Characterization of sub-nuclear changes in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> embryos exposed to brief,intermediate and long-term anoxia to analyze anoxia-induced cell cycle arrest

Background  

The soil nematode C. elegans survives oxygen-deprived conditions (anoxia; <.001 kPa O₂) by entering into a state of suspended animation in which cell cycle progression reversibly arrests. The majority of blastomeres of embryos exposed to anoxia arrest at interphase, prophase and metaphase. The spindle checkpoint proteins SAN-1 and MDF-2 are required for embryos to survive 24 hours of anoxia. To further investigate the mechanism of cell-cycle arrest we examined and compared sub-nuclear changes such as chromatin localization pattern, post-translational modification of histone H3, spindle microtubules, and localization of the spindle checkpoint protein SAN-1 with respect to various anoxia exposure time points. To ensure analysis of embryos exposed to anoxia and not post-anoxic recovery we fixed all embryos in an anoxia glove box chamber.     

Anoxia-Reoxygenation Regulates Mitochondrial Dynamics through the Hypoxia Response Pathway,SKN-1/Nrf,and Stomatin-Like Protein STL-1/SLP-2

Many aerobic organisms encounter oxygen-deprived environments and thus must have adaptive mechanisms to survive such stress. It is important to understand how mitochondria respond to oxygen deprivation given the critical role they play in using oxygen to generate cellular energy. Here we examine mitochondrial stress response in C. elegans, which adapt to extreme oxygen deprivation (anoxia, less than 0.1% oxygen) by entering into a reversible suspended animation state of locomotory arrest. We show that neuronal mitochondria undergo DRP-1-dependent fission in response to anoxia and undergo refusion upon reoxygenation. The hypoxia response pathway, including EGL-9 and HIF-1, is not required for anoxia-induced fission, but does regulate mitochondrial reconstitution during reoxygenation. Mutants for egl-9 exhibit a rapid refusion of mitochondria and a rapid behavioral recovery from suspended animation during reoxygenation; both phenotypes require HIF-1. Mitochondria are significantly larger in egl-9 mutants after reoxygenation, a phenotype similar to stress-induced mitochondria hyperfusion (SIMH). Anoxia results in mitochondrial oxidative stress, and the oxidative response factor SKN-1/Nrf is required for both rapid mitochondrial refusion and rapid behavioral recovery during reoxygenation. In response to anoxia, SKN-1 promotes the expression of the mitochondrial resident protein Stomatin-like 1 (STL-1), which helps facilitate mitochondrial dynamics following anoxia. Our results suggest the existence of a conserved anoxic stress response involving changes in mitochondrial fission and fusion.     

Patterns of ubiquitylation and SUMOylation associated with exposure to anoxia in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus acquire extreme tolerance to anoxia during embryonic development. These embryos can survive environmental and cellular conditions that would likely result in death in the majority of vertebrate cells, despite experiencing a massive loss of ATP. It is highly likely that the initial response to anoxia must quickly alter cellular physiology to reprogram cell signaling and metabolic pathways to support anaerobiosis. Covalent protein modifications are a mechanism that can quickly act to effect large-scale changes in protein structure and function and have been suggested by others to play a key role in mammalian ischemia tolerance. Using Western blot analysis, we explored patterns of protein ubiquitylation and SUMOylation in embryos of A. limnaeus exposed to anoxia and anoxic preconditioning. Surprisingly, we report stage-specific protein ubiquitylation patterns that suggest different mechanisms for altering protein turnover in dormant and actively developing embryos that both survive long-term anoxia. Anoxic preconditioning does not appear to alter levels of ubiquitin conjugates in a unique manner. Global SUMOylation of proteins does not change in response to anoxia, but there are stage-specific changes in SUMOylation of specific protein bands. Contrary to other systems, global changes in protein SUMOylation may not be required to support long-term tolerance to anoxia in embryos of A. limnaeus. These data lead us to conclude that embryos of A. limnaeus respond to anoxia in a unique manner compared to other vertebrate models of anoxia tolerance and may provide novel mechanisms for engineering vertebrate tissues to survive long-term anoxia.     

