Nuclear aggregates induce local disruptions of the nuclear envelope and nuclear lamina. (A) Schematic representation of the use of DNA sensing cGAS expression as a tool for detecting NE rupture localization. (B) Time-lapse images of representative U2OS cell expressing cGAS (magenta) and polyQ74-NLS (green), showing nuclear cGAS entry (white arrowhead) around a nuclear aggregate. (C) Intensity profile of cGAS accumulation around aggregate shown in B. (D) Time-lapse images of representative U2OS-RFP-NLS cell (cyan) expressing polyQ74-NLS (green) and eRFP-cGAS (emiRFP670; magenta). Zooms show blebbing (orange arrowhead) and subsequent eRFP-cGAS accumulation (orange arrows). (E) Graph depicting increase in eRFP-cGAS signal (magenta curve) after rupture indicated by loss of RFP-NLS enrichment (cyan curve). Duration of the blebbing event is shown in gray. (F) Live-cell imaging of representative U2OS-RFP-NLS (gray) cells expressing polyQ74-NLS (green) and HaloTag-lamin A (magenta) showing local lamin A depletion (arrowhead) and scar formation post-rupture (black arrowhead). (G) Zooms and intensity profiles along the NE of rupture site shown in F pre- and post-rupture. (H) Representative images of U2OS cells expressing polyQ23-NLS, polyQ74-NLS, or polyQ74 (green) immunostained for lamin B1 (magenta), showing lamin B1 disruption (white arrows) or unifocal (white arrowheads) lamin B1 deformation. (I and J) Percentage of U2OS control cells, or cells expressing polyQ23-NLS, polyQ74, or polyQ74-NLS that show unifocal deformation (I) or disruption (J) of lamin B1. Horizontal bars represent mean ± SD and dots represent independent replicates (ncells = 589, 480, 253, 289; N = 3). PolyQ (B, C, and F) and cGAS (B and C) signals were gamma-adjusted (γ = 0.75). Scale bars represent 10 μm (B, D, F, and H) and 5 μm (Bzoom).**P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, assessed by Fischer’s exact test (I) or one-way ANOVA with Tukey’s multiple comparisons test (J).
