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Takahiro Kikawada - One of the best experts on this subject based on the ideXlab platform.
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combined metabolome and transcriptome analysis reveals key components of complete desiccation tolerance in an anhydrobiotic insect
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Alina Ryabova, Richard Cornette, Takahiro Kikawada, Takashi Okuda, Alexander Cherkasov, Elena I Shagimardanova, Masahiko Watanabe, Oleg GusevAbstract:Some organisms have evolved a survival strategy to withstand severe dehydration in an ametabolic state, called Anhydrobiosis. The only known example of Anhydrobiosis among insects is observed in larvae of the chironomid Polypedilum vanderplanki. Recent studies have led to a better understanding of the molecular mechanisms underlying Anhydrobiosis and the action of specific protective proteins. However, gene regulation alone cannot explain the rapid biochemical reactions and independent metabolic changes that are expected to sustain Anhydrobiosis. For this reason, we conducted a comprehensive comparative metabolome–transcriptome analysis in the larvae. We showed that anhydrobiotic larvae adopt a unique metabolic strategy to cope with complete desiccation and, in particular, to allow recovery after rehydration. We argue that trehalose, previously known for its anhydroprotective properties, plays additional vital roles, providing both the principal source of energy and also the restoration of antioxidant potential via the pentose phosphate pathway during the early stages of rehydration. Thus, larval viability might be directly dependent on the total amount of carbohydrate (glycogen and trehalose). Furthermore, in the anhydrobiotic state, energy is stored as accumulated citrate and adenosine monophosphate, allowing rapid reactivation of the citric acid cycle and mitochondrial activity immediately after rehydration, before glycolysis is fully functional. Other specific adaptations to desiccation include potential antioxidants (e.g., ophthalmic acid) and measures to avoid the accumulation of toxic waste metabolites by converting these to stable and inert counterparts (e.g., xanthurenic acid and allantoin). Finally, we confirmed that these metabolic adaptations correlate with unique organization and expression of the corresponding enzyme genes.
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New group of transmembrane proteins associated with desiccation tolerance in the anhydrobiotic midge Polypedilum vanderplanki
Scientific Reports, 2020Co-Authors: Taisiya A Voronina, Richard Cornette, Oleg Gusev, Takahiro Kikawada, Alexander Nesmelov, Sabina A. Kondratyeva, Ruslan Deviatiiarov, Yugo Miyata, Shoko Tokumoto, Elena I ShagimardanovaAbstract:Larvae of the sleeping chironomid Polypedilum vanderplanki are known for their extraordinary ability to survive complete desiccation in an ametabolic state called "Anhydrobiosis". The unique feature of P. vanderplanki genome is the presence of expanded gene clusters associated with Anhydrobiosis. While several such clusters represent orthologues of known genes, there is a distinct set of genes unique for P. vanderplanki. These include Lea-Island-Located (LIL) genes with no known orthologues except two of LEA genes of P. vanderplanki, PvLea1 and PvLea3. However, PvLIL proteins lack typical features of LEA such as the state of intrinsic disorder, hydrophilicity and characteristic LEA_4 motif. They possess four to five transmembrane domains each and we confirmed membrane targeting for three PvLILs. Conserved amino acids in PvLIL are located in transmembrane domains or nearby. PvLEA1 and PvLEA3 proteins are chimeras combining LEA-like parts and transmembrane domains, shared with PvLIL proteins. We have found that PvLil genes are highly upregulated during Anhydrobiosis induction both in larvae of P. vanderplanki and P. vanderplanki-derived cultured cell line, Pv11. Thus, PvLil are a new intriguing group of genes that are likely to be associated with Anhydrobiosis due to their common origin with some LEA genes and their induction during Anhydrobiosis.
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identification of a master transcription factor and a regulatory mechanism for desiccation tolerance in the anhydrobiotic cell line pv11
PLOS ONE, 2020Co-Authors: Takahiro Yamada, Richard Cornette, Oleg Gusev, Takahiro Kikawada, Elena I Shagimardanova, Yusuke Hiki, Noriko Hiroi, Akira FunahashiAbstract:Water is essential for living organisms. Terrestrial organisms are incessantly exposed to the stress of losing water, desiccation stress. Avoiding the mortality caused by desiccation stress, many organisms acquired molecular mechanisms to tolerate desiccation. Larvae of the African midge, Polypedilum vanderplanki, and its embryonic cell line Pv11 tolerate desiccation stress by entering an ametabolic state, Anhydrobiosis, and return to active life after rehydration. The genes related to desiccation tolerance have been comprehensively analyzed, but transcriptional regulatory mechanisms to induce these genes after desiccation or rehydration remain unclear. Here, we comprehensively analyzed the gene regulatory network in Pv11 cells and compared it with that of Drosophila melanogaster, a desiccation sensitive species. We demonstrated that nuclear transcription factor Y subunit gamma-like, which is important for drought stress tolerance in plants, and its transcriptional regulation of downstream positive feedback loops have a pivotal role in regulating various Anhydrobiosis-related genes. This study provides an initial insight into the systemic mechanism of desiccation tolerance.