NPP-16/Nup50 Function and CDK-1 Inactivation Are Associated with Anoxia-induced Prophase Arrest in Caenorhabditis elegans

Oxygen, an essential nutrient, is sensed by a multiple of cellular pathways that facilitate the responses to and survival of oxygen deprivation. The Caenorhabditis elegans embryo exposed to severe oxygen deprivation (anoxia) enters a state of suspended animation in which cell cycle progression reversibly arrests at specific stages. The mechanisms regulating interphase, prophase, or metaphase arrest in response to anoxia are not completely understood. Characteristics of arrested prophase blastomeres and oocytes are the alignment of condensed chromosomes at the nuclear periphery and an arrest of nuclear envelope breakdown. Notably, anoxia-induced prophase arrest is suppressed in mutant embryos lacking nucleoporin NPP-16/NUP50 function, indicating that this nucleoporin plays an important role in prophase arrest in wild-type embryos. Although the inactive form of cyclin-dependent kinase (CDK-1) is detected in wild-type–arrested prophase blastomeres, the inactive state is not detected in the anoxia exposed npp-16 mutant. Furthermore, we found that CDK-1 localizes near chromosomes in anoxia-exposed embryos. These data support the notion that NPP-16 and CDK-1 function to arrest prophase blastomeres in C. elegans embryos. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.     

Cell cycle arrest associated with anoxia-induced quiescence, anoxic preconditioning, and embryonic diapause in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus can enter into dormancy associated with diapause and anoxia-induced quiescence. Dormant embryos are composed primarily of cells arrested in the G(1)/G(0) phase of the cell cycle based on flow cytometry analysis of DNA content. In fact, most cells in developing embryos contain only a diploid complement of DNA, with very few cells found in the S, G(2), or M phases of the cell cycle. Diapause II embryos appear to be in a G(0)-like state with low levels of cyclin D1 and p53. However, the active form of pAKT is high during diapause II. Exposure to anoxia causes an increase in cyclin D1 and p53 expression in diapause II embryos, suggesting a possible re-entry into the cell cycle. Post-diapause II embryos exposed to anoxia or anoxic preconditioning have stable levels of cyclin D1 and stable or reduced levels of p53. The amount of pAKT is severely reduced in 12?dpd embryos exposed to anoxia or anoxic preconditioning. This study is the first to evaluate cell cycle control in embryos of A. limnaeus during embryonic diapause and in response to anoxia and builds a foundation for future research on the role of cell cycle arrest in supporting vertebrate dormancy.     

Long-term anoxia in encysted embryos of the crustacean, Artemia franciscana: viability, ultrastructure, and stress proteins

Cells of encysted embryos of Artemia franciscana, the brine shrimp, are among the most resistant of all animal cells to extremes of environmental stress. We focus here on their ability to survive continuous anoxia for periods of years, during which their metabolic rate is undetectable. We asked whether their impressive tolerance was reflected in changes at the ultrastructural level. The ultrastructure of encysted embryos previously experiencing 38 days and 3.3 years of anoxia was compared with those not undergoing anoxia (controls). Rough endoplasmic reticulum was abundant in anoxic embryos, in spite of the absence of protein biosynthesis in their cells. Other cytoplasmic changes had occurred in the anoxic cells, but overall their structure was remarkably intact, in view of their 3 years of continuous anoxia. A major difference was the presence of abundant electron-dense granules in the nuclei of anoxic embryos; these were present but rare in nuclei of controls. Biochemical fractionation and Western immunoblotting confirmed previous observations that substantial amounts of the small heat shock/alpha-crystallin protein (p26) translocated into nuclei of anoxic embryos. We have no evidence that the dense granules contain this protein, but that remains a possibility. In contrast, and contrary to expectation, proteins of the hsp70 and 90 families did not undergo anoxia-induced nuclear translocation, an unusual result since such translocations have been widely observed in cells from a variety of organisms.     

