GRAVITATIONAL AND SPACE BIOLOGY BULLETIN

Volume 13, Number 2 June 2000 Publication of the American Society for Gravitational and Space Biology ISSN 1089-988X

ASGSB GOVERNING BOARD EDITOR-IN-CHIEF

Jay C. Buckey Marian L. Lewis Mary E. Musgrave President President-Elect University of Massachusetts Dartmouth Medical School University of Alabama Amherst, MA Hanover, NH Huntsville,AL

Kenneth A. Souza Norman G. Lewis SCIENCE EDITORS Secretary Treasurer Immediate Past President Washington State University NASA Ames Research Center Karl H. Hasenstein Pullman, WA Moffett Field, CA University of Southwestern Louisiana Lafayette, LA

Patricia Russell Janet Braam Mary E. Musgrave Executive Director Rice University University of Massachusetts USRA Houston, TX Amherst, MA Washington, DC

Thomas W. Dreschel Charles Fuller Dynamac Corporation University of California PUBLISHING EDITOR Kennedy Space Center, FL Davis, CA Mary E. Musgrave University of Massachusetts Karl H. Hasenstein John Z. Kiss Amherst, MA University of Southwestern Louisiana Miami University Lafayette, LA Oxford, OH

ASSISTANT EDITOR Bonnie J. McClain Danny A. Riley Kathleen Chick Colorado State University Medical College of Wisconsin University of Massachusetts Fort Collins, CO Milwaukee, WI Amherst MA

Tom K. Scott Michael Wiederhold University of North Carolina University of Texas Health Science Center Chapel Hill, NC San Antonio, TX

CORPORATE MEMBERS ASSOCIATE CORPORATE MEMBERS

Boeing Company Lockheed-Martin, Inc. SHOT, Inc. Mains Associates, Inc. ORBITEC, Inc. Seal Beach, CA Moffett Field, CA Floyd Knobs, IN Berkeley, CA Madison, WI

GENERAL INFORMATION

Gravitational and Space Biology Bulletin (ISSN 1089-988X) is a journal devoted to research in gravitational and space biology. It is published by the American Society for Gravitational and Space Biology, a non-profit organization whose members share a common goal of furthering the understanding of the biological effects of gravity and the use of the unique environment of spaceflight for biological research. The Bulletin is overseen by a steering committee consisting of the Publications Committee, the Bulletin Editor, the President, and the Secretary- Treasurer of the ASGSB. This issue of Gravitational and Space Biology Bulletin was printed at Common Wealth Printing Co., Inc., Hadley, Massachusetts..

The American Society for Gravitational and Space Biology was created in 1984 to provide an avenue for scientists interested in gravitational and space biology to share information and join together to speak with a united voice in support of this field of science. The biological effects of gravity have been acknowledged since Galileo's time, but only in this century has gravitational biology begun to attract attention. With the birth of the space age, the opportunity for experimentation over the full spectrum of gravity finally became a reality, and a new environment and research tool became available to probe biological phenomena and expand scientific knowledge. Space and spaceflight introduced new questions about space radiation and the physiological and psychological effects of the artificial environment of spacecraft.

The objectives of ASGSB are:

· To promote research, education, training, and development in the areas of gravitational and space biology and to apply the knowledge gained to a better understanding of the effect of gravity and space environmental factors on the flora and fauna of Earth. · To disseminate information on gravitational and space biology research and the application of this research to the solution of terrestrial and space biological problems. · To provide a forum for communication among professionals in academia, government, business, and other segments of society involved in gravitational and space biological research and application. · To promote the study of concepts and the implementation of programs that can achieve these ends and further the advancement and welfare of humankind.

A Collaborative Production: This issue of the Gravitational and Space Biology Bulletin was produced through a collaboration with the Professional Writing and Technical Communication Program at the University of Massachusetts Amherst. Under the direction of Dr. John Nelson, the PWTC Program has trained students for a variety of professions that demand excellent technical writing and editing skills. Since its inception in 1990, the program has placed nearly 100% of its graduates. The American Society for Gravitational and Space Biology is pleased to sponsor an editorial fellowship for students in the PWTC program at the University of Massachusetts, and gratefully acknowledges the contributions of its students and directors to the production of our journal.

MEMBERSHIP: The American Society for Gravitational and Space Biology welcomes individual, organizational, and corporate members in all of the basic and applied fields of the space and gravitational life sciences. Members are active in the fields of space medicine, plant and animal gravitational physiology, cell and developmental biology, biophysics, and space hardware and life support system development. Membership is open to nationals of all countries. Members must have education or research or applied experience in areas related to the Society's purposes: i.e., Doctorate, Masters with 2 years experience, Bachelors with 4 years experience (student members must be actively enrolled in an academic curriculum leading toward a career related to the Society's purposes), or special appointment by the Board of Directors. Membership applications may be obtained by writing the American Society for Gravitational and Space Biology, P.O. Box 12247, Rosslyn, VA 22219.

Gravitational and Space Biology Bulletin is sent to all members of the American Society for Gravitational and Space Biology. Requests for copies, information about subscriptions and membership, changes of address, questions on permission to reproduce parts of this volume, and other correspondence should be sent to the American Society for Gravitational and Space Biology, P.O. Box 12247, Rosslyn, VA 22219.

Copyright © 2000 by the American Society for Gravitational and Space Biology

2 Gravitational and Space Biology Bulletin 13(2), June 2000

TABLE OF CONTENTS

LIFE IN EXTREME ENVIRONMENTS

Extremophiles in Astrobiology: Per Ardua ad Astra. Jonathan D. Trent ...... 5

Metazoans in Extreme Environments: Adaptations of Hydrothermal Vent and Hydrocarbon Seep Fauna. Erin R. McMullin, Derk C. Bergquist and Charles R. Fisher ...... 13

Life at Body Temperatures below 0°C: The Physiology and Biochemistry of Antarctic Fishes. Bruce D. Sidell ...... 25

Life in Extreme Environments: How Will Humans Perform on Mars? Dava J. Newman...... 35

Bold Endeavors: Behavioral Lessons from Polar and Space Exploration. Jack W. Stuster ...... 49

CELLULAR CYBERNETICS: THE ROLE OF THE CYTOSKELETON

Cortical Microtubules Form a Dynamic Mechanism That Helps Regulate the Direction of Plant Growth. Clive W. Lloyd, Regina Himmelspach, Peter Nick and Carol Wymer ...... 59

Mechanical Forces in Plant Growth and Development. Deborah D. Fisher and Richard J. Cyr ...... 67

The Actin Cytoskeleton May Control the Polar Distribution of an Auxin Transport Protein. Gloria K. Muday, Shiquan Hu and Shari R. Brady ...... 75

Control of Development and Motility in the Spermatozoids of Lower Plants. Stephen M. Wolniak, Vincent P. Klink, Peter E. Hart and Chia-Wei Tsai...... 85

Columella Cells Revisited: Novel Structures, Novel Properties, and a Novel Gravisensing Model. L. Andrew Staehelin, Hui Qiong Zheng, Thomas L. Yoder, Jeffrey D. Smith and Paul Todd...... 95

Gravitational and Space Biology Bulletin 13(2), June 2000 3

4 Gravitational and Space Biology Bulletin 13(2), June 2000

Extremophiles in Astrobiology: Per Ardua ad Astra Jonathan D. Trent Astrobiology Technology Branch, NASA Ames Research Center, Moffett Field CA

ABSTRACT · terrestrial and submarine hot springs, where As we consider the possibilities of finding life on other thermophiles grow at temperatures above 100°C planets, it behooves us to evaluate what we know about the limits (the current highest temperature observed is for life on planet Earth. In our continued exploration of Earth, we 113°C) (Blöchl et al., 1997). are finding microbes in a variety of unexpected habitats. In Microbes have also ingeniously adapted to extremes of radiation, geothermal hot springs, we have discovered organisms thriving at toxin concentrations, low nutrients (starvation), water activity, temperatures near the boiling point of water and at pH values longevity, and other seemingly bizarre conditions (Kushner, 1981; down to 0.5; in the deepest parts of the oceans, those that grow Scheie, 1970). optimally at pressures above 1000 bars and die at pressures below Here the focus will not be on cataloging the esoteric extremes 500 bars; and at the poles, those that grow below the freezing point to which life has adapted, but rather on how research on of water and die at temperatures above 10°C. All of these extremophiles has provided insights into fundamental problems in organisms are living proof that the biochemical “machinery” of life biology; in particular, how studying the hyperthermophilic can be adapted to conditions that, from our anthropocentric acidophile Sulfolobus shibatae (an organism that grows at 85°C and perspective, appear to be extreme. pH 3) has contributed to an understanding of protein folding and By studying the molecular adaptations of extremophiles, we membrane stabilization. begin to identify the critical cellular components that expand the envelope for life. As an example, I will discuss what we have HOW TO BEAT THE HEAT learned about the role of the proteins we call “heat shock proteins” Thermophiles is the name given to organisms that grow in pushing the upper temperature limit of life and how our studies optimally at temperatures above 50°C; hyperthermo-philes to have provided a new perspective on the function of these proteins. those that grow optimally above 80°C (Figure 1). Although the ASTRO-EXTREMO-BIOLOGY existence of these high- temperature organisms has been known

One of the prominent goals of astrobiology is to discover life since the early twentieth century (Brock, 1978), research on their or signs of life on planets beyond Earth. To approach this goal, it structural and functional adaptations to these extreme temperatures will be useful to know the physical and chemical limits for life on is still in its early stages. There is currently considerable interest in Earth and, perhaps more importantly, to understand the underlying applying biophysical characteristics of life that set these limits. Such knowledge would allow us to make educated guesses— based on remote measurements of physical and chemical parameters alone— about the likelihood of finding life on other planets. Although an inventory of life on Earth is far from complete, it is clear that microbes (bacteria and archaea) dominate most habitats, especially extreme habitats. In this context , extreme means habitats that radically deviate from the very limited physical and chemical conditions that a human being would consider normal. In cold, deep-sea habitats, with temperatures between 2-4°C and pressures up to 1100 bars, specially adapted microbes known as “barophiles” or “piazophiles” thrive (Yayanos, 1995). These organisms require low temperatures and high pressures to grow, and they die at temperatures above 15°C and pressures below 200 bars (Yayanos et al., 1981). Other extreme habitats of microbes include:

· sulfuric acid springs that emerge from mines Figure 1. The Temperature Range in the Universe (°K), the associated with pyrite deposits, where acidophiles Envelope in Which Life Is Known to Be Metabolically Active thrive by iron-sulfur metabolism at pH 0 (Edwards (°C), and the Current Ranges for Each of the Recognized et al., 2000); Phylogenetic Divisions of Life—Eukarya, Bacteria, and · salt deposits of evaporation ponds, where the Archaea. The terms used to distinguish groups of organisms halophiles grow in saturated salt solutions and based on their temperatures for optimum growth: withstand desiccation for long periods of time (Kates, 1993; Vreeland, 1993); · psychrophiles = 15°C or below · the Siberian permafrost, where psychrophiles · mesophiles = between 15 and 50 °C metabolize at temperatures down to –20°C (Mazur, · thermophiles = above 50°C 1980); · hyperthermophile = above 80°C

Gravitational and Space Biology Bulletin 13(2), June 2000 5

EXTREMOPHILES IN ASTROBIOLOGY what we learn about the molecular basis of thermophily to research different species, different HSPs appear to be critical. In the in biotechnology, bioremediation, and molecular biology; and, more bacterium Escherichia coli, 20 different HSPs are correlated to recently, in astrobiology (Clark and Kelly, 1989; Kelly and thermotolerance, but their respective roles haven’t yet been Deming, 1988). In biotechnology, interest is based in part on the clarified (VanBogelen et al., 1987). In the yeast Saccharomyces need to discover or develop more stable and effective enzyme cerevisiae, thermotolerance depends on the synthesis of a 104-kDa systems (Adams and Kelly, 1995). In bioremediation, research protein (HSP104) (Sanchez and Lindquist, 1990); and in the fruit focuses on understanding and engineering microbes that can thrive fly Drosophila melanogaster, it depends on a 70 kDa protein in the harsh conditions associated with environmental restoration. (HSP70) (Sanders et al., 1986). In mammalian cells, a variety of In molecular and astrobiology, the interest is twofold: (1) to HSPs, especially the small HSPs, appear to be important (Welch determine the repertoire of critical molecular adaptations that and Mizzen, 1988). In hyperthermophilic archaea, in particular the transform the familiar heat-labile biomolecules into thermostable hyperthermophilic/acidophilic archaeon Sulfolobus shibatae, it variants, and (2) to understand how the integrated living system seems that only two 60 kDa HSPs (HSP60s á & â) are critical for can cope with temperatures near boiling (Jaenicke, 1991). thermotolerance (Trent, 1996). Thermophiles are living proof that all fundamental life The actual role of HSPs in acquired thermotolerance is not processes can be adapted to high temperatures. How are the yet clear. Since the early 1980s, most research has focused on the macromolecules (e.g., nucleic acids, lipids, and proteins) stabilized? hypothesis that HSPs interact with damaged proteins. Hightower, How does the living system as a whole remain coordinated and who first observed that the heat and chemical agents inducing HSP balanced? How far can biological systems be pushed before kinetic synthesis also cause proteins to unfold and aggregate, suggested energy tears them apart? These are critical questions that current that HSPs may help cells cope with unfolded proteins—either by research is attempting to address. Two partial answers are binding to them to prevent aggregation, marking them for emerging: proteolysis, or assisting in their refolding (Hightower, 1991). In

vitro experiments with pure proteins indicate that some HSPs can · Macromolecules in thermophiles are modified indeed recognize and bind unfolded proteins, thereby preventing in a variety of subtle ways that intrinsically aggregation and assisting in the process of refolding (Georgopoulos increase their thermostability (natural selection et al., 1994; Hendrick and Hartl, 1993). These in vitro observations is working at the level of function, so there are of protein folding, combined with in vivo observations that some many possibilities for thermostabilizing HSPs (in particular HSP60 and HSP70) are present in cells under structures as long as the function is normal growth conditions, led to the hypothesis that these HSPs uncompromised). may be involved in de novo protein folding (Pelham, 1986). In this · Intracellular conditions can be modified in ways role, HSPs have been referred to as "molecular chaperones” (Ellis et that extrinsically increase thermostability al., 1993). As chaperones, HSPs guard against inappropriate (Hensel, 1993; Perutz, 1978). interactions between proteins damaged by heat or chemical stresses, and they also function as midwives in the process of de These modifications include changes in intracellular salts, novo protein folding. This hypothesis not only provides a production of compatible solutes, and the synthesis of specific functional link between HSP synthesis and thermotolerance, but it proteins. Research is being conducted on a class of proteins also addresses one of the most fundamental questions in cell known as “heat shock proteins” (HSPs), so called because their biology: How do proteins fold in the highly concentrated and synthesis markedly increases with increasing temperatures apparently chaotic mixture of macromolecules in the living cell? (Lindquist and Craig, 1988; Watson, 1990). HSPs, abundantly While the molecular chaperone hypothesis still has many produced by hyperthermophiles, appear to be essential for the supporters, there is a growing body of evidence that it is thermophilic lifestyle (Trent, 1996). Investigation has provided fundamentally wrong. The critical HSPs in cells are not involved in insights into the function of thermophilic HSPs in vivo (Trent et folding or refolding proteins in vivo, and the underlying assumption al., 1998). of the hypothesis—that the interior of the cell is “chaotic” and molecules need specific chaperones to function—is misleading.

HSPs, THERMOTOLERANCE, AND MOLECULAR CHAPERONES THE CHAPERONE CONCEPT: VICTORIOUS OR VICTORIAN? All organisms produce HSPs. It has been known for many years that the production of HSPs correlates with a physiological Current investigation at Ames Research Center on HSPs in phenomenon known as “acquired thermotolerance.” This the hyperthermophilic acidophile Sulfolobus shibatae has led to the designation derives from the observation that an organism exposed conclusion that HSP60 is the most critical of the HSPs. In S. to a lethal temperature tends to survive much better if it has first shibatae, the HSP60s predominate. Other heat-inducible proteins been exposed to a near-lethal temperature for a short time (Gerner are either absent or present at very low concentrations. For and Schneider, 1975). Enhanced survival after a heat shock is example, HSP70, which is abundant in some organisms, appears to observed in nearly all organisms, including thermophiles, and it be absent from S. shibatae, as well as from other crenarcheaota for correlates with the increased synthesis of HSPs (Laszlo, 1988). In which whole genome sequences are now available (Klenk et al.,

6 Gravitational and Space Biology Bulletin 13(2), June 2000 EXTREMOPHILES IN ASTROBIOLOGY

1997). In the molecular chaperone model, HSP60 plays a central role by providing the core particle known as the “chaperonin,” within which protein folding is believed to occur (Figure 2). The chaperonin system has been best studied in E. coli, in which the chaperonin (GroEL) interacts with a co-chaperonin (GroES) and, in an ATP-dependent process, does indeed influence the refolding of a select set of model proteins in vitro. It has been observed, however, that many proteins do not interact with chaperonins, which brings into question their universality as "protein-folding machines" (Hartl and Martin, 1995; Horwich et al., 1993). Co- immunoprecipitation experiments indicate that fewer than 15% of E. coli proteins bind to GroEL in cell lysates—i.e., 85% of E. coli proteins do not bind (Ewait et al., 1997). The rates of protein folding and the abundance of GroEL + GroES in cells indicate that not more than 2% of E. coli proteins could be folded by chaperonins in vivo—i.e., 98% are folding without using the chaperonin (Lorimer, 1996). In addition, the model for the function Figure 2. The Putative Chaperonin Cycle, Beginning with of GroEL + GroES indicates that folding occurs within the central the Production of the Polypeptide at the Ribosome and cavity of the chaperonin (Weissman et al., 1996). The high- Ending with a Finally Folded Protein. The polypeptide is resolution structural information that is now available for GroEL + thought to be escorted by DnaK (peanut-shaped blobs) as it GroES (Xu et al., 1997) provides precise information about the emerges from the ribosome and is transferred to the cavity in size of this central cavity. This information can be combined with GroEL; folding to take place within the cavity under the cover structural information about E. coli proteins to determine the size formed by GroES. The process requires energy from ATP of proteins that will physically fit within the central cavity of the hydrolysis, which causes conformational changes in GroEL to chaperonin (Figure 3), an exercise demonstrating that only small accommodate GroES and ultimately release the protein. proteins (<40 kDa) can be accommodated. In summary, the data indicate that only a small fraction of small proteins can be folded by the chaperonin system in E. coli. This finding is inconsistent with the underlying rationale for the chaperonin system—i.e., that chaperonins are needed to mediate protein folding within the crowded, chaotic intracellular environment. At this point one may well ask: Why only some proteins? And why, in particular, the smaller ones, when the larger ones may be slower and more problematic to fold?

THE MEMBRANE IS THE THING

Recent observations reveal that, in some bacteria, the majority of chaperonins are not present in the cell cytoplasm

(where most protein folding is believed to occur); rather, they are associated with the plasma membranes (Garduno et al., 1998; Figure 3. The Crystallographic Structures of the E. Coli Török et al., 1997). Our investigations of chaperonins in Chaperonins GroEL and GroES, with Structures of hyperthermophilic archaea reveal that most of the chaperonin Proteins Placed Inside. The size of GroES is outlined and proteins are associated with the plasma membrane in these superimposed on GroEL to indicate the maximum size to which the organisms, too (Trent, Yaoi, and Kagawa, unpublished). Some cavity of GroEL can be enlarged. The protein structures shown are years ago it was discovered that the archaeal chaperonin is more drawn to scale, indicating the severe space limitations of the closely related to a group of eukaryotic proteins known as TCP1s putative protein-folding cavity of the chaperonin. than to the bacterial chaperonins (Trent et al., 1991). TCP1s are present in the cytoplasm of eukaryotes and are purified from cells (A) RNase H = 17 kDa as a supramolecular structure similar to the chaperonin. There are (B) malate dehydrogenase = 32 kDa claims that TCP1 may be involved in specifically folding actin (C) isocitrate dehydrogenase = 45 kDa (D) glycerol kinase = 56 kDa (E) fragment of topoisomerase = 67 kDa

Gravitational and Space Biology Bulletin 13(2), June 2000 7

EXTREMOPHILES IN ASTROBIOLOGY and tubulin (Vinh and Drubin, 1994; Yaffe et al., 1992), but genetic clear that nascent membrane proteins penetrate lipid bilayers and manipulations of yeast do not support this interpretation (Ursic that the lipid hydrophobic phase is essential to the folding process. and Culbertson, 1991). In addition, the abundance and localization The mechanism of penetration and folding, not yet completely of the eukaryotic cytosolic chaperonins in yeast suggest a role for understood, is currently the subject of intense interest. TCP1 other than protein folding (Ursic et al., 1994). In both yeast Surprisingly, the presence of a folded protein in a lipid and mammalian cells, TCP1-chaperonins are associated with bilayer ordinarily does not lead to an increased ionic conductance membranes and are involved in cytoskeleton organization (Ursic across the bilayer, unless the protein happens to produce an ion- and Culbertson, 1991; Ursic and Culbertson, 1992; Ursic et al., conducting channel by a specific folding of transmembrane alpha 1994), cell division (Brown et al., 1996), and exocytosis (Creutz et helices. On the other hand, non-specific penetration of a lipid al., 1994). This apparent structural and membrane-associated role bilayer by hydrophobic peptides can markedly affect bilayer for chaperonins supports the hypothesis that the membrane, not permeability. Oliver and Deamer (1994) demonstrated that alpha the cytoplasm, is the site of chaperonin activity. helical strands of polyalanine and polyleucine incorporate themselves into lipid bilayers, both in the form of liposomes and AN ALTERNATE HYPOTHESIS: planar membranes. It was discovered that, under these conditions, HSP60-CHAPERONINS MEDIATE MEMBRANE liposomes and planar membranes became leaky to ionic flux. PERMEABILITY Significantly, the leaks showed channel-like behavior in planar bilayers, with a remarkable specificity for protons. This finding It has been known for many years that bacteria, eukarya, and confirmed earlier studies indicating that synthetic hydrophobic archaea adapt their membranes to different environmental peptides produced proton-specific ion-conducting channels under temperatures by reconstituting their lipids (de Mendoza and certain conditions (Deamer, 1992). If heat shock can produce even Cronan, 1983). Such reconstitution includes increasing the chain a small number of denatured proteins with exposed hydrophobic length of the lipid acyl chains, the ratio of iso- to anteiso-branching, residues that penetrate lipid bilayers and thereby introduce ion or the degree of saturation (Prado et al., 1988). It has also been leaks, it follows that HSP-binding to these peptides may protect demonstrated that membrane permeability to protons is maintained the membrane from proton leaks. within a narrow range in bacteria and archaea, and that both natural Since the proton-permeability of membranes increases with membranes and liposomes become highly permeable to protons temperature, the problem of maintaining an effective proton when cells are exposed to high-temperature stress (Peeples and gradient is exaggerated for hyperthermophiles (Driessen et al., Kelly, 1995). The correlation between the loss of membrane 1996). If this hypothesis is correct, increases in membrane proton integrity and the synthesis of HSPs has not gone unnoticed (Mejia permeability can be compensated for by increases in the level of et al., 1995). However, under the influence of the prevailing HSP60—a possible explanation for the extraordinary abundance of chaperonin theory, the increased membrane permeability has been HSP60 in the hyperthermophilic archaea. HSP60 reaches 12% of interpreted as a signal to the cell to initiate HSP synthesis in order total protein in the S. shibatae at its upper growth temperature; to prepare itself for damaged proteins, not so that the HSPs will and, in one of the most extreme hyperthermophiles, Pyrodictium influence the membrane itself (Török et al., 1997). occultum, HSP60 reportedly reaches 76% of total protein near the We suggest that chaperonin interactions with membranes organism's upper growth temperature (Phipps et al., 1991). allow cells to rapidly adjust membrane permeability and respond to short-term fluctuations in their environment. The reconstitution of CONCLUSION membrane lipids is a secondary, more permanent adjustment the cells make in response to long-term environmental changes. Thus, As indicated above, the HSP60s are currently under intense chaperonin-mediated membrane stabilization allows cells to survive investigation to determine their role in protein folding. Our research rapid changes in their environments and gives them time to "decide" seeks to clarify their potential role in membrane stabilization. Our if they should modify their lipids to accommodate the hypothesis, based on empirical data showing that chaperonins are environmental change. associated with membranes in vivo, suggests that this membrane This hypothesis explains the localization of chaperonins at interaction is the primary function of chaperonins in vivo, but it the cell membrane and is consistent with observations that most of does not exclude a secondary or indirect role for chaperonins in the stresses that induce cells to produce HSPs are suspected or protein folding and assembly. The chaperonin-membrane known to damage membranes. HSP-inducing stresses, such as interaction may influence membrane integrity and thereby affect alcohol, peroxide, and heavy metals, clearly impact membranes; but membrane permeability, which may influence not only the even unfolded proteins, with their exposed hydrophobic and conditions in the cytoplasm but also protein folding indirectly. We hydrophilic peptide chains, may impact membranes (this remains are not questioning observations that chaperonins influence protein to be demonstrated). It has long been known, however, that folding in vitro. It has been firmly established that chaperonins proteins are surface-active, with the ability to form monolayers at (like BSA, PEG, urea, detergents, and a variety of other reagents) air-water interfaces (Deamer, 1992). More recently, it has become influence the folding of a limited number of proteins in vitro, which

8 Gravitational and Space Biology Bulletin 13(2), June 2000 EXTREMOPHILES IN ASTROBIOLOGY is a capability that may be of significant value in biotechnology. REFERENCES

Still unanswered are questions about how cells live at Adams, M.W.W. and Kelly, RM. 1995. Enzymes from hyperthermophilic temperatures and, more generally, how all cells microorganisms in extreme environments. Chemical and cope with the fluctuating temperatures in their natural habitats. It Engineering News:32-42. may be time to abandon the prevailing protein-folding hypothesis in favor of a membrane stabiliza-tion hypothesis if we are to Blöchl, E., Rachel, R., Burggraf, S., Hafenbradl D., Jannasch, H.W. understand the role of HSP60 in vivo. Fundamental questions arise and Stetter, K.O. 1997. Pyrolobus fumarii, gen and sp. nov. about the assumptions inherent in the molecular chaperone model. represents a novel group of archaea, extending the upper For example: temperature limit for life at 113°C. Extremophiles 1:14-21.

· Are the protein-protein, protein-substrate, or, Brock, T.D. 1978. Thermophilic Microorganisms and Life at High more generally, protein-macromolecule Temperatures. New York: Springer Verlag. interactions that inevitably occur in the crowded

confines of the cell leading to non-productive Brown, C.R., Doxsey, S.J., Hong-Brown, L.Q., Martin, R.L. and aggregations? Welch, W.J. 1996. Molecular chaperones and the centrosome: A · Are macromolecular interactions part of the role for TCP-1 in microtubles nucleation. Journal of Biological natural intracellular dynamics and could they Chemistry 271:824-832. even be productive?

· Are chaperones necessary? Clark, D.S. and Kelly, R.M. 1989. Microorganisms at extreme Besides, if a protein is forced into a non-productive interaction temperatures and pressures: Engineering insights. Chemtech 18:12- under what may be rare circumstances, cells seem well equipped 18. with proteases for dealing with this mistake. The alternative hypothesis—that the weak link in the cell is Creutz, C.E., Liou, A., Snyder, S.L., Brownawell, A. Willison, K. the plasma membrane, not proteins, and that HSP60s effectively 1994. Identification of the major chromaffin granule-binding stabilize the cell membrane—provides a new perspective for protein, chromobindin A, as the cytosolic chaperonin CCT understanding HSP60 function in vivo. Our research on the (chaperonin containing TCP-1). Journal of Biological Chemistry extremophile S. shibatae certainly supports this model. Perhaps it is 269:32035-32038. serendipitous that we chose that organism, living as it does at low pH and high temperatures. With neutral pH inside a cell that is de Mendoza, D. and Cronan, J.E. 1983. Thermal regulation of under constant threat of membrane heat damage, it may be no membrane lipid fluidity in bacteria. Trends in Biochemical Sciences surprise that 12% of total cellular protein is HSP60, a protein that 8:49-52. limits the permeability of the membrane. Extremophiles are revealing a diversity of subtle biochemical Deamer, D.W. 1992. Role of water in proton flux mechanisms. In adaptations that allow them to thrive under extreme conditions. On Biomembrane Structure & Function: The State of Art (Gaber, B.P. the molecular level, these adaptations include specific modifications and Eswaren, K.R.K., Eds.) New York: Adenine Press, pp. 209-226. of their essential biomolecules, such as lipids, nucleic acids, and proteins. On the cellular, they include production or regulation of Driessen, A.J.M., van de Vossenberg, J.L.C.M. and Konings, W.N. compounds such as organic solutes, salts, and proteins that interact 1996. Membrane composition and ion-permeability in with essential biomolecules to help stabilize specific macromolecules extremophiles. FEMS Microbiological Reviews 18:139-148. and/or the organization of the organism as a whole. This may be one of the advantages of studying extremophiles: the opportunity to Edwards, K.J., Bond, P.L., Gihring, T.M. and Banfield, J.F. 2000. look at the biochemical and molecular adaptations that allow them to An archaeal iron-oxidizing extreme acidophile important in acid live under extreme conditions, thereby expanding our perspective on mine drainage. Science 287:1796-1798. the physical and chemical limits for life on Earth and enhancing our ability to rationally explore for signs of life on planets beyond Earth. Ellis, R.J., Laskey, R.A. and Lorimer, G.H. (eds.). 1993. Molecular Chaperones. London: Chapman and Hall. Acknowledgments Ewaitm K.L., Hendrickm J.P., Hourym W.A. and Hartl, F.U. 1997. I thank Drs. Hiromi Kagawa and Takuro Yaoi for providing In vivo observation of polypeptide flux through the bacterial many of the results that have led the ideas and conclusions chaperonin system. Cell 90:491-500. presented here, Drs. Laszlo Vigh and Iboya Horvath for stimulating discussion, Susanne Johansen Trent and Karen Bunn for editorial Garduno, R.A., Faulkner, G., Trevors, M.A., Vats N. and assistance, and DOE/BES grant DE-AI03-99ER20354 for financial Hoffman, P.S. 1998. Immumolocalization of Hsp60 in Legionella support. I also thank Dr. Mary Musgrave for organizing the pneumophilia. Journal of Bacteriology 180(3):505-513. Extremophile workshop and inviting me to participate. Georgopoulos, C., Liberek, K., Zylicz, M., and Ang, D. 1994.

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Properties of the heat shock proteins of Escherichia coli and the Lorimer, G.H. 1996. A quantitative assessment of the role of autoregulation of the heat shock response. In: The Biology of Heat chaperonin proteins in protein folding in vivo. FASEB Journal Shock Proteins and Molecular Chaperones (Morimoto, R.I., 10:5-9. Tissiéres, A. and Georgopoulos, C., Eds.) Plainview NY: Cold Spring Harbor Laboratory Press. pp. 209-249. Mazur, P. 1980. Limits to life at low temperatures and at reduced water contents and water activities. Origins of Life 10:137-159. Gerner, E.W. and Schneider, M.J. 1975. Induced thermal resistance in HeLa cells. Nature 256:500-502. Mejia, R., Gomez-Eichelmann, M.C. and Fernandez, M.S. 1995. Membrane fluidity of Escherischia coli during heat-shock. Hartl, F.U. and Martin, J. 1995. Molecular chaperones in cellular Biochemica et Biophysica Acta 1239:195-200. protein folding. Current Opinion in Structural Biology 5:92-102. Oliver, A. and Deamer D.W. 1994. Alpha helical hydrophobic Hendrick, J.P. and Hartl, F.-U. 1993. Molecular chaperone peptides form proton-selective channels in lipid bilayers. functions of heat-shock proteins. Annual Review of Biochemistry Biophysical Journal 66:1364-1379. 69:349-384. Peeples, T.L. and Kelly, R.M. 1995. Bioenergetic response of the Hensel, R. 1993. Proteins of extreme thermophiles. In: The extreme thermoacidophile Metallosphaera sedula to thermal and Biochemistry of Archaea (Archaebacteria) (Kates, M., Kushner, nutritional stresses. Applied Environmental Microbiology 61:2314- D.J. and Matheson, A.T., Eds.) Amsterdam: Elsevier Science 2321. Publishers B.V., pp. 209-222. Pelham, H. 1986. Speculations on the functions of the major heat Hightower, L.E. 1991. Heat shock, stress proteins, chaperones, and shock and gluocose-regulated proteins. Cell 46:959-961. proteotoxity. Cell 69:191-197. Perutz, M.F. 1978. Electrostatic effects in proteins. Science Horwich, A.L., Low, K.B., Fenton W.A., Hirshfield I.N. and 201:1187-1191. Furtak, K. 1993. Folding in vivo of bacterial cytoplasmic proteins: role of GroEL. Cell 74:909-917. Phipps, B.M., Hoffmann, A., Stetter, K.O. and Baumeister, W. 1991. A novel ATPase complex selectively accumulated upon heat Jaenicke, R. 1991. Protein stability and molecular adaptation to shock is a major cellular component of thermophilic archaebacteria. extreme conditions. European Journal of Biochemistry 202:715- The EMBO Journal 10(7):1711-1722. 728. Prado, A., Da Costa, M.S. and Madeira, V.M.C. 1988. Effect of Kates, M. 1993. Biology of halophilic bacteria, Part II growth temperature on the lipid composition of two strains of Membrane lipids of extreme halophiles: biosynthesis, function and Thermus sp. Journal of General Microbiology 134:1653-1660. evolutionary significance. Experientia 49(6/7):1027-1036. Sanchez, Y. and Lindquist, S.L. 1990. HSP104 required for induced Kelly, R.M. and Deming, J.W. 1988. Extremely thermophilic thermotolerance. Science 248:1112-1115. archaebacteria: biological and engineering considerations. Biotechnology Progress 4(2):47-62. Sanders, M.M., Triemer, D.F. and Olsen, A.S. 1986. Regulation of protein synthesis in heat-shocked Drosophila cells. Journal of Klenk, H., Clayton, R., Tomb, J., White, O., Nelson, K., Ketchum, Biological Chemistry 261(5):2189-2196. K., Dodson, R., Gwinn, M., Hickey, E., Peterson, J., et al. 1997. The complete genome sequence of the hyperthermophilic, Scheie, P.O. 1970. Environmental limits of cellular existance. sulphate-reducing archaeon Archaeoglobus fulgidus. Nature Journal of Theoretical Biology 28:315-325. 390:364-370. Török, Z., Horvath, I., Goloubinoff, P., Kovacs, E., Glatz, A., Kushner, D. 1981. Extreme environments: are there any limits to Balogh, G. and Vigh, L. 1997. Evidence for a lipochaperonin: life? In: Comets and the Origin of Life (Ponnamperuma, C., Ed.) association of active protein-folding GroESL oligomers with lopids New York: Springer Verlag. pp. 241-248. can stabilize membranes under heat shock conditions. Proceedings of the National Academy of Sciences, USA 94:2192-2197. Laszlo, A. 1988. The relationship of heat-shock proteins, thermotolerance, and protein synthesis. Experimental Cell Trent, J.D. 1996. A review of acquired thermotolerance, heat shock Research 178:401-414. proteins, and molecular chaperones in archaea. FEMS Microbiological Reviews 18:249-258. Lindquist, S. and Craig, E.A. 1988. The heat-shock proteins. Annual Review of Genetics 22:631-677. Trent, J.D., Kagawa, H.K. and Yaoi, T. 1998. The role of chaperonins in vivo: the next frontier. Annals of the New York

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Metazoans in Extreme Environments: Adaptations of Hydrothermal Vent and Hydrocarbon Seep Fauna Erin R. McMullin, Derk C. Bergquist and Charles R. Fisher* Department of Biology, 208 Mueller Lab, Pennsylvania State University, University Park PA

ABSTRACT (reviewed in Somero, 1998). High pressure can alter enzyme Some of the most extreme environments where animals kinetics and efficiency, change protein structure (reviewed in survive are associated with active vents and seeps in the deep sea. Gibbs, 1997). The sheer distance of much of the ocean floor from a In addition to the extreme pressure, low temperatures, and lack of source of photosynthetic production makes it largely a nutrient- light that characterize the deep sea in general, a variety of other poor environment, where any carbon consumed by an organism factors that are hostile to most animals prevail in these must be strictly conserved (Sibuet, 1992; Gage and Tyler, 1996). A environments. Hydrothermal vent regions show extremes in diverse array of metazoans have responded to this combination of temperature, areas of very low oxygen, and the presence of toxic environmental extremes by developing specialized proteins and hydrogen sulfide and heavy metals. Hydrocarbon seeps, though lipids, capable of functioning under temperatures and pressures much cooler than vents, also have regions of very low oxygen and high hydrogen sulfide, as well as other potentially harmful that would hinder shallower-dwelling species, and depressed substances such as crude oil and supersaturated brine. Specially metabolic rates and activity that conserve a limited energy supply adapted animals not only tolerate these conditions, they often (reviewed in Gibbs, 1997 and Somero, 1998). thrive under them. In most cases this tolerance is due to a Hydrothermal vent environments have the same high combination of physiological and behavioral adaptations that allow pressure and continuous darkness characteristic of the deep sea in animals to avoid the extremes of their habitats and yet benefit from general, but they differ in nutrient supply, temperature variation, the chemoautotrophic production characteristic of these oxygen concentration, pH, and levels of such potentially toxic environments. chemicals as sulfide and heavy metals (Childress and Fisher, 1992). In active venting regions, entrained seawater is superheated by

deep, subsurface, hot basalt, converting geothermal energy into the INTRODUCTION chemical energy of hot, highly reduced hydrothermal fluid Metazoans are multicellular animals and, as such, are more (Jannasch, 1989). This superheated fluid rises through a system of complex in their body plans and often have stricter physiological interconnected subterranean cracks and fissures in the newly requirements than unicellular animals or bacteria. Individual cells in formed sea floor, resulting in multiple flow sites and flow these organisms perform specialized tasks, and cells of similar manifestations at the surface. In areas where the effluent reaches function are typically organized into layers or compartments. In temperatures up to 400°C and actively mixes with the cold oxygen- most cases, this means that materials cannot simply diffuse from bearing ambient seawater, chimneys or mounds may form when the environment to all cells. Therefore, systems must be in place to chemicals rapidly precipitate out of solution. Hydrothermal fluids transport materials throughout the body, to allow communication may also mix with cooler water as it rises through the basalt, between different compartments, and to regulate the internal emerging as diffuse flow fields at temperatures ranging from environment in which these processes occur. Strictly speaking, all slightly above ambient to about 100°C (Delaney et al., 1992) known metazoans are heterotrophic and must depend on the (Figure 1). oxidation of autotrophically produced organic carbon compounds Hydrocarbon seeps in the Gulf of Mexico provide a chemical for energy. This metabolic process can occur in the absence of environment similar to that of hydrothermal vents, though differing oxygen (anaerobically). However, in the presence of oxygen significantly in temperature, sulfide concentrations, and types of (aerobically), the amount of energy harnessed per unit of fixed toxins. Sulfide in seep areas is produced when organic carbon carbon oxidized increases dramatically. As metazoans often have (crude oil and natural gas) migrates to the sea floor from deep relatively large body sizes and high energy demands, they require reservoirs. Sediment layers at the sea floor diffuse and retain the not only ample supplies of fixed carbon and oxygen but also seeping oil, and sulfate reducers in the upper meters of sediment environmental conditions under which their complex biochemical produce hydrogen sulfide. The upward migration of fluids at cold pathways may function. An extreme environment for a metazoan, seeps is to some extent thermally driven (Kennicutt et al., 1992). then, may be one in which fixed carbon or oxygen is scarce or Unlike the process in vents, however, this process is more passive, absent, or where environmental conditions—such as temperature, and does not result in vigorous mixing of seep fluid and ambient pressure, or the presence of toxins—hinder or prevent bottom water. Seep fluids are normally at ambient water physiological processes. The deep sea is a relatively inhospitable environment for temperatures by the time they reach the sediment/water interface. metazoans: ambient temperature is constantly low (~2o C), Sulfide levels found in emitted seep fluid are generally much lower pressure is high, light is absent, and organic carbon is scarce. Low and more stable temporally and spatially, than levels found in temperatures may slow or impede many biochemical reactions and vents, (MacDonald et al., 1989; Julian et al., 1999). However, decrease the fluidity of lipids, a factor of primary importance to interstitial sulfide concentrations can be comparable to those found cell membrane function at hydrothermal vents (Kennicutt et al, 1989). Unlike

*Correspondence to: Charles R. Fisher: e-mail: [email protected]

Gravitational and Space Biology Bulletin 13(2), June 13 METAZOANS IN EXTREME ENVIRONMENTS

