Molecular interactions that stabilize antarctic fish microtubules at low temperatures H. WILLIAM DETRICH, III Department of Biology Northeastern University Boston, Massachusetts 02115 The cold-adapted antarctic fishes diverged from a temperate fish fauna approximately 40 million years ago as the southern ocean began to cool (DeWitt 1971). Over time, the antarctic fishes evolved cellular and biochemical adaptations that main- tain appropriate reaction rates and equilibria at their cold body temperatures (- 1.9 to + 2 °C). The goal of my project is to determine the molecular adaptations that enable the micro- tubules of antarctic fishes to assemble and function efficiently in their extreme thermal environment. Microtubules are a major component of the cytoskeleton of most eukaryotic cells. They participate in many fundamental processes, including mitosis, nerve growth and regeneration, the intracellular transport of organelles, and the determination of cell shape. The formation of microtubules from their subunit proteins, tubulin alpha-beta dimers and microtubule-associ- ated proteins (MAPs), is an entropically driven process that is favored by high temperatures (Correia and Williams 1983). Thus, the microtubule proteins of vertebrate homeotherms po- lymerize at temperatures near 37 °C, but these microtubules are cold-labile; they disassemble to their subunits at low tem- peratures (0-4 °C). How, then, do the microtubules of cold- living poikilotherms (e.g., the fishes of the antarctic marine ecosystem) assemble and function at body temperatures as low as —1.9°C? During the past year, we completed studies of the poly- merization energetics of pure antarctic fish tubulins at near- physiological and supraphysiological temperatures (Detrich, Johnson, and Marchese-Ragona 1989). The figure presents a representative electron micrograph of the microtubules that formed when a solution of brain tubulin from an antarctic cod, Notothenia coriiceps neglecta, was warmed from 0 to 20 °C. We found that the critical (i.e., minimal) concentrations of fish tubulin necessary to support microtubule assembly, deter- mined by a quantitative sedimentation assay, ranged from 0.87 milligrams/milliliter at 0 °C to 0.02 milligrams/milliliter at 18 °C. By contrast, critical concentrations for pure marimalian tubulins at like temperatures are estimated to be two orders of magnitude larger (Williams, Correia, and DeVries 1985). Clearly, antarctic fish tubulins form microtubules efficiently at low temperatures. A van't Hoff analysis of the data for the antarctic fish tubulins gave a standard enthalpy change for polymerization of + 26.9 kilocalories/mole and a standard en- tropy change of + 123 entropy units. These values, which are substantially larger than those for polymerization of tubulins from temperate poikilotherms or from homeotherms, suggest that an increase in the proportion of hydrophobic interactions (relative to other bond types) at sites of tubulin-tubulin contact is the major functional adaptation of the antarctic fish tubulins. Many, if not most, of these alterations are likely to reside in AA V ' ft I All Electron micrograph of microtubule polymer assembled in vitro from the brain tubulin of N. corilceps neglecta. A solution of tubulin (0.64 milligrams per milliliter in a polymerization buffer containing 1 milllmolar guanosine 5'-triphosphate) was warmed from 0 to 20 °C, and a negatively stained specimen was prepared 30 minutes later. Microtubules of normal morphology were the predominant product of assembly. The protofilaments of these microtubules are readily apparent. The bar represents 100 nanometers. Reprinted from Detrich, Johnson, and Marchese-Ragona (1989) with permis- sion. Copyright 1989 American Chemical Society. their structurally divergent alpha chains (Detrich and Overton 1986; Detrich, Prasad, and Ludueña 1987). The results presented above indicate that much of the cold stability of antarctic fish microtubules results from alterations to their tubulins. Nevertheless, one may ask whether the MAPs of these fishes make additional contributions to the energetics of microtubule formation at low temperatures. To address this question, we compared the capacities of MAPs from antarctic fishes and from a mammal (the cow) to promote the poly- merization of antarctic fish tubulins at temperatures near 0 °C (Detrich et al. 1990). Compared on a weight basis, both bovine and fish MAPs induced comparable extents of microtubule formation. Thus, it appears unlikely that the MAPs of antarctic fishes possess major functional adaptations that are absent in 218 ANTARCTIC JOURNAL
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Molecular interactionsthat stabilize
antarctic fish microtubulesat low temperatures
H. WILLIAM DETRICH, III
Department of BiologyNortheastern University
Boston, Massachusetts 02115
The cold-adapted antarctic fishes diverged from a temperatefish fauna approximately 40 million years ago as the southernocean began to cool (DeWitt 1971). Over time, the antarcticfishes evolved cellular and biochemical adaptations that main-tain appropriate reaction rates and equilibria at their cold bodytemperatures (- 1.9 to + 2 °C). The goal of my project is todetermine the molecular adaptations that enable the micro-tubules of antarctic fishes to assemble and function efficientlyin their extreme thermal environment.