Respiration of Artemia franciscana embryos after continuous anoxia over 1-year period

Encysted embryos of the brine shrimp, Artemia franciscana, exhibit extraordinary longevity when exposed to continuous anoxia. To explore the metabolic basis of this ability, the post-anoxic respiration of embryos exposed to anoxia for periods exceeding 1 year was measured. Since anoxic metabolism might result in the accumulation of metabolic end products, an O₂ debt would be expected. Contrary to that expectation, post-anoxic embryos exhibited a marked depression in respiration rate whether embryos were hydrated under anoxic conditions or were exposed to a previous aerobic incubation and then placed under anoxia. These results, and those of previous studies, suggest that extended anoxia may bring the metabolism of these embryos to a reversible standstill.     

Environmental and genetic preconditioning for long-term anoxia responses requires AMPK in Caenorhabditis elegans

Background

Preconditioning environments or therapeutics, to suppress the cellular damage associated with severe oxygen deprivation, is of interest to our understanding of diseases associated with oxygen deprivation. Wildtype C. elegans exposed to anoxia enter into a state of suspended animation in which energy-requiring processes reversibly arrest. C. elegans at all developmental stages survive 24-hours of anoxia exposure however, the ability of adult hermaphrodites to survive three days of anoxia significantly decreases. Mutations in the insulin-like signaling receptor (daf-2) and LIN-12/Notch (glp-1) lead to an enhanced long-term anoxia survival phenotype.

Methodology/Principal Findings

In this study we show that the combined growth environment of 25°C and a diet of HT115 E. coli will precondition adult hermaphrodites to survive long-term anoxia; many of these survivors have normal movement after anoxia treatment. Animals fed the drug metformin, which induces a dietary-restriction like state in animals and activates AMPK in mammalian cell culture, have a higher survival rate when exposed to long-term anoxia. Mutations in genes encoding components of AMPK (aak-2, aakb-1, aakb-2, aakg-2) suppress the environmentally and genetically induced long-term anoxia survival phenotype. We further determine that there is a correlation between the animals that survive long-term anoxia and increased levels of carminic acid staining, which is a fluorescent dye that incorporates in with carbohydrates such as glycogen.

Conclusions/Significance

We conclude that small changes in growth conditions such as increased temperature and food source can influence the physiology of the animal thus affecting the responses to stress such as anoxia. Furthermore, this supports the idea that metformin should be further investigated as a therapeutic tool for treatment of oxygen-deprived tissues. Finally, the capacity for an animal to survive long bouts of severe oxygen deprivation is likely dependent on specific subunits of the heterotrimeric protein AMPK and energy stores such as carbohydrates.     

Protection of cellular and mitochondrial functions against anoxic damage by fructose in perfused liver.

In anoxic perfused liver, conversion of fructose to lactate was greatly increased to about 3 mumol/min per g liver. This increase in lactate implied that the same amount of ATP was also produced. The rate of metabolism of glucose was less than 10% of that of fructose, as judged by rate of production of lactate. In anoxic liver perfused with fructose, the ATP levels of both the tissue and mitochondria remained high, despite lack of oxygen, thus preventing enzyme leakage and preserving processes requiring ATP, such as bile excretion and urea formation. The mitochondrial oxidative phosphorylation capacity of anoxic liver perfused with fructose was also unimpaired. Spectral analysis of light transmitted through the liver revealed that the mitochondrial electron transfer system was in the completely reduced state during anoxia, indicating that the mitochondria were incapable of synthesizing ATP. These results suggest that fructose metabolism during anoxia resulted in sufficient production of ATP for maintaining the physiological functions of the cells and the oxidative phosphorylation capacity of their mitochondria.     

