Surviving extreme radiation.

Tiny animals known as tardigrades use a combination of DNA repair machinery and a novel protein to mend their genome after intense ionizing radiation.

Disordered proteins interact with the chemical environment to tune their protective function during drying.

The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins combined with the exposure of their residues accounts for this sensitivity. One context in which IDPs play important roles that is concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family, synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results demonstrate that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet, the mechanisms underlying this synergy differ between IDP families.

Water content, transition temperature and fragility influence protection and anhydrobiotic capacity.

Water is essential for metabolism and all life processes. Despite this, many organisms distributed across the kingdoms of life survive near-complete desiccation or anhydrobiosis. Increased intracellular viscosity, leading to the formation of a vitrified state is necessary, but not sufficient, for survival while dry. What properties of a vitrified system make it desiccation-tolerant or -sensitive are unknown. We have analyzed 18 different in vitro vitrified systems, composed of one of three protective disaccharides (trehalose, sucrose, or maltose) and glycerol, quantifying their enzyme-protective capacity and their material properties in a dry state. Protection conferred by mixtures containing maltose correlates strongly with increased water content, increased glass-transition temperature, and reduced glass former fragility, while the protection of glasses formed with sucrose correlates with increased glass transition temperature and the protection conferred by trehalose glasses correlates with reduced glass former fragility. Thus, in vitro different vitrified sugars confer protection through distinct material properties. Next, we examined the material properties of a dry desiccation tolerant and intolerant life stage from three different organisms. The dried desiccation tolerant life stage of all organisms had an increased glass transition temperature and reduced glass former fragility relative to its dried desiccation intolerant life stage. These results suggest in nature organismal desiccation tolerance relies on a combination of various material properties. This study advances our understanding of how protective and non-protective glasses differ in terms of material properties that promote anhydrobiosis. This knowledge presents avenues to develop novel stabilization technologies for pharmaceuticals that currently rely on the cold-chain. Statement of significance: For the past three decades the anhydrobiosis field has lived with a paradox, while vitrification is necessary for survival in the dry state, it is not sufficient. Understanding what property(s) distinguishes a desiccation tolerant from an intolerant vitrified system and how anhydrobiotic organisms survive drying is one of the enduring mysteries of organismal physiology. Here we show in vitro the enzyme-protective capacity of different vitrifying sugars can be correlated with distinct material properties. However, in vivo, diverse desiccation tolerant organisms appear to combine these material properties to promote their survival in a dry state.

Helicity of a tardigrade disordered protein contributes to its protective function during desiccation.

To survive extreme drying (anhydrobiosis), many organisms, spanning every kingdom of life, accumulate intrinsically disordered proteins (IDPs). For decades, the ability of anhydrobiosis-related IDPs to form transient amphipathic helices has been suggested to be important for promoting desiccation tolerance. However, evidence empirically supporting the necessity and/or sufficiency of helicity in mediating anhydrobiosis is lacking. Here, we demonstrate that the linker region of CAHS D, a desiccation-related IDP from the tardigrade Hypsibius exemplaris, that contains significant helical structure, is the protective portion of this protein. Perturbing the sequence composition and grammar of the linker region of CAHS D, through the insertion of helix-breaking prolines, modulating the identity of charged residues, or replacement of hydrophobic amino acids with serine or glycine residues results in variants with different degrees of helical structure. Importantly, correlation of protective capacity and helical content in variants generated through different helix perturbing modalities does not show as strong a trend, suggesting that while helicity is important, it is not the only property that makes a protein protective during desiccation. These results provide direct evidence for the decades-old theory that helicity of desiccation-related IDPs is linked to their anhydrobiotic capacity.

Water content, transition temperature and fragility influence protection and anhydrobiotic capacity.

