Nucleolar and Chromatin Remodeling during γ-Irradiation-Induced Mitotic Catastrophe: Live-Cell Imaging Correlated with UBTF and Fibrillarin 3D Redistribution

Study explores the detailed chronology of nuclear and nucleolar pre-mortal 2D dynamics and 3D structural remodeling during 30 Gy γ-irradiation-induced mitotic catastrophe and apoptosis. Using post-irradiation time-lapse imaging over 72 hours, we analyzed nuclear deformation, nucleolar components and nucleolus-associated condensed chromatin remodeling in histone H2B-GFP tagged He-La cells. To assess the 3D redistribution of major nucleolus-specific proteins and related resident sub-compartments we visualized the most stress-sensing nucleolar components using anti-UBTF and anti-fibrillarin immunolabeling. Post-irradiation time-lapse imaging revealed a three stages of multinucleation: (i) progressive nuclear invagination leading to a lobulated shape, (ii) asymmetric nuclear fragmentation into unequal-sized micronucleoli, via amitosis and (iii) endomitotic division of nuclear fragments, followed by apoptotic nuclear degradation. γ-Irradiation-induced nucleolar changes resembled nucleolus associated chromatin rearrangements observed with chemical inhibitors. Unlike classical pattern of rRNA synthesis inhibition, nucleolar segregation or capping were not observed. Even after 12 and 24 hours, nucleoli remained large, irregularly shaped, revealing decrease of UBTF-positive nucleolar components number accompanied with sufficient enlargement of their sizes. These changes suggest a pre-segregated state rather than complete nucleolar segregation. During 48-72 hours we observed emergence of giant UBTF-positive structures, however typical pattern that finalizes nucleolar segregation by formation of crescent like UBTF- and fibrillarin-positive caps, have never been registered. Our findings provide new look into the impact of γ-irradiation on nuclear and nucleolar architecture, with implications for understanding chromatin and major nucleolar components dynamics in radiation-induced damage response leading to MC and apoptosis.