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involvement of heat shock proteins in invertebrate Anhydrobiosis
2018Co-Authors: Alexander Nesmelov, Elena I Shagimardanova, Takahiro Kikawada, Oleg GusevAbstract:The Anhydrobiosis is a unique state notable for a complete lack of detectable metabolic activity and an ability to withstand extreme stresses. It is induced, as its name suggests, by severe water loss, extending up to complete desiccation. Despite of severe stress, organisms entering Anhydrobiosis normally retain their viability. This phenomena is ensured by a variety of protective mechanisms, including expression of protective proteins. The latter include Heat shock proteins (Hsp), which have long been recognized for their importance in a wide range of stress protection mechanisms. This chapter summarizes the theory and available experimental data, both suggesting the importance of Hsp in invertebrate Anhydrobiosis. However, most of experimental data are the results of expression studies. We show that they are insufficient to make robust conclusions on the role of Hsp in Anhydrobiosis. To date, only two robust evidences based on loss of-function-experiments are available, leaving for the future research the complete elucidation of Hsp function in Anhydrobiosis.
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Current findings on the molecular mechanisms underlying Anhydrobiosis in Polypedilum vanderplanki.
Current Opinion in Insect Science, 2017Co-Authors: Yoichiro Sogame, Takahiro KikawadaAbstract:Water is an essential molecule for living organisms. However, some organisms can survive in environments which receive no rainfall for months and in which ordinary life cannot survive. How do they endure the extended dry season? The sleeping chironomid Polypedilum vanderplanki, which inhabits sub-Saharan Africa, exhibits extreme tolerance to complete desiccation, a process termed Anhydrobiosis. During Anhydrobiosis these organisms dry up and entirely shut down their metabolism. However, when the dried larvae are immersed in water, their metabolism is resumed. Interestingly, Anhydrobiosis allows these organisms to tolerate not only desiccation but also high and low temperatures, the absence of oxygen, radiation, and chemical stresses. Here, we describe the mechanisms by which P. vanderplanki achieves Anhydrobiosis revealed in our recent research.
Takashi Okuda - One of the best experts on this subject based on the ideXlab platform.
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combined metabolome and transcriptome analysis reveals key components of complete desiccation tolerance in an anhydrobiotic insect
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Alina Ryabova, Richard Cornette, Takahiro Kikawada, Takashi Okuda, Alexander Cherkasov, Elena I Shagimardanova, Masahiko Watanabe, Oleg GusevAbstract:Some organisms have evolved a survival strategy to withstand severe dehydration in an ametabolic state, called Anhydrobiosis. The only known example of Anhydrobiosis among insects is observed in larvae of the chironomid Polypedilum vanderplanki. Recent studies have led to a better understanding of the molecular mechanisms underlying Anhydrobiosis and the action of specific protective proteins. However, gene regulation alone cannot explain the rapid biochemical reactions and independent metabolic changes that are expected to sustain Anhydrobiosis. For this reason, we conducted a comprehensive comparative metabolome–transcriptome analysis in the larvae. We showed that anhydrobiotic larvae adopt a unique metabolic strategy to cope with complete desiccation and, in particular, to allow recovery after rehydration. We argue that trehalose, previously known for its anhydroprotective properties, plays additional vital roles, providing both the principal source of energy and also the restoration of antioxidant potential via the pentose phosphate pathway during the early stages of rehydration. Thus, larval viability might be directly dependent on the total amount of carbohydrate (glycogen and trehalose). Furthermore, in the anhydrobiotic state, energy is stored as accumulated citrate and adenosine monophosphate, allowing rapid reactivation of the citric acid cycle and mitochondrial activity immediately after rehydration, before glycolysis is fully functional. Other specific adaptations to desiccation include potential antioxidants (e.g., ophthalmic acid) and measures to avoid the accumulation of toxic waste metabolites by converting these to stable and inert counterparts (e.g., xanthurenic acid and allantoin). Finally, we confirmed that these metabolic adaptations correlate with unique organization and expression of the corresponding enzyme genes.
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a novel dry preservation technology inspired by desiccation tolerant insect polypedilum vanderplanki
2012Co-Authors: Takashi OkudaAbstract:Some multi-cellular organisms can stand complete desiccation. This phenomenon called“Anhydrobiosis” iindicates that theoretically our cells could be stored in a dry form at room temperature. An African insect, P. vanderplanki is going to teach us how to preserve cells and tissues at room temperature.
