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1.
Cell Stress Chaperones ; 29(3): 425-436, 2024 Apr 10.
Article in English | MEDLINE | ID: mdl-38608858

ABSTRACT

Anhydrobiotic species can survive virtually complete water loss by entering a reversible ametabolic glassy state that may persist for years in ambient conditions. The Pv11 cell line was derived from the egg mass of the anhydrobiotic midge, Polypedilum vanderplanki, and is currently the only available anhydrobiotic cell line. Our results demonstrate that the necessary preconditioning for Pv11 cells to enter anhydrobiosis causes autophagy and reduces mitochondrial respiration by over 70%. We speculate that reorganizing cellular bioenergetics to create and conserve energy stores may be valuable to successfully recover after rehydration. Furthermore, mitochondria in preconditioned cells lose their membrane potential during desiccation but rapidly restore it within 30 min upon rehydration, demonstrating that the inner mitochondrial membrane integrity is well-preserved. Strikingly, the nucleolus remains visible immediately upon rehydration in preconditioned cells while absent in control cells. In contrast, a preconditioning-induced membraneless organelle reformed after rehydration, demonstrating that membraneless organelles in Pv11 cells can be either stabilized or recovered. Staining the endoplasmic reticulum and the Golgi apparatus revealed that these organelles fragment during preconditioning. We hypothesize that this process reduces sheering stress caused by rapid changes in cellular volume during desiccation and rehydration. Additionally, preconditioning was found to cause the filamentous-actin (F-actin) network to disassemble significantly and reduce the fusion of adjacent plasma membranes. This study offers several exciting avenues for future studies in the animal model and Pv11 cell line that will further our understanding of anhydrobiosis and may lead to advancements in storing sensitive biologics at ambient temperatures for months or years.

2.
Proc Natl Acad Sci U S A ; 121(14): e2317254121, 2024 Apr 02.
Article in English | MEDLINE | ID: mdl-38551840

ABSTRACT

Pv11 is the only animal cell line that, when preconditioned with a high concentration of trehalose, can be preserved in the dry state at room temperature for more than one year while retaining the ability to resume proliferation. This extreme desiccation tolerance is referred to as anhydrobiosis. Here, we identified a transporter that contributes to the recovery of Pv11 cells from anhydrobiosis. In general, the solute carrier 5 (SLC5)-type secondary active transporters cotransport Na+ and carbohydrates including glucose. The heterologous expression systems showed that the transporter belonging to the SLC5 family, whose expression increases upon rehydration, exhibits Na+-dependent trehalose transport activity. Therefore, we named it STRT1 (sodium-ion trehalose transporter 1). We report an SLC5 family member that transports a naturally occurring disaccharide, such as trehalose. Knockout of the Strt1 gene significantly reduced the viability of Pv11 cells upon rehydration after desiccation. During rehydration, when intracellular trehalose is no longer needed, Strt1-knockout cells released the disaccharide more slowly than the parental cell line. During rehydration, Pv11 cells became roughly spherical due to osmotic pressure changes, but then returned to their original spindle shape after about 30 min. Strt1-knockout cells, however, required about 50 min to adopt their normal morphology. STRT1 probably regulates intracellular osmolality by releasing unwanted intracellular trehalose with Na+, thereby facilitating the recovery of normal cell morphology during rehydration. STRT1 likely improves the viability of dried Pv11 cells by rapidly alleviating the significant physical stresses that arise during rehydration.