Figure 1. Hydrothermal Vents Manifest on the Sea Floor as Chimney Structures and Diffuse Flow Fields. Superheated seawater carries highly reduced compounds to the surface, where they rapidly precipitate out of solution or fuel extensive biological communities based on chemoautotrophic production. (Modified from Jannash, 1989) the fauna in vent regions, fauna in seeps experience temperatures concentrations for significant periods of time (Millero, 1986; that do not vary appreciably from ambient deep-sea temperatures Johnson et al., 1986). The needs of the symbiont or (~8°C). Methane and crude oil may also bubble through overlying free-living bacteria for sulfide and oxygen, and of the animal for sediments at these sites, and dense anoxic brines can form pools on additional oxygen, limit vent fauna to areas where hot, sulfide- the seafloor (MacDonald, 1990; MacDonald, 1998). bearing vent water and cold, oxygen-bearing ambient water actively The abundant reduced chemicals found in both hydrothermal mix (Childress and Fisher, 1992). Animals in such regions therefore vent and hydrocarbon seep fluids, hydrogen sulfide in particular, experience rapid shifts in temperature that coincide with changes in can be used by chemosynthetic prokaryotes as an energy source oxygen and sulfide concentration. Seep fauna are also limited by for carbon fixation. For metazoan life, this offers the potential for a access to sulfide and, as sulfide is present in areas of active large food source more or less independent from the overlying seepage, are exposed to potentially toxic levels of hydrocarbons. photic zone (Jannasch, 1989). Most vent and many seep organisms The extraordinarily high level of chemosynthetic-based rely entirely on this chemosynthetic primary production by primary production that is supported by venting and seepage is actively feeding on free-living bacteria or by forming symbiotic one of the outstanding differences between these regions and the relationships with chemosynthetic bacteria for the bulk of their average deep-sea environment. Metazoans are found at high food supply (Childress and Fisher, 1992; Kennicutt and Burke, densities in vent and seep regions, but they are of low diversity and 1995; Fisher, 1996). The sulfide-containing hydrothermal vent fluid have a high degree of endemism (Tunnicliffe et al., 1998; Sibuet and on which primary production relies is hot, extremely deficient in Olu, 1998). Organisms of taxonomic and functional similarity are oxygen, and laden with toxic chemicals. Moreover, sulfide and found at both seeps and vents. It appears that vents and seeps are oxygen react spontaneously and do not coexist in significant both areas of significant resources and of extreme environmental

14 Gravitational and Space Biology Bulletin 13(2), June 2000

METAZOANS IN EXTREME ENVIRONMENTS demands that are largely exploited by a limited group of animals (Chevaldonne et al., 1992). Temperatures of 20-80°C have been with specialized physiological and biochemical adaptations measured on surfaces colonized by P. sulfincola and P. palmiformis (Childress and Fisher, 1992; Fisher, 1996). (Juniper et al., 1992), and temperature measurements taken in alvinellid tubes have recorded temperatures of 68°C, with spikes as TEMPERATURE high as 80°C (Desbruyères et al., 1998). These recordings have led Temperature variation is one of the striking characteristics of some authors to conclude that alvinellids regularly experience such hydrothermal vent environments. Water temperature can range high temperatures, and that alvinellid tubes may open to vent fluid from 2°C to 400°C within a centimeter, and animals may have at the back, allowing warm vent water to flow outward over the occasional brief contact with 100°C+ water (Chevaldonne et al., animals (Cary et al., 1998). 1992; Delaney et al., 1992; Cary et al., 1998). In areas of diffuse Measurements of temperature effects on alvinellid proteins, flow, a vent animal may experience water temperatures of 2, 20 and however, indicate that these animals cannot survive body 40+°C in rapid succession, or even simultaneously over the length temperatures over 50°C for extended periods. For example, both of its body (Johnson et al., 1988; Cary et al., 1998). Additionally, Alvinella pompejana and A. caudata have hemoglobin that is at least one life stage of these organisms must be able to withstand unstable at 50°C, with highest oxygen binding affinities occurring at extended periods of cold (~2°C) during dispersal. roughly 15° and 25°C, respectively (Toulmond et al., 1990). More Many biological structures, such as enzymes and lipid striking are the melting temperatures of alvinellid collagens, which bilayer membranes, depend on a particular degree of molecular in A. pompejana are 40°C for cuticle collagen and 46°C for instability or fluidity, which is directly affected by temperature. interstitial collagen (Gaill et al., 1991). Based on these Increasing temperatures can increase reaction rates and affect measurements, these animals likely live at temperatures averaging reaction equilibria through higher kinetic energy, and high 30 to 35°C rather than at the higher temperatures proposed by temperatures can cause protein denaturation, resulting in complete others (Childress and Fisher, 1992; Fisher, 1998). The conflict and often irretrievable loss of function (Hochachka and Somero, between observed temperature probe readings (Chevaldonne et al., 1984). Adaptation to the deep sea requires more “fluid” proteins 1992; Cary et al., 1998) and the in vitro physiological limits and lipids to compensate for the stabilizing forces of high pressure probably reflects both the steep temperature gradient that exists and low temperature (Hochachka and Somero, 1984). High within alvinellid tubes and the difference between a temperature temperature, on the other hand, tends to destabilize molecules, and probe reading and actual body temperature. Even if these animals selection in such environments is for more stable forms. At vent do not experience body temperatures of 60+°C, they live at regions, however, temperature can vary by almost 100°C within an temperatures much higher than ambient and may even take animal’s habitat (Cary et al., 1998; Desbruyères et al., 1998). How, advantage of the large temperature gradient present over the lengths then, do vent organisms maintain proper function in such extremes? of their bodies—from 22°C near the gills to 60+°C at the body As with all metazoan challenges, this threat can be met trunk (Cary et al., 1998) (see discussion of alvinellid hemoglobin). through morphological, physiological, and behavioral adaptations. Animal survival in extreme temperatures depends as much on Biochemically, changes that favorably affect reaction rate equilibria behavior as on physical adaptations. Different species in vent and molecular stability become fixed in a population. A number of fields have different environmental requirements, and they will molecules show a particularly strong correlation between functional either settle in or migrate to regions that meet their particular needs. and denaturation temperatures and the temperature of an Within specific habitats, animals may also have behavioral organism’s environment (Hochachka and Somero, 1984). Molecules mechanisms for modifying environmental conditions to their of particular interest are enzymes, collagen, and lipids. Enzymes benefit. Paralvinella sulfonicola colonizes young sulfide chimneys quickly lose function above and below an optimal temperature. The in the early stages of sulfide mineralization, where both subunits of the structural polymer collagen, the most abundant temperature and sulfide levels have begun to decrease (Juniper and animal protein, often show a melting temperature (Tm) close to the Martineau, 1995). Additionally, P. sulfonicola has been found to upper lethal limit for an animal, though the polymer itself is have a 2-mm-thick layer of FeS2 below its tubes, which forms a somewhat more stable. Lipids of cell membrane bilayers must be barrier between hot vent water and cold sea water—a barrier that both fluid and structurally coherent to form a functional membrane, may be formed by the activity of the animal itself (Juniper and a characteristic that is also is very sensitive to temperature change Martineau, 1995). Alvinellids, in general, may also actively cool (Hochachka and Somero, 1984). their tubes by pumping in ambient seawater (Chevaldonne et al., Many hydrothermal vent animals must have a wide 1991). temperature tolerance (especially compared to ambient deep-sea Though the vent animals described here likely withstand fauna). Of particular interest are such chimney-dwelling body temperatures that rival those of the most thermotolerant polychaetes as Alvinella pomejana, A. caudata, and Paralvinella metazoans, the best-documented thermotolerant animals are found sulfincola, which live on newly formed vent walls very near the in other, more easily studied environments. Desert-adapted bees super-hot vent fluid. Photographic data from one such site show an fly with a sustained internal temperature of 46°C (Willmer and alvinellid crawling over a temperature probe that is reading 105°C Stone, 1997), and desert ants can survive even hotter temperatures.

Gravitational and Space Biology Bulletin 13(2), June 2000 15 METAZOANS IN EXTREME ENVIRONMENTS

The Australian ant Melophorus bagoti actively forages at soil do not normally co-occur (Millero, 1986). temperatures above 70°C and has a critical thermal maximum of Vent and seep animals show a variety of behavioral and 56.7°C, surviving for one hour at 54°C (Christian and Morton, physiological adaptations to low and variable oxygen tensions. 1992). Unlike desert insects, however, vent organisms live in a Behavioral mechanisms that help to maintain an aerobic metabolism in these environments include: world of rapidly shifting temperature extremes: within minutes, water temperatures vary in tens of degrees at a given location and · spatially spanning an oxic/anoxic transition zone; in hundreds of degrees over centimeters. Vent organisms must · temporally spanning the transition zone by moving therefore be adapted to extreme temperature variation as well as to from oxic to anoxic water; extreme temperatures. · acquiring oxygen by pumping oxic water into an anoxic burrow or tube.

HYPOXIA/ANOXIA Alvinellids build tubes that protrude from chimney walls, allowing Metazoan metabolic energy is produced via the release and their gills access to oxygen-bearing ambient water. Mobile transfer of electrons from reduced-carbon electron donor molecules predators, such as Bythograea thermydron, may move between to more oxidized electron acceptors. In aerobic metabolism, the pools of oxic and anoxic water. Hesiocaeca methanicola, a electron acceptor is oxygen; in metazoan anaerobic metabolism, the polychaete that can live on buried anoxic methane hydrates in the electron acceptor is an organic molecule such as lactate or fumarate. cold seeps of the Gulf of Mexico, appears to increase circulation of Aerobic respiration yields 36 ATP/mol glucose oxidized, while oxygenated water in its habitat using its parapodia (Fisher et al., anaerobic respiration yields 2-8 ATP/mol glucose, depending on 2000), as might the alvinellid polychaetes that live in tubes on the pathway and electron acceptor utilized (Fenchel and Finlay, hydrothermal chimneys (Desbruyères, 1998). 1995). Many organisms found in hypoxic environments are able to Metazoans rely primarily on the efficient, high-energy maintain aerobic respiration and normal metabolic rate even at very output of aerobic respiration to maintain a normal level of low oxygen tensions (oxyregulation of respiration). A number of metabolism. Metazoans forced to use inefficient fermentation for physiological adaptations are seen in oxyregulators, including large energy production over extended periods need a large and surface areas for gas exchange, short diffusion distances from constantly replenished food source to maintain normal bodily external surface to blood spaces, a well-developed function and reproduction, a situation not common in nature, although endoparasites are a possible exception (Bryant, 1991). Oxygen is also involved in a number of critical biosynthetic pathways, and no metazoan has yet been conclusively documented to complete its entire lifecycle without its presence, though obligate anaerobe eukaryotic protozoa do exist (Fenchel and Finlay, 1995). Some nematode and oligochaete meiofauna, which occur deep in the sulphidic zone of sediments, may prove to be capable of growth and reproduction in the absence of oxygen (these environments have not yet been established as completely anaerobic) (Fenchel and Finlay, 1995). Yet numerous metazoans from diverse groups and environments (e.g., nematodes, annelids, amphipods, and goldfish) can withstand extended periods of anoxia (Bryant, 1991; Panis et al., 1996; Hagerman et al., 1997). Vent and seep fluids are highly reduced and contain significant levels of sulfide. Oxygen in vent habitats varies inversely with temperature, and organisms in areas of actively mixing hydrothermal and ambient water may experience rapid fluctuations in both (Childress and Fisher, 1992) (Figure 2). At hydrocarbon seeps, oxygen concentration decreases with proximity to the substrate and with increased depth in the sediment (Kennicutt et al., 1989). Seep organisms colonize both brine pools Figure 2. Temperature, Oxygen, and Sulfide and methane hydrates, which are habitats of particularly low Concentrations Measured in Situ at the Galapagos Rift. The oxygen (MacDonald, 1990; Fisher et al., 2000). Metazoans with sampling probe was moved from ambient bottom water to within a chemoautotrophic sulfide-oxidizing symbionts at vent and seep vent mussel bed and then back, a distance of about 50 cm. Oxygen sites require oxygen for aerobic respiration, as well as both oxygen and sulfide react spontaneously, and do not coexist for significant and sulfide to power chemoautotrophic carbon fixation. This dual amounts of time. In areas where hot, sulfide-rich vent water mixes requirement can prove a challenge for metazoans that are dependent with cold, oxygen-rich ambient water, oxygen concentration upon chemoautotrophic symbionts, because they will require decreases as water temperature and sulfide increase. (Johnson et substantial amounts of both oxygen and sulfide, two chemicals that al., 1986)

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METAZOANS IN EXTREME ENVIRONMENTS circulatory system, and the presence of respiratory pigments where roughly two-thirds of the oxygen consumption occurs (Weber, 1978; Bryant, 1991). Some vent fauna, such as B. (reviewed in Childress and Fisher, 1992). The utility of the thermydron, have been shown to oxyregulate to very low levels of Bohr effect for oxygen offloading to the trophosome was environmental oxygen (Mickel and Childress, 1982). A seep- questioned by Childress and Fisher (1992) because CO2 endemic orbiniid and the methane hydrate polychaete H. consumption by the symbionts in the trophosome would methanicola can also oxyregulate to very low oxygen tensions overshadow tissue CO2 production. However, Goffredi et al. (Fisher et al., 2000; Hourdez et al., personal communication). (1999) have recently demonstrated a net H+ ion production One common adaptation to improve oxygen exchange is the by autotrophic Riftia, indicating H+ ion production in the development of large gills or specialized surfaces for gas exchange, trophosome, which could explain the utility of the Bohr such as the plume of R. pachyptila (Jones, 1981, 1988; Arp et al., effect in this symbiosis. 1985) and the hypertrophied gills of Alvinellids (Jones, 1981; In the absence of oxygen, some organisms can use anaerobic Jouin and Gaill, 1990). Decreased diffusion distances between gas metabolism for extended periods. Indeed, sulfide may poison exchange surfaces and the blood supply, another common aerobic respiration at the electron transport chain, forcing the adaptation that facilitates uptake of dissolved gases, has been organism to rely on anaerobic metabolism even in the presence of documented in both alvinellids (Jouin and Gaill, 1990) and the seep oxygen (Bryant, 1991). In general, annelids and molluscs are able to orbiniid (Hourdez et al., 2000). Finally, well-developed and highly use more efficient mitochondrial pathways of fermentation vascularized circulatory systems have been found in alvinellids, (Bryant, 1991; Fenchel and Finlay, 1995; Tielens and Van vestimentiferans, and a seep orbiniid (Jones, 1981, 1988; Jouin et Hellemond, 1998), whereas no crustacean has been found that can al., 1996; Hourdez et al., 2000). Respiratory pigments, such as use a pathway beyond glycolysis, which yields only two to three hemoglobin or hemocyanin, with high affinities and ATP/glucose (reviewed in: DeZwaan and Putzer, 1985; and capacities for oxygen, are a particularly useful adaptation for Bryant, 1991). Again, only selected vent and seep species an organism experiencing low and/or variable oxygen have been tested for anaerobic tolerance. The vent crab tensions. Although the respiratory pigments of most vent Bythograea thermydron can survive only about 12 hours in and seep fauna have not been characterized, both Riftia the absence of oxygen, and the glycolytic endproduct pachyptila and Alvinella spp. contain circulating lactate is accumulated during this time (Mickel and hemoglobins with very high oxygen affinities (Terwilliger et Childress, 1982). However, Riftia tolerates anoxia up to 60 al., 1980; Arp and Childress, 1981; Terwilliger and Terwilliger, hours and accumulates succinate when kept under anaerobic 1984; Toulmond et al., 1990), which allows them to take up conditions, indicating the use of a modified citric acid cycle oxygen from very low concentrations and accumulate it to for fermentation (Arndt et al., 1998). Similarly, both the help withstand short periods of anoxia. B. thermydron hydrate worm Hesiocaeca methanicola and a seep orbiniid hemocyanin affinity is increased by the presence of can survive four to five days in the absence of oxygen thiosulfate (a sulfide detoxification product) and lactate (a (Fisher et al., 2000; Hourdez et al., 2000). byproduct of anaerobic metabolism) (Sanders and Childress, Thus we see the same pattern with respect to oxygen 1992). Riftia hemoglobins (R. pachyptila has three different that we saw with temperature. The vent and seep fauna that Hb's) bind sulfide, as well as oxygen, with high affinity and have been investigated are not significantly more tolerant of at high capacity, allowing simultaneous transport of both anoxia or high temperature than the best-adapted fauna from gasses to the symbionts in their internal trophosome while other environments, but are certainly very well adapted for preventing the reaction of sulfide and oxygen in the blood extremes in these parameters. (Arp and Childress, 1983; Childress et al., 1984). Alvinellid hemoglobin oxygen affinity is reduced by low pH (normal TOXICITY Bohr effect) and high temperature (Toulmond et al., 1990), Potentially toxic chemicals abound in hydrothermal vent and which may facilitate the uptake and delivery of oxygen from cold seep environments (Corliss et al., 1979; Johnson et al., 1986; the plume, normally extended outside of the tube in cooler McDonald, 1990; Nix et al., 1995). Of these, sulfide is perhaps the waters, to the body, which is often bathed in highly reduced most abundant and well studied, and its consequences for biological high-temperature fluids (Desbruyères et al, 1998; Cary et al., systems have been well documented in many other reducing 1998). environments, including mud flats, mangrove swamps, and sewage Riftia hemoglobins have such a high affinity for outfalls (see reviews in Somero et al., 1989 and Grieshaber and oxygen that study of the binding properties is difficult, Volkel, 1998). Heavy metals may also occur in extremely high which has led to some variation among the results of concentrations at hydrothermal vents, where they precipitate out different investigators. Overall, the data suggest that oxygen of solution to form chimneys and sometimes coat tubeworm tubes binding by Riftia hemoglobins shows a moderate normal and mollusk shells. In spite of our limited knowledge regarding Bohr and temperature effect that may assist offloading to the specific adaptations to these toxins in vent and seep organisms, more posterior animal tissues, particularly in the trophosome, what we know of their physiologies and those of their shallower-

Gravitational and Space Biology Bulletin 13(2), June 2000 17 METAZOANS IN EXTREME ENVIRONMENTS dwelling relatives should allow us to investigate some potential are exposed to sulfide in the surrounding water. mechanisms for detoxifying these substances. Likewise, oxidation of sulfide to more benign sulfur compounds, most commonly thiosulfate, may occur within the animal by a variety of means, including sulfide-oxidase enzymes Sulfide and mitochondrial oxidation. The vent crab Bythograea thermydron maintains aerobic metabolism by steadily increasing its rate of Sulfide is a toxin that, in just micromolar amounts, is capable oxygen consumption up to environmental sulfide concentrations of of impairing biological processes necessary to metazoan function. about 800mM and apparently detoxifying sulfide via a sulfide- Its most important physiological effect may be to severely inhibit oxidase (Vetter et al., 1987; Childress and Fisher, 1992). Sulfide- aerobic respiration by interfering with cellular respiration and blood oxidizing activity has also been found in tissues from several other oxygen transport (reviewed in: Somero et al., 1989; Vismann, 1991; vent species, including the crab Munidopsis subsquamosa, the Grieshaber and Völkel, 1998). In the mitochondria, sulfide may shrimp Alvinocaris lusca, Riftia pachyptila, and Calyptogena poison the respiratory enzyme cytochrome c oxidase, thus magnifica (Vetter et al., 1987; Powell and Somero, 1986b), as well inhibiting ATP production by the electron transport chain. Sulfide as a host of species living in non-vent, reducing habitats (Lee et al., may also bind to the hemoglobin molecule in blood, reducing its 1996; Grieshaber and Völkel, 1998). Oxidation of sulfide may also capacity to carry oxygen and, in high concentrations, rendering it be coupled to energy production directly in the mitochondria of nonfunctional. In addition, a recent study found that sulfide is some animals or indirectly by providing reduced sulfur capable of inhibiting muscular contraction independent of its intermediates to symbionts. Solemya reidi, a clam that inhabits effects on aerobic metabolism (Julian et al., 1998). areas organically enriched by sewage and paper mill effluent, links To avoid these toxic effects, an organism has several practical sulfide oxidation to ATP production in its mitochondria at low-to- options: avoid sulfide, switch to anaerobic metabolism, exclude moderate sulfide concentrations, but this ability becomes inhibited sulfide from sensitive tissues, or oxidize sulfide to more benign at high concentrations (Powell and Somero, 1986a). Similarly, the forms. Most inhabitants of vent and seep environments do not intertidal lugworm Arenicola marina possesses the ability to realistically have the option of avoiding sulfide altogether. Species oxidize sulfide in its mitochondria even at very high sulfide containing sulfide-oxidizing bacteria must supply this chemical to concentrations (Volkel and Grieshaber, 1996). It is not known their symbionts, thus requiring them to inhabit areas where sulfide whether sulfide oxidation by animal tissues of the vent mussel is abundant. Nonsymbiotic endemic heterotrophs not only must Bathymodiolus thermophilus is linked directly to ATP production, forage in areas where at least brief exposure is likely, but some but the resultant thiosulfate is supplied to its bacterial must also consume symbiotic or free-living sulfide oxidizers that endosymbionts, where it is further oxidized to fuel often contain high levels of sulfide (Somero et al., 1989). To chemoautotrophic carbon fixation (Powell and Somero, 1986a; prevent poisoning of the electron transport chain in the presence of Fisher et al., 1987; Nelson and Fisher, 1995). In this way, sulfide high concentrations of sulfide, many invertebrates temporarily oxidation in the animal’s tissues is indirectly linked to energy switch from aerobic to anaerobic metabolism. (Grieshaber and production. As research continues at vents and seeps, sulfide Völkel, 1998). As discussed above, several vent and seep species oxidation mediated by mitochondria may prove to be a common have considerable anaerobic capacity; whether this occurs in the method of detoxification. presence of sulfide has not been directly tested. Exclusion from Metazoans hosting sulfide-oxidizing bacterial symbionts sensitive tissues and oxidation within the body are the two best- must not only tolerate this potential toxin but must also acquire documented strategies to prevent sulfide poisoning among vent and both sulfide and oxygen from the environment and transport them seep animals, and symbiont-containing species often use them in to the symbionts. In many cases, these animals employ specialized conjunction. blood proteins that bind sulfide reversibly to prevent inhibition of Exclusion of sulfide from tissues may involve physical, oxygen transport, poisoning of cytochrome c oxidase in the biological, or chemical barriers around or within an animal. Thick animals' tissues, and spontaneous reaction of sulfide with oxygen. tubes or cuticles may reduce or prevent exposure of some external In Riftia pachyptila, two different extracellular hemoglobins in the tissues to sulfide, and epibiotic bacteria and abundant metal ions vascular blood and one in the coelomic fluid bind sulfide and may oxidize sulfide before it makes contact with external tissues. oxygen simultaneously and reversibly, with high affinity (Arp et The Pompeii worm, Alvinella pompejana, resides on active al., 1985). Cysteine residues and disulfide groups on these chimney structures where sulfide is abundant, within a secreted hemoglobins apparently provide the sulfide-binding mechanism proteinaceous tube that it shares with epibiotic bacteria (Zal et al., 1998). Their presence as well on the extracellular (Desbruyères et al., 1998). Although specific data is lacking, the hemoglobin of A. pompejana indicates that they may be a common tube is thought to provide a regulated environment (potentially adaptation to sulfide-rich vent habitats (Zal et al., 1997; Zal et al., lower in sulfide than the immediately surrounding vent fluid), and 1998). These hemoglobins also bind sulfide with a high enough the bacteria to supply both nutrition and a means of sulfide affinity to prevent the sulfide poisoning of cytochrome c oxidase detoxification for A. pompejana (Desbruyères et al., 1998). Several (Powell and Somero, 1983). Deep-sea clams in the family other worms living in direct contact with vent fluid, including the Vesicomyidae are also capable of binding both vestimentiferans, also secrete tubes and only expose portions of their bodies directly to sulfidic fluids. These structures may, in part, serve to limit what tissues and how much tissue surface area

18 Gravitational and Space Biology Bulletin 13(2), June 2000

METAZOANS IN EXTREME ENVIRONMENTS

extracellular sulfide binding factor that binds with high affinity (Arp et al., 1984; Childress et al., 1991). Sulfide binding can only protect sensitive tissues when a sink for the bound sulfide is available to remove sulfide and maintain free binding sites. In these species, internal chemoautotrophic microbial symbionts oxidize sulfide, acting as an internal sink while providing a source of fixed carbon for the host (Figure 3). All vestimentiferans and vesicomyid clams appear to utilize a similar system to tolerate and exploit the sulfide-rich environments in which they live. This system is characterized by the exclusion of sulfide from sensitive tissues via high-affinity binding to blood components, transport to symbionts via the blood, and the oxidation of sulfide by intracellular chemoautotrophic bacteria.

Metals

Due to the interactions between circulating crustal water and hot basalts, dissolved heavy metals are particularly abundant in hydrothermal vent systems. Metals, in general, may interfere with a wide array of biological processes, including respiration, muscular function, osmoregulation, reproduction, development, and protein utilization (Luoma and Carter, 1991). Metals can also cause morphological abnormalities, histopathological problems, and instability of genetic material (Luoma and Carter, 1991). Metazoans typically detoxify absorbed or ingested metals by using metal-binding proteins (metallothioneins) and forming subcellular inclusions. These mechanisms often act jointly to consolidate and enclose excess metals, which then accumulate within tissues and/or skeletal structures over time (Beeby, 1991; Luoma and Carter, 1991). The few investigations into potential metal detoxi-fication in vent and seep fauna indicate that strategies used by these animals are not very different from those studied elsewhere. Polychaetes of the genus Paralvinella secrete mucus, rich in metallothionien-like proteins, that removes inorganic material from the epidermis and may also remove metals from the immediate external environment (Juniper et al., 1986). Metallothionien-like proteins and metal-rich inclusions have been found within the tissues of R. pachyptila, A. pompejana, C. magnifica, and a Bathymodiolid mussel from the Mid-Atlantic Ridge, and elevated levels of metals have been found within the shells of C. magnifica and B. thermophilus (reviewed in Childress

and Fisher, 1992; Geret et al., 1998). What may separate vent and Figure 3. Mechanisms Allowing Vent Tubeworms to seep species from shallower marine taxa is not the detoxification Tolerate and Exploit Their Sulfide-rich Environments. (A) mechanism, per se, but rather the ability of the mechanism to Using its plume, the only part of its body that protrudes from the function effectively at high metal concentrations and over long open end of the chitinous tube it inhabits, Riftia pachyptila acquires exposure times. sulfide and oxygen from areas where vent fluids actively mix with ambient sea water. (B) Specialized hemoglobins in the blood of R. pachyptila simultaneously and reversibly bind both sulfide and SUMMARY AND CONCLUSIONS oxygen, and (C) transport them to internally housed, sulfur- Metazoans colonizing vent and seep habitats must tolerate oxidizing, chemoautotrophic bacteria that act as a sink for the not only the already extreme characteristics of the deep sea but also potentially poisonous sulfide. (Modified from Arp et al., 1985) a wide range of additional conditions that result from the complex

geologic and microbiological processes driving these environments. Ironically, high concentrations of hydrogen sulfide, one of the oxygen and sulfide simultaneously. However, unlike R. pachyptila, primary characteristics that should make these environments they utilize two different binding molecules: (1) an intracellular inhospitable to metazoan life, also drives biological production to hemoglobin that binds oxygen with moderate affinity, and (2) an

Gravitational and Space Biology Bulletin 13(2), June 2000 19 METAZOANS IN EXTREME ENVIRONMENTS levels far exceeding those of the surrounding deep sea. To exploit Acknowledgements the energetic abundance of vents and seeps, metazoans must The authors are supported by the National Science tolerate not only the sulfide but also a whole suite of factors Foundation, the Mineral Management Service, and the NOAA intrinsically correlated with its presence. At vents, the presence of National Undersea Research program. sulfide corresponds directly to the high temperatures, absence of oxygen, and presence of heavy metals characteristic of the effluent REFERENCES waters. At seeps, as at vents, the presence of sulfide corresponds to the absence of oxygen; but, unlike the situation at vents, it also Arndt, C., Schiedek, D., and Felbeck, H. 1998. Metabolic corresponds to the presence of potentially toxic hydrocarbons. responses of the hydrothermal vent tube worm Riftia pachyptila to Although not well studied in seep fauna, crude oil and its individual severe hypoxia. Marine Ecology Progress Series 174: 151-158. chemical components display fouling and mutagenic effects on a host of biological functions, including feeding, respiration, Arp, A.J. and Childress, J.J., 1981. Blood function in the excretion, reproduction, development, and chemoreception (Bayne hydrothermal vent vestimentiferan tube worm. Science 213:342- et al., 1982; Suchanek, 1993). 344. To cope with the numerous, potentially interacting extremes of these environments, the denizens of vents and seeps employ and Arp, A.J. and Childress, J.J. 1983. Sulfide binding properties of the combine a vast array of morphological, physiological, and blood of the hydrothermal vent tube worm Riftia pachyptila. behavioral adaptations. 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24 Gravitational and Space Biology Bulletin 13(2), June 2000

Life at Body Temperatures below 0°C: The Physiology and Biochemistry of Antarctic Fishes Bruce D. Sidell School of Marine Sciences, University of Maine, Orono ME

ABSTRACT unusual and presumably adaptive features of the physiology and biochemistry that have enabled the success of Antarctic fishes in Fishes of the Southern Ocean surrounding Antarctica are cold polar waters. Finally, I will argue that examining traits of some dominated by species of the suborder Notothenoidei. For ~14MY, Antarctic fishes—traits that are permitted by the unique features these highly successful fishes have evolved under stable thermal of the Antarctic marine environment and that probably would be conditions that result in a body temperature of ca. 0°C throughout lethal in any other marine environment on earth—may yield as their life histories. Evolution in this chronically cold environment much insight into processes of evolution as does the study of traits has led to unusual physiological and biochemical characteristics. In arrived at through strong natural selection. To illustrate this point, I some cases, these characteristics are essential to survival and will describe several aspects of the unique physiology of one normal biological function at cold body temperature (e.g., family of Antarctic notothenioid fishes, the hemoglobinless and development of antifreeze glycoproteins, structural modification of sometimes myoglobinless Channichthyid icefishes. enzymes, cold-stable microtubules, and cardiovascular adaptations). In other instances, mutations that probably would THE TECTONIC AND CLIMATIC HISTORY OF have been lethal in warmer, less oxygen-rich environments than the ANTARCTICA Southern Ocean have been retained in Antarctic fishes (e.g., loss of hemoglobin production and variable expression of myoglobin in one A single landmass, Pangea, and a single world ocean, notothenioid family, the Channichthyidae). These unique animals Panthalassa, existed throughout the Paleozoic. By the mid- offer opportunities for insight into evolutionary processes leading Mesozoic (Jurassic, ca. 200 MY ago), the current cycle of seafloor to physiological and biochemical characteristics that either arise spreading and plate tectonic movement had begun, Pangea having from strong selective pressure or persist through relaxation of spawned the hemispheric supercontinents—Gondwana in the selective pressure. After briefly describing the Antarctic marine south and Laurasia in the north. Although the Antarctic region of environment, I discuss several unique aspects of the physiology Gondwana had probably attained a south polar position by some and biochemistry of Antarctic fishes, specifically emphasizing our time in the Cretaceous, the breakup of this supercontinent into laboratory’s recent studies of an unusual pattern of myoglobin crustal plates continued through the early Tertiary, when East expression in the Channichthyid icefishes. Antarctica and West Antarctica separated from the Australian and South American landmasses. Despite the southern polar position INTRODUCTION of these Antarctic landmasses, the sea temperatures surrounding them were relatively mild during this period, with bottom water In an absolute sense, the thermal range encompassing the cell temperatures ranging from 12° to 16°C (Kennett, 1977, 1982). The temperatures of metazoan organ-isms on earth is quite narrow: fish fauna of these warm shallow waters showed a complex from approximately +35° to 40°C (in endothermic mammals and diversity typical of other contemporary temperate oceans birds, and in ectothermic desert reptiles and tropical fishes) to (Eastman, 1990, 1993). temperatures approaching -2°C (in marine invertebrates and fishes It was not until ca. 25 MY ago, when the deep ocean that inhabit polar seas). Yet, the challenges for maintenance of trenches of the Drake Passage opened between Antarctica and normal cellular function over this range are considerable. Rates of South America, that the circumpolar currents that influence most physiological and biochemical processes show a change of 2- Antarctica’s modern climate began to form. This process led to to 3-fold for every 10°C change in temperature (i.e. Q10 = 2-3). In progressive cooling of the Southern Ocean. Figure 1 shows that sea the absence of compensatory mechanisms, one would expect the temperatures of the Southern Ocean have been well below 5°C for biological processes of polar ectotherms at -2°C to proceed at rates 10 to 14 MY and that they presently approach -2°C at the more between 16- and 91-fold lower than those of their endothermic or southerly boundaries of the shelf (Littlepage, 1965). During this tropical counterparts. In addition, the severely cold body period, evolution of fish taxa that dominate the Southern Ocean has temperatures of ectothermic animals from the polar seas are, in occurred under chronically cold seawater conditions. some cases, below the colligative freezing point of their body fluids and below the normal (from our anthropocentric viewpoint) range MARINE HABITAT AND FISH FAUNA OF of structural stability of important cellular macromolecules. The MODERN ANTARCTICA abundant and successful species of fishes and marine invertebrates that have evolved to occupy the Southern Ocean surrounding The present polar ocean surrounding Antarctica is the most Antarctica, Earth’s coldest marine environment, provide clear severely cold and thermally stable marine environment on earth. evidence that these constraints have been overcome. Sea temperatures near the Ross Ice Shelf at McMurdo Station, In this paper, I will briefly describe the geological and Antarctica, never vary from approximately –1.9°C (Littlepage, paleoclimatic history of the Antarctic marine environment that has 1965), and even those in the more northerly reaches of the set the stage for evolutionary change. I will also describe several Antarctic Peninsula range only between summer temperatures of

Gravitational and Space Biology Bulletin 13(2), June 2000 25 PHYSIOLOGY AND BIOCHEMISTRY OF ANTARCTIC FISHES

occurred under a very unusual set of conditions:

· relative oceanographic isolation from other faunas due to circumpolar currents and deep ocean trenches surrounding the continent; · chronically, severely cold water temperature; · very high oxygen availability; · very low levels of niche competition in a Southern Ocean depauperate of species.

The last point is important, and I will return to it. The features described above make Antarctic notothenioid fishes an uniquely attractive group for the study of physiological Figure 1. Estimated Temperature of the Southern Ocean and biochemical adaptations to cold body temperature. In fact, during the Tertiary Period. Estimates presented are mean Antarctic notothenioids are so specialized for life in the cold that values inferred from oxygen isotope compositions of planktonic and they cannot survive exposure to water temperatures above benthic species of foraminfera from sediment cores. (Data and approximately +5°C (Somero and DeVries, 1967). figures redrawn from Kennett, 1977) PHYSIOLOGICAL AND BIOCHEMICAL ADAPTATIONS TO COLD +1.5°C to winter temperatures of –1.8°C (DeWitt, 1971). The literature of thermal biology has recognized two general Vertically, the water column south of the Antarctic Polar Front is classes of adaptation to extreme temperatures—mechanisms exceedingly well mixed, and all depths are close to complete oxygen necessary for survival of the organism in the face of potentially saturation. Because oxygen solubility in seawater is inversely lethal thermal insult, and mechanisms that permit normal biological proportional to temperature, the cold Antarctic seas are an function under the extreme temperature regime. Antarctic fishes exceptionally oxygen-rich aquatic habitat. display excellent examples of each. Despite the taxonomically diverse composition of fishes in the warm seas surrounding early Antarctic landmasses, the present Antifreeze Glycoproteins fish fauna surrounding the continent is dominated by six families of a single perciform suborder, the Notothenoidei. Nototheniods Like other marine teleosts, Antarctic notothenioids account for approximately 35% of the total fish species known to hypoosmotically regulate body fluids at an osmotic concentration occur south of the Antarctic Polar Front, but more significantly, of approximately one-third that of the seawater in which they they comprise as much as 90% of the biomass of fish captures swim. Their blood osmotic concentration of 400-500 mOsm, around the continent (Ekau, 1990). The suborder is highly endemic although somewhat higher than temperate zone teleosts, still to Antarctica, with only a handful of notothenioid representatives corresponds to a colligative freezing point depression of only being found in waters north of the Antarctic Polar front in New approximately -1°C (DeVries, 1988). Notothenioids thus find Zealand, Australia, and South America. How did such a themselves in the seemingly untenable position of spending their taxonomically restricted group of fishes come to dominate the entire lives at a body temperature (equivalent to sea temperature) Southern Ocean, and what became of the rich mix of species that that is nearly a full °C below the predicted freezing point of their originally inhabited Antarctic coastal waters? The simple answer is body fluids, based on solute concentration. The extent of this that we do not know. challenge is underscored by the fact that many notothenioid species The fossil record for notothenioid fishes is inaccessible, encounter ice crystals, either at the underside of sea surface ice- locked under the massive ice sheets that cover Antarctica. All that cover, in the water column, or in association with “anchor ice” that we can say with certainty is that, sometime between the mid- forms on the sediments of the coldest Antarctic waters. Colligative Tertiary and the present, there was a massive crash of species properties of solution chemistry alone would predict that contact diversity that left a sluggish, demersal stock of ancestral with an ice crystal should nucleate very rapid crystal growth and notothenioids to colonize the vast Southern Ocean. Although it is result in death by freezing. Yet, obviously, this does not happen. tempting to attribute this collapse of species diversity to decreases This set of observations formed the basis for research that has told in water temperature, the slow (in biological terms) geologic pace of the story of one of the best-known and most fascinating ocean cooling has led Eastman and Clarke (1998) to conclude that biochemical adaptations found in Antarctic fishes: antifreeze the loss of shallow water habitat that accompanied Antarctica’s glycopeptides (AFGPs). Arthur DeVries discovered the chemical glaciation may have been of equal or greater importance in causing identity of these compounds in the mid-to-late 1960s. With his the ext inction. Despite our inability to resolve these factors, it is colleagues, he has continued a multifaceted program of research clear that the considerable subsequent radiation of notothenioid during the last 30 years that has contributed most of what we have species come to know about them.

26 Gravitational and Space Biology Bulletin 13(2), June 2000 PHYSIOLOGY AND BIOCHEMISTRY OF ANTARCTIC FISHES

The blood of Antarctic notothenioids contains high concentrations (up to 35 mg×ml-1) of a family of eight glycopeptides ranging in mass from ca. 2,600 to 33,700 daltons (Cheng and DeVries, 1991). AFGPs are distributed in blood and extracellular fluids throughout the body, with the exception of the central nervous system. All share a common and very regular structure that underlies their function as “antifreezes.” The peptide backbone of AFGPs consists of a repeating tripeptide sequence of alanine-alanine-threonine, with proline occasionally substituted for the first alanine in the smaller classes of AFGPs. A disaccharide moiety of b-D-galactopyranosyl-(1®3)-2-deoxy -a-D- galactopyranose is glycosidically linked to each threonine residue in the repeating structure (Figure 2).

