Cell Biology in the Antarctic: Studying Life in the Freezer

commentary Cell biology in the Antarctic: studying life in the freezer

Karin Römisch and Tom Matheson

Many classical biologists working in Antarctica have ‘gone molecular’ to study the physiological basis for life on or below the ice. Investigating the remarkable adaptations of specific cells, organelles and molecules to this extreme environment can provide new perspectives on the processes studied in conventional experimental organisms.

iological research in the Antarctic focuses on survival mechanisms in Bextreme conditions. Low temperature slows all physiological processes, changes protein–protein interactions, reduces membrane fluidity and increases the viscosity of water. Ice formation can rapidly destroy cell membranes and disrupts osmotic balance. The examination of how cells and organisms respond to such challenges requires a wide range of sophisticated techniques. The development of suitable facilities in the Antarctic has contributed to an explosion of research at molecular and cellular levels. For example, the US Antarctic Program maintains three stations, Palmer on the Antarctic peninsula, the Amundsen-Scott South Pole station and McMurdo on Ross Island. The McMurdo station (Fig. 1; http://www.nsf.gov/od/opp/antarct/usap.ht m; also see British Antarctic Survey, http://www.antarctica.ac.uk) is by far the largest of the three, with a summer popula- Figure 1 McMurdo Station on Hut Point, Ross Island. In the summer, the base runs around the clock tion of 1,200. The Crary Laboratory at with a busy heliport (front left) and an ice-pier (rear centre) that becomes the focus of activity when McMurdo (Fig. 1) is well equipped and icebreakers and resupply ships make their essential visits. The main science facility is the Crary Lab allows scientists to investigate the same top- (three-tier building in the centre). The base has a hospital (red roof), fire station and central galley ics as they would in their home labs, but on (large beige building, centre-right), as well as dormitories (ranks of brown buildings in the back) and the uniquely adapted Antarctic organisms. numerous other maintenance and support buildings. This approach may help identify limiting steps in complex cellular processes, such as protein secretion or neuronal function. is not understood, but our sequence data sug- Antifreeze gest that similar to cold-adapted enzymes, the Fish from the polar regions thrive at tem- amino acid substitutions in the channel pro- peratures close to freezing point (the mini- Protein secretion and membrane fluidity tein designate hinge regions that allow a con- mum water temperature at McMurdo is − Protein secretion is an essential process that is formational change essential for protein 1.9 °C; freezing occurs at −1.91 °C). To extremely cold-sensitive1. The majority of life translocation across the endoplasmic reticu- avoid death by freezing, fish in these envi- on earth, however, exists well below the tem- lum membrane2,4. ronments have evolved antifreeze molecules peratures at which model organisms, such as Membranes from organisms that exist in that they secrete into their blood at high tissue culture cells and yeast2,3, are usually cold habitats contain a high proportion of concentrations (30–60 mg ml−1; refs 5, 6). grown. But how does protein secretion work unsaturated fatty acids, which can keep Four classes of antifreeze proteins have in the cold? We found that at low tempera- membranes fluid at low temperatures; how- been characterized: types I and IV are tures, the first step of protein secretion — ever, this correlation is limited5. For exam- α-helical, whereas types II and III are small translocation into the endoplasmic reticulum ple, in endoplasmic reticulum membranes globular proteins7. In addition, a fifth — was more efficient in cold-adapted organ- from an Antarctic fish, we found a high group, antifreeze glycopeptides, is common isms when compared with temperate species. concentration of unsaturated fatty acids, in Antarctic fish. These glycopeptides have We identified a limited number of amino acid but no concomitant increase in membrane relative molecular masses (Mr) ranging changes in conserved positions of the translo- fluidity (Römisch et al., unpublished obser- from 2,400–34,000 (2.4–34K) and a highly cation channel-forming protein that may vations). Our data suggest that membrane repetitive α-helical structure, (Ala-Ala- constitute cold-adaptation (Römisch et al., rigidity is not a limiting factor for protein Thr)n, which arose from multiplication of unpublished observations). The channel- translocation across the endoplasmic retic- an exon–intron boundary in trypsinogen8. opening mechanism for protein translocation ulum membrane in the cold. The poly-protein precursor has a cleavable