Subject(s)
Chironomidae , Desiccation , Animals , Trehalose/metabolism , Larva/metabolism , Chironomidae/genetics , Insecta/metabolism , Cell Line
3.
Mitochondrion ; 73: 84-94, 2023 11.
Article in English | MEDLINE | ID: mdl-37956777

ABSTRACT

The sleeping chironomid (Polypedilum vanderplanki) is the only insect capable of surviving complete desiccation in an ametabolic state called anhydrobiosis. Here, we focused on the role of oxidative stress and we observed the production of reactive oxygen species (ROS) in desiccating larvae and in those exposed to salinity stress. Oxidative stress occurs to some extent in desiccating larvae, inducing carbonylation of proteins. Oxidative stress overcomes the antioxidant defenses of the larvae during the first hour following rehydration of anhydrobiotic larvae. It facilitates the oxidation of DNA and cell membrane lipids; however, these damages are quickly repaired after a few hours. In addition to its deleterious effects, we demonstrated that artificial exposure to oxidative stress could induce a response similar to desiccation stress, at the transcriptome and protein levels. Furthermore, the response of anhydrobiosis-related genes to desiccation and salinity stress was inhibited by antioxidant treatment. Thus, we conclude that oxidative stress is an essential trigger for inducing the expression of protective genes during the onset of anhydrobiosis in desiccating of P. vanderplanki larvae.


Subject(s)
Chironomidae , Animals , Chironomidae/genetics , Chironomidae/metabolism , Desiccation , Antioxidants/metabolism , Oxidative Stress , Larva/genetics , Larva/metabolism
4.
Cytotechnology ; 75(6): 491-503, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37841960

ABSTRACT

Pv11 was derived from embryos of the sleeping chironomid Polypedilum vanderplanki, which displays an extreme form of desiccation tolerance known as anhydrobiosis. Pre-treatment with a high concentration of trehalose allows Pv11 cells to enter anhydrobiosis. In the dry state, Pv11 cells preserve transgenic luciferase while retaining its activity. Thus, these cells could be utilized for dry-preserving antibodies, enzymes, signaling proteins or other valuable biological materials without denaturation. However, Pv11 cells grow in suspension, which limits their applicability; for instance, they cannot be integrated into microfluidic devices or used in devices such as sensor chips. Therefore, in this paper, we developed an effective immobilization system for Pv11 cells that, crucially, allows them to maintain their anhydrobiotic potential even when immobilized. Pv11 cells exhibited a very high adhesion rate with both biocompatible anchor for membrane (BAM) and Cell-Tak coatings, which have been reported to be effective on other cultured cells. We also found that Pv11 cells immobilized well to uncoated glass if handled in serum-free medium. Interestingly, Pv11 cells showed desiccation tolerance when trehalose treatment was done prior to immobilization of the cells. In contrast, trehalose treatment after immobilization of Pv11 cells resulted in a significant decrease in desiccation tolerance. Thus, it is important to induce anhydrobiosis before immobilization. In summary, we report the successful development of a protocol for the dry preservation of immobilized Pv11 cells. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00592-0.

5.
Genome Biol Evol ; 15(10)2023 Oct 06.
Article in English | MEDLINE | ID: mdl-37708413

ABSTRACT

The sleeping chironomid Polypedilum vanderplanki is capable of anhydrobiosis, a striking example of adaptation to extreme desiccation. Tolerance to complete desiccation in this species is associated with emergence of multiple paralogs of protective genes. One of the gene families highly expressed under anhydrobiosis and involved in this process is protein-L-isoaspartate (D-aspartate) O-methyltransferases (PIMTs). Recently, another closely related midge was discovered, Polypedilum pembai, which is able not only to tolerate desiccation but also to survive multiple desiccation-rehydration cycles. To investigate the evolution of anhydrobiosis in these species, we sequenced and assembled the genome of P. pembai and compared it with P. vanderplanki and also performed a population genomics analysis of several populations of P. vanderplanki and one population of P. pembai. We observe positive selection and radical changes in the genetic architecture of the PIMT locus between the two species, including its amplification in the P. pembai lineage. In particular, PIMT-4, the most highly expressed of these PIMTs, is present in six copies in the P. pembai; these copies differ in expression profiles, suggesting possible sub- or neofunctionalization. The nucleotide diversity of the genomic region carrying these new genes is decreased in P. pembai, but not in the orthologous region carrying the ancestral gene in P. vanderplanki, providing evidence for a selective sweep associated with postduplication adaptation in the former. Overall, our results suggest an extensive relatively recent and likely ongoing adaptation of the mechanisms of anhydrobiosis.

