Fig. 2: Mechano-osmotic nuclear remodelling is a rapid response to FGF2 removal.
From: Mechano-osmotic signals control chromatin state and fate transitions in pluripotent stem cells
a, Quantification of change in nuclear volume upon exposure to culture medium/growth factors indicated (n = 4 independent experiments with 953 (Pluripotency; Pluri), 1,157 (Basal), 851 (FGF2) and 882 (TGF-β1) nuclei per condition; minimum-to-maximum box plots show 75th, 50th and 25th percentiles; Kruskal–Wallis/Dunn’s). b, Representative projections of nuclear envelope fluctuations as a function of time upon pluripotency factor removal and adding back specific growth factors. Note that removal of pluripotency factors triggers fluctuations that can be rescued by adding back TGF-β1 and FGF2 (scale bars, 5 µm; n = 301 (Pluri), 268 (Basal), 320 (Basal + FGF), 382 (Basal + TGF-β1) and 386 (Basal + FGF2/TGF-β1) nuclei pooled across 3 independent experiments; ANOVA/Dunnett’s). c, Representative snapshots and line scans of live imaging videos of LaminB1-RFP-tagged hiPS cells in basal medium, stained with FastAct and memGlow to label actin and plasma membrane, respectively. Left: perinuclear actin rings surrounding nuclei and intercellular cavities corresponding nuclear deformation. Right: blebs derived from a mitotic cell deforming the nucleus of a neighbouring cell (scale bars, 10 µm; images representative of 5 videos). d, Quantification of nuclear fluctuations from cells in basal medium with or without inhibitor treatments as indicated (n = 812 (Basal), 267 (Calyculin A (Calyc)), 357 (CytochalasinD (CytoD)), 522 (CytoD + Nocodazole (Nocod.)) and 998 (ATP-depleted) nuclei per condition pooled across 3 independent experiments; ANOVA/Dunnett’s). e,f, A schematic of the experimental outline, representative images (e) and quantification (f) of nuclear envelope fluctuations in cells compressed (Comp) in pluripotency or basal medium for timepoints indicated. Note decreased fluctuations in pluripotency condition and an increase in basal medium (scale bars, 10 µm; n = 3 independent experiments with 318, 337, 355 and 207 (Pluripotency 0, 5, 15 and 30 min, respectively) and 350,188, 320 and 151 (Basal 0, 5, 15 and 30 min, respectively) total nuclei per condition; ANOVA/Fischer’s). g, Quantification of nuclear volume dynamics from of Sox2-GFP-tagged hiPS cells live imaged directly after a media change into pluripotency or basal medium, followed by compression. Line represents median volume and individual dots are average colony volumes at indicated timepoints (n = 10 colonies per condition pooled across 6 independent experiments). h, AFM force indentation experiments of iPS cell nuclei within 20 min of media switch. Note increased elastic modulus of cells in basal media conditions, restored by adding FGF2 (n = 69 (Pluri), 71 (Basal), 76 (Basal + FGF2), 85 (Basal + TGF-β1) and 74 (Basal + FGF + TGF-β1) nuclei pooled across 5 independent experiments; Kruskal–Wallis/Dunn’s). i, Representative tracks of nucGEM particles. Colours represent average rate of diffusion per tracked particle (scale bars, 5 µm). j,k, Quantification of mean squared displacement (MSD) versus lag time (tau τ/s per nucGEM particle (j) and nucGEM diffusion (Deff) and diffusivity exponent β (k) (n = 260 (Pluri) and 370 (Basal) cells pooled across 4 independent experiments; mean ± s.d.; Kruskal–Wallis/Dunn’s). l,m, Representative snapshots of live imaging and quantification of HALO-tagged endogenous YAP localization (l) and nuclear height (m) in cells compressed to 5 µm height in pluripotency or basal medium. Note YAP nuclear entry in pluripotency condition but not in basal medium upon compression (scale bars, 30 µm; l, n = 3 independent experiments with 127 (Pluri) and 101 (Basal) total cells per condition; m, n = 56 cells (Pluri uncompressed), 42 (Basal uncompressed), 34 (5 µm Pluri), 47 (5 µm Basal), 37 (3 µm Pluri) and 41 (5 µm Basal) cells pooled across 3 independent experiments; ANOVA/Friedman).