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signal peptide and its threonine residues are but most probably by oxygen availability, as Communication between neurons at synaps- heavily O-glycosylated. The glycoprotein is discussed below. es depends on the rates of neurotransmitter proteolytically processed into characteristi- release, diffusion and binding, and subse- cally sized antifreeze glycopeptides before quent neurotransmitter recycling or break- or after secretion. Arctic fish secrete similar Oxygen uptake and delivery down. glycopeptides into their blood, but these are The solubility of oxygen in water is Striking differences in neuronal function derived from a different gene — a classic inversely correlated to the water tempera- and network architecture between the example of convergent evolution9. ture, and the oxygen saturation in Antarctic mollusc Clione antarctica and a Biological antifreezes adsorb to the sur- McMurdo Sound can reach 100%. related temperate species have been identi- faces of ice crystals in the blood of these fish Therefore, oxygen uptake and transport fied (J. Rosenthal, personal communica- and prevent the association of additional are not limiting for Antarctic fish. tion). The voltage dependence of sodium water molecules, and thus growth of the ice Erythrocyte counts of red-blooded channel opening is shifted positively in C. crystals6,7,10. For example, Type III antifreeze Antarctic notothenioid fish are an order antarctica, which raises the neuronal spike has a flat, amphipathic ice-binding surface of magnitude lower than related temper- threshold and makes it more difficult for with polar residues arranged such that they ate fish and can be reduced by three the neurons to carry signals (as sodium can hydrogen-bond to oxygen on a specific orders of magnitude in white-blooded channels drive regenerative action poten- prism plane of the ice crystal11,12. Antarctic icefish20. This may be advanta- tials). However, calcium channels operate Subsequently, Van der Waal’s interactions geous in coping with increased viscosity over their usual voltage range, and may between other residues in the ice-binding of body fluids at low temperature and is therefore have an enhanced role in spike surface and the ice itself strengthen to partially compensated for by increased generation in the Antarctic species. exclude water near the ice surface11,12. blood volume and higher cardiac out- Our work includes studies of neuronal Recent work suggests the key feature of all put21,22. The haemoglobin content of ery- function in Antarctic marine crustacea protein antifreezes is complementarity throcytes is variable in Antarctic fish and (http://www.zoo.cam.ac.uk/zoostaff/math- between the ice-binding protein surfaces is positively correlated to swimming eson/antarc1.html). At all temperatures and the ice crystals13. Antifreeze molecules activity20,23,24. Haemoglobin can be com- examined, the peripheral nerves of the can also adsorb to biological membranes pletely absent in icefish, which can also Antarctic isopod G. antarcticus (Fig. 2, and protect them by maintaining lipid lack the oxygen binding protein of mus- right) conduct more rapidly than those of a bilayer structure during cooling through cle, myoglobin25,26. temperate species, but this is not related to their phase transition temperatures14,15. Reduced levels of oxygen-binding pro- differences in axon diameter or myelina- teins in Antarctic icefish correlate with an tion. Examination of touch-sensitive increase in the percentage of cell volume mechanoreceptors in G. antarcticus demon- Heat shock containing mitochondria27,28. Furthermore, strate that sensory transduction is strongly At high temperatures, cells increase the oxygen solubility is higher in membrane affected by temperature. The number of expression of heat shock proteins (Hsp), lipids than in aqueous solutions, and there- action potentials elicited by a stimulus specific molecular chaperones that prevent fore mitochondrial membranes may act as increases with temperature from 1.8–7 °C, the aggregation of thermally unfolded pro- conduits for oxygen and partially compen- but then decreases and fails at 21 °C. teins. What constitutes heat stress depends sate for the lack of haemoglobin and myo- In Antarctic fish, nerve conduction fails on the habitat temperature of the organism globin24,28. Antarctic budding yeast often when freezing sets in (at around −5 °C); and the maximal temperature variation. contain giant mitochondria (Fig. 2, cen- conversely, the velocity of propagation Marine organisms in McMurdo Sound are tre), which may support the hypothesis increases with temperature (up to 28 °C) in adapted to survive extremely low tempera- that intracellular membranes are critical a linear fashion31. At low temperature, tures (minimum −1.9 °C) and a narrow for adequate oxygen delivery at low tem- nerves from Antarctic fish conduct substan- temperature range; even in the austral sum- perature (Römisch et al., unpublished tially faster and their neuromuscular junc- mer, the water temperature rises maximally observations). tions have higher rates of spontaneous neu- by 1.5 °C16. As a result of adapting to this A further consequence of high oxygen rotransmitter release (miniature endplate environment, Antarctic marine inverte- availability in cold water is gigantism of potentials) than those of temperate species, brates induce heat shock proteins at 0 °C invertebrates29. Increased oxygen content of indicating that their presynaptic terminals (National Science Foundation Marine the blood allows a longer circulatory sys- are relatively closer to neurotransmitter Biology Course, 2000). An Antarctic teleost tem, and thus a larger body30. For example, release. This is probably a mechanism for fish, Trematomus bernacchii, has lost its heat the volume of the Antarctic giant isopod reducing transmission failures at low tem- shock response altogether. T. bernacchii Glyptonotus antarcticus (Fig. 2, right), a rel- perature. However, individual endplate (Fig. 2, left) fails to increase expression of ative of woodlice (about 250 µl volume), potentials decay more rapidly in the heat shock proteins in response to either can excede 50 ml. Antarctic fish, thus limiting the post-synap- thermal or chemical stress17. This may be a tic response amplitude at low temperatures. result of living in a thermal environment In contrast, the responses of vestibular (bal- that has been extremely stable for the past Neurobiological adaptations ance and orientation) neurons in the central 14–25 million years17. The Hsp70 family, in Neuronal function is dependent on the ability nervous system of Antarctic fish are remark- particular, may be dispensable, because they of cell membranes to establish, maintain and ably similar to those of temperate species. interact primarily with hydrophobic patch- rapidly change transmembrane electrical es of unfolded proteins18.Hydrophobic potential. Ion pumps generate electrochemical interactions are relatively weak at low tem- gradients across membranes, and the ability of Structural adaptations of enzymes peratures, so the Hsp70 family will proba- neurons to generate trains of action potentials Life exists even in the most inhospitable bly not be functional in the cold, nor will is influenced by the kinetics of channel open- regions of Antarctica, such as brine canals hydrophobic patches be important for ing and closing. Channel gating, which is in sea ice and snow at the South Pole32.The aggregation of misfolded proteins2. T. dependent on conformational changes of pro- organisms that live in these extreme envi- bernacchii die at temperatures of 5–6 °C19. teins, is more strongly affected by changes in ronments have enzymes with either higher The upper lethal temperature is not deter- temperature than those of channel conduc- catalytic activity over a temperature range, mined by thermal unfolding of proteins, tance, which depends primarily on diffusion. or specifically at low temperature. The

