Cryopreserved red blood cells maintain allosteric control of oxygen binding when utilizing trehalose as a cryoprotectant.

One of the most common life-saving medical procedures is a red blood cell (RBC) transfusion. Unfortunately, RBCs for transfusion have a limited shelf life after donation due to detrimental storage effects on their morphological and biochemical properties. Inspired by nature, a biomimetics approach was developed to preserve RBCs for long-term storage using compounds found in animals with a natural propensity to survive in a frozen or desiccated state for decades. Trehalose was employed as a cryoprotective agent and added to the extracellular freezing solution of porcine RBCs. Slow cooling (-1 °C min-1) resulted in almost complete hemolysis (1 ± 1 % RBC recovery), and rapid cooling rates had to be used to achieve satisfactory cryopreservation outcomes. After rapid cooling, the highest percentage of RBC recovery was obtained by plunging in liquid nitrogen and thawing at 55 °C, using a cryopreservation solution containing 300 mM trehalose. Under these conditions, 88 ± 8 % of processed RBCs were recovered and retained hemoglobin (14 ± 2 % hemolysis). Hemoglobin's oxygen-binding properties of cryopreserved RBCs were not significantly different to unfrozen controls and was allosterically regulated by 2,3-bisphosphoglycerate. These data indicate the feasibility of using trehalose instead of glycerol as a cryoprotective compound for RBCs. In contrast to glycerol, trehalose-preserved RBCs can potentially be transfused without time-consuming washing steps, which significantly facilitates the usage of cryopreserved transfusible units in trauma situations when time is of the essence.

Liquid-liquid phase separation promotes animal desiccation tolerance.

Proteinaceous liquid-liquid phase separation (LLPS) occurs when a polypeptide coalesces into a dense phase to form a liquid droplet (i.e., condensate) in aqueous solution. In vivo, functional protein-based condensates are often referred to as membraneless organelles (MLOs), which have roles in cellular processes ranging from stress responses to regulation of gene expression. Late embryogenesis abundant (LEA) proteins containing seed maturation protein domains (SMP; PF04927) have been linked to storage tolerance of orthodox seeds. The mechanism by which anhydrobiotic longevity is improved is unknown. Interestingly, the brine shrimp Artemia franciscana is the only animal known to express such a protein (AfrLEA6) in its anhydrobiotic embryos. Ectopic expression of AfrLEA6 (AWM11684) in insect cells improves their desiccation tolerance and a fraction of the protein is sequestered into MLOs, while aqueous AfrLEA6 raises the viscosity of the cytoplasm. LLPS of AfrLEA6 is driven by the SMP domain, while the size of formed MLOs is regulated by a domain predicted to engage in protein binding. AfrLEA6 condensates formed in vitro selectively incorporate target proteins based on their surface charge, while cytoplasmic MLOs formed in AfrLEA6-transfected insect cells behave like stress granules. We suggest that AfrLEA6 promotes desiccation tolerance by engaging in two distinct molecular mechanisms: by raising cytoplasmic viscosity at even modest levels of water loss to promote cell integrity during drying and by forming condensates that may act as protective compartments for desiccation-sensitive proteins. Identifying and understanding the molecular mechanisms that govern anhydrobiosis will lead to significant advancements in preserving biological samples.

LEAfing through literature: late embryogenesis abundant proteins coming of age-achievements and perspectives.

To deal with increasingly severe periods of dehydration related to global climate change, it becomes increasingly important to understand the complex strategies many organisms have developed to cope with dehydration and desiccation. While it is undisputed that late embryogenesis abundant (LEA) proteins play a key role in the tolerance of plants and many anhydrobiotic organisms to water limitation, the molecular mechanisms are not well understood. In this review, we summarize current knowledge of the physiological roles of LEA proteins and discuss their potential molecular functions. As these are ultimately linked to conformational changes in the presence of binding partners, post-translational modifications, or water deprivation, we provide a detailed summary of current knowledge on the structure-function relationship of LEA proteins, including their disordered state in solution, coil to helix transitions, self-assembly, and their recently discovered ability to undergo liquid-liquid phase separation. We point out the promising potential of LEA proteins in biotechnological and agronomic applications, and summarize recent advances. We identify the most relevant open questions and discuss major challenges in establishing a solid understanding of how these intriguing molecules accomplish their tasks as cellular sentinels at the limits of surviving water scarcity.

