Cell-cycle-resolved τSTED nanoscopy reveals nanoscale reorganization of TGN46-positive structures.

The Golgi apparatus is a highly organized membrane system whose structure is remodeled during cell-cycle progression, but nanoscale changes in individual Golgi subdomains during interphase remain incompletely understood. Here, we used Fucci-compatible fluorescence lifetime-based STED (τSTED) nanoscopy to examine cell-cycle-associated reorganization of cis-Golgi and trans-Golgi network (TGN)-associated structures in fixed HeLa cells. By combining Fucci-SA reporters with red/far-red Golgi labeling, large-field sequential imaging, Abberior STAR RED nanobody labeling, and reproducible field-wise lifetime-trajectory-based τSTED reconstruction, we correlated Fucci-defined cell-cycle state with nanoscale Golgi morphology in individual cells. Automated morphometry showed that GM130-positive cis-Golgi and TGN46-positive trans/TGN-associated distribution domains were relatively compact in G1 cells, whereas TGN46-positive structures exhibited an expanded spatial distribution in G2-enriched cells. Optimized τSTED imaging resolved this expansion as dispersed submicron TGN46-positive structures rather than uniform enlargement of the Golgi apparatus. These findings reveal cell-cycle-associated spatial reorganization of TGN46-positive trans/TGN-associated structures and demonstrate the utility of cell-cycle-resolved super-resolution imaging for analyzing organelle architecture.Key words: Golgi apparatus, cell cycling, super-resolution microscopy, τSTED, Fucci.