Nuclear Fragility in Radiation-Induced Senescence: Blebs and Tubes Visualized by 3D Electron Microscopy - PubMed (original) (raw)

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

Benjamin M Freyter et al. Cells. 2022.

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.

Keywords: cGAS-STING signaling; cellular senescence; chromatin reorganization; cytosolic chromatin fragments (CCF); ionizing radiation; nuclear blebbing; radiation-induced senescence; serial block-face scanning electron microscopy (SBF-SEM); transmission electron microscopy (TEM).

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1

Figure 1

Cellular senescence following IR. (A) Increased numbers of SA-β-Gal-positive and p21-positive cells following IR. (B) Decrease in Ki-67-positive and BrdU-positive cells. (C) Lamin B1 loss in nuclear envelope following IR exposure visualized by IFM (left) and TEM (right). Quantification of lamin B1 in WI-38 fibroblasts by IFM (top middle), and MS (bottom middle). Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Figure 2

Radiation-induced changes in protein expression. (A) Volcano plot showing differential protein expression following IR. (B) Senescence-related pathway enrichment results were generated using ReactomePA R package. (C) Visualization of enriched pathways components by enrichplot R package.

Figure 3

Figure 3

IR-induced morphological changes in WI-38 fibroblasts. (A) Visualization of SAHF formation by IFM. (B) Visualization of CCF formation by IFM. (C) Visualization of dense chromatin fragments (marked by red arrows) by TEM. (D) Visualization of nuclear groove in senescent fibroblast by TEM.

Figure 4

Figure 4

SBF-SEM: 3D reconstruction of senescent fibroblast. (A) Original SBF-SEM sections were used for 3D reconstruction. (B) Serial SBF-SEM sections were segmented for structures of interest: nucleus (light-blue), nucleoli (red), lysosomes (blue), and CCF (light-red), nanotubes (yellow), cytosol (gray) and used for 3D visualization (bottom row: transparent view).

Figure 5

Figure 5

SBF-SEM: Separation process of CCF. (A) Original micrographs presenting detaching CCF. (B) Segmented micrographs of the same regions. (C) 3D reconstruction of detaching CCF: nucleus (light-blue), nucleoli (red), lysosomes (blue), and CCF (light-red), nanotubes (yellow).

Figure 6

Figure 6

Nucleo-cytoplasmic nanotube. (A) Original micrographs showing cross-sections of the nanotube. (B) Segmented micrographs for the same area: nucleus (light-blue), nucleoli (red), nanotubes (yellow). (C) Models showing nanotube’s appearance from within the nucleus (1st and 2nd image from top), segmented volume showing the nanotube from side (3rd and 4th image from top), and original micrograph of the same region. (D) Visualization of the nanotube’s interior.

Similar articles

Cited by

References

    1. Salama R., Sadaie M., Hoare M., Narita M. Cellular senescence and its effector programs. Genes Dev. 2014;28:99–114. doi: 10.1101/gad.235184.113. - DOI - PMC - PubMed
    1. Kumari R., Jat P. Mechanisms of Cellular Senescence: Cell Cycle Arrest and Senescence Associated Secretory Phenotype. Front. Cell Dev. Biol. 2021;9:645593. doi: 10.3389/fcell.2021.645593. - DOI - PMC - PubMed
    1. Isermann A., Mann C., Rube C.E. Histone Variant H2A.J. Marks Persistent DNA Damage and Triggers the Secretory Phenotype in Radiation-Induced Senescence. Int. J. Mol. Sci. 2020;21:9130. doi: 10.3390/ijms21239130. - DOI - PMC - PubMed
    1. Campisi J., Robert L. Cell senescence: Role in aging and age-related diseases. Interdiscip. Top. Gerontol. 2014;39:45–61. doi: 10.1159/000358899. - DOI - PMC - PubMed
    1. Rube C.E., Baumert C., Schuler N., Isermann A., Schmal Z., Glanemann M., Mann C., Scherthan H. Human skin aging is associated with increased expression of the histone variant H2A.J. in the epidermis. NPJ Aging Mech. Dis. 2021;7:7. doi: 10.1038/s41514-021-00060-z. - DOI - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources