B.A. 1962 Carleton College - Biology
Ph.D. 1967 Stanford University - Biology
2008-current Associate Director—Hopkins Marine Station
2005-current Senior Fellow—Woods Institute for the Environment, Stanford University
2000-2008 Director—Hopkins Marine Station, Stanford University
1995-current David and Lucile Packard Professor of Marine Science, Stanford University
1991-1995 Wayne and Gladys Valley Professor of Marine Biology, Oregon State University
1980-1991 Professor, Scripps Institution of Oceanography, University of California, San Diego
1984-1989 John Dove Isaacs Chair in Natural Philosophy, Scripps Institution of Oceanography
1983-1989 Chair, Marine Biology Research Division, Scripps Institution of Oceanography
1976-1980 Associate Professor, Scripps Institution of Oceanography
1970-1976 Assistant Professor, Scripps Institution of Oceanography
1967-1970 Postdoctoral Fellow, University of British Columbia
1963-1966 Member-U.S. Antarctic Research Program
National Science Foundation Predoctoral Fellowship
National Science Foundation Postdoctoral Fellowship
Isaac Walton Killam Postdoctoral Fellowship
John Simon Guggenheim Memorial Fellowship
Member of U.S. National Academy of Sciences
Fellow of the American Association for the Advancement of Science
Honorary Doctor of Science Degree-Carleton College
Helsinki Medal-University of Helsinki
Member of California Academy of Sciences
American Journal of Physiology, Journal of Comparative Physiology, Journal of Experimental Biology, Comparative Biochemistry and Physiology
National Science Foundation—Panel on Ecological and Evolutionary Physiology (1992-1995)
National Research Council—Ocean Studies Board (1997-1999)
National Research Council—Committee on Evaluation, Design, and Monitoring of Marine
Reserves and Protected Areas (1998-2000)
National Research Council—Frontiers in Polar Biology (2002)
National Research Council—International Polar Year committee (2003)
National Research Council—Committee on Ocean Acidification (chair: 2012)
Current Research and Scholarly Interests
RESEARCH INTERESTS: The unifying theme in our research is adaptation of organisms to the environment. We seek to determine how different environmental factors, notably temperature and the threat of desiccation, affect organisms, and how organisms respond adaptively to these perturbations. Proteins are a primary study system in most of our work. We are documenting how adaptive change in protein sequence (primary structure) achieves the conservation of critical functional and structural characteristics of enzymatic and structural proteins. These studies exploit homologous (orthologous) proteins from differently adapted species, frequently congeneric species adapted to only slightly different temperatures. We are using these comparative studies not only to examine adaptation to environment, but also to deduce basic structure-function relationships in proteins. For example, we are delineating the sites in the primary and higher orders of protein structure where adaptive change is permissible. Mapping of adaptively important changes in orthologs of closely related congeners supports the hypothesis that much of the protein molecule contributes to the energy changes that accompany catalysis.
Our studies of proteins are performed in solution conditions that mimic the intracellular conditions encountered by the proteins. The use of in vitro media that simulate in vivo conditions has enabled us to demonstrate that the “micromolecules” of the cell, that is, the small solutes that bathe macromolecules, contribute importantly to the establishment of the functional and structural properties of proteins. Macromolecular and micromolecular evolution play complementary roles in adaptation to environment.
Our field studies focus on “real world” effects of temperature on protein systems. A primary focus of these studies is to determine how changes in environmental temperature affect the latitudinal and depth distribution patterns of marine organisms. These studies demonstrate that many organisms live close to the upper thermal limits of protein structure and function, and suggest that global warming may have pronounced effects on ectothermic (“cold-blooded”) animals.
Studies of thermal effects at the molecular level are complemented by physiological investigations, for example, of heart function. The results of the physiological studies also indicate that only slight increases in maximal habitat temperature are likely to have profound negative effects on many marine animals. Paradoxically, the most warm-adapted species appear in many cases to be the most threatened by further increases in temperature, such as those that may result from continued global climate change. Our physiological studies also focus on an invasive species of mussel which has replaced a native mussel along much of the California coast. We have identified differences in cardiac function and enzymatic activity that may account for the competitive advantage of the invader and allow predictions to be made of how effectively it will colonize habitats to the north of its present distribution range.
We are using DNA microarray (“gene chip”) technology to monitor shifts in gene expression in response to environmental change (alterations in oxygen availability, salinity, and temperature). Among our goals in using these new approaches is the elucidation of the molecular bases of the different capacities of species to respond adaptively to environmental changes.
- Oceanic Biology
BIOHOPK 163H (Win)
Independent Studies (11)
- Advanced Research Laboratory in Experimental Biology
BIO 199 (Aut, Win, Spr, Sum)
- Directed Individual Study in Earth Systems
EARTHSYS 297 (Aut, Win, Spr, Sum)
- Directed Reading in Biology
BIO 198 (Aut, Win, Spr, Sum)
- Directed Research
EARTHSYS 250 (Aut, Win, Spr, Sum)
- Graduate Research
BIO 300 (Aut, Win, Spr, Sum)
- Honors Program in Earth Systems
EARTHSYS 199 (Aut, Win, Spr, Sum)
EARTHSYS 260 (Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Sum)
- Out-of-Department Directed Reading
BIO 198X (Spr, Sum)
- Out-of-Department Graduate Research
BIO 300X (Sum)
- Teaching of Biology
BIO 290 (Aut, Win, Spr)
- Advanced Research Laboratory in Experimental Biology
Prior Year Courses
- Frontiers in Marine Biology
BIO 3 (Aut)
- Oceanic Biology
BIOHOPK 163H, BIOHOPK 263H (Win)
- Physiology of Global Change
BIOHOPK 152H, BIOHOPK 252H (Spr)
- Frontiers in Marine Biology
Thermal stress and cellular signaling processes in hemocytes of native (Mytilus californianus) and invasive (M. galloprovincialis) mussels: Cell cycle regulation and DNA repair
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY
2013; 165 (2): 159-168
In a previous study using hemocytes from native and invasive congeners of Mytilus (Mytilus californianus and Mytilus galloprovincialis, respectively) we showed that DNA damage and cell signaling transduction processes related to the cellular stress response and apoptosis were induced by acute temperature stress. The present study extends this work by examining effects of acute heat- and cold stress on total hemocyte counts (THCs) and expression of key regulatory molecules involved in responding to stress: tumor suppressor factor (p53), cell cycle arrest activator (p21), and a DNA base excision repair enzyme (apurinic/apyrimidinic endonuclease (APE)). Hyperthermia (28 °C, 32 °C) led to significant decreases of THCs in both species. The extent of decrease in THC was temperature-, time-, and species-dependent; lower THC values were found in M. californianus, the more cold-adapted species. Western blot analyses of hemocyte extracts with antibodies specific for p53 protein, several site-specific phosphorylation states of p53, p21 protein, and APE indicated that heat- and cold (2 °C) stress induced a time-dependent activation of stress-related proteins in response to DNA damage; these stress-induced changes could govern cell cycle arrest or DNA damage repair. Our results show that the downstream regulatory response to temperature-induced cell damage may play an important role in deciding cellular fate following heat- and cold stress. Compared to M. californianus, the more warm-adapted M. galloprovincialis appears to have a higher temperature tolerance due to a lesser reduction in THC, faster signaling activation and transduction, and stronger DNA repair ability following heat stress.
View details for DOI 10.1016/j.cbpa.2013.02.024
View details for Web of Science ID 000319233300007
View details for PubMedID 23454628
Behavior and survival of Mytilus congeners following episodes of elevated body temperature in air and seawater
JOURNAL OF EXPERIMENTAL BIOLOGY
2013; 216 (3): 502-514
Coping with environmental stress may involve combinations of behavioral and physiological responses. We examined potential interactions between adult mussels' simple behavioral repertoire - opening/closing of the shell valves - and thermal stress physiology in common-gardened individuals of three Mytilus congeners found on the West Coast of North America: two native species (M. californianus and M. trossulus) and one invasive species from the Mediterranean (M. galloprovincialis). We first continuously monitored valve behavior over three consecutive days on which body temperatures were gradually increased, either in air or in seawater. A temperature threshold effect was evident between 25 and 33°C in several behavioral measures. Mussels tended to spend much less time with the valves in a sealed position following exposure to 33°C body temperature, especially when exposed in air. This behavior could not be explained by decreases in adductor muscle glycogen (stores of this metabolic fuel actually increased in some scenarios), impacts of forced valve sealing on long-term survival (none observed in a second experiment), or loss of contractile function in the adductor muscles (individuals exhibited as many or more valve adduction movements following elevated body temperature compared with controls). We hypothesize that this reduced propensity to seal the valves following thermal extremes represents avoidance of hypoxia-reoxygenation cycles and concomitant oxidative stress. We further conjecture that prolonged valve gaping following episodes of elevated body temperature may have important ecological consequences by affecting species interactions. We then examined survival over a 90 day period following exposure to elevated body temperature and/or emersion, observing ongoing mortality throughout this monitoring period. Survival varied significantly among species (M. trossulus had the lowest survival) and among experimental contexts (survival was lowest after experiencing elevated body temperature in seawater). Surprisingly, we observed no cumulative impact on survival of 3 days relative to 1 day of exposure to elevated body temperature. The delayed mortality and context-specific outcomes we observed have important implications for the design of future experiments and for interpretation of field distribution patterns of these species. Ultimately, variation in the catalog of physiological and behavioral capacities among closely related or sympatric species is likely to complicate prediction of the ecological consequences of global change and species invasions.
View details for DOI 10.1242/jeb.076620
View details for Web of Science ID 000313740600026
View details for PubMedID 23038732
Functional Determinants of Temperature Adaptation in Enzymes of Cold- versus Warm-Adapted Mussels (Genus Mytilus)
MOLECULAR BIOLOGY AND EVOLUTION
2012; 29 (10): 3061-3070
Temperature is a strong selective force on the evolution of proteins due to its effects on higher orders of protein structure and, thereby, on critical protein functions like ligand binding and catalysis. Comparisons among orthologous proteins from differently thermally adapted species show consistent patterns of adaptive variation in function, but few studies have examined functional adaptation among multiple structural families of proteins. Thus, with our present state of knowledge, it is difficult to predict what fraction of the proteome will exhibit adaptive variation in the face of temperature increases of a few to several degrees Celsius, that is, temperature increases of the magnitude predicted by models of global warming. Here, we compared orthologous enzymes of the warm-adapted Mediterranean mussel Mytilus galloprovincialis and the cold-adapted Mytilus trossulus, a native of the North Pacific Ocean, species whose physiologies exhibit significantly different responses to temperature. We measured the effects of temperature on the kinetics (Michaelis-Menten constant-K(m)) of five enzymes that are important for ATP generation and that represent distinct protein structural families. Among phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (GTP) (PEPCK), and isocitrate dehydrogenase (NADP) (IDH), only IDH orthologs showed significantly different thermal responses of K(m) between the two species. The K(m) of isocitrate of M. galloprovincialis-IDH was intrinsically lower and more thermally stable than that of M. trossulus-IDH and thus had higher substrate affinity at high temperatures. Two amino acid substitutions account for the functional differences between IDH orthologs, one of which allows for more hydrogen bonds to form near the mobile region of the active site in M. galloprovincialis-IDH. Taken together, our findings cast light on the targets of adaptive evolution in the context of climate change; only a minority of proteins might adapt to small changes in temperature, and these adaptations may involve only small changes in sequence.
View details for DOI 10.1093/molbev/mss111
View details for Web of Science ID 000309927900018
View details for PubMedID 22491035
The Physiology of Global Change: Linking Patterns to Mechanisms
ANNUAL REVIEW OF MARINE SCIENCE, VOL 4
2012; 4: 39-61
Global change includes alterations in ocean temperature, oxygen availability, salinity, and pH, abiotic variables with strong and interacting influences on the physiology of all taxa. Physiological stresses resulting from changes in these four variables may cause broad biogeographic shifts as well as localized changes in distribution in mosaic habitats. To elucidate these causal linkages, I address the following questions: What types of physiological limitations can alter species' distributions and, in cases of extreme stress, cause extinctions? Which species are most threatened by these physiological challenges--and why? How do contents of genomes establish capacities to respond to global change, notably in the case of species that have evolved in highly stable habitats? How fully can phenotypic acclimatization offset abiotic stress? Can physiological measurements, including new molecular ("-omic") approaches, provide indices of the degree of sublethal stress an organism experiences? And can physiological evolution keep pace with global change?
View details for DOI 10.1146/annurev-marine-120710-100935
View details for Web of Science ID 000300634900004
View details for PubMedID 22457968
Comparative physiology: a "crystal ball" for predicting consequences of global change
AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY
2011; 301 (1): R1-R14
Comparative physiology offers powerful approaches for developing causal, mechanistic explanations of shifts in biogeographic patterning occurring in concert with global change. These analyses can identify the cellular loci and intensities of stress-induced perturbation and generate predictions about ecosystem alterations in a changing world. Congeneric species adapted to different abiotic conditions offer excellent study systems for these purposes. Several findings have emerged from such comparative studies: 1) In aquatic and terrestrial habitats, the most heat-tolerant ectotherms may be most threatened by further increases in temperature, due to proximity of these species' thermal optima and tolerance limits to current maximal ambient temperatures and limited capacities for acclimatization to higher temperatures. 2) Cardiac function is a "weak link" in acute thermal tolerance. 3) Stress-induced changes in gene expression comprise a graded response involving genes linked to damage repair, lysis of irreversibly damaged molecules, and downregulation of cell proliferation. Transcriptomic and proteomic analyses provide "biomarkers" for diagnosing degrees of stress. 4) Different abiotic stresses may have synergistic or opposing effects on gene expression, a complexity needing consideration when developing integrated pictures of effects of global change. 5) Adaptation of proteins can result from one to a few amino acid substitutions, which can occur at many sites in a protein, a discovery with implications for rates of adaptive evolution. 6) Greater thermal tolerance of invasive species may favor their replacement of natives. 7) Losses of protein-coding genes and temperature-responsive gene regulatory abilities in stenothermal ectotherms of the Southern Ocean may lead to broad extinctions.
View details for DOI 10.1152/ajpregu.00719.2010
View details for Web of Science ID 000292319800001
View details for PubMedID 21430078
Effects of thermal acclimation on transcriptional responses to acute heat stress in the eurythermal fish Gillichthys mirabilis (Cooper)
AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY
2011; 300 (6): R1373-R1383
The capacities of eurythermal ectotherms to withstand wide ranges of temperature are based, in part, on abilities to modulate gene expression as body temperature changes, notably genes encoding proteins of the cellular stress response. Here, using a complementary DNA microarray, we investigated the sequence in which cellular stress response-linked genes are expressed during acute heat stress, to elucidate how severity of stress affects the categories of genes changing expression. We also studied how prior acclimation history affected gene expression in response to acute heat stress. Eurythermal goby fish (Gillichthys mirabilis) were acclimated to 9 ± 0.5, 19 ± 0.5, and 28 ± 0.5°C for 1 mo. Then fish were given an acute heat ramp (4°C/h), and gill tissues were sampled every +4°C to monitor gene expression. The average onset temperature for a significant change in expression during acute stress increased by ?2°C for each ?10°C increase in acclimation temperature. For some genes, warm acclimation appeared to obviate the need for expression change until the most extreme temperatures were reached. Sequential expression of different categories of genes reflected severity of stress. Regardless of acclimation temperature, the gene encoding heat shock protein 70 (HSP70) was upregulated strongly during mild stress; the gene encoding the proteolytic protein ubiquitin (UBIQ) was upregulated at slightly higher temperatures; and a gene encoding a protein involved in cell cycle arrest and apoptosis, cyclin-dependent kinase inhibitor 1B (CDKN1B), was upregulated only under extreme stress. The tiered, stress level-related expression patterns and the effects of acclimation on induction temperature yield new insights into the fundamental mechanisms of eurythermy.
View details for DOI 10.1152/ajpregu.00689.2010
View details for Web of Science ID 000291532000014
View details for PubMedID 21411771
Transcriptomic responses to salinity stress in invasive and native blue mussels (genus Mytilus)
2011; 20 (3): 517-529
The invasive marine mussel Mytilus galloprovincialis has displaced the native congener Mytilus trossulus from central and southern California, but the native species remains dominant at more northerly sites that have high levels of freshwater input. To determine the extent to which interspecific differences in physiological tolerance to low salinity might explain limits to the invasive species' biogeography, we used an oligonucleotide microarray to compare the transcriptional responses of these two species to an acute decrease in salinity. Among 6777 genes on the microarray, 117 genes showed significant changes that were similar between species, and 12 genes showed significant species-specific responses to salinity stress. Osmoregulation and cell cycle control were important aspects of the shared transcriptomic response to salinity stress, whereas the genes with species-specific expression patterns were involved in mRNA splicing, polyamine synthesis, exocytosis, translation, cell adhesion, and cell signalling. Forty-five genes that changed expression significantly during salinity stress also changed expression during heat stress, but the direction of change in expression was typically opposite for the two forms of stress. These results (i) provide insights into the role of changes in gene expression in establishing physiological tolerance to acute decreases in salinity, and (ii) indicate that transcriptomic differences between M. galloprovincialis and M. trossulus in response to salinity stress are subtle and involve only a minor fraction of the overall suite of gene regulatory responses.
View details for DOI 10.1111/j.1365-294X.2010.04973.x
View details for Web of Science ID 000286254700007
View details for PubMedID 21199031
Phosphorylation Events Catalyzed by Major Cell Signaling Proteins Differ in Response to Thermal and Osmotic Stress among Native (Mytilus californianus and Mytilus trossulus) and Invasive (Mytilus galloprovincialis) Species of Mussels
PHYSIOLOGICAL AND BIOCHEMICAL ZOOLOGY
2010; 83 (6): 984-996
Sharp environmental gradients encountered within the intertidal zone have driven the evolution of physiological adaptations that allow its inhabitants to maintain cellular function in the presence of fluctuating abiotic factors. These adaptations are mediated by gene-regulatory networks that, despite their inherent complexity, must remain evolvable and capable of responding to different selection pressures associated with specific ecological niches. Phosphorylation events catalyzed by cell-signaling enzymes represent a parsimonious mechanism to integrate new functional or regulatory properties into these gene-regulatory networks. In this study, proteins phosphorylated on consensus sequences for protein kinases A, B, and C; cyclin-dependent kinases; and mitogen-activated protein kinases, as well as the abundance of phosphorylated stress-activated protein kinase (phospho-SAPK/JNK), were quantified in order to ascertain whether phosphorylation events are divergent among native (Mytilus californianus and Mytilus trossulus) and invasive (Mytilus galloprovincialis) species of mussels that differ in their tolerance toward environmental stress. Abundances of phosphorylated substrate proteins for each of the major signaling proteins that were investigated, as well as the abundance of phospho-SAPK/JNK, differed both within and between species during thermal and osmotic stress. These data suggest that modulating protein function via phosphorylation may be an important mechanism to integrate novel properties into stress-regulatory networks. In turn, differential phosphorylation during environmental stress may contribute to species-specific tolerances toward abiotic stress, interspecies dynamics, and biogeographic patterns in Mytilus congeners.
View details for DOI 10.1086/656192
View details for Web of Science ID 000283992100009
View details for PubMedID 20946068
Transcriptomic responses to heat stress in invasive and native blue mussels (genus Mytilus): molecular correlates of invasive success
JOURNAL OF EXPERIMENTAL BIOLOGY
2010; 213 (20): 3548-3558
Invasive species are increasingly prevalent in marine ecosystems worldwide. Although many studies have examined the ecological effects of invasives, little is known about the physiological mechanisms that might contribute to invasive success. The mussel Mytilus galloprovincialis, a native of the Mediterranean Sea, is a successful invader on the central and southern coasts of California, where it has largely displaced the native congener, Mytilus trossulus. It has been previously shown that thermal responses of several physiological traits may underlie the capacity of M. galloprovincialis to out-compete M. trossulus in warm habitats. To elucidate possible differences in stress-induced gene expression between these congeners, we developed an oligonucleotide microarray with 8874 probes representing 4488 different genes that recognized mRNAs of both species. In acute heat-stress experiments, 1531 of these genes showed temperature-dependent changes in expression that were highly similar in the two congeners. By contrast, 96 genes showed species-specific responses to heat stress, functionally characterized by their involvement in oxidative stress, proteolysis, energy metabolism, ion transport, cell signaling and cytoskeletal reorganization. The gene that showed the biggest difference between the species was the gene for the molecular chaperone small heat shock protein 24, which was highly induced in M. galloprovincialis and showed only a small change in M. trossulus. These different responses to acute heat stress may help to explain--and predict--the invasive success of M. galloprovincialis in a warming world.
View details for DOI 10.1242/jeb.046094
View details for Web of Science ID 000282541800018
View details for PubMedID 20889835
Transcriptional responses to thermal acclimation in the eurythermal fish Gillichthys mirabilis (Cooper 1864)
AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY
2010; 299 (3): R843-R852
Thermal acclimation (acclimatization) capacity may be critical for determining how successfully an ectotherm can respond to temperature change, and adaptive shifts in gene expression may be pivotal for mediating these acclimatory responses. Using a cDNA microarray, we examined transcriptional profiles in gill tissue of a highly eurythermal goby fish, Gillichthys mirabilis, following 4 wk of acclimation to 9 degrees C, 19 degrees C, or 28 degrees C. Overall, gill transcriptomes were not strikingly different among acclimation groups. Of the 1,607 unique annotated genes on the array, only 150 of these genes (9%) were significantly different in expression among the three acclimation groups (ANOVA, false discovery rate < 0.05). Principal component analysis revealed that 59% of the variation in expression among these genes was described by an expression profile that is upregulated with increasing acclimation temperature. Gene ontology analysis of these genes identified protein biosynthesis, transport, and several metabolic categories as processes showing the greatest change in expression. Our results suggest that energetic costs of macromolecular turnover and membrane-localized transport rise with acclimation temperature. The upregulation of several classes of stress-related proteins, e.g., heat shock proteins, seen in the species' response to acute thermal stress was not observed in the long-term 28 degrees C-acclimated fish. The transcriptional differences found among the acclimation groups thus may reflect an acclimation process that has largely remedied the effects of acute thermal stress and established a new steady-state condition involving changes in relative energy costs for different processes. This pattern of transcriptional alteration in steady-state acclimated fish may be a signature of eurythermy.
View details for DOI 10.1152/ajpregu.00306.2010
View details for Web of Science ID 000281537500014
View details for PubMedID 20610827
The physiology of climate change: how potentials for acclimatization and genetic adaptation will determine 'winners' and 'losers'
JOURNAL OF EXPERIMENTAL BIOLOGY
2010; 213 (6): 912-920
Physiological studies can help predict effects of climate change through determining which species currently live closest to their upper thermal tolerance limits, which physiological systems set these limits, and how species differ in acclimatization capacities for modifying their thermal tolerances. Reductionist studies at the molecular level can contribute to this analysis by revealing how much change in sequence is needed to adapt proteins to warmer temperatures--thus providing insights into potential rates of adaptive evolution--and determining how the contents of genomes--protein-coding genes and gene regulatory mechanisms--influence capacities for adapting to acute and long-term increases in temperature. Studies of congeneric invertebrates from thermally stressful rocky intertidal habitats have shown that warm-adapted congeners are most susceptible to local extinctions because their acute upper thermal limits (LT(50) values) lie near current thermal maxima and their abilities to increase thermal tolerance through acclimation are limited. Collapse of cardiac function may underlie acute and longer-term thermal limits. Local extinctions from heat death may be offset by in-migration of genetically warm-adapted conspecifics from mid-latitude 'hot spots', where midday low tides in summer select for heat tolerance. A single amino acid replacement is sufficient to adapt a protein to a new thermal range. More challenging to adaptive evolution are lesions in genomes of stenotherms like Antarctic marine ectotherms, which have lost protein-coding genes and gene regulatory mechanisms needed for coping with rising temperature. These extreme stenotherms, along with warm-adapted eurytherms living near their thermal limits, may be the major 'losers' from climate change.
View details for DOI 10.1242/jeb.037473
View details for Web of Science ID 000275002600013
View details for PubMedID 20190116
Heterologous hybridization to a complementary DNA microarray reveals the effect of thermal acclimation in the endothermic bluefin tuna (Thunnus orientalis)
2009; 18 (10): 2092-2102
The temperature stress that pelagic fishes experience can induce physiological and behavioural changes that leave a signature in gene expression profiles. We used a functional genomics approach to identify genes that were up- or down-regulated following thermal stress in the Pacific bluefin tuna. Following the acclimation period, 113, 81 and 196 genes were found to be differentially expressed between the control (20 degrees C) and cold (15 degrees) treatment groups, in ventricle, red muscle and white muscle, respectively. The genes whose expression levels were responsive to thermal acclimation varied according to muscle fibre type, perhaps reflecting the tissue-specific degrees of endothermy characteristic of this species.
View details for DOI 10.1111/j.1365-294X.2009.04174.x
View details for Web of Science ID 000265774300005
View details for PubMedID 19389180
Temperature adaptation of cytosolic malate dehydrogenases of limpets (genus Lottia): differences in stability and function due to minor changes in sequence correlate with biogeographic and vertical distributions
JOURNAL OF EXPERIMENTAL BIOLOGY
2009; 212 (2): 169-177
We characterized functional and structural properties of cytoplasmic malate dehydrogenases (cMDHs) from six limpets of the genus Lottia that have different vertical and latitudinal distributions. Particular attention was given to the cryptic species pair Lottia digitalis (northern occurring) and L. austrodigitalis (southern occurring) because of recent contraction in the southern range of L. digitalis and a northward range extension of L. austrodigitalis. As an index of adaptation of function, we measured the effects of temperature on the apparent Michaelis-Menten constant (K(m)) of the cofactor NADH (K(m)(NADH)). K(m)(NADH) values of cMDHs from the mid- to high-intertidal, low-latitude species L. scabra and L. gigantea were less sensitive to high temperature than those of cMDHs from the low- and mid-intertidal, high-latitude species L. scutum and L. pelta. cMDH of L. digitalis was more sensitive to high temperatures than the cMDH ortholog of L. austrodigitalis. Thermal stability (rate of loss of activity at 42.5 degrees C) showed a similar pattern of interspecific variation. Comparison of the deduced amino acid sequences showed that interspecific differences ranged from one to as many as 17 residues. Differences in K(m)(NADH) and thermal stability between orthologs of L. digitalis and L. austrodigitalis result from a single amino acid substitution. At position 291, the glycine residue in cMDH of L. digitalis is replaced by a serine in cMDH of L. austrodigitalis, a change that favors additional hydrogen bonding and reduced conformational entropy. This difference between closely related congeners demonstrates the role of minor alterations in protein sequence in temperature adaptation and suggests that such variation is important in governing shifts in biogeographic range in response to climate change.
View details for DOI 10.1242/jeb.024505
View details for Web of Science ID 000262011600008
View details for PubMedID 19112135
Protein-protein interactions enable rapid adaptive response to osmotic stress in fish gills.
Communicative & integrative biology
2009; 2 (2): 94-96
Cells respond to changes in osmolality with compensatory adaptations that re-establish ion homeostasis and repair disturbed aspects of cell structure and function. These physiologically complex processes can be separated into two functionally distinct cellular phases. The first phase operates to temporarily minimize cellular damage and stabilize critical cell functions necessary for survival. This phase is contingent upon the ability to generate a rapid adaptive response. For this reason, it occurs largely in the absence of de novo protein synthesis and instead relies upon modifying the activity of existing cellular proteins through protein-protein interactions and post-translational modifications. The second phase of the osmotic stress response is centered upon adjusting the expression of specific effector proteins required to re-establish cellular homeostasis. This phase is dependent on the completion of signal transduction events; as well the transcription and translation of target genes, and is therefore characterized by a significant temporal delay and not detected until several hours post exposure. Osmotic effector proteins central to the second phase, such as ion transporting proteins and organic osmolyte generating enzymes, have been studied in considerable detail. However, knowledge surrounding the first phase of the osmotic stress response is limited. This article focuses on recent insights into the players and interactions governing the first phase of the osmotic stress response with specific emphasis on protein-protein interactions.
View details for PubMedID 19704899
A microarray-based transcriptomic time-course of hyper- and hypo-osmotic stress signaling events in the euryhaline fish Gillichthys mirabilis: osmosensors to effectors
JOURNAL OF EXPERIMENTAL BIOLOGY
2008; 211 (22): 3636-3649
Cells respond to changes in osmolality with compensatory adaptations that re-establish ion homeostasis and repair disturbed aspects of cell structure and function. These physiological processes are highly complex, and require the coordinated activities of osmosensing, signal transducing and effector molecules. Although the critical role of effector proteins such as Na+, K+-ATPases and Na+/K+/Cl(-) co-transporters during osmotic stress are well established, comparatively little information is available regarding the identity or expression of the osmosensing and signal transduction genes that may govern their activities. To better resolve this issue, a cDNA microarray consisting of 9207 cDNA clones was used to monitor gene expression changes in the gill of the euryhaline fish Gillichthys mirabilis exposed to hyper- and hypo-osmotic stress. We successfully annotated 168 transcripts differentially expressed during the first 12 h of osmotic stress exposure. Functional classifications of genes encoding these transcripts reveal that a variety of biological processes are affected. However, genes participating in cell signaling events were the dominant class of genes differentially expressed during both hyper- and hypo-osmotic stress. Many of these genes have had no previously reported role in osmotic stress adaptation. Subsequent analyses used the novel expression patterns generated in this study to place genes within the context of osmotic stress sensing, signaling and effector events. Our data indicate multiple major signaling pathways work in concert to modify diverse effectors, and that these molecules operate within a framework of regulatory proteins.
View details for DOI 10.1242/jeb.022160
View details for Web of Science ID 000260540700020
View details for PubMedID 18978229
Heat-Shock Protein 70 (Hsp70) Expression in Four Limpets of the Genus Lottia: Interspecific Variation in Constitutive and Inducible Synthesis Correlates With in situ Exposure to Heat Stress
2008; 215 (2): 173-181
Limpets of the genus Lottia occupy a broad vertical distribution on wave-exposed rocky shores, a range that encompasses gradients in the frequency and severity of thermal and desiccation stress brought on by aerial emersion. Using western blot analysis of levels of heat-shock protein 70 (Hsp70), we examined the heat-shock responses of four Lottia congeners: Lottia scabra and L. austrodigitalis, which occur in the high-intertidal zone, and L. pelta and L. scutum, which are restricted to the low- and mid-intertidal zones. Our results suggest distinct strategies of Hsp70 expression in limpets occupying different heights and orientations in the rocky intertidal zone. In freshly field-collected animals and in specimens acclimated at ambient temperature ( approximately 14 degrees C) for 14 days, the two high-intertidal species had higher constitutive levels of Hsp70 than the low- and mid-intertidal species. During aerial exposure to high temperatures, the two low-shore species and L. austrodigitalis exhibited an onset of Hsp70 expression at 28 degrees C; no induction of Hsp70 occurred in L. scabra. Our findings suggest that high-intertidal congeners of Lottia employ a "preparative defense" strategy involving maintenance of high constitutive levels of Hsp70 in their cells as a mechanism for protection against periods of extreme and unpredictable heat stress.
View details for Web of Science ID 000260049800007
View details for PubMedID 18840778
The cellular response to heat stress in the goby Gillichthys mirabilis: a cDNA microarray and protein-level analysis
JOURNAL OF EXPERIMENTAL BIOLOGY
2006; 209 (14): 2660-2677
The cellular response to stress relies on the rapid induction of genes encoding proteins involved in preventing and repairing macromolecular damage incurred as a consequence of environmental insult. To increase our understanding of the scope of this response, a cDNA microarray, consisting of 9207 cDNA clones, was used to monitor gene expression changes in the gill and white muscle tissues of a eurythermic fish, Gillichthys mirabilis (Gobiidae) exposed to ecologically relevant heat stress. In each tissue, the induction or repression of over 200 genes was observed. These genes are associated with numerous biological processes, including the maintenance of protein homeostasis, cell cycle control, cytoskeletal reorganization, metabolic regulation and signal transduction, among many others. In both tissues, the molecular chaperones, certain transcription factors and a set of additional genes with various functions were induced in a similar manner; however, the majority of genes displayed tissue-specific responses. In gill, thermal stress induced the expression of the major structural components of the cytoskeleton, whereas these same genes did not respond to heat in muscle. In muscle, many genes involved in promoting cell growth and proliferation were repressed, perhaps to conserve energy for repair and replacement of damaged macromolecules, but a similar repression was not observed in the gill. Many of the observed changes in gene expression were similar to those described in model species whereas many others were unexpected. Measurements of the concentrations of the protein products of selected genes revealed that in each case an induction in mRNA synthesis correlated with an increase in protein production, though the timing and magnitude of the increase in protein was not consistently predicted by mRNA concentration, an important consideration in assessing the condition of the stressed cell using transcriptomic analysis.
View details for DOI 10.1242/jeb.02292
View details for Web of Science ID 000239640800011
View details for PubMedID 16809457
Following the heart: temperature and salinity effects on heart rate in native and invasive species of blue mussels (genus Mytilus)
JOURNAL OF EXPERIMENTAL BIOLOGY
2006; 209 (13): 2554-2566
The three species of blue mussels, Mytilus trossulus Gould 1850, M. edulis Linnaeus 1758 and M. galloprovincialis Lamarck 1819, have distinct global distribution patterns that are hypothesized to reflect differences in their tolerances of temperature and salinity. We examined effects on heart rate (beats min(-1)) of acute exposure and acclimation to different combinations of temperature and salinity to test this hypothesis and, in the context of the invasive success of M. galloprovincialis, to gain insights into the factors that may explain the replacement of the temperate Pacific native, M. trossulus, by this Mediterranean Sea invader along much of the California coast. Heart rate of M. trossulus was significantly higher than that of M. galloprovincialis, consistent with evolutionary adaptation to a lower habitat temperature (temperature compensation) in the former species. Heart rates of M. trossulus/M. galloprovincialis hybrids were intermediate between those of the parental species. Following acclimation to 14 degrees C and 21 degrees C, heart rates of all species exhibited partial compensation to temperature. Heart rate increased with rising temperature until a high temperature was reached at which point activity fell sharply, the high critical temperature (H(crit)). H(crit) increased with increasing acclimation temperature and differed among species in a pattern that reflected their probable evolutionary adaptation temperatures: M. galloprovincialis is more heat tolerant than the other two congeners. Ability to sustain heart function in the cold also reflected evolutionary history: M. trossulus is more cold tolerant than M. galloprovincialis. Heart rates for all three congeners decreased gradually in response to acute reductions in salinity until a low salinity (S(crit)) was reached at which heart rate dropped precipitously. S(crit) decreased with decreasing salinity of acclimation and was generally lowest for M. galloprovincialis. Mortality during acclimation under common garden conditions was greatest in M. trossulus and was highest at high acclimation temperatures and salinities. These intrinsic differences in basal heart rate, thermal and salinity responses, acclimatory capacity, and survivorship are discussed in the contexts of the species' biogeographic patterning and, for the invasive species M. galloprovincialis, the potential for further range expansion along the Pacific coast of North America.
View details for DOI 10.1242/jeb.02259
View details for Web of Science ID 000238421800025
View details for PubMedID 16788038
Evolutionary and acclimation-induced variation in the thermal limits of heart function in congeneric marine snails (Genus Tegula): Implications for vertical zonation
2005; 208 (2): 138-144
We analyzed the thermal limits of heart function for congeneric species of the marine snail Tegula that have different patterns of vertical zonation. T. funebralis is found in the low to mid-intertidal zone, and T. brunnea and T. montereyi live in the low-intertidal or subtidally. As indices of thermal limits of heart function, we used the temperature at which heart rate initially decreased rapidly during heating (the Arrhenius break temperature, or ABT) and the temperature at which heart ceased to beat with either heating or cooling (the flatline temperature, or FLT(hot) or FLT(cold), respectively). These three indices provide an estimate of the thermal range within which Tegula heart function is maintained. For field-acclimatized specimens, the thermal range of the high-intertidal T. funebralis was greater than those of its two lower-occurring congeners (higher ABT, higher FLT(hot), lower FLT(cold)). We also demonstrated the effects of constant thermal acclimation on the heart rate response to heat stress. Acclimation to 14 degrees C and 22 degrees C resulted in increases in ABT and FLT(hot), with the largest changes in T. brunnea and T. montereyi. Although T. funebralis is more heat tolerant and eurythermal than its two lower-occurring congeners, it can encounter field body temperatures that exceed ABT, indicating that T. funebralis faces a larger threat from heat stress, in situ. These findings are consistent with recent studies on other taxa of marine invertebrates that have shown, somewhat paradoxically, that warm-adapted, eurythermal intertidal species may be more impacted by global warming than congeneric subtidal species that are less heat tolerant.
View details for Web of Science ID 000228618100008
View details for PubMedID 15837963
Peter Hochachka: Adventures in biochemical adaptation
ANNUAL REVIEW OF PHYSIOLOGY
2005; 67: 25-37
Peter Hochachka was one of the most creative forces in the field of comparative physiology during the past half-century. His career was truly an exploratory adventure, in both intellectual and geographic senses. His broad comparative studies of metabolism in organisms as diverse as trout, tunas, oysters, squid, turtles, locusts, hummingbirds, seals, and humans revealed the adaptable features of enzymes and metabolic pathways that provide the biochemical bases for diverse lifestyles and environments. In its combined breadth and depth, no other corpus of work better illustrates the principle of "unity in diversity" that marks comparative physiology. Through his publications, his stimulating mentorship, his broad editorial services, and his continuous-and highly infectious-enthusiasm for his field, Peter Hochachka served as one of the most influential leaders in the transformation of comparative physiology.
View details for Web of Science ID 000228406800003
View details for PubMedID 15709951
Linking biogeography to physiology: Evolutionary and acclimatory adjustments of thermal limits.
Frontiers in zoology
2005; 2 (1): 1
Temperature-adaptive physiological variation plays important roles in latitudinal biogeographic patterning and in setting vertical distributions along subtidal-to-intertidal gradients in coastal marine ecosystems. Comparisons of congeneric marine invertebrates reveal that the most warm-adapted species may live closer to their thermal tolerance limits and have lower abilities to increase heat tolerance through acclimation than more cold-adapted species. In crabs and snails, heart function may be of critical importance in establishing thermal tolerance limits. Temperature-mediated shifts in gene expression may be critical in thermal acclimation. Transcriptional changes, monitored using cDNA microarrays, have been shown to differ between steady-state thermal acclimation and diurnal temperature cycling in a eurythermal teleost fish (Austrofundulus limnaeus). In stenothermal Antarctic notothenioid fish, losses in capacity for temperature-mediated gene expression, including the absence of a heat-shock response, may reduce the abilities of these species to acclimate to increased temperatures. Differences among species in thermal tolerance limits and in the capacities to adjust these limits may determine how organisms are affected by climate change.
View details for PubMedID 15679952
Adaptation of enzymes to temperature: searching for basic "strategies"
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY
2004; 139 (3): 321-333
The pervasive influence of temperature on biological systems necessitates a suite of temperature--compensatory adaptations that span all levels of biological organization--from behavior to fine-scale molecular structure. Beginning about 50 years ago, physiological studies conducted with whole organisms or isolated tissues, by such pioneers of comparative thermal physiology as V.Ya. Alexandrov, T.H. Bullock, F.E.J. Fry, H. Precht, C.L. Prosser, and P.F. Scholander, began to document in detail the abilities of ectothermic animals to sustain relatively similar rates of metabolic activity at widely different temperatures of adaptation or acclimation. These studies naturally led to investigation of the roles played by enzymatic proteins in metabolic temperature compensation. Peter Hochachka's laboratory became an epicenter of this new focus in comparative physiology. The studies of the enzyme lactate dehydrogenase (LDH) that he initiated as a PhD student at Duke University in the mid-1960s and continued for several years at the University of British Columbia laid much of the foundation for subsequent studies of protein adaptation to temperature. Studies of orthologs of LDH have revealed the importance of conserving kinetic properties (catalytic rate constants (kcat) and Michaelis-Menten constants (Km) and structural stability during adaptation to temperature, and recently have identified the types of amino acid substitutions causing this adaptive variation. The roles of pH and low-molecular-mass organic solutes (osmolytes) in conserving the functional and structural properties of enzymes also have been elucidated using LDH. These studies, begun in Peter Hochachka's laboratory almost 40 years ago, have been instrumental in the development of a conceptual framework for the study of biochemical adaptation, a field whose origin can be traced largely to his creative influences. This framework emphasizes the complementary roles of three "strategies" of adaptation: (1) changes in amino acid sequence that cause adaptive variation in the kinetic properties and stabilities of proteins, (2) shifts in concentrations of proteins, which are mediated through changes in gene expression and protein turnover; and (3) changes in the milieu in which proteins function, which conserve the intrinsic properties of proteins established by their primary structure and modulate protein activity in response to physiological needs. This theoretical framework has helped guide research in adaptational biochemistry for many years and now stands poised to play a critical role in the post-genomic era, as physiologists grapple with the challenge of integrating the wealth of new data on gene sequences (genome), gene expression (transcriptome and proteome), and metabolic profiles (metabolome) into a realistic physiological context that takes into account the evolutionary histories and environmental relationships of species.
View details for DOI 10.1016/j.cbpc.2004.05.003
View details for Web of Science ID 000225554900005
View details for PubMedID 15544958
Changes in gene expression associated with acclimation to constant temperatures and fluctuating daily temperatures in an annual killifish Austrofundulus limnaeus
JOURNAL OF EXPERIMENTAL BIOLOGY
2004; 207 (13): 2237-2254
Eurythermal ectotherms commonly thrive in environments that expose them to large variations in temperature on daily and seasonal bases. The roles played by alterations in gene expression in enabling eurytherms to adjust to these two temporally distinct patterns of thermal stress are poorly understood. We used cDNA microarray analysis to examine changes in gene expression in a eurythermal fish, Austrofundulus limnaeus, subjected to long-term acclimation to constant temperatures of 20, 26 and 37 degrees C and to environmentally realistic daily fluctuations in temperature between 20 degrees C and 37 degrees C. Our data reveal major differences between the transcriptional responses in the liver made during acclimation to constant temperatures and in response to daily temperature fluctuations. Control of cell growth and proliferation appears to be an important part of the response to change in temperature, based on large-scale changes in mRNA transcript levels for several key regulators of these pathways. However, cell growth and proliferation appear to be regulated by different genes in constant versus fluctuating temperature regimes. The gene expression response of molecular chaperones is also different between constant and fluctuating temperatures. Small heat shock proteins appear to play an important role in response to fluctuating temperatures whereas larger molecular mass chaperones such as Hsp70 and Hsp90 respond more strongly to chronic high temperatures. A number of transcripts that encode for enzymes involved in the biosynthesis of nitrogen-containing organic osmolytes have gene expression patterns that indicate a possible role for these 'chemical chaperones' during acclimation to chronic high temperatures and daily temperature cycling. Genes important for the maintenance of membrane integrity are highly responsive to temperature change. Changes in fatty acid saturation may be important in long-term acclimation and in response to fluctuating temperatures; however cholesterol metabolism may be most critical for short-term acclimation to fluctuating temperatures. The variable effect of temperature on the expression of genes with daily rhythms of expression indicates that there is a complex interaction between the temperature cycle and daily rhythmicity in gene expression. A number of new hypotheses concerning temperature acclimation in fish have been generated as a result of this study. The most notable of these hypotheses is the possibility that the high mobility group b1 (HMGB1) protein, which plays key roles in the assembly of transcription initiation and enhanceosome complexes, may act as a compensatory modulator of transcription in response to temperature, and thus as a global gene expression temperature sensor. This study illustrates the utility of cDNA microarray approaches in both hypothesis-driven and 'discovery-based' investigations of environmental effects on organisms.
View details for DOI 10.1242/jeb.01016
View details for Web of Science ID 000222727900016
View details for PubMedID 15159429
Evolutionary convergence in adaptation of proteins to temperature: A(4)-Lactate dehydrogenases of pacific damselfishes (Chromis spp.)
MOLECULAR BIOLOGY AND EVOLUTION
2004; 21 (2): 314-320
We have compared the kinetic properties (Michaelis-Menten constant [K(m)] and catalytic rate constant [k(cat)]) and amino acid sequences of orthologs of lactate dehydrogenase-A (A(4)-LDH) from congeners of Pacific damselfishes (genus Chromis) native to cold-temperate and tropical habitats to elucidate mechanisms of enzymatic adaptation to temperature. Specifically, we determined whether the sites of adaptive variation and the types of amino acids involved in substitutions at these sites were similar in the Chromis orthologs and other orthologs of warm-adapted and cold-adapted A(4)-LDH previously studied. We report striking evolutionary convergence in temperature adaptation of this protein and present further support for the hypothesis that enzyme adaptation to temperature involves subtle amino acid changes at a few sites that affect the mobility of the portions of the enzyme that are involved in rate-determining catalytic conformational changes. We tested the predicted effects of differences in sequence using site-directed mutagenesis. A single amino acid substitution in a key hinge region of the A(4)-LDH molecule is sufficient to change the kinetic characteristics of a temperate A(4)-LDH to that of a tropical ortholog. This substitution is at the same location that was identified in previous studies of adaptive variation in A(4)-LDH and was hypothesized to be important in adjusting K(m) and k(cat). Our results suggest that certain sites within an enzyme, notably those that establish the energy changes associated with rate-limiting movements of protein structure during catalysis, are "hot spots" of adaptation and that common types of amino acid substitutions occur at these sites to adapt structural "flexibility" and kinetic properties. Thus, despite the wide array of options that proteins have to adjust their structural stabilities in the face of thermal stress, the adaptive changes that couple "flexibility" to alterations of function may be limited in their diversity.
