Figure 4.
Figure 4. High-curvature pores favor nuclear envelope rupture and lamin-B dilution, especially for rapid distension. (A) Latrunculin-treated U2OS cells transfected with mCherry-cGAS and GFP-lamin-B1 detached from the coverslip and were pulled under controlled pressure into micropipettes of varying diameter, Dp. The probability of nuclear rupture, as indicated by nuclear entry of mCherry-cGAS, increases with 1/Dp (we ignore a factor of 2 in the conventional definition of curvature). Rupture probability shows the same functional dependence on curvature for active migration through Transwell pores, where nuclear blebs demark rupture. Fit function and parameters are per the single filament model (see Materials and methods); also, y = Axm/(B + xm) fits equally well with m ≈ 4, consistent with a continuum model (Pfeifer et al., 2018; six or more cells per Dp; ≥50 cells per Transwell pore diameter; Mean ± SEM). Inset: Derivatives of fits indicate a critical pipette/pore diameter of ∼4 µm, consistent with Fig. 3 E. (B) Smaller pores increase nuclear bleb size and lamin-B dilution (i.e., nucleus-to-bleb lamin-B intensity ratio minus 1). Dashed line is fitted to first two points, and the origin (≥11 cells per Transwell pore (TW) diameter; SEM). Images: Nuclear blebs indicated by arrows. Scale bars: 10 µm. (C) Accumulation of mCherry-cGAS at the aspirated tip of the nucleus (arrow) indicates nuclear rupture, which coincides with GFP-LMNB1 dilution. sec, second. (D) Tip dilution of lamin-B1 (α) is relative to inside intensity (inset), and mCherry-cGAS accumulation is normalized (norm.) to both cytoplasmic intensity and pipette cross section (as a maximum area). Binned points show that higher α correlates with more cGAS inside the nucleus at t ≈ 5 min. Each point is labeled by mean pipette diameter for cells within that bin (36 cells total, ≥5 cells per bin; SEM). (Ei) For every aspirated cell at t ≈ 15 s, nuclear extension ΔL into the pipette shows that low or slow ΔL associates with low α (unshaded region), and small diameter (2–4 µm) is needed for high α (shaded region). Filled portions of data points indicate the fraction of nuclei within that bin that rupture by t ≈ 5 min, with upper heatmap indicating rupture probabilities (Prob.) for low or high ΔL (36 cells total, three or more cells per bin; SEM). (Eii) Data for all cells aspirated with Dp = 4 µm at t ≈ 1 s (light red) and at ΔL = 10 µm (i.e., Transwell filter thickness). Fast nuclear entry and rupture occurs at ΔP = 4.6 ± 1.2 kPa, whereas slow entry and no rupture occurs at ΔP = 3.2 ± 1.0 kPa (six cells total, three cells per bin; SEM). (F) Lamin-B filaments are too stiff to bend along high-curvature nuclear membranes, and are thus depleted, but can stably interact with low-curvature membranes.

High-curvature pores favor nuclear envelope rupture and lamin-B dilution, especially for rapid distension. (A) Latrunculin-treated U2OS cells transfected with mCherry-cGAS and GFP-lamin-B1 detached from the coverslip and were pulled under controlled pressure into micropipettes of varying diameter, Dp. The probability of nuclear rupture, as indicated by nuclear entry of mCherry-cGAS, increases with 1/Dp (we ignore a factor of 2 in the conventional definition of curvature). Rupture probability shows the same functional dependence on curvature for active migration through Transwell pores, where nuclear blebs demark rupture. Fit function and parameters are per the single filament model (see Materials and methods); also, y = Axm/(B + xm) fits equally well with m ≈ 4, consistent with a continuum model (Pfeifer et al., 2018; six or more cells per Dp; ≥50 cells per Transwell pore diameter; Mean ± SEM). Inset: Derivatives of fits indicate a critical pipette/pore diameter of ∼4 µm, consistent with Fig. 3 E. (B) Smaller pores increase nuclear bleb size and lamin-B dilution (i.e., nucleus-to-bleb lamin-B intensity ratio minus 1). Dashed line is fitted to first two points, and the origin (≥11 cells per Transwell pore (TW) diameter; SEM). Images: Nuclear blebs indicated by arrows. Scale bars: 10 µm. (C) Accumulation of mCherry-cGAS at the aspirated tip of the nucleus (arrow) indicates nuclear rupture, which coincides with GFP-LMNB1 dilution. sec, second. (D) Tip dilution of lamin-B1 (α) is relative to inside intensity (inset), and mCherry-cGAS accumulation is normalized (norm.) to both cytoplasmic intensity and pipette cross section (as a maximum area). Binned points show that higher α correlates with more cGAS inside the nucleus at t ≈ 5 min. Each point is labeled by mean pipette diameter for cells within that bin (36 cells total, ≥5 cells per bin; SEM). (Ei) For every aspirated cell at t ≈ 15 s, nuclear extension ΔL into the pipette shows that low or slow ΔL associates with low α (unshaded region), and small diameter (2–4 µm) is needed for high α (shaded region). Filled portions of data points indicate the fraction of nuclei within that bin that rupture by t ≈ 5 min, with upper heatmap indicating rupture probabilities (Prob.) for low or high ΔL (36 cells total, three or more cells per bin; SEM). (Eii) Data for all cells aspirated with Dp = 4 µm at t ≈ 1 s (light red) and at ΔL = 10 µm (i.e., Transwell filter thickness). Fast nuclear entry and rupture occurs at ΔP = 4.6 ± 1.2 kPa, whereas slow entry and no rupture occurs at ΔP = 3.2 ± 1.0 kPa (six cells total, three cells per bin; SEM). (F) Lamin-B filaments are too stiff to bend along high-curvature nuclear membranes, and are thus depleted, but can stably interact with low-curvature membranes.

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