Fig. 5

Lysosomal degradation of TOP1cc occurs in interphase cells through the nuclear envelope (A) LysoIP performed after 6 h of 50 nM CPT in unsynchronized or S-phase HeLa cells (n = 3). SE, short exposure; LE, long exposure. (B) Live-cell imaging started 3 min after treatment with 50 nM CPT. Normalized fluorescence intensity measured along the dashed line for both mCherry and GFP. Scale bar, 5 μm; zoom panel, 1 μm. (C) Live-cell imaging. Arrows pointing at the alteration of Lamin A/C-GFP integrity at lysosomal contact sites (n = 3). Rendering by TrackMate of lysosomes and nuclear envelope integrity. Scale bar, 10 μm. (D) Volume electron microscopy images with nuclei segmentation and three-dimensional (3D) rendering after 30 min of 50 nM CPT. INM, inner nuclear membrane; ONM, outer nuclear membrane; Nu, nucleoplasm; Cy, cytoplasm; A, autophagosome. Scale bar, 500 nm. Blister-positive cells present at least 2 enlarged spaces between the INM and the ONM of more than 150 nm, close to a membrane invagination region. (E) Cryo-focused ion beam (FIB) milling and cryo-fluorescence correlation using LysoTracker after 30 min of 50 nM CPT. (Top) Computational slice through a tomogram; (middle) segmentation of the tomogram; (bottom) overlay. Scale bar, 200 nm. See also Figure S5 and Videos S1, S2, S3, and S4.