View details for DOI 10.1093/molbev/msh021
View details for Web of Science ID 000220083300014
View details for PubMedID 14660697
Influences of thermal acclimation and acute temperature change on the motility of epithelial wound-healing cells (keratocytes) of tropical, temperate and Antarctic fish
JOURNAL OF EXPERIMENTAL BIOLOGY
2003; 206 (24): 4539-4551
The ability to heal superficial wounds is an important element in an organism's repertoire of adaptive responses to environmental stress. In fish, motile cells termed keratocytes are thought to play important roles in the wound-healing process. Keratocyte motility, like other physiological rate processes, is likely to be dependent on temperature and to show adaptive variation among differently thermally adapted species. We have quantified the effects of acute temperature change and thermal acclimation on actin-based keratocyte movement in primary cultures of keratocytes from four species of teleost fish adapted to widely different thermal conditions: two eurythermal species, the longjaw mudsucker Gillichthys mirabilis (environmental temperature range of approximately 10-37 degrees C) and a desert pupfish, Cyprinodon salinus (10-40 degrees C), and two species from stable thermal environments, an Antarctic notothenioid, Trematomus bernacchii (-1.86 degrees C), and a tropical clownfish, Amphiprion percula (26-30 degrees C). For all species, keratocyte speed increased with increasing temperature. G. mirabilis and C. salinus keratocytes reached maximal speeds at 25 degrees C and 35 degrees C, respectively, temperatures within the species' normal thermal ranges. Keratocytes of the stenothermal species continued to increase in speed as temperature increased above the species' normal temperature ranges. The thermal limits of keratocyte motility appear to exceed those of whole-organism thermal tolerance, notably in the case of T. bernacchii. Keratocytes of T. bernacchii survived supercooling to -6 degrees C and retained motility at temperatures as high as 20 degrees C. Mean keratocyte speed was conserved at physiological temperatures for the three temperate and tropical species, which suggests that a certain rate of motility is advantageous for wound healing. However, there was no temperature compensation in speed of movement for keratocytes of the Antarctic fish, which have extremely slow rates of movement at physiological temperatures. Keratocytes from all species moved in a persistent, unidirectional manner at low temperatures but at higher temperatures began to take more circular or less-persistent paths. Thermal acclimation affected the persistence and turning magnitude of keratocytes, with warmer acclimations generally yielding more persistent cells that followed straighter paths. However, acclimation did not alter the effect of experimental temperature on cellular speed. These findings suggest that more than one temperature-sensitive mechanism may govern cell motility: the rate-limiting process(es) responsible for speed is distinct from the mechanism(s) underlying directionality and persistence. Keratocytes represent a useful study system for evaluating the effects of temperature at the cellular level and for studying adaptive variation in actin-based cellular movement and capacity for wound healing.
View details for Web of Science ID 000187394300018
View details for PubMedID 14610038
Protein adaptations to temperature and pressure: complementary roles of adaptive changes in amino acid sequence and internal milieu
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY
2003; 136 (4): 577-591
Retention of required structural and functional properties of proteins in species adapted to different temperatures and pressures is achieved through variation in amino acid sequence and accumulation of small organic solutes that stabilize protein traits. Conservation of ligand binding and catalytic rate can be achieved by minor differences in sequence. For orthologs of lactate dehydrogenase-A (A(4)-LDH) temperature adaptation may involve only a single amino acid substitution. Adaptation involves changes in conformational mobility of regions of A(4)-LDH that undergo movement during ligand binding, movements that are rate-limiting to catalysis. A model that integrates adaptations in sequence and intracellular milieu is developed on the basis of conformational microstates. Although orthologs of different thermally adapted species vary in stability, at physiological temperatures it is hypothesized that a similar ensemble of conformational microstates exists for all orthologs. Organic solutes stabilize this ensemble of microstates. Differences among orthologs in responses to organic solutes at a common temperature lead to similar responses at normal body temperatures. Because protein stability increases at high protein concentrations, intrinsic stabilities of proteins may reflect the protein concentrations of the cellular compartments in which they occur. Protein-stabilizing solutes like trimethylamine-N-oxide (TMAO) conserve protein function and structure at elevated hydrostatic pressures.
View details for DOI 10.1016/S1096-4959(03)00215-X
View details for Web of Science ID 000187366400002
View details for PubMedID 14662287
Local selection and latitudinal variation in a marine predator-prey interaction
2003; 300 (5622): 1135-1137
Although pairs of species often interact over broad geographic ranges, few studies have explored how interactions vary across these large spatial scales. Surveys along 1500 kilometers of the Pacific coast of North America documented marked variation in the frequency of predation by the snail Nucella canaliculata on the intertidal mussel Mytilus californianus. Laboratory rearing experiments suggest that regional differences in drilling behavior have a genetic basis, and mitochondrial sequence variation confirms that gene flow is low among these snail populations. Marine communities separated by hundreds of kilometers may have intrinsically different dynamics, with interactions shaped by restricted gene flow and spatially varying selection.
View details for Web of Science ID 000182886500042
View details for PubMedID 12750518
Base compositions of genes encoding alpha-actin and lactate dehydrogenase-A from differently adapted vertebrates show no temperature-adaptive variation in G+C content
MOLECULAR BIOLOGY AND EVOLUTION
2003; 20 (1): 105-110
There is a long-standing debate in molecular evolution concerning the putative importance of GC content in adapting the thermal stabilities of DNA and RNA. Most studies of this relationship have examined broad-scale compositional patterns, for example, total GC percentages in genomes and occurrence of GC-rich isochores. Few studies have systematically examined the GC contents of individual orthologous genes from differently thermally adapted species. When this has been done, the emphasis has been on comparing large numbers of genes in only a few species. We have approached the GC-adaptation temperature hypothesis in a different manner by examining patterns of base composition of genes encoding lactate dehydrogenase-A (ldh-a) and alpha-actin (alpha-actin) from 51 species of vertebrates whose adaptation temperatures ranged from -1.86 degrees C (Antarctic fishes) to approximately 45 degrees C (desert reptile). No significant positive correlation was found between any index of GC content (GC content of the entire sequence, GC content of the third codon position [GC(3)], and GC content at fourfold degenerate sites [GC(4)]) and any index of adaptation temperature (maximal, mean, or minimal body temperature). For alpha-actin, slopes of regression lines for all comparisons did not differ significantly from zero. For ldh-a, negative correlations between adaptation temperature and total GC content, GC(3), and GC(4) were observed but were shown to be due entirely to phylogenetic influences (as revealed by independent contrast analyses). This comparison of GC content across a wide range of ectothermic ("cold-blooded") and endothermic ("warm-blooded") vertebrates revealed that frogs of the genus Xenopus, which have commonly been used as a representative cold-blooded species, in fact are outliers among ectotherms for the alpha-actin analyses, raising concern about the appropriateness of choosing these amphibians as representative of ectothermic vertebrates in general. Our study indicates that, whereas GC contents of isochores may show variation among different classes of vertebrates, there is no consistent relationship between adaptation temperature and the percentage of thermal stability-enhancing G + C base pairs in protein-coding genes.
View details for DOI 10.1093/molbev/msg008
View details for Web of Science ID 000180243700014
View details for PubMedID 12519912
Obituary: Peter W. Hochachka (1937-2002).
Comparative biochemistry and physiology. Toxicology & pharmacology : CBP
2002; 133 (4): 471-473
View details for PubMedID 12841218
Thermal physiology and vertical zonation of intertidal animals: Optima, limits, and costs of living
OXFORD UNIV PRESS INC. 2002: 780-789
Temperature's pervasive effects on physiological systems are reflected in the suite of temperature-adaptive differences observed among species from different thermal niches, such as species with different vertical distributions (zonations) along the subtidal to intertidal gradient. Among the physiological traits that exhibit adaptive variation related to vertical zonation are whole organism thermal tolerance, heart function, mitochondrial respiration, membrane static order (fluidity), action potential generation, protein synthesis, heat-shock protein expression, and protein thermal stability. For some, but not all, of these thermally sensitive traits acclimatization leads to adaptive shifts in thermal optima and limits. The costs associated with repairing thermal damage and adapting systems through acclimatization may contribute importantly to energy budgets. These costs arise from such sources as: (i) activation and operation of the heat-shock response, (ii) replacement of denatured proteins that have been removed through proteolysis, (iii) restructuring of cellular membranes ("homeoviscous" adaptation), and (iv) pervasive shifts in gene expression (as gauged by using DNA microarray techniques). The vertical zonation observed in rocky intertidal habitats thus may reflect two distinct yet closely related aspects of thermal physiology: (i) intrinsic interspecific differences in temperature sensitivities of physiological systems, which establish thermal optima and tolerance limits for species; and (ii) 'cost of living' considerations arising from sub-lethal perturbation of these physiological systems, which may establish an energetics-based limitation to the maximal height at which a species can occur. Quantifying the energetic costs arising from heat stress represents an important challenge for future investigations.
