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Live cell imaging of mitochondria following targeted irradiation in situ reveals rapid and highly localized loss of membrane potential.

ABSTRACT: The reliance of all cell types on the mitochondrial function for survival makes mitochondria an interesting target when trying to understand their role in the cellular response to ionizing radiation. By harnessing highly focused carbon ions and protons using microbeams, we have performed in situ live cell imaging of the targeted irradiation of individual mitochondria stained with Tetramethyl rhodamine ethyl ester (TMRE), a cationic fluorophore which accumulates electrophoretically in polarized mitochondria. Targeted irradiation with both carbon ions and protons down to beam spots of <1??m induced a near instant loss of mitochondrial TMRE fluorescence signal in the targeted area. The loss of TMRE after targeted irradiation represents a radiation induced change in mitochondrial membrane potential. This is the first time such mitochondrial responses have been documented in situ after targeted microbeam irradiation. The methods developed and the results obtained have the ability to shed new light on not just mitochondria's response to radiation but to further elucidate a putative mechanism of radiation induced depolarization and mitochondrial response.

SUBMITTER: Walsh DWM

PROVIDER: S-EPMC5404225 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Live cell imaging of mitochondria following targeted irradiation in situ reveals rapid and highly localized loss of membrane potential.

Walsh Dietrich W M DWM Siebenwirth Christian C Greubel Christoph C Ilicic Katarina K Reindl Judith J Girst Stefanie S Muggiolu Giovanna G Simon Marina M Barberet Philippe P Seznec Hervé H Zischka Hans H Multhoff Gabriele G Schmid Thomas E TE Dollinger Guenther G

Scientific reports 20170425

The reliance of all cell types on the mitochondrial function for survival makes mitochondria an interesting target when trying to understand their role in the cellular response to ionizing radiation. By harnessing highly focused carbon ions and protons using microbeams, we have performed in situ live cell imaging of the targeted irradiation of individual mitochondria stained with Tetramethyl rhodamine ethyl ester (TMRE), a cationic fluorophore which accumulates electrophoretically in polarized m ...[more]

PMID: 28440317

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Project description:Standard cytotoxic cancer treatments, such as radiation, can damage and deplete the supply of oocytes stored within the ovary, which predisposes females to infertility and premature menopause later in life. The mechanisms by which radiation induces oocyte damage have not been completely elucidated. The objective of this study was to determine if γ-irradiation changes mitochondrial characteristics in oocytes, possibly contributing to a reduction in oocyte number and quality. Immature oocytes were collected from postnatal day (PN) 9-11 C57Bl6 mice 3, 6 and 24 hours after 0.1 Gy γ-irradiation to monitor acute mitochondrial changes. Oocytes were classified as small (>20 µm) or growing (40-60 µm). Mitochondrial membrane potential was lost in 20% and 44% of small oocytes (~20 µm) at 3 and 6 hours after γ-irradiation, respectively, consistent with the induction of apoptosis. However, mitochondrial mass, distribution and membrane potential in the surviving small oocytes were similar to the non-irradiated controls at both time points. At 24 hours after γ-irradiation, all mitochondrial parameters analysed within immature oocytes were similar to untreated controls. Mitochondrial parameters within growing oocytes were also similar to untreated controls. When mice were superovulated more than 3 weeks after γ-irradiation, there was a significant reduction in the number of mature oocytes harvested compared to controls (Control 18 ± 1 vs 0.1 Gy 4 ± 1, n = 6/16 mice, p < 0.05). There was a slight reduction in mitochondrial mass in mature oocytes after γ-irradiation, though mitochondrial localization, mtDNA copy number and ATP levels were similar between groups. In summary, this study shows that γ-irradiation of pre-pubertal mice is associated with loss of mitochondrial membrane potential in a significant proportion of small immature oocytes and a reduction in the number of mature oocytes harvested from adult mice. Furthermore, these results suggest that immature oocytes that survive γ-irradiation and develop through to ovulation contain mitochondria with normal characteristics. Whether the oocytes that survive radiation and eventually undergo meiosis can support fertility remains to be determined.

Dataset Information

Live cell imaging of mitochondria following targeted irradiation in situ reveals rapid and highly localized loss of membrane potential.

Publications

Live cell imaging of mitochondria following targeted irradiation in situ reveals rapid and highly localized loss of membrane potential.

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