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increased thermolability accompanying these adaptations may be a consequence of increased protein flexibility conferred by amino acid changes that allow cold-adapted enzymes to undergo conformational changes required for activity at low temper- ature2–4. Alternatively, thermolability may be caused simply by a lack of selective pressure, as temperatures in cold habitats are often very stable2,3,33. Increased thermolability means that enzymes from psy- Figure 2 Experimental organisms. The Antarctic teleost fish T. bernacchii (left; 15 cm total length), chrophiles (organisms that can only live in budding yeast Cryptococcus antarcticus (centre; 5 µm diameter) and isopod crustacean G. antarcti- the cold) are often difficult to crystallize. cus (right; 7 cm body width). Most structural data available are derived from cloning, sequencing, homology mod- elling strategies, and recently from nuclear magnetic resonance (NMR) studies. The associate laterally to form a hollow cylinder, Molecular cell biology research in the changes that make enzymes work in the the microtubule. Tubulins from Antarctic Antarctic is still in its infancy. At a molecu- cold are subtle and often challenging to fish contain a small number of amino acid lar level, the majority of biological phe- identify, as it can be difficult to discern what substitutions that map to the lateral inter- nomena specific to this environment are changes evolved under selective pressure, protofilament surfaces and to the interior of just beginning to be understood. Much and which are functionally neutral33.For the α- and β-subunits, but surprisingly, not remains to be discovered for anyone inter- these reasons, directed evolution in the lab to the longitudinal inter-dimer surfaces36. ested in life from the cold habitats of our is now being used to understand what pro- These alterations seem to stabilize the tubu- planet. tein features are important for adaptation lin monomers in a conformation that Karin Römisch is in the Cambridge Institute of to specific parameters33. favours polymerization and strengthens Medical Research and the Department of Clinical Protein flexibility, and hence enzymatic protofilament interactions. In addition, Biochemistry University of Cambridge, Hills Road, activity, is determined by intramolecular dynamic instability of microtubules from Cambridge, CB2 2XY, UK bonds, interactions between enzyme sub- Antarctic fish is strongly reduced, presum- Tom Matheson is in the Department of Zoology, units, substrate binding and interactions ably because the amino acid changes in the University of Cambridge, Downing Street, with the solvent2. These interactions change tubulin monomers slow the conformation- Cambridge CB2 3EJ, UK with decreasing temperature: hydrogen al change in the tubulin dimer preceding e-mail: [email protected] 36 bonds and electrostatic interactions depolymerization . Microtubules from 1. Baker, D., Hicke, L., Rexach, M., Schleyer, M. & Schekman, R. become stronger and hydrophobic interac- Saccharomyces cerevisiae also display little Cell 54, 335–344 (1988). tions are weakened. Enzymes with low opti- dynamic instability when compared with 2. Russell, N. J. 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