Sean P. Place

Regulation of heat shock genes in isolated hepatocytes from an Antarctic fish, Trematomus bernacchii.

SUMMARY The Antarctic fishes, isolated over evolutionary history in the sub-zero waters of the Southern Ocean, are an ideal group for studying the processes of cold adaptation. One species of Antarctic notothenioid fish, Trematomus bernacchii, has lost the ability to induce heat shock proteins (Hsps) in response to exposure to acute thermal stress or to the toxic heavy metal cadmium, an important part of the cellular defense response to such stressors. To elucidate the mechanism responsible for the lack of Hsp induction, we examined several stages of the hsp gene expression pathway, including transcription factor activity, Hsp70 mRNA production and protein synthesis patterns, in hepatocytes from T. bernacchii. Hsp70 mRNA was detected, as was heat shock factor 1 (HSF1) with DNA-binding activity. However, exposure to elevated temperature and to chemical inducers of the heat shock response failed to increase Hsp70 mRNA levels, HSF1 activity or the concentration of any size class of Hsps. These results suggest that Hsps, inducible in nearly every other species, are expressed constitutively in the cold-adapted T. bernacchii.

Molecular Chaperones in Ectothermic Marine Animals: Biochemical Function and Gene Expression

Abstract The intertidal zone has historically functioned as an important natural laboratory for testing ideas about how physical factors such as temperature influence organismal physiology and in turn influence the distribution patterns of organisms. Key to our understanding of how the physical environment helps structure organismal distribution is the identification of physiological processes that have ecological relevance. We have focused on biochemical- and molecular-level physiology that would contribute to thermal tolerance and maintenance of a functional intracellular protein pool in the face of extreme and fluctuating environmental temperatures. Past research has addressed processes central to protein homeostasis (e.g., protein ubiquitination) and the molecular ecology of molecular chaperones, a.k.a. heat shock proteins (Hsps), in ectothermic animals. In this presentation, we focus on two new developments regarding the biology of heat shock proteins as molecular chaperones in intertidal organisms. First, we present data on the functional characteristics of the transcriptional factor, HSF1 and discuss how these data relate to the plasticity of Hsp gene expression observed in intertidal organisms in nature. Second, we present data on the biochemical function of heat shock proteins purified from our non-model study organisms and discuss the temperature relationships of these molecules as they assist in protein folding in situ.

High latitude fish in a high CO2 world: Synergistic effects of elevated temperature and carbon dioxide on the metabolic rates of Antarctic notothenioids.

Although the physiological response of teleost fishes to increased temperature has been well documented, there is only a small body of literature that examines the effects of ocean acidification on fish under ecologically relevant scenarios. Furthermore, little data exists which examines the possible synergistic effects of increased sea surface temperatures and pCO(2) levels, although it is well established that both will co-committedly change in the coming centuries. In this study we examined the effects of increased temperature, increased pCO(2), and a combination of these treatments on the resting metabolic rate (RMR) of four species of notothenioid fish, Trematomus bernacchii, T. hansoni, T. newnesi, and Pagothenia borchgrevinki, acclimated to treatment conditions for 7, 14 or 28days. While most species appear capable of rapidly acclimating to increased pCO(2), temperature continues to impact RMRs for up to 28days. One species in particular, T. newnesi, displayed no acclimatory response to any of the treatments regardless of acclimation time and may have a reduced capacity to respond to environmental change. Furthermore, we present evidence that temperature and pCO(2) act synergistically to further elevate the RMR and slow acclimation when compared to temperature or pCO(2) increases alone.

Is warmer better? Decreased oxidative damage in notothenioid fish after long-term acclimation to multiple stressors

Antarctic fish of the suborder Notothenioidei have evolved several unique adaptations to deal with subzero temperatures. However, these adaptations may come with physiological trade-offs, such as an increased susceptibility to oxidative damage. As such, the expected environmental perturbations brought on by global climate change have the potential to significantly increase the level of oxidative stress and cellular damage in these endemic fish. Previous single stressor studies of the notothenioids have shown they possess the capacity to acclimate to increased temperatures, but the cellular-level effects remain largely unknown. Additionally, there is little information on the ability of Antarctic fish to respond to ecologically relevant environmental changes where multiple variables change concomitantly. We have examined the potential synergistic effects that increased temperature and ṖCO2 have on the level of protein damage in Trematomus bernacchii, Pagothenia borchgrevinki and Trematomus newnesi, and combined these measurements with changes in total enzymatic activity of catalase (CAT) and superoxide dismutase (SOD) in order to gauge tissue-specific changes in antioxidant capacity. Our findings indicate that total SOD and CAT activity levels displayed only small changes across treatments and tissues. Short-term acclimation to decreased seawater pH and increased temperature resulted in significant increases in oxidative damage. Surprisingly, despite no significant change in antioxidant capacity, cellular damage returned to near-basal levels, and significantly decreased in T. bernacchii, after long-term acclimation. Overall, these data suggest that notothenioid fish currently maintain the antioxidant capacity necessary to offset predicted future ocean conditions, but it remains unclear whether this capacity comes with physiological trade-offs.

Effects of seawater acidification on cell cycle control mechanisms in Strongylocentrotus purpuratus embryos.

Previous studies have shown fertilization and development of marine species can be significantly inhibited when the pH of sea water is artificially lowered. Little mechanistic understanding of these effects exists to date, but previous work has linked developmental inhibition to reduced cleavage rates in embryos. To explore this further, we tested whether common cell cycle checkpoints were involved using three cellular biomarkers of cell cycle progression: (1) the onset of DNA synthesis, (2) production of a mitotic regulator, cyclin B, and (3) formation of the mitotic spindle. We grew embryos of the purple sea urchin, Strongylocentrotus purpuratus, in seawater artifically buffered to a pH of ∼7.0, 7.5, and 8.0 by CO2 infusion. Our results suggest the reduced rates of mitotic cleavage are likely unrelated to common cell cycle checkpoints. We found no significant differences in the three biomarkers assessed between pH treatments, indicating the embryos progress through the G1/S, G2/M and metaphase/anaphase transitions at relatively similar rates. These data suggest low pH environments may not impact developmental programs directly, but may act through secondary mechanisms such as cellular energetics.