Protein synthesis by rice coleoptiles during prolonged anoxia: implications for glycolysis, growth and energy utilization

BACKGROUND AND AIMS: Anoxia-tolerant plant tissues synthesize a number of proteins during anoxia, in addition to the 'classical anaerobic proteins' involved in glycolysis and fermentation. The present study used a model system of rice coleoptile tips to elucidate patterns of protein synthesis in this anoxia-tolerant plant tissue. METHODS: Coleoptile tips 7-11 mm long were excised from intact seedlings exposed to anoxia, or excised from hypoxically pre-treated seedlings and then exposed to anoxia for 72 h. Total proteins or 35S-labelled proteins were extracted, separated using two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis and analysed using mass spectrometry. KEY RESULTS: The coleoptile tips excised after intact seedlings had been exposed to anoxia for 72 h had a similar proteome to tips that were first excised and then exposed to anoxia. After 72 h anoxia, Bowman-Birk trypsin inhibitors and a glycine-rich RNA-binding protein decreased in abundance, whereas a nucleoside diphosphate kinase and several proteins with unknown functions were strongly enhanced. Using [35S]methionine as label, proteins synthesized at high levels in anoxia, and also in aeration, included a nucleoside diphosphate kinase, a glycine-rich RNA-binding protein, a putative elicitor-inducible protein and a putative actin-depolymerizing factor. Proteins synthesized predominately in anoxia included a pyruvate orthophosphate dikinase (PPDK), alcohol dehydrogenase 1 and 2, fructose 1,6-bisphosphate aldolase and a protein of unknown function. CONCLUSION: The induction of PPDK in anoxic rice coleoptiles might, in combination with pyruvate kinase (PK), enable operation of a 'substrate cycle' producing PPi from ATP. Production of PPi would (a) direct energy to crucial transport processes across the tonoplast (i.e. the H+-PPiase); (b) be required for sucrose hydrolysis via sucrose synthase; and (c) enable acceleration of glycolysis, via pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) acting in parallel with phosphofructokinase (PFK), thus enhancing ATP production in anoxic rice coleoptiles; ATP production would need to be increased if there was a substantial requirement for PPi.     

Diguanosine nucleotide metabolism and the survival of artemia embryos during years of continuous anoxia.

Encysted embryos of the primitive crustacean, Artemia franciscana, are remarkably resistant to a variety of harsh environmental conditions, including continuous anoxia for periods of years at physiological temperatures and water contents. Previous study produced no evidence of an ongoing anoxic metabolism, suggesting that these embryos remained viable in spite of the lack of detectable free energy flow and biosynthesis. That seeming violation of a major axiom of cell biology and biochemistry prompted us to re-examine the nucleotide pool of encysted embryos during prolonged anoxia. We found that the nucleotide Gp(4)G, present initially in very large amounts, decreased slowly as anoxia continued over the 5.6-year period examined. Studies on other nucleotides and associated enzymes, including results from previous papers, provide a plausible metabolic pathway leading to the provision of ATP and GTP to meet the needs of endergonic processes in anoxic embryos. Exactly what those processes are is not obvious. One possibility involves the extensive anoxia-induced nuclear translocation of the stress protein, molecular chaperone p26, whose large molecular mass (approximately 500 kDa) most likely requires a supply of free energy to cross the nuclear envelope. Support for this possibility comes from our finding here that p26 is also a GTPase.     

Nucleotide Levels Do Not Critically Determine Survival of Maize Root Tips Acclimated to a Low-Oxygen Environment

We tested the hypothesis that ATP levels and energy charge determine the resistance of maize (Zea mays) root tips to anoxia. We focused on root tips of whole maize seedlings that had been acclimated to low O2 by exposure to an atmosphere of 3% (v/v) O2 in N2. Acclimated anoxic root tips characteristically have higher ATP levels and energy charge and survive longer under anoxia than nonacclimated tips. We poisoned intact, acclimated root tips with either fluoride or mannose, causing decreases in ATP and energy charge to values similar to or, in most cases, below those found in nonacclimated anoxic tips. With the exception of the highest fluoride concentration used, the poisoned, acclimated tips remained much more tolerant of anoxia than nonacclimated root tips. We conclude that high ATP and energy charge are not components critical for the survival of acclimated root tips during anoxia. The reduced nucleotide status in poisoned, acclimated root tips had little effect on cytoplasmic pH regulation during anoxia. This result indicates that in anoxic, acclimated root tips either cytoplasmic pH regulation is not dominated by ATP-dependent processes or these processes can continue in vivo largely independently of any changes in ATP levels in the physiological range. The role of glycolytic flux in survival under anoxia is discussed.     