Selection on dispersal drives evolution of metabolic capacities for energy production in female wing-polymorphic sand field crickets, Gryllus firmus.

Life history and metabolism covary, but the mechanisms and individual traits responsible for these linkages remain unresolved. Dispersal capability is a critical component of life history that is constrained by metabolic capacities for energy production. Conflicting relationships between metabolism and life histories may be explained by accounting for variation in dispersal and maximal metabolic rates. We used female wing-polymorphic sand field crickets, Gryllus firmus, selected either for long wings (LW, flight-capable) or short wings (SW, flightless) to test the hypothesis that selection on dispersal capability drives the evolution of metabolic capacities. While resting metabolic rates were similar, long-winged crickets reached higher maximal metabolic rates than short-winged crickets, resulting in improved running performance. We further provided insight into the mechanisms responsible for covariation between life history and metabolism by comparing mitochondrial content of tissues involved in powering locomotion and assessing the function of mitochondria isolated from long- and short-winged crickets. Our results demonstrated that larger metabolic capacities in long-winged crickets were underpinned by increases in mitochondrial content of dorsoventral flight muscle and enhanced bioenergetic capacities of mitochondria within the fat body, a tissue responsible for fuel storage and mobilization. Thus, selection on flight capability correlates with increases in maximal, but not resting metabolic rates, through modifications of tissues powering locomotion at the cellular and organelle levels. This allows organisms to meet high energetic demands of activity for life history. Dispersal capability should therefore explicitly be considered as a potential factor driving the evolution of metabolic capacities.

Seasonal changes in mitochondrial bioenergetics and physiological performance of the bluegill sunfish, Lepomis macrochirus, from a shallow, Midwest river.

As global temperature shifts due to anthropogenic impacts, seasonal temperatures in shallow aquatic ecosystems are expected to increase. Previous studies on freshwater fishes that experience significant temperature changes during the annual seasons found pronounced physiological restructuring not observed in animals inhabiting more thermally stable environments. Studies evaluating mitochondrial bioenergetics in fish are often performed on animals acclimated to constant temperatures in the laboratory. However, natural habitats are much more complex. Fishes may experience substantial daily and seasonal variation in temperature, energy requirements and resource availability, which are impossible to emulate on acclimation studies. Our study explores the effects of these more complex natural environments on whole-organism thermal tolerance and mitochondrial bioenergetics in bluegill sunfish (Lepomis macrochirus), a native fish to the temperate zone of North America. Compensatory mechanisms and variations in physiological thresholds were observed in specimens acclimatized to the fall season compared to specimens acclimatized to spring and summer seasons. Somatic indices, such as relative weights and hepatosomatic indices, showed significant differences across seasons and critical thermal maxima significantly decreased in the cold acclimatized specimens. Liver mitochondria from L. macrochirus also showed significantly higher uncoupled proton conductance, cytochrome c oxidase (COX) activity, and reduced respiratory control ratios in individuals sampled in the colder season. These findings suggest that mechanisms regulating proton conductance and COX activity modulate mitochondrial function across seasons to sustain physiological fitness in ectotherms inhabiting shallow, inland aquatic habitats.

The Mitochondrial Protein MitoNEET as a Probe for the Allostery of Glutamate Dehydrogenase.

The proteins glutamate dehydrogenase (GDH) and mitoNEET are both targets of drug development efforts to treat metabolic disorders, cancer, and neurodegenerative diseases. However, these two proteins differ starkly in the current knowledge about ligand binding sites. MitoNEET is a [2Fe-2S]-containing protein with no obvious binding site for small ligands observed in its crystal structures. In contrast, GDH is known to have a variety of ligands at multiple allosteric sites thereby leading to complex regulation in activity. In fact, while GDH can utilize either NAD(H) or NADP(H) for catalysis at the active site, only NAD(H) binds at a regulatory site to inhibit GDH activity. Previously, we found that mitoNEET forms a covalent bond with GDH in vitro and increases the catalytic activity of the enzyme. In this study we evaluated the effects of mitoNEET binding on the allosteric control of GDH conferred by inhibitors. We examined all effectors using NAD or NADP as the coenzyme to determine allosteric linkage by the NAD-binding regulatory site. We found that GDH activity, in the presence of the inhibitory palmitoyl-CoA and EGCG, can be rescued by mitoNEET, regardless of the coenzyme used. This suggests that mitoNEET rescues GDH by stabilizing the open conformation.