The non-colligative action of AFGPs in retarding freezing is Figure 2. Repeating Structural Unit of Glycoprotein revealed by a disparity between the freezing point and the melting Antifreezes (AFGP) from Antarctic Notothenioid Fishes. point of a solution that contains the compounds, a behavior termed Eight size classes of AFGP, ranging from 2.6 to 33.7 kDa in “thermal hysteresis.” Body fluids removed from Antarctic molecular size, are composed of this fundamental repeating notothenioids often resist freezing until temperatures of <-2°C are structure. In the smaller size classes, proline residues are attained, yet they will not melt on warming until ca. –0.6°C, the occasionally substituted for the alanine in position 1 of the melting point predicted by their osmotic concentration. tripeptide. Disaccharide moieties are linked to each threonine The mechanism of antifreeze action by AFGPs is known as residue in position 3 of the repeat. (Redrawn from DeVries, 1988) adsorption-inhibition (Raymond and DeVries, 1977). AFGPs adhere to the exposed lattices of small ice crystals and prevent their further growth by excluding new water molecules from the lattice. catalytically active enzyme proteins and the assembly of Very regular secondary molecular structure positions polar macromolecular protein complexes. hydroxyl groups of the disaccharide moieties and the carbonyl Cold temperature influences the rate of enzymatic catalysis oxygens of the peptide backbone in nearly perfect register with in more than one way. The first is by simply lowering the kinetic hydrogen-bonding sites of the ice lattice. The result is a series of energy of the system. Comparing physiological temperatures of small, highly curved fronts that form on the surface of the crystal Antarctic species even with those of mammals, however, reveals and interrupt its growth until the temperature is further lowered. that this range (ca. 40°C) accounts for a change of only a few There is little doubt that AFGP development in Antarctic percentage points in terms of absolute temperature. A potentially notothenioids was the pivotal evolutionary “innovation” that greater area of influence is the effect of cooling on the ease with enabled the group to radiate to dominance in the polar sea which proteins undergo conformational changes that are required temperatures of the Southern Ocean. Indeed, recent molecular during substrate binding, catalysis, and the release of products. The evidence from DeVries’ laboratory indicates that AFGPs originally thermal sensitivity of the conformational flexibility of proteins is derived from the gene for trypsinogen via a mutational event that rooted in the effect of temperature on the weak bonding forces that dates back roughly 10-15 million years, coinciding with accepted stabilize higher orders of protein structure. Decreases in estimates of ocean cooling to subzero temperatures (Chen et al., temperature tend to stabilize Van der Waals interactions, hydrogen- 1997). Although this fascinating group of molecules explains how bonding, and electrostatic interactions, due to the negative enthalpy notothenioid fishes were able to survive subzero temperatures, changes associated with their formation. On the other hand, cooling AFGPs do not provide a biochemical explanation of how these destabilizes hydrophobic interactions, because enthalpy change animals maintain normal rates of biological function. Recent during their formation has a positive value (Hochachka and Somero, findings have begun to shed light on some of the structural and 1984). Weak bonding forces underlie both the conformational functional characteristics, at both molecular and cellular levels, that change of enzymatic proteins and the macromolecular assembly of enable life at cold body temperature. multimeric protein complexes. We can expect, therefore, that tens

of millions of years of evolutionary history at cold body

temperature may have resulted in the selection of specific protein Structure and Function of Proteins structures that ensure both of these processes in polar species. Ectothermic animals, such as Antarctic fish, are in nearly Indeed, mounting evidence suggests that this is the case. perfect thermal equilibrium with their habitat. Thus, in the case of At the grossest level, the conformational flexibility of temperature, the environment-organism interface resides at the proteins can be probed by examining their thermal denaturation molecular level. Cold temperature (low heat content) can temperatures. Numerous studies over the past 20 years have profoundly affect the function of shown that proteins from normally cold-bodied animals denature at significantly lower temperatures

Gravitational and Space Biology Bulletin 13(2), June 2000 27 PHYSIOLOGY AND BIOCHEMISTRY OF ANTARCTIC FISHES

When adjusted to a common temperature, the rate at which LDHs catalyze conversion of pyruvate to lactate is highly correlated with the normal cell temperature of the source animal. Figure 3 shows a directly inverse relationship between normal

body temperature and the catalytic rate constant, kcat, estimated at 0°C for LDHs from a wide array of vertebrate species. Clearly the rate of catalysis at 0°C is substantially higher for LDHs from Antarctic notothenioids (points 1-4) than from warmer-bodied animals. The enzymes from Antarctic species even show slightly but significantly higher rates than those from phylogenetically closely related notothenioids from southern South America (points 5-7), whose normal body temperatures are only a few degrees C warmer. These differences strongly suggest that the enzymes from polar species are most capable of undergoing necessary conformational change during the catalytic process at cold temperature—i.e., their structure is inherently more flexible. Yet, Figure 3. The Relationship between Normal Body Temper- no similar correlation is observed when the kinetics of thermal ature and the Catalytic Rate Constant, kcat, Estimated at 0ºC denaturation are compared between LDHs from the closely related for A4-Lactate Dehydrogenases from the White Skeletal Antarctic and South American species (Fields and Somero, 1998). Muscle of a Wide Variety of Vertebrate Animals. Open It appears that structural changes resulting in these functional boxes (1-4) are Antarctic fish species. differences must be much subtler than can be detected by thermal

1 = Parachaenichthys charcoti disruption of the gross protein structure. 2 = Lepidonotothen nudifrons Fields and Somero (1998) used reverse transcription-PCR to 3 = Champsocephalus gunnari obtain cDNA sequences for LDH-A from 12 species of Antarctic 4 = Harpagifer antarcticus and South American notothenioid fishes. Careful comparison of 5 = Patagonotothen tesselata amino acid sequences deduced from these cDNAs divulged that 6 = Eleginops maclovinus 7 = Sebastes mystinus (rockfish) · different primary structures characterize orthologs 8 = Hippoglossus stenolepis (halibut) that display similar kinetic characteristics; 9 = Sphyraena argentea (barracuda) · very minor changes in primary structure can yield significant differences in kinetics. 10 = Squalus acanthias (spiny dogfish) 11 = Sphyraena lucasana In addition, these minor changes could even be located at sections 12 = Gillichthys mirabilis (goby) of the protein’s primary structure that were far removed from the 13 = Thunnus thynnus (bluefin tuna) active site, as long as they ultimately exerted an influence on the 14 = Sphyraena ensis conformational flexibility of the active site through tertiary 15 = Bos taurus (cow) structure of the enzyme. Gallus gallus 16 = (chicken) Fields and Somero reasoned that higher values of kcat, 17 = Meleagris gallopavo (turkey) reflecting an inherently more flexible structure, should indicate that 18 = Dipsosaurus dorsalis (desert iguana) a statistically greater number of alternative configurations will characterize enzymes from the Antarctic species. Since only a (Redrawn from Fields and Somero, 1998) limited subset of these conformations would be expected to bind the substrate optimally, Fields and Somero further hypothesized that greater flexibility will concomitantly lower enzyme-substrate affinity (i.e., elevation of the enzyme’s Michaelis-Menton

constant, KM) when comparisons are made at the same than do homologous molecules from warmer-bodied species temperature. Figure 4 shows that estimates of KM for pyruvate for (Somero, 1995). These observations suggest that adaptation to LDHs from Antarctic, sub-Antarctic and Temperate Zone fish different body temperatures results in selection for fairly profound species bear out this prediction. In other words, at any given alterations in aggregate weak bonding forces and conformational temperature of measurement, increases in both kcat and KM are flexibility. Although this overall concept remains valid, we now observed to accompany decreases in normal body temperature. The know that changes in protein structure arising from temperature- other notable feature of these data is that, when estimates of KM driven selection can be much subtler than those revealed by thermal (pyruvate) are compared at physiological temperatures, values for denaturation studies. The recent work of George Somero and the constant are conserved in a range from approximately 0.15 to colleagues with orthologous homologs of muscle-specific A4- 0.30 mM among all the species examined, regardless of normal lactate dehydrogenases (LDHs) provides an excellent illustration body temperature. Thus, the evolution of Antarctic fish species (Somero et al., 1998, Fields and Somero, 1998). has subtly modified enzyme structure to conserve rates of catalysis

28 Gravitational and Space Biology Bulletin 13(2), June 2000 PHYSIOLOGY AND BIOCHEMISTRY OF ANTARCTIC FISHES by increasing flexibility of the protein, while the very same structural changes have assured maintenance of absolute enzyme- substrate affinity within a narrow range of tolerance. The same thermally sensitive, weak bonding forces that influence the structural pliancy of enzymes also strongly affect significant polymerization reactions of biological macromolecules. Not surprisingly, important protein components of the cellular cytoskeleton in Antarctic fishes also show clear evidence of evolutionary adaptation to cold body temperature. In the case of polymerization of G-actin monomers to F-actin microfilaments, enhanced flexibility of the protein and the attendant lowering of enthalpic requirements for polymerization seem to parallel the situation described above for enzymes from Antarctic species (Swezey and Somero, 1982). Greater reliance of polymerization on polar bonding forces, which are strengthened by cold temperature, appears to confer stability to the filaments. This is in contrast to findings regarding the polymerization of a- and b-tubulin subunits to form microtubules in the same animals. Figure 4. The Relationship between Apparent KM for

Microtubules are essential elements of the cytoskeleton in Pyruvate of A4- Lactate Dehydrogenases from Antarctic, eukaryotic organisms. These structures are involved in such critical Sub-Antarctic and Temperate Zone Fish Species. Shaded cellular functions as transport of organelles, separation of areas encompass 95% confidence limits for each group. (Data for chromosomes during mitosis and meiosis, determination of cell Antarctic and sub-Antarctic species from Fields and Somero, 1998; shape and growth, and regeneration of neural tissues. Among the data for Temperate Zone species from Somero et al., 1998) notable characteristics of microtubules from mammals is their destabilization by temperatures below ca. 10°C. In fact, cold disassembly of mammalian microtubules to their constituent Cellular Ultrastructure tubulins is so widely recognized that it often is exploited as an experimental manipulation in vitro. Similar behavior by One of the most striking features of aerobic muscular tissues microtubular proteins in Antarctic fishes would be devastating to from very cold-bodied animals is often extraordinarily dense normal cellular function and incompatible with life. populations of mitochondria (Sidell, 1998). Tissues from both Extensive studies by H. W. Detrich and coworkers at seasonally cold-acclimated Temperate Zone fishes and Northeastern University have established that cold-stable evolutionarily adapted Antarctic species share this characteristic. microtubules successfully polymerize in Antarctic fishes at their The percentage of cell volume displaced by mitochondria in the physiological temperature of ~0°C, well below the temperature at oxidative skeletal muscles of Antarctic fish species can reach 45- which mammalian microtubules would disassemble (reviewed by 50%, the upper limit considered possible for permitting a Detrich, 1997). Deduced amino acid sequences of b-tubulins from contractile role for the tissue (Table 1). Two potentially adaptive an Antarctic fish species reveal several substitutions in a section of benefits of very high mitochondrial volume in cold-bodied fishes primary structure that is localized at a surface domain where have been identified. First, the increased cellular density of tubulin dimers contact during microtubule assembly. Detrich mitochondria results in elevating the intracellular concentration of interprets these substitutions as possibly conferring greater enzymes associated with pathways of aerobic energy metabolism. flexibility, which would enhance addition of tubulins to the growing Greater intracellular concentration of enzymes compensates for the ends of microtubules. If correct, this interpretation describes a depressing effect of cold temperature on the catalytic rate of trend toward greater conformational flexibility similar to that individual enzyme molecules, causing an increase in the aggregate observed in the enzymes of Antarctic fish species. What is more metabolic potential of these pathways in the tissue. Second, the surprising, however, is that analysis of the polymerization mean diffusional path length is reduced for both the water-soluble thermodynamics of microtubules in Antarctic fishes indicates that small metabolites that exchange between cytoplasmic and the process is very strongly entropy-driven, suggesting greater mitochondrial compartments and the oxygen that must diffuse from reliance on hydrophobic interactions than is seen in warmer-bodied adjacent capillaries to the cell’s mitochondria. Reduction in animals. Because hydrophobic interactions are unique among weak- diffusional path length may be particularly important, because the bonding forces in being destabilized by cold temperature, such a very high aqueous viscosity that is caused by the cold cellular conclusion seems at least superficially paradoxical. It clearly temperatures of these ectotherms can have a profound effect on the distinguishes adaptations in microtubular polymerization from diffusion rates of small molecules and gases (Sidell and Hazel, those of actins from Antarctic fishes that are more reliant on polar 1987). bonding forces (pun intended).

Gravitational and Space Biology Bulletin 13(2), June 2000 29 PHYSIOLOGY AND BIOCHEMISTRY OF ANTARCTIC FISHES

Table 1. Mitochondrial Densities of Oxidative Muscles from Selected Antarctic Fish Species Species and Tissue % Cellular Volume Displaced by Mitochondria

Family Channichthyidae Chaenocephalus aceratus Heart Ventriclea 36.53 ± 2.07 Oxidative Skeletal Musclec 49.0 ± 1.0 Chionodraco rastrospinosus Heart Ventriclea 20.10 ± 0.74 Oxidative Skeletal Musclec 39.0 ± 3.0

Family Nototheniidae Gobionotothen gibberifrons Heart Ventriclea 15.87 ± 0.74 Oxidative Skeletal Muscleb 24.9 ± 0.7 Trematomus newnesi Oxidative Skeletal Muscleb 34.8 ± 1.2

(Data from: aO’Brien and Sidell, 2000; bLondraville and Sidell, 1990; cO’Brien and Sidell, unpublished results)

In each of the examples I have selected to discuss, I have mammal populations of the Southern Ocean during the nineteenth attempted to illustrate features that clearly contribute to the century, reports filtered back of a strange group of white-blooded survival and normal function of southern polar fishes. Other fishes—referred to as devilfishes or icefishes—found in the waters characteristics of the physiology and biochemistry of Antarctic surrounding Antarctica. It was not until 1954, however, that the fishes (e.g., biophysical properties of cellular and subcellular first modern physiological description of these unique animals membrane systems) also fall into this category. Clearly, millions of appeared in the scientific literature (Ruud, 1954). We now know of years of evolution at chronically cold body temperature has fifteen species in this family of notothenioid fishes resulted in selection of traits that have finely tuned this group to (Channichthyidae). their thermal environment. In addition to these distinctly adaptive All channichthyid icefishes share the remarkable characteristics, however, some Antarctic fishes display unique characteristic of lacking hemoglobin and red cells in their attributes whose adaptive significance is, at best, obscure. Indeed, circulation. We have learned recently that this state is the result of there is every reason to believe that the mutations that led to these an apparently complete deletion of the gene for b-globin and partial traits were harmful, rather than advantageous. We will now turn to loss of the gene for a-globin (Cocca et al. 1995). Channichthyid consideration of some of these physiological characteristics, and icefishes are the only known vertebrate animals to display this attempt to understand why they have been maintained over attribute as adults, and they have received considerable attention evolutionary time. from comparative biologists interested in determining how adequate oxygenation of tissues can be accomplished in the absence of a PHYSIOLOGICAL CHARACTERISTICS PERMITTED circulating oxygen-binding protein. BY COLD: THE CHANNICHTHYID Several fairly draconian modifications of the cardio-vascular ICEFISHES OF ANTARCTICA system of icefishes may help compensate for the lack of a circulating oxygen-carrier: Current population genetics theory holds that, due to diminished viability and/or reproductive success, · Icefishes possess very large hearts compared to “disadvantageous” traits will be subject to negative selection and be red-blooded fishes of equivalent body size, and large eliminated. Implicit in the conceptual underpinnings of this idea is heart size results in a weight-specific cardiac output that disadvantage accrues through competition with other that is four- to five-fold greater than that of red- individuals that possess more beneficial homologous traits. This blooded species (Hemmingsen et al., 1972) implicit assumption is critical, and one to which I will return. Its · The blood volumes of icefishes are up to four-fold importance will become apparent as I briefly describe a very those of red-blooded teleosts, and the diameter of their unique family of Antarctic fishes whose physiological capillaries is unusually large (Fitch et al., 1984) characteristics have no parallel in any other group of vertebrate This combination of cardiovascular features permits a large volume animals. of blood to circulate throughout the bodies of icefishes at a high

flow rate. Yet, because of decreased peripheral resistance, vascular The Hemoglobinless State of Channichthyid Icefishes pressure is relatively low. Combined with the very high oxygen As British sealers and whalers exploited the rich marine content of Antarctic waters and the relatively low absolute metabolic rate of these animals, alluded to above,

30 Gravitational and Space Biology Bulletin 13(2), June 2000 PHYSIOLOGY AND BIOCHEMISTRY OF ANTARCTIC FISHES

Figure 5. Pattern of Myoglobin Protein Expression in the Heart Ventricles of 13 of the 15 Known Species of Antarctic Channichthyid Icefishes. Presence (+) or absence (-) of myoglobin is shown in relation to phylogenetic positions of the species, based on combined morphological characters (Iwami, 1985) and molecular characters (mitochondrial DNA; Bargelloni et al., 1994; Chen et al., 1998).

these circulatory features apparently ensure delivery of sufficient compensate for the lack of circulating hemoglobin in icefishes, it is oxygen to tissues (Hemmingsen, 1991). These traits are most likely difficult to envision how they could counteract the absence of the adaptive, in the sense that they at least partially compensate for intracellular oxygen-binding protein, myoglobin. In recent years, the lack of hemoglobin in the circulation of these animals. our laboratory has been addressing questions of when and how Can loss of hemoglobin expression itself be considered an myoglobin expression was lost during the evolution of the adaptive feature in icefishes? I suspect not, even though a Antarctic icefish family, and whether loss of the protein has consensus answer to this question has not emerged. Despite functional consequences to the physiology of these animals. arguments made by some about the benefits of the lowered blood We have examined oxidative muscle tissues from thirteen of viscosity and cardiac work that accompany loss of red cells, the fifteen known species of channichthyid icefishes, and have reduction in blood oxygen-carrying capacity to <10% that of established that myoglobin, in fact, is present in the heart ventricles hemoglobin-expressing fishes would seem to more than offset this in eight of these species, but absent from the same tissue in five advantage. As Eastman (1993) pointed out, the very existence of other icefish species (Sidell et al., 1997; Moylan and Sidell, 2000). rather remarkable features of cardiovascular anatomy and One of the most striking aspects of this pattern of variable physiology (heart size, capillary diameter, etc.) that appear to have myoglobin expression is that loss of myoglobin has apparently been selected to compensate for loss of a circulating oxygen-carrier occurred through at least four independent mutational events during testifies that mutation(s) leading to this loss probably had the evolution of this family. Figure 5 maps the presence/absence of deleterious consequences. myoglobin in the hearts of icefishes, based on the best available

phylogeny of the family, and clearly shows that the majority of Myoglobin Expression in Icefishes is Variable species lacking myoglobin are more closely related to species that produce the protein than to those that do not. Subsequent studies It is generally deemed that, in addition to lacking hemoglobin, have established that the mutational mechanisms leading to loss of icefishes are devoid of the intracellular oxygen-binding protein, myoglobin in at least three of these species are discretely different myoglobin, in their oxidative muscles. Myoglobin is widely (Small et al., 1998; Vayda et al., 1997). Such a pattern, at least distributed in aerobically poised tissues of animals, and has been superficially, suggests that myoglobin might not be functional at ascribed roles in both intracellular storage and diffusion of oxygen the cold body temperatures of these animals, and that loss of its (Wittenberg and Wittenberg, 1989). Although each of the expression might not carry functional significance. Recent results, cardiovascular adaptations described above potentially could help however, argue strongly against this view.

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Using stopped-flow spectral studies, we have been able to establish that oxygen binds and dissociates from icefish myoglobin more rapidly than from mammalian my oglobins at cold temperature (Cashon et al., 1997). Even more compelling evidence of myoglobin functionality in icefishes has been obtained from isolated, perfused heart studies (Acierno et al., 1997). Figure 6 shows that hearts from Chionodraco rastrospinosus, an icefish that possesses myoglobin, are capable of sustaining cardiac output against the challenge of a greater afterload pressure than those from the closely related myoglobin-lacking Chaenocephalus aceratus. Recognizing that, although provocative, these results only correlated mechanical performance with the presence of myoglobin, we sought to resolve the issue more definitively. We repeated experiments with perfused hearts, incorporating 5 mM NaNO2 into the perfusate to poison myoglobin function selectively. Figure 7 shows that selective ablation of myoglobin function results in the loss of mechanical performance by hearts Figure 6. The Ability of Isolated, Saline-Perfused Hearts that express the protein, but not by hearts that naturally lack from Icefish Species to Maintain Cardiac Output When myoglobin. In fact, comparison of the results shown in Figures 6 Challenged by Increasing Afterload Pressure. C. aceratus and 7 reveals that hearts from Chaenocephalus aceratus that hearts are naturally devoid of myoglobin protein, while those of C. naturally lack myoglobin can meet an even greater pressure-work rastrospinosus contain myoglobin. (Data from Acierno et al., 1997) challenge than those of Chionodraco rastrospinosus with myoglobin function chemically ablated. Taken together, these results strongly indicate that myoglobin is functional when present, and that hearts lacking the protein have been altered through evolutionary selection to compensate partially for its loss. Recent studies, in fact, have revealed that ventricular tissue from myoglobin-lacking C. aceratus has greater mitochondrial density and a shorter intracellular diffusion distance for oxygen than does tissue from myoglobin-containing C. rastrospinosus (O’Brien and Sidell, 2000).

The observations above clearly suggest that losses of oxygen- binding proteins, hemoglobin and myoglobin, that have occurred in the Antarctic channichthyid icefishes are not inherently adaptive. In fact, these losses may have resulted in a decrement in the Figure 7. Ability of Isolated, Perfused Hearts from Icefish physiological performance of these animals. Yet, current prevailing Species to Maintain Cardiac Output in the Face of evolutionary theory suggests that selective pressure should then Increasing Afterload Pressure Challenge When 5 Mm lead to retention of hemoglobin and myoglobin expression, and that NaNo2 Is Incorporated into the Perfusion Solution. This mutations causing their loss should be subject to negative selection concentration of NaNO2 selectively poisons myoglobin, preventing and eliminated from the population. In Antarctic icefishes, the protein from binding oxygen. See legend for Figure 6 for further however, loss of the ability to express these physiologically explanation of species. (Data from Acierno et al., 1997) important proteins apparently has not been lethal. A combination of environmental and organismal characteristics may help explain why hemoglobin and myoglobin tissues of channichthyid ancestors despite the loss of oxygen- losses are not lethal at the level of the individual organism. As binding proteins. This reasoning, however, does not address the mentioned earlier, the very cold temperature and extensive vertical more difficult question of why these apparently “disadvantageous” mixing of the Southern Ocean results in the exceptionally high traits were maintained at the population level. oxygen content of its waters. Because of their cold body At the beginning of this section, I mentioned the implicit temperature, the absolute metabolic rates of Antarctic fishes are assumption that, if not acutely lethal, a trait is disadvantageous relatively low. The unique convergence of these features may have only in reference to competition with other organisms. The ensured that sufficient oxygen to sustain life was available to extremely unusual evolutionary history of the Antarctic fish fauna that I described earlier makes it reasonable to ask whether

32 Gravitational and Space Biology Bulletin 13(2), June 2000 PHYSIOLOGY AND BIOCHEMISTRY OF ANTARCTIC FISHES competitive disadvantage, in fact, can exist in the absence of Cashon, R.E., Vayda, M.E., and Sidell, B.D. 1997. Kinetic competition. The massive crash of species diversity among fishes characterization of myoglobins from vertebrates with vastly in the Southern Ocean, occurring between the mid-Tertiary and the different body temperatures. Comparative Biochemistry and present, left an ancestral stock of demersal notothenioids to Physiology. 117B:613-620. colonize approximately 10% of the world’s ocean volume. Although the proximate cause is not known with certainty, most Chen, L., DeVries, A.L. and Cheng, C.-H. 1997. Evolution of polar biologists think that this event explains the ultimate antifreeze-glycoprotein gene from a trypsinogen gene in Antarctic dominance of notothenioid species in the Antarctic seas (Eastman, notothenioid fish. Proceedings of the National Academy of Sciences 1993). In a marine habitat of low species diversity and a low of the United States 94:3811-3816. population density of fishes, weak niche competition may also explain why populations of icefish species lacking oxygen-binding Chen, W.-J., Bonillo, C., Lecointre, G. 1998. Phylogeny of the proteins have persisted and become abundant members of the Channichthyidae (Notothenioidei, Teleostei) based on two Southern Ocean’s fish community. mitochondrial genes. In: Fishes of Antarctica (di Prisco, G., Pisano, E. and Clarke, A.., Eds.) Heidelberg: Springer-Verlag, pp. 287-298.

In the topics covered, I hope that I have succeeded in Cheng, C.C. and A.L. DeVries. 1991. The role of antifreeze illustrating that highly unusual variations of life can arise and glycopeptides and peptides in the freezing avoidance of cold-water thrive, even in the most extreme of the earth’s biological habitats. fish. In: Life Under Extreme Conditions: Biochemical Adaptation. Indeed, environmental extremes can contribute to uniquely adaptive (di Prisco, G., Ed.) Berlin and Heidelberg: Springer-Verlag,. pp. 1- characteristics through processes of natural selection. In some 14. unusual cases, they also can permit persistence of traits that might even be lethal in environments that we view as normal from our Cocca, E., Ratnayake-Lecamwasam, M., Parker, S.K., Camardella, own limited perspective. L., Ciaramella, M., diPrisco, G. and Detrich, H.W. III. 1995. Genomic remnants of a-globin genes in the hemoglobinless Acknowledgements Antarctic icefishes. Proceedings of the National Academy of Science of the United States 92:1817-1821. The author’s research is supported by grants from the National Science Foundation (OPP 94-21657 and OPP 99-09055). Detrich, H.W., III. 1997. Microtubule assembly in cold-adapted I thank my colleagues—Thomas Moylan, Kristin O’Brien, organisms: Functional properties and structural adaptations of Michael Vayda, and Raffaele Acierno—for their scientific and tubulins from Antarctic fishes. Comparative Biochemistry and conceptual contributions to our work, described in this paper. Physiology 118A:501-513. Much of the additional information presented on fascinating adaptations displayed by Antarctic fishes is from results of work DeVries, A.L. 1988. The role of antifreeze glycopeptides and by Arthur DeVries, Chris Cheng-DeVries, H. William Detrich III , peptides in the freezing avoidance of Antarctic fishes. Comparative George Somero, and Peter Fields. All of us are in the debt of Biochemistry and Physiology 90B: 611-621. Joseph Eastman for the seminal thoughts that he and his colleagues have presented about the evolution of notothenioid fishes in Dewitt, H.H. 1971. Folio 15. In: Antarctic Map Folio Series Antarctic waters. Finally, the work that my colleagues and I have (Bushnell, V.C., Ed.) New York: American Geographical Society, conducted in the challenging environment of Antarctica would not pp. 1-10. have been successful were it not for the excellent support provided by the Masters and crew of the R/Vs Polar Duke and L.M. Gould Eastman, J.T. 1993. Antarctic Fish Biology: Evolution in a Unique and the many support staff of Antarctic Support Associates at Environment. New York: Academic Press. Palmer Station, Antarctica. Eastman, J.T. 1990. The biology and physiological ecology of REFERENCES notothenioid fishes. Fishes of the Southern Ocean. (Gon, O. and Acierno, R., Agnisola, C., Tota, B. and Sidell, B.D. 1997. P.C. Heemstra, Eds.) Grahamstown, South Africa: J.L.B. Smith Myoglobin enhances cardiac performance in Antarctic species that Institute of Ichthyology, pp. 34-51. express the protein. American Journal of Physiology. 273:R100- R106. Eastman, J.T. and Clarke, A.C. 1998. A comparison of adaptive radiations of Antarctic fish with those of non-Antarctic fish. In: Bargelloni, L., Ritchie, P.A., Patarnello, T., Battaglia, B., Lambert, Fishes of Antarctica: A Biological Overview. (di Prisco, G., Pisano, D.M., and Meyer, A. 1994. Molecular evolution at subzero E. and Clarke, A.C., Eds.) Springer-Verlag, Heidelberg, pp. 3-26. temperatures: mitochondrial and nuclear phylogenies of fishes from Antarctica (Suborder Notothenioidei), and the evolution of Ekau, W. 1990. Demersal fish fauna of the Weddell Sea, Antarctica. antifreeze glyco-proteins. Molecular Biology and Evolution 11:854- Antarctic Science 2:129-137. 863.

Gravitational and Space Biology Bulletin 13(2), June 2000 33 PHYSIOLOGY AND BIOCHEMISTRY OF ANTARCTIC FISHES

Fields, P.A. and G.N. Somero. 1998. Hot spots in cold adaptation: Ruud, J.T. 1954. Vertebrates without erythrocytes and blood Localized increases in conformational flexi-bility in lactate pigment. Nature 173:848-850. dehydrogenase A4 orthologs of Antarctic notothenioid fishes. Proceedings of the National Academy Sidell, B.D. 1998. Intracellular oxygen diffusion: The roles of of Sciences of the United States 95:11476-11481. myoglobin and lipid at cold body temperature. Journal of Experimental Biology 201:1118-1127. Fitch, N.A., Johnston, I.A. and Wood, R.E. 1984. Skeletal muscle capillary supply in a fish that lacks respirtory pigments. Sidell, B.D. and Hazel, J. R. 1987. Temperature affects the Respiration Physiology 57: 201-211. diffusion of small molecules through cytosol of fish muscle. Journal of Experimental Biology 129:191-203. Hemmingsen, E.A. 1991. Respiratory and cardio-vascular adaptation in hemoglobin-free fish: resolved and unre-solved Sidell, B.D., Vayda, M.E., Small, D.J., Moylan, T.J., Londraville, problems. In: Biology of Antarctic Fish (di Prisco, G., Maresca, B. R.L., Yuan, M.-L., Rodnick, K.J., Eppley, Z.A. and Costello, L. and Tota, B., Eds.) New York: Springer-Verlag, pp. 191-203. 1997. Variation in the expression of myoglobin among species of hemoglobinless Antarctic icefishes. Proceedings of the National Hemmingsen, E.A., Douglas, E.L., Johansen, K. and Millard, R. W. Academy of Sciences of the United States 94:3420-3424. 1972. Aortic blood flow and cardiac output in the hemoglobin-free fish Chaenocephalus aceratus. Comparative Biochemistry and Small, D.J., Vayda, M.E. and Sidell, B.D. 1998. A novel vertebrate Physiology 43A:1045-1051. myoglobin gene containing three A+T– rich introns is conserved among Antarctic teleost species, which differ in myoglobin Hochachka, P.W. and Somero, G.N. 1984. Biochemical Adaptation. expression. Journal of Molecular Evolution 47:156-166. Princeton, New Jersey: Princeton University Press. Somero, G.N. 1995. Proteins and temperature. Annual Review of Iwami, T. 1985. Osteology and relationships of the family Physiology 57:43-68. Channichthyidae. Memoranda of the National Institute for Polar Research Ser. 36E:1-69. Somero, G.N. and DeVries, A.L. 1967. Temperature tolerance of some Antarctic fishes. Science 156:257-258. Kennett, J.P. 1977. Cenozoic evolution of Antarctic glaciation, the circum-Antarctic Ocean and their impact on global Somero, G.N., Fields, P.A., Hofmann, G.E., Weinstein, R.B. and paleooceanography. Journal of Geophysical Research. 82:3843- Kawall, H. 1998. Cold adaptation and stenothermy in Antarctic 3860. notothenioid fishes: What has been gained and what has been lost? In: Fishes of Antarctica: A biological overview. (di Prisco, G., Kennett, J.P. 1982. Marine Geology. Englewood Cliffs, New Pisano, E. and Clarke, A., Eds.) Heidelberg: Springer-Verlag, pp. Jersey: Prentice-Hall. 97-109.

Littlepage, J.L. 1965. Oceanographic investigation in McMurdo Swezey, R. R. and Somero, G.N. 1982. Polymerization Sound, Antarctica. In: Biology of the Antarctic Seas, vol. II (Llano, thermodynamics and structural stabilities of skeletal muscle actins G.A., Ed.) Washington, DC: American Geophysical Union, pp. 1- from vertebrates adapted to different temperatures and hydrostatic 37. pressures. Biochemistry 21:4496-4503.

Londraville, R.L. and Sidell, B.D. 1990. Ultra-structure of aerobic Vayda, M.E., Small, D.J., Yuan, M.-L. and Sidell, B.D. 1997. muscle in Antarctic fishes may contribute to maintenance of Conservation of the myoglobin gene among notothenioid fishes. diffusive fluxes. Journal of Experimental Biology 150:205-220. Molecular Marine Biology and Bio-technology 6:207-216.

Moylan, T.J. and Sidell, B.D. 2000. Concentrations of myoglobin Wittenberg, B.A., and Wittenberg, J.B. 1989. Transport of oxygen and myoglobin mRNA in heart ventricles from Antarctic fishes. in muscle. Annual Review of Physiology. 51:857-878. Journal of Experimental Biology 203:1277-1286.

O’Brien, K.M and Sidell, B.D. 2000. The interplay among cardiac ultrastructure, metabolism and the expression of oxygen binding proteins in Antarctic fishes. Journal of Experimental Biology 203:1287-1297.

Raymond, J.A. and A.L. DeVries. 1977. Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Proceedings of the National Academy of Sciences of the United States 74:2589-2593.

34 Gravitational and Space Biology Bulletin 13(2), June 2000

Life in Extreme Environments: How Will Humans Perform on Mars? Dava J. Newman Department of Aeronautics and Astronautics, Massachusetts Institute of Technology, Cambridge MA

ABSTRACT capabilities relative to the effects of partial gravity on human locomotion will enhance the integration of humans and machines This review of astronaut extravehicular activity for future missions. (EVA) and the details of American and Soviet/Russian spacesuit design focuses on design recommendations to THE SPACE SHUTTLE AND MIR SPACE STATION enhance astronaut safety and effectiveness. Innovative Many of the tasks accomplished onboard the Space spacesuit design is essential, given the challenges of Shuttle—the world's first reusable spacecraft and one of future exploration-class missions in which astronauts will NASA's foremost projects—have furthered space exploration be called upon to perform increasingly complex and and enhanced the quality of life on Earth. The Space Shuttle is physically demanding tasks in the extreme environments the first U.S. vehicle with a standard sea-level atmospheric of microgravity and partial gravity. pressure and composition. (Mercury, Gemini, and Apollo all

operated at 33.4 kPa [5 psi or 0.33 atm] pressure and 100% oxygen composition.) The Space Shuttle’s capabilities allow INTRODUCTION scientists routinely to conduct experiments that explore the Since the beginning of human exploration above Earth’s effects of the space environment, particularly microgravity, on atmosphere, our main challenge has been to supply the explorer human physiology under conditions that cannot be duplicated on with the basic necessities for life support that nature normally Earth. provides. Unprotected by a spacecraft or spacesuit, anyone Between March 1995 and May 1998, NASA astronauts encountering the near-vacuum of space would survive only a few flew onboard the Russian space station Mir in a collaborative minutes. Body fluids would vaporize in the absence of pressure effort with the Russian space program. The NASA program that and an atmosphere, and gas that would quickly expand in the has supported this endeavor, commonly known as International lungs and other tissues would prevent circulation and Space Station Phase 1 (or Shuttle-Mir), has encompassed 11 respiration. Space Shuttle and joint Soyuz flights. The international program This paper focuses on the demands faced by astronauts has resulted in joint space experience for the crew and the start when they leave their spacecrafts and perform extravehicular of joint scientific research. Shuttle-Mir participants (crew activities (EVA) in space, and on the evolutionary design of members, principal investigators, and mission control staff) spacesuits to meet these needs. These suits comprise a investigated vital questions about the future of human life in necessary operational resource for the long-duration missions space. Mir has been a test site for three main areas of experience that will establish human presence beyond Earth. The different and investigation: spacesuit choices pursued by the American and Soviet/Russian · Designing, Building, and Staffing the Inter- space programs provide the basis for case studies relative to the national Space Station design of human space exploration systems. Many factors Participants have drawn from the experience and bearing on the challenge of keeping humans alive and functioning resources of many nations to learn from one another, optimally in space will be considered in the following discussion, and also to learn how to work together. including atmosphere composition and pressure, thermal control, · Investigation radiation protection, human physiology, and human performance Mir has offered a unique opportunity for long-duration in partial gravity. data gathering. Station designers have used Mir as a test Human presence on space missions offers many advantages site for space station hardware, materials, and to ensure mission success: flexibility and dexterous construction methods. Mir crew members have utilized manipulation, human visual interpretation and cognitive ability, the microgravity environment to conduct scientific and real-time approaches to problems. However, there are investigations into biological and materials studies. factors that may degrade human performance. These include · Operation pressure-suit encumbrance, prebreathe requirements, insufficient In the almost 40-year history of human spaceflight, no working volume, limited duration, sensory deprivation, and poor previous program has required so many transport task or tool design (NASA, 1989). In addition to microgravity vehicles and so much interdependent operation between performance, the partial-gravity environments of the moon and organizations. Shuttle-Mir experience has given Mars require advanced technology, hardware, and performance participants an opportunity to prepare for the capabilities for successful space endeavors. While EVA, as well formidable cooperative effort required on the as robotics and automation, expand the scope of space International Space Station. operations, a more thorough understanding of astronaut

Gravitational and Space Biology Bulletin 13(2), June 2000 35 ASTRONAUT PERFORMANCE FROM MIR TO MARS

A Case Study: MIT’s Enhanced Dynamic Load Sensors (EDLS) Experiment on Mir

One of the key missions of the International Space Station (ISS) is to perform microgravity experiments that require a quiescent environment (~10-4 to 10-7 g); that is, to perform experiments that make use of the almost complete absence of any accelerations as a vehicle orbits the Earth. For this reason, we conduct spaceflight experiments for the ISS program that investigate how astronauts move around in space and how they may disturb the spacecraft microgravity environment. While a some microgravity experiments can be fully automated, many require astronauts to execute or supervise them. We have wanted to ensure that these astronauts, who play a critical role in the success of the experiments, are not a significant source of disturbance to the spacecraft acceleratory environment. When astronauts move inside the cabin of a spacecraft, they impart impulses to the vehicle. From vehicle and environmental parameters, we can estimate external disturbances, such as aerodynamic drag and solar pressure, quite easily. Similarly, we can predict interior disturbances caused by operating mechanical equipment, such as pumps and fans. However, the inherent randomness of astronaut-induced disturbances makes their analysis a far more challenging task. Phase I of the ISS program gave seven American astronauts the opportunity to conduct long-duration spaceflight experiments on the Russian space station Mir, and within the b framework of the program, Massachusetts Institute of . Technology (MIT) conducted the Enhanced Dynamic Load Sensors (EDLS) experiment on Mir to quantify astronaut- induced disturbances to the microgravity environment. The Figure 1. MIT’s Enhanced Dynamic Load Sensors (EDLS) experiment was designed with two objectives: on Mir. (a) A Shuttle-Mir crew member using the EDLS

1. Primarily, to assess nominal astronaut- handhold on Mir. (b) Four EDLS, force sensors that were induced forces and torques during long- used in the Priroda and Mir Base Block modules. duration space station missions by measuring everyday activities and induced loads (using smart sensors, called Whenever the computer detected that the measured forces and “restraints”). torques exceeded a specified threshold force, data were recorded 2. Secondarily, to gain a detailed on the storage medium (Figures1a and b). The experiment was understanding of the how astronauts devise conducted during the stay of U.S. astronauts Shannon Lucid strategies for moving around in (March–September 1996) and Jerry Linenger (January–May microgravity as they propel themselves 1997) aboard Mir. The overall data recording time was 133 hours with their hands and float from module to over the two periods. The storage media with the data were module. returned to Earth via the Space Shuttle in 1998. Table 1 shows the seven typical astronaut motions used for locomotion The experimental set-up consisted of four load sensors and a (including floating) in microgravity. These motions are quite specially-designed computer. The sensors included an different from the standing, walking, and running that constitute instrumented handhold and two instrumented foot restraints, bipedal motion on the Earth. which provided the same functionality as the hand rail and foot Video recordings of astronauts moving in the modules and using loops built into the Space Shuttle Orbiter and the Mir orbital restraint and mobility aids on the NASA 2 and NASA 4 complex, and an instrumented push-off pad envisioned as the missions let us identify several typical astronaut motions and kind of flat surface from which astronauts propel themselves quantify the associated load levels exerted on the spacecraft. It with their hands or feet. was found that The astronauts were instructed to activate the computer and go about their regular on-orbit activities. · for 2,806 astronaut activities recorded by the foot restraints and handhold sensor, the highest force magnitude was 137 N;

36 Gravitational and Space Biology Bulletin 13(2), June 2000 ASTRONAUT PERFORMANCE FROM MIR TO MARS

Table 1. Characteristic Astronaut Motions

Characteristic Motion Description

Landing Flying across module and landing Push off Pushing off and flying Flexion/Extension Flexing or extending limb Single Support Using one limb for support Double Support Using two limbs for support Twisting Twisting body motion Reorienting Usually small corrections for posture control

· ~99% of the time, the maximum force magnitude was will learn how to live and work “off planet” in an international below 90 N; ~96% of the time, the maximum force way. magnitude was below 60 N; The Skylab and Mir space station experiences · for 95% of the astronaut motions, the root mean demonstrated that crew members become very skilled in square force level was below 9.0 N; performing tasks on long-duration missions. After approxi- · the average momentum imparted by the astronauts on mately 60 days in orbit, a crew member’s knowledge the Mir space station was 83±228 kg·m/s. encompasses the laboratory, stowage locations, procedures,

personal dynamics among colleagues, and many other elements. It can be concluded that expected astronaut-induced loads on the This experience-based knowledge and understanding is ISS from usual astronaut intravehicular activity are considerably considerable when compared to what can be learned in missions less than previously thought and will not significantly disturb that last only two weeks or less. Crew members on long- the ISS microgravity environment (Amir and Newman, 2000). duration missions also have time to fully adapt to space (e.g., to These are very low forces when compared to typical Earth sleep well, eat well, and exercise regularly). forces. Actually, they are an order of magnitude less. (Consider The completed ISS will be powered by almost an acre of that a person with a mass of 52 kg exerts 1 BW, or 510 N with solar panels and have a mass of almost one million pounds, and every step he or she takes, and then think about how many the station’s pressurized volume will be roughly equivalent to steps a person takes every day.) Essentially, the data prove that the space inside two jumbo jets. The U.S. habitation module to astronauts in microgravity adopt the appropriate strategy for to be delivered by the final ISS assembly mission will have their new weightless environment and use “finger push-offs” and enhanced accommo-dations and will provide for as many as “toe-offs” as they move about in space. After living in space for seven crew members. months, astronauts, like highly trained athletes or professional The ISS is where key biomedical, life support, and human ballet dancers, move about with grace and control. They go factors questions must be answered to ensure crew health, well- about their daily activities exerting very low forces on the being, and productivity for future exploration missions. microgravity environment they inhabit.

EXTRAVEHICULAR ACTIVITY (EVA) THE INTERNATIONAL SPACE STATION Human space exploration is epitomized by extravehicular The ISS will offer a world-class research laboratory in low activity (EVA)—that is, space walks. In March of 1965, earth orbit. Once assembled, it will afford scientists, engineers, cosmonaut Alexei Leonov became the first human to walk in and entrepreneurs an unprecedented platform on which to space. Attached to a 5-meter long umbilical that supplied him perform complex, long-duration, and repeatable experiments in with air and communications, Leonov floated free of the the unique environment of space. The ISS’s invaluable assets Voskhod spacecraft for over ten minutes. In June of the same include opportunities for prolonged exposure to microgravity year, Edward White became the first American astronaut to leave and the presence of human experimenters in the research a spacecraft while in orbit. White performed his spectacular process. Yet the ISS is much more than a state-of-the-art lab- space walk during the third orbit of the Gemini-Titan 4 flight. oratory in a novel environment; it is an international human Figure 2 summarizes Russian and U. S. EVA to date, as a experiment—an exciting city in space—and a place where we

Gravitational and Space Biology Bulletin 13(2), June 2000 37 ASTRONAUT PERFORMANCE FROM MIR TO MARS baseline for comparison to future EVA entailed by the ISS paramount flexibility that humans performing EVA offer toward assembly and Mars exploration (to be discussed later). the success of space mission operations and scientific endeavors. Although some early EVA efforts were plagued with Cosmonauts performed critical EVAs on Salyut to examine and problems, the feasibility of placing humans in free space was replace a docking unit and returned experimental equipment to demonstrated. The Gemini EVAs revealed the need for adequate Earth that had been subjected to solar radiation for ten months. body restraints and the value of neutral buoyancy simulation for The Salyut 7 space station program saw successful astronaut extended-duration training in weightlessness. During the Apollo EVAs to study cosmic radiation and the methods and equipment program, EVA became a useful mode of functioning in space, for assembly of space structures. On 25 July 1984, during her rather than just an experimental activity. Twelve crew members second spaceflight (her first was in August 1982), cosmonaut spent a total of 160 hours in spacesuits on the moon, covering Svetlana Savitskaya became the first woman to perform an EVA, 100 kilometers (60 miles) on foot and with the lunar rover as during which she used a portable electron beam device to cut, they collected 2196 soil and rock samples. The EVA spacesuits weld, and solder metal plates. were pressurized to 26.2 kPa (3.9 psi) with 100% oxygen, and EVAs performed during Space Shuttle missions and Mir the Apollo cabin pressure was 34.4 kPa (5 psi) with 100% long-duration missions have accomplished many significant oxygen. During pre-launch, the Apollo cabin was maintained at tasks. During these missions, trained crew members have 101.3 kPa (14.7 psi) with a normal air (21% oxygen and 79% responded in real time to both planned mission objectives and nitrogen) composition. Just before liftoff, the cabin was unplanned contingencies. depressurized to 34.4 kPa (5 psi). To counteract the risk of decompression sickness after this depressurization, the SPACESUITS astronauts prebreathed 100% oxygen for three hours prior to To date, crew members have accomplished successful launch. EVAs wearing a variety of spacesuits that have evolved from the The potential benefits of EVA were nowhere more evident umbilical models of the Voskhod and Gemini era into today’s than in the Skylab missions. When the crew first entered Skylab, self-contained, modular designs. Advanced spacesuit concepts the internal temperature was up to 71oC (160oF), rendering the incorporate self-contained life-support systems (both the spacecraft nearly uninhabitable. The extreme temperatures American and Russian spacesuits) and modular components (the resulted from the loss of a solar panel and a portion of the American spacesuit). Modularity allows for ease of resizing to vehicle’s outer skin. After the failure of a second solar panel fit humans ranging in size from fifth percentile females to ninety- deployment and a consequent loss of power and cooling fifth percentile males, a distinct advantage over the custom-fitted capability, astronauts salvaged the entire project by rigging a suits previously used. Further evolution will yield spacesuits solar shade through the science airlock and freeing the remaining solar panel during EVA. The Skylab experience demonstrated the

300 2500 RUSSIAN Rev. E Assembly Sequence SHUTTLE / HST / DTO / ISS 2000 With HST and Surface Exploration APOLLO / SKYLAB

200 GEMINI Assumptions: MARS (Scale on 1500 12 US ISS Maintenance EVA/yr post assembly complete the right) 6 Russian ISS Maintenance EVA/yr post assembly complete 8-Hour Lunar EVAs commencing in 2010 250 8-Hour Mars EVAs in 2015 1000 100

500

0 0 TOTAL EVA DURATION (Clock Hours) 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93 95 97 99 01 03 05 07 09 11 13 15 CALENDAR YEAR

Figure 2. The “Wall of EVA.” Illustrating the history of EVA, the three-fold anticipated increase in EVAs for ISS assembly, and the possible 40-fold increase for planetary EVA. HST=Hubble Space Telescope; DTO=detailed test objective (additional EVA opportunities).

38 Gravitational and Space Biology Bulletin 13(2), June 2000 ASTRONAUT PERFORMANCE FROM MIR TO MARS for microgravity, lunar, and Martian environments. dioxide-scrubbing capability for nominal metabolic rates. In case of an emergency, a secondary oxygen pack, located The Space Shuttle Extravehicular Mobility Unit (EMU) at the bottom of the PLSS, provides an additional 30 minutes, minimum, of oxygen at a reduced pressure of The current Space Shuttle EVA system, known as the 26.9 kPa (3.9 psi). Between EVAs, the silver-zinc cell bat- Extravehicular Mobility Unit (EMU), consists of a spacesuit tery that powers the LSS machinery and communications assembly (SSA), an integrated life-support system (LSS), and is recharged in place. the EMU support equipment. · Displays and Controls. All of the displays and controls · Space Suit Assembly. The SSA is a 29.6 kPa (4.3 psi), that a crew member activates and monitors are mounted on 100% oxy gen spacesuit made of multiple fabric layers the front of the HUT. The temperature control valve is on attached to an aluminum-fiberglass hard upper torso unit the crew member’s upper left, and the oxygen control (HUT). The SSA retains the oxygen pressure required for actuator is on the lower right. The large controls are breathing and ventilation and protects against bright designed to be simple to operate, even by a crew member sunlight and temperature extremes. wearing pressurized spacesuit gloves.

· Life Support System. The LSS controls the internal There are numerous fabric layers in the EMU: oxygen pressure, makes up oxy gen losses due to leakage and metabolism, and circulates ventilation gas flow and 1. The liquid cooling and ventilation garment (LCVG) cooling water to the crew member. The LSS also removes is innermost. It is made of nylon/spandex lined with tricot the crew member’s released carbon dioxide, water vapor, and resembles a pair of long underwear. Ethylene-vinyl- and trace contaminants. The spacesuit and its life-support acetate plastic tubing is woven throughout the spandex to

system weigh approximately 117 kg (258 lbm) when fully route water close to the crew member’s skin for body charged with consumables for EVA (Wilde, 1984). The cooling. spacesuit is equipped with a disposable urine collection 2. The spacesuit’s pressure-garment modules come next. device. These retain pressure over the arms, legs, and feet. They · Support Equipment. The EMU support equipment, are made of urethane-coated nylon, covered by a woven which stays in the airlock during an EVA, functions dacron restraint layer. Sizing strips are used to adjust the mainly to replenish consumables and assist the crew length of the restraint layer. member with EMU donning and doffing. 3. The thermal meteoroid protection garment (TMG) The SSA’s Hard Upper Torso Unit (HUT) is the primary comprises the final layers of the EMU’s fabric structural member of the EMU. The helmet, arms, lower torso components. The TMG liner is neoprene-coated ripstop assembly (LTA), and the primary life-support system (PLSS) nylon, and it provides puncture, abrasion, and tear both mount to the HUT, which incorporates scye bearings to protection. accommodate a wide range of shoulder motions. 4. Aluminized mylar thermal insulation, designed to · Helmet. The spacesuit helmet is a transparent prevent radiant heat transfer, make up the spacesuit’s next polycarbonate bubble that protects the crew member and five layers (Wilde, 1984). directs ventilation flow over the head for cooling. The 5. The familiar white covering comes last. This sunlight- neck-ring disconnect of the helmet mounts to the HUT, reflecting outer layer is made of ortho fabric, which and the helmet is equipped with a visor that has a consists of a woven blend of kevlar and nomex synthetic moveable sunshade as well as camera and light mounts. fibers. The ortho fabric itself is very strong and resistant The crew member’s earphones and microphones are held to puncture, abrasion, and tearing, and it is coated with in place by a fabric head cover, known as the “Snoopy teflon to stay clean during training on Earth. cap.”

· Arms . The spacesuit arms are fabric comp onents Metabolic expenditures and crew performance during EVA equipped with wrist-, elbow-, and upper-arm bearings that are integrally tied to the mobility of the spacesuit and the allow for elbow extension and flexion in addition to elbow capabilities of the life-support system. The LCVG, the and wrist rotation. innermost layer of the spacesuit, provides thermal control by · Lower Torso Assembly. The LTA, which includes circulating air and water, cooled by a sublimator, over the crew boots and fabric legs that permit hip and knee flexion, is member’s body. (This concept was initially used by English equipped with a bearing that allows waist rotation. fighter pilots and later adopted by the Russian and American space programs.) The LCVG can handle peak loads of up to 500 · Primary Life Support System. The PLSS, or backpack, kcal/hr (2000 Btu/hr) for 15 minutes, 400 kcal/hr (1600 Btu/hr) houses most of the LSS and a two-way AM radio for

communications and bioinstrumenta-tion monitoring. Typically, EVA is scheduled for up to six hours, but the PLSS is equipped with a seven hour oxygen and carbon-

Gravitational and Space Biology Bulletin 13(2), June 2000 39 ASTRONAUT PERFORMANCE FROM MIR TO MARS

Figure 3. The NASA Spacesuit, or Extravehicular Mobility Unit (EMU). (Courtesy of Hamilton Standard, rev. 2/95)

40 Gravitational and Space Biology Bulletin 13(2), June 2000 ASTRONAUT PERFORMANCE FROM MIR TO MARS

for up to one hour, or 250 kcal/hr (1000 Btu/hr) for up to seven Table 2. Average Metabolic Rates for Past Space Missions (Waligora et al., 1991) hours. (See Table 2) The TMG covers the entire EMU, except for the helmet, controls, displays, and glove fingertips. The TMG and LSS Spacesuit Gravity (G) Metabolic Rate (kcal/hr) cooling system limit skin-contact temperature to the range of 10oC to 45oC (50oF to 113oF), and additional thermal mittens are used for grasping objects with temperatures that can range from - Apollo 1/6 235 (suited) o o o 118 C (-180 F) on the shadow side of an orbit to +113 C microgravity 151 (cabin) (+235oF) on the light side of an orbit. Gloves are the crew member’s interface with the equipment Skylab microgravity 238 and tools he or she uses. The EMU gloves, which connect to the Space Shuttle microgravity 197 arms at the wrist joints, have jointed fingers and jointed palms. Each glove also includes a pressure bladder, a restraint layer, and a protective thermal outer layer. To enhance tactility, fingertips are made from silicone rubber caps. The gloves present the most difficult engineering problem in spacesuit design. A dexterous The spacesuit has self-contained, integrated pressure and spacesuit glove that provides ideal finger motion and feedback O2 systems in a backpack-type PLSS that can be maintained on- has not yet been realized. orbit. The oxygen supply system includes reserve oxygen storage Throughout the EVA, monitoring of carbon-dioxide and equipment for controlling and maintaining the pressure. The concentration and other suit parameters occurs via telemetry to ventilation system and environmental gas-composition control the ground, with updates every two minutes. Carbon dioxide is system include CO2 and contaminant-removal units along with kept below 0.99 kPa (0.15 psi) and is absorbed by lithium- gas circulation control equipment. The spacesuit has no umbilical hydroxide canisters. Electrocar-diographic leads are worn to also lines. Oxygen, water supplies, pumps, and blowers are located in allow constant monitoring of heart rate and rhythm. For the cover of the rear hatch. sustenance, the crew member is provided with a food bar and up Adequate microclimate conditions in the suit are provided to 21 ounces of water in the EMU. by a closed-loop, regenerative life-support system. The suit’s The EMU, EVA support tools (i.e., foot restraints, thermal control system maintains the cosmonaut’s body handholds, and specialized tools), and EVA training are credited temperature and humidity level within acceptable limits and with the reduction that has occurred in Shuttle mission workload utilizes an efficient sublimating heat exchanger. The cosmonaut over the course of time. Most EVA training takes place wears the liquid-cooled garment described earlier (LCVG), underwater in the neutral buoyancy laboratory (NBL) at comprised of a network of plastic tubes, that allows the NASA’s Johnson Space Center in Houston, Texas. Training crew temperature to be maintained manually on a comfort basis or members extensively practice scheduled EVAs in the neutral automatically by the spacecraft temperature-regulation system. buoyancy setting to simulate weightlessness. The heat exchanger and LCVG provide a nominal thermal mode for sustained operation at practically any metabolic workload. Materials and colors which reflect strong solar radiation are used, and the spacesuit has layers of protection against extreme The Russian Spacesuit temperatures. The nonhermetically sealed outside layer is a The current spacesuit used for Mir Space Station EVAs is a protective vacuum insulator, while the hermetically sealed inside derivative of the semi-rigid suit used in the Salyut-Soyuz layer is a special rubber suit that retains the pressure. program. The Orlan suit has undergone continuous modification, In summary, the spacesuit’s designer, Guy Severin of and a fifth-generation model is currently used for EVA Svezda, lists the following seven attributes of the semi-rigid Orlan spacesuit (Severin, 1990): operations. Similar to the American EMU, the Orlan spacesuit has an integrated life-support system to enable EVA operations · Minimal overall dimensions of suit torso in a from Mir. As stated, the 100% oxygen spacesuit nominally pressurized state operates at 40.6 kPa (5.88 psi). Weighing ~105 kg (231 lbm)— · Rapid donning and doffing which does not include a fully charged PLSS—this is an · Easy handling capabilities and improved reliability of adjustable, universally sized suit with a metal upper torso and lines connecting the life-support system fabric arms and legs. Metal ball bearings and sizing adjustments · Reliability of the hatch sealing system are notable features. An advancement and difference from the · Suitability for crew members of different EMU is found in the Orlan’s rear hatch entry, which allows an anthropometric dimensions unassisted spacesuit entry that requires only two to three · Easy replacement of consumable elements minutes (Bluth and Helppie, 1987). · Easy maintainability through convenient access to units

Gravitational and Space Biology Bulletin 13(2), June 2000 41 ASTRONAUT PERFORMANCE FROM MIR TO MARS

Future Russian spacesuit research and development Both the EMU and the Orlan spacesuits meet these design activities are aimed toward improving suit performance requirements, although there is room for much improvement in characteristics (specifically mobility), extending spacesuit glove design and the matter of weight. Each suit has its own operating life, using microprocessors to control and monitor strengths and limitations (see Table 3). For example, the EMU spacesuit systems, and decreasing the payload weight that is provides better mobility than the Orlan, primarily because its delivered to orbit in the process of replenishing spacesuit con- lower operating pressure permits more joint and glove motion. sumables. Ideas to decrease payload weight include regenerating The incorporation of advanced materials into the EMU’s design

CO2 absorbers, removing heat without evaporative water loss, also extends its design life. However, when it comes to donning decreasing spacesuit O2 leak rates, and using advanced O2 and doffing, the Orlan design is clearly superior. Its rear hatch supplies. entry allows the crew member to don and doff the spacesuit unassisted. (Only in theory is this possible with the two-piece EMU.) The lower mass, hence lower weight, of the Orlan system PHILOSOPHICAL DIFFFERENCES UNDERLYING also offers a distinct advantage over the EMU. For space station DESIGN CHOICES operations in a weightless environment, designers tend to discard mass as a critical spacesuit design requirement, but for a future Many instructive design lessons emerge from comparing planetary spacesuit, a suit with low mass will most likely offer U.S. and Russian spacesuits. NASA has used a different the most promising design. spacesuit design for each of its main human spaceflight programs

(except Apollo/Skylab and Shuttle/ ISS), whereas the Russians have used essentially one spacesuit design that has evolved SPACESUITS FOR FUTURE MISSIONS throughout their program history. This difference is a matter of design choice—there is no right or wrong—and the U.S. and Planetary EVA and the extensive construction and Russian approaches to spacesuit design selection may reflect maintenance of future space stations will require increased levels underlying philosophical differences. of EVA capability. To meet a three-fold increase in the number of In NASA’s case, each human spaceflight program has EVAs needed for ISS assembly and a possible forty-fold increase offered many different industrial and academic partners new for planetary EVA (Figure 2) revolutionary spacesuit design opportunities to influence spacesuit design. In Russia, on the concepts should be considered. An advanced spacesuit might be other hand, one spacesuit designer provided the spacesuit, more like everyday clothing or something radically different, such which, once adopted, has been enhanced for various programs. as a pod with robotic actuators. No design rule dictates that NASA’s approach promotes creativity and cost savings, as, with future spacesuits must look like current ones, only that spacesuit each new program, designers with the most unique designs and requirements be met through innovative design. Ideally, the lowest bids compete for a contract. The Russians have advanced spacesuits will provide the crew member with a pursued their goal—to create a robust, reliable system—by protective, mobile, regenerable life-support system for use in relying on the master designer’s original design, which has been orbit and on planetary surfaces. Advanced spacesuits should tested over time and altered very little. (The NASA and Russian provide: space programs have followed similar design philosophies for · Working pressures for shirtsleeve mobility space vehicles, where NASA typically designs and builds a new craft for each major program and the Russians rely · Dexterous gloves or actuators more on mass production of similarly designed, · Longevity evolutionary spacecraft.) The need always drives the design requirement; and design · Easy maintenance requirements are met by a successful design. Both the EMU and · Adequate environmental protection Orlan spacesuits meet the crew member’s need for life support during EVA. Among the design requirements for a spacesuit are Mobile joint systems must allow for minimum energy · life support for the extravehicular crew expenditures during EVA tasks; gloves should be certified for member, including pressure, oxygen supply, long-duration use at high pressures; and improved technology and carbon dioxide and trace gas removal, materials should insure spacesuit durablity. Primary life-support humidity and temperature control, and systems should be regenerable, low-mass, and modular. A broad environmental protection; metabolic loading range between 63-625 kcal/hr (250-2500 Btu/hr) should be achieved with automatic thermal control · mobility and dexterity—especially in the systems; a modular, evolvable design is advantageous. gloves—for successfully accomplishing EVA Technological advances should lead to real-time environmental tasks; monitoring and innovative display and vision systems. It is clear · a system that is as light as possible; that a radical new approach to spacesuit design will best meet such future challenges as Martian EVAs likely to entail repelling · continuous operation during of the EVA down shear cliffs and traversing monumental canyons. excursion.

42 Gravitational and Space Biology Bulletin 13(2), June 2000 ASTRONAUT PERFORMANCE FROM MIR TO MARS

Future Space Walks suits that should be heeded. Recounting lunar astronauts’ Microgravity EVA has been admirably demon-strated. frequent falls, Jones and Schmitt (1992) suggested that While significant improvements are necessary for long-term space improvements in mobility and suit flexibility would have a station EVA, quantum improvements are required for planetary significant impact on astronaut productivity. They emphasized, EVA. To move about in microgravity, the crew member primarily however, that the greatest impact would come from uses the small musculature of the upper body, rather than the improvements both for increased manual dexterity and for large musculature of the lower body. Planetary EVA, however, reduced muscle fatigue and abrasion-induced damage to the hands. will dictate a true locomotion spacesuit, because the large muscles Noting that the fine dust particles of lunar regolith caused of the legs will be used for locomotion in the 3/8-g environment, problems with the Apollo suits, Jones and Schmitt (1992) also and the upper body muscles will be called upon for EVA tasks predicted that dust from lunar and Martian habitats would other than self-locomotion. Apollo 17 EVA astronaut Harrison present an obstacle to EVA performance on a continuous, daily Schmitt praised the Apollo spacesuits for working without a basis. It is clear from these comments that the design and serious malfunction for up to 22 hours of exposure to the lunar development of future planetary spacesuits will be challenging. environment, but he also made recommendations for future planetary space-

Table 3. Comparisons between the U.S. and Russian Space Suits (Asker, 1995)

NASA Space Shuttle Extravehicular Mobility Orlan-DMA Space Suit Unit (EMU)

Manufacturer United Technologies, HamiltonSundstrand, Zvezda Research, Development, and Windsor Locks, CT Production Enterprise Tomilio, Russia

Suit Operating Pressure 30 kPa (4.3 psi) differential 40 and 26 kPa (5.8 and 3.8 psi) differential

Nominal Maximum 7 hours 6 hours Mission Duration

Emergency Life Support 30 minutes 30 minutes Useful Life

Sizing · Modular assembly to 5 percentile female · One adjustable size with axial restraint to 95 percentile male system allowing on-orbit sizing · 11 suit assembly items · Two glove sizes

Construction of suit · Urethane-coated nylon pressure bladder · Semi-rigid with latex rubber dual Assembly · Polycarbonate helmet and visors pressure bladder in arms and legs · Ball-bearing joints · Dual-layer helmet · Liquid cooling/ventilation undergarment · Dual-seal bearings in shoulder and · Fiberglass hard upper torso wrist · Ortho fabric and aluminized mylar · Liquid cooling undergarment thermal/meteoroid garment · Rear-entry suit design

· On-orbit limb sizing

Construction of Life- · Closed-loop, pure oxygen generative · Closed-loop, pure oxygen generative Support System · 7 interchangeable subsystem modules · On-orbit servicing through rear entry · Expendables replaceable or rechargeable door on orbit · Redundant life-critical features

Donning 15 minutes (typically with assistance) Self-donning, rapid

Weight 117 kg (258 lb ) 105 kg (231 lb ) m m Design Life Up to 30 years with maintenance 4 years/10 missions

Gravitational and Space Biology Bulletin 13(2), June 2000 43 ASTRONAUT PERFORMANCE FROM MIR TO MARS

LOCOMOTION IN PARTIAL GRAVITY cm displacement. The anteroposterior flexion of the trunk reveals maximum backward flexion at the beginning of the Some Characteristics Associated with Walking support phase and maximum forward flexion toward the end of Basic hypotheses relating to human movement involve the support phase, resulting in small 1- to 2-cm deflections. notions about minimizing energy expenditure and forces. The functional significance of the determinants of gait is to minimize vertical and lateral oscillations of the center of gravity (CoG) In sum, the characteristics of walking described above are during walking, thus to minimize both energy expenditure and, seen to minimize oscillations of the CoG and optimize efficiency perhaps, the generation of muscular force. The design of future during locomotion, due to minimum energy expenditure. Many locomotion spacesuits ideally will incorporate what we know of the characteristics of gait absorb shock during a stride cycle, about the following six desirable characteristics of walking: which effectively reduces the force exerted on the ground and,

equally, the reactionary force on the skeletal system and the · Pelvic rotation whole human body. Recommendations based on the · Pelvic tilt understanding of gait determinants suggest that spacesuit design · Knee flexion during the stance phase should provide a waist bearing that permits pelvic rotation and · Heel strike and heel-off interactions with the knee tilt; a knee joint that enables flexion; an ankle joint for plantar · Trunk lateral flexion and dorsi-flexion; and a hip/waist/upper-body capability that · Trunk anteroposterior flexion accommodates trunk flexion. Pelvic rotation describes the pelvis rotating from side-to-side around the body’s longitudinal (vertical) axis for normal walking. During the leg’s swing phase, medial rotation at the weight- bearing (stance) hip advances the contralateral (swing-phase) hip (Figure 4). Pelvic rotation effectively increases leg length, thereby step length, and flattens out the arcuate trajectory of the CoG, reducing energy expenditure by insuring a smoother ride as the radii of the arcs of the hip increase. The pelvis is tilted downward about five degrees on the swing phase side. This occurs with pelvic adduction at the hip joint on the stance phase side.

Pelvic tilt further flattens the arcs of the hip, allowing for a smooth ride during walking. Figure 4. Pelvic Rotation during Walking. The pelvis is rotated from side-to-side about the longitudinal axis of Knee flexion occurs during the stance (support) phase of the body. walking. The knee is extended at heel strike, but then begins to flex. At heel-off, just prior to the middle of the support phase, the knee extends again. This extension-flexion-extension sequence reduces the excursion of the CoG’s arcuate trajectory and absorbs shock during a stride cycle. If the knee joint is absent, the travel of the CoG is not reduced, which is very costly in terms of energy expenditure.

Heel strike and heel-off interactions with the knee comprise the fourth characteristic of gait. At heel strike, the foot plantar flexes (rotates downward around an axis formed at heel contact), thus lowering the ankle as the foot makes full contact with the ground (Figure 5). A fused (immobile) ankle joint without plantar flexion would cause the CoG to rise as if the leg were a stilt. Ankle plantar flexion affects gait in a manner similar to ankle flexion—i.e., the trajectory of the CoG is reduced and shock absorption is noted at heel strike. The heel-off phase provides a horizontal CoG trajectory as the ankle rotates upwards around an axis formed at the ball of the foot.

Figure 5. Heel Strike and Heel-off. Top: Heel strike. The foot plantar flexes which lowers the ankle as the foot Trunk lateral and anteroposterior flexion make up the final contacts the ground. Bottom: Heel-off interactions with characteristics under discussion. The ipsilateral flexion of the the knee. Heel-off keeps the excursion of the center of vertebral column toward the stance phase side causes a 1- to 2- gravity to a minimum.

44 Gravitational and Space Biology Bulletin 13(2), June 2000 ASTRONAUT PERFORMANCE FROM MIR TO MARS

Human Performance in Partial-Gravity Environments

Quantifying partial-gravity performance allows for efficient spacesuit and life-support system designs. The three primary techniques to simulate partial gravity (before we make it back to the moon or Mars) are

· underwater immersion, · parabolic flight, · suspension.

Underwater Immersion. During tests, a neutrally buoyant subject is ballasted to simulate the desired partial gravity loading. For example, one-sixth of the subject’s body mass is added in ballast if a lunar simulation is desired. Water immersion offers the subject freedom from time constraints and freedom of movement, but the hydrodynamic drag is disadvantageous for movement studies.

Parabolic Flight. NASA KC-135 aircraft or Russian IL-76 aircraft are typically used to simulate partial gravity by flying Keplerian trajectories through the sky. This technique provides approximately 20, 30, and 40 seconds for microgravity, lunar gravity, and Martian gravity tests, respectively. Parabolic flight is the only way to effect true partial gravity on Earth, but experiments are expensive and of limited duration.

Suspension. Many partial-gravity suspension systems have been designed and used since the Apollo program. The cable suspension method typically uses vertical cables to suspend the major segments of the body and relieve some of the weight exerted by the subject on the ground, thus simulating partial gravity. Suspension systems often afford the most economical partial-gravity simulation technique, but limit freedom of movement.

Force traces help quantify the peak force exerted by a crew member during locomotion. These data pertain to spacesuit design, as well as to the human physiologic effects of musculoskeletal deconditioning during long-duration spaceflight. There is a significant reduction in peak force during locomotion in partial gravity and a general trend toward loping (between running and skipping) as gravity decreases from 1 g (Figure 6). Figure 7 shows actual data from the Apollo 11 lunar mission. Figure 6: A Comparison of Partial Gravity Locomotion Stepping frequency is displayed for the Apollo 11 data, with Earth Gravity. The data reveal a significant underwater-simulated lunar gravity data, and 1-g data. There is reduction of (P<0.001) in peak force, fmax, for a scatter in the Apollo data, but the simulated lunar stepping rates decrease in gravity level. There is a 50% reduction from are seen to correlate well with the actual Apollo data. The 1 g to Martian gravity (3/8 g) and a 74% reduction in stepping frequencies at 1 g are significantly higher than the lunar peak force from 1 g to lunar gravity (1/6 g). The contact stepping frequencies (P<0.05). Since the time available to apply time is the duration of the support foot’s contact with the muscular force to the ground during locomotion is constant ground (tc). The time for a single stride (tstride) increases across gravity levels, a reduction in metabolic costs for low as the gravity level decreases; thus, a decrease in stride gravity levels is anticipated (peak-force results reveal that less frequency (strides/min) is seen for a reduction in gravity muscular force is required for locomotion at reduced gravity level. A significant aerial time (ta—time between toe-off levels). The combination of decreases in stride frequency and and ground contact of the opposite foot), exists for constant values of contact time also partial gravity locomotion, whereas terrestrial locomotion elicits no significant aerial phase at this velocity.

Gravitational and Space Biology Bulletin 13(2), June 2000 45 ASTRONAUT PERFORMANCE FROM MIR TO MARS

telescope. He or she suits up in the airlock, assembles the Earth gravity necessary hand tools to carry by hand to the worksite (at this 3 Apollo 11 lunar data* point you may well ask, “What about construction equipment— Simulated lunar gravity bulldozers, loaders, cranes, etc.?”), leaves the lunar habitat 2.5 1 g through the airlock, and begins the day’s task. Whether driving or using self-locomotion to get to the site, the crew member 2 needs a light, mobile spacesuit and LSS. Once at the site, an initial survey of the lunar terrain requires agility, traction, tools, 1.5 Lunar g and possibly illumination. Before starting to assemble the telescope platform, the crew member probably has to move 1 some lunar regolith and flatten the desired plot. No doubt there is dust everywhere, fouling the spacesuit bearings and hampering 0.5 the rover’s machinery. Once the platform is assembled and leveled, work on the telescope begins. The telescope’s assembly

Stepping Frequency (steps/sec) 0 and adjustments require extreme finger dexterity. 0 0.5 1 1.5 2 2.5 Clearly, the simple task of deploying a telescope requires Velocity (m/s) an involved EVA. Planetary EVAs for building habitats, setting * Stone, R.W. Celobeka b Kosmos , 1971 * Stone, R.W. (1971) Man in Space. up laboratories, and conducting field science will be a great deal more complicated, demanding EVA systems and crew member Firgure 7. Stepping Frequency for Apollo 11 and skills that do not currently exist. Simulated Lunar Gravity. Stepping frequency for Whatever the EVA task may be, the crew member must terrestrial locomotion is also plotted. The Apollo data have adequate life support, protection from the environment, and simulated lunar data show a reduction in stepping and appropriate tools and equipment. In addition to meeting the frequency as compared to 1 g, especially for locomotion design requirements already mentioned, spacesuit design for our at velocities of 1.5 m/s and 2.3 m/s. (Stone, 1971) hypothetical planetary EVA must assure

· adequate mobility;

suggest an increase in aerial time for partial-gravity locomotion · natural, efficient locomotion; locomotion. A significantly extended aerial phase typifies loping, · correct balance and orientation; in which subjects essentially propel themselves into an aerial trajectory for a few hundred milliseconds during the stride · reasonable physical loads on the crew member, the (Newman et al., 1994). spacesuit, and the life-support system; Since the functional significance of the characteristics of · adequate lighting; gait is to minimize energy expenditures, bioenergetics (oxygen consumption) data drive the design requirements for planetary · adequate power; EVA life-support systems. Results show surprising information · gloves that support maximum manual dexterity. for partial-gravity locomotion. There is a well-documented

optimal cost of transport for terrestrial walking at the speed of 1 Again, such requirements will be met only through extensive m/s (Margaria, 1976). In terms of metabolic expenditure, it costs research and design efforts. Perhaps a model that incorporates about half of the amount of energy to walk 1.67 km (1 mile) that mechanical pressure rather than air pressure will provide the it costs to run 1.67 km. However, walking at 1 m/s is not the crew member with a light, form-fitting spacesuit. On the other optimal method of transporting one kg of body mass over one hand, if an optimal-locomotion spacesuit cannot be realized, the meter in partial gravity. Cost of transport for the lunar (1/6 g) concept of a full-body enclosure with manipulators may prove and Martian (3/8 g) environments decreases as speed increases, successful. At this early stage in the conceptual design of future suggesting that quicker locomotion is cheaper in terms of the spacesuits, the field is wide open and all designs and cost of transport. Results from underwater immersion and methodologies should be considered. suspension simulators indicate that above 1/2 g, walking has a

lower cost of transport than running, but running is cheaper than CONCLUSION walking from 1/4 g to 1/2 g (Farley and McMahon, 1992; Newman and Alexander, 1993). The successful design of future planetary spacesuits depends on providing improved mobility, improved glove A DAY IN THE LIFE OF A LUNAR CONSTRUC-TION performance, adequate operating pressures, improved radiation WORKER shielding, mass reductions, regenerable life-support systems, and improved human/machine interfaces. Locomotion spacesuits Try to imagine what a day in the life of a lunar should incorporate current research efforts, findings that pertain astronaut/construction worker might involve. One of the to the altered mechanics for locomotion in partial gravity, and simplest tasks confronting a crew member might be to set up a

46 Gravitational and Space Biology Bulletin 13(2), June 2000 ASTRONAUT PERFORMANCE FROM MIR TO MARS suggestions from past Apollo experience. Medical risks to crew members will be another driving force in planetary spacesuit Waligora, J., Horrigan, D. and Nicogossian, A. 1991. The design. Finally, the relationship between humans and machines is physiology of spacecraft and spacesuit atmosphere selection. still undefined in EVA operations, and further research could 8th IAA Man in Space Symposium. Acta Astronautica 23: lead to optimal mission planning with EVA crew members 171-77. assisted by robotics. Wilde, R. 1984. EMU—a human spacecraft. Proceedings of REFERENCES the 14th International Symposium on Space Technology and Science. Tokyo, Japan: Hamilton Standard, pp. 1565-76. Amir, A.R. and Newman, D.J. 2000. Research into the effects of astronaut motion on the spacecraft: A review. Acta

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Asker, J. 1995. U.S., Russian suits serve diverse EVA goals.

Aviation Week & Space Technology. 142(3):40-46.

Bluth, B. J. and Helppie, M. 1987. Soviet Space Station

Analogs (2nd Ed.). Report under National Aeronautics and

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Jones, E. and Schmitt, H. 1992. Pressure suit requirements for the moon and Mars EVA’s. Paper Number LA-UR-91-3083.

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48 Gravitational and Space Biology Bulletin 13(2), June 2000

BOLD ENDEAVORS: BEHAVIORAL LESSONS FROM POLAR AND SPACE EXPLORATION Jack W. Stuster Anacapa Sciences, Inc., Santa Barbara, CA

ABSTRACT Material in this article was drawn from several chapters of the author’s book, Bold Endeavors: Lessons from Polar and Space Anecdotal comparisons frequently are made between Exploration. (Annapolis, MD: Naval Institute Press. 1996). expeditions of the past and space missions of the future. the crew gradually became afflicted with a strange and persistent Spacecraft are far more complex than sailing ships, but melancholy. As the weeks blended one into another, the from a psychological perspective, the differences are few condition deepened into depression and then despair. between confinement in a small wooden ship locked in the Eventually, crew members lost almost all motivation and found polar ice cap and confinement in a small high-technology it difficult to concentrate or even to eat. One man weakened and ship hurtling through interplanetary space. This paper died of a heart ailment that Cook believed was caused, at least in discusses some of the behavioral lessons that can be part, by his terror of the darkness. Another crewman became learned from previous expeditions and applied to facilitate obsessed with the notion that others intended to kill him; when human adjustment and performance during future space he slept, he squeezed himself into a small recess in the ship so expeditions of long duration. that he could not easily be found. Yet another man succumbed to hysteria that rendered him temporarily deaf and unable to speak. Additional members of the crew were disturbed in other ways. It In many ways, the Belgian Antarctic Expedition of 1898 was to this dismal condition that Roald Amundsen referred when to 1899 was a precursor of things to come. It was the first he later wrote, “Insanity and disease stalked the decks of the expedition to camp, although briefly, on the Antarctic continent, Belgica that winter.” and the first to spend an entire year locked in its icy embrace. Dr. Cook believed the malady was caused more by lack of Most important was the international composition of its crew— light than by the scurvy they were experiencing. Whatever the eighteen men isolated together in one of the most challenging actual cause, it is clear that the problem was also psychological. environments on Earth. In an era when expeditions were The dreaded polar night is not really that dreadful¾it has been expressions of nationalistic tendencies, the cosmopolitan endured without ill effects by many explorers and countless makeup of the Belgian Antarctic Expedition was truly modern, indigenous inhabitants of the Arctic regions¾but it took a consisting of nine Belgians, six Norwegians, two Poles, a terrible toll on the crew of the Belgica. The men suffered from Romanian, and an American, the ship’s physician, Dr. Frederick poor circulation, heart troubles, and impaired digestion. Their A. Cook. diet was low in fiber and probably certain vitamins. Although Dr. Cook had responded to a newspaper advertisement vitamins had not been discovered yet, Dr. Cook believed that the that was placed when the expedition’s original physician backed diet lacked some important element. He attempted to remedy the out only a few days before the ship sailed. Commandant Adrien condition by encouraging the men to eat fresh penguin meat, but de Gerlache, organizer of the expedition, selected Cook on the many found it unpalatable. He also prescribed an exercise basis of his previous Arctic experience. In October 1897, Cook program to counter growing symptoms of insanity among the joined the expedition in the roadstead of Rio de Janeiro, Brazil. crew, but walks on the ice devolved into a circular path around The Belgica, the expedition’s ship, arrived in the Antarctic the ship that came to be known as the “madhouse promenade.” during January 1898. Though late in the season, the crew was Cook’s journal entries reflect the depression into which this able to make several landings to collect geological specimens, small society had fallen. The following is an example: lichens, moss, and insects. They conducted more scientific work The darkness grows daily a little deeper, and than had any previous Antarctic expedition, but they probably the night soaks hourly a little more color spent too much time on shore. In March, the ship became from our blood. Our gait is now careless, the trapped in the frozen Bellinghausen Sea and, locked in by pack step non-elastic, the foothold uncertain . . . . ice, drifted there for more than a year. The crew was not fully Most of us in the cabin have grown prepared for the experience. decidedly gray within two months, though The medical officers of polar expeditions, and later at few are over thirty. Our faces are drawn, and Antarctic research stations, usually experienced considerable there is an absence of jest and cheer and frustration because they found few professional duties to hope in our make-up which, in itself, is one perform. However, this was not to be the case for Dr. Cook. The of the saddest incidents in our existence. . . . thirty-two-year-old physician was occupied during the The novelty of life has been worn out. . . . remainder of the expedition with a problem that started when the We miss the usual poetry and adventure of ship became locked in the ice and grew increasingly acute home on winter nights. We miss the flushed throughout the long winter night: almost every member of maidens, the jingling bells, the spirited horses, the inns, the crackling blaze of the

country fire. We miss much of life which makes it worth the trouble of existence. (Cook [1900] 1980, 319)

Gravitational and Space Biology Bulletin 13(2), June 2000 49 BEHAVIORAL LESSONS FROM POLAR AND SPACE EXPLORATION

In desperation, Cook devised a method that he called the Lieutenant George Washington De Long, that sailed onboard “baking treatment,” in which the most seriously ill sat with the Jeannette out of San Francisco Bay on 8 July 1879. their bodies exposed to the warm glow of the ship’s stove for Telegraph Hill and the Embarcadero swarmed with well- an hour each day. This therapy, combined with enforced wishers, and more were afloat on the yachts, tugs, and portions of fresh penguin meat, seemed to help, but Cook launches that filled the bay as the barque-rigged coal burner observed that “surely one of the most important things was to steamed through the Golden Gate and set a course for the raise the patients’ hopes and instill a spirit of good humor” Arctic. Two months later, the Jeannette was beset by ice (Cook [1900] 1980). This he did consciously and persistently and trapped, as the Erebus and the Terror and countless throughout the remainder of the expedition. other ships had been over the centuries. The crew stayed with The crew’s spirits began to improve in the spring, but the the ship for nearly two years until she was crushed, then ice floe that trapped the Belgica gave no indication that it made their way to shore and through the Siberian wilderness. would ever break up. It was necessary to escape the Antarctic Only thirteen men survived the ordeal. because, as each man knew, to stay another year would be Three years after De Long and his party abandoned their fatal. Laboring with large ice saws, axes, and explosives, the ship, pieces of the Jeannette’s wreckage were found on the crew eventually blasted the ship free, but the Belgica did not southwest coast of Greenland, thousands of miles from where, reach open water for another month. In November 1899, the crushed by the ice, she had sunk. This information contributed ship arrived in Europe, where crew members were greeted as if to a theory that the far north was covered with ice, and that they had been to the moon and back. this ice cap moved in a westerly direction across the Arctic. Frederick Cook described life onboard the Belgica as a Dr. Fridtjof Nansen, a young scientist, outdoorsman, and “hellish existence,” but he rose to the occasion and is credited curator at Christiania University, developed a bold plan to with saving the expedition from psychological disaster. test the hypothesis. Nansen recently had returned from The cause of the malady that affected the Belgian making the first successful crossing of the Greenland plateau, Antarctic Expedition remains a mystery. The diet and lack of a remarkable accomplishment that would prove to be only a sunlight could have caused anemia and depression, as Cook prelude to one of the world’s boldest endeavors. surmised, or perhaps the crew suffered from a shared The genius of Nansen’s plan was to build a special ship hysterical reaction or some other psychological group for the expedition instead of converting an existing vessel. phenomenon. Simple boredom and depression may have This ship would be designed to rise up out of the ice as the affected all the members of the party and driven some beyond floes pressed against her hull, rather than to resist the full the limits of their endurance. Like most complex phenomena, force of the pressure. Critics scoffed at Nansen for his theory the crew’s experience was probably caused by a combination and predicted that his expedition would end in failure. of factors. Certainly it was of considerable relevance to plans However, he persevered and obtained an initial grant from the for future long-duration expeditions. Norwegian government. There were cost overruns, just as It is increasingly difficult for people to imagine what life there are in modern programs, when the design was changed to was like in the closing years of the 19th century. Today we increase the ship’s capacity and the margin for crew safety. take for granted the air transportation network and wireless The Fram—the name means “onward” in Norwegian— communications that cover the globe, but much of Earth was was heavily built, but constructed with no edges below the still inaccessible in the 1800s. The polar regions were among water line that might give ice a purchase on the ship. The keel the most consuming mysteries of the natural world yet to be was recessed, and all fittings could be removed to create a explained. No one knew what conditions to expect—whether smooth and rounded profile. Departing the beautiful Hanseatic land, ice, or sea covered the poles. port of Bergen on 1 July 1893, she sailed north and east, Many efforts had been made to reach the farthest north. crossed the Barents and Kara seas, and skirted the northern Most notably, in 1845, the British Admiralty dispatched Sir coast of Siberia. Three months later, at a point closer to John Franklin to locate and navigate the Northwest Passage, Alaska than Norway, she headed into the ice pack, where she and it was assumed that Franklin and his carefully selected was intentionally locked in the ice just north of 78º latitude. party would succeed where others had failed. Two ships, the As the floes encroached and the forces on the Fram’s hull Erebus and the Terror, were loaded with supplies to increased, the sturdy little ship rose out of the ice and support a crew of 129 for four years. After departing England, remained cradled above the pressure ridges, drifting with the they hailed a group of whalers off Greenland on their course ice pack across the top of the world for nearly three years. north, then vanished without a trace. Nansen’s design worked according to plan, and the theory of During most of the next two decades, polar exp loration polar drift was confirmed. When it appeared that the Fram’s was dedicated to finding and, perhaps, rescuing any survivors course would take her no farther north across the polar ice of the Franklin Expedition. Among the attempts was an cap, Nansen selected Hjalmar Johansen to accompany him on American expedition, thirty-one men commanded by young a dash to the pole with kayaks, sledges, and dogs.