Water is essential for metabolism and all life processes. Despite this, many organisms distributed across the kingdoms of life survive near-complete desiccation or anhydrobiosis (Greek for "life without water"). Increased intracellular viscosity, leading to the formation of a vitrified state is necessary, but not sufficient, for survival while dry. What properties of a vitrified system make it desiccation-tolerant or -sensitive are unknown. We have analyzed 18 different in vitro vitrified systems, composed of one of three protective disaccharides (trehalose, sucrose, or maltose) and varying amounts of glycerol, quantifying their enzyme-protective capacity and their material properties in a dry state. We find that protection conferred by mixtures containing maltose correlates strongly with increased water content, increased glass-transition temperature, and reduced glass former fragility, while the protection of glasses formed with sucrose correlates with increased glass transition temperature and the protection conferred by trehalose glasses correlates with reduced glass former fragility. Thus, in vitro different vitrified sugars confer protection through distinct material properties. Extending on this, we have examined the material properties of a dry desiccation tolerant and intolerant life stage from three different organisms. In all cases, the dried desiccation tolerant life stage of an organism had an increased glass transition temperature relative to its dried desiccation intolerant life stage, and this trend is also seen in all three organisms when considering reduced glass former fragility. These results suggest that while drying of different protective sugars in vitro results in vitrified systems with distinct material properties that correlate with their enzyme-protective capacity, in nature organismal desiccation tolerance relies on a combination of these properties. This study advances our understanding of how protective and non-protective glasses differ in terms of material properties that promote anhydrobiosis. This knowledge presents avenues to develop novel stabilization technologies for pharmaceuticals that currently rely on the cold-chain.

The tardigrade protein CAHS D interacts with, but does not retain, water in hydrated and desiccated systems.

Tardigrades are a group of microscopic animals renowned for their ability to survive near complete desiccation. A family of proteins, unique to tardigrades, called Cytoplasmic Abundant Heat Soluble (CAHS) proteins are necessary to mediate robust desiccation tolerance in these animals. However, the mechanism(s) by which CAHS proteins help to protect tardigrades during water-loss have not been fully elucidated. Here we use thermogravimetric analysis to empirically test the proposed hypothesis that tardigrade CAHS proteins, due to their propensity to form hydrogels, help to retain water during desiccation. We find that regardless of its gelled state, both in vitro and in vivo, a model CAHS protein (CAHS D) retains no more water than common proteins and control cells in the dry state. However, we find that while CAHS D proteins do not increase the total amount of water retained in a dry system, they interact with the small amount of water that does remain. Our study indicates that desiccation tolerance mediated by CAHS D cannot be simply ascribed to water retention and instead implicates its ability to interact more tightly with residual water as a possible mechanism underlying its protective capacity. These results advance our fundamental understanding of tardigrade desiccation tolerance which could provide potential avenues for new technologies to aid in the storage of dry shelf-stable pharmaceuticals and the generation of stress tolerant crops to ensure food security in the face of global climate change.

Natural and engineered mediators of desiccation tolerance stabilize Human Blood Clotting Factor VIII in a dry state.

Biologics, pharmaceuticals containing or derived from living organisms, such as vaccines, antibodies, stem cells, blood, and blood products are a cornerstone of modern medicine. However, nearly all biologics have a major deficiency: they are inherently unstable, requiring storage under constant cold conditions. The so-called 'cold-chain', while effective, represents a serious economic and logistical hurdle for deploying biologics in remote, underdeveloped, or austere settings where access to cold-chain infrastructure ranging from refrigerators and freezers to stable electricity is limited. To address this issue, we explore the possibility of using anhydrobiosis, the ability of organisms such as tardigrades to enter a reversible state of suspended animation brought on by extreme drying, as a jumping off point in the development of dry storage technology that would allow biologics to be kept in a desiccated state under not only ambient but elevated temperatures. Here we examine the ability of different protein and sugar-based mediators of anhydrobiosis derived from tardigrades and other anhydrobiotic organisms to stabilize Human Blood Clotting Factor VIII under repeated dehydration/rehydration cycles, thermal stress, and long-term dry storage conditions. We find that while both protein and sugar-based protectants can stabilize the biologic pharmaceutical Human Blood Clotting Factor VIII under all these conditions, protein-based mediators offer more accessible avenues for engineering and thus tuning of protective function. Using classic protein engineering approaches, we fine tune the biophysical properties of a protein-based mediator of anhydrobiosis derived from a tardigrade, CAHS D. Modulating the ability of CAHS D to form hydrogels make the protein better or worse at providing protection to Human Blood Clotting Factor VIII under different conditions. This study demonstrates the effectiveness of tardigrade CAHS proteins and other mediators of desiccation tolerance at preserving the function of a biologic without the need for the cold-chain. In addition, our study demonstrates that engineering approaches can tune natural products to serve specific protective functions, such as coping with desiccation cycling versus thermal stress. Ultimately, these findings provide a proof of principle that our reliance on the cold-chain to stabilize life-saving pharmaceuticals can be broken using natural and engineered mediators of desiccation tolerance.

Trehalose and tardigrade CAHS proteins work synergistically to promote desiccation tolerance.

