Nuclear aggregates induce NE rupture and impair recovery. (A) Schematic representation of experimental design probing NE integrity in U2OS-RFP-NLS (gray) cells expressing nuclear (polyQ74-NLS), non-expanded (polyQ23-NLS), or non-targeted (polyQ74) polyQ protein. (B) Representative images of localization and aggregate formation in cells expressing various polyQ constructs. (C) Time-lapse images of NE rupture in representative U2OS-RFP-NLS (magenta) cell expressing polyQ74-NLS (green), resulting in transient loss of nuclear RFP-NLS enrichment. Solid arrowhead marks nuclear bleb, open arrowhead marks cytosolic RFP-NLS. (D) Nuclear (magenta curve) and cytosolic (black curve) RFP-NLS intensity of cell in C. (E and F) Percentage of cells showing NE rupture (E) and NE blebbing (F) in U2OS-RFP-NLS control cells and cells expressing polyQ23-NLS, polyQ74 and polyQ74-NLS (n_cells = 1,069, 506, 163, 325; N = 3, 3, 4, 4). Dots represent independent replicates. (G) Percentage of ruptures in polyQ74-NLS expressing cells that were preceded by blebbing events (n = 107 ruptures; N = 4). (H) Time-lapse images of NE rupture in representative U2OS-RFP-NLS cells expressing polyQ74-NLS, showing permanent loss of RFP-NLS enrichment after NE rupture. (I) Fraction of U2OS-RFP-NLS cells that recovered nuclear enrichment of RFP-NLS signal after NE rupture (n = 21, 37, 13, 103; N = 3, 3, 4, 4). (J) Recovery of normalized nuclear RFP-NLS enrichment after rupture (t = 0) in control cells (black curve) and cells expressing polyQ23-NLS (orange curve) and polyQ74-NLS cells with (green curve) or without recovery (cyan curve; n = 18, 21, 17, 31; N = 3, 3, 4, 4). (K) Graph of time until 50% recovery after NE rupture of individual control cells or cells expressing polyQ23-NLS or polyQ74-NLS shown in J. Horizontal bars represent mean ± SEM. Scale bars are 10 μm (B) or 15 μm (C and G). *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, assessed by Fischer’s exact test.