High-curvature probes rapidly rupture nuclei without disrupting plasma membrane. (A) Probing nuclei in living WT U2OS cells at constant force (∼10–20 nN) with medium-curvature beads (diameter = 4.5 µm) shows no YFP-NLS mislocalization (inverse grayscale), whereas lamin A knockdown (siLMNA) causes frequent mislocalization (bar graph). (B) High-curvature tips (diameter < 0.1 µm) rupture WT nuclei, based on mislocalization of YFP-NLS or GFP-53BP1 into cytoplasm within minutes (10/15 ruptured for GFP-53BP1 and 4/6 ruptured for YFP-NLS). Intensity profiles show decreased nuclear signal and higher cytoplasmic signal. Inset: Cytoplasmic GFP accumulates even after probe tip is removed. (C) Various DNA repair proteins may be affected when the nuclear envelope ruptures. (D) DNA binding protein cGAS (dark gray) enters the nucleus under a high-curvature tip. (E) DNA repair factor GFP-KU80 mislocalizes to cytoplasm simultaneously with cGAS accumulation under the probe tip. Total GFP and mCherry in the cell (bottom numbers) remain constant during the entire export, indicating plasma membrane integrity. (F) A gap in nuclear envelope GFP-LBR coincides spatiotemporally with cGAS accumulation under a probe tip, indicating nuclear envelope rupture. Bars, 10 µm. Arrows in A, B, and D–F point to site of AFM tip indentation of nucleus. All data are presented as mean ± SEM.