50 Gravitational and Space Biology Bulletin 13(2), June 2000 BEHAVIORAL LESSONS FROM POLAR AND SPACE EXPLORATION

effects, and pressed on eagerly in their kayaks. Walruses attacked them, and at one point the explorers nearly lost their kayaks and equipment when their small craft drifted away from the ice flow onto which they had climbed. A month after departing their hut, they encountered the English explorer Frederick Jackson in one of history’s most remarkable chance meetings. They stayed with the Jackson Expedition nearly two months, waiting for its relief ship. The day that Nansen and Johansen set foot on Norwegian soil, the Fram broke free from the ice on the opposite side of the Arctic and headed for Spitzbergen and then Tromsö, Norway. Here her crew was reunited after seventeen months of separation. A few weeks later, on 9 September 1896, the Fram steamed up Christiania Fjord three years and three months following her departure. Nansen and his crew were greeted as if they had just returned from another planet. During his isolation and confinement, Fridtjof Nansen experienced a lethargy that was similar to that described a few years later by Dr. Cook of the Belgica. Nansen described his feelings in his journal:

My mind is confused; the whole thing has got into a tangle; I am a riddle to myself. I am worn out, and yet I do not feel any special tiredness. Is it because I sat up reading last night? Everything around us is

emptiness, and my brain is a blank. I look at the Figure 1. “Fram in the Ice” by William Gilkerson, 1894. home pictures and I am moved by them in a curious, (Courtesy of the author) dull way; I look into the future, and feel as if it does

not much matter to me whether I get home in the autumn of this year or next. So long as I get home in By 7 April 1895, Nansen and Johansen were making only the end, a year or two seem almost nothing. I have a mile headway each day over rough ice, so they turned back at never thought this before. I have no inclination to 86º13' north latitude—160 miles farther north than any explorer read, nor to draw, nor to do anything else whatever. had previously achieved. Navigating with erroneous charts and The only thing that helps me is writing, trying to caught by an early winter, they made it to Franz Josef Land and express myself on these pages, and then looking at built a small hut out of stones and walrus hides, in which they myself, as it were, from the outside. (Nansen 1897, would live in complete isolation and confinement. Life in the six- vol. 1, 372-73) by-ten-foot hut was unpleasant in many ways, not the least being the decline in personal hygiene they endured because they Thanks to better equipment, procedures, leadership, and, most lacked supplies, including fuel for melting snow. For the entire important, the extensive planning that preceded the Norwegian nine-month Arctic winter, they cooked their food and Polar Expedition, the malaise onboard the Fram was short-lived illuminated their world with the small flame of a blubber lamp, and more effectively contained than that suffered by the crew of coating the hut and themselves with soot and grease. The best the Belgica. How did the Norwegian Polar Expedition endure way they found to clean themselves, scraping their skin with more than three years with scarcely a problem, while the Belgian their knife blades, produced usable quantities of fuel that they Antarctic Expedition nearly collapsed within its first year? recycled in their lamp. Conditions were about as bad as humans The primary characteristic that distinguished Nansen from can reasonably endure. Nansen and Johansen’s dreams were most other polar explorers was that he approached all aspects of filled with Turkish baths and visits to clothing stores. expedition planning with scientific precision. He started by The two explorers lived together as one might imagine reading accounts of previous expeditions to learn from the Neolithic hunters who had ventured too far and become stranded experiences of his predecessors. Nansen remarked in his diary by an early winter storm; but Nansen and Johansen survived the that, to his surprise, most of the problems confronting him experience. They suffered from the mind-numbing sameness of already had been addressed and, in many instances, solved by their days and the other health-threatening conditions, but previous explorers: wear appropriate clothing, pay special emerged from their den early in the spring of 1896 to expertly attention to the food, select crew members who can get along, perform all of the technical tasks necessary to fight their way and keep the crew busy and entertained. Nansen developed through pack ice to the safety and comforts of civilization. They special high-calorie rations and systematically tested every item survived the extreme austerity of their life with no apparent ill of food; he developed and evaluated sledges, harnesses,

Gravitational and Space Biology Bulletin 13(2), June 2000 51 BEHAVIORAL LESSONS FROM POLAR AND SPACE EXPLORATION protective clothing, and other equipment; and he invented solutions to equipment problems that still are used by polar travelers. He evenequipped his ship with wind-powered electric lights to illuminate the winter darkness at a time when electricity was still a novelty, and he fostered group solidarity with an egalitarian approach to his crew during an era when expeditions were managed autocratically. Modern exploration really began with Fridtjof Nansen and his Norwegian Polar Expedition. All who came after him benefitted immensely from his experience, and his experience is relevant to the full range of behavioral issues confronting expedition teams of the future. The Norwegian Polar Expedition provides an appropriate Figure 2. Models of the Nina, Pinta, and Santa Maria in model for modern explorers in many ways. Nansen’s systematic Front of Launch Pad 39A. Like NASA, Columbus believed in triple redundancy. With fewer than three hulls the simulation, testing, and evaluation of every item of equipment expedition might not have survived, as the Santa Maria and his meticulous attention to every detail and possible went aground on Christmas Day, 1492. (Courtesy of contingency set him apart from all previous and most NASA) subsequent explorers. But, most important, Nansen recognized that the physical and psychological well-being of his crew could make the difference between success and failure. Accordingly, he BEHAVIORAL THEMES provided a well-designed habitat, insightful procedures, and exceptional leadership to a qualified and compatible crew. “The I began a chronological review of past expeditions with human factor is three quarters of any expedition,” wrote Roald accounts of Columbus’s first voyage to the New World in 1492. Amundsen, the most successful of all explorers. Before Although the outward-bound trip for Columbus's three small ships took only thirty-three days and the total voyage lasted Amundsen, Nansen knew that human factors were the critical about seven months, accounts of this expedition have component in any expedition; in Nansen’s words, “It is the man considerable relevance. Columbus faced many of the same that matters.” problems, including strong-willed and independent subordinates, Despite superficial similarities to other space missions and that will confront leaders of future expeditions. My review also Earth-bound analogues, lunar and Martian missions—involving included accounts of Charles Darwin’s famous 1831–36 voyage extended durations and astronomical distances—will be far more onboard the Beagle, of commercial whaling and sealing voyages, difficult and dangerous. Crowded conditions, logistics and and of more recent adventures (e.g., Thor Heyerdahl’s Kon-Tiki equipment problems, radiation concerns, communication lag and The Ra Expedition). Although my range was broad, times, workloads, language and cultural differences, and a variety late-nineteenth- and twentieth-century accounts of polar of other issues will conspire to impair the performance and expeditions predominate. Notable among these are Fridtjof affect the behavior of crew personnel. Above all stressors, Nansen's Norwegian Polar Expedition (1893–97); the Belgian however, the long durations of missions will impose the greatest Antarctic Expedition (1898–99); the Amundsen and Scott race burdens and extract the most severe tolls on the humans to the South Pole (1910–12); Ernest Shackleton’s British involved. On long-duration space missions, time is likely to be Trans-Antarctic Expedition (1914–16); Admiral Richard E. the factor that will compound all issues, however trivial, into Byrd’s two expeditions to Antarctica (1928–30 and 1933–35); serious problems. and the International Biomedical Expedition to the Antarctic, or IBEA (1980–81). I also studied other examples of human Anecdotal comparisons frequently are made between experience characterized by isolation and confinement, including future space missions and expeditions of the past. From an such underwater habitats as Sealab and Tektite; offshore oil engineering perspective, spacecraft are far more complex than platforms; saturation chambers; submarines; Skylab; and sailing ships, and one of the factors that drives spacecraft remote-duty military and scientific environments. Recently, I complexity is the requirement to support the crew in the hostile completed an analysis of diaries maintained by the leaders and environment of space. The technological differences are physicians at French remote-duty stations, providing the first significant. From a behavioral or psychological perspective, quantitative data on the relative importance of behavioral issues. however, the differences between confinement in a small wooden My research methods have resulted in an alternative to the ship locked in the polar icecap and confinement in a small, traditional behavioral science perspective on life in isolation and high-technology ship hurtling through interplanetary space are confinement. This alternative perspective places new emphasis probably few. on the many examples in which humans have operated successfully for long durations despite their austere, isolated, and confined conditions. The well-known disasters are instructive, because they remind us of the need to be careful in

52 Gravitational and Space Biology Bulletin 13(2), June 2000 BEHAVIORAL LESSONS FROM POLAR AND SPACE EXPLORATION the design of habitats, equipment, and procedures and in the Remaining comments address the most salient category, “Group selection of personnel for special duty. The successes, however, Interaction.” are perhaps more instructive, providing considerable Mark Twain said that the best way to learn if you like encouragement to those who will be called upon to endure the someone is to travel with that person. However, the crews of inevitable stressors associated with space station missions and future space expeditions will experience interpersonal problems life on future lunar bases and interplanetary spacecraft. even if friendship and compatibility have been established The main themes to emerge from my research are as through years of selection, simulation, and training together. follows: While individuals cause some difficulties, most interpersonal

problems within isolated and confined groups are rather the · There are highly predictable behavioral inevitable result of fundamental forces and processes that are responses to isolation and confinement. characteristic of the experience. Sustained, close personal contact · Minor interpersonal and psychological can be extremely stressful, and interpersonal problems are problems are common, but serious problems exacerbated by additional sources of stress, such as danger, time are avoidable if proper precautions are taken. pressure, equipment malfunctions, and heavy workloads (or, · Future long-duration space expeditions will conversely, boredom). This stress is cumulative, and behavioral more closely resemble sea voyages than the consequences are likely if there is no way to eliminate its test flights that have served as models up to source—for example, by removing oneself from the group now. temporarily. But, as the physician of the Belgian Antarctic · Valuable lessons concerning the design and Expedition described in the following diary entry, it is conduct of future expeditions can be learned impossible to get away from one’s comrades when living in from studying the experiences of remote-duty isolation and confinement: personnel and previous explorers. · Humans can endure almost anything. 20 May 1898: I do not mean to say that we are

more discontented than other men in similar Twenty-two categories of behavioral issues have emerged conditions. This part of the life of polar from my research. All of these issues are involved, to varying explorers is usually suppressed in the degrees, in an individual’s adjustment to living and working in narratives. An almost monotonous discontent isolation and confinement. They are listed below in their order of occurs in every expedition through the polar salience, as determined by the content analysis of remote-duty night. It is natural that this should be so, for diaries (Stuster, Bachelard and Suedfeld, 1999): when men are compelled to see one another’s Group Interaction faces, encounter the few good and the many Outside Communications bad traits of character for weeks, months, and Workload years, without any outer influence to direct the Recreation & Leisure mind, they are apt to remember only the rough Medical Support edges which rub up against their own bumps of Adjustment misconduct. If we could only get away from Leadership each other for a few hours at a time, we might Events learn to see a new side and take a fresh interest Food Preparation in our comrades; but this is not possible. The Organization & Management truth is, that we are at this moment as tired of Equipment each other’s company as we are of the cold Sleep monotony of the black night and of the Safety unpalatable sameness of our food. Now and Personnel Selection then we experience affectionate moody spells Waste Management and then we try to inspire each other with a Internal Communications sort of superficial effervescence of good cheer, Exercise but such moods are short-lived. Physically, Habitat Aesthetics mentally, and perhaps morally, then, we are Hygiene depressed, and from my past experience in the Personal Hygiene Arctic I know that this depression will increase Privacy/Personal Space with the advance of the night, and far into the Clothing increasing dawn of next summer. (Cook 1980

[1900], 290-91) Recommendations range from special theme dinners—to promote group solidarity and help mark the passage of time—to Imagine living in a medium-sized motor home, locked in private quarters designed to mitigate the cumulative stress that with five other adults for a period of three years. Socially, this results from the unrelenting proximity of one’s comrades. situation approximates a mission to Mars. The crew will be excited following departure from Earth orbit and extremely busy

Gravitational and Space Biology Bulletin 13(2), June 2000 53 BEHAVIORAL LESSONS FROM POLAR AND SPACE EXPLORATION with important technical tasks, contingency planning, and mission-abort rehearsal activities. But a change will occur as the excitement dissipates and the days begin to blend into weeks, then months—as the crew makes the transition to the cruise phase of the voyage. Each crew member’s repertoire of jokes, anecdotes, personal experiences, and opinions will become well known to the other members of the tiny, closed society (if this has not already occurred during years of premission training and simulations). Nothing that anyone says or does will seem new, and previously innocuous mannerisms will be magnified into intolerable flaws as crew members become increasingly weary of each other. The lavatory and the small compartments that serve as private sleep chambers offer the only escapes from others. Interpersonal friction and overt conflicts among crew members are the inevitable consequences of these conditions. The stresses associated with isolation and confinement consistently result in minor interpersonal problems; sometimes Figure 3. Card Games During Richard E. Byrd’s First major conflicts occur, but they are rare. Typically, exaggeration Expedition to Antarctica Helped the Men Pass the Long of trivial issues causes most of the interpersonal conflicts that Winter Night. (Courtesy of the National Archives) occur within isolated and confined groups—issues that under normal conditions would be considered inconsequential. The most trivial of issues are predictably exaggerated beyond reasonable proportions by the relentless proximity of comrades and by the other stresses of isolated and confined living that On the twentieth day of the seventy-one-day motorized accumulate over time. Dr. Desmond Lugg’s final, predeparture traverse that began near the Dumont d’Urville station, one words to Australian Antarctic personnel concern what he has member of the expedition had to be evacuated for psychological named “The Rule of 10”: that is, when one is isolated, the reasons. The others endured the entire mission but returned from strength of one’s initial reaction—be it to someone within the the traverse “humorless, tired, despondent, and resentful.” None group or to a communication from the outside—should be of the participants found their Antarctic experience enjoyable, divided by ten to achieve the appropriate measure before not due to climate or hardships but to the “inconsiderate and responding. selfish behavior” of colleagues. Most of the interpersonal An account of the International Biomedical Expedition to problems were precipitated by disagreements over the the Antarctic (IBEA), written in 1988 by Jean Rivolier and his performance of necessary communal work and camp chores. colleagues, provides the most relevant examples of interpersonal These trivial issues were aggravated by underlying rivalries and problems. The IBEA, composed of a total of twelve scientists cultural and language differences among members of the party. from five nations, was conducted, in part, to obtain information Despite the efforts of the organizers, the group was fragmented about group interaction that might be useful to future space and lacked a unifying spirit or sense of mission. Fortunately, no missions. This objective was achieved; the interpersonal serious emergency occurred that would have required a problems experienced during the IBEA are extremely relevant to coordinated response. plans for future expeditions. Rivolier et al. describe the If trivial issues are inevitably, sometimes dangerously, problems: blown out of proportion, it seems clear that a way to minimize the potential for this phenomenon would be to eliminate, to the There were times such as at the onset of the extent possible, differences among the members of an expedition. laboratory programme in Sydney and at the In this regard, it is important to note that the most successful arrival of the group in Antarctica when the (i.e., remarkable) expeditions have been conducted by relatively group worked with a will as a team to unpack homogeneous groups or groups that have been organized and test their gear. But the harmony was specifically on the basis of compatibility. The most salient short-lived. Individuals asserted themselves. examples are Fridtjof Nansen’s group of thirteen Norwegians They competed with each other for status and who sailed onboard the Fram (Norwegian Polar Expedition, responsibility, and they drew apart in their 1893–96), and the twenty-seven men carefully selected by national groups. Occasionally they regrouped Ernest Shackleton to conduct an ambitious expedition to according to their antipathy to particular Antarctica onboard the Endurance (Imperial Trans-Antarctic experimenters, and even less occasionally Expedition, 1914–15). The Fram’s crew endured three years of they forgot their differences to enjoy each isolation and confinement and, in the process, reached what was other’s company. (1988, 91) then the point farthest north achieved by humans, an accomplishment of such magnitude at the time that modern readers might find it difficult to comprehend. In contrast, the

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Endurance never even reached Antarctica; but the performance this station would you choose first?” A fourth variable, of Shackleton’s crew in surviving the loss of their ship may have friendship, was included in the analysis to serve as a control. been an even greater achievement than that of the Norwegians. It Table 1 presents the three behavioral clusters and the control is true that both of these exemplary expeditions experienced variable in rank order of importance, as indicated by the some interpersonal problems, but not nearly to the extent of magnitudes of the correlations with the criterion. Civilians judged contemporary expeditions composed of heterogeneous crews. social compatibility to be the most important cluster of traits, It is not feasible to select a homogeneous crew for future whereas military personnel favored emotional stability. Social space expeditions because of the social and economic realities of compatibility refers to an individual’s ability to get along with these endeavors. International cooperation will be necessary to others, a difficult process for some in the tension-filled finance such large-scale undertakings as lunar bases and environment of a remote-duty station. Similarly, emotional interplanetary voyages. Thus, many future space crews will be stability refers to an individual’s capacity for avoiding extreme composed of individuals from different countries and cultures. In moods and behavior. It is essential to note that both groups short, it appears inevitable that cultural differences, such as found personality traits, rather than task performance, the most those that contributed to divisiveness during the Belgian important factors determining the kind of individual with whom Antarctic Expedition and the IBEA, will be a component in experienced personnel would want to share another year in future space expeditions. What can be done to mitigate the isolation and confinement. These results are as statistically and disruptive effects of these differences? intuitively valid today as they were when the studies were It would be prudent to develop countermeasures to conducted, and they could be applied to the development of minimize the possibility of conflict in crews composed of personnel selection criteria for other remote-duty environments, individuals of different genders, technical specialties, ages, and such as future long-duration space expeditions. cultural and national backgrounds. Personnel selection The following is a list of personal characteristics required procedures, training programs, formal policies, and informal for successful adaptation to isolation and confinement. It is practices and customs could greatly reduce the potential for based on the Navy research and on my review of original and serious interpersonal problems. The ideal personnel selection secondary sources concerning expeditions and voyages of system would identify those candidates who are both willing and discovery. capable of working with others under special conditions, and it would actually select crews, at least in part, on the basis of Likability specific intracrew compatibilities. Emotional control An extensive program of behavioral research at early U.S. Patience Antarctic stations was precipitated by a severe psychosis that Tolerance emerged among the Navy crew that was preparing a base for the Self-confidence (without egotism or arrogance) International Geophysical Year (IGY) in 1957. The research, largely conducted by Eric Gunderson and Paul Nelson, involved A team approach (willingness to subordinate one’s several hundred winter-over personnel and the identification of interest to that of the group) three clusters of behavioral traits that were highly correlated Sense of humor with effective performance at Antarctic stations. Gunderson Social resourcefulness (easily entertained) labeled the clusters (1) emotional stability, (2) task performance, Technical competence and (3) social compatibility. Emotional stability involves an individual’s ability to maintain control of his or her emotions, Participants in future long-duration expeditions should despite the stresses of isolated and confined living; “calm” and receive instruction in the behavioral and psycho-logical problems “even-tempered” are the ideal characteristics. Task that can occur during an expedition and in techniques to help deal performance refers to both task motivation and proficiency; with circumstances as they arise. The astronauts who returned “industrious” and “hard-working” describe the ideal traits in this category. Social compatibility includes a number of personal characteristics, such as likability, cheerfulness, and consideration for others; “friendly” and “popular” are the ideal characteristics. Table 1. Relative Importance of Behavioral Traits to Navy psychologists and psychiatrists have used these categories Successful Performance at U.S. Antarctic Stations for the past three decades to guide the screening of volunteers for Antarctic duty. Order Navy Personnel Civilian Personnel

Gunderson and his colleagues at the Naval Health Research Emotional stability Center estimated the relative importance of the three behavioral 1 Social compatibility clusters to overall performance at U.S. Antarctic stations, as 2 Task performance Emotional stability perceived by Navy and civilian winter-over personnel (Doll and 3 Social compatibility Task performance Gunderson, 1970; Gunderson, 1973b). Crew ratings of their 4 Friendship Friendship colleagues on the three behavioral traits were correlated with responses to a criterion item: “If you were given the task of selecting men to winter over at a small station, which men from

Gravitational and Space Biology Bulletin 13(2), June 2000 55 BEHAVIORAL LESSONS FROM POLAR AND SPACE EXPLORATION from Mir recognized this requirement, and they convinced their inordinate incidence of these problems is a normal consequence colleagues that long-duration isolation and confinement is of living in close proximity to others with no opportunity for qualitatively different from previous experiences, such as shuttle variety or escape. Interpersonal problems are certainly common, missions. As a consequence, a training program has been but serious problems are not inevitable, especially if the developed and pilot-tested with a group of twelve astronauts individuals are particularly compatible or if their solidarity is who are members of NASA’s Expedition Corps, astronauts who essential to their survival. For example, Lansing ([1959] 1994) are candidates for missions to the International Space Station. writes of Shackleton and the crew of the Endurance adrift on The two-day training program covers a broad range of issues and their ice floe: includes specific examples of the habitability and behavioral principles that have been identified in the analogue and It was remarkable that there were not more experimental literature. The training program also offers fairly cases of friction among the men, especially simple guidelines, such as the following: after the Antarctic night set in. The gathering darkness and the unpredictable weather limited · Avoid controversial subjects. their activities to an ever-constricting area around the ship. There was very little to · Consider the possible consequences before you say occupy them, and they were in closer contact or do something. with one another than ever. But instead of · Do more than your share of communal tasks. getting on each other’s nerves, the entire party seemed to become more close knit. (42) · Be considerate; more than that, try to avoid being annoying in any way. Individual compatibility and recognition of the need to maintain solidarity are among the ingredients of a successful · Consciously attempt to be cheerful and supportive of long-duration expedition. Perhaps it was one or both of these your teammates. factors that permitted Fridtjof Nansen and Hjalmar Johansen to · Be polite and respectful. endure nine months of confinement together in a crude Arctic hut without a single argument:

A particularly divisive source of interpersonal problems Our spirits were good the whole time; we occurs when the normal tendency for subgroups to form looked serenely towards the future, and escalates into the development of cliques. Although the tendency rejoiced in the thought of all the delights it had for subgroups to form is unavoidable, the environment should be in store for us. We did not even have recourse structured to encourage maximum communication across to quarrelling to while away the time. (Nansen subgroups to offset, to some extent, the increased 1897, vol. 2, 464) communication among members within subgroups. Subgroups serve as coping mechanisms for some individuals, but they can After their return to Norway, Johansen was asked how be disruptive and dangerous, because one person (or more) they had gotten along during the winter, and whether they had inevitably is excluded. quarreled. Reporters were as eager for controversy 100 years ago Meals offer an opportunity for the type of communication as they are now and they recognized it would be a severe test for that will help to mitigate the tendency for subgroup formation two men to live so long together in perfect isolation. Johansen among members of an isolated, confined crew. Eating together as replied, “Oh no, we didn’t quarrel; the only thing was that I had a group is a natural activity that most people seem to enjoy; the the bad habit of snoring in my sleep, and then Nansen used to benefits to group solidarity of eating together are so well known kick me in the back.” He would shake himself a little then sleep as to be a behavioral cliché. The requirement for daily nutrition calmly. Nansen was shocked when he read Johansen’s comment and the apparent human tendency to find some pleasure in in a newspaper. Nansen admitted to giving Johansen many a dining together offer valuable opportunities to encourage well-meant kick, but it was a surprise to learn so long afterward interpersonal communication that will foster group solidarity that Johansen had awakened sufficiently to realize that he had and counter the potentially negative effects of subgroup been kicked. formation. Some crew members are bound to find reasons to eat by themselves and withdraw from the group in other ways. It is REFERENCES important, however, that the design of equipment and Amundsen, R. [1912] 1976. The South Pole: An Account of procedures encourages eating together at least once each day, as the Antarctic Expedition in the Fram. London: John well as at frequent special dinners (e.g., theme dinners and Murray,. celebrations of holidays and mission milestones).

Cook, F. A. [1900] 1980. Through the First Antarctic Night A FINAL NOTE 1898-1899: A Narrative of the Voyage of the Belgica The point is made in the preceding discussion that among Newly Discovered Lands and over an Unknown interpersonal problems are inevitable among individuals living in Sea about the South Pole. Montreal: McGill-Queen’s isolation and confinement for long periods, and that the University Press.

56 Gravitational and Space Biology Bulletin 13(2), June 2000 BEHAVIORAL LESSONS FROM POLAR AND SPACE EXPLORATION

Doll, R.E. and Gunderson, E.K.E. 1970. The relative importance of selected behavioral characteristics of group members in an extreme environment. Journal of Psychology 75: 231-37.

Gunderson, E.K.E. 1966. Adaptation to Extreme Environments: Prediction of Performance. San Diego, Calif.: U.S. Navy Medical Neuropsychiatric Research Unit. Unit Report No. 66-17.

Gunderson, E.K.E. 1973. Individual behavior in confined or isolated groups. In: Man in Isolation and Confinement (Rasmussen, J.E., Ed.) Chicago: Aldine, pp. 145-66.

Lansing, A. [1959] 1994. Endurance: Shackleton’s Incredible Voyage. New York: Carroll and Graf.

Nansen, F. 1897. Farthest North. New York: Harper and Brothers.

Rivolier, J., Goldsmith, R., Lugg, D.J. and Taylor, A.J.W. 1988. Man in the Antarctic: The Scientific Work of the International Biomedical Expedition to the Antarctic (IBEA). New York: Taylor & Francis.

Stuster, J., Bachelard, C. and Suedfeld, P. 1999. In the Wake of the Astrolabe: Review and Analysis of Diaries Maintained by the Leaders and Physicians at French Remote-Duty Stations. Technical Report 1159 for the National Aeronautics and Space Administration. Santa Barbara, CA: Anacapa Sciences, Inc.

Stuster, J. 1996. Bold Endeavors: Lessons from Polar and Space Exploration. Annapolis, MD: Naval Institute Press.

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58 Gravitational and Space Biology Bulletin 13(2), June 2000 Cortical Microtubules Form a Dynamic Mechanism That Helps Regulate the Direction of Plant Growth Clive W. Lloyd1*, Regina Himmelspach1, 2, Peter Nick2 and Carol Wymer1, 3 1Department of Cell Biology, John Innes Centre, Colney, Norwich UK 2Institut für Biologie II, Albert-Ludwigs Universität, Freiburg, Germany 3Department of Biological and Environmental Sciences, 103 Lappin Hall, Morehead State University, Morehead KY

ABSTRACT MICROTUBULES Plants form an axis by controlling the direction of cell Microtubules are usually composed of 13 protofilaments expansion; this depends on the way in which cellulose microfibrils folded into a hollow tube, approximately 25nm in diameter (see in the wall resist stretching in particular directions. In turn, the reviews in Hyams and Lloyd, 1994). Each protofilament is alignment of cellulose microfibrils correlates strongly with the composed of alternating dimers of á and áâ tubulin, which, alignment of plasma membrane-associated microtubules, which since they all enter the polymer with the same sense (áâ, áâ, áâ. . therefore seem to act as templates for laying down the wall fibrils. .), imparts a polarity to the microtubule. This is reflected in the Microtubules are now known to be quite dynamic, and to reorient fact that subunits add on faster to one end (the plus end) than to themselves between transverse and longitudinal alignments. Plants the other. Microtubules, therefore, have an intrinsic polarity that “steer” the direction of growth by reorienting the imparts directionality to the cytoplasm, which is at the core of the cellulose/microtubule machinery. For example, the model predicts many structural properties of these cytoskeletal elements. that a transverse reorientation on one flank of an organ and a Molecules—such as the microtubule motor proteins, kinesin and longitudinal orientation on the other should lead to bending. This dynein—“read” the directionality of microtubules and help response has recently been observed in living, gravistimulated transport vesicles and organelles to one end or the other. maize coleoptiles microinjected with fluorescent microtubule Microtubules are found in virtually all eukaryotic cells, and the protein. This paper reviews the idea of the dynamic relatedness of the tubulin proteins across large phylogenetic microtubule template and discusses possible mechanisms of distances is demonstrated by the fact that tubulin from vertebrate reorientation. Recent biochemical work has shown that brains is readily incorporated into the microtubule structures of microtubules are decorated with different classes of associated plants. proteins, whose potential roles are outlined. Microtubules are employed in constructing the mitotic spindle for the separation of the chromosomal material. However,

when the cell is not dividing, microtubules disassemble and reconstruct other assemblies, such as the cilia and flagella of INTRODUCTION unicellular algae and the bundles that support the long processes of The ability of wooden artefacts to maintain their shape nerve cells. In higher land plants, the microtubules are found depends on the organization and chemistry of the cellulose-based immediately beneath the plasma membrane (to which they are walls that surround dead cells. In the living state, however, plants attached by poorly characterized cross-bridges). Spread along the change their shape over time, and this plasticity depends on an entire cell, hundreds of microtubules of variable length important contribution from the living cytoplasm in remodelling circumnavigate the cell in overlapping relays. They are generally the structure of the wall. The cell wall can yield and grow in pictured as encircling the cell. However, as described below, the different directions. Although the chemistry of the wall is an microtubule array is highly dynamic and can adopt different important key to understanding how this happens, it is not the orientations. only one. The texture of the wall is also important. The wall is composed of several, sometimes many, layers of cellulose MICROTUBULES AND DIRECTIONAL CELL microfibrils. The fibrils follow the same overall direction within GROWTH any one layer, but they can change from layer to layer, and it seems One of the key features of plant development is the ability that the direction of the layer(s) nearest the plasma membrane both to direct growth up into the atmosphere—to display determines the direction in which the cell will grow. The direction photosynthetic and reproductive organs—and to send roots down in which that innermost layer is deposited is believed to depend on into the ground. This necessarily involves limiting growth to a cues from a complementary set of proteinaceous fibres just inside particular axis, and it is in this capacity that microtubules are the plasma membrane. It is now known that the fibres that believed to have an important role. Naked protoplasts placed in a constitute this so-called cytoskeleton—particularly the hypotonic solution will swell isotropically. However, when the cell microtubules—are highly dynamic. This dynamicity, which wall grows back, the turgor pressure is directed along an axis, appears to be linked to the ability of the cell to respond to factors producing an asymmetric cell (Wymer et al., 1996). Only the such as light and gravity, is the subject of this article. inelastic cellulose microfibrils in the wall are believed to have the necessary strength to resist turgor pressure. It was established a long time ago that the cellulose microfibrils in the wall share the same direction as the cortical microtubules, and it is this *Correspondence to: Clive W. Lloyd: fax: +44-1603-501771; e-mail: [email protected]

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hypothesized to track along the underlying microtubules, spinning out cellulose microfibrils as they move (for review see Giddings and Staehelin, 1991). Over the years, it has been difficult to test this hypothesis, because its credibility rests entirely on the coincidence of microtubule and microfibril parallelism. There is a large weight of evidence for this parallelism. However, it has not been possible to fully reconstitute cellulose biosynthesis in vitro or to completely identify the components of the multi-enzyme complexes in higher plants. This situation has contributed to the lack of probes to test exactly how the cellulose synthases interact (if at all) with the microtubules. One idea is that the microtubules merely define lanes within which the intramembranous synthesizing complexes glide, propelled by the act of extruding the cellulose fibre. However, the possibility cannot be formally excluded that the synthases are somehow linked through the membrane to either the contractile actin (“muscle”) filaments or to microtubules, which—if they could use microtubule motor proteins like kinesin and dynein to slide against each other—could generate movement of the cellulose synthases in a more direct way.

Figure 1. Parallelism Between the Cortical Microtubules, MICROTUBULES REORIENT THEMSELVES Here Circumnavigating the Cell as Transverse “Hoops,” and the Cellulose Microfibrils in the Cell Wall. Although Microtubules can be transverse, oblique, or parallel to the these two linear elements are separated by the plasma membrane, organ axis, and it is known that they must reorient themselves they are thought to be functionally related by microtubules that form between these different configurations. Reorientation can be tracks for the movement of the plasma-membranous cellulose that affected by a range of environmental agents (like gravity) and by synthesizes enzyme complexes. Transversely arrayed cellulose plant growth regulators. This is illustrated in Figure 2, which microfibrils resist increase in cell girth and convert turgor pressure shows that factors work antagonistically to stimulate transverse or into an elongating force. longitudinal microtubules and to shift the direction of cell expansion. For instance, the plant growth regulator ethylene is known to stunt growth and, when added to pea stem cells, to cause 0 the microtubules to reorient through 90 so they become parallel to co-linearity that forms the basis of directed growth (Ledbetter and the cell’s long axis (Roberts, Lloyd and Roberts, 1985). Dwarf peas Porter, 1963). Transverse “hoops” of cytoplasmic microtubules with the le mutation have short internodes and an insufficiency of follow the same general direction as the cellulose microfibrils in the the hormone gibberellic acid. Experimental addition of exogenous wall (Figure 1). When cellulose microfibrils are wound around a cell gibberellic acid to segments of pea stems causes the cells to in transverse layers like this, their lack of stretch acts like a girdle elongate measurably within two hours, during which the originally that resists increases in diameter. However, because the swelling longitudinal microtubules reorient to the transverse (Duckett and force of turgor pressure can tease apart adjacent microfibrils in any Lloyd, 1994). one layer, the cell stretches perpendicularly to the direction of the As templates for the co-alignment of cellulose microfibrils, microfibrils. (In an oversimplified way, this can be thought of as longitudinal microtubules should lead to the deposition of new the stretching of a coiled spring in which adjacent windings are layers of wall in which the longitudinal cellulose microfibrils resist pulled apart). On the other hand, cellulose microfibrils can be cell elongation. From examining the pattern of cellulose in arranged parallel to the long axis of the cell. Lack of stretch along successive layers of the cell wall, Shibaoka’s group has suggested this axis should lead to inhibition of cell elongation and encourage that cells with a cross-ply structure (alternating layers of lateral (radial) expansion instead. The alignment of the cellulose longitudinal and transverse microfibrils) should display a fibrils therefore influences the direction in which the turgid cell corresponding rhythmic alternation of the microtubular template expands. If the microfibrils in epidermal cells are longitudinal on (Shibaoka, 1994; Mayumi and Shibaoka, 1996). This implicit one flank of a shoot and transverse on the other, subsequent reorientation was also long-suspected from immunofluorescence unequal growth should lead to bending. As discussed below, this is and electron-microscopic images of fixed cells, but the mechanism what seems to occur in gravitropic and phototropic responses. was unclear. In the 1990s, Peter Hepler’s colleagues (Wasteneys, Returning to the question of microtubule/microfibril Gunning and Hepler, 1993; Hush et al., 1994) microinjected parallelism, the microtubules on the inner face of the plasma fluorescently tagged vertebrate brain tubulin into plants to study membrane are believed to form the template for the deposition of microtubule dynamics in living plant cells. In this way, Hepler and cellulose microfibrils on the external face. In brief, large multi- his colleagues showed that the fluorescent protein cycled through enzyme complexes that sit within the plasma membrane are some of the other assemblies (mitotic spindle and cytokinetic

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two ways to account for this phenomenon. (1) Plant microtubules undergo dynamic instability (Mitchison and Kirschner, 1984). This concept, first introduced in animal cell biology, speaks in terms of the biased behavior of microtubule growth, mentioned earlier. Tubulin subunits increase at the plus end faster than at the minus end. (2) Individual microtubules show stochastic transitions between growing and shrinking phases—i.e., some microtubules grow while others shrink. However, depending on the conditions, microtubules tend not to shrink completely, but to undergo “rescue,” whereby they stop and regrow. The rapid recovery of fluorescence within bleach zones of the plant cortical array can therefore be ascribed to dynamic instability—the addition of new fluorescent tubulin to the ends of microtubules within the area. Because we cannot be sure that a fluorescent element under the microscope is actually a single microtubule, there is the formal possibility, which we cannot yet rule out, that some contribution to the fluorescence recovery is made by groups of microtubules Figure 2. Antagonistic Factors Affect the Alignment of sliding against each other (“telescoping”) in the zone. Nevertheless, Microtubules and the Direction of Cell Expansion. Red light the microtubules must be turning over rapidly, given the speed of and the plant hormones, gibberellic acid and auxin, encourage the initial labelling. Certainly, the half-times for recovery from transverse microtubules, which are generally found in elongating photobleaching put plant microtubules ahead of most figures for tissue. Blue light, ethylene, and abscissic acid encourage dynamic animal microtubules. This apparently paradoxical longitudinal microtubules, which are often found in cells that are behavior of highly dynamic microtubules in stationary cells could swelling laterally instead of elongating. There may be a rhythmic account for the ability of sessile organisms to respond rapidly to switching between these two configurations in some cells, building changing environmental conditions. up a cross-ply pattern of wall lamellation.

