Theories and models on the biology of cells in space

Adv. Space Rev. Vol. 17. No. 6/7. pp. (6/7)3-(6/7)10, 1996 Copyright 0 1995 COSPAR Printed in Great Britain. All rights reserved. 0273-I 177196 $9.50 + 0.00 0273-1177(95)00606-0

THEORIES AND MODELS ON THE BIOLOGY OF CELLS IN SPACE P. Todd* and D. M. Klaus** * Department of Chemical Engineering, Campus Box 424, University of Colorado, Boulder, CO 80309-0424, U.S.A. ** Department of Aerospace Engineering Sciences, Campus Box 424, University of Colorado, Boulder, CO 80309-0424, U.S.A.

ABSTRACT

A wide variety of observations on cells in space, admittedly made under constraining and unnatural conditions in many cases, have led to experimental results that were surprising or unexpected. Reproducibility, freedom from artifacts, and plausibility must be considered in all cases, even when results are not surprising. The papers in the symposium on ‘Theories and Models on the Biology of Cells in Space” are dedicated to the subject of the plausibilityof cellular responses to gravity -- inertial accelerations between 0 and 9.8 m/s2 and higher. The mechanical phenomena inside the cell, the gravitactic locomotion of single eukaryotic and prokaryotic cells, and the effects of inertial unloading on cellular physiology are addressed in theoretical and experimental studies. INTRODUCTION Attempts to unify the field of gravitational cell biology could take many directions. Theories involving extracellular transport /2/, stretch-sensitive membrane channels /2/, organelle sedimentation /3/, center of gravity in swimming cells /4/, cytoskeletal tensegrity /5/ or elasticity /6/, symmetry breaking (bifurcation theory) /7/, and electrical phenomena /8,9/ have been offered. It is proposed that these theories are in some cases subsets of one another, and in some cases related to the “gravitational taxonomy” of cells with different specific functions. Therefore, the subjects of gravitational taxonomy of cells, symmetry breaking and signal transduction are treated as unifying principles in gravitational cell biology. In the following discussion, two specific examples are emphasized: The extracellular transport model applied to bacterial cells in unstirred suspensions as an example of signal transduction and the symmetry-breaking model applied to the eukaryotic cytoskeleton. A GRAVITATIONAL

TAXONOMY OF CELLS

Attempts have been made to divide the effects of gravity on cells into “direct” and “indirect”. These categories are difficult to define operationally and seem to have different meanings in different biological systems. The existence of a variety of gravitational responses that are organism dependent suggests that a “taxonomy” of biological responses exists, in which there is a limited number of cell-environment combinations that can be placed in categories. The

WV3

P. Todd and D. M. Klaus

WJM

following list is offered: 1. Prokaryotic unicellular organisms 2. Swimming unicellular eukaryotes 3. Plant cells that respond to forces and gradients 4, Suspended cells from m~ticellul~ animals and plants 5. Attached animal cells in vitro 6. Attached animal cells in vivo For example, cases 2 and 3 could be called “intrinsically” gravisensing cells, while all others might be considered “fortuitous“ gravity sensors /lo/. All but case 3 might be considered to be dominated by extracellular transport. Cases 1 and 2 may be capable of selfpropulsion and tactic motions. In some of these categories there exist cell types with specific adaptations to gravity: negatively geotactic bacteria (1) and protista (2), root-tip columella cells (3), and the cells of gravisensing organs in invertebrates (6). In surveying the above list it is also easy to see that different responses to gravity require different models for their explanation. A list that nearly matches the above taxonomic cases might be the following (where numerals correspond to members of the taxonomic list): 1. Extracellular transport model 2. Center of gravity model 3a. Segmenting organelle model 3b. Cytoskeletal elasticity model 3c. Electrical signal model 3d, 4a. Activated charmels model 4b, 5a. Tensegrity model 6. Electrical and chemical signals model A survey of this list suggests two basic principles that are not mutually exclusive: ~~~e~ breaking and signal tr~~~~t~~~. SY~ME~Y

BREAKING AND THE CYTCXXELETGN

The basic principles of bifurcation theory are represented graphically in Figure 1. It remains to attach an actual biological process to this model with numbers. Three cases of symmetry breaking in cytoskeletal systems are reviewed briefly. At least three models have been applied to the ~a~~tion~ responses of the cytoskeleton: cytoskeletal elasticity /6/, tensegrity /S/ and s~et~ breaking 17,111. These are not degenerate, nor even complementary, models but represent different levels of abstraction. Consider the cytoskeletal elasticity model as an example. Elasticity is a component of tensegrity in the most classical meaning of tensegrity. When a civil engineer builds a tensegrity structure, whether it be a ‘pop” tent or the new Denver International Airport, the elastic modulus of the tensile members (fabric) and the bending modulus of the stiff members (beams) are factors that must be known before designing can proceed. The bifurcation between failure and success depends on the state, intact or broken, of any member of the structure, thus tensile strength and breaking strength are important classical variables in civil engineering. In addition, tensegrity structures can have multiple stable states without breaking any members /5/, and hand-held tensegrity structures with this property can be purchased at toy stores for the fascination of “children”. While a convincing case can be made for the existence of tensile members in the cytoskeleton /12,13/, only recently-has evidence been presented for the existence of stiff members, and this consists

