Extended Data Fig. 8: Biophysical perturbation of cellular mass density modulates spindle scaling.
From: Cell state-specific cytoplasmic density controls spindle architecture and scaling
a. Hypoosmotic treatment of ESCs for live optical diffraction tomography (ODT) or confocal imaging. b. ODT-derived cellular mass densities after hypoosmotic treatment (left: 80/20, right: 75/25, % medium/H2O) of ESCs. Boxes show interquartile ranges, horizontal lines show medians, whiskers show minima and maxima. Data points show individual cells (80/20: n isosmotic: 52 cells, n hypoosmotic: 51 cells pooled from 3 independent experiments, 75/25: n isosmotic: 107 cells, n hypoosmotic: 96 cells pooled from 4 independent experiments). Welch’s t-test (two-sided), Iso vs. Hypo (80%): P = 0.02; Iso vs. Hypo (75%): P = 6.2 x 10-6. c. Confocal live-cell imaging (central slices) of control ESCs (isosmotic treatment) versus increasing hypoosmotic challenges. Tubulin::GFP in grey, chromatin in blue. Dotted lines show cell boundaries. Scale bar: 5 µm. d. Cell volume-normalized spindle volumes during hypoosmotic challenge of ESCs, fold change relative to control ESCs (Iso). Boxes show interquartile ranges, horizontal lines show medians, whiskers show minima and maxima. Data points show individual cells (n isosmotic: 175 cells, n Hypo (80%): 97 cells, n Hypo (75%): 71 cells, and n Hypo (50%): 37 cells from 5 independent experiments). Welch’s ANOVA and two-sided Games-Howell post-hoc testing, Iso vs. Hypo (80%): P = 0.37; Iso vs. Hypo (75%): P = 1.3 x 10-7; Iso vs. Hypo (50%): P = 1.7 x 10-13. e. Data from (d) re-analysed, showing spindle volumes within cell volume bins (n = 24, 17, 2, 0; 69, 49, 15, 7; 63, 25, 32, 20; 15, 6, 16, 8; 4, 0, 6, 2 for Iso, Hypo (80%), Hypo (70%), and Hypo (50%) conditions, respectively, binned from 2,000 µm3 through 4,000 µm3 in 500 µm3 bins). Circles show means, error bars show standard deviation. Horizontal grey lines indicate mean value of the control ESCs (Iso). f. Boxplots and data as (d) but re-analysed showing spindle aspect ratios (length/width). Welch’s ANOVA and two-sided Games-Howell post-hoc testing, Iso vs. Hypo (80%): P = 0.15; Iso vs. Hypo (75%): P = 2.9 x 10-9; Iso vs. Hypo (50%): P < 1.0 x 10-100. g. Hyperosmotic treatment of DIF for live optical diffraction tomography (ODT) or confocal imaging. h. ODT-derived cellular mass densities after hyperosmotic treatment (left: +50 mM Sorbitol, right: +100 mM Sorbitol) of DIF, untreated ESCs as additional control. Boxes show the interquartile ranges, horizontal lines show medians, whiskers show minima and maxima. Data points show individual cells (+50 mM Sorbitol: n ESCs: 12 cells, n isosmotic DIF: 13 cells, n hyperosmotic DIF: 13 cells pooled from 1 independent experiment, +100 nM Sorbitol: n isosmotic DIF: 15 cells, n hyperosmotic: 15 cells from 1 independent experiment) Welch’s ANOVA and two-sided Games-Howell post-hoc testing, ESCs vs. DIFIso: P = 6.3 x 10-3; ESCs vs. DIF50mM Sorbitol: P = 0.60; ESCs vs. DIF100mM Sorbitol: P = 0.04. i. Cell volume-normalized spindle volumes during hyperosmotic challenge of DIF (fold change relative to ESCs). Boxes show interquartile ranges, horizontal lines show medians, whiskers show minima and maxima. Data points show individual cells (n ESCs: 58 cells, n isosmotic DIF: 91 cells, n hyperosmotic DIF (50 mM): 100, n hyperosmotic DIF (100 mM): 22 pooled from 2 independent experiments). Welch’s ANOVA and two-sided Games-Howell post-hoc testing, ESCs vs. DIFIso: P = 5.3 x 10-4; ESCs vs. DIF50mM Sorbitol: P = 0.99; ESCs vs. DIF100mM Sorbitol: P = 0.90. j. Composite chart showing the cell volume-normalized spindle volumes (fold changes relative to controls) as a function of the mean cellular mass density (as fold change relative to controls). Circles show means, error bars show standard error of the mean. Data merged from this figure and Fig. 6 (n ESCs: 181 cells and n DIF: 119 cells pooled from 5 independent experiments; n ESCs+Iso: 175, n ESCs+Hypo80%: 97, n ESCs+Hypo75%: 71, n ESCs+Hypo50%: 37 pooled from 5 independent experiments; n DIF+Iso: 91 cells, n DIF+50mM: 100 cells, n DIF+100mM: 22 cells pooled from 2 experiments). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, n.s.: not significant, P > 0.05.