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Expression of heat shock protein-coding genes associated with Anhydrobiosis in an African chironomid Polypedilum vanderplanki
Cell Stress and Chaperones, 2011Co-Authors: Oleg Gusev, Richard Cornette, Takahiro Kikawada, Takashi OkudaAbstract:In order to survive in extreme environments, organisms need to develop special adaptations both on physiological and molecular levels. The sleeping chironomid Polypedilum vanderplanki , inhabiting temporary water pools in semi-arid regions of Africa, is the only insect to have evolutionarily acquired the ability to withstand prolonged complete desiccation at larval stage, entering a state called Anhydrobiosis. Even after years in a dry state, larvae are able to revive within a short period of time, completely restoring metabolism. Because of the possible involvement of stress proteins in the preservation of biomolecules during the Anhydrobiosis of the sleeping chironomid, we have analyzed the expression of genes encoding six heat shock proteins ( Pv-hsp90 , Pv-hsp70 , Pv-hsc70 , Pv-hsp60 , Pv-hsp20 , and Pv-p23 ) and one heat shock factor ( Pv-hsf1 ) in dehydrating, rehydrating, and heat-shocked larvae. All examined genes were significantly up-regulated in the larvae upon dehydration and several patterns of expression were detected. Gene transcript of Pv-hsf1 was up-regulated within 8 h of desiccation, followed by large shock proteins expression reaching peak at 24–48 h of desiccation. Heat-shock-responsive Pv-hsp70 and Pv-hsp60 showed a two-peak expression: in dehydrating and rehydrating larvae. Both small alpha-crystallin heat shock proteins (sHSP) transcripts were accumulated in the desiccated larvae, but showed different expression profiles. Both sHSP-coding genes were found to be heat-inducible, and Pv-hsp20 was up-regulated in the larvae at the early stage of desiccation. In contrast, expression of the second transcript, corresponding to Pv-p23 , was limited to the late stages of desiccation, suggesting possible involvement of this protein in the glass-state formation in anhydrobiotic larvae. We discuss possible roles of proteins encoded by these stress genes during the different stages of Anhydrobiosis in P. vanderplanki .
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Anhydrobiosis associated nuclear dna damage and repair in the sleeping chironomid linkage with radioresistance
PLOS ONE, 2010Co-Authors: Oleg Gusev, Richard Cornette, Yuichi Nakahara, Takahiro Kikawada, Veronica Vanyagina, Ludmila Malutina, Tetsuya Sakashita, Nobuyuki Hamada, Yasuhiko Kobayashi, Takashi OkudaAbstract:Anhydrobiotic chironomid larvae can withstand prolonged complete desiccation as well as other external stresses including ionizing radiation. To understand the cross-tolerance mechanism, we have analyzed the structural changes in the nuclear DNA using transmission electron microscopy and DNA comet assays in relation to Anhydrobiosis and radiation. We found that dehydration causes alterations in chromatin structure and a severe fragmentation of nuclear DNA in the cells of the larvae despite successful Anhydrobiosis. Furthermore, while the larvae had restored physiological activity within an hour following rehydration, nuclear DNA restoration typically took 72 to 96 h. The DNA fragmentation level and the recovery of DNA integrity in the rehydrated larvae after Anhydrobiosis were similar to those of hydrated larvae irradiated with 70 Gy of high-linear energy transfer (LET) ions (4He). In contrast, low-LET radiation (gamma-rays) of the same dose caused less initial damage to the larvae, and DNA was completely repaired within within 24 h. The expression of genes encoding the DNA repair enzymes occurred upon entering Anhydrobiosis and exposure to high- and low-LET radiations, indicative of DNA damage that includes double-strand breaks and their subsequent repair. The expression of antioxidant enzymes-coding genes was also elevated in the anhydrobiotic and the gamma-ray-irradiated larvae that probably functions to reduce the negative effect of reactive oxygen species upon exposure to these stresses. Indeed the mature antioxidant proteins accumulated in the dry larvae and the total activity of antioxidants increased by a 3–4 fold in association with Anhydrobiosis. We conclude that one of the factors explaining the relationship between radioresistance and the ability to undergo Anhydrobiosis in the sleeping chironomid could be an adaptation to desiccation-inflicted nuclear DNA damage. There were also similarities in the molecular response of the larvae to damage caused by desiccation and ionizing radiation.