MICROTUBULE REORIENTATION IN LIVING CELLS phragmoplast) that microtubules form in dividing plant cells. By microinjecting rhodamine-tubulin into pea epidermal cells Our group microinjected rhodamine-conjugated pig brain we have seen how such reorientations might occur. Yuan et al. tubulin into pea epidermal cells to see whether this would label the (1994) reported that new microtubules could first be seen along the cortical microtubules of nondividing cells (Yuan et al., 1994). Some edge of a cell—at the junction between the outer circumferential of these cells are very large—several hundred micrometers long— face of the epidermal cell and the radial side wall. These but the cortical microtubule array was labelled as soon as the stem microtubules could be newly nucleated at that locus, or they could tissue could be transferred from the microscope used for injection have grown along the radial wall and then spilled over onto the to the confocal laser-scanning microscope. This suggests that the outer surface. Either way, they do not share the alignment of the soluble tubulin is incorporated into the polymer and that the existing array. These “discordant” microtubules increase in number microtubules must be exchanging subunits with the cytoplasmic in the new direction, while the existing microtubules either pool. One possibility, however, is that this may label only a depolymerize or re-grow in the new direction. Normally, dynamic subset, so that any stable microtubules would go microtubules are sufficiently well organized that populations can unlabelled. To test this, we correlated the image obtained from be classified as “transverse,” “oblique,” or “longitudinal.” That is, microinjected cells with the image of the same area fixed and there is an approximate, general uniformity. We now believe that subsequently labelled with anti-tubulin antibodies that should the transitional stages of the reorganization represent the random- recognise dynamic and stable microtubules alike (Wymer et al., looking mixtures of new and previously unclassified, old 1997). The limited resolution of light microscopy is unable to microtubules (Lloyd, 1994). After the microtubules in the new distinguish between single microtubules and small groups of direction have replaced those in the old, the array must undergo parallel microtubules. Nevertheless, the coincidence of the some kind of smoothing process to restore overall order. We microinjected and the fixed patterns indicates that there is no hypothesize that proteins involved in the parallel packing of hidden subset of stable microtubules in a different orientation, microtubules are likely to play an important role in this process. demonstrating that microinjection does produce a representative By taking serial optical sections through microinjected cells, then picture of the array. rotating the 3-D computer reconstructions, it is possible to see that Photobleaching small patches of fluorescence in microinjected microtubules on the outer face of pea epidermal cells can cells has made it possible to show that the fluorescence recovers sometimes be in a different orientation to the microtubules along rather rapidly (Hush et al., 1994; Yuan et al., 1994). That the the radial side walls (Yuan et al., 1995). This tends to suggest that fluorescence is “killed” when the spot is bleached implies that new reorientation occurs cell face by cell face (although there is no rule fluorescence must invade from beyond the bleach zone. There are

Gravitational and Space Biology Bulletin 13(2), June 2000 61 DYNAMIC MICROTUBULES AND PLANT GROWTH that microtubules need always share the same orientation on attachment process. However, it was formally possible that different facets of the same cell). However, oblique arrays have attachment could set up stresses that could themselves trigger the been seen in microinjected cells where microtubules wind in 45º microtubule reorientation. To control for this, coleoptiles were helices around all surfaces of the cell. Since these arrays tend to attached to slides, exactly as they would have been for occur in the older parts of the internode, it is possible that they microinjection, then placed vertically, i.e., without gravitropic represent the final stage of the reorganization process, whereby the stimulation. No effect could be detected on microtubule parallelization process imposes an order that spreads to all cell reorientation, indicating that gravity was the trigger. Using this set- surfaces. They could represent the “best-fit” configuration for cells up, it was possible to microinject gravistimulated epidermal cells no longer subjected to the strains of cell elongation. on the upper surface of the coleoptile and observe them undergo This proposed method of reorientation—in which one set transverse-to-longitudinal reorientation in response to 1-g. The appears to be replaced by another in a different orientation—may microtubule response could be subdivided into three categories: not be the only method. It is important to keep open the possibility that other mechanisms may occur in cells not · About 30 minutes after the onset of stimulation, traumatized by excision from the stem, artificial increase in the size longitudinal microtubules appeared among the of the tubulin pool through microinjection, physical damage from previously transverse microtubules, giving the the microneedle, horizontal placement, etc. Yuan et al. (1994) and array a patchwork appearance. Wymer and Lloyd (1996) have seen another kind of reorientation. This occurs in long epidermal cells where microtubules at one end · The microtubules sorted out a new oblique of the outer circumferential cell face are oriented differently from orientation in which the mixed order became those at the other end. In such cases, we have sometimes seen one replaced by a new parallel organization in the set gradually adopt the alignment of the other. At the moment, we new direction. do not know whether this is an alternative mechanism of reorientation or part of the “smoothing” process that restores order · The angle of microtubule alignment often after reorientation. increased smoothly over time to give a steeper, near-longitudinal pattern. This sequence was similar to that seen in the earlier microinjection MICROTUBULES AND GRAVITY studies on pea epidermis (Yuan et al., 1994) and in the gibberellic acid-induced reorientation Because the reorientation of microtubules in microinjected described below (except that this was in the cells occurs spontaneously and sporadically, it has been important opposite longitudinal-transverse direction) to find factors that trigger this process. Plant growth regulators are (Lloyd, et al., 1996). known to do this (the effect of one of them is discussed below), but more recently we have obtained data on the effect of gravity, As discussed, the plant growth regulator gibberellic acid can based on photo- and gravitropic studies pursued in Peter Nick’s stimulate stem elongation and convert longitudinal microtubules to laboratory. Himmelspach et al. (1999) germinated maize seed under the transverse axis. Using pea epidermal cells microinjected with continuous red light, and the coleoptiles were excised at three days. fluorescent tubulin, Lloyd et al. (1996) first identified living cells Fixation and immunofluorescence studies have shown that cortical with longitudinal microtubules and then added exogenous gibberellic microtubules are generally transverse to the organ axis in vertically acid to the cells under the microscope. A reverse transition to the grown coleoptiles; but that, when they are gravistimulated by transverse orientation resulted. As before, transverse “discordant” horizontal placement of the coleoptile, they reorient themselves microtubules appeared, and the existing longitudinal microtubules from transverse to longitudinal on the upper side and adopt a seemed to depolymerize as these microtubules grew. That is, transverse alignment on the lower side (Nick et al., 1990). This is microtubules appear in the new direction, and there is a mixed consistent with the increased elongation of the cells on the lower alignment for a while before the new, parallel array sorts out the side, such that the growing coleoptile then bends upwards. new direction. In all cases, it seems paradoxical that one set of Figure 3 shows how this bending is thought to occur. When microtubules grows as the old set shrinks. Therefore, to understand maize coleoptiles grown in red light are placed horizontally, the the mechanism involved, it will probably be necessary to coleoptile bends upward in response to gravity. The microtubules understand the molecular basis of microtubule stabilization. The in the epidermis on the upper side reorient themselves to the long literature indicates that gibberellic acid physically stabilizes cell axis (resulting in inhibited cell elongation), and the transverse microtubules. For instance, Mita and Shibaoka (1984) showed that microtubules on the lower side encourage the elongation that results pretreatment of onion leaf sheath cells with gibberellic acid in the bending. For the purposes of microinjection, so that this stabilized the cortical microtubules against the depolymerizing reorientation could be observed in living cells, Himmelspach et al. effects of cold or of an anti-microtubule herbicide. In dwarf pea, the (1999) glued coleoptiles horizontally to microscope slides using gibberellic acid-induced, longitudinal-to-transverse reorientation is medical adhesive. Conventional fixation studies showed that more accompanied by modification of the tubulin polypeptides (Duckett than 75% of the microtubules in epidermal cells on the upper flank and Lloyd, 1994). Immunoblotting 2-D gels with reoriented to steeply oblique/longitudinal within one hour of gravistimulation—hence the graviresponse was not inhibited by the

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respectively) could also be seen to be purified. Since then, other MAPs have been identified. For instance, the microtubule motor protein kinesin is now a key player in microtubule biology. However, it is noteworthy that this protein was not isolated by the classical methods. Accordingly, definitions should be sufficiently flexible to include, for example, enzymes that could attach to microtubules and modify their functionality. Plant MAPs may be sufficiently different from the well- known animal MAPs to make searching for sequence homologies in the database unproductive. Our own approach has been to biochemically identify proteins that associate with the cytoskeleton. By extracting protoplasts with detergent, we have produced resistant fibrous cytoskeletons that contain microtubules, actin bundles, and the nucleus. We can then depolymerize the cytoskeletal proteins in cold, calcium-containing buffer and isolate any potential plant MAPs by pelleting them on taxol-stabilized Figure 3. Different Microtubule Orientations on Opposite brain microtubules (Chan et al., 1996). In this way, we have Flanks Are Found in Maize Coleoptiles Bending in identified two groups of carrot proteins homologous to those Response to Gravity. Maize coleoptiles placed horizontally bend isolated by Jiang and Sonobe (1993) from tobacco cells. The so- upwards. The epidermal cells on the lower side of the coleoptile called 65kDa proteins are a family of three or more polypeptides elongate more than those on the upper side. When the coleoptile is from 62-68kDa that appear to be antigenically related. In placed horizontally, transverse microtubules in the vertically grown immunofluorescence studies, anti-MAP65 antibodies stain the four 0 tissue reorient themselves through 90 on the upper flank, microtubule arrays around the cell cycle. Recently, we have consistent with the decreased rate of cell elongation. separated the 65kDa MAPs from the 120kDa MAP in our carrot MAP fraction and, in add-back experiments, shown that MAP65 causes brain microtubules to form into tight, parallel bundles (Chan et al., 1999). In the electron microscope, one can see the microtubules cross-bridged by 25-30nm filaments, which are antibodies specific for particular tubulin epitopes has shown that regularly spaced along the length of the microtubule. In EM studies gibberellic acid causes the probable detyrosination of an alpha of plant cells, this is the spacing between microtubules, and it tubulin. Throughout eukaryotic cell biology, this posttranslational would appear that MAP65 is responsible for the parallel modification usually accompanies conversion of microtubules to arrangement in vivo. A combination of 1) attachment to the plasma the stable state. The idea is that stable tubules are a good substrate membrane and 2) maintenance of a regular intermicrotubule distance for a carboxypeptidase that removes the terminal tyrosine residue by MAP65 goes a long way to explaining the typical organization more efficiently than it does from dynamic microtubules. More of the plant cell’s cortical array. Microtubules from the recently, it has been shown that pretreatment of a maize cell microinjection studies appear in a mixed organization before they suspension with gibberellic acid stabilizes the protoplast realign and become smoothed in the new direction, and it is microtubules against being chilled (Huang and Lloyd, 1999). This probable that MAP65 plays a part in this parallelization process. effect was accompanied by acetylation of a tubulin isotype— We have seen three, sometimes four polypeptides in another posttranslational modification associated with microtubule immunoblots of the 65kDa family, and further studies are required stabilization. Neither modification is thought to directly affect the to determine the individual role of the members. We were microtubule; instead such modifications are thought to indirectly previously unable to demonstrate a bundling effect with the 60kDa indicate changes in dynamicity imposed by other factors, such as MAP, and it is therefore unclear at present whether microtubule-associated proteins. · only one or some of the proteins can cross-link MICROTUBULE-ASSOCIATED PROTEINS (MAPs) microtubules,

· their activity is modified by posttranslational MAPs are generally defined as the proteins that co- modification, polymerize with microtubules as they go through rounds of temperature-dependent cycles of assembly/dis-assembly. This · they form complexes, definition originally came from the animal field, where microtubules · their patterns of expression and activity change were first biochemically isolated. Brains contain a high proportion according to the stage of the cell cycle. of tubulin, and it was found that—when calcium was chelated and temperature increased—the tubulin in brain homogenates self- For instance, with the onset of cell division, cortical microtubules assembled into microtubules that could be pelleted at low speed. disappear from the ends of the cell and bunch up into the After several rounds of assembly/dissembly, MAPs (these include preprophase band (which foretells the plane of cell division), and it the low- and high-molecular-weight MAPs, tau and MAP2, seems likely that some modification of the cross-linking activity of

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MAP65 must occur in the process. Concerning the interphase arranged filamentous cross-bridges between microtubules. array itself, the ability of MAP65 to form stable microtubule Proceedings of the National Academy of Sciences USA 96:14931- bundles in vitro is ostensibly inconsistent with the observed 14936. dynamicity of the cortical microtubules in vivo. For this reason, it is possible that some process, such as posttranslational Duckett, C.M. and Lloyd, C.W. 1994. Gibberellic acid-induced modification, regulates the activity of the 65kDa MAPs, switching microtubule reorientation in dwarf peas is accompanied by rapid them on and off. Such signalling processes may be part not only of modification of an á-tubulin isotype. Plant Journal 5:363-372. cell cycle progression but also of the response to plant growth regulators, such as gibberellic acid, which are known to modulate Giddings, T.H. and Staehelin, L.A. 1991. Microtubule-mediated the behavior of plant microtubules. control of microfibril deposition: a re-examination of the Models of the influence of microtubules on cellulose hypothesis. In: The Cytoskeletal Basis of Plant Growth and Form biosynthesis must be updated to account for the presence of (Lloyd, C. W., Ed.) London: Academic Press, pp. 85-99. filamentous MAPs on the cortical array (see Giddings and Staehelin,1991). We know too little to decide definitively between Himmelspach, R., Wymer, C.L., Lloyd, C.W. and Nick, P. 1999. direct models (in which microtubules and cellulose synthases are Gravity-induced reorientation of cortical microtubules observed in directly linked) and indirect models (in which microtubules form vivo. Plant Journal 18:449-453. the channels above which the synthases freely move). However, in both models the cross-bridging of adjacent microtubules would Huang, R.F. and Lloyd, C.W. 1999. Gibberellic acid stabilises have an effect on the hypothetical mechanisms. According to the microtubules in maize suspension cells to cold and stimulates indirect models, synthases move in the membranous lanes between acetylation of á-tubulin. FEBS Letters 443:317-320. microtubules. In principle, there could be two sorts of “lanes,” depending on the precise organization of the microtubules. Hush, J.M., Wadsworth, P., Callaham, D.A. and Hepler, P.K. According to the first model, the synthases could move in lanes 1994. Quantification of microtubule dynamics in living plant cells between adjacent bundles of microtubules. In this case, the using fluorescence redistribution after photobleaching. Journal of interbundle channels would almost certainly be variable if the Cell Science 107: 775-784. bundles are not bridged to each other. In such a loose model, there would be an approximate relationship, instead of a 1:1 relationship, Hyams, J.S. and Lloyd, C.W. (eds.). 1994. Microtubules. New between tubule and microfibril. However, it is possible to envisage York: Alan R. Liss. a tight version of the indirect model, in which synthases move in the narrower lanes directly above pairs of microtubules bridged by Jiang, C-J. and Sonobe, S. 1993. Identification and preliminary MAP65. The ca. 25nm cellulose-synthesizing rosettes would be characterization of a 65kDa higher plant microtubule-associated accommodated by the 25-30nm intermicrotubule space maintained protein. Journal of Cell Science 105: 891-901. by MAP65 cross-bridges. In this tight, indirect model, paired microtubules form a defined “railroad track,” for which MAP65 Ledbetter, M.C. and Porter, K.R. 1963. A “microtubule” in plant molecules form the ties (or sleepers). This version predicts that cell fine structure. Journal of Cell Biology 19:239-250. exact 1:1 matches will be found between pairs of microtubules and individual microfibril as they are being deposited. However, this Lloyd, C.W. 1994. Why should stationary plant cells have such will be sensitive to the visualization technique. It is unlikely that dynamic microtubules? Molecular Biology of the Cell 5:1277-1280. such a relationship would be revealed by simply comparing an already-deposited lamella’s pattern of fibrils with a fixed image of Lloyd, C.W., Shaw, P.J., Warn, R.M. and Yuan, M. 1996. the microtubule array. The latter, we now know, is so dynamic Gibberellic acid-induced reorientation of cortical microtubules in that, by the time an entire lamella is deposited, fine details of living plant cells. Journal of Microscopy 181:140-144. patterning will have been lost by turnover. Although it is still too early to decide between direct and Mayumi, K. and Shibaoka, H. 1996. The cyclic reorientation of indirect models for the influence of microtubules on cellulose cortical microtubules on walls with a crossed polylamellate alignment, it seems increasingly likely that MAPs play an structure: effects of plant hormones and an inhibitor of protein important part in the determination of wall texture. kinases on the progression of the cell cycle. Ptotoplasma 195:112- 122. REFERENCES

Chan, J., Rutten, T. and Lloyd, C.W. 1996. Isolation of Mitchison, T. and Kirschner, M. 1984. Dynamic instability of microtubule-associated proteins from carrot cytoskeletons: a microtubule growth. Nature, London 312: 237-242. 120kDa MAP decorates all four microtubule arrays and the nucleus. Plant Journal 10:251-259. Nick, P., Bergfeld, R., Schäfer, E. and Schopfer, P. 1990. Unilateral

reorientation of microtubules at the outer epidermal wall during Chan, J., Jensen, C.G., Jensen, L.C.W., Bush, M. and Lloyd, C.W. photo- and gravitropic curvature of 1999. The 65kDa microtubule-associated protein forms regularly- maize coleoptiles and sunflower hypocotyls. Planta 181:162-168.

64 Gravitational and Space Biology Bulletin 13(2), June 2000 DYNAMIC MICROTUBULES AND PLANT GROWTH

Mita, T. and Shibaoka, H. 1984. Gibberellin stabilizes microtubules in onion leaf sheath cells. Protoplasma 119:100-109.

Roberts, I.N., Lloyd, C. W. and Roberts, K. 1985. Ethylene-induced microtubule reorientations: mediation by helical arrays. Planta 164:439-447.

Shibaoka, H. 1994. Plant-hormone-induced changes in the orientation of cortical microtubules: alterations in the cross-linking between microtubules and the plasma membrane. Annual Review of Plant Physiology and Plant Molecular Biology 45:527-544.

Wasteneys, G.O., Gunning, B.E.S. and Hepler, P.K. 1993. Microinjection of fluorescent brain tubulin revelas dynamic properties of cortical microtubules in living plant cells. Cell Motility and the Cytoskeleton 24:205-213.

Wymer, C.L., Fisher, D.D., Moore, R.C. and Cyr, R.J. 1996. Elucidating the mechanism of cortical microtubule reorientation in plant cells. Cell Motility and the Cytoskeleton 35:162-173.

Wymer, C. and Lloyd, C.W. 1996. Dynamic microtubules: implications for cell wall patterns. Trends in Plant Sciences 7:222- 228.

Wymer, C.L., Shaw, P.J., Warn, P.J. and Lloyd, C.W. 1997. Microinjection of fluorescent tubulin into plant cells provides a representative picture of the cortical microtubule array. Plant Journal 12:229-234.

Yuan, M., Shaw, P.J., Warn, R.M. and Lloyd, C.W. 1994. Dynamic reorientation of cortical microtubules from transverse to longitudinal, in living plant cells. Proceedings of the National Academy of Sciences USA 9:6050-6053.

Yuan, M., Warn, R.M., Shaw, P.J. and Lloyd, C.W. 1995. Dynamic microtubules under the radial and outer tangential walls of microinjected pea epidermal cells observed by computer reconstruction. Plant Journal 7:17-23.

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66 Gravitational and Space Biology Bulletin 13(2), June 2000

Mechanical Forces in Plant Growth and Development Deborah D. Fisher and Richard J. Cyr* Department of Biology, 208 Mueller Lab, Pennsylvania State University, University Park PA

ABSTRACT GRAVITY WORKS ON MOVEABLE OBJECTS Plant cells perceive forces that arise from the environment External factors are used as developmental cues by and from the biophysics of plant growth. These forces provide growing plants (e.g., electromagnetic radiation, gravity, and meaningful cues that can affect the development of the plant. temperature; Hangarter, 1997). Undoubtedly, these same Seedlings of Arabidopsis thaliana were used to examine the elements have had a profound impact on plant evolution cytoplasmic tensile character of cells that have been implicated (Barlow, 1995; Niklas, 1998). Although the entire plant is in the gravitropic response. Laser-trapping technology revealed continuously subjected to gravitational acceleration, probably that the starch-containing statoliths of the central columella cells only specialized cells have the ability to perceive this force in in root caps are held loosely within the cytoplasm. In contrast, angiosperms (Sack, 1991). Perception appears to be based the peripheral cells have starch granules that are relatively largely on cellular rigidity. Basically, if a cellular object is held resistant to movement. The role of the actin cytoskeleton in tightly within the cytoplasm (i.e., if the tensile strength holding a affecting the tensile character of these cells is discussed. To particular object is greater than the product of the object’s explore the role that biophysical forces might play in generating gravitational acceleration and its mass) the object remains developmental cues, we have developed an experimental model stationary. However, if the object is loosely suspended within system in which protoplasts, embedded in a synthetic agarose the cell, the gravitationally induced force will cause it to fall matrix, are subjected to stretching or compression. We have downward. This downward movement, in specialized cells, is found that protoplasts subjected to these forces from five transduced to affect the growth of both shoots and roots minutes to two hours will subsequently elongate either at right (Masson, 1995; Volkmann et al., 1999). angles or parallel to the tensive or compressive force vector. Among the mass types that gravity affects are the heavy Moreover, the cortical microtubules are found to be organized starch grains (statoliths) located within the root caps of either at right angles or parallel to the tensive or compressive angiosperm cells (Figure 1). Genetic and morphological data force vector. We discuss these results in terms of an interplay of indicate that the starch grains within the root cap are major sites information between the extracellular matrix and the underlying of gravity perception (Kiss et al., 1989; Kiss and Sack, 1989; cytoskeleton. Sack and Kiss, 1989). Amyloplasts likely play a similar role within the graviresponsive cells of shoots (Kiss et al., 1997; INTRODUCTION Weise and Kiss, 1999). It appears that only certain cells within Land plants are relatively sessile organisms, and rigid cell the root cap are involved in the gravity response. Laser ablation walls interconnect all the cells within the plant thallus. These of the more centrally located columella cells abolishes a root’s traits have two important consequences. First, plants generally ability to respond to gravity; however, cannot move quickly in response to a changing environment; rather they must adapt to where they are. Second, due to the physical coupling of rigidly bound cells within the plant tissues, mechanical events in one area of the plant can be transmitted to other areas. Cells are subjected to a variety of forces during plant growth and development. Exogenous forces arise from the environment and endogenous forces stem from the biophysics of plant growth. The overall objective of this article is to provide the reader with an appreciation of how the plant can use these forces as meaningful developmental cues. In the first part of the article, we will describe how specialized cells perceive gravitational forces. In the second part, we will discuss how endogenous forces might be used by plant cells to help monitor their own growth status—in particular, the relationship between the microtubule cytoskeleton and the cell wall. We will present data to support the hypothesis that plants have the ability to continually use a variety of mechanical cues to monitor and adjust morphogenetically. These mechanical cues, derived from the action of exogenous and endogenous forces, are integrated by the plant to insure that optimal growth occurs under a variety of Figure 1. The Root Tip is Highly Differentiated. A root tip conditions. from Arabidopsis thaliana is shown with outlined central columella cells, which contain loose statoliths. *Correspondence to: Richard J. Cyr: fax: 814-865-9131; e-mail: [email protected]

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Figure 2. Loose Starch Grains Demonstrated with Laser Trapping. Central columella cells are visualized within an Arabidopsis thaliana root tip and a starch-containing statolith. (a) The infrared optical trap is turned on, and a starch grain is trapped (arrow). (b) The steering mirror for the laser trap has been adjusted to reposition the focal point of the laser and the entrapped starch grain to a new position. Note the upward displacement of the starch grain here, compared with the same grain’s position in a.

ablation of the more laterally located peripheral cells does not statoliths of the central columella cells would predictably move (Blancaflor et al., 1998). The approximate location of these faster, therefore generating more force in response to gravity, and central columella cells is outlined in Figure 1. These ablation thus would be a more sensitive transducer of gravity’s vector. results correlate well with differences in sedimentation velocities What cellular component would allow the starch grains to of amyloplasts in the central cells, versus the peripheral cells, of move more freely in the central columella cells? The actin the root cap (Sack et al., 1986; Blancaflor et al., 1998). moiety of the cytoskeleton is probably the most likely Therefore, it appears that there are differences between central candidate. In most plant cells, the actin cytoskeleton forms a and peripheral columella cells. One difference might be the tight network that acts to suspend organelles and, in conjunction tensile strength of the cytoplasm holding the statoliths (Baluska with myosin, to affect intracellular motility (Williamson, 1993). and Hasenstein, 1997). Baluska et al. (1997) report that the central columella cells are To address this possibility, we used laser-trapping devoid of cytoskeletal elements radiating into the cell interior, technology to explore the tensile character of the cytoplasm while the peripheral columella cells are rich in these filaments. within the root cap cells. The technology of laser trapping, also Failure to demonstrate cytoskeletal elements in the more interior known as laser tweezers, is based on the behavior of high- locations of central columella cells indicates that the cytoplasm intensity light as it passes through a highly refractive dielectric in these cells is structurally unique. During differentiation of sphere. Basically, as light passes into a dielectric sphere with a central columella cells, it is likely that actin is either down- higher refractive index than the medium, the light ray is refracted regulated and/or that actin-severing/depolymerizing proteins are and reflected, which leads to a change in photonic momentum. up-regulated. Further experimentation will undoubtedly reveal As a result, a major restoring force vector is generated towards the molecular basis for this loose state, which appears to be the focal point, which creates a physical trap (Ashkin, 1998). ideally suited for detecting the movement of heavy organelles in We grew Arabidopsis seedlings on agarose-coated response to gravity. coverslips oriented at 45°C. This orientation insures that the tip of the root will grow along the optical surface of the coverslip, ENDOGENOUS FORCES WORK ON THE CELL thereby providing the best possible image. Figure 2a shows Compared to the cells found within a developing animal, central columella cells within an Arabidopsis thaliana root tip plant cells develop in a somewhat constrained environment and and statolith (see arrow) that have been trapped by the infrared do not migrate relative to one another (Fosket, 1994). Hence, all laser trap. In Figure 2b, the steering mirror has been moved, morphogenetic processes must occur within a relatively rigid which displaces the trap along with the statolith. milieu. To appreciate how plants grow under these constraints, Physically trapping intercellular organelles, such as starch consider that osmotic forces cause plant cells to develop high statoliths, enabled us to determine the relative tensile character water pressures. Thus, a turgid plant cell is under pressure, and of the cytoplasm in different cells of the root cap. We this force is exerted isotropically within the cell (Cosgrove, systematically scanned across the root cap and determined how 1993). How does the cell, or a file of cells, elongate in a vectoral loosely the statoliths were held. The space in which statoliths manner when driven by isotropic forces? The strong cellulose freely move within the cells coincides with the central columella microfibrils within a growing cell (Figure 3a) are not arranged cells outlined in Figure 1. The outline shows that only the central randomly, rather they are highly oriented at right angles to the columella cells have starch grains that are held loosely. Those in axis of elongation. Cellulose reinforces the wall, much as hoops the cap’s periphery are held relatively tightly, and cannot be strengthen a barrel, allowing little lateral expansion. However, moved easily. These results indicate that the starch-containing

68 Gravitational and Space Biology Bulletin 13(2), June 2000 FORCES AND PLANT DEVELOPMENT growth is unconstrained in the axis perpendicular to cellulose alignment (Green and Poethig, 1982). Therefore, as a consequence of growth, a major strain axis develops along this axis. This strain axis arises when the isotropic forces of turgor are permitted to work solely at right angles to the orientation of cellulose microfibrils (Gertel and Green, 1977; Green and Selker, 1991). The orderly deposition of cellulose occurs during its synthesis. Cellulose synthase complexes are found in complexes within the plasma membrane (Delmer and Amor, 1995). The synthesis of cellulose microfibrils can be minimally described as a two-step process: glucose is first assembled into a b1,4 polyglucan chain, which then combines with 30-100 other polyglucan chains to crystallize into a microfibril (Brett and Waldron, 1990). Crystallization is an exergonic reaction that can work to move the entire cellulose synthase complex into the plane of the fluid membrane. However, the movement of these complexes is not random. Somehow, the cortical microtubules, which are attached to the inner face of the plasma membrane, restrict the gliding cellulose synthase complexes, as well as nascent microfibrils, along a particular path (Cyr and Palevitz, 1995). Cortical microtubules flanking the cellulose synthase complexes act like railroad tracks to guide the complex (Figure 3b; Giddings and Staehelin, 1991; Cyr, 1994). One weakness of this so-called “microtubule/microfibril paradigm” is that the molecular nature of how microtubules interact with the plasma membrane is not understood, nor is it clear whether the cellulose synthase complexes can interact directly with the underlying microtubules. Although it is clear that microtubules influence the Figure 3. Organization of Cellulose in Elongating Cells. deposition of microfibrils in many, if not most, elongating plant (a) Turgor pressure, coupled with wall relaxation, drives cellular cells, how microtubules become organized remains uncertain. It expansion that is limited to one major axis by the organized has been proposed that biophysical forces orient microtubules cellulose microfibrils. (b) The organized deposition of cellulose is (Green et al., 1970). The evidence that supports this hypothesis affected by cortical microtubules, which are attached to the is largely circumstantial (Williamson, 1990; Williamson, 1991; underside of the plasma membrane. Cyr, 1994) and somewhat controversial (Nick, 1999). We have taken an experimental approach to verify or refute this hypothesis. · Can newly isolated protoplasts have the future To understand the possible role that mechanical forces axis of elongation manipulated? play in orienting microtubules, Nagata et al. (1992) used a · Simultaneously, do the same manipulations that cultured tobacco cell line, designated BY-2. This cell line is easy provide an axial “cue” also affect the organizational to culture and, depending upon the culture conditions, will grow status of the cortical microtubules? either as rapidly dividing cells or as elongating cells (Hasezawa and Syono, 1983). As with many plant cells, it is relatively easy Wymer et al. previously showed that a brief centrifugation to remove the cell wall via enzymatic digestion. This is of newly isolated protoplasts could cue the future axis of important, because the rigid nature of the cell wall makes it elongation (1996). However, in those studies, it was difficult to difficult to know what forces are perceived when an external know precisely what forces the cells were experiencing. To force is applied. By removing the wall, we can directly measure further extend the studies, we used a modified, commercially the effect of the force as a deformation of the protoplast. Once available apparatus marketed by Flexcell Corporation (Banes et the cell wall is removed, a spherical protoplast is released that al., 1985) to manipulate protoplasts embedded in agarose on a contains cortical microtubules that usually are very randomly flexible sheet of silastic. The agarose served as an artificial elastic oriented. Upon culturing, protoplasts regenerate a wall, matrix that allowed us to study the response of the cell to a reorganize their cortical microtubules, and within 24-48 hours given amount of applied force. We poured a protoplast/ agarose begin to elongate (Figure 4). Our experiments address the suspension onto the membrane. After the agarose solidified, we following questions: placed the silicon membrane (held tightly in a sterile chamber) over a vacuum manifold containing an immobile post.

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indicating that the agarose matrix behaved as a completely elastic membrane and that the applied force had no demonstrable plastic-deforming effects (data not shown). Freshly isolated protoplasts were deformed for two hours, then released and cultured to favor elongation. Typically, during the first 24 hours after force application they remained somewhat spherical (if asymmetrical, there was no dominant asymmetric axis). However, within 72 hours, the majority of cells showed some anisodiametric growth; within seven days, their length-to-width ratio was typically between three and six. In areas of the agarose sheet where cells were exposed to biaxial forces (i.e., the center of the round loading post), the elongative axes were random. However, in areas where the cells were exposed to a uniaxial tensive force, the majority had a nonrandom elongative axis parallel to the tension vector (Figure 5). Similarly, in areas that were under uniaxial compression, we observed a nonrandom elongative axis—but it was at right angles to the compression vector (data not shown). Finding that a brief uniaxial tensive or compressive force can cue elongative axes raises questions about how these forces are perceived and about the nature of the memory once perception has occurred. Recall that force application occurs 22- 44 hours before any morphological change is observed. Cortical

microtubules are involved in the deposition of cellulose Figure 4. Experimental System for Studying Elongation. microfibrils and, because the ordered deposition of cellulose is Top row: Cultured tobacco cells grow in elongate cell files, and required for elongation in these cells, the microtubules are each cell contains an organized cortical microtubule (MTs) array candidates for both force perception and the memory that can be visualized with anti-tubulin antibodies. Middle row: component. That is, random microtubules might perceive the Upon enzymatic removal of the wall, spherical protoplasts with force, become aligned, and commence orienting cellulose disorganized cortical microtubules are produced.. Bottom row: deposition, which consequently affects the axis of elongation. To Upon reculturing in a medium that supports elongation, the cells test this hypothesis, we subjected cells to compressive and reorganize their microtubules and elongate, as seen at 7 days tensive forces, then fixed and processed them for post-protoplasting. immunolocalization of microtubules (Figure 6). The cortical microtubules in this cell are not random; rather they show a net organization in the horizontal direction similar to the tension When we drew a vacuum on the manifold, the silicon membrane force vector (Figure 6, arrow). Also, when protoplasts were first deformed around the post and the attached protoplasts/agarose subjected to compressive forces, then fixed and processed for matrix was stretched or compressed. By changing the geometry cytoskeletal visualization, the microtubules in many cells of the loading post, we exposed the cells to different forces. showed alignment. However, this alignment was at right angles Note that we set up this apparatus for microscopic examination, to the compressive force vector (data not shown). which permitted visualization of the cells as a force was applied. Also, by adding fluorescent reference beads, we could quantify CONCLUSION the nature of applied force on the matrix and embedded The above data support the hypothesis that physical forces can protoplasts. influence axis determination in a plant cell. The finding that both Immediately after isolation, cells appeared mostly compressive and tensive forces can elicit this response is spherical (if slightly aspherical, there was no preferential axis of consistent with observations reported in whole-plant studies. asymmetry). During the application of force, the cells appeared For example, it has been reported that physically bending maize either deformed at right angles to the compression force vector or coleoptiles induces microtubule alignment at right angles to the parallel to the tension vector (data not shown). Significantly, tension vector (Zandomeni and Schopfer, 1994). The role of after the stretching period, the cells returned to a spherical shape compression in affecting microtubule alignment might also (if non-spherical, there was no preferential axis of asymmetry). explain the change in microtubule orientation observed in We directly quantified the magnitude of applied force by graviresponding roots. In vertically grown roots, microtubules measuring the displacement of fluorescent microbeads, which within the epidermis and cortical cells are aligned at right angles confirmed the major compressive or tensive axis. It is to the root axis. When roots are tilted horizontally, the cells noteworthy that, once the applied force was released, the within the upper epithelium show the typical transverse reference beads returned to their original positions, thereby alignment of microtubules regularly observed in rapidly growing

70 Gravitational and Space Biology Bulletin 13(2), June 2000 FORCES AND PLANT DEVELOPMENT

compressed, and high-resolution growth studies have reported a negative growth rate in this region (Ishikawa et al., 1991). If the tissue is shrinking, then it is also compressing; and the lower epidermal cells could perceive this force, which would explain their transverse alignment. We suggest that the relationship between microtubules, microfibrils, and elongative growth is functionally interrelated. This relationship can be summarized as an extension of the microtubule/ microfibril paradigm, in which cortical microtubules serve as templates to affect the deposition of cellulose microfibrils. The microfibril’s high tensile strength then restricts isotropic turgor forces to one axis, and the resulting biophysical forces act as cues that the cells use to affect microtubule alignment. The alignment cue has previously been inferred from work with cellulose synthesis inhibitors (Fisher and Cyr, 1998). Here, we provide data indicating that both tensive and compressive forces can orient microtubules. The molecular details of how microtubules relay information to the cellulose

synthase are currently speculative. Therefore, we do not know Figure 5. Stretching Cues the Axis of Elongation. Tobacco the sequence of events by which biophysical forces might be protoplasts that were stretched for 2 hours were visualized after perceived and transmitted to the microtubules. It has been 7 days of culture. The majority of cells have elongative axes proposed that this process may be indirect and that it may parallel to the direction of stretching. involve the molecular alignment of linking microtubule

nucleators, which then serve to orient microtubules (Williamson, 1990). Conversely, it has been proposed that microtubules themselves may be the strain sensors (Green et al., 1970). All models will remain speculative until we determine the molecular nature of microtubule interaction with the plasma membrane and the cellulose synthase. The challenge for future research will be to identify these components and study their behavior under conditions that are known to affect microtubule alignment and the resulting elongative growth.

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Blancaflor, E., Fasano, J., Gilroy, S. 1998. Mapping the functional roles of cap cells in the response of Arabidopsis Hangarter, R.P. 1997. Gravity, light and plant form. Plant Cell primary roots to gravity. Plant Physiology 116:213-222. and Environment 20:796-800.

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72 Gravitational and Space Biology Bulletin 13(2), June 2000 FORCES AND PLANT DEVELOPMENT

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Wymer, C.L., Wymer, S.A., Cosgrove, D.J. and Cyr, R.J. 1996. Plant cell growth responds to external forces and the response requires intact microtubules. Plant Physiology 110:425-430.

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74 Gravitational and Space Biology Bulletin 13(2), June 2000 The Actin Cytoskeleton May Control the Polar Distribution of an Auxin Transport Protein Gloria K. Muday*, Shiquan Hu and Shari R. Brady Department of Biology, Wake Forest University, Winston-Salem NC

ABSTRACT al., 1995). The auxin efflux carrier is thought to control The gravitropic bending of plants has long been linked to the amount and direction of polar auxin transport. It has the changes in the transport of the plant hormone auxin. To also been proposed that the basal localization of an auxin understand the mechanism by which gravity alters auxin efflux carrier determines the polarity of IAA transport in movement, it is critical to know how polar auxin transport is plant tissues (Rubery and Sheldrake, 1974; Jacobs and initially established. In shoots, polar auxin transport is basipetal Gilbert, 1983; Muller et al., 1998). (i.e., from the shoot apex toward the base). It is driven by the In addition to moving by polar transport down the basal localization of the auxin efflux carrier complex. One length of plant tissues, auxin can move laterally across mechanism for localizing this efflux carrier complex to the basal gravity-stimulated shoots and roots. The Cholodny-Went membrane may be through attachment to the actin cytoskeleton. hypothesis, originally proposed in 1937, suggests that the The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds lateral transport of auxin across gravity-stimulated plant auxin transport inhibitors, such as naphthylphthalamic acid tissues drives differential gravitropic growth (Evans, (NPA). Several lines of experimentation have been used to 1991; Trewavas, 1992). Lateral redistribution of determine if the NPA binding protein interacts with actin radiolabeled IAA has been measured in both shoots filaments. The NPA binding protein has been shown to partition (Parker and Briggs, 1990) and roots (Young et al., 1990), with the actin cytoskeleton during detergent extraction. Agents and the redistribution of IAA has been shown to precede that specifically alter the polymerization state of the actin differential growth and the gravity response (Parker and cytoskeleton change the amount of NPA binding protein and Briggs, 1990). Applying auxin transport inhibitors to actin recovered in these cytoskeletal pellets. Actin-affinity growing plants leads to an inhibition of the gravity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a response. These synthetic inhibitors inhibit auxin efflux single peak from the actin filament column. Cytochalasin D, (Rubery, 1990), and culturing plants on these compounds which fragments the actin cytoskeleton, was shown to reduce completely inhibits gravity response in the roots of a polar auxin transport in zucchini hypocotyls. The interaction of number of plant species, under conditions where growth the NPA binding protein with the actin cytoskeleton may still occurs (Katekar and Geissler, 1980; Muday and localize it in one plane of the plasma membrane, and thereby Haworth, 1994, Rashotte et al., 2000). The effect of the control the polarity of auxin transport. auxin transport inhibitor naphthylphthalamic acid (NPA) on gravity response is very rapid, with application at the time of gravitropic stimulation completely inhibiting INTRODUCTION gravitropic bending (Rashotte et al., 2000).