Tardigrades are microscopic animals renowned for their ability to survive extreme desiccation. Unlike many desiccation-tolerant organisms that accumulate high levels of the disaccharide trehalose to protect themselves during drying, tardigrades accumulate little or undetectable levels. Using comparative metabolomics, we find that despite being enriched at low levels, trehalose is a key biomarker distinguishing hydration states of tardigrades. In vitro, naturally occurring stoichiometries of trehalose and CAHS proteins, intrinsically disordered proteins with known protective capabilities, were found to produce synergistic protective effects during desiccation. In vivo, this synergistic interaction is required for robust CAHS-mediated protection. This demonstrates that trehalose acts not only as a protectant, but also as a synergistic cosolute. Beyond desiccation tolerance, our study provides insights into how the solution environment tunes intrinsically disordered proteins' functions, many of which are vital in biological contexts such as development and disease that are concomitant with large changes in intracellular chemistry.

Production of reactive oxygen species and involvement of bioprotectants during anhydrobiosis in the tardigrade Paramacrobiotus spatialis.

Water unavailability is an abiotic stress causing unfavourable conditions for life. Nevertheless, some animals evolved anhydrobiosis, a strategy allowing for the reversible organism dehydration and suspension of metabolism as a direct response to habitat desiccation. Anhydrobiotic animals undergo biochemical changes synthesizing bioprotectants to help combat desiccation stresses. One stress is the generation of reactive oxygen species (ROS). In this study, the eutardigrade Paramacrobiotus spatialis was used to investigate the occurrence of ROS associated with the desiccation process. We observed that the production of ROS significantly increases as a function of time spent in anhydrobiosis and represents a direct demonstration of oxidative stress in tardigrades. The degree of involvement of bioprotectants, including those combating ROS, in the P. spatialis was evaluated by perturbing their gene functions using RNA interference and assessing the successful recovery of animals after desiccation/rehydration. Targeting the glutathione peroxidase gene compromised survival during drying and rehydration, providing evidence for the role of the gene in desiccation tolerance. Targeting genes encoding glutathione reductase and catalase indicated that these molecules play roles during rehydration. Our study also confirms the involvement of aquaporins 3 and 10 during rehydration. Therefore, desiccation tolerance depends on the synergistic action of many different molecules working together.

Membrane and lipid metabolism plays an important role in desiccation resistance in the yeast Saccharomyces cerevisiae.

BACKGROUND: Anhydrobiotes, such as the yeast Saccharomyces cerevisiae, are capable of surviving almost total loss of water. Desiccation tolerance requires an interplay of multiple events, including preserving the protein function and membrane integrity, preventing and mitigating oxidative stress, maintaining certain level of energy required for cellular activities in the desiccated state. Many of these crucial processes can be controlled and modulated at the level of organelle morphology and dynamics. However, little is understood about what organelle perturbations manifest in desiccation-sensitive cells as a consequence of drying or how this differs from organelle biology in desiccation-tolerant organisms undergoing anhydrobiosis. RESULTS: In this study, electron and optical microscopy was used to examine the dynamic changes of yeast cells during the desiccation process. Dramatic structural changes were observed during the desiccation process, including the diminishing of vacuoles, decrease of lipid droplets, decrease in mitochondrial cristae and increase of ER membrane, which is likely caused by ER stress and unfolded protein response. The survival rate was significantly decreased in mutants that are defective in lipid droplet biosynthesis, or cells treated with cerulenin, an inhibitor of fatty acid synthesis. CONCLUSION: Our study suggests that the metabolism of lipid droplets and membrane may play an important role in yeast desiccation tolerance by providing cells with energy and possibly metabolic water. Additionally, the decrease in mitochondrial cristae coupled with a decrease in lipid droplets is indicative of a cellular response to reduce the production of reactive oxygen species.

The biology of tardigrade disordered proteins in extreme stress tolerance.

Disordered proteins have long been known to help mediate tolerance to different abiotic stresses including freezing, osmotic stress, high temperatures, and desiccation in a diverse set of organisms. Recently, three novel families of intrinsically disordered proteins were identified in tardigrades, microscopic animals capable of surviving a battery of environmental extremes. These three families include the Cytoplasmic-, Secreted-, and Mitochondrial- Abundant Heat Soluble (CAHS, SAHS, and MAHS) proteins, which are collectively termed Tardigrade Disordered Proteins (TDPs). At the level of sequence conservation TDPs are unique to tardigrades, and beyond their high degree of disorder the CAHS, SAHS, and MAHS families do not resemble one another. All three families are either highly expressed constitutively, or significantly enriched in response to desiccation. In vivo, ex vivo, and in vitro experiments indicate functional roles for members of each TDP family in mitigating cellular perturbations induced by various abiotic stresses. What is currently lacking is a comprehensive and holistic understanding of the fundamental mechanisms by which TDPs function, and the properties of TDPs that allow them to function via those mechanisms. A quantitative and systematic approach is needed to identify precisely what cellular damage TDPs work to prevent, what sequence features are important for these functions, and how those sequence features contribute to the underlying mechanisms of protection. Such an approach will inform us not only about these fascinating proteins, but will also provide insights into how the sequence of a disordered protein can dictate its functional, structural, and dynamic properties. Video Abstract.