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Vitrification is essential for Anhydrobiosis in an African chironomid, Polypedilum vanderplanki
Proceedings of the National Academy of Sciences, 2008Co-Authors: Minoru Sakurai, Yuichi Nakahara, Takahiro Kikawada, Masahiko Watanabe, Takao Furuki, Ken-ichi Akao, Daisuke Tanaka, Takashi OkudaAbstract:Anhydrobiosis is an extremely dehydrated state in which organisms show no detectable metabolism but retain the ability to revive after rehydration. Thus far, two hypotheses have been proposed to explain how cells are protected during dehydration: (i) water replacement by compatible solutes and (ii) vitrification. The present study provides direct physiological and physicochemical evidence for these hypotheses in an African chironomid, Polypedilum vanderplanki, which is the largest multicellular animal capable of Anhydrobiosis. Differential scanning calorimetry measurements and Fourier-transform infrared (FTIR) analyses indicated that the anhydrobiotic larvae were in a glassy state up to as high as 65°C. Changing from the glassy to the rubbery state by either heating or allowing slight moisture uptake greatly decreased the survival rate of dehydrated larvae. In addition, FTIR spectra showed that sugars formed hydrogen bonds with phospholipids and that membranes remained in the liquid-crystalline state in the anhydrobiotic larvae. These results indicate that larvae of P. vanderplanki survive extreme dehydration by replacing the normal intracellular medium with a biological glass. When entering Anhydrobiosis, P. vanderplanki accumulated nonreducing disaccharide trehalose that was uniformly distributed throughout the dehydrated body by FTIR microscopic mapping image. Therefore, we assume that trehalose plays important roles in water replacement and intracellular glass formation, although other compounds are surely involved in these phenomena.
Oleg Gusev - One of the best experts on this subject based on the ideXlab platform.
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cas9 mediated genome editing reveals a significant contribution of calcium signaling pathways to Anhydrobiosis in pv11 cells
Scientific Reports, 2021Co-Authors: Yugo Miyata, Yuki Yoshida, Richard Cornette, Oleg Gusev, Ruslan Deviatiiarov, Shoko Tokumoto, Hiroto Fuse, Yusuke Hiki, Takahiro Yamada, Elena I ShagimardanovaAbstract:Pv11 is an insect cell line established from the midge Polypedilum vanderplanki, whose larval form exhibits an extreme desiccation tolerance known as Anhydrobiosis. Pv11 itself is also capable of Anhydrobiosis, which is induced by trehalose treatment. Here we report the successful construction of a genome editing system for Pv11 cells and its application to the identification of signaling pathways involved in Anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established Pv11 cells that stably expressed GCaMP3 to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase in cytosolic Ca2+ concentration, and further experiments revealed that the calmodulin–calcineurin–NFAT pathway contributes to tolerance of trehalose treatment as well as desiccation tolerance, while the calmodulin–calmodulin kinase–CREB pathway conferred only desiccation tolerance on Pv11 cells. Thus, our results show a critical contribution of the trehalose-induced Ca2+ surge to Anhydrobiosis and demonstrate temporally different roles for each signaling pathway.
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cas9 mediated genome editing reveals a significant contribution of calcium signaling pathways to Anhydrobiosis in pv11
bioRxiv, 2020Co-Authors: Yugo Miyata, Yuki Yoshida, Richard Cornette, Oleg Gusev, Ruslan Deviatiiarov, Shoko Tokumoto, Hiroto Fuse, Yusuke Hiki, Takahiro Yamada, Elena I ShagimardanovaAbstract:Abstract Pv11 is an insect cell line established from the midge Polypedilum vanderplanki that exhibits an extreme desiccation tolerance known as Anhydrobiosis. Pv11 has also an anhydrobiotic ability which is induced by trehalose treatment. Here we report the successful construction of the genome editing system for Pv11 cells and its application for identifying the signaling pathways in the Anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established GCaMP3-stably expressing Pv11 cells to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase of cytosolic Ca2+ concentration, and further experiments indicated the contribution of the calmodulin – calcineurin – NFAT pathway to the tolerance for trehalose treatment as well as the desiccation tolerance, while the calmodulin – calmodulin Kinase – CREB pathway conferred only the desiccation tolerance on Pv11 cells. Thus, our results show the critical contribution of the trehalose–induced Ca2+ surge to the Anhydrobiosis and the temporal different roles of each signaling pathway.
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combined metabolome and transcriptome analysis reveals key components of complete desiccation tolerance in an anhydrobiotic insect
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Alina Ryabova, Richard Cornette, Takahiro Kikawada, Takashi Okuda, Alexander Cherkasov, Elena I Shagimardanova, Masahiko Watanabe, Oleg GusevAbstract:Some organisms have evolved a survival strategy to withstand severe dehydration in an ametabolic state, called Anhydrobiosis. The only known example of Anhydrobiosis among insects is observed in larvae of the chironomid Polypedilum vanderplanki. Recent studies have led to a better understanding of the molecular mechanisms underlying Anhydrobiosis and the action of specific protective proteins. However, gene regulation alone cannot explain the rapid biochemical reactions and independent metabolic changes that are expected to sustain Anhydrobiosis. For this reason, we conducted a comprehensive comparative metabolome–transcriptome analysis in the larvae. We showed that anhydrobiotic larvae adopt a unique metabolic strategy to cope with complete desiccation and, in particular, to allow recovery after rehydration. We argue that trehalose, previously known for its anhydroprotective properties, plays additional vital roles, providing both the principal source of energy and also the restoration of antioxidant potential via the pentose phosphate pathway during the early stages of rehydration. Thus, larval viability might be directly dependent on the total amount of carbohydrate (glycogen and trehalose). Furthermore, in the anhydrobiotic state, energy is stored as accumulated citrate and adenosine monophosphate, allowing rapid reactivation of the citric acid cycle and mitochondrial activity immediately after rehydration, before glycolysis is fully functional. Other specific adaptations to desiccation include potential antioxidants (e.g., ophthalmic acid) and measures to avoid the accumulation of toxic waste metabolites by converting these to stable and inert counterparts (e.g., xanthurenic acid and allantoin). Finally, we confirmed that these metabolic adaptations correlate with unique organization and expression of the corresponding enzyme genes.
