Fig. 6: Cytoplasmic dilution shifts spindle architecture by increasing centrosomal nucleation capacity.
From: Cell state-specific cytoplasmic density controls spindle architecture and scaling
a, Osmotic perturbation of ESCs (i) and liberating CPAP from its inhibitory binding to tubulin in ESCs (ii). b, Average (Avg) z-projections of 3D RI maps derived from optical diffraction tomography (ODT) imaging of mitotic ESCs after adding isotonic medium (Iso, 337 mOsmol kg−1) or 25% ultrapure water (Hypo, 250 mOsmol kg−1) (top). Colour-coded according to RI. Maximum-projected epi-fluorescent micrographs showing tubulin::GFP signal (bottom). Dotted lines show cell boundaries. Scale bars, 5 µm. c, ODT-derived cell volumes after hypo-osmotic treatment of ESCs. Boxes show interquartile ranges, black lines inside boxes denote medians, whiskers show the minima and maxima. Data points show individual cells (n isosmotic: 107, n hypo-osmotic: 96 cells pooled from four independent experiments). Welch’s t-test (two-sided), P = 0.02. d, As c but showing mitotic cellular mass density. P = 6.2 × 10−6. e, Maximum-projected confocal micrographs showing immunostained γ-tubulin signals (yellow, or inverted grey (cropped images), respectively) in fixed ESCs, tubulin::GFP in grey, chromatin in blue. Cells after hypo-osmotic challenge (‘Hypo’) versus control (‘Iso’) (top), cells after CCB02 treatment versus control (dimethylsulfoxide ‘DMSO’) (bottom). Scale bars, 5 µm. f, Fraction of total γ-tubulin signals residing at centrosomes, comparing iso- versus hypo-osmotically treated ESCs (n = 71 and 60 cells from iso- and hypo-osmotically treated ESCs, respectively, pooled from two independent experiments, normalized to isosmotic control) and comparing DMSO- versus CCB02-treated ESCs (n = 109 and 121 cells from DMSO- and CCB02-treated ESCs, respectively, pooled from three independent experiments, normalized to DMSO control) and comparing ESCs (n = 188 cells) with DIF (n = 182 cells) (pooled from six independent experiments (Fig. 3b), normalized to ESCs). Boxes show interquartile ranges, black lines inside boxes denote medians, whiskers show the minima and maxima. Welch’s t-test (two-sided), Iso versus Hypo, P = 2.7 × 10−4; DMSO versus CCB02, P = 1.4 × 10−5; ESCs versus DIF, P = 2.9 × 10−8. g, Left: Sum-projected videos (20 s) of mitotic ESCs expressing EB1::tdTomato, after iso- or hypo-osmotic treatment (top), or after treatment with CCB02 or DMSO (bottom). Rectangle indicates region for sum intensity profiles, subdivided into spindle pole and central spindle. Boxplots as in f but showing fraction of EB1::tdTomato sum at spindle poles (right). Iso- (n = 53 cells) or hypo-osmotically treated (n = 67 cells) ESCs (pooled from n = 2 independent experiments), and of ESCs after CCB02 treatment (n = 40 cells) (versus DMSO control, n = 49 cells) (pooled from two independent experiments) and ESCs (n = 158 cells) or DIF (n = 98 cells) (same as Fig. 2g, pooled from six independent experiments). Iso versus Hypo, P = 7.2 × 10−4; DMSO versus CCB02, P = 8.7 × 10−4; ESCs versus DIF, P = 2.0 × 10−7. h, Confocal micrographs (max-projected) of metaphase control (‘Iso’) ESCs or after hypo-osmotic treatment (‘Hypo’) (top row) or CCB02- or DMSO-treated ESCs (bottom row), stained with anti-tubulin antibodies (grey). Chromatin in blue. Dotted lines show cell boundaries. Scale bar, 5 µm. i, Boxplots as in f but showing number of astral microtubules, comparing iso- (n = 37 cells) versus hypo-osmotically treated ESCs (n = 37 cells) (pooled from two independent experiments, normalized to isosmotic control), and comparing DMSO- (n = 54 cells) versus CCB02-treated (n = 70 cells) ESCs (pooled from two independent experiments, normalized to DMSO control), and comparing ESCs (n = 115 cells) with DIF (n = 122 cells) (pooled from three independent experiments (same data as Fig. 2k), normalized to ESCs). Iso versus Hypo, P = 1.1 × 10−7; DMSO versus CCB02, P = 1.1 × 10−6; ESCs versus DIF, P = 2.5 × 10−12. j, Boxplots as in f but showing percentage of cell volume occupied by spindle, comparing iso- (n = 67 cells) versus hypo-osmotically treated ESCs (n = 71 cells) (pooled from n = 2 experiments), and comparing DMSO- (n = 136 cells) versus CCB02-treated (n = 119 cells) ESCs (pooled from three independent experiments), and comparing ESCs (n = 181 cells) with DIF (n = 119 cells) (pooled from five independent experiments (data as Fig. 5e)). Iso versus Hypo, P = 1.3 × 10−5; DMSO versus CCB02, P = 3.8 × 10−20; ESCs versus DIF, P = 2.3 × 10−6. *P < 0.05; ***P < 0.001; ****P < 0.0001.