The effects of elevated temperature and ocean acidification on the metabolic pathways of notothenioid fish

Abstract Organisms inhabiting the sub-zero waters surrounding Antarctica display remarkably narrow tolerances for environmental change. This study assessed three closely related fish exposed to simultaneous changes in oceanic conditions to ascertain the impact additive stress has on their capacity to acclimate and whether or not these fish employ similar metabolic responses.

RNA-seq reveals a diminished acclimation response to the combined effects of ocean acidification and elevated seawater temperature in Pagothenia borchgrevinki

PURPOSE The IPCC has reasserted the strong influence of anthropogenic CO2 contributions on global climate change and highlighted the polar-regions as highly vulnerable. With these predictions the cold adapted fauna endemic to the Southern Ocean, which is dominated by fishes of the sub-order Notothenioidei, will face considerable challenges in the near future. Recent physiological studies have demonstrated that the synergistic stressors of elevated temperature and ocean acidification have a considerable, although variable, impact on notothenioid fishes. The present study explored the transcriptomic response of Pagothenia borchgrevinki to increased temperatures and pCO2 after 7, 28 and 56days of acclimation. We compared this response to short term studies assessing heat stress alone and foretell the potential impacts of these stressors on P. borchgrevinkis ability to survive a changing Southern Ocean. RESULTS P. borchgrevinki did demonstrate a coordinated stress response to the dual-stressor condition, and even indicated that some level of inducible heat shock response may be conserved in this notothenioid species. However, the stress response of P. borchgrevinki was considerably less robust than that observed previously in the closely related notothenioid, Trematomus bernacchii, and varied considerably when compared across different acclimation time-points. Furthermore, the molecular response of these fish under multiple stressors displayed distinct differences compared to their response to short term heat stress alone. CONCLUSIONS When exposed to increased sea surface temperatures, combined with ocean acidification, P. borchgrevinki demonstrated a coordinated stress response that has already peaked by 7days of acclimation and quickly diminished over time. However, this response is less dramatic than other closely related notothenioids under identical conditions, supporting previous research suggesting that this notothenioid species is less sensitive to environmental variation.

Temperature differentially affects adenosine triphosphatase activity in Hsc70 orthologs from Antarctic and New Zealand notothenioid fishes

Abstract To test the temperature sensitivity of molecular chaperones in poikilothermic animals, we purified the molecular chaperone Hsc70 from 2 closely related notothenioid fishes—the Antarctic species Trematomus bernacchii and the temperate New Zealand species Notothenia angustata—and characterized the effect of temperature on Hsc70 adenosine triphosphatase (ATPase) activity. Hsc70 ATPase activity was measured using [α-32P]-adenosine triphosphate (ATP)–based in vitro assays followed by separation of adenylates by thin-layer chromatography. For both species, a significant increase in Hsc70 ATPase activity was observed across a range of temperatures that was ecologically relevant for each respective species. Hsc70 from T bernacchii hydrolyzed 2-fold more ATP than did N angustata Hsc70 at 0°C, suggesting that the Antarctic molecular chaperone may be adapted to function more efficiently at extreme cold temperatures. In addition, Q10 measurements indicate differential temperature sensitivity of the ATPase activity of Hsc70 from these differentially adapted fish that correlates with the temperature niche inhabited by each species. Hsc70 from T bernacchii was relatively temperature insensitive, as indicated by Q10 values calculated near 1.0 across each temperature range measured. In the case of Hsc70 purified from N angustata, Q10 values indicated thermal sensitivity across the temperature range of 0°C to 10°C, with a Q10 of 2.714. However, Hsc70 from both T bernacchii and N angustata exhibited unusually high thermal stabilities with ATPase activity at temperatures that far exceeded temperatures encountered by these fish in nature. Overall, as evidenced by in vitro ATP hydrolysis, Hsc70 from T bernacchii and N angustata displayed biochemical characteristics that were supportive of molecular chaperone function at ecologically relevant temperatures.

Impact of ocean acidification on the hypoxia tolerance of the woolly sculpin, Clinocottus analis

Global climate change may exacerbate naturally occurring stressors encountered by intertidal organisms. This in turn, may reduce the ability of these organisms to withstand the daily stress of life in the intertidal zone. Here we investigated how elevated CO2 might impact low oxygen tolerance of a marine teleost.

Single-point mutation in a conserved TPR domain of Hip disrupts enhancement of glucocorticoid receptor signaling

The Hsp70-interacting protein Hip has been identified as a transient participant in the assembly of both glucocorticoid (GR) and progesterone receptor complexes. Although it has been difficult to identify a physiological role for Hip, it is believed to have intrinsic chaperoning properties and has been identified as a potential anti-apoptotic target of Granzyme B. In vitro assays have provided evidence that Hip may interact with GR complexes in an Hsp70 independent manner and can enhance the function of GR in hormone based reporter assays. In this study, a cDNA for human Hip was used in mutational analysis to map Hip function to critical structural elements. A single amino acid substitution (L211S) resulted in a loss of Hip function. This mutation also appears to disrupt the interaction of Hip with Hsp70 in vitro. Failure to recover Hip-L211S constructs in co-immunoprecipitation assays with an Hsp70 monoclonal antibody suggests that the mutation is unlikely to result in a misfolded substrate.