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New group of transmembrane proteins associated with desiccation tolerance in the anhydrobiotic midge Polypedilum vanderplanki
Scientific Reports, 2020Co-Authors: Taisiya A Voronina, Richard Cornette, Oleg Gusev, Takahiro Kikawada, Alexander Nesmelov, Sabina A. Kondratyeva, Ruslan Deviatiiarov, Yugo Miyata, Shoko Tokumoto, Elena I ShagimardanovaAbstract:Larvae of the sleeping chironomid Polypedilum vanderplanki are known for their extraordinary ability to survive complete desiccation in an ametabolic state called "Anhydrobiosis". The unique feature of P. vanderplanki genome is the presence of expanded gene clusters associated with Anhydrobiosis. While several such clusters represent orthologues of known genes, there is a distinct set of genes unique for P. vanderplanki. These include Lea-Island-Located (LIL) genes with no known orthologues except two of LEA genes of P. vanderplanki, PvLea1 and PvLea3. However, PvLIL proteins lack typical features of LEA such as the state of intrinsic disorder, hydrophilicity and characteristic LEA_4 motif. They possess four to five transmembrane domains each and we confirmed membrane targeting for three PvLILs. Conserved amino acids in PvLIL are located in transmembrane domains or nearby. PvLEA1 and PvLEA3 proteins are chimeras combining LEA-like parts and transmembrane domains, shared with PvLIL proteins. We have found that PvLil genes are highly upregulated during Anhydrobiosis induction both in larvae of P. vanderplanki and P. vanderplanki-derived cultured cell line, Pv11. Thus, PvLil are a new intriguing group of genes that are likely to be associated with Anhydrobiosis due to their common origin with some LEA genes and their induction during Anhydrobiosis.
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identification of a master transcription factor and a regulatory mechanism for desiccation tolerance in the anhydrobiotic cell line pv11
PLOS ONE, 2020Co-Authors: Takahiro Yamada, Richard Cornette, Oleg Gusev, Takahiro Kikawada, Elena I Shagimardanova, Yusuke Hiki, Noriko Hiroi, Akira FunahashiAbstract:Water is essential for living organisms. Terrestrial organisms are incessantly exposed to the stress of losing water, desiccation stress. Avoiding the mortality caused by desiccation stress, many organisms acquired molecular mechanisms to tolerate desiccation. Larvae of the African midge, Polypedilum vanderplanki, and its embryonic cell line Pv11 tolerate desiccation stress by entering an ametabolic state, Anhydrobiosis, and return to active life after rehydration. The genes related to desiccation tolerance have been comprehensively analyzed, but transcriptional regulatory mechanisms to induce these genes after desiccation or rehydration remain unclear. Here, we comprehensively analyzed the gene regulatory network in Pv11 cells and compared it with that of Drosophila melanogaster, a desiccation sensitive species. We demonstrated that nuclear transcription factor Y subunit gamma-like, which is important for drought stress tolerance in plants, and its transcriptional regulation of downstream positive feedback loops have a pivotal role in regulating various Anhydrobiosis-related genes. This study provides an initial insight into the systemic mechanism of desiccation tolerance.
Richard Cornette - One of the best experts on this subject based on the ideXlab platform.
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cas9 mediated genome editing reveals a significant contribution of calcium signaling pathways to Anhydrobiosis in pv11 cells
Scientific Reports, 2021Co-Authors: Yugo Miyata, Yuki Yoshida, Richard Cornette, Oleg Gusev, Ruslan Deviatiiarov, Shoko Tokumoto, Hiroto Fuse, Yusuke Hiki, Takahiro Yamada, Elena I ShagimardanovaAbstract:Pv11 is an insect cell line established from the midge Polypedilum vanderplanki, whose larval form exhibits an extreme desiccation tolerance known as Anhydrobiosis. Pv11 itself is also capable of Anhydrobiosis, which is induced by trehalose treatment. Here we report the successful construction of a genome editing system for Pv11 cells and its application to the identification of signaling pathways involved in Anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established Pv11 cells that stably expressed GCaMP3 to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase in cytosolic Ca2+ concentration, and further experiments revealed that the calmodulin–calcineurin–NFAT pathway contributes to tolerance of trehalose treatment as well as desiccation tolerance, while the calmodulin–calmodulin kinase–CREB pathway conferred only desiccation tolerance on Pv11 cells. Thus, our results show a critical contribution of the trehalose-induced Ca2+ surge to Anhydrobiosis and demonstrate temporally different roles for each signaling pathway.