6.
Arch Insect Biochem Physiol ; 112(1): e21971, 2023 Jan.
Article in English | MEDLINE | ID: mdl-36205078

ABSTRACT

Aphids harbor proteobacterial endosymbionts such as Buchnera aphidicola housed in specialized bacteriocytes derived from host cells. The endosymbiont Buchnera supplies essential amino acids such as arginine to the host cells and, in turn, obtains sugars needed for its survival from the hemolymph. The mechanism of sugar supply in aphid bacteriocytes has been rarely studied. It also remains unclear how Buchnera acquires its carbon source. The hemolymph sugars in Acyrthosiphon pisum are composed of the disaccharide trehalose containing two glucose molecules. Here, we report for the first time that trehalose is transported and used as a potential carbon source by Buchnera across the bacteriocyte plasma membrane via trehalose transporters. The current study characterized the bacteriocyte trehalose transporter Ap_ST11 (LOC100159441) using the Xenopus oocyte expression system. The Ap_ST11 transporter was found to be proton-dependent with a Km value ≥700 mM. We re-examined the hemolymph trehalose at 217.8 mM using a fluorescent trehalose sensor. The bacteriocytes did not obtain trehalose by facilitated diffusion along the gradient across cellular membranes. These findings suggest that trehalose influx into the bacteriocytes depends on the extracellular proton-driven secondary electrochemical transporter.


Subject(s)
Aphids , Buchnera , Animals , Aphids/metabolism , Protons , Trehalose/metabolism , Hemolymph , Symbiosis , Buchnera/metabolism , Carbon/metabolism
7.
iScience ; 25(8): 104639, 2022 Aug 19.
Article in English | MEDLINE | ID: mdl-36039361

ABSTRACT

African chironomid (Polypedilum vanderplanki) larvae can suspend their metabolism by undergoing severe desiccation and then resume this activity by simple rehydration. We present a microdevice using interdigital comb electrodes to detect the larval motion using the natural surface charge of the living larvae in water. The larvae were most active 2 h after soaking them in water at 30°C; they exhibited motions with 2 Hz frequency. This was comparable to the signal obtained from the microdevice via fast Fourier transform (FFT) processing. The amplitude of the voltage and current were 0.11 mV and 730 nA, respectively. They would be enough to be detected by a low power consumption microcomputer. Temperature and pH sensing were demonstrated by detecting the vital motions of the revived larvae under different conditions. This multi-functional biosensor will be a useful microdevice to search for survivable locations under extreme environmental conditions like those on other planets.

8.
Insects ; 13(7)2022 Jul 15.
Article in English | MEDLINE | ID: mdl-35886811

ABSTRACT

Tardigrades are small micrometazoans able to resist several environmental stresses in any stage of their life cycle. An integrated analysis of tardigrade specimens collected in Tsukuba (Japan) revealed a peculiar morphology and a new sensory field in the cloaca. Molecular taxonomy and phylogenetic analysis on different genes (COI, ITS2, 18S and 28S) confirmed that this population is a new species, Macrobiotus kyoukenus sp. nov., belonging to the widespread Macrobiotus hufelandi group. The stress resistance capabilities of M. kyoukenus sp. nov. have been tested by submitting animals to extreme desiccation, rapid freezing, and high levels of ultraviolet radiations (UVB and UVC). Animals were able to survive desiccation (survivorship 95.71 ± 7.07%) and freezing up to -80 °C (82.33 ± 17.11%). Both hydrated and desiccated animals showed a high tolerance to increasing UV radiations: hydrated animals survived to doses up to 152.22 kJ m-2 (UVB) and up to 15.00 kJ m-2 (UVC), while desiccated specimens persisted to radiations up to 165.12 kJ m-2 (UVB) and up to 35.00 kJ m-2 (UVC). Present data contribute to the discovery of a larger tardigrade biodiversity in Japan, and the tolerance capabilities of M. kyoukenus sp. nov. show that it could become a new emerging model for stress resistance studies.