Sonoporation enables high-throughput loading of trehalose into red blood cells.

Despite recent advances in biostabilization, clinical blood supplies still experience shortages and storage limitations for red blood cells (RBCs) have not yet been sufficiently addressed. Storing RBCs in a frozen or dried state is an appealing solution to address storage limitations, but many promising cryoprotectants, including the non-reducing sugar trehalose, are impermeant to mammalian cell membranes and cannot be utilized effectively using currently available compound-loading methods. We found that transient pore formation induced by ultrasound and microbubbles (sonoporation) offers an effective means of loading trehalose into RBCs to facilitate long-term storage in a frozen or desiccated state. The protective potential of trehalose loading was demonstrated by freezing processed RBCs at -1 °C/min to -80 °C, then either storing the cells at -80 °C or lyophilizing them. RBCs were either thawed or rehydrated after 42 days of storage and evaluated for membrane integrity and esterase activity to estimate recovery and cell viability. The intracellular concentration of trehalose reached 40 mM after sonoporation and over 95% of treated RBCs were recovered after loading. Loading of trehalose was sufficient to maintain RBC morphology and esterase activity in most cells during freezing (>90% RBC recovery) and to a lower degree after lyophilization and rehydration (>20% recovery). Combining sonoporation with an integrated fluidics device allowed for rapid loading of up to 70 mM trehalose into RBCs. These results demonstrate the potential of sonoporation-mediated trehalose loading to increase recovery of viable RBCs, which could lead to effective methods for long-term stabilization of RBCs.

Acoustofluidic-mediated molecular delivery to human T cells with a three-dimensional-printed flow chamber.

Cell-based therapies have garnered significant interest to treat cancer and other diseases. Acoustofluidic technologies are in development to improve cell therapy manufacturing by facilitating rapid molecular delivery across the plasma membrane via ultrasound and microbubbles (MBs). In this study, a three-dimensional (3D) printed acoustofluidic device was used to deliver a fluorescent molecule, calcein, to human T cells. Intracellular delivery of calcein was assessed after varying parameters such as MB face charge, MB concentration, flow channel geometry, ultrasound pressure, and delivery time point after ultrasound treatment. MBs with a cationic surface charge caused statistically significant increases in calcein delivery during acoustofluidic treatment compared to MBs with a neutral surface charge (p < 0.001). Calcein delivery was significantly higher with a concentric spiral channel geometry compared to a rectilinear channel geometry (p < 0.001). Additionally, calcein delivery was significantly enhanced at increased ultrasound pressures of 5.1 MPa compared to lower ultrasound pressures between 0-3.8 MPa (p < 0.001). These results demonstrate that a 3D-printed acoustofluidic device can significantly enhance intracellular delivery of biomolecules to T cells, which may be a viable approach to advance cell-based therapies.

4-Hydroxynonenal and 4-Oxononenal Differentially Bind to the Redox Sensor MitoNEET.

MitoNEET is a CDGSH iron-sulfur protein that has been a target for drug development for diseases such as type-2 diabetes, cancer, and Parkinson's disease. Functions proposed for mitoNEET are as a redox sensor and regulator of free iron in the mitochondria. We have investigated the reactivity of mitoNEET toward the reactive electrophiles 4-hydroxynonenal (HNE) and 4-oxononenal (ONE) that are produced from the oxidation of polyunsaturated fatty acid during oxidative stress. Proteomic, electrophoretic, and spectroscopic analysis has shown that HNE and ONE react in a sequence selective manner that was unexpected considering the structure similarity of these two reactive electrophiles.

Binding of thiazolidinediones to the endoplasmic reticulum protein nutrient-deprivation autophagy factor-1.