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cas9 mediated genome editing reveals a significant contribution of calcium signaling pathways to Anhydrobiosis in pv11
bioRxiv, 2020Co-Authors: Yugo Miyata, Yuki Yoshida, Richard Cornette, Oleg Gusev, Ruslan Deviatiiarov, Shoko Tokumoto, Hiroto Fuse, Yusuke Hiki, Takahiro Yamada, Elena I ShagimardanovaAbstract:Abstract Pv11 is an insect cell line established from the midge Polypedilum vanderplanki that exhibits an extreme desiccation tolerance known as Anhydrobiosis. Pv11 has also an anhydrobiotic ability which is induced by trehalose treatment. Here we report the successful construction of the genome editing system for Pv11 cells and its application for identifying the signaling pathways in the Anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established GCaMP3-stably expressing Pv11 cells to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase of cytosolic Ca2+ concentration, and further experiments indicated the contribution of the calmodulin – calcineurin – NFAT pathway to the tolerance for trehalose treatment as well as the desiccation tolerance, while the calmodulin – calmodulin Kinase – CREB pathway conferred only the desiccation tolerance on Pv11 cells. Thus, our results show the critical contribution of the trehalose–induced Ca2+ surge to the Anhydrobiosis and the temporal different roles of each signaling pathway.
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combined metabolome and transcriptome analysis reveals key components of complete desiccation tolerance in an anhydrobiotic insect
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Alina Ryabova, Richard Cornette, Takahiro Kikawada, Takashi Okuda, Alexander Cherkasov, Elena I Shagimardanova, Masahiko Watanabe, Oleg GusevAbstract:Some organisms have evolved a survival strategy to withstand severe dehydration in an ametabolic state, called Anhydrobiosis. The only known example of Anhydrobiosis among insects is observed in larvae of the chironomid Polypedilum vanderplanki. Recent studies have led to a better understanding of the molecular mechanisms underlying Anhydrobiosis and the action of specific protective proteins. However, gene regulation alone cannot explain the rapid biochemical reactions and independent metabolic changes that are expected to sustain Anhydrobiosis. For this reason, we conducted a comprehensive comparative metabolome–transcriptome analysis in the larvae. We showed that anhydrobiotic larvae adopt a unique metabolic strategy to cope with complete desiccation and, in particular, to allow recovery after rehydration. We argue that trehalose, previously known for its anhydroprotective properties, plays additional vital roles, providing both the principal source of energy and also the restoration of antioxidant potential via the pentose phosphate pathway during the early stages of rehydration. Thus, larval viability might be directly dependent on the total amount of carbohydrate (glycogen and trehalose). Furthermore, in the anhydrobiotic state, energy is stored as accumulated citrate and adenosine monophosphate, allowing rapid reactivation of the citric acid cycle and mitochondrial activity immediately after rehydration, before glycolysis is fully functional. Other specific adaptations to desiccation include potential antioxidants (e.g., ophthalmic acid) and measures to avoid the accumulation of toxic waste metabolites by converting these to stable and inert counterparts (e.g., xanthurenic acid and allantoin). Finally, we confirmed that these metabolic adaptations correlate with unique organization and expression of the corresponding enzyme genes.
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New group of transmembrane proteins associated with desiccation tolerance in the anhydrobiotic midge Polypedilum vanderplanki
Scientific Reports, 2020Co-Authors: Taisiya A Voronina, Richard Cornette, Oleg Gusev, Takahiro Kikawada, Alexander Nesmelov, Sabina A. Kondratyeva, Ruslan Deviatiiarov, Yugo Miyata, Shoko Tokumoto, Elena I ShagimardanovaAbstract:Larvae of the sleeping chironomid Polypedilum vanderplanki are known for their extraordinary ability to survive complete desiccation in an ametabolic state called "Anhydrobiosis". The unique feature of P. vanderplanki genome is the presence of expanded gene clusters associated with Anhydrobiosis. While several such clusters represent orthologues of known genes, there is a distinct set of genes unique for P. vanderplanki. These include Lea-Island-Located (LIL) genes with no known orthologues except two of LEA genes of P. vanderplanki, PvLea1 and PvLea3. However, PvLIL proteins lack typical features of LEA such as the state of intrinsic disorder, hydrophilicity and characteristic LEA_4 motif. They possess four to five transmembrane domains each and we confirmed membrane targeting for three PvLILs. Conserved amino acids in PvLIL are located in transmembrane domains or nearby. PvLEA1 and PvLEA3 proteins are chimeras combining LEA-like parts and transmembrane domains, shared with PvLIL proteins. We have found that PvLil genes are highly upregulated during Anhydrobiosis induction both in larvae of P. vanderplanki and P. vanderplanki-derived cultured cell line, Pv11. Thus, PvLil are a new intriguing group of genes that are likely to be associated with Anhydrobiosis due to their common origin with some LEA genes and their induction during Anhydrobiosis.