9.
Biology (Basel) ; 11(4)2022 Mar 22.
Article in English | MEDLINE | ID: mdl-35453687

ABSTRACT

Anhydrobiosis, an adaptive ability to withstand complete desiccation, in the nonbiting midge Polypedilum vanderplanki, is associated with the emergence of new multimember gene families, including a group of 27 genes of late embryogenesis abundant (LEA) proteins (PvLea). To obtain new insights into the possible functional specialization of these genes, we investigated the expression and localization of PvLea genes in a P. vanderplanki-derived cell line (Pv11), capable of anhydrobiosis. We confirmed that all but two PvLea genes identified in the genome of P. vanderplanki are expressed in Pv11 cells. Moreover, PvLea genes are induced in Pv11 cells in response to anhydrobiosis-inducing trehalose treatment in a manner highly similar to the larvae of P. vanderplanki during the real induction of anhydrobiosis. Then, we expanded our previous data on PvLEA proteins localization in mammalian cells that were obtained using C-terminal fusions of PvLEA proteins and green fluorescent protein (GFP). We investigated PvLEA localization using N- and C-terminal fusions with GFP in Pv11 cells and the Sf9 insect cell line. We observed an inconsistency of PvLEA localization between different fusion types and different cell cultures, that needs to be taken into account when using PvLEA in the engineering of anhydrobiotic cell lines.

10.
NAR Genom Bioinform ; 4(2): lqac029, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35387384

ABSTRACT

Non-biting midges (Chironomidae) are known to inhabit a wide range of environments, and certain species can tolerate extreme conditions, where the rest of insects cannot survive. In particular, the sleeping chironomid Polypedilum vanderplanki is known for the remarkable ability of its larvae to withstand almost complete desiccation by entering a state called anhydrobiosis. Chromosome numbers in chironomids are higher than in other dipterans and this extra genomic resource might facilitate rapid adaptation to novel environments. We used improved sequencing strategies to assemble a chromosome-level genome sequence for P. vanderplanki for deep comparative analysis of genomic location of genes associated with desiccation tolerance. Using whole genome-based cross-species and intra-species analysis, we provide evidence for the unique functional specialization of Chromosome 4 through extensive acquisition of novel genes. In contrast to other insect genomes, in the sleeping chironomid a uniquely high degree of subfunctionalization in paralogous anhydrobiosis genes occurs in this chromosome, as well as pseudogenization in a highly duplicated gene family. Our findings suggest that the Chromosome 4 in Polypedilum is a site of high genetic turnover, allowing it to act as a 'sandbox' for evolutionary experiments, thus facilitating the rapid adaptation of midges to harsh environments.

11.
Genes (Basel) ; 13(3)2022 02 24.
Article in English | MEDLINE | ID: mdl-35327960

ABSTRACT

Genomic safe harbors (GSHs) provide ideal integration sites for generating transgenic organisms and cells and can be of great benefit in advancing the basic and applied biology of a particular species. Here we report the identification of GSHs in a dry-preservable insect cell line, Pv11, which derives from the sleeping chironomid, Polypedilum vanderplanki, and similar to the larvae of its progenitor species exhibits extreme desiccation tolerance. To identify GSHs, we carried out genome analysis of transgenic cell lines established by random integration of exogenous genes and found four candidate loci. Targeted knock-in was performed into these sites and the phenotypes of the resulting transgenic cell lines were examined. Precise integration was achieved for three candidate GSHs, and in all three cases integration did not alter the anhydrobiotic ability or the proliferation rate of the cell lines. We therefore suggest these genomic loci represent GSHs in Pv11 cells. Indeed, we successfully constructed a knock-in system and introduced an expression unit into one of these GSHs. We therefore identified several GSHs in Pv11 cells and developed a new technique for producing transgenic Pv11 cells without affecting the phenotype.