View details for Web of Science ID 000180793500010
View details for PubMedID 21708776
Temperature adaptation in Gillichthys (Teleost : Gobiidae) A(4)-lactate dehydrogenases: identical primary structures produce subtly different conformations
JOURNAL OF EXPERIMENTAL BIOLOGY
2002; 205 (9): 1293-1303
Alternative conformations of proteins underlie a variety of biological phenomena, from prion proteins that cause spongiform encephalopathies to membrane channel proteins whose conformational changes admit or exclude specific ions. In this paper, we argue that conformational differences within globular 'housekeeping' enzymes may allow rapid adaptation to novel environments. Muscle-type lactate dehydrogenases (A(4)-LDHs) from the gobies Gillichthys seta and G. mirabilis have identical amino acid sequences but show potentially adaptive differences in substrate affinity (apparent Michaelis constants for pyruvate, K(m)(PYR)) as well as differences in thermal stability. We examined the A(4)-LDH of each species using fluorescence spectroscopy, near- and far-ultraviolet circular dichroism (CD) spectroscopy and hydrogen/deuterium exchange (H/D) Fourier-transform infrared spectroscopy to determine whether structural differences were apparent, the extent to which structural differences could be related to differences in conformational flexibility and whether specific changes in secondary or tertiary structure could be defined. The fluorescence spectra and far-ultraviolet CD spectra of the A(4)-LDH from the two species were indistinguishable, suggesting that the two conformations are very similar in secondary and tertiary structure. Apparent melting temperatures (T(m)) followed by fluorescence and CD spectroscopy confirmed that the G. mirabilis A(4)-LDH is more thermally stable than the G. seta form. H/D exchange kinetics of Gillichthys A(4)-LDH was described using double-exponential regression; at 20 degrees C, G. seta A(4)-LDH has a higher exchange constant, indicating a more flexible and open structure. At 40 degrees C, the difference in H/D exchange constants disappears. Second-derivative analysis of H/D exchange infrared spectra indicates that alpha-helical, but not beta-sheet structure, differs in conformational flexibility between the two forms. Second-derivative ultraviolet spectra indicate that at least one of the five tyrosyl residues in the Gillichthys LDH-A monomer is located in a more hydrophobic environment in the G. mirabilis form. Homology models of A(4)-LDH indicate that Tyr246 is the most likely candidate to experience a modified environment because it is involved in subunit contacts within the homotetramer and sits in a hinge between a static alpha-helix and one involved in catalytic conformational changes. Subtle differences in conformation around this residue probably play a role both in altered flexibility and in the potentially adaptive differences in kinetics between the two A(4)-LDH forms.
View details for Web of Science ID 000175725600011
View details for PubMedID 11948206
Interspecific- and acclimation-induced variation in levels of heat-shock proteins 70 (hsp70) and 90 (hsp90) and heat-shock transcription factor-1 (HSF1) in congeneric marine snails (genus Tegula): implications for regulation of hsp gene expression
JOURNAL OF EXPERIMENTAL BIOLOGY
2002; 205 (5): 677-685
In our previous studies of heat-shock protein (hsp) expression in congeneric marine gastropods of the genus Tegula, we observed interspecific and acclimation-induced variation in the temperatures at which heat-shock gene expression is induced (T(on)). To investigate the factors responsible for these inter- and intraspecific differences in T(on), we tested the predictions of the 'cellular thermometer' model for the transcriptional regulation of hsp expression. According to this model, hsps not active in chaperoning unfolded proteins bind to a transcription factor, heat-shock factor-1 (HSF1), thereby reducing the levels of free HSF1 that are available to bind to the heat-shock element, a regulatory element upstream of hsp genes. Under stress, hsps bind to denatured proteins, releasing HSF1, which can now activate hsp gene transcription. Thus, elevated levels of heat-shock proteins of the 40, 70 and 90 kDa families (hsp 40, hsp70 and hsp90, respectively) would be predicted to elevate T(on). Conversely, elevated levels of HSF1 would be predicted to decrease T(on). Following laboratory acclimation to 13, 18 and 23 degrees C, we used solid-phase immunochemistry (western analysis) to quantify endogenous levels of two hsp70 isoforms (hsp74 and hsp72), hsp90 and HSF1 in the low- to mid-intertidal species Tegula funebralis and in two subtidal to low-intertidal congeners, T. brunnea and T. montereyi. We found higher endogenous levels of hsp72 (a strongly heat-induced isoform) at 13 and 18 degrees C in T. funebralis in comparison with T. brunnea and T. montereyi. However, T. funebralis also had higher levels of HSF1 than its congeners. The higher levels of HSF1 in T. funebralis cannot, within the framework of the cellular thermometer model, account for the higher T(on) observed for this species, although they may explain why T. funebralis is able to induce the heat-shock response more rapidly than T. brunnea. However, the cellular thermometer model does appear to explain the cause of the increases in T(on) that occurred during warm acclimation of the two subtidal species, in which warm acclimation was accompanied by increased levels of hsp72, hsp74 and hsp90, whereas levels of HSF1 remained stable. T. funebralis, which experiences greater heat stress than its subtidal congeners, consistently had higher ratios of hsp72 to hsp74 than its congeners, although the sum of levels of the two isoforms was similar for all three species except at the highest acclimation temperature (23 degrees C). The ratio of hsp72 to hsp74 may provide a more accurate estimate of environmental heat stress than the total concentrations of both hsp70 isoforms.
View details for Web of Science ID 000174507900011
View details for PubMedID 11907057
Phylogenetic relationships and biochemical properties of the duplicated cytosolic and mitochondrial isoforms of malate dehydrogenase from a teleost fish, Sphyraena idiastes
JOURNAL OF MOLECULAR EVOLUTION
2002; 54 (1): 107-117
Unlike birds and mammals, teleost fish express two paralogous isoforms (paralogues) of cytosolic malate dehydrogenase (cMDH; EC 184.108.40.206; NAD+: malate oxidoreductase) whose evolutionary relationships to the single cMDH of tetrapods are unknown. We sequenced complementary DNAs for both cMDHs and the mitochondrial isoform (mMDH) of the fish Sphyraena idiastes (south temperate barracuda) and compared the sequences, kinetic properties, and thermal stabilities of the three isoforms with those of mammalian orthologues. Both fish cMDHs comprise 333 residues and have subunit masses of approximately 36 kDa. One cytosolic isoform, cMDH-S, was significantly more heat-stable than either the other cMDH (cMDH-L) or mMDH. In contradiction to the generally accepted model of vertebrate cMDH evolution, our phylogenetic analysis indicates that the duplication of the fish cytosolic paralogues occurred after the divergence of the lineages leading to teleosts and tetrapods. cMDH-L and cMDH-S differed in optimal concentrations of substrates and cofactors and apparent Michaelis-Menten constants, suggesting that the two paralogues may play distinct physiological roles. Differences in intrinsic thermal stability among MDH paralogues may reflect different degrees of stabilization in vivo by extrinsic stabilizers, notably protein concentration in the case of mMDH. Thermal stabilities of porcine mMDH and cMDH-L, but not cMDH-S, were significantly increased when denaturation was measured at a high protein (bovine serum albumin; BSA) concentration, but the BSA-induced stabilization reduced the catalytic activity.
View details for Web of Science ID 000172518200013
View details for PubMedID 11734904
Intrinsic versus extrinsic stabilization of enzymes - The interaction of solutes and temperature on A(4)-lactate dehydrogenase orthologs from warm-adapted and cold-adapted marine fishes
EUROPEAN JOURNAL OF BIOCHEMISTRY
2001; 268 (16): 4497-4505
We examined the effects of temperature and stabilizing solutes on A4-lactate dehydrogenase (A4-LDH) from warm- and cold-adapted fishes, to determine how extrinsic stabilizers affect orthologs with different intrinsic stabilities. Conformational changes during substrate binding are rate-limiting for A4-LDH, thus stabilization due to intrinsic or extrinsic factors leads to decreased activity. A4-LDH from a warm-temperate goby (Gillichthys mirabilis), which has lower values for kcat and the Michaelis constant for pyruvate ( K m PYR), was intrinsically more stable than the orthologs of the cold-adapted Antarctic notothenioids Parachaenichthys charcoti and Chionodraco rastrospinosus, as shown by a higher apparent transition ('melting') temperature (Tm(APP)). We used four solutes, glycerol, sucrose, trimethylamine-N-oxide and poly(ethylene glycol) 8000, which stabilize proteins through different modes of preferential exclusion, to study temperature-solute interactions of the three orthologs. Changes in Tm(APP) were similar for all orthologs in each solute tested, but the catalytic rate of G. mirabilis A4-LDH was decreased most by solutes and increased most by temperature. In contrast, the K m PYR values of the Antarctic orthologs were more affected than that of the goby by both solutes and temperature. We conclude that (a) preferential exclusion of solutes functions within the native state of A4-LDH to favor conformational microstates with minimal surface area; (b) the varied effects of the different solutes on the kinetic properties are due to the interaction between this nonspecific stabilization and the differing intrinsic stabilities of the orthologs; (c) the catalytic rates of A4-LDH orthologs are equally affected by stabilizing solutes, if measurements are made at physiologically appropriate temperatures; and (d) global stability and localized flexibility of these A4-LDH orthologs may evolve independently.