Nutrient-deprivation autophagy factor-1 (NAF-1, miner1; gene cisd2) is part of the [2Fe-2S]-containing protein family which includes mitoNEET (gene cisd1) and MiNT (miner2; gene cisd3). These proteins are redox active and are thought to play an important role in cellular energy homeostasis with NAF-1 playing a critical role in calcium regulation and aging. To date, no studies have investigated potential ligand interaction with NAF-1. Here we show that the thiazolidinediones pioglitazone and rosiglitazone along with the mitoNEET ligand, NL-1, bind to NAF-1 with low micromolar affinities. Further, we show that overexpression of NAF-1 in hepatocellular carcinoma (HepG2) cells reduces inhibition of mitochondrial respiration by pioglitazone. Our findings support the need for further efforts of the rational design of selective NAF-1 ligands.

Role of Intrinsic Disorder in Animal Desiccation Tolerance.

This review compares the molecular strategies employed by anhydrobiotic invertebrates to survive extreme water stress. Intrinsically disordered proteins (IDPs) play a central role in desiccation tolerance in all species investigated. Various hypotheses about the functions of anhydrobiosis-related intrinsically disordered (ARID) proteins, including late embryogenesis abundant (LEA) and tardigrade-specific intrinsically disordered proteins, are evaluated by broad sequence characterization. A surprisingly wide range in sequence characteristics, including hydropathy and the frequency and distribution of charges, is discovered. Interestingly, two clusters of similar proteins are found that potentially correlate with distinct functions. This may indicate two broad groups of ARID proteins, composed of one group that folds into functional conformations during desiccation and a second group that potentially displays functions in the hydrated state. A broad range of physiochemical properties suggest that folding may be induced by factors such as hydration level, molecular crowding, and interactions with binding partners. This plasticity may be required to fine-tune the ARID-proteome response at different hydration levels during desiccation. Furthermore, the sequence properties of some LEA proteins share qualities with IDPs known to undergo liquid-liquid phase separations during environmental challenges.

Modulation of cellular energetics by galactose and pioglitazone.

The Warburg effect is ameliorated by culturing transformed cells in the presence of galactose instead of glucose as the primary carbon source. However, metabolic consequences may occur in addition to sensitizing the cells to mitochondrial toxins. The screening of pharmaceutical agents against transformed cells while using galactose must therefore be carefully evaluated. Pioglitazone is employed in clinical applications to treat type-2 diabetes but clearly has other off-target effects. Human hepatocellular carcinoma cells (HepG2) were cultured in glucose or galactose-containing medium to investigate the role of pioglitazone on cellular bioenergetics by calorimetry and respirometry. Compared with cells cultured in 10 mM glucose, HepG2 cells cultured in the presence of 10 mM galactose showed decreased metabolic activity as measured by cellular heat flow. Interestingly, cellular heat flow increased after the addition of pioglitazone for cells cultured in glucose, but not for cells cultured in galactose. Our calorimetric data indicated that a reduction in cellular capacity for glycolysis was the mechanism responsible for the increase in sensitivity to pioglitazone, and possibly to mitochondrial toxins in general, for cells cultured in galactose. Furthermore, oxygen consumption rates were decreased after the addition of pioglitazone to cells grown in glucose but remained unchanged for cells grown in the presence of galactose. We have demonstrated that pioglitazone induces a reduction in mitochondrial activity that is partially compensated via an increase in glycolysis in the presence of glucose.

Effect of trehalose as an additive to dimethyl sulfoxide solutions on ice formation, cellular viability, and metabolism.

Cryopreservation is the only established method for long-term preservation of cells and cellular material. This technique involves preservation of cells and cellular components in the presence of cryoprotective agents (CPAs) at liquid nitrogen temperatures (-196 °C). The organic solvent dimethyl sulfoxide (Me2SO) is one of the most commonly utilized CPAs and has been used with various levels of success depending on the type of cells. In recent years, to improve cryogenic outcomes, the non-reducing disaccharide trehalose has been used as an additive to Me2SO-based freezing solutions. Trehalose is a naturally occurring non-toxic compound found in bacteria, fungi, plants, and invertebrates which has been shown to provide cellular protection during water-limited states. The mechanism by which trehalose improves cryopreservation outcomes remains not fully understood. Raman microspectroscopy is a powerful tool to provide valuable insight into the nature of interactions among water, trehalose, and Me2SO during cryopreservation. We found that the addition of trehalose to Me2SO based CPA solutions dramatically reduces the area per ice crystals while increasing the number of ice crystals formed when cooled to -40 or -80 °C. Differences in ice-formation patterns were found to have a direct impact on cellular viability. Despite the osmotic stress caused by addition of 100 mM trehalose, improvement in cellular viability was observed. However, the substantial increase in osmotic pressure caused by trehalose concentrations above 100 mM may offset the beneficial effects of changing the morphology of the ice crystals achieved by addition of this sugar.