Microtubules are a major component of the cytoskeleton ofmost eukaryotic cells. They participate in many fundamentalprocesses, including mitosis, nerve growth and regeneration,the intracellular transport of organelles, and the determinationof cell shape. The formation of microtubules from their subunitproteins, tubulin alpha-beta dimers and microtubule-associ-ated proteins (MAPs), is an entropically driven process that isfavored by high temperatures (Correia and Williams 1983).Thus, the microtubule proteins of vertebrate homeotherms po-lymerize at temperatures near 37 °C, but these microtubulesare cold-labile; they disassemble to their subunits at low tem-peratures (0-4 °C). How, then, do the microtubules of cold-living poikilotherms (e.g., the fishes of the antarctic marineecosystem) assemble and function at body temperatures as lowas —1.9°C?
During the past year, we completed studies of the poly-merization energetics of pure antarctic fish tubulins at near-physiological and supraphysiological temperatures (Detrich,Johnson, and Marchese-Ragona 1989). The figure presents arepresentative electron micrograph of the microtubules thatformed when a solution of brain tubulin from an antarctic cod,Notothenia coriiceps neglecta, was warmed from 0 to 20 °C. Wefound that the critical (i.e., minimal) concentrations of fishtubulin necessary to support microtubule assembly, deter-mined by a quantitative sedimentation assay, ranged from 0.87milligrams/milliliter at 0 °C to 0.02 milligrams/milliliter at 18°C. By contrast, critical concentrations for pure marimaliantubulins at like temperatures are estimated to be two ordersof magnitude larger (Williams, Correia, and DeVries 1985).Clearly, antarctic fish tubulins form microtubules efficiently atlow temperatures. A van't Hoff analysis of the data for theantarctic fish tubulins gave a standard enthalpy change forpolymerization of + 26.9 kilocalories/mole and a standard en-tropy change of + 123 entropy units. These values, which aresubstantially larger than those for polymerization of tubulinsfrom temperate poikilotherms or from homeotherms, suggestthat an increase in the proportion of hydrophobic interactions(relative to other bond types) at sites of tubulin-tubulin contactis the major functional adaptation of the antarctic fish tubulins.Many, if not most, of these alterations are likely to reside in
AA
V'
ft I All
Electron micrograph of microtubule polymer assembled in vitrofrom the brain tubulin of N. corilceps neglecta. A solution of tubulin(0.64 milligrams per milliliter in a polymerization buffer containing1 milllmolar guanosine 5'-triphosphate) was warmed from 0 to 20°C, and a negatively stained specimen was prepared 30 minuteslater. Microtubules of normal morphology were the predominantproduct of assembly. The protofilaments of these microtubules arereadily apparent. The bar represents 100 nanometers. Reprintedfrom Detrich, Johnson, and Marchese-Ragona (1989) with permis-sion. Copyright 1989 American Chemical Society.
their structurally divergent alpha chains (Detrich and Overton1986; Detrich, Prasad, and Ludueña 1987).
The results presented above indicate that much of the coldstability of antarctic fish microtubules results from alterationsto their tubulins. Nevertheless, one may ask whether the MAPsof these fishes make additional contributions to the energeticsof microtubule formation at low temperatures. To address thisquestion, we compared the capacities of MAPs from antarcticfishes and from a mammal (the cow) to promote the poly-merization of antarctic fish tubulins at temperatures near 0 °C(Detrich et al. 1990). Compared on a weight basis, both bovineand fish MAPs induced comparable extents of microtubuleformation. Thus, it appears unlikely that the MAPs of antarcticfishes possess major functional adaptations that are absent in
218
ANTARCTIC JOURNAL
the MAPs of homeotherms. With respect to polymerization atcold temperatures, the major locus of adaptation appears tobe the tubulin dimer.
At Palmer Station we also made substantial progress in otherproject objectives. As part of our effort to specify the structuraladaptations of antarctic fish tubulins, we employed reverse-phase high-performance liquid chromatography to isolate pep-tides from chvmotryptic and cyanogen-bromide digests of thealpha and beta tubulins of N. coriiceps neglecta. The amino acidsequences of these peptides will be determined by automatedEdman degradation on a gas-liquid solid-phase protein se-quencer. In addition, we examined the assembly properties oftubulin purified from eggs of N. coriiceps neglecta. We also com-pared the domain structures of native brain tubulins from ant-arctic fishes and from the cow. The results of these studies arecurrently being analyzed.