View details for Web of Science ID 000170467000014
View details for PubMedID 11502210
Hypoxia-induced gene expression profiling in the euryoxic fish Gillichthys mirabilis
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2001; 98 (4): 1993-1998
Hypoxia is important in both biomedical and environmental contexts and necessitates rapid adaptive changes in metabolic organization. Mammals, as air breathers, have a limited capacity to withstand sustained exposure to hypoxia. By contrast, some aquatic animals, such as certain fishes, are routinely exposed and resistant to severe environmental hypoxia. Understanding the changes in gene expression in fishes exposed to hypoxic stress could reveal novel mechanisms of tolerance that may shed new light on hypoxia and ischemia in higher vertebrates. Using cDNA microarrays, we have studied gene expression in a hypoxia-tolerant burrow-dwelling goby fish, Gillichthys mirabilis. We show that a coherent picture of a complex transcriptional response can be generated for a nonmodel organism for which sequence data were unavailable. We demonstrate that: (i) although certain shifts in gene expression mirror changes in mammals, novel genes are differentially expressed in fish; and (ii) tissue-specific patterns of expression reflect the different metabolic roles of tissues during hypoxia.
View details for Web of Science ID 000166949200121
View details for PubMedID 11172064
A comparative analysis of the evolutionary patterning and mechanistic bases of lactate dehydrogenase thermal stability in porcelain crabs, genus Petrolisthes
JOURNAL OF EXPERIMENTAL BIOLOGY
2001; 204 (4): 767-776
The kinetic properties of orthologous homologs (orthologs) of enzymes are typically correlated with environmental temperatures in species adapted to different thermal regimes, but correlations between adaptation temperature and enzyme thermal stability are less clear. Although the thermal stability of a protein is related chiefly to its primary structure (including post-translational modification), thermal stability can also be altered by extrinsic factors present in the intracellular milieu. Here, we present a comparative analysis of the thermal stability of lactate dehydrogenase (LDH) orthologs from 22 congeneric species of porcelain crab (genera Petrolisthes and Allopetrolisthes) from a broad range of thermal habitats. Interspecific diversity of LDH stability is high: temperatures required for a 50 % loss of activity in 10 min ranged from 65 to 75.5 degrees C, corresponding to half-lives of less than 1 min to more than 3 h at 70 degrees C. Although stability is positively correlated with maximal habitat temperature in some sister taxa, phylogenetic comparative analysis incorporating all 22 species does not indicate that the interspecific diversity of LDH stability represents an adaptive response to current thermal habitats. Examination of the mechanistic bases of LDH stabilization indicates that differences in stability are related both to properties of the LDH molecule itself (intrinsic stability) and to the effects of extrinsic protein(s). Intrinsic differences were shown by the unfolding of structure during heating, as measured by circular dichroism spectroscopy. Stabilizing effects of extrinsic proteins are implied by the results of cellular fractionation experiments that removed low-molecular-mass solutes and proteins from the muscle homogenates. We conclude that the overall structural stability and functional properties of proteins can evolve independently and that in vivo protein-protein interactions can provide another means to regulate protein stability selectively.
View details for Web of Science ID 000167441000016
View details for PubMedID 11171359
Time course and magnitude of synthesis of heat-shock proteins in congeneric marine snails (Genus Tegula) from different tidal heights
PHYSIOLOGICAL AND BIOCHEMICAL ZOOLOGY
2000; 73 (2): 249-256
The time course and magnitude of the heat-shock response in relation to severity of thermal stress are important, yet poorly understood, aspects of thermotolerance. We examined patterns of protein synthesis in congeneric marine snails (genus Tegula) that occur at different heights along the subtidal to intertidal gradient after a thermal exposure (30 degrees C for 2.5 h, followed by 50 h recovery at 13 degrees C) that induced the heat-shock response. We monitored the kinetics and magnitudes of protein synthesis by quantifying incorporation of 35S-labeled methionine and cysteine into newly synthesized proteins and observed synthesis of putative heat-shock proteins (hsp's) of size classes 90, 77, 70, and 38 kDa. In the low- to mid-intertidal species, Tegula funebralis, whose body temperature frequently exceeds 30 degrees C during emersion, synthesis of hsp's commenced immediately after heat stress, reached maximal levels 1-3 h into recovery, and returned to prestress levels by 6 h, except for hsp90 (14 h). In contrast, in the low-intertidal to subtidal species, Tegula brunnea, for which 2.5 h at 30 degrees C represents a near lethal heat stress, synthesis of hsp's commenced 2-14 h after heat stress; reached maximal levels after 15-30 h, which exceeded magnitudes of synthesis in T. funebralis; and returned to prestress levels in the case of hsp90 (50 h) and hsp77 (30 h) but not in the case of hsp70 and hsp38. Exposures to 30 degrees C under aerial (emersion) and aquatic (immersion) conditions resulted in differences in hsp synthesis in T. brunnea but not in T. funebralis. The different time courses and magnitudes of hsp synthesis in these congeners suggest that the vertical limits of their distributions may be set in part by thermal stress.
View details for Web of Science ID 000087276400013
View details for PubMedID 10801403
- Unity in diversity: A perspective on the methods, contributions, and future of comparative physiology ANNUAL REVIEW OF PHYSIOLOGY 2000; 62: 927-937
Evolutionary and acclimation-induced variation in the heat-shock responses of congeneric marine snails (genus Tegula) from different thermal habitats: implications for limits of thermotolerance and biogeography.
The Journal of experimental biology
1999; 202 (Pt 21): 2925-2936
Heat stress sufficient to cause cellular damage triggers the heat-shock response, the enhanced expression of a group of molecular chaperones called heat-shock proteins (hsps). We compared the heat-shock responses of four species of marine snails of the genus Tegula that occupy thermal niches differing in absolute temperature and range of temperature. We examined the effects of short-term heat stress and thermal acclimation on the synthesis of hsps of size classes 90, 77, 70 and 38 kDa by measuring incorporation of (35)S-labeled methionine and cysteine into newly synthesized proteins in gill tissue. Temperatures at which enhanced synthesis of hsps first occurred (T(on)), temperatures of maximal induction of hsp synthesis (T(peak)) and temperatures at which hsp synthesis was heat-inactivated (T(off)) were lowest in two low-intertidal to subtidal species from the temperate zone, T. brunnea and T. montereyi, intermediate in a mid- to low-intertidal species of the temperate zone, T. funebralis, and highest in a subtropical intertidal species from the Gulf of California, T. rugosa. Synthesis of hsps and other classes of protein by T. brunnea and T. montereyi was heat-inactivated at temperatures commonly encountered by T. funebralis during low tides on warm days. In turn, protein synthesis by T. funebralis was blocked at the upper temperatures of the habitat of T. rugosa. Acclimation of snails to 13 degrees C, 18 degrees C and 23 degrees C shifted T(on) and T(peak) for certain hsps, but did not affect T(off). The heat-shock responses of field-acclimatized snails were generally reduced in comparison with those of laboratory-acclimated snails. Overall, despite the occurrence of acclimatory plasticity in their heat-shock responses, genetically fixed differences in T(on), T(peak) and T(off) appear to exist that reflect the separate evolutionary histories of these species and may play important roles in setting their thermal tolerance limits and, thereby, their biogeographic distribution patterns.
View details for PubMedID 10518474
Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A(4) orthologs of Antarctic notothenioid fishes
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1998; 95 (19): 11476-11481
To elucidate mechanisms of enzymatic adaptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A4 (A4-LDH) from nine Antarctic (-1.86 to 1 degree C) and three South American (4 to 10 degree C) notothenioid teleosts. Higher Michaelis-Menten constants (Km) and catalytic rate constants (kcat) distinguish orthologs of Antarctic from those of South American species, but no relationship exists between adaptation temperature and the rate at which activity is lost because of heat denaturation. In all species, active site residues are conserved fully, and differences in kcat and Km are caused by substitutions elsewhere in the molecule. Within geographic groups, identical kinetic properties are generated by different substitutions. By combining our data with A4-LDH sequences for other vertebrates and information on roles played by localized conformational changes in setting kcat, we conclude that notothenioid A4-LDHs have adapted to cold temperatures by increases in flexibility in small areas of the molecule that affect the mobility of adjacent active-site structures. Using these findings, we propose a model that explains linked temperature-adaptive variation in Km and kcat. Changes in sequence that increase flexibility of regions of the enzyme involved in catalytic conformational changes may reduce energy (enthalpy) barriers to these rate-governing shifts in conformation and, thereby, increase kcat. However, at a common temperature of measurement, the higher configurational entropy of a cold-adapted enzyme may foster conformations that bind ligands poorly, leading to high Km values relative to warm-adapted orthologs.
View details for Web of Science ID 000075957100085
View details for PubMedID 9736762