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. 2016 Sep 20;7:12808.
doi: 10.1038/ncomms12808.

Extremotolerant Tardigrade Genome and Improved Radiotolerance of Human Cultured Cells by Tardigrade-Unique Protein

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Free PMC article

Extremotolerant Tardigrade Genome and Improved Radiotolerance of Human Cultured Cells by Tardigrade-Unique Protein

Takuma Hashimoto et al. Nat Commun. .
Free PMC article

Abstract

Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus, one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ∼40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms.

Conflict of interest statement

T. Kunieda and T.H. declare competing financial interests, as a part of the work described in this publication has been applied for a patent (Japanese patent application number 2015-032209). All other authors declare no competing financial interests.

Figures

Figure 1
Figure 1. The extremotolerant tardigrade R. varieornatus and taxonomic origins of its gene repertoire.
(a,b) Scanning electron microscopy images of the extremotolerant tardigrade, R. varieornatus, in the hydrated condition (a) and in the dehydrated state (b), which is resistant to various physical extremes. Scale bars, 100 μm. (c) Classification of the gene repertoire of R. varieornatus, according to their putative taxonomic origins and distribution of best-matched taxa in putative HGT genes.
Figure 2
Figure 2. Selective loss of stress responsive signalling to mTORC1 downregulation.
Gene networks involved in the regulation of mTORC1 activity. Magenta indicates genes absent in the tardigrade genome and green indicates retained genes. The interconnected eight genes mediating environmental stress stimuli to downregulate mTORC1 were selectively lost, whereas all components involved in sensing and mediating physiologic demands were present.
Figure 3
Figure 3. Co-localization with nuclear DNA and mobility shift of DNA by Dsup.
(a) Subcellular localization of Dsup-GFP fusion proteins transiently expressed in HEK293T cells. Nuclear DNA was visualized by Hoechst 33342. Scale bars, 10 μm. (b) Mobility shift of DNA by bacterially expressed Dsup protein in a dose-dependent manner (10, 50, 75 or 100 ng). Black arrowhead indicates the predicted size of the probe DNA (3 kbp, 10 ng). Red arrowhead indicates the position of the extremely slowly migrating DNA in the presence of Dsup protein. A similar extensive mobility shift was observed with histone H1.
Figure 4
Figure 4. Dsup protein suppresses stress-induced DNA fragmentation in human cultured cells.
(a) The effects of Dsup on SSBs by 10 Gy X-ray irradiation in alkaline comet assays. The irradiated cells were immediately subjected to the assay. Representative images are shown for each condition. In the pseudo-coloured images in the inset, red to blue circles indicate nuclear DNA and magenta indicates fragmented DNA in tail. DNA fragmentation was assessed by the proportion of DNA detected in the tail region (% of DNA in Comet Tail). At least 281 comets were analysed for each condition. **P<0.01 and ***P<0.001 (Welch’s t-test: non-irradiated, t-value=−3.199, P-value=0.0015; irradiated: t-value=8.599, P-value<1.0E−15). (b) The effects of Dsup on SSBs caused by hydrogen peroxide (H2O2) treatment in alkaline comet assays. Cells were treated with 100 μM H2O2 for 30 min at 4 °C, to induce DNA damage with or without pretreatment with 10 mM NAC as an antioxidant for 30 min. At least 203 comets were analysed for each condition. ***P<0.001 (Tukey–Kramer’s test). (c) The effects of Dsup on DSBs by 5 Gy X-ray irradiation in neutral comet assays. Three hundred comets were analysed for each condition. **P<0.01 and ***P<0.001 (Welch’s t-test: non-irradiated, t-value=2.758, P-value=0.0060; irradiated; t-value=7.406, P-value=4.7E−13). Values represent mean±s.d. in all panels. Scale bars, 100 μm.
Figure 5
Figure 5. Reduced formation of γ-H2AX foci in human cultured cells depending on Dsup expression.
(a) Distribution of the numbers of γ-H2AX foci per nucleus is shown. Each dot represents an individual nucleus of a HEK293 cell (Control) or a Dsup-expressing cell (Dsup) under non-irradiated and irradiated conditions. ***P<0.001; NS, not significant (Welch’s t-test). (b) Significant decrease of Dsup transcript in shRNA-introduced cells (Dsup+shDsup) compared with that in untreated Dsup-expressing cells (Dsup shRNA(−)). n=3. Values represent mean±s.e.m. ***P<0.001 (Student’s t-test). (c) Quantitative comparison of γ-H2AX foci number among untransfected HEK293 cells (Control), Dsup-expressing cells (Dsup) and Dsup-knockdown cells (Dsup+shDsup) under non-irradiated and 1 Gy X-ray irradiated conditions. At least 70 cells were analysed for each condition. Values represent mean±s.d. **P<0.01; NS indicates not significant (Tukey–Kramer’s test). (d) Representative images detecting γ-H2AX foci in each condition. Fluorescent images were converted to binary images for automatic counting of foci. Scale bar, 10 μm.
Figure 6
Figure 6. Improved viability and proliferative ability of Dsup-expressing cells after irradiation.
(a) Representative microscopic images with phase contrast at 8, 10 and 12 dps, of untransfected HEK293 cells (Control), Dsup-expressing cells (Dsup) and Dsup-knockdown cells (Dsup+shDsup) irradiated with 4 Gy X-ray at 1 dps. Scale bar, 200 μm. (b) Comparison of growth curves of untransfected cells (Control), Dsup-expressing cells (Dsup) and Dsup-knockdown cells (Dsup+shDsup) in non-irradiated and irradiated conditions. Values represent mean±s.d.

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