Extended Data Fig. 2: Spindle size subscaling in differentiating embryonic stem cells (ESCs) is independent of confluency and cell geometry.
From: Cell state-specific cytoplasmic density controls spindle architecture and scaling
a. Volumetric cell analysis, based on training pixel-classifier models in Ilastik52 to distinguish mitotic cytoplasm via tubulin::GFP. Spindle volume masks were generated via Spindle3D51, based on adaptive thresholding of tubulin::GFP. b. Spindle length after binning data into cell volume bins (bin size 500 µm3). Numbers inside the bins show respective n of cells (data sets as in Fig. 1h, n = 5, 208; 57, 1051; 280, 1017; 445, 543; 238, 91; 59, 10 cells for ESCs, DIF conditions binned from 1000 µm3 through 3500 µm3 in 500 µm3 bins, pooled from 9 independent replicates). Boxes denote interquartile ranges, horizontal lines inside boxes show medians, whiskers indicate minima and maxima. Welch’s t-test (two-sided). c. Same as c) but showing spindle width. d. Spindle aspect ratio (spindle length / spindle width). Boxes denote interquartile ranges, horizontal lines inside boxes show medians, whiskers indicate minima and maxima. Data points show individual cells (ESCs n = 1,084 cells, DIF n = 2,920 cells pooled from 9 independent experiments, see Fig. 1h). Welch’s t-test (two-sided), P = 9.3 x 10-45. e. Mitotic cell volume is independent of the local cell confluency. Single data points represent individual cells (ESCs n = 110 cells (yellow) and DIF n = 151 cells (blue) from 3 experiments). rs: Spearman’s correlation coefficient (PESCs = 0.07, PDIF = 0.21). f. Osmolality of the ESCs culturing medium and of the differentiation medium. g. Cell volume occupied by the spindle in ESCs (yellow) and DIF (blue) per differentiation time bin. The circles show the means, error bars show the standard deviation. Welch’s t-test (two-sided). n: data points in each bin. h. Mitotic cells in both differentiation states are spherical. Each data point represents an individual cell (ESCs n = 1,084 (yellow) and DIF n = 2,920 (blue), data pooled from 9 independent experiments). Big circles represent the median of each cell volume bin, the error bars show the interquartile range. The dotted line represents the behaviour of a perfect sphere. i. In cells with comparable cell surface area : cell volume ratios, spindle volume subscales in DIF (blue) when compared with ESCs (yellow). Boxes show interquartile ranges, white lines inside boxes denote medians, whiskers show minima and maxima. n = 395, 176; 697, 1647; 67, 1080; 445, 543; 238, 91; 59, 10 cells for ESCs, DIF conditions binned from 0.3 µm-1 through 0.45 µm-1 in 0.05 µm-1 bins, pooled from 9 independent replicates. d: Cohen’s d. Welch’s t-test (two-sided) for data in each bin. j. Top: Maximum-projected confocal images of mitotic ESCs or DIF (tubulin::GFP in grey) acquired during our automated live-cell imaging protocol (Fig. 1e). Bottom: tubulin::GFP-based intensity profiles of a cross-section between spindle equator and pole in maximum projected confocal images (top, dotted white lines). For consistency, cells were randomly drawn from a pool of cells within the 2500-3000 µm3 cell volume bin (ESCs n = 75 cells (yellow) and DIF n = 75 cells (blue) pooled from 6 independent experiments). Lines denote the means, shaded areas denote the 95% confidence intervals. *: P < 0.05, ***: P < 0.001, ****: P < 0.0001, n.s.: not significant, P > 0.05. d: Cohen’s d.