To support our research, we obtained specimens of twonototheniids, N. coriiceps neglecta and N. gibberifrons, and oneice fish, Chaenocephalus aceratus, by bottom trawling from RIVPolar Duke near Low Island and in Dailman Bay near BrabantIsland. Additional specimens of N. coriiceps neglecta were caughtat Arthur Harbor by fishing with baited hook-and-line. Thefishes were transported to Palmer Station where they weremaintained in seawater aquaria at 0 to +2 °C.
Field studies were conducted at Palmer Station from midMarch to mid May 1990. I am deeply indebted to Sandra K.Parker and Marianne A. Farrington of Northeastern Univer-sity, to Silvio P. Marchese-Ragona of Pennsylvania State Uni-versity, and to Laurie B. Connell of the Massachusetts Instituteof Technology for their participation in the field research pro-
gram. I gratefully acknowledge the assistance provided to theproject by the captains and crews of RIV Polar Duke, by thepersonnel of ITT Antarctic Services, Inc., and of Antarctic Sup-port Associates, and by the scientists of Palmer Station. Thisresearch was supported by National Science Foundation grantDPP 86-14788.
References
Correia, J.J., and R.C. Williams, Jr.. 1983. Mechanisms of assemblyand disassembly of microtubules. Annual Review of Biophysics andBioengineering, 12, 211-235.
Detrich, H.W., III, K.A. Johnson, and S.P. Marchese-Ragona. 1989.Polymerization of antarctic fish tubulins at low temperatures: En-ergetic aspects. Biochemistry, 28(26), 10,085-10,093.
Detrich, H.W., III, B.W. Neighbors, R.D. Sloboda, and R.C. Williams,Jr. 1990. Microtubule-associated proteins from antarctic fishes. CellMotility and the Cytoskeleton, 17(3), 174-186.
Detrich, H.W., III, and S.A. Overton. 1986. Heterogeneity and struc-ture of brain tubulins from cold-adapted antarctic fishes: Compar-ison to brain tubulins from a temperate fish and a mammal. Journalof Biological Chemistry, 261(23), 10,922-10,930.
Detrich, H.W., III, V. Prasad, and R.F. Ludueña. 1987. Cold-stablemicrotubules from antarctic fishes contain unique alpha tubulins.Journal of Biological Chemistry, 262(17), 8,360-8,366.
DeWitt, H.H. 1971. Coastal and deep-water benthic fishes of the ant-arctic. In V.C. Bushnell (Ed.), Antarctic map folio series, (folio 15).New York: American Geographical Society.
Williams, R.C., Jr., J.J. Correia, and A.L. DeVries. 1985. Formation ofmicrotubules at low temperatures by tubulin from antarctic fish.Biochemistry, 24(11), 2,790-2,798.
Natural historyof emperor penguinsat Cape Washington
GERALD L. KOOYMAN, SCOTT E. ECKERT, and CARSTEN A.KOOYMAN
Physiological Research LaboratoryScripps Institution of Oceanography
University of CaliforniaLa Jolla, California 92093
MARKUS HORNING
Max-Planck-lnstitut fur VerhaltensphysiologieAbteilung Wickler
D-8131-Seewiesen, West Germany
The study at Cape Washington was a continuation of a pro-gram begun in 1986 (Kooyman and Croll 1987). It will continuethrough 1990 to obtain some measure of interannual variationin the breeding population, reproductive success, predationpressure, and ice conditions, to mention a few. In addition to
these major objectives, we also sought to determine foragingbehavior, fledging mass and time of fledging.
To conduct these studies, we established a remote camp atCape Washington which is 300 kilometers north of McMurdoStation. We were put in by LC-130 at Priestly Glacier, thenmen, machines, and science equipment were transferred toTerra Nova Bay by UHIN helicopters. The camp was estab-lished on 27 October. At this time and for the remainder ofthe season, there were six large icebergs trapped near the capein such a conformation that they protected the sea ice whichwas fast for 3 kilometers offshore from the cape.
Weather was monitored continuously with a Squirrel datalogger. Distribution of the birds was charted from the top ofthe cape. Group sizes and total colony size was done by aground count on 9 December. Mass determinations of chickswere obtained with a load-cell type of platform scale. Leopardseal predation behavior was assessed by many hours of ice-edge observations. Several aspects of foraging behavior weremonitored ranging from the general characteristics of cycleduration to the specifics of dive depths and duration. Cycledurations were determined from radio transmitters attachedto the birds. Dive behavior was recorded with attached mi-croprocessor units.
Similar to 1986, the weather was mild during the time ofour stay. The ice conditions showed no evidence of severewinter storms as they did in 1986. There were about the samenumber of groups, but the total chick count was larger by about
1990 REVIEW 219
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