Regulation of tail muscle arginine kinase by reversible phosphorylation in an anoxia-tolerant crayfish

Freshwater crayfish, Orconectes virilis, can experience periodic exposures to hypoxia or anoxia due to low water flow (in summer) or ice cover (in winter) in their natural habitat. Hypoxia/anoxia disrupts energy metabolism and triggers mechanisms that to support ATP levels while often also suppressing ATP use. Arginine kinase (AK) (E.C. 2.7.3.3) is a crucial enzyme involved in energy metabolism in muscle, gating the use of phosphagen stores to buffer ATP levels. The present study investigated AK from tail muscle of O. virilis identifying changes to kinetic properties, phosphorylation state and structural stability between the enzyme from aerobic control and 20 h anoxic crayfish. Muscle AK from anoxia-exposed crayfish showed a significantly higher (by 59%) K _m for l-arginine and a lower I₅₀ value for urea than the aerobic form. Several lines of evidence indicated that AK was converted to a high phosphate form under anoxia: (a) aerobic and anoxic forms of AK showed well-separated elution peaks on DEAE ion exchange chromatography, (b) ProQ Diamond phosphoprotein staining showed a 64% higher bound phosphate content on anoxic AK compared with the aerobic form, and (c) treatment of anoxic AK with alkaline phosphatase reduced K _m l-arginine to aerobic levels whereas incubation of aerobic AK with protein kinase A catalytic subunit raised the K _m to anoxic levels. The physiological consequence of anoxia-induced AK phosphorylation may be to suppress AK activity in the phosphagen-synthesizing direction and, together with reduced cellular pH and ATP levels, promote the phosphagen-catabolizing direction under anoxic conditions. This is first time that AK has been shown to be regulated by reversible phosphorylation.     

Effects of anoxia and the mitochondrion on expression of aerobic nuclear COX genes in yeast: evidence for a signaling pathway from the mitochondrial genome to the nucleus

Eucaryotic cells contain at least two general classes of oxygen-regulated nuclear genes: aerobic genes and hypoxic genes. Hypoxic genes are induced upon exposure to anoxia while aerobic genes are down-regulated. Recently, it has been reported that induction of some hypoxic nuclear genes in mammals and yeast requires mitochondrial respiration and that cytochrome-c oxidase functions as an oxygen sensor during this process. In this study, we have examined the role of the mitochondrion and cytochrome-c oxidase in the expression of yeast aerobic nuclear COX genes. We have found that the down-regulation of these genes in anoxic cells is reflected in reduced levels of their subunit polypeptides and that cytochrome-c oxidase subunits I, II, III, Vb, VI, VII, and VIIa are present in promitochondria from anoxic cells. By using nuclear cox mutants and mitochondrial rho(0) and mit(-) mutants, we have found that neither respiration nor cytochrome-c oxidase is required for the down-regulation of these genes in cells exposed to anoxia but that a mitochondrial genome is required for their full expression under both normoxic and anoxic conditions. This requirement for a mitochondrial genome is unrelated to the presence or absence of a functional holocytochrome-c oxidase. We have also found that the down-regulation of these genes in cells exposed to anoxia and the down-regulation that results from the absence of a mitochondrial genome are independent of one another. These findings indicate that the mitochondrial genome, acting independently of respiration and oxidative phosphorylation, affects the expression of the aerobic nuclear COX genes and suggest the existence of a signaling pathway from the mitochondrial genome to the nucleus.     

Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog,Rana sylvatica

The wood frog (Rana sylvatica) can survive the winter in a frozen state, in which the frog’s tissues are also exposed to dehydration, ischemia, and anoxia. Critical to wood frog survival under these conditions is a global metabolic rate depression, the accumulation of glucose as a cryoprotectant, and a reliance on anaerobic glycolysis for energy production. Pyruvate kinase (PK) catalyzes the final reaction of aerobic glycolysis, generating pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP. This study investigated the effect of each stress condition experienced by R. sylvatica during freezing, including dehydration and anoxia, on PK regulation. PK from muscle of frozen and dehydrated frogs exhibited a lower affinity for PEP (K_m = 0.098 ± 0.003 and K_m = 0.092 ± 0.008) than PK from control and anoxic conditions (K_m = 0.065 ± 0.003 and K_m = 0.073 ± 0.002). Immunoblotting showed greater serine phosphorylation on muscle PK from frozen and dehydrated frogs relative to control and anoxic states, suggesting a reversible phosphorylation regulatory mechanism for PK activity during freezing stress. Furthermore, PK from frozen animals exhibited greater stability under thermal and urea-induced denaturing conditions than PK from control animals. Phosphorylation of PK during freezing may contribute to mediating energy conservation and maintaining intracellular cryoprotectant levels, as well as increase enzyme stability during stress.     

Suspended Animation Extends Survival Limits of Caenorhabditis elegans and Saccharomyces cerevisiae at Low Temperature

The orderly progression through the cell division cycle is of paramount importance to all organisms, as improper progression through the cycle could result in defects with grave consequences. Previously, our lab has shown that model eukaryotes such as Saccharomyces cerevisiae, Caenorhabditis elegans, and Danio rerio all retain high viability after prolonged arrest in a state of anoxia-induced suspended animation, implying that in such a state, progression through the cell division cycle is reversibly arrested in an orderly manner. Here, we show that S. cerevisiae (both wild-type and several cold-sensitive strains) and C. elegans embryos exhibit a dramatic decrease in viability that is associated with dysregulation of the cell cycle when exposed to low temperatures. Further, we find that when the yeast or worms are first transitioned into a state of anoxia-induced suspended animation before cold exposure, the associated cold-induced viability defects are largely abrogated. We present evidence that by imposing an anoxia-induced reversible arrest of the cell cycle, the cells are prevented from engaging in aberrant cell cycle events in the cold, thus allowing the organisms to avoid the lethality that would have occurred in a cold, oxygenated environment.     

Mitochondrial physiology of diapausing and developing embryos of the annual killifish Austrofundulus limnaeus: implications for extreme anoxia tolerance

Diapausing embryos of the annual killifish Austrofundulus limnaeus have the highest reported anoxia tolerance of any vertebrate and previous studies indicate modified mitochondrial physiology likely supports anoxic metabolism. Functional mitochondria isolated from diapausing and developing embryos of the annual killifish exhibited VO₂, respiratory control ratios (RCR), and P:O ratios consistent with those obtained from other ectothermic vertebrate species. Reduced oxygen consumption associated with dormancy in whole animal respiration rates are correlated with maximal respiration rates of mitochondria isolated from diapausing versus developing embryos. P:O ratios for developing embryos were similar to those obtained from adult liver, but were diminished in mitochondria from diapausing embryos suggesting decreased oxidative efficiency. Proton leak in adult liver corresponded with that of developing embryos but was elevated in mitochondria isolated from diapausing embryos. In metabolically suppressed diapause II embryos, over 95% of the mitochondrial oxygen consumption is accounted for by proton leak across the inner mitochondrial membrane. Decreased activity of mitochondrial respiratory chain complexes correlates with diminished oxidative capacity of isolated mitochondria, especially during diapause. Respiratory complexes exhibited suppressed activity in mitochondria with the ATP synthase exhibiting the greatest inhibition during diapause II. Mitochondria isolated from diapause II embryos are not poised to produce ATP, but rather to shuttle carbon and electrons through the Kreb’s cycle while minimizing the generation of a proton motive force. This particular mitochondrial physiology is likely a mechanism to avoid production of reactive oxygen species during large-scale changes in flux through oxidative phosphorylation pathways associated with metabolic transitions into and out of dormancy and anoxia.