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identification of a master transcription factor and a regulatory mechanism for desiccation tolerance in the anhydrobiotic cell line pv11
PLOS ONE, 2020Co-Authors: Takahiro Yamada, Richard Cornette, Oleg Gusev, Takahiro Kikawada, Elena I Shagimardanova, Yusuke Hiki, Noriko Hiroi, Akira FunahashiAbstract:Water is essential for living organisms. Terrestrial organisms are incessantly exposed to the stress of losing water, desiccation stress. Avoiding the mortality caused by desiccation stress, many organisms acquired molecular mechanisms to tolerate desiccation. Larvae of the African midge, Polypedilum vanderplanki, and its embryonic cell line Pv11 tolerate desiccation stress by entering an ametabolic state, Anhydrobiosis, and return to active life after rehydration. The genes related to desiccation tolerance have been comprehensively analyzed, but transcriptional regulatory mechanisms to induce these genes after desiccation or rehydration remain unclear. Here, we comprehensively analyzed the gene regulatory network in Pv11 cells and compared it with that of Drosophila melanogaster, a desiccation sensitive species. We demonstrated that nuclear transcription factor Y subunit gamma-like, which is important for drought stress tolerance in plants, and its transcriptional regulation of downstream positive feedback loops have a pivotal role in regulating various Anhydrobiosis-related genes. This study provides an initial insight into the systemic mechanism of desiccation tolerance.
Teymuras V. Kurzchalia - One of the best experts on this subject based on the ideXlab platform.
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c elegans possess a general program to enter cryptobiosis that allows dauer larvae to survive different kinds of abiotic stress
Scientific Reports, 2020Co-Authors: Vamshidhar R Gade, Sofia Traikov, Jana Oertel, Karim Fahmy, Teymuras V. KurzchaliaAbstract:All organisms encounter abiotic stress but only certain organisms are able to cope with extreme conditions and enter into cryptobiosis (hidden life). Previously, we have shown that C. elegans dauer larvae can survive severe desiccation (Anhydrobiosis), a specific form of cryptobiosis. Entry into Anhydrobiosis is preceded by activation of a set of biochemical pathways by exposure to mild desiccation. This process called preconditioning induces elevation of trehalose, intrinsically disordered proteins, polyamines and some other pathways that allow the preservation of cellular functionality in the absence of water. Here, we demonstrate that another stress factor, high osmolarity, activates similar biochemical pathways. The larvae that acquired resistance to high osmotic pressure can also withstand desiccation. In addition, high osmolarity significantly increases the biosynthesis of glycerol making larva tolerant to freezing. Thus, to survive abiotic stress, C. elegans activates a combination of genetic and biochemical pathways that serve as a general survival program.
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the glyoxylate shunt is essential for desiccation tolerance in c elegans and budding yeast
eLife, 2016Co-Authors: Cihan Erkut, Vamshidhar R Gade, Sunil Laxman, Teymuras V. KurzchaliaAbstract:Many organisms can survive losing all the water from their body in periods of severe drought by suspending their life. This ability is called Anhydrobiosis (from the Greek for ‘life without water’). When the desiccated organisms encounter water again, they resume life as normal. Two organisms commonly used in research, a roundworm called Caenorhabditis elegans and a yeast called Saccharomyces cerevisiae, are anhydrobiotes. To survive without water, anhydrobiotes alter the chemical reactions that sustain their life, and so change their metabolic state. The organisms also produce molecules that preserve the structure of their cells. One such essential molecule is a sugar called trehalose. However, both worms and yeast can only enter Anhydrobiosis during particular stages of life where they do not eat. So where does the trehalose come from? Erkut et al. have now addressed this question by studying the metabolism of C. elegans and S. cerevisiae as these species entered Anhydrobiosis. The experiments revealed that while preparing for desiccation, both species change their metabolism to favor creating sugars rather than releasing energy. In this process, the worms and yeast use a biochemical pathway called the glyoxylate shunt, which can convert fat or acetic acid into sugar. Genetic mutations that deactivate this pathway severely reduce the ability of both organisms to produce trehalose and tolerate desiccation. From these findings, Erkut et al. conclude that the source of trehalose in non-feeding worms is their fat deposits, while in yeast it is acetate: a molecule that is derived from ethanol, the end-product of the fermentation process. The glyoxylate shunt had been thought only to be a non-essential biochemical shortcut of another well-known metabolic pathway called the Krebs cycle. Now that Erkut et al. have shown that the glyoxylate shunt has its own specific biological role, further investigation is needed to understand how it is activated to act as a metabolic switch. The molecules that regulate similar metabolic transitions will also need to be identified in future studies. Ultimately, understanding these processes could present new ways of diagnosing and treating metabolic diseases such as diabetes and cancer.