Subject(s)
Chironomidae , Animals , Cell Line , Chironomidae/genetics , Genomics , Insecta , Larva
12.
Sci Rep ; 11(1): 19698, 2021 10 05.
Article in English | MEDLINE | ID: mdl-34611198

ABSTRACT

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.


Subject(s)
CRISPR-Cas Systems , Calcium Signaling , Dehydration , Gene Editing , Animals , Calcium/metabolism , Cell Line , Computational Biology/methods , Gene Expression Profiling , Gene Knock-In Techniques , Gene Ontology , Insecta , Larva , RNA, Guide, Kinetoplastida , Stress, Physiological , Trehalose/metabolism , Trehalose/pharmacology
13.
Biology (Basel) ; 10(7)2021 Jun 30.
Article in English | MEDLINE | ID: mdl-34209345

ABSTRACT

Until now, the ability to reversibly halt cellular processes has been limited to cryopreservation and several forms of anabiosis observed in living organisms. In this paper we show that incubation of living cells with a solution containing ~50 mM neodymium induces a rapid shutdown of intracellular organelle movement and all other evidence of active metabolism. We have named this state REEbernation (derived from the terms REE (rare earth elements) and hibernation) and found that the process involves a rapid replacement of calcium with neodymium in membranes and organelles of a cell, allowing it to maintain its shape and membrane integrity under extreme conditions, such as low pressure. Furthermore, phosphate exchange is blocked as a result of non-dissolvable neodymium salts formation, which "discharged" the cell. We further showed that REEbernation is characterized by an immediate cessation of transcriptional activity in observed cells, providing an intriguing opportunity to study a snapshot of gene expression at a given time point. Finally, we found that the REEbernation state is reversible, and we could restore the metabolism and proliferation capacity of the cells. The REEbernation, in addition to being an attractive model to further investigate the basic mechanisms of cell metabolism control, also provides a new method to reversibly place a cell into "on-hold" mode, opening opportunities to develop protocols for biological samples fixation with a minimum effect on the omics profile for biomedical needs.

14.
Int J Mol Sci ; 22(11)2021 May 28.
Article in English | MEDLINE | ID: mdl-34071490

ABSTRACT

The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.


Subject(s)
Adaptation, Physiological/genetics , Chironomidae/genetics , Desiccation , Gene Expression Regulation , Genome-Wide Association Study/methods , Heat Shock Transcription Factors/genetics , Insect Proteins/genetics , Animals , Cell Line , Chironomidae/cytology , Cluster Analysis , Gene Expression Profiling/methods
15.
Insects ; 11(11)2020 Nov 11.
Article in English | MEDLINE | ID: mdl-33187095

ABSTRACT

The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 (Aequorea coerulescens green fluorescent protein) was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, the avian myeloblastosis virus reverse transcriptase α subunit (AMV RTα), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins.

16.
Insects ; 11(9)2020 Sep 16.
Article in English | MEDLINE | ID: mdl-32947792

ABSTRACT

Multiple co-localized paralogs of genes in Polypedilum vanderplanki's genome have strong transcriptional response to dehydration and considered to be a part of adaptation machinery at the larvae stage. One group of such genes represented by L-isoaspartate O-methyltransferases (PIMT). In order to highlight specific role of PIMT paralogization in desiccation tolerance of the larvae we annotated and compared S-adenosylmethionine (SAM) dependent methyltransferases of four insect species. From another side we applied co-expression analysis in desiccation/rehydration time course and showed that PIMT coding genes could be separated into five clusters by expression profile. We found that among Polypedilum vanderplanki's PIMTs only PIMT1 and PIMT2 have enzymatic activity in normal physiological conditions. From in silico analysis of the protein structures we found two highly variable regions outside of the active center, but also amino acid substitutions which may affect SAM stabilization. Overall, in this study we demonstrated features of Polypedilum vanderplanki's PIMT coding paralogs related to different roles in desiccation tolerance of the larvae. Our results also suggest a role of different SAM-methyltransferases in the adaptation, including GSMT, JHAMT, and candidates from other classes, which could be considered in future studies.