Mechanisms and evolution of resistance to environmental extremes in animals.

When animals are exposed to an extreme environmental stress, one of three possible outcomes takes place: the animal dies, the animal avoids the environmental stress and survives, or the animal tolerates the environmental stress and survives. This review is concerned with the third possibility, and will look at mechanisms that rare animals use to survive extreme environmental stresses including freezing, desiccation, intense heat, irradiation, and low-oxygen conditions (hypoxia). In addition, an increasing understanding of the molecular mechanisms involved in environmental stress tolerance allows us to speculate on how these tolerances arose. Uncovering the mechanisms of extreme environmental stress tolerance and how they evolve has broad implications for our understanding of the evolution of early life on this planet, colonization of new environments, and the search for novel forms of life both on Earth and elsewhere, as well as a number of agricultural and health-related applications.

Protecting activity of desiccated enzymes.

Protein-based biological drugs and many industrial enzymes are unstable, making them prohibitively expensive. Some can be stabilized by formulation with excipients, but most still require low temperature storage. In search of new, more robust excipients, we turned to the tardigrade, a microscopic animal that synthesizes cytosolic abundant heat soluble (CAHS) proteins to protect its cellular components during desiccation. We find that CAHS proteins protect the test enzymes lactate dehydrogenase and lipoprotein lipase against desiccation-, freezing-, and lyophilization-induced deactivation. Our data also show that a variety of globular and disordered protein controls, with no known link to desiccation tolerance, protect our test enzymes. Protection of lactate dehydrogenase correlates, albeit imperfectly, with the charge density of the protein additive, suggesting an approach to tune protection by modifying charge. Our results support the potential use of CAHS proteins as stabilizing excipients in formulations and suggest that other proteins may have similar potential.

Desiccation of Hypsibius exemplaris.

Many species of tardigrades can survive severe water loss, but different species tolerate different desiccation conditions. Hypsibius exemplaris is able to survive desiccation after an initial period of slow drying, as described here. This protocol will likely work for other tardigrade species as well. Drying of tardigrades can be used for probing the mechanistic underpinnings of desiccation tolerance, as well as for practical purposes such as shipping and long-term storage of the animals.

Total RNA Extraction from Tardigrades.

Purification of high-quality total RNA from specimens is essential for many molecular techniques. In tardigrades (water bears), disruption of the cuticle is an important step in obtaining a good yield that is representative of all tissues of the animal. As with all single-stranded RNA methods, sterile technique, proper storage conditions, and handling are required for maintaining the quality and integrity of the material. This procedure will result in high-quality total RNA suitable for downstream applications such as cDNA synthesis, reverse transcriptase-polymerase chain reaction (RT-PCR), RNAseq library generation, and northern blots.

LITE microscopy: Tilted light-sheet excitation of model organisms offers high resolution and low photobleaching.

Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.

Intrinsically Disordered Proteins and Desiccation Tolerance: Elucidating Functional and Mechanistic Underpinnings of Anhydrobiosis.

Over 300 years ago the father of microscopy, Antonie van Leeuwenhoek, observed dried rotifers (tiny animals) "coming back to life" upon rehydration. Since then, scientists have been fascinated by the enduring mystery of how certain organisms survive losing essentially drying out completely. Historically sugars, such as the disaccharide trehalose, have been viewed as major functional mediators of desiccation tolerance. However, some desiccation tolerant organisms do not produce this sugar, hinting that additional mediators, and potentially novel mechanisms exist. It has become apparent that a common theme among such organisms is the production and use of intrinsically disordered proteins (IDPs) to mediate survival in this dry state. However, the basic biology of these proteins - which unlike globular proteins lack persistent three-dimensional structure - is poorly understood, as are the functional mechanisms utilized by these enigmatic proteins that allow them to mediate desiccation tolerance. We purpose that probing the biochemical and biophysical nature of stress-related IDPs will provide mechanistic insights into these fascinating proteins.