-AE or ROCK-inhibitor-injected embryos. White dotted lines highlight cell contours. Scale bar, 10 μm. (C) 3D reconstructed cells, before mitotic entry, at metaphase (yellow boxes, 0 s), during cytokinesis, and after sister cell abscission. The time (in seconds) relative to metaphase is given below. The top row illustrates the dramatic cell-shape change from columnar to round in a WT cell upon mitotic entry. The middle and bottom rows illustrate a cell division occurring perpendicular to the plane of the tissue in a ROCK-inhibitor-injected embryo (middle) or in a sqh1; sqh-AE mutant embryo (bottom). In both cases, the cells do not round up at metaphase. White arrows indicate the cleavage furrow. (D) Quantification of cell aspect ratio in metaphase for the indicated genotype or drug injections. n = 9 cells, 1 WT embryo; n = 13 cells, 2 sqh1; sqh-AE embryos; and n = 10 cells, 1 ROCK-inhibitor-injected embryo. Individual data points, as well as box-and-whisker plots, are shown. (E) Quantification of cell length along the apical-basal axis at metaphase for the same cells as in (D). Individual data points as well as box-and-whisker plots are shown. Current Biology 2017 27, 3350-3358.e3DOI: (10.1016/j.cub.2017.09.039) Copyright © 2017 Terms and Conditions

Figure 4 Spindle Misorientation Is Correlated with Defective Cell Rounding (A) Apical-basal cross-section views of dividing cells in metaphase. A significant number of cells remained elongated along the apical-basal axis in moe-RNAi and Cyto-D-injected embryos. Arrowheads indicate the cleavage furrows. Scale bar, 10 μm. (B) Quantification of division angles for the indicated genotypes or drug/solvent injections. n = 53 divisions, 7 WT embryos; n = 92 divisions, 4 moe-RNAi embryos; n = 50 divisions, 2 DMSO-injected embryos; and n = 90 divisions, 4 Cyto-D-injected embryos. ∗∗∗p < 0.01, two-sample Kolmogorov-Smirnov test. (C) Quantification of cell aspect ratio in metaphase cells for the indicated genotype or drug injections. n = 9 cells, 1 WT embryo; n = 10 cells, 2 moe-RNAi embryos; n = 12 cells, 2 Cyto-D-injected embryos; and n = 10 cells, 2 Dlg-RNAi embryos. Individual data points as well as box-and-whisker plots are shown. ∗∗p < 0.05; ∗∗∗p < 0.01, two-sample Kolmogorov-Smirnov test. (D) Division angles are plotted depending on the cell’s aspect ratio at metaphase for individual cells. Individual data are color coded depending on the genotype or drug treatment. The regression line is shown. We found a correlation coefficient ρ = 0.8, p < 0.01. (E) Division angles are plotted depending on the cell’s aspect ratio at metaphase for individual Dlg-RNAi cells. Cells round up at metaphase, but the division angle is random; there is not a significant correlation between the two variables (ρ = 0.43, p = 0.218). n.s., not significant. Current Biology 2017 27, 3350-3358.e3DOI: (10.1016/j.cub.2017.09.039) Copyright © 2017 Terms and Conditions