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

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.

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Three-dimensional microtissues as an in vitro model for personalized radiation therapy

The Analyst ◽

10.1039/c7an00794a ◽

2017 ◽

Vol 142 (19) ◽

pp. 3605-3612 ◽

Cited By ~ 1

Author(s):

Yuting Qiu ◽

Dandan Ning ◽

Peipei Zhang ◽

Stephanie Curly ◽

Yong Qiao ◽

...

Keyword(s):

Cell Culture ◽

Dna Damage ◽

Animal Model ◽

Radiation Therapy ◽

In Vitro Model ◽

Three Dimensional ◽

Intermediate Model ◽

Radiation Induced ◽

Vitro Model

This paper describes the use of 3D microtissues as an intermediate model between the 2D cell culture and the animal model to assess radiation-induced cellular and DNA damage in the context of personalized radiation therapy.

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Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

Veterinary and Comparative Orthopaedics and Traumatology ◽

10.1055/s-0038-1632561 ◽

1997 ◽

Vol 10 (01) ◽

pp. 6-11 ◽

Cited By ~ 3

Author(s):

R. F. Rosenbusch ◽

L. C. Booth ◽

L. A. Dahlgren

Keyword(s):

Electron Microscopy ◽

Extracellular Matrix ◽

Light Microscopy ◽

Light And Electron Microscopy ◽

Tendon Healing ◽

Matrix Proteins ◽

Isolated Cells ◽

Superficial Digital Flexor Tendon ◽

Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

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Faculty Opinions recommendation of Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.7630956.7936054 ◽

2011 ◽

Author(s):

Linda Amos

Keyword(s):

Electron Microscopy ◽

High Sensitivity ◽

3D Electron Microscopy ◽

3D Electron

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Establishment of an in vitro model for analyzing mitochondrial ultrastructure in PRKN-mutated patient iPSC-derived dopaminergic neurons

Molecular Brain ◽

10.1186/s13041-021-00771-0 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Mutsumi Yokota ◽

Soichiro Kakuta ◽

Takahiro Shiga ◽

Kei-ichi Ishikawa ◽

Hideyuki Okano ◽

...

Keyword(s):

Electron Microscopy ◽

Dopaminergic Neurons ◽

Structural Changes ◽

In Vitro Model ◽

Induced Pluripotent Stem Cell ◽

Substantia Nigra Pars Compacta ◽

Ultrastructural Changes ◽

Mitochondrial Morphology ◽

Vitro Model

AbstractMitochondrial structural changes are associated with the regulation of mitochondrial function, apoptosis, and neurodegenerative diseases. PRKN is known to be involved with various mechanisms of mitochondrial quality control including mitochondrial structural changes. Parkinson’s disease (PD) with PRKN mutations is characterized by the preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, which has been suggested to result from the accumulation of damaged mitochondria. However, ultrastructural changes of mitochondria specifically in dopaminergic neurons derived from iPSC have rarely been analyzed. The main reason for this would be that the dopaminergic neurons cannot be distinguished directly among a mixture of iPSC-derived differentiated cells under electron microscopy. To selectively label dopaminergic neurons and analyze mitochondrial morphology at the ultrastructural level, we generated control and PRKN-mutated patient tyrosine hydroxylase reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines. Correlative light-electron microscopy analysis and live cell imaging of GFP-expressing dopaminergic neurons indicated that iPSC-derived dopaminergic neurons had smaller and less functional mitochondria than those in non-dopaminergic neurons. Furthermore, the formation of spheroid-shaped mitochondria, which was induced in control dopaminergic neurons by a mitochondrial uncoupler, was inhibited in the PRKN-mutated dopaminergic neurons. These results indicate that our established TH-GFP iPSC lines are useful for characterizing mitochondrial morphology, such as spheroid-shaped mitochondria, in dopaminergic neurons among a mixture of various cell types. Our in vitro model would provide insights into the vulnerability of dopaminergic neurons and the processes leading to the preferential loss of dopaminergic neurons in patients with PRKN mutations.

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Crocetin Mitigates Irradiation Injury in an In Vitro Model of the Pubertal Testis: Focus on Biological Effects and Molecular Mechanisms

Molecules ◽

10.3390/molecules26061676 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1676

Author(s):

Giulia Rossi ◽

Martina Placidi ◽

Chiara Castellini ◽

Francesco Rea ◽

Settimio D'Andrea ◽

...

Keyword(s):

In Vitro Model ◽

Molecular Mechanisms ◽

Biological Effects ◽

Cellular Damage ◽

X Rays ◽

Adolescent Cancer ◽

Radiation Induced ◽

Vitro Model ◽

Underlying Mechanisms

Infertility is a potential side effect of radiotherapy and significantly affects the quality of life for adolescent cancer survivors. Very few studies have addressed in pubertal models the mechanistic events that could be targeted to provide protection from gonadotoxicity and data on potential radioprotective treatments in this peculiar period of life are elusive. In this study, we utilized an in vitro model of the mouse pubertal testis to investigate the efficacy of crocetin to counteract ionizing radiation (IR)-induced injury and potential underlying mechanisms. Present experiments provide evidence that exposure of testis fragments from pubertal mice to 2 Gy X-rays induced extensive structural and cellular damage associated with overexpression of PARP1, PCNA, SOD2 and HuR and decreased levels of SIRT1 and catalase. A twenty-four hr exposure to 50 μM crocetin pre- and post-IR significantly reduced testis injury and modulated the response to DNA damage and oxidative stress. Nevertheless, crocetin treatment did not counteract the radiation-induced changes in the expression of SIRT1, p62 and LC3II. These results increase the knowledge of mechanisms underlying radiation damage in pubertal testis and establish the use of crocetin as a fertoprotective agent against IR deleterious effects in pubertal period.

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