Membraneless and membrane-bound organelles in an anhydrobiotic cell line are protected from desiccation-induced damage.

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%.

A sodium-dependent trehalose transporter contributes to anhydrobiosis in insect cell line, Pv11.

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.

Oxidative stress is an essential factor for the induction of anhydrobiosis in the desiccation-tolerant midge, Polypedilum vanderplanki (Diptera, Chironomidae).

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.

Effective methods for immobilization of non-adherent Pv11 cells while maintaining their desiccation tolerance.

Unlabelled: Pv11 was derived from embryos of the sleeping chironomid , 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.

Population Genomics of Two Closely Related Anhydrobiotic Midges Reveals Differences in Adaptation to Extreme Desiccation.

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).

Proton gradient mediates hemolymph trehalose influx into aphid bacteriocytes.

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.

Anhydrobiotic chironomid larval motion-based multi-sensing microdevice for the exploration of survivable locations.

African chironomid () 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.

Resistance to Extreme Stresses by a Newly Discovered Japanese Tardigrade Species, (Eutardigrada, Macrobiotidae).

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, sp.

Intracellular Localization and Gene Expression Analysis Provides New Insights on LEA Proteins' Diversity in Anhydrobiotic Cell Line.

Anhydrobiosis, an adaptive ability to withstand complete desiccation, in the nonbiting midge , is associated with the emergence of new multimember gene families, including a group of 27 genes of late embryogenesis abundant (LEA) proteins (). To obtain new insights into the possible functional specialization of these genes, we investigated the expression and localization of genes in a -derived cell line (Pv11), capable of anhydrobiosis. We confirmed that all but two genes identified in the genome of are expressed in Pv11 cells.

High quality genome assembly of the anhydrobiotic midge provides insights on a single chromosome-based emergence of extreme desiccation tolerance.

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 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.

Identification of Genomic Safe Harbors in the Anhydrobiotic Cell Line, Pv11.

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, , 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.

Cas9-mediated genome editing reveals a significant contribution of calcium signaling pathways to anhydrobiosis in Pv11 cells.

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.

Life-On-Hold: Lanthanoids Rapidly Induce a Reversible Ametabolic State in Mammalian Cells.

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.

Genome-Wide Role of HSF1 in Transcriptional Regulation of Desiccation Tolerance in the Anhydrobiotic Cell Line, Pv11.

The Pv11, an insect cell line established from the midge , 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.

Development of a Tet-On Inducible Expression System for the Anhydrobiotic Cell Line, Pv11.

The Pv11 cell line established from an African chironomid, , 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.

Diversity and Regulation of S-Adenosylmethionine Dependent Methyltransferases in the Anhydrobiotic Midge.

Multiple co-localized paralogs of genes in 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.

Combined metabolome and transcriptome analysis reveals key components of complete desiccation tolerance in an anhydrobiotic insect.

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 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.

New group of transmembrane proteins associated with desiccation tolerance in the anhydrobiotic midge Polypedilum vanderplanki.

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.

Group 3 LEA Protein Model Peptides Suppress Heat-Induced Lysozyme Aggregation. Elucidation of the Underlying Mechanism Using Coarse-Grained Molecular Simulations.

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.

Identification of a master transcription factor and a regulatory mechanism for desiccation tolerance in the anhydrobiotic cell line Pv11.

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.

Trehalose uptake and dehydration effects on the cryoprotection of CHO-K1 cells expressing TRET1.

Chinese hamster ovary cells (CHO-K1 cells) in which the trehalose transporter (TRET1) is expressed can have greater cryoprotection than ordinary CHO-K1 cells. This study examines the uptake characteristics of trehalose into cells via TRET1 and determines the influence of intracellular trehalose on the freeze-thaw viabilities. In our experiments, the intracellular trehalose concentration is controlled by the extracellular trehalose concentration and the immersion time in a freezing solution.

Identification of a novel strong promoter from the anhydrobiotic midge, Polypedilum vanderplanki, with conserved function in various insect cell lines.

Larvae of the African midge Polypedilum vanderplanki (Diptera: Chironomidae) show a form of extreme desiccation tolerance known as anhydrobiosis. The cell line Pv11 was recently established from the species, and these cells can also survive under desiccated conditions, and proliferate normally after rehydration. Here we report the identification of a new promoter, 121, which has strong constitutive transcriptional activity in Pv11 cells and promotes effective expression of exogenous genes.

Transcriptome analysis of the anhydrobiotic cell line Pv11 infers the mechanism of desiccation tolerance and recovery.

The larvae of the African midge, Polypedilum vanderplanki, can enter an ametabolic state called anhydrobiosis to overcome fatal desiccation stress. The Pv11 cell line, derived from P. vanderplanki embryo, shows desiccation tolerance when treated with trehalose before desiccation and resumes proliferation after rehydration.

A LEA model peptide protects the function of a red fluorescent protein in the dry state.

We tested whether a short model peptide derived from a group 3 late embryogenesis abundant (G3LEA) protein is able to maintain the fluorescence activity of a red fluorescent protein, mKate2, in the dry state. The fluorescence intensity of mKate2 alone decreased gradually through repeated dehydration-rehydration treatments. However, in the presence of the LEA model peptide, the peak intensity was maintained almost perfectly during such stress treatments, which implies that the three dimensional structure of the active site of mKate2 was protected even under severe desiccation conditions.

The Antioxidant System in the Anhydrobiotic Midge as an Essential, Adaptive Mechanism for Desiccation Survival.

One of the major damaging factors for living organisms experiencing water insufficiency is oxidative stress. Loss of water causes a dramatic increase in the production of reactive oxygen species (ROS). Thus, the ability for some organisms to survive almost complete desiccation (called anhydrobiosis) is tightly related to the ability to overcome extraordinary oxidative stress.