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The C. elegans dauer larva as a paradigm to study metabolic suppression and desiccation tolerance
Planta, 2015Co-Authors: Cihan Erkut, Teymuras V. KurzchaliaAbstract:Main conclusion The hypometabolic, stress-resistant dauer larva of Caenorhabditis elegans serves as an excellent model to study the molecular mechanisms of desiccation tolerance, such as maintenance of membrane organization, protein folding, xenobiotic and ROS detoxification in the dry state. Many organisms from diverse taxa of life have the remarkable ability to survive extreme desiccation in the nature by entering an ametabolic state known as Anhydrobiosis (life without water). The hallmark of the anhydrobiotic state is the achievement and maintenance of an exceedingly low metabolic rate, as well as preservation of the structural integrity of the cell. Although described more than three centuries ago, the biochemical and biophysical mechanisms underlying this phenomenon are still not fully comprehended. This is mainly due to the fact that Anhydrobiosis in animals was studied using non-model organisms, which are very difficult, if not impossible, to manipulate at the molecular level. Recently, we introduced the roundworm (nematode) Caenorhabditis elegans as a model for Anhydrobiosis. Taking advantage of powerful genetic, biochemical and biophysical tools, we investigated several aspects of Anhydrobiosis in a particular developmental stage (the dauer larva) of this organism. First, our studies allowed confirming the previously suggested role of the disaccharide trehalose in the preservation of lipid membranes. Moreover, in addition to known pathways such as reactive oxygen species defense, heat-shock and intrinsically disordered protein expression, evidence for some novel strategies of Anhydrobiosis has been obtained. These are increased glyoxalase activity, polyamine and polyunsaturated fatty acid biosynthesis. All these pathways may constitute a generic toolbox of Anhydrobiosis, which is possibly conserved between animals and plants.
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molecular strategies of the caenorhabditis elegans dauer larva to survive extreme desiccation
PLOS ONE, 2013Co-Authors: Cihan Erkut, Andrej Vasilj, Sebastian Boland, Bianca Habermann, Andrej Shevchenko, Teymuras V. KurzchaliaAbstract:Massive water loss is a serious challenge for terrestrial animals, which usually has fatal consequences. However, some organisms have developed means to survive this stress by entering an ametabolic state called Anhydrobiosis. The molecular and cellular mechanisms underlying this phenomenon are poorly understood. We recently showed that Caenorhabditis elegans dauer larva, an arrested stage specialized for survival in adverse conditions, is resistant to severe desiccation. However, this requires a preconditioning step at a mild desiccative environment to prepare the organism for harsher desiccation conditions. A systems approach was used to identify factors that are activated during this preconditioning. Using microarray analysis, proteomics, and bioinformatics, genes, proteins, and biochemical pathways that are upregulated during this process were identified. These pathways were validated via reverse genetics by testing the desiccation tolerances of mutants. These data show that the desiccation response is activated by hygrosensation (sensing the desiccative environment) via head neurons. This leads to elimination of reactive oxygen species and xenobiotics, expression of heat shock and intrinsically disordered proteins, polyamine utilization, and induction of fatty acid desaturation pathway. Remarkably, this response is specific and involves a small number of functional pathways, which represent the generic toolkit for Anhydrobiosis in plants and animals.
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trehalose renders the dauer larva of caenorhabditis elegans resistant to extreme desiccation
Current Biology, 2011Co-Authors: Cihan Erkut, Teymuras V. Kurzchalia, Karim Fahmy, Sider Penkov, Hassan Khesbak, Daniela Vorkel, Jean Marc VerbavatzAbstract:Summary Water is essential for life on Earth. In its absence, however, some organisms can interrupt their life cycle and temporarily enter an ametabolic state, known as Anhydrobiosis [1]. It is assumed that sugars (in particular trehalose) are instrumental for survival under anhydrobiotic conditions [2]. However, the role of trehalose remained obscure because the corresponding evidence was purely correlative and based mostly on in vitro studies without any genetic manipulations of trehalose metabolism. In this study, we used C. elegans as a genetic model to investigate molecular mechanisms of Anhydrobiosis. We show that the C. elegans dauer larva is a true anhydrobiote: under defined conditions it can survive even after losing 98% of its body water. This ability is correlated with a several fold increase in the amount of trehalose. Mutants unable to synthesize trehalose cannot survive even mild dehydration. Light and electron microscopy indicate that one of the major functions of trehalose is the preservation of membrane organization. Fourier-transform infrared spectroscopy of whole worms suggests that this is achieved by preserving homogeneous and compact packing of lipid acyl chains. By means of infrared spectroscopy, we can now distinguish a "dry, yet alive" larva from a "dry and dead" one.