17.
Proc Natl Acad Sci U S A ; 117(32): 19209-19220, 2020 08 11.
Article in English | MEDLINE | ID: mdl-32723826

ABSTRACT

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.


Subject(s)
Diptera/metabolism , Insect Proteins/metabolism , Adenosine Monophosphate/metabolism , Animals , Desiccation , Diptera/chemistry , Diptera/genetics , Droughts , Glycogen/genetics , Glycogen/metabolism , Insect Proteins/chemistry , Insect Proteins/genetics , Larva/chemistry , Larva/genetics , Larva/metabolism , Metabolome , Transcriptome , Trehalose/metabolism , Water/metabolism
18.
Sci Rep ; 10(1): 11633, 2020 07 15.
Article in English | MEDLINE | ID: mdl-32669703

ABSTRACT

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.


Subject(s)
Cell Membrane/metabolism , Chironomidae/physiology , Dehydration , Insect Proteins/metabolism , Membrane Proteins/metabolism , Amino Acid Motifs , Animals , Cell Line , Cloning, Molecular , Cluster Analysis , Computer Simulation , Gene Expression Profiling , Gene Expression Regulation , Larva/physiology , Multigene Family , Phylogeny , Protein Domains , RNA-Seq
19.
PLoS One ; 15(3): e0230218, 2020.
Article in English | MEDLINE | ID: mdl-32191739

ABSTRACT

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.


Subject(s)
Insect Proteins/genetics , Transcription Factors/genetics , Animals , Biological Phenomena/genetics , Cell Line , Chironomidae/genetics , Dehydration/genetics , Desiccation/methods , Drosophila melanogaster/genetics , Gene Expression Regulation/genetics , Larva/genetics , Stress, Physiological/genetics
20.
J Phys Chem B ; 124(14): 2747-2759, 2020 04 09.
Article in English | MEDLINE | ID: mdl-32192343

ABSTRACT

We investigated experimentally whether a short peptide, PvLEA-22, which consists of two tandem repeats of an 11-mer motif of Group 3 late embryogenesis abundant proteins, has a chaperone-like function for denatured proteins. Lysozyme was selected as a target protein. Turbidity measurements indicated that the peptide suppresses the heat-induced aggregation of lysozyme when added at a molar ratio of PvLEA-22/lysozyme >40. Circular dichroism and differential scanning calorimetry measurements confirmed that the lysozyme was denatured on heating but spontaneously refolded on subsequent cooling in the presence of the peptide. As a result, up to 80% of the native catalytic activity of lysozyme was preserved. Similar chaperone-like activity was also observed for a peptide with the same amino acid composition as PvLEA-22 but whose sequence is scrambled. To elucidate the underlying mechanism of the chaperone function of these peptides, we performed coarse-grained molecular dynamics simulations. This revealed that a denatured lysozyme molecule is shielded by several peptide molecules in aqueous solution, which acts as a physical barrier, reducing the opportunities for collision between denatured proteins. An important finding was that a peptide bound to the denatured protein is very rapidly replaced by another; due to such rapid exchange, peptide-protein contact time is very short, that is, on the order of ∼200 ns. Therefore, the peptide does not constrain the behavior of the denatured protein, which can refold freely.


Subject(s)
Hot Temperature , Muramidase , Circular Dichroism , Peptides , Protein Denaturation , Protein Folding
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