Nuclear Fragility in Radiation-Induced Senescence: Blebs and Tubes Visualized by 3D Electron Microscopy.

Benjamin M Freyter,Anne Dietz,Maria Gomolka,Anna Isermann,Omid Azimzadeh,Claudia E Rübe,Mutaz A Abd Al-Razaq,Liesbeth Hekking

doi:10.3390/cells11020273

Abstract

Irreparable DNA damage following ionizing radiation (IR) triggers prolonged DNA damage response and induces premature senescence. Cellular senescence is a permanent state of cell-cycle arrest characterized by chromatin restructuring, altered nuclear morphology and acquisition of secretory phenotype, which contributes to senescence-related inflammation. However, the mechanistic connections for radiation-induced DNA damage that trigger these senescence-associated hallmarks are poorly understood. In our in vitro model of radiation-induced senescence, mass spectrometry-based proteomics was combined with high-resolution imaging techniques to investigate the interrelations between altered chromatin compaction, nuclear envelope destabilization and nucleo-cytoplasmic chromatin blebbing. Our findings confirm the general pathophysiology of the senescence-response, with disruption of nuclear lamin organization leading to extensive chromatin restructuring and destabilization of the nuclear membrane with release of chromatin fragments into the cytosol, thereby activating cGAS-STING-dependent interferon signaling. By serial block-face scanning electron microscopy (SBF-SEM) whole-cell datasets were acquired to investigate the morphological organization of senescent fibroblasts. High-resolution 3-dimensional (3D) reconstruction of the complex nuclear shape allows us to precisely visualize the segregation of nuclear blebs from the main nucleus and their fusion with lysosomes. By multi-view 3D electron microscopy, we identified nanotubular channels formed in lamin-perturbed nuclei of senescent fibroblasts; the potential role of these nucleo-cytoplasmic nanotubes for expulsion of damaged chromatin has to be examined.

Highlights

Cellular senescence is a complex stress response leading to permanent cell cycle arrest with resistance to mitogenic or apoptotic signals [1]
Senescent cells are characterized by extensive chromatin restructuring with formation of senescence-associated heterochromatic foci (SAHF) and altered nuclear morphology
As cellular senescence is characterized as the basis for aging in organisms, culturing of normal human fibroblasts to mimic the in vivo aging processes has been developed as major methods to investigate cellular and molecular events involved in aging [6]

Summary

Introduction

Cellular senescence is a complex stress response leading to permanent cell cycle arrest with resistance to mitogenic or apoptotic signals [1]. Normal cells undergo premature senescence in response to severe or irreparable DNA damage, induced, e.g., by ionizing radiation (IR) [3]. Radiation-induced DNA damage, such as double-strand breaks (DSBs), triggers DNA damage response (DDR) mechanisms to coordinate DSB repair activities within the chromatin context [7,8]. During this DDR, multiple repair proteins are recruited to DSB sites, forming radiation-induced foci (e.g., γH2AX- and 53BP1-foci) [9,10]. Focal accumulation of DSB repair factors around persisting DNA lesions is a characteristic feature of cellular senescence [11]

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Conclusion