Physiological performance of warm-adapted marine ectotherms: Thermal limits of mitochondrial energy transduction efficiency.

Thermal regimes in aquatic systems have profound implications for the physiology of ectotherms. In particular, the effect of elevated temperatures on mitochondrial energy transduction in tropical and subtropical teleosts may have profound consequences on organismal performance and population viability. Upper and lower whole-organism critical temperatures for teleosts suggest that subtropical and tropical species are not susceptible to the warming trends associated with climate change, but sub-lethal effects on energy transduction efficiency and population dynamics remain unclear. The goal of the present study was to compare the thermal sensitivity of processes associated with mitochondrial energy transduction in liver mitochondria from the striped mojarra (Eugerres plumieri), the whitemouth croaker (Micropogonias furnieri) and the palometa (Trachinotus goodei), to those of the subtropical pinfish (Lagodon rhomboides) and the blue runner (Caranx crysos). Mitochondrial function was assayed at temperatures ranging from 10 to 40°C and results obtained for both tropical and subtropical species showed a reduction in the energy transduction efficiency of the oxidative phosphorylation (OXPHOS) system in most species studied at temperatures below whole-organism critical temperature thresholds. Our results show a loss of coupling between O2 consumption and ATP production before the onset of the critical thermal maxima, indicating that elevated temperature may severely impact the yield of ATP production per carbon unit oxidized. As warming trends are projected for tropical regions, increasing water temperatures in tropical estuaries and coral reefs could impact long-term growth and reproductive performance in tropical organisms, which are already close to their upper thermal limit.

Molecular approaches for improving desiccation tolerance: insights from the brine shrimp Artemia franciscana.

MAIN CONCLUSION: We have evaluated the endogenous expression and molecular properties of selected Group 3 LEA proteins from Artemia franciscana , and the capacity of selected Groups 1 and 3 proteins transfected into various desiccation-sensitive cell lines to improve tolerance to drying. Organisms inhabiting both aquatic and terrestrial ecosystems frequently are confronted with the problem of water loss for multiple reasons--exposure to hypersalinity, evaporative water loss, and restriction of intracellular water due to freezing of extracellular fluids. Seasonal desiccation can become severe and lead to the production of tolerant propagules and entry into the state of anhydrobiosis at various stages of the life cycle. Such is the case for gastrula-stage embryos of the brine shrimp, Artemia franciscana. Physiological and biochemical responses to desiccation are central for survival and are multifaceted. This review will evaluate the impact of multiple late embryogenesis abundant proteins originating from A. franciscana, together with the non-reducing sugar trehalose, on prevention of desiccation damage at multiple levels of biological organization. Survivorship of desiccation-sensitive cells during water stress can be improved by use of the above protective agents, coupled to metabolic preconditioning and rapid cell drying. However, obtaining long-term stability of cells in the dried state at room temperature has not been accomplished and will require continued efforts on both the physicochemical and biological fronts.

Protective effects of osmolytes in cryopreserving adherent neuroblastoma (Neuro-2a) cells.

A simple method to cryopreserve adherent monolayers of neuronal cells is currently not available, but the development of this technique could facilitate numerous applications in the field of biomedical engineering, cell line development, and drug screening. However, complex tissues of some exceptional animals survive freezing in nature. These animals are known to accumulate several small molecular weight solutes prior to freezing. Following a similar strategy, we investigated the effects of osmolytes such as trehalose, proline, and sucrose as additives to the traditional cryoprotectant dimethyl sulfoxide (Me2SO) in modulating the cryopreservation outcome of mouse neuroblastoma (Neuro-2a) cells. Neuro-2a cells adhered to cell culture plates were incubated for 24 h at varying concentrations of trehalose, proline, sucrose and combinations of these compounds. Cells were cryopreserved for 24 h and cell viability post-freezing and thawing was quantified by trypan blue exclusion assay. On average, only 13.5% of adherent cells survived freezing in the presence of 10% Me2SO alone (control). Pre-incubation of cells with medium containing both trehalose and proline severely decreased cell proliferation, but increased cell recovery to about 53% of control. Furthermore, characterization using Raman microspectroscopy revealed that the addition of both trehalose and proline to 10% Me2SO substantially increased the size, and altered the nature, of ice crystals formed during freezing. Our results suggest that pre-incubation of Neuro-2a cells with trehalose and proline in combination provides cell protection along with alterations of ice structure in order to increase cell survival post-freezing.

Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation.

Expression of late embryogenesis abundant (LEA) proteins is highly correlated with desiccation tolerance in anhydrobiotic animals, selected land plants, and bacteria. Genes encoding two LEA proteins, one localized to the cytoplasm/nucleus (AfrLEA2) and one targeted to mitochondria (AfrLEA3m), were stably transfected into human HepG2 cells. A trehalose transporter was used for intracellular loading of this disaccharide. Cells were rapidly and uniformly desiccated to low water content (<0.12 g H(2)O/g dry weight) with a recently developed spin-drying technique. Immediately on rehydration, control cells without LEA proteins or trehalose exhibited 0% membrane integrity, compared with 98% in cells loaded with trehalose and expressing AfrLEA2 or AfrLEA3m; surprisingly, AfrLEA3m without trehalose conferred 94% protection. Cell proliferation across 7 d showed an 18-fold increase for cells dried with AfrLEA3m and trehalose, compared with 27-fold for nondried controls. LEA proteins dramatically enhance desiccation tolerance in mammalian cells and offer the opportunity for engineering biostability in the dried state.

LEA proteins during water stress: not just for plants anymore.

Late embryogenesis abundant (LEA) proteins are extremely hydrophilic proteins that were first identified in land plants. Intracellular accumulation is tightly correlated with acquisition of desiccation tolerance, and data support their capacity to stabilize other proteins and membranes during drying, especially in the presence of sugars like trehalose. Exciting reports now show that LEA proteins are not restricted to plants; multiple forms are expressed in desiccation-tolerant animals from at least four phyla. We evaluate here the expression, subcellular localization, biochemical properties, and potential functions of LEA proteins in animal species during water stress. LEA proteins are intrinsically unstructured in aqueous solution, but surprisingly, many assume their native conformation during drying. They are targeted to multiple cellular locations, including mitochondria, and evidence supports that LEA proteins stabilize vitrified sugar glasses thought to be important in the dried state. More in vivo experimentation will be necessary to fully unravel the multiple functional properties of these macromolecules during water stress.

Cryopreservation of hepatocyte (HepG2) cell monolayers: impact of trehalose.

A simple method to cryogenically preserve hepatocyte monolayers is currently not available but such a technique would facilitate numerous applications in the field of biomedical engineering, cell line development, and drug screening. We investigated the effect of trehalose and dimethyl sulfoxide (Me2SO) in cryopreservation of human hepatocellular carcinoma (HepG2) cells in suspension and monolayer formats. HepG2 cell monolayers were incubated for 24h at varying concentrations of trehalose (50-150 mM) prior to cryopreservation to identify the optimum concentration for such preincubation. When trehalose alone was used as the cryoprotective agent (CPA), cells in monolayer format did not survive freezing while cells in suspension demonstrated 14% viability 24h after thawing. Only 6-13% of cells in monolayers survived freezing in cell culture medium supplemented with 10% Me2SO, but 42% of cells were recovered successfully if monolayers were preincubated with 100 mM trehalose prior to freezing in the Me2SO supplemented medium. Interestingly, for cells frozen in suspension in presence of 10% Me2SO, metabolic activity immediately following thawing did not change appreciably compared to unfrozen control cells. Finally, Raman spectroscopy techniques were employed to evaluate ice crystallization in the presence and absence of trehalose in freezing solutions without cells because crystallization may alter the extent of injury observed in cell monolayers. We speculate that biomimetic approaches of using protective sugars to preserve cells in monolayer format will facilitate the development of techniques for long-term preservation of human tissues and organs in the future.

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%. 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.