Auxins are a class of plant hormones that control elongation, development, and the response of plants to gravity and other environmental signals. Auxins, of which indole-3-acetic acid (IAA) is the predominant naturally occurring hormone, move through plants by a unique polar transport mechanism (as reviewed in Goldsmith, 1977; Lomax et al., 1995). This polar movement of auxin is from the shoot meristem towards the base of stems, and is a cell-to-cell movement. Polar auxin transport results in an auxin gradient down the length of the plant, with the highest auxin concentrations found in the regions of greatest elongation (Ortuno et al., 1990). Auxin is not transported at constant rates; rather its transport is regulated, and it changes during development and in response to environmental stimuli (Lomax et al., 1995). Several proteins are believed to control polar auxin transport. There are two protein complexes, the auxin uptake carrier and the auxin efflux carrier, that control auxin movement into and out of cells (Figure 1). IAA can Figure 1. Schematic Model of the Chemiosmotic Hypothesis move into cells both passively, since it is hydrophobic for Polar Auxin Transport. Protonated IAA in the cell wall when protonated, and through an influx carrier (Lomax et space can enter the cell either by diffusion or via an uptake carrier. Once in the more basic cytoplasm, the IAA dissociates Portions of this article have been reproduced from Muday and can exit only via the auxin efflux carrier. The names of the (2000) with permission from Kluwer Academic Publishers. proteins that may constitute auxin transporters are in parentheses. (Reprinted from Muday, 2000, with kind *Correspondence to: Gloria Muday: fax: 336-758-6008; e-mail: permission from Kluwer Academic Publishers) [email protected]

Gravitational and Space Biology Bulletin 13(2), June 2000 75 AN AUXIN TRANSPORT PROTEIN INTERACTS WITH ACTIN FILAMENTS

Although the validity of the Cholodny-Went control both polar and gravity-induced lateral auxin hypothesis has been debated (Trewavas, 1992), recent transport. For example, it is not yet clear whether the molecular and genetic evidence has provided additional same protein complex controls auxin efflux during both support (Chen et al., 1999). One powerful test has been polar and lateral movement of IAA. If multiple auxin through the construction of transgenic plants with an efflux carrier complexes are used, then gravity-induced auxin-responsive promoter that drives the expression of â- lateral auxin transport could require expression of a gene glucuronidase. Redistribution of auxin-induced gene that encodes another carrier, or it could be mediated by expression across a gravity-stimulated shoot (Li et al., differential activation of one gene product. Alternatively, 1991) and root (Li et al., 1999) is consistent with changes if a single efflux carrier complex controls both polar and in lateral auxin transport. This finding supports the lateral transport, we must alter either its localization or the Cholodny-Went hypothesis. The ability of auxin transport activity of a subset of these carriers in order to change the inhibitors to block both differential auxin-regulated gene directionality of auxin movement. Once we identify all expression and gravitropic bending suggests that lateral the proteins that control auxin transport and obtain the auxin transport, rather than a change in auxin sensitivity, molecular tools to study the position and activity of these is the mechanism leading to differential gene expression. proteins, we can elucidate the mechanisms by which Another approach that has shown the dependence of auxin transport changes to allow gravity response. gravity response on auxin transport has been to isolate plants with mutations in auxin transport proteins that BIOCHEMICAL CHARACTER OF THE AUXIN result in an agravitropic phenotype. The aux1 and allelic EFFLUX CARRIER eir1/agr1/pin2 mutants have agravitropic roots (Chen et The biochemical dissection of the auxin efflux al., 1999). The recent cloning of the genes that are carrier will increase our understanding of how this protein mutated in these plants suggests that these genes are auxin complex is regulated and how its localization to the basal transporters. It has been suggested that AUX1 may plasma membrane controls the polarity of auxin encode the auxin uptake carrier (Bennett et al., 1996), movement. The efflux carrier complex appears to be which transports auxin into cells, and that composed of more than one polypeptide. Composition EIR1/AGR1/PIN2 and PIN1 may encode transmembrane includes: protein of the auxin efflux carrier (Chen et al., 1998; Luschnig et al., 1998; Galweiler et al., 1998; Muller et al., · an integral membrane transporter encoded by a 1998), which pumps auxin out of cells. That roots of agr1 member of the PIN gene family; accumulate more radiolabeled IAA than wild-type roots is · a NPA binding protein (NBP) that may act consistent with an inhibition of auxin efflux (Chen et al., as a regulatory polypeptide; 1998). The recent development of assays to measure polar · perhaps a third, rapidly turned-over protein auxin movement in the roots of Arabidopsis has revealed that connects these two subunits a reduction in polar auxin transport in the roots of eir1 (Morris et al., 1991). (Rashotte et al., 2000). Together, these results suggest that auxin transport plays an important role in controlling Several members of the PIN gene family in Arabidopsis plant gravity response. have been identified (Galweiler et al., 1998; Muller et al., Although these results link lateral auxin transport to 1998), indicating that there are multiple auxin efflux gravity response, there are some experimental results that carriers with distinct expression patterns. Plants with do not easily fit the simple interpretation of the Cholodny- mutations in two genes of this family have phenotypes Went hypothesis. The growth characteristics of roots in consistent with tissue-specific alterations in auxin response to gravitropic stimulation have been carefully transport (Okada et al., 1991; Galweiler et al., 1998; examined through computerized image analysis, and the Muller et al., 1998), and alterations in auxin transport pattern of root growth is not as simple as initially occur in the affected tissues (Okada et al., 1991; Chen et predicted (Evans, 1991). The root over-responds to al., 1998; Rashotte et al., 2000). PIN genes encode gravity, turning from horizontal to vertical to beyond proteins with ten membrane-spanning domains that are vertical; then growth switches from one root side to the similar to other membrane transport proteins (Chen et al., other, allowing reorientation to the vertical (Ishikawa et 1999). The protein products of these genes show an al., 1991). In addition, roots grown on high concentrations asymmetric localization in the plasma membrane that is of auxin can still respond to gravity, even when growth is consistent with controlling the polarity of auxin almost totally inhibited (Ishikawa and Evans, 1993; movement (Galweiler et al., 1998; Muller et al., 1998). Muday and Haworth, 1994). These results, which appear Therefore, it has been suggested that the PIN genes contradictory to the Cholodny-Went hypothesis, indicate encode one polypeptide of the auxin efflux carrier. that the role of auxin in root growth and gravity response Until the PIN proteins were identified, most studies is complex, and that additional experimentation will be of the auxin efflux carrier focused on the NPA binding protein. The activity of this protein can be followed using required to completely understand how gravitropic growth 3 is controlled (Trewavas, 1992). a binding assay with [H]-NPA: radiolabeled NPA is Identifying the proteins that transport auxin is the incubated with membrane vesicles or solublilized protein, first step in understanding the molecular mechanisms that and the protein and ligand complexes are recovered by

76 Gravitational and Space Biology Bulletin 13(2), June 2000 AN AUXIN TRANSPORT PROTEIN INTERACTS WITH ACTIN FILAMENTS filtration or centrifugation. The NPA ligand binds with NPA BINDING PROTEIN PARTITIONS WITH THE high affinity to a single class of NPA binding proteins ACTIN CYTOSKELETON DURING DETERGENT associated with the zucchini plasma membrane (Muday et EXTRACTION al., 1993). Therefore, this assay has allowed extensive To purify integral membrane proteins, the first step biochemical characterization of the NPA binding protein. is to treat them with detergent, which solubilizes the Several lines of evidence suggest that the protein protein and releases it from the membrane. As it was that binds inhibitors of auxin efflux is distinct from the initially assumed that the NPA binding activity and the PIN gene products. Treatments with inhibitors of protein auxin efflux carrier activity were localized on the same translation and protein processing in the Golgi reduce the polypeptide, most investigators initiated experiments regulation of auxin transport by NPA without altering the using detergent solubilization with the goal of releasing amount of NPA binding activity. (Morris et al., 1991; NPA binding activity. Although several reports in the Wilkinson and Morris, 1994; Morris and Robinson, literature indicate that NPA binding activity can be 1998). These results (1) suggest that the NPA binding and released from the membrane by detergent treatment, all of auxin efflux activities are on separate proteins, and (2) these procedures resulted in very low yields of soluble support the idea that a third protein may connect them NPA binding activity (Sussman and Gardner, 1980; (Morris et al., 1991). Jacobs and Gilbert, 1983; Cox and Muday, 1994; It also appears that the NBP is peripherally Bernasconi et al., 1996). In two of these reports, the associated with the plasma membrane. Treatment of amount of NPA binding activity in the detergent-insoluble plasma membrane vesicles with potassium iodide (KI) or pellet was quantified, and in both cases, the majority of sodium bicarbonate released the NPA binding protein into the activity was in the detergent-insoluble pellet (Sussman the supernatant after ultracentrifugation, suggesting that and Gardner, 1980; Cox and Muday, 1994). The NPA binds to a peripheral protein (Cox and Muday, insolubility of the NPA binding protein during detergent 1994). Furthermore, the NBP is still active in detergent- extraction may be due to interaction with the insoluble pellets. These pellets should be almost free of cytoskeleton, as proteins associated with the cytoskeleton lipids, yet the majority of NPA binding activity was show this behavior (Carraway, 1992). recovered, suggesting that the NBP does not require a Cox and Muday (1994) reported the first study that lipophilic environment for activity (Cox and Muday, addressed whether the detergent insolubility of the NPA 1994; Butler et al., 1998). Most integral membrane binding was due to association with the cytoskeleton. proteins would lose activity under these conditions. After purified zucchini plasma membranes were treated Therefore, in our current model, the NBP is a peripheral with Triton X-100, NPA binding activity and both actin membrane regulatory protein. This model is consistent and tubulin polypeptides were examined in the pellet and with the results of Morris et al. (1991) and Wilkinson and supernatant fractions after ultracentrifugation (Cox and Morris (1994), which indicate that NPA binding activity Muday, 1994). Actin, tubulin, and NPA binding activity and auxin efflux activity are on two distinct polypeptides. all partitioned preferentially into the detergent-insoluble Biochemical evidence suggests that NPA binding pellet. Treatment of the detergent-insoluble or activity is localized to the cytoplasmic face of the plasma cytoskeletal pellet with cytochalasin B, a drug that membrane. Several investigators have examined the fragments the filamentous form of actin (F-actin), protease sensitivity of NPA binding activity in plasma 3 membranes isolated from zucchini hypocotyls. Consistent released [H]-NPA binding activity into the supernatant with a cytoplasmic localization, treatment of intact right- after ultracentrifugation. Use of this drug in vitro caused side-out vesicles with protease does not lead to loss of the release of both actin and tubulin cytoskeletal NPA binding activity (Dixon et al., 1996, Bernasconi et fragments, so these experiments could not differentiate al., 1996). In contrast, disruption of membranes by between association with actin and association with detergent, followed by protease treatment, results in a tubulin (Cox and Muday, 1994). total loss of NPA binding activity (Bernasconi et al., Although this initial study supported the argument 1996). Furthermore, plasma membrane vesicles have been that the NPA binding protein interacts with the subjected to several different treatments that should have cytoskeleton, the use of purified plasma membranes with converted them to inside-out orientation (Hertel et al., treatments designed to alter actin polymerization was not 1983; Dixon et al., 1996; Bernasconi et al., 1996). The optimal. Rather, the use of fresh and relatively crude effectiveness of these treatments, however, was only extracts of zucchini hypocotyl proteins proved to be a verified by analysis of marker enzymes in one case better method for determining whether the NPA binding (Dixon et al., 1996). When the ability of treatments to protein was interacting with the cytoskeleton, and for convert vesicles to an inside–out orientation was verified, determining which cytoskeletal polymer was the site of both NPA binding activity and the protease sensitivity of interaction (Butler et al., 1998). Butler et al. (1998) also that activity had increased in inside-out vesicles (Dixon et found that NPA binding activity and actin partitioned into al., 1996). Therefore, the NPA binding site appears to be the cytoskeleton pellet after detergent extraction of fresh localized to the cytoplasmic face of the membrane and extracts. There was very little tubulin polypeptide found positioned for interaction with the cytoskeleton. in these extracts, suggesting an interaction between the NPA binding protein and actin (Butler et al., 1998).

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To more directly test for an interaction between the (Cox and Muday, 1994). The KI was removed to allow NPA binding protein and actin, intact zucchini hypocotyls actin filaments to reform, and the resulting sample was were treated in vivo with one of three drugs known to subjected to centrifugation. After this treatment, the pellet alter cytoskeletal organization—phalloidin, cytochalasin and supernatant were collected, and both actin filaments D, or taxol (Butler et al., 1998). After drug treatment, and NPA binding activity were found predominantly in extracts were prepared and treated with detergent, and the the pellet. Although NPA binding activity was amount of NPA binding activity in the detergent-insoluble preferentially partitioned into the samples that were cytoskeletal pellets was measured. Phalloidin and enriched in actin, there was very low recovery. cytochalasin D act on the actin cytoskeleton to stabilize Furthermore, both actin and tubulin were enriched in polymers or to fragment polymers, respectively. these samples, suggesting that this procedure was not a Treatment with phalloidin increased both the amount of very specific way to recover actin polymers (Cox and pelletable actin and NPA binding activity, while treatment Muday, 1994). Therefore, an alternative approach was with cytochalasin D decreased both pelletable actin developed to more directly examine actin interactions. polypeptide and NPA binding activity. In contrast, taxol To directly test the interaction of the NPA binding treatment stabilized microtubules, resulting in increased protein with purified actin filaments, F-actin affinity pelletable tubulin after detergent solubilization, but not columns were prepared and their ability to retain binding increased pelletable NPA binding activity (Butler et al., activity was examined. There are a number of actin genes 1998). in plants, encoding as many as 20 different actin Butler et al (1998) used one additional treatment to polypeptides (Kandasamy et al., 1999), for which there depolymerize actin. The buffer Tris has been previously are specific tissue-specific expression patterns. It was reported to lead to actin depolymerization (Pinder et al., therefore critical to obtain actin isoforms from the tissues 1995). Butler and colleagues (1998) found that using Tris that are known to transport auxin and to possess NPA to treat detergent-insoluble pellets (from either fresh binding activity. extracts or plasma membranes isolated from zucchini There are no procedures in the literature for hypocotyls) led to a dose-dependent decrease in pelletable purifying actin that is competent for polymerization from actin and NPA binding activity (Butler et al., 1998). plant vegetative tissues, although procedures to purify Initial experiments using Tris did not result in a maize pollen actin have been published (Liu and Yen, concomitant increase in NPA binding activity in the 1992; Ren et al., 1997). However, there are numerous detergent supernatant, so it was not clear whether Tris reports in the literature of purifying animal actin using was releasing NPA binding activity or denaturing the affinity columns prepared with the enzyme DNase I NPA binding activity. To stabilize the NPA binding coupled to a solid support (Sheterline et al., 1998; Zechel, protein during its release, the NPA ligand was included 1980), so this approach was chosen to purify zucchini during the detergent extraction and Tris treatment. The hypocotyl actin. DNase I binds G-actin with a 1:1 ratio Tris was removed, and the pH was lowered to a proton with high affinity. It is also commercially available, and it concentration more optimal for NPA binding, resulting in has previously been used to partially purify actin from pea the recovery of NPA binding activity in the supernatant roots (Andersland et al., 1992). (Butler et al., 1998). Therefore, it appeared that Tris Using DNase I chromatography followed by released NPA binding activity, and did not denature the ultracentrifugation, Hu et al. (in review) purified actin protein (Butler et al., 1998). Together, these results were from zucchini hypocotyls to electrophoretic homogeneity. consistent with an association of the NPA binding protein Since actin was eluted from the DNase I resin with with the actin cytoskeleton, although the interactions were formamide, which can denature proteins, it was only indirectly demonstrated. The next step to particularly critical to demonstrate that this actin was demonstrate this interaction was to show that the NPA native. First, the profilin binding activity of purified binding protein could bind in vitro to homogenous and zucchini hypocotyl actin was compared to purified and purified actin filaments. native maize pollen actin that had been isolated according to Ren et al. (1997). The profilin binding ability was INTERACTION OF THE NPA BINDING PROTEIN compared using two isoforms of maize profilin, one WITH ACTIN FILAMENTS IN VITRO expressed in pollen (ZmPRO1) and the other expressed predominantly in vegetative tissues (ZmPRO5). The The first approach to demonstrate the interaction of resulting K values for these two actin pools were not the NBP with actin filaments was to subject detergent- d statistically different under these conditions (Hu et al., in insoluble cytoskeletal pellets to rounds of polymerization review). The native structure of the actin was also and depolymerization. Throughout these cycles of actin confirmed by the ability of the purified actin to bind and polymerization, the location of both the actin polypeptide inhibit DNase I activity (Hu et al., in review). and the NPA binding activity were followed. Initially, The ability of purified zucchini hypocotyl actin to actin and NPA binding activity were recovered in the form filaments was demonstrated by sedimentation of F- detergent-insoluble pellet. Upon treatment with potassium actin during ultracentrifugation, by decreased mobility of iodide (KI), both actin and NPA binding activity moved F-actin on native gels, and by examination of actin into the supernatant as filaments were depolymerized filaments with electron microscopy (Hu et al., in review).

78 Gravitational and Space Biology Bulletin 13(2), June 2000 AN AUXIN TRANSPORT PROTEIN INTERACTS WITH ACTIN FILAMENTS

Ultrastructural examination of in vitro polymerized actin cytoskeleton. The next question was whether these showed helical filaments with a width of 6.8 nm (Hu et columns would retain sufficient quantities of the NPA al., in review), consistent with the conformation and size binding protein to allow the protein’s isolation for amino of maize actin filaments assembled in vitro (Ren et al., acid sequence analysis. The pattern of proteins retained 1997). Together, these results demonstrated that purified by the column was examined by subjecting samples to zucchini hypocotyl actin was native and competent for SDS-PAGE, followed by silver staining. There was no polymerization. consistent band found in samples that contained NPA Purified, native zucchini hypocotyl actin was then binding activity (Hu and Muday, unpublished result). In used to prepare both G- and F-actin columns. BSA was contrast, two proteins were routinely eluted from the F- used to create a third affinity matrix to test for nonspecific actin column with high salt concentrations (higher than protein interactions (Hu et al., in review). Examining the those required to elute NPA binding activity) (Hu et al., in binding of vertebrate á-actinin to the F-actin column review). These two proteins of 30-35 kDA were confirmed the selectivity of the F-actin column. Purified recognized by annexin antisera (Hu et al., in review). As á-actinin was shown to bind tightly to the F-actin, but annexins are plasma membrane proteins that have been weakly to the G-actin column (Hu et al., in review). shown to interact with the actin cytoskeleton (Calvert et Since the NPA binding protein is associated with the al., 1996), this result provides further evidence that these plasma membrane, isolated plasma membranes were used columns can retain F-actin binding proteins in a specific as the starting sample for chromatography on the actin fashion. columns. Treating plasma membranes with Triton X-100 and Tris resulted in the recovery of NPA binding activity DRUGS THAT FRAGMENT THE ACTIN CYTO- in the supernatant after ultracentrifugation. This soluble SKELETON REDUCE POLAR AUXIN TRANSPORT sample was applied to the actin or BSA columns. Eluted The interaction of the NPA binding protein with the protein samples were analyzed for NPA binding activity, actin cytoskeleton may be necessary either for movement and were examined by silver stain after SDS-PAGE. NPA of auxin across the membrane or for the polar localization binding activity was retained by the F-actin column and of the efflux carrier complex. If either of these hypotheses reproducibly eluted with high salt concentrations. In five is correct, then disruption of the actin cytoskeleton would separate experiments, NPA binding activity was localized be predicted to reduce polar auxin transport. Treatment of to one or two fractions eluted from the F-actin column. either corn coleoptiles (Cande et al., 1973) or zucchini This activity was significantly greater than the activity hypocotyls (Butler et al., 1998) with cytochalasins, drugs eluted from a BSA column or an F-actin column to which which fragment the actin cytoskeleton, have led to the no solubilized proteins had been applied (Hu et al., in reduction of auxin transport. review). The effect of cytochalasin D on auxin transport in To date, the elution of NPA binding activity from zucchini hypocotyls was measured using a modification the F-actin column is the strongest evidence to indicate of previously published assays (summarized in Figure 2). associa tion of the NPA binding protein with the actin Zucchini hypocotyl segments treated with and without 3 cytochalasin D were simultaneously loaded with [H]- IAA and [14C]-benzoic acid, and the amount of radioactivity transported out of each end of the segment was recovered in agar blocks. When zucchini hypocotyls were treated with cytochalasin D, there was a statistically significant reduction in basipetal auxin transport, as shown in Table I (data from Butler et al., 1998). This reduction in transport is not at the level of diffusion, as there are no changes in the amount of either basipetal benzoic acid movement or acropetal auxin transport. Because this assay measures passive diffusion from the segment as well as polar transport, each measurement contains some background diffusion. The level of background diffusion can be assessed by examining the percentage of acropetal auxin movement or the percentage of benzoic acid diffusion. Basipetal IAA Figure 2. Representation of the Assay Used to Measure IAA transport can be normalized by subtracting the amount of Transport. For cytochalasin D treatments, samples were diffusion (calculated by averaging the percentage of BA treated with 200 ìM cytochalasin D for 1 hour before the diffusion, both acropetal and basipetal, and the percentage incubation with [3H]IAA and [14C]BA. The % transport is the number of counts in each agar block divided by the total number of acropetal IAA diffusion) from the basipetal auxin of counts in both agar blocks and the segment multiplied by transport. The magnitude of the effect of cytochalasin D 100%. treatment increases to two-fold when the normalized values are compared.

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Table I. Cytochalasin D Reduces Polar Auxin Transport

%Transporta

-Cytochalasin D +Cytochalasin D p valueb Basipetal IAA 19.0 ± 1.2 13.3 ± 1.1 <0.005 Acropetal IAA 8.0 ± 0.4 8.8 ± 0.8 >0.2 Basipetal BA 7.0 ± 0.9 7.0 ± 0.8 >0.2 Normalized Basipetal IAAc 11.3 5.6

a The % transport is the average and standard error of 12 separate experiments. b The % transport in the absence and presence of 200 ìM cytochalasin D is compared by student t-test. c Normalized basipetal IAA transport was calculated by subtracting the background diffusion (the average of the % of acropetal IAA and % basipetal BA transport) from the % basipetal IAA transport. (Reprinted from Muday, 2000, with kind permission from Kluwer Academic Publishers)

If an intact actin cytoskeleton is required for subunit of the auxin efflux carrier, the NPA binding localization of the auxin efflux carrier complex, should protein, binds actin filaments (Hu et al., in review). fragmentation of actin filaments with cytochalasin lead to Other studies, in both plants and animals, have a total loss of auxin transport? This question can be demonstrated the importance of the actin cytoskeleton in considered by examining the model in Figure 3 (opposite establishing and maintaining cell polarity. In yeast and the page). If the efflux carriers are totally randomized, then brown alga, Fucus, initial establishment of cell polarity transport should be at the level of diffusion. Basipetal requires an intact actin cytoskeleton (Goodner and IAA transport is not reduced to the level of diffusion by Quantrano, 1993; Li et al., 1995) and is preceded by cytochalasin. However, the cytochalasin D treatment was changes in the organization of the actin cytoskeleton for only one hour, perhaps not enough time to allow all (Ayscough and Drubin, 1996; Kropf et al., 1989; Alessa the efflux carriers to randomize. Also, there may have and Kropf, 1999; Chant, 1999). To preserve cellular been partial recovery of polar auxin transport capacity polarity, a number of plasma membrane proteins with during the 1.5-hour transport period. These results are asymmetric localization maintain their distribution by consistent with the model shown in Figure 3, although attaching themselves to the actin cytoskeleton. In the more complex possibilities cannot yet be eliminated. developing zygotes of Fucus, the dihydropyridine Finally, it should be noted that the treatment of roots does not completely abolish root gravitropism, suggesting that lateral auxin transport may not depend on the actin cytoskeleton (Blandoflor and Hasenstein, 1997; Staves et al., 1997).

CONCLUSIONS The results from these studies indicate that a regulatory subunit of the auxin efflux carrier, the NPA binding protein, binds directly to actin filaments. The actin association of the NPA binding protein may localize the auxin efflux carrier complex in one plane of the plasma membrane and thereby control the polarity of auxin transport. Galweiler et al. (1998) and Muller et al. (1998) have used antibodies that recognize two different isoforms of an integral membrane protein of the auxin efflux carrier to show that these proteins are localized to one plane of the plasma membrane. Butler et al. (1998) Figure 3. Model for the Effect of Cytochalasin D on Polar and Cande et al. (1973) have also shown that Auxin Transport. A file of untreated cells (left) compared to cytochalasin-D-treatment of zucchini hypocotyls and corn those treated with cytochalasin D (right). The fragmentation of actin filaments by cytochalasin D is shown, as well as the coleoptiles, respectively, reduces polar auxin transport, a randomization of the auxin efflux carrier complex as a result of result that is consistent with the actin cytoskeleton’s role the loss of actin structure, which may serve to localize this in maintaining the polar distribution of auxin transport protein. (Reprinted from Muday, 2000, with kind permission proteins. Our studies have shown that a key regulatory from Kluwer Academic Publishers)

80 Gravitational and Space Biology Bulletin 13(2), June 2000 AN AUXIN TRANSPORT PROTEIN INTERACTS WITH ACTIN FILAMENTS receptor has been shown to develop asymmetry that also Andersland, J.M., Jagendorf, A.T. and Parthasarathy, requires an intact actin cytoskeleton (Shaw and Quatrano, M.V. 1992. The isolation of actin from pea roots by 1996). Both the acetylcholine receptor of neurons and the DNase I affinity chromatography. Plant Physiology Na+, K+-ATPase of epithelial cells have polar 100:1716-1723. distributions that are required for their function (Froehner, 1993; Apel and Merlie, 1995). Evidence for both of these Apel, E.D. and Merlie, J.P. 1995. Assembly of the protein complexes indicates that attachment to the actin postsynaptic apparatus. Current Opinion in Neurobiology cytoskeleton controls their localization (Nelson and 5:62-67. Hammerton, 1989; Froehner, 1993). The acetylcholine receptor is a particularly interesting example, because one Ayscough, K.R. and Drubin, D.G. 1996. ACTIN: general of the proteins in the complex is a 43 kDa peripheral principles from studies in yeast. Annual Review of Cell membrane protein (rapsyn), which is associated with the and Development Biology 12:129-160. actin cytoskeleton. In mice that are deficient in this protein, the acetylcholine receptor fails to properly Bennett, M.J., Marchant, A., Green H.G., May S.T., Ward localize (Gautam et al., 1995). The NPA binding protein S.P., Millner, P.A., Walker, A.R., Schulz, B. and may function in a similar way to localize the efflux Feldmann, K.A. 1996. Arabidopsis AUX1 gene: a carrier. permease-like regulator of root gravitropism. Science Differential actin association of the NPA binding 273:948-950. protein may also provide a level of auxin transport regulation. Morris and Johnson (1990) suggest that, in Bernasconi, P., Bhavesh C.P., Reagan, J.D. and tissues that have reduced polar auxin transport, the Subramanian, M.V. 1996. The N-1-naphthylph-thalamic randomized location of the efflux carrier—rather than acid-binding protein is an integral membrane protein. changes in its abundance—may cause transport reduction. Plant Physiology 111:427-432. This could be mediated by a decrease in the actin association of the NPA binding protein. Additionally, Blancaflor, E.B. and Hasenstein, K.H. 1997. The auxin transport is reduced in response to ethylene organization of the actin cytoskeleton in vertical and treatment (Suttle, 1988), and this reduction may be graviresponding primary roots of maize. Plant Physiology controlled by changes in the cytoskeletal attachment of 113:1447-1455. the NBP (Ebenezer, 1997). The ethylene-induced reduction in auxin transport is accompanied by a Butler, J.H., Hu, S., Brady, S.R., Dixon, M.W. and statistically significant (p<0.01) decrease in the amount of Muday, G.K. 1998. In vitro and in vivo evidence for actin NPA binding activity in detergent-insoluble cytoskeletal association of the naphthylphthalamic acid-binding pellets, although the abundance of the protein in crude protein from zucchini hypocotyls. Plant Journal 13:291- extracts is constant (Ebenezer, 1997). Consequently, it 301. may be that alteration in the cytoskeletal association of the NPA binding protein acts to control the amount of Calvert, C.M., Gant, S.J. and Bowles, D.J. 1996. Tomato polar auxin transport. annexins p34 and p35 bind to F-actin and display Together, these results suggest that the NPA binding nucleotide phosphodiesterase activity inhibited by protein is associated with the actin cytoskeleton in vitro, phospholipid binding. Plant Cell 8: 333-342. and that this association is required for maximal auxin transport and its regulation by NPA in vivo. Although the Cande, W.Z., Goldsmith, M.H. and Ray, P.M. 1973. Polar significance of this association is not yet clear, it may be auxin transport and auxin-induced elongation in the that attachment of the NBP to the actin cytoskeleton absence of cytoplasmic streaming. Planta 111:279-296. serves to localize this protein in one plane of the plasma membrane and thereby to control the polarity of auxin Carraway, C. 1992. Association of cytoskeletal proteins transport. with membranes. In: The Cytoskeleton: A Practical Approach. (Carraway, K. and Carraway, C., Eds.) Acknowledgements Oxford: IRL Press, pp. 123-150.

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84 Gravitational and Space Biology Bulletin 13(2), June 2000

Control of Development and Motility in the Spermatozoids of Lower Plants Stephen M. Wolniak*, Vincent P. Klink, Peter E. Hart and Chia -Wei Tsai. Department of Cell Biology and Molecular Genetics, University of Maryland, College Park MD

ABSTRACT , Equisetum The spermatozoids of lower plants have long been (e.g. [Sharp, 1912; Duckett, 1973]), the recognized as remarkably complex motile gametes. spermatozoids have a spiral cell body and as few as 40 to Spermatozoids differ markedly from the other gametophyte cells as many as 200 cilia (Mizukami and Gall, 1966; Rice and that surround or give rise to them. Their differentiation process Laetsch, 1967; Hepler, 1976; Myles and Hepler, 1977; involves the synthesis and assembly of a complex cytoskeleton Marc and Gunning, 1986). Among the gymnosperms, and a motile apparatus that can be simple or complex, having as only the cycads and Ginkgo biloba possess motile male few as two to as many as thousands of ciliary axonemes. An gametes; these spherically shaped spermatozoids have important aspect of spermiogenesis involves the de novo 1,200 to 30,000 cilia (Mizukami and Gall, 1966; Norstog, synthesis of basal bodies in a cytoplasmic particle known as the 1967, 1986; Gifford and Lin, 1975). blepharoplast: that is, the cells that produce spermatocytes do not contain centrioles. Thus, these cells provide an ideal system There is a direct relationship between gamete size in which to study the formation of basal bodies. The and the complexity of the motile apparatus. The smallest cytoskeletons of spermatozoids from different organisms display and simplest of the gametes are usually produced in the a common architecture, with a multilayered structure (MLS) at largest numbers. The spermatozoids of bryophytes and the anterior end of the cell and a dorsally situated planar ribbon lycopods are smaller than those of the ferns. These, in of crosslinked microtubules extending the length of the turn, are smaller than the spermatozoids of Ginkgo and elongated gamete. The function of the MLS is not known, but it the cycads. The spermatozoids of these gymnosperms are could be involved in cell-body elongation during development rare cells: usually only two are produced by each male and in the control of ciliary motility in the mature gamete, gametophyte. Ironically, the giant motile apparatus of particularly during chemotaxis. The application of modern techniques on these cells can shed light on long-standing these spermatozoids is used to propel the gamete for problems relating to spermiogenesis and motility. remarkably short distances as they travel from the ruptured pollen tube to the archegonium of the female INTRODUCTION gamete. In contrast, the spermatozoids of lower plants can swim freely for several hours to several days. In that time, Plant spermatozoids are motile male gametes that they can travel considerable distances, up to dozens of develop from non-motile cells within the antheridia of meters (Wolniak, unpublished observations). lower plants. By about 1900, the existence of these unusual cells in several organisms had been documented. DE NOVO SYNTHESIS OF BASAL BODIES —THE Within 15 years, characterizations of the developmental BLEPHAROPLAST IS A BASAL BODY FACTORY sequence giving rise to plant spermatozoids had both raised and resolved questions concerning the formation of Incumbent in the developmental program for a motile apparatus in cells that had previously lacked spermiogenesis in the above-mentioned organisms is the centrioles (Wilson, 1911; Sharp, 1912, 1914, 1934). Plant de novo formation of basal bodies that serve as templates spermatozoids differ dramatically from the cells that for axonemal assembly during this process. These basal produce them: they are highly streamlined, elongated, and bodies presumably also function in the coordination of often-coiled cells that possess no walls. These gametes ciliary beat in the mature gamete. Basal bodies are have an elaborate microtubule-based cytoskeleton and a structurally similar to centrioles; and in animals they arise motile apparatus comprising varying numbers of basal from centrioles. In the lower plants, however, they appear bodies with ciliary axonemes. The motile apparatus of the in cells that initially possess no centrioles. They arise in plant spermatozoid varies in size, depending on the these cells from an aggregation of flocculent material in organism. Although most plant spermatozoids have often the cytosol, forming an organized precursor structure been described in the literature as multiflagellate, an known as a blepharoplast (Webber, 1897; Sharp, 1912, analysis of the beat shape reveals that the axonemes are 1914; Mizukami and Gall, 1966; Hepler, 1976). In ferns more properly described as ciliary (Sharp, 1934; Wolniak such as M. vestita, where the blepharoplast’s formation and Cande, 1980; Wolniak, unpublished observations). In and development have been best described, at least the mosses, liverworts, and some of the lower vascular structurally (Sharp, 1914; Mizukami and Gall, 1966; plants (e.g., the bryophytes), mature spermatozoids are Hepler, 1976; Myles and Bell, 1975; Myles and Hepler, shaped like fishhooks and possess only two ciliary 1977), the blepharoplast is a discrete site where axonemes (Wilson, 1911; Carothers and Kreitner, 1967, procentriolar (i.e., pro-basal body) material is assembled 1968; Carothers and Rushing, 1988). In ferns such as (Mizukami and Gall, 1966; Hepler, 1976). After the pro- Marsilea (Sharp, 1914) and other lower vascular plants basal body cores are formed, there is successive addition of tubulin-containing subfibers of the microtubule triplets. *Correspondence to: Stephen M. Wolniak: fax: 301-314-9081; e-mail: [email protected]

Gravitational and Space Biology Bulletin 13(2), June 2000 85 DEVELOPMENT AND MOTILITY IN PLANT SPERMATOZOIDS

PLANT SPERMATOZOIDS EXHIBIT A NUMBER Recent EM-immunogold labeling in spermatozoids OF COMMON CYTOSKELETAL FEATURES of the fern Ceratopteris richardii revealed that the lower strata of the MLS contains centrin or a centrinlike antigen The cytoskeleton in all plant spermatozoids has a (Vaughn et al., 1993). In C. richardii, centrin has also distinctive “multilayered structure” (MLS) (Carothers and been immunolocalized in the osmiophilic material that Kreitner, 1967, 1968; Norstog, 1967; Duckett, 1973; separates basal bodies from one another just on the distal Myles and Hepler, 1977), found in close association with side of the microtubule ribbon (Vaughn et al., 1993). In a mitochondrion at the anterior end of the cell. A M. vestita, centrin probably resides in both the MLS and signature structure of all lower-plant motile male gametes, the rootlet material that links basal bodies on the distal the MLS consists of several sets of stacked vanes and fins side of the microtubule ribbon of mature spermatozoids. that underlie a planar ribbon of crosslinked microtubules. Centrin is a small acidic protein, abundant in microtubule In early ultrastructural characterizations (Paolillo, 1965; organizing centers (MTOCs) (Baron et al.,1992; Carothers and Kreitner, 1967, 1968), there was a dispute Salisbury, 1995). It is thought to function in the about the number of layers of fins and vanes present in microtubule nucleation process (Salisbury, 1995; Levy et the structure. It is now widely accepted that the MLS al., 1998). Centrin may also control the function of intact usually comprises four strata, and that the size, shape and microtubules where its phosphorylation or calcium length of the array is specific to the organism (Carothers binding is manifested as changes in microtubule activity. and Rushing, 1988). The overall size of the MLS is Additionally, it may serve as part of a scaffold that links somewhat related to the size of the motile apparatus. The microtubules with other cytoskeletal proteins, thereby uppermost stratum of the MLS is a planar ribbon of facilitating their mechanochemical interactions (Salisbury microtubules that extends proximally along the dorsal et al., 1984). In animal cells, centrin associated with the side of the elongated gamete. The ribbon of microtubules, centrosome is localized in the distal lumen of centrioles frequently called a spline (Carothers and Rushing, 1988), (Paoletti et al., 1996), but its function there is unknown. It is subtended by an elongated nucleus. The nuclear is reasonable to suspect that centrin plays multiple roles in envelope is attached to the microtubules by a series of both the developing spermatid and the mature arm-like projections. The basal bodies are positioned spermatozoid. During plant spermiogenesis, centrin may above the spline microtubules and at the dorsal side of the be involved in the formation and maturation of basal elongated or coiled cell body, just beneath the plasma bodies, and thereafter in the separation of basal bodies membrane. Thus, the spline microtubules are attached to during blepharoplast fragmentation. In the late stage of both the nuclear envelope and to the basal bodies. development, centrin may have a role in the placement of The function of the lower strata of the MLS is not basal bodies along the microtubule ribbon during the known, though it is likely to play an organizing role in assembly of the motile apparatus (Wright et al., 1985, controlling the size and shape of the microtubule ribbon 1989). during spermiogenesis. Apparently it also links the microtubule ribbon to the anterior mitochondrion in the SPERMIOGENESIS IN MARSILEA—M. VESTITA gamete. In this paper, we pose another hypothetical role AS A MODEL SYSTEM for the MLS in mature spermatozoids, namely, in the control of ciliary beat. Control at this level of organization Marsilea vestita is a heterosporous, aquatic fern. provides a mechanism for the regulation of swimming After meiosis, its spores are stored in hardened structures behavior during, for instance, chemotaxis. known as sporocarps, and they can remain there for many years without loss of viability. Megaspores give rise to COMPOSITION OF THE MLS female gametes, and microspores give rise to spermatozoids (Rice and Laetsch, 1967; Laetsch, 1967). Tubulin is an obvious component in both the MLS Microspores can be separated from megaspores either and the planar ribbon of plant spermatozoids (Myles and when they are dry (Hepler, 1976) or after they have been Hepler, 1977; Marc and Gunning, 1986; Hoffman and imbibed in culture medium (Pennell et al., 1986, 1988). Vaughn, 1995a, 1995b) It is an equally obvious Gametogenesis occurs after the spores are placed into an component of basal bodies and ciliary axonemes. A aqueous medium (Sharp, 1914; Mizukami and Gall, 1966; number of years ago, one of us (S.W.) devised ways to Laetsch, 1967; Rice and Laetsch, 1967; Hepler, 1976); the obtain biochemical fractions of bracken fern process is synchronous in a population of spores and is spermatozoids that were enriched in the MLS. extremely rapid (only 11 hours is required for Ultrastructural analyses of these isolates revealed spermiogenesis at 20oC) (Hepler, 1976; Wick and Hepler, considerable nuclear and other contamination, but the 1980). Male gametophyte development occurs entirely distinctive four-tiered MLS was clearly present. within the microspore wall, and the spermatogenous mass Electrophoretic analysis of the proteins present in the represents approximately 60% of the total cell volume isolates at the time showed that there were <50 prominent within the spore. With the majority of cellular activities in bands, but two were clearly distinguishable as á- and â- the spore directed towards spermatozoid formation, M. tubulins. Because of limitations encountered in the vestita provides an unusual opportunity to assess synthetic continued isolation and characterization of proteins at that processes and biochemical events that are largely aimed at time, little additional progress was made on the project. a single developmental end. At the time of imbibition and

86 Gravitational and Space Biology Bulletin 13(2), June 2000 DEVELOPMENT AND MOTILITY IN PLANT SPERMATOZOIDS for a short time thereafter, drugs, labels, and molecules of · a heavily crosslinked microtubule-based varying size can be taken up through the spore wall into cytoskeleton attached to the dorsal side of the the cells of the gametophyte (Hart and Wolniak, 1998). nucleus; Thus, we found that it was possible to perform labeling, · a motile apparatus comprising ~140 ciliary activation, and inhibition experiments without having to axonemes with their subtending basal bodies resort to invasive procedures. (Myles and Hepler, 1977, 1982), the basal bodies attached to the microtubule ribbon. STRUCTURAL ANALYSIS OF DEVELOPMENT IN M. VESTITA THE BLEPHAROPLAST OF MARSILEA Much of our understanding of the blepharoplast has In M. vestita, the blepharoplast first forms midway come from cytological and ultrastructural studies of M. through gametophyte development, in the spermatocyte vestita (Sharp, 1914; Mizukami and Gall, 1966; Hepler, mother cells, as a single particle (Hepler, 1976) that splits 1976; Myles and Hepler, 1977; Hart and Wolniak, 1998, into two. It functions as a centrosome for mitotic spindles 1999). This plant produces single-celled haploid spores in the last two mitotic divisions that culminate in the that germinate almost immediately upon immersion into production of the spermatids. Then the blepharoplasts water or aqueous culture medium. During the first 2.5 disperse. However, for the ninth mitotic division, which hours following immersion, the single cell undergoes five produces the spermatids, they reform and function as mitotic divisions. Four of these divisions are asymmetric, prominent centrosomal-like particles at spindle poles. producing seven smaller sterile jacket cells and two larger Thereafter, the blepharoplast in each spermatid increases central, undifferentiated antheridial initials. Subsequently, in size and develops a set of electron-lucent channels within the next two hours, both antheridial initials (Hepler, 1976) that appear to be sites of procentriolar undergo four symmetric divisions, producing a total of 32 (pro-basal body) assembly. Early stages of pro-basal body undifferentiated spermatids. During the seventh division, assembly do not reveal any discrete structural features the blepharoplast arises transiently, serving as a focused common to basal bodies (Hepler, 1976; Pennell et al., MTOC. During the ninth division, it arises again to 1986, 1988). Later, however, microtubule triplet assembly synthesize procentrioles within electron-lucent channels is obvious in the periphery of each of the electron-lucent (Mizukami and Gall, 1966; Hepler, 1976). In the next 5.5 channels (Hepler, 1976; Doonan et al., 1986; Pennell et hours, the undifferentiated spermatids undergo a massive, al., 1988). This assembly links the blepharoplast and its synchronous transformation into 32 spermatozoids, each channels with basal body formation. It appears that basal having ~140 cilia (Sharp 1914; Hepler 1976). During this body assembly requires the initial accumulation of transformation, the newly made basal bodies are deployed specific components to form the channels. Then, with the onto the multilayered structure (MLS) from which cilia assembly of microtubule triplets, the basal bodies become emerge (Myles and Hepler, 1977, 1982). The MLS is a recognizable. As the basal bodies form, the blepharoplast series of four parallel striated layers. One function of expands to the point at which it is barely recognizable. In these layers is to organize the microtubules that laminate the nearby cytoplasm, a MLS begins to assemble; and on the mitochondrion and nucleus on one side of the MLS the distal surface of the MLS, the spline microtubules with the basal bodies and cilia on the other side. This start to elongate. Soon thereafter, basal bodies begin to organizational process occurs during the genesis of nine associate with the spline microtubules as the motile or ten cilia-containing gyres at the anterior end of the apparatus forms (Myles and Hepler, 1977). spermatid. Thus, both the blepharoplast and the MLS serve as MTOCs (Hart and Wolniak, 1998). AXONEMAL STRUCTURE AND CILIARY BEAT About 30-40 minutes after imbibition, the single gametophytic cell in the fern microspore undergoes The motile apparatus comprises the basal bodies mitosis and cytokinesis, producing a germ cell and a with their attached ciliary axonemes. The basal bodies are prothallial cell. The prothallial cell, a presumptive attached at their bases to the microtubule ribbon and remnant of a vegetative gametophytic thallus, does not probably to each other by rootlet material, which appears divide further. The germ cell, however, begins to divide, like a floccular, osmiophilic matrix among the basal and its progeny undergo eight successive mitotic divisions bodies. These basal bodies and ciliary axonemes exhibit during the next five hours, ultimately producing 32 all of the features common to homologous “9 + 0”, and “9 spermatids and a set of sterile jacket cells (Sharp, 1914). + 2” arrays in other eukaryotes, except that they lack During the second 5.5 hours after imbibition, each outer dynein arms in their axonemes (Wolniak and Cande, spermatid differentiates into a motile spermatozoid. 1980; Hyams, 1985; Hyams and Campbell, 1985). Each spermatozoid has Inasmuch as ciliary beat frequency is directly related to the abundance of outer dynein arms in a number of motile · a coiled cell body that contains one or more cells (e.g., Gibbons and Gibbons, 1973), it is not mitochondria and an elongated nucleus with surprising that 6-12 Hz ciliary beat frequency in highly condensed chromatin; spermatozoids of Pteridium aquilinum (Wolniak and Cande, 1980) and Marsilea vestita (Hyams and Campbell, 1985) is considerably lower than the beat

Gravitational and Space Biology Bulletin 13(2), June 2000 87 DEVELOPMENT AND MOTILITY IN PLANT SPERMATOZOIDS frequency observed in axonemes that possess this motor gametophytes remain in the single cell stage for extended protein. Hyams' (1985) dynein add-back experiment with periods after imbibition. In contrast, imbibition in á- M. vestita indicates that the axonemal microtubule amanitin does not block the cell division phase of doublets of the fern can properly bind dynein arms and gametophyte development, and each of the spermatids use them to increase beat frequency. undergoes normal elongation and coiling of the cell body. Elongation and coiling are morphogenetic events that REGULATORY CONTROL OVER involve the organized expansion of the cytoskeleton, its DEVELOPMENT OF A MOTILE APPARATUS association with the nucleus and the anterior mitochondrion, and the segregation of nonessential For the past several years, we have been interested cytoplasmic components from those that will reside in the in identifying the components of a basal body and mature gamete. Because we have never seen mature discerning the steps involved in blepharoplast spermatozoids released from the microspore wall that formation/maturation and basal body assembly. Because contains them, it is not yet clear if a fully functional microspores of M. vestita are formed in cells that lack motile apparatus is formed in the presence of á-amanitin. preexisting centrioles, and thus de novo in blepharoplasts Since spermatozoid release fails to occur in these (Sharp, 1914; Mizukami and Gall, 1966; Hepler, 1976; gametophytes, we infer that some transcription is Pennell et al. 1986, 1988), they provide a unique necessary for the late stages of gametophyte development. opportunity to investigate how basal bodies are made. It is obvious, then, to ask just what new proteins are Structural work from several laboratories during the past made, and when. Since tubulin is a major component of 90 years (Sharp, 1914, Mizukami and Gall, 1966; Hepler, the cytoskeleton and motile apparatus of the mature 1976; Myles and Hepler, 1977) has established when spermatozoid, it seemed reasonable to assess whether these events take place and how these structures appear at there were increases in tubulin abundance that could be specific stages during development. linked to specific developmental transitions during Recent work in our laboratory has established that spermiogenesis. If these increases were apparent, then one the microspore is packed with both stored protein and could infer that the synthesis of a particular isoform of stored mRNA (Hart and Wolniak, 1998, 1999). In our tubulin might regulate progression of spermatozoid initial studies—extensions of work from the Hyams lab— maturation. In the late 1980s, Pennell and coworkers we treated populations of microspores with the (1988) showed maximal tubulin abundance in M. vestita transcriptional inhibitor, á-amanitin, or with the at the end of the middle stages of spermiogenesis that translational inhibitor cycloheximide. In a fashion similar preceded the assembly of ciliary axonemes. By to the earlier reports (Pennell et al., 1986, 1988), we performing their experiments at 30oC, they were able to found that no spermatozoids were released for extended compress the process of spermiogenesis into about 5.5 to periods. We isolated proteins that were present in the 6 hours. We repeated their experiments, but we incubated microspores at various stages of development, and we our cells at 20oC, thus doubling the time of development. could not detect any significant differences on our In a series of pulse-labeling studies (Hart and Wolniak, electrophoresis gels throughout the entire 11-hour period 1998), our autoradiograms of (35S)-labeled proteins that of spermiogenesis. Surprisingly, we also saw no were isolated from gametophytes at specific intervals differences in proteins that were isolated at various times after imbibition revealed measurable increases in the from microspores that were inbibed with either á- abundance of newly made á- and â-tubulin only during amanitin or cycloheximide. We then asked whether any late stages of spermiogenesis, which coincided with the new proteins were being made in the male gametophytes. assembly of ciliary axomenes. For gametophytes imbibed We treated the microspores with [35½]-methionine during with cycloheximide, we saw no new protein labeling. For imbibition and isolated proteins at various time points gametophytes imbibed with á-amanitin, tubulin labeling thereafter. We saw only approximately 30 discrete bands (and accumulation) was evident, revealing that new that were labeled on the autoradiograms, and we observed translation utilized mRNAs already present in the spores. the same banding pattern in cells imbibed in the presence An alternative to an absolute increase in total á- or â- of á-amanitin. As expected, there were no labeled protein tubulin abundance during spermiogenesis through new bands in isolates obtained from microspores imbibed in translation could be some sort of posttranslational the presence of cycloheximide. From these results, we modifications of existing proteins that would confer concluded that there is considerable stored protein and physiological differences to specific tubulins, so that they mRNA in the dry microspore. We also concluded that a could participate in particular events during spermatid relatively small number of proteins are synthesized during development. Recently, Hoffman and Vaughn (1995b) spermiogenesis, and most of these new proteins are found posttranslational differences in tubulins located in translated from stored mRNAs (Hart and Wolniak, 1998). different structures of the spermatozoids of Ceratopteris Clearly, certain specific mRNAs are translated at richardii. We (Hart and Wolniak, unpublished) screened precise times during development, and their presence in our immunoblots with some of the same antibodies, but the spermatogenous cell mass appears to be essential for were unable to detect measurable changes in acetylation continued development of the gametes. Recently, we or tyrosination that correlated with the formation of the (Klink and Wolniak, unpublished) have found that cytoskeleton or the MLS. With the level of resolution cycloheximide blocks development completely, and

88 Gravitational and Space Biology Bulletin 13(2), June 2000 DEVELOPMENT AND MOTILITY IN PLANT SPERMATOZOIDS provided by our antibodies and labeling experiments, it content from microspores imbibed with cycloheximide or appears that there is abundant á - and â -tubulin present in á-amanitin and found that the marked increase in centrin the dry spore from the onset of gametophyte protein abundance arises from the translation of stored development. New á- and â-tubulin synthesis seems to mRNA rather than from the translation of newly made rely on translation from stored transcript, and the bulk of transcripts. this new translation occurs during axonemal formation. We then made a cDNA library from mRNA that had How then, are proper amounts of these already abundant been isolated from microgametophytes at all stages of tubulins recruited into the blepharoplast for basal body development (Hart and Wolniak, 1999). A major, and assembly, or onto the MLS for the formation of the most tedious part of this effort was devising methods of spline? mRNA isolation from this organism (Hart, unpublished). We (Hart and Wolniak, 1998) looked into increases By screening the library with a C. reinhardtii centrin in ã-tubulin that could be linked with specific stages of cDNA (from J. Salisbury), we were able to isolate a clone spermiogenesis. This protein has been localized in that encoded a full-length centrin, named MvCen1 (Hart centrosomes and at the foci of microtubule organizing and Wolniak, 1999). In a variety of organisms, sequence activity (Zheng et al., 1995), having been linked with the analysis of MvCen1 revealed a putative protein of proper development of the MLS in plant spermatozoids molecular weight and with all of the characteristic (Hoffman et al.,1994). On immunoblots, we found that signature motifs and EF-hands diagnostic of centrins. We here, too, the abundance of ã-tubulin (antibodies provided then asked if MvCen1 could be translated in vitro from by B. Oakley) remained essentially constant during the mRNA isolates obtained from gametophytes imbibed for entire process, and that neither cycloheximide nor á- varying lengths of time. We used a wheat-germ extract to amanitin had any effect on the abundance of this antigen translate proteins, and immunoblots revealed that an anti- in the gametophytes. Thus, we concluded that changes in centrin antibody would bind to a single low-molecular the abundance of these ã-tubulins were not involved in weight polypeptide band, present in mRNA isolates of regulating the rate of spermatozoid formation or microspores, but only if the mRNAs came from spores maturation. that had been imbibed for more than 30 minutes. This With the tubulins exhibiting uniform abundance result suggests that some sort of mRNA processing through time and development, it became necessary to ask probably takes place during the first 30 minutes after what other microtubule-associated or basal-body- imbibition. We currently have no clear indication of what associated proteins might be present in and around the kind of RNA processing occurs during the interval prior motile apparatus, whose presence might control or even to the first mitotic division. limit the rate of spermatid differentiation. We looked for likely candidates, hypothesizing that the synthesis of FUTURE DIRECTIONS FOR DEVELOPMENTAL relatively few regulatory proteins could control the STUDIES WITH MARSILEA timing, extent, and size of important structures like the Marsilea vestita provides us with a unique blepharoplast and the MLS. Hoffman and Vaughn opportunity to study the rapid formation of basal bodies (1995a) had shown that centrin—a protein long known to and a complex ciliary array in cells that originally had no be associated with the basal bodies, flagellar rootlets, and centrioles. With populations of cells that develop microtubule organizing centers in a variety of cells synchronously, we can look into how the incorporation of (Weich et al., 1996)—accumulated around blepharoplasts newly made proteins regulates the rate and extent of basal and basal bodies of the spermatozoids of Ceratopteris body assembly and the formation of ciliary axonemes. richardii. We were able to obtain several anti-centrin Radiolabeling newly made proteins (Hart and Wolniak, antibodies (from J. Salisbury) directed against the centrin 1998) reveals that only about 40-60 new proteins appear from Chlamydomonas reinhardtii. We observed a striking in sufficient abundance to be seen as discrete bands on increase in the centrin antibody binding of a ~20 kDa electrophoretic gels, but that many of the new proteins protein band on our western blots, coinciding with the present in low-copy numbers can be detected by appearance of blepharoplasts about four hours after immunoblotting (Klink and Wolniak, unpublished). We imbibition (Hart and Wolniak, 1998). This result, which have now screened approximately 50 different antibodies contrasted to the uniform tubulin abundance found in that are directed against a variety of centrosomal, protein isolates from M. vestita gametophytes imbibed for cytoskeletal, and axonemal antigens. On immunoblots, a different lengths of time, provided an implicit indication sizable number of these antibodies exhibit binding to that increased centrin protein is linked to blepharoplast single polypeptide bands, and some reveal increases in formation. In gametophytes imbibed less than four hours, antigen abundance that correlate with development during we saw the weak anti-centrin antibody labeling of a band spermiogenesis. with a molecular weight that was slightly greater than that We have generated a variety of additional nucleotide of the major centrin band that appears later during probes and reagents to examine the impact of new spermiogenesis. This minor band may be a translation on the formation of the motile apparatus in M. phosphorylated centrin. Further investigation showed that vestita. Using assorted probes, we screened our cDNA the major centrin protein accumulation resulted from the library from microspores at all stages of development. translation of stored mRNAs. We analyzed protein Initial screens enabled us to characterize a centrin cDNA

Gravitational and Space Biology Bulletin 13(2), June 2000 89 DEVELOPMENT AND MOTILITY IN PLANT SPERMATOZOIDS that was present in the cells (Hart and Wolniak, 1999). A change their swimming behavior with shifts in free series of in vitro translation experiments established the calcium concentration but uniform levels of L-malate. A efficacy of our approach using heterologous rapid, uniform shift of calcium or L-malate was found to immunoprobes. Recently, we have found that antisense induce subtle changes in the helical swimming path of technologies can be used to investigate how the randomly swimming populations of these cells, while an translation of particular mRNAs affects abrupt increase in free calcium or L-malate resulted in a microgametophyte development. We can use the specific more tightly coiled helical swimming pattern (Wolniak, inhibition of the translation of stored mRNA as an assay unpublished observations). The tightening of the helical to determine how a particular gene product controls swimming pattern was not caused by a change in ciliary formation of the blepharoplast and the motile apparatus beat frequency or apparent beat shape (Wolniak and (Klink and Wolniak, 1999, Tsai and Wolniak, 1999). Cande, 1980; Wolniak, unpublished observations). Through antisense strategies, we have determined that Instead, swimming changes appear to relate to changes in centrin plays a key role in the formation of the the moment of gamete rotation: faster cell rotation blepharoplast (Klink and Wolniak, 1999), and that resulting in tighter swimming patterns. A likely blepharoplast formation—but not centrin synthesis— mechanism for altered periods of rotation may come from depends on mitotic divisions and partitioning of the a change in the coiling of the cell body, which would spermatogenous mass from the sterile jacket cells of the result in a change in the spatial relationship of adjacent gametophyte (Tsai and Wolniak, 1999). We have cilia along the dorsal side of the cell. This change in cell performed a series of cDNA isolations from the library, body coiling would effectively change the “pitch on the and sequenced these inserts. We have over 100 of these propeller,” thereby creating a change in swimming. clones sequenced, and ~45% encode proteins of known A mechanism that reasonably explains changes in function in cell cycle regulation, in the development of cell body coiling is observed in the microtubule ribbon cytoskeletal arrays, and in the formation of a motile that overlies the nucleus. Sliding of adjacent microtubules apparatus. These probes set the stage for the future work in this ribbon could be manifested as a change in cell of identifying specific translational events at particular body coiling, and consequently as major changes in times during spermiogenesis for the formation of particles rotation and swimming. In bracken fern, rapid changes in or structures that we recognize as the blepharoplast, basal the concentration of L-malic acid (in the presence of 1 bodies, the MLS, and ciliary axonemes. mM calcium) results in changes in swimming patterns, but without any alteration of ciliary beat frequency or beat CHEMOTAXIS AND THE CONTROL OF shape (Wolniak and Cande, 1980; Wolniak, unpublished SWIMMING IN PLANT SPERMATOZOIDS observations). If changes in cell body coiling accompany chemoattraction, they can easily be detected in bracken The spermatozoid is a remarkable cell that can swim fern spermatozoids. The positioning of basal bodies along considerable distances through films of water after its the dorsal side of the bracken spermatozoid can be viewed release, altering its swimming path in response to certain by immunofluorescence microscopy (Marc and Gunning, kinds of chemical stimuli. Pfeffer, in his classic studies of 1986). In addition to a linear array of basal bodies placed bracken fern spermatozoids (1884), coined the word along one edge of the ribbon, there are two additional chemotaxis for the rapid changes in the swimming axonemes positioned along the ribbon at regular intervals. direction and patterns exhibited by these gametes in Three basal bodies are thus aligned with each other in an response to L-malic acid, a compound he found present in axis perpendicular to that of the microtubules of ribbon. If archegonial exudates. Pfeffer postulated that chemicals sliding is occurring in the microtubule ribbon, then secreted by the female gamete (or gametophyte) acted as chemotactically-induced changes in cell body coiling attractants for the motile spermatozoids. Throughout the should be detectable by shifts in the relative positions of last century, the study of chemotaxis in fern these basal bodies. These kinds of analyses are planned spermatozoids has continued at irregular intervals for Pteridium aquilinum as spores become available. (Rothschild, 1956, Brokaw, 1957, 1958, 1974; Wolniak Alternatively, the same processes can be addressed in and Cande, 1980). Marsilea vestita, where gametes are available on a year- Bracken fern spermatozoids swim in a helical path, around basis. and the cells rotate as they swim. Pfeffer (1884) showed that concentration gradients of L-malic acid are sufficient CONCLUSIONS AND FUTURE OUTLOOK to induce aggregations of spermatozoids in randomly swimming populations of cells. Rothschild (1956) Plant spermatozoids provide unusual opportunities presented a compelling case suggesting that chemotactic to study the formation of a motile apparatus and the swimming behavior cannot simply result from faster or regulation of a complex motile cell that responds to even directed swimming. Instead, it involves two signals in its environment. By studying the precisely components: a vectorial pattern of swimming that results timed developmental process of spermiogenesis in in the cell getting closer to the attractant source, followed Marsilea vestita, we have taken advantage of prior by a reduced rate of swimming, essentially a “hovering structural studies and applied modern molecular and response,” that keeps the cell in close proximity to the structural techniques to address questions that focus on source. Brokaw (1974) showed that the cells would how basal bodies are formed. In the microspores, there

90 Gravitational and Space Biology Bulletin 13(2), June 2000 DEVELOPMENT AND MOTILITY IN PLANT SPERMATOZOIDS are stores of proteins and mRNAs. We have found that Doonan, J.H., Lloyd, C.W. and Duckett, J.G. 1986. Anti- development appears to rely on the translation of tubulin antibodies locate the blepharoplast during particular stored mRNAs at specific times. It is reasonable spermatogenesis in the fern Platyzoma microphyllum to hypothesize that the synthesis of proteins like centrin R.BR.: a correlated immunofluorescence and electron- controls the rates and extents of development during microscopic study. Journal of Cell Science 81:243-265. critical stages of spermiogenesis. Mature fern spermatozoids exhibit profound changes in swimming Duckett, J.G. 1973. An ultrastructural study of the patterns in response to their environment. These cells may differentiation of the spermatozoid of Equisetum. Journal provide insight on mechanisms of signal reception and the of Cell Science 12:95-129. subsequent regulation of motility in a complex, ciliated eukaryote. Gibbons, B.H. and Gibbons, I.R. 1973. The effect of partial extraction of dynein arms on the movement of Acknowledgements reactivated sea urchin sperm. Journal of Cell Science 13:337-358. We gratefully acknowledge support for this work from a National Science Foundation Grant (MCB- Gifford, E.M., Jr., and Lin, J. 1975. Light microscope and 9809950). We appreciate the generous provision of ultrastructural studies of the male gametophyte of Ginkgo probes and antibodies from many members of the biloba: the spermatogenous cell. American Journal of cytoskeletal, centrosomal, and axonemal research Botany 62:974-981. communities. For studies mentioned in this paper, we make a special note to thank Jeff Salisbury and Berl Hart, P.E. and Wolniak, S.M. 1998. Spermiogenesis in Oakley for probes, and Peter Hepler for supplying us with Marsilea vestita: a temporal correlation between centrin our initial batch of sporocarps. Finally, we thank Kevin expression and blepharoplast differentiation. Cell Motility Vaughn, Peter Hepler, Zane Carothers, Zac Cande, and the Cytoskeleton 41:39-48. Jeremy Hyams, Sue Wick, and Diana Myles for input and ideas over the years that contributed importantly to the Hart, P.E. and Wolniak, S.M. 1999. Molecular cloning of initiation of our new studies on spermiogenesis in a centrin homolog from Marsilea vestita and evidence for Marsilea. its translational control during spermiogenesis.

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Columella Cells Revisited: Novel Structures, Novel Properties, and a Novel Gravisensing Model L. Andrew Staehelin1*, Hui Qiong Zheng1, Thomas L. Yoder2,4, Jeffrey D. Smith2,5 and Paul Todd3 1Departments of Molecular, Cellular and Developmental Biology; 2Aerospace Engineering Science and 3Chemical Engineering, University of Colorado, Boulder CO; 4present address: USAF Academy, Department of Aeronautical Engineering, USAFA, CO; 5present address: NASA Ames Research Center, Moffett Field CA

ABSTRACT Despite these advances, we are still far from understanding in mechanistic terms how the physical A hundred years of research has not produced a clear process of statolith sedimentation is converted into a understanding of the mechanism that transduces the energy associated with the sedimentation of starch-filled amyloplast biochemical signal that can be used to bring about a statoliths in root cap columella cells into a growth response. growth response. While reviewing this state of affairs, we Most models postulate that the statoliths interact with have become aware that many of the critical observations microfilaments (MF) to transmit signals to the plasma underlying current assumptions about columella cell membrane (or ER), or that sedimentation onto these organelles structure and function are based on experiments that are produces the signals. However, no direct evidence for statolith- no longer state-of-the-art. To this end, we have MF links has been reported, and no asymmetric structures of undertaken a series of studies to reevaluate the structural columella cells have been identified that might explain how a and physiological foundations upon which current o root turned by 90 knows which side is up. To address these and theories of columella cell function are built. These studies other questions, we have (1) quantitatively examined the effects have included of microgravity on the size, number, and spatial distribution of statoliths; (2) re-evaluated the ultrastructure of columella cells in · microgravity experiments designed to deter- high-pressure frozen/freeze-substituted roots; and (3) followed mine if plants do indeed “sense” the absence of the sedimentation dynamics of statolith movements in reoriented gravity, as evidenced by measurable compensa- root tips. The findings have led to the formulation of a new tory growth responses (Smith et al., 1997); model for the gravity-sensing apparatus of roots, which envisages the cytoplasm pervaded by an actin-based cytoskeletal · experiments in which we have observed and network. This network is denser in the ER-devoid central region quantitatively analyzed the dynamics and of the cell than in the ER-rich cell cortex and is coupled to trajectories of sedimenting statoliths in living receptors in the plasma membrane. Statolith sedimentation is cells (Yoder, 1999; Yoder et al., personal postulated to disrupt the network and its links to receptors in communication); some regions of the cell cortex, while allowing them to reform · experiments in which we have reassessed the in other regions and thereby produce a directional signal. architecture of columella cells and how this

architecture is affected by cytoskeleton- disrupting drugs (Zheng and Staehelin, sub- INTRODUCTION mitted).

Columella cells have occupied a central position in These experimental studies, as well as a series of physical gravitropism research ever since Nemec (1900) and modeling experiments, have resulted in a number of Haberlandt (1900) reported on the gravity-dependent unexpected findings that cannot be reconciled with location of their starch-rich amyloplasts. Since then, current models of columella cell function. This, in turn, numerous researchers have reported on columella cell has led us to formulate the new model of the gravisensory structure, development, physiological properties and their apparatus of columella cells described in this report. responses to cytoskelton-disrupting drugs, and on how these features are related to columella cell gravity-sensing CHANGES IN STATOLITH MASS AND functions (reviewed in Björkman, 1988; Sievers et al., GROUPING IN COLUMELLA CELLS OF PLANTS 1991; Sack, 1991,1994, and 1997; Konings, 1995; GROWN IN MICROGRAVITY Baluska and Hasenstein, 1997; Chen et al., 1999). These studies have shown that The signaling capability of amyloplast statoliths is based on the principle that their buoyant weight must be · columella cells arise from the calyptrogen cells sufficiently large to overcome other interfering forces, of the root apical meristem; such as thermal motion, cytoskeletal tension, and · columella cells possess a polar organization; cytoplasmic streaming (Björkman, 1988). At the same · amyloplasts do indeed act as statoliths; time, it is obvious that—with amyloplasts occupying only · the distribution and the mobility of a small volume of the gravity-sensing cells—these cells amyloplasts is dependent on the cytoskeleton; do not produce the largest possible amyloplast statoliths. · sedimentation of the statoliths onto sheets of ER Thus, plants appear to regulate the buoyant weight of their cisternae could play a role in gravity perception statoliths for optimal sensory perception. This hypothesis and/or the gravity signaling response. can be tested by changing the magnitude and direction of gravitational stimulation and determining if the cells respond by altering the mass or number of their statoliths *Correspondence to: L. Andrew Staehelin: e-mail: We have performed such experiments by comparing the [email protected]

Gravitational and Space Biology Bulletin 13(2), June 2000 95 COLUMELLA CELLS REVISITED

differences were seen in plants grown the same length of time under different simulated gravity conditions.

In addition, serial section reconstruction experi- ments provided quantitative information about the size, shape, volume, and spatial relationships of groups of columella cells, as well as the size and spatial distribution of statoliths and the size and position of the nuclei (Figure. 1). The most striking and potentially most important finding was that, in plants of equivalent age, the Figure 1. Three-dimensional Reconstruction of Contiguous size of the amyloplasts increased in microgravity relative Clover Root Columella Cells Grown under (a) 1-g Control, to the amyloplasts of ground-grown plants, but remained (b) Microgravity, and (c) 2 Days of Growth in 1-g, Followed constant in seedlings grown on the clinostat. The number by a Third Day of Clinorotation. The light spheres correspond to the amyloplast statoliths; the dark spheres to the nuclei. Note of amyloplasts per cell was found to be proportional to that in the microgravity-grown cells, the statoliths are clustered cell volume in both ground and microgravity-grown towards the cell center and individual statoliths appear bigger plants, and to be decreased in clinorelated specimens. than those in the 1-g control cells. (From Smith et al., 1997) Moreover, under none of the conditions tested did the amyloplasts become randomly organized. Thus, in microgravity, they were grouped near the cell centers; in statoliths and their cellular distribution in clover seedlings clinostat-treated cells, they appeared more dispersed but grown in 1 g, microgravity, and on the clinostat (Smith et still retained some grouping. al., 1997; Smith et al., 1999). Based on these findings, it appears that plants do We germinated and grew seedlings of white clover sense the absence of gravity stimulation when grown (Trifolium repens)—contained in the Fluid Processing under microgravity conditions, as evidenced by their Apparatus developed by BioServe Space Technologies attempt to compensate for this loss by increasing the mass and described by Smith et al. (1999)—between water- of the statoliths. These findings support the hypothesis moistened filter paper for 24, 40 and 72 hours. The Fluid that the size of amyloplasts in columella cells is regulated Processing Apparatus let us hydrate the seedlings with by a feedback control system that is integrated into the distilled water and grow them in orbit under sterile gravity-sensing/signaling pathway. The observed group- conditions, then chemically fix them with 1% ing of statoliths suggests that plants might increase the glutaraldehyde at the end of the growth period. For signal-to-noise ratio of their amyloplast statolith gravity- clinostat experiments, we rotated the seedlings only sensing system by maintaining the statoliths in a grouped during the final 24 hours of their growth (since longer configuration. The mechanism that promotes this exposure to the clinostat has led to severe perturbations in grouping has yet to be elucidated, but could involve some kind of tethering between the statoliths or their exclusion the architecture of the columella cells). When the glutaraldehyde-fixed samples were removed from the apparatus, the root tips were excised, fixed in osmium tetroxide, dehydrated, and embedded in Spurr's resin. We serially sectioned the root tips (0.5 µm thick), digitized the light microscopic images of the columella cells, and reconstructed groups of adjacent cells using the Reconstruction of Serial Sections (ROSS) software developed at the Biocomputation Center (NASA Ames Research Center, Moffett Field, CA) (Figure 1). Some root tips were also sectioned for electron microscopic analysis to determine the starch content and the ultrastructural features of the statolith amyloplasts. The principal findings of these studies were as follows:

1. Statolith amyloplasts differed in their architecture from starch-storage amyloplasts by having a 0.1- to 0.3-µm-thick boundary layer Figure 2. Electron Micrograph of a Cryofixed/Freeze- around the starch granules. Substituted Tobacco Columella Cell. (Membranous organelles 2. The boundary layer appeared to link the different are traced to highlight their distribution.) The tubular ER is starch granules into a single structural unit confined to a tight band underlying the plasma membrane. With within the amyloplasts. the exception of the mitochondria (M), all of the other 3. Plants grown 40 hours contained more starch per organelles are limited to the central, ER-devoid region of the amyloplast than those grown 72 hours; but no cell. Am=amyloplast statolith; G=Golgi; V=vacuole.

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substituted columella cell in which all of the membranous organelles have been traced to highlight their spatial distribution. Most striking is the presence of a tubular ER network in the cell periphery that exhibits a very sharp transition to the ER-free central region of the cell. More- over, due to the compactness of this tubular ER network, all of the major membranous organelles—such as amyloplasts, Golgi, and vacuoles—are excluded from the ER-rich cell cortex and confined to the central region. Only mitochondria are observed in both of these regions. The cytosol of the central region is also unusual in that it occupies more of the cytoplasmic volume than other root

tip cells and is devoid of actin filament bundles. Instead, it Figure 3. Electron Micrograph of Two Interconnected Nodal appears to be composed of randomly oriented single actin ER Membrane Domains in a Cryofixed/Freeze-substituted filaments that, together with other molecules, form a Tobacco Columella Cell. Sheetlike rough ER cisternae appear network-like cytoskeletal matrix. attached along their edges to the ~100 nm-in-dia. nodal rods. A novel form of ER discovered in our cryofixed Am=amyloplast statolith; G=Golgi stack; M=mitochondrion. specimens is illustrated in Figure 3. Based on its

architecture, we have termed this "nodal ER." These unique domains consist of a central "nodal rod" element from a gel-like cytosolic cytokeletal matrix. We postulate to which three to eight (usually seven) rough ER cisternae that the latter type of system is responsible for the are attached. The edge-on attachment of the ER grouping behavior (discussion follows). Our data also membranes to the rod appears to be partly responsible for provide further evidence that clinostats constitute a less the sheetlike organization of the ER membranes, an than ideal system for mimicking microgravity, since the organization that stimulates the binding of polysomes. We chronic gravity signaling the overload to which plants have mapped the spatial distribution of these nodal ER grown on clinostats are subjected leads to harmful domains within central and flanking columella cells and changes in columella cell architecture, as already reported found that, in both cell types, most of these domains are by Hensel and Sievers (1980) and Guikema et al. (1993). localized into cortical patches near the equator of the cells. In flanking cells, they also occur in the basal region. NODAL ER, A NOVEL FORM OF ER FOUND Careful analysis of the spatial relationship among nodal EXCLUSIVELY IN COLUMELLA CELLS ER domains, the tubular ER in the cell cortex, and the Root cap columella cells exhibit a distinct amyloplasts has shown that these specialized ER domains architectural polarity, postulated to be related to their (1) are located at the interface between the cortical tubular gravity-sensing function. This polarity manifests itself in ER and the central domain, and (2) constitute a physical vertically oriented roots with the nuclei located adjacent barrier to the amyloplasts, which prevents them from to the top (proximal side) and the sedimented amyloplast approaching the tubular ER and the plasma membrane. statoliths concentrated close to the bottom (distal end) of On a more global scale, the highest density of nodal ER the cells. Electron micrographs of columella cells have domains is seen along the outer walls of the flanking colu- also demonstrated that most of the ER is located in the mella cells (Figure 4). This type of asymmetric arrange- cell periphery (Sack and Kiss, 1989). The observed sedi- ment is what one might expect of a structural system that mentation of statoliths onto the surface of sheets of distal ER membranes has led to the hypothesis that statolith-ER interactions could mediate the gravity response. Chemical fixation is known to frequently produce artifactual images of cellular structures (Gilkey and Staehelin, 1986). Therefore, because virtually all of our structural knowledge of columella cells has come from studies of chemically fixed cells, we have reexamined the ultrastructure of columella cells of tobacco (Nicotiana tabaccum) seedlings preserved by high-pressure freezing/ freeze-substitution techniques (Kiss and Staehelin, 1995). These studies have not only produced a number of new insights into the spatial distribution of organelles and the nature of the cytosolic matrix, but they have also led to Figure 4. Distribution of Nodal ER Domains (Stars) in a the discovery of a new form of ER that is unique to Reconstructed 0.2-µM-Thick Cross-section through a columella cells and has not been reported previously. Tobacco Root Tip at the Level of the Second-Tier Columella Cells. Note that the majority of the nodal ER domains in the Figure 2 depicts an electron micrograph of a columella cells (central white cells) are found in the outer cortex longitudinal section through a cryofixed/freeze- region of the flanking cells.

Gravitational and Space Biology Bulletin 13(2), June 2000 97 COLUMELLA CELLS REVISITED

Figure 5. Tensegrity-based Model of the Gravisensing Apparatus of Columella Cells. The model shows an actin-based cytoskeletal network (crosshatched lines) that

· pervades the entire cytoplasm; · is denser in the cell center than in the cell cortex; · is coupled to stretch-sensitive receptors in the plasma membrane.

We postulate that the model’s numbered statoliths are not linked to the cytoskeletal network. They function to activate/inactivate the receptors in the plasma membrane by locally disrupting the network as they sediment to a new location, allowing new connections to be formed at sites they vacate. The asymmetrically distributed nodal ER domains shield local plasma membrane sites from approaching statoliths, and may thereby provide a directionality vector to the sensing system. The interface between the ER-rich cortical and the ER-devoid central regions of the cell constitutes a plane of weakness in the cytoskeletal network, and the statoliths travel preferentially within this region. In side-to-side sedimentation experiments (e, f, and g), the statoliths first move horizontally towards forming "channels" before they pass through the channels to the lower side of the cell.

enables the columella cells to produce an asymmetric vertically through these channels (Figure 6B). gravisensing signal, leading to expansive growth on the Furthermore, unlike the distal-to-side sedimenting upper side and to a reduction in cell expansion on the statoliths that displayed their highest velocity at the lower side of a root turned by 90o. This role has been beginning and then slowed down monotonically as they incorporated into the new gravisensing model depicted in approached the lower plasma membrane, statoliths that Figure 5. crossed the central region were slowest at the beginning and then accelerated once they reached a channel. In the AMYLOPLAST SEDIMENTATION DYNAMICS IN presence of the actin-disrupting drug cytochalasin D at a MAIZE COLUMELLA CELLS concentration of 100 µM (which does not completely

disrupt all actin filaments), the channeling effect was less We have used videomicroscopy to analyze the pronounced, suggesting that the channeling is related to sedimentation behavior of amyloplast statoliths in living the presence of an actin-based cytoskeletal network in the columella cells of Zea mays seedlings (Yoder, 1999; central region of the cell. In such cells, the statoliths were Yoder et al., personal communication). Control cells also seen to accelerate sooner than in the control cells, displayed no cytoplasmic streaming, and their statoliths suggesting that the actin-based network resists penetration exhibited only random movements characteristic of by the statoliths and that the slow initial acceleration was Brownian motion. In roots rotated by 90o, the statoliths related to the formation of the channels. Ultrastructural moved downward along the distal wall at an average studies of high-pressure frozen/freeze-substituted cells velocity of 1.7 µm/min and then spread out along the new revealed that maize columella cells possess the same type cell bottom (Figure 6A). After this settling, the roots were of tubular cortical ER system as the tobacco cells rotated along their longitudinal axis by 180o to position discussed earlier. This finding suggests that the low- the statoliths along the top wall. When forced to traverse resistance statolith pathway in the cell periphery, which the complete width of the cells, the statoliths displayed an gives rise to the horizontal motion in side-to-side unexpected behavior. Instead of settling as individual sedimentation experiments, could be associated with the units across the cytoplasm, the majority initially moved interface between the ER-rich cortical and the ER-devoid horizontally towards "channels" and then translocated central region of these cells.

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A NEW MODEL OF THE GRAVITY-SENSING APPARATUS OF COLUMELLA CELLS

The sedimentation behavior of statoliths in maize columella cells described in the preceding section is difficult to reconcile with current hypotheses for the gravisensing mechanism of columella cells—most notably with models in which the statoliths are postulated to be tethered to receptors in the ER or the plasma membrane through actin filaments (Björkman, 1988; Sievers et al., 1991; Sack, 1994; Baluska and Hasenstein, 1997). Our findings lead us to formulate a new hypothesis to explain how the sedimentation of statoliths in reoriented columella cells may locally stimulate or inhibit stretch-activated receptors in the plasma membrane to produce a gravity signal. The model in Figure 5 envisions the entire cytoplasm pervaded by an actin-based cytoskeletal network that is denser in the ER-devoid central region of the cell than in the ER-rich peripheral cytoplasm. This network is postulated to be attached to stretch-sensitive receptors in the plasma membrane and to be associated with microtubules and membranous organelles to form a tensegrity-based force interaction system (Ingber, 1993 and 1998). In this scheme, the sedimentable amyloplast statoliths are not bound to any of the components of the cytoskeletal network; instead, they are postulated to function by locally disrupting the network. Thus, Figure 6. Tracings of Sedimenting Amyloplast Statoliths in displacement of the statoliths is envisioned to produce a Maize Columella Cells. (A) Typical distal-to-side movement signal by disrupting links from the cytoskeletal network to profile of 7 statoliths with 15 seconds between frames. (B) the plasma membrane in some places and simultaneously Typical side-to-side movement profile with 9 statoliths falling allowing such links to form elsewhere (depicted in Figure the entire width of the columella cell with 30 seconds between 5). Since all of the tensional elements are mechanically frames. Note that most but not all statoliths pass through a coupled to each other, this redistribution of cytoskeletal- common vertical channel. to-plasma membrane links also simultaneously produces more global tensional changes throughout the network, CONCLUSIONS which may aid in the integration of the signals of individual receptors. Consistent with this idea of the While confirming many classical ideas about how importance of actin filaments for gravitropic signaling, columella cells use amyloplast statoliths to sense gravity, Guikenia and Gallegos (1992) have reported that, when the studies described in this paper have also yielded cytochalasin D is applied in agar blocks at greater than findings that challenge several proposals concerning the 40ìg/ml to maize root caps, the roots lose their ability to actual energy transduction mechanism. In particular, our directionally reorient themselves in response to a change data are difficult to reconcile with the concept of tethered in the gravitational field. Furthermore, since the primary statoliths, including how such statoliths may transmit the changes would involve redistribution of links to the physical force of sedimentation to stretch receptors in the plasma membrane, the directionality of the signaling plasma membrane. Most notably, our data suggest that could be refined by the distribution of nodal ER domains that may locally shield some links from the statoliths’ 1. the statoliths produce a signal by locally effects on the cortical cytoskeleton. This model makes a disrupting a tensegrity-based cytoskeletal number of predictions that can be experimentally tested. network; For example, the observed clustering of amyloplasts near 2. specialized ER domains could modify the the center of columella cells of microgravity-grown network disruption signals to add a direction- ality component to the sensing system. seedlings is predicted to result from the exclusion of the statoliths from the surrounding cytoskeletal network and Thus, when these statoliths are induced to sediment to a not from the tethering of the statoliths to each other via new location, the translocation causes a disruption of the actin-filament links. This and other predictions will be cytoskeletal network-to-plasma membrane-receptor links tested by using optical tweezers to measure the forces in some places and to the reformation of such links in needed to displace individual and grouped statoliths in others. The combination of these two sets of stimuli then different regions of central and flanking columella cells.

Gravitational and Space Biology Bulletin 13(2), June 2000 99 COLUMELLA CELLS REVISITED would form the basis of an integrated gravisensing Nemec, B. 1900. Über die Art der Wahrnehmung des response. Schwerkraftes bei den Pflanzen. Berichte der Deutschen Botanischen Gesellschaft 18:241-245. Acknowledgements

Sack, F.D. 1991. Plant gravity sensing. International This work was supported by grants NAG 5-3967 and Review of Cytology 127:193-252. NCC 8-131 from the National Aeronautics and Space

Administration Sack, F.D. 1994. Cell biology of plant cell gravity

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