Theories of CeEs in Space

of estimations of flexural rigidity of microtubules undergo thermal fluctuations in shape /13/.

wm

and actin filaments in vitro as they

APPROACH >

Figure 1. Graphical representation of a bifurcating process. As a system progresses from left to right (in time, space, metabolic progress, steps through the cell cycle, etc.) random fluctuations are imposed on the steady-state condition of variable x. As an end-point is approached, a choice is made between two energy wells, which may, in turn, be shifting depths in absolute terms or relative to one another 1’71. Thus, bifurcation theory, or symmetry breaking, is a broader principle that governs the final state of a structure. At least three examples have been identified and published in the recent cytoskeletal literature that might be relevant to cellular gravitational biology. Bi~rcation theory is especially applicable to self-assembly processes, an example of which is protein crystal growth /14/, which is also affected by gravity at small dimensions /15/. Tabony and Job /ll/ studied the formation of birefringent gels in the polymerization of tubulin and found that the structure that formed could be interpreted as consisting of microtubules parallel to the gravity vector. Rotation of the reaction container in the presence of gravity at a critical time during pol~e~zation subst~tially modifies the geometry of self-assembly of microtubules. A very sensitive example of cytoskeletal symmetry breaking is found in experiments with microtubules attached to immobilized kinesin molecules /16/. When a microtubule is attached to one immobilized kinesin molecule, it is translated& it is free to rotate. But when at~chment to a second kinesin molecule occurs, rotation stops, and tra~lation continues. This effect is shown diagra~atically in Figure 2. A third example is the cytoskeletal elasticity model, which states that intracellular organelles travel through a viscous medium /17/ due to a motor force /18,19/ and a gravitational force /3,10/ opposed by the elastic deformation of the cytoskeletal assembly through which they must be pushed /6/. In cells that respond to gravity in roots and rhizoids, strong evidence exists that the elastic modulus of the force-resisting cytoskeleton is greatly reduced compared to that of cells that do not have organelles that sediment in response to gravity /6,20/. The sounding-rocket experiments of Sievers et a1./21/ “broke into the circle” of the

WY5

P. Todd and D. M. Khs

organelle force dilemma by providing fairly accurate data from which to independently estimate the elastic force on statoliths in cress-root columella cells and in Cham rhizoids. When columella cells are treated with cytochalasin, the nucleus also falls down /20/; when

Rotation Translation <

Translation

only

<

Substratum Figure 2. Sketch showing rotation and translation of a microtubule captured by a single kinesin molecule (left) and translation only of a microtubule captured by two immobilized kinesin molecules /16/. these cells are maintained in low gravity, the anisotropic distribution of amyloplasts is lost, These treatments suggest that the and the direction of root growth is randomized. cytoskeleton of some cells is poised very close to the limit of the elastic forces required to hold organelles in place, and, when this elastic force is decreased only slightly the expected gravitational effect takes over. Such a condition is an example of symmetry breaking: organelles do or do not move freely in the cytoplasm depending on a high or low elastic modulus of the cytoskeleton (see Figure 1). SIGNAL TRANSDUCTION The modern view of intracellular signal transduction consist of cell surface receptors sensitive to a chemical or a force in the environment causing the catalysis of a chemical reaction just “under” the membrane of the cell. Such a reaction leads typically to the phosphorylation of an enzyme to activate it, .or, if genetic expression is required, the transmission of a second messenger to the cell nucleus. Stretch-activated channels /2,22/ and electrical currents /8/ have been associated with the responses of cells to extracellular forces. In addition, all single cells in a modified gravity vector are subjected to changes in the extracellular chemical environment as a consequence of modified transport processes. In “zero” gravity, for example, all solute transport becomes diffusive, and buoyancy-driven flows (convection, sedimentation) are absent or negligible. Such extracellular transport modifications should affect the growth of suspended prokaryotic cells. EXTRACELLULAR

TRANSPORT

AND BACTERIAL

GROWTH

Several investigators, irrespective of their research goals, have noted an increased growth of bacterial cells in microbial experiments conducted on board orbiting spacecraft /23,24,25,26/. These results might be interpreted as due to an increased exponential balanced growth rate, p, or an increased final cell concentration N,, at stationary phase. The latter, higher final biomass, is commonly achieved in bioreactor engineering by feeding reduced carbon source (glucose) at a low concentration, thereby averting excessive accumulation of acetate, especially when oxygen is limiting and glucose is not /27/. According to calculations applied to the popular biotechnology bacterium E. cd, maximal growth rate is achieved at around 4 or 5 g/liter of glucose, while the limiting concentration,

Theories of Cellsin Space

Km7

which gives half-maximal growth rate (the “Monad” constant, K) is about 0.02 g/l 128,‘. Holding glucose constant at 0.1 - 0.4 g/l results in reduced production of acetate with a corresponding increase in final biomass of about a factor of 2 1271. The extracellular transport model, in a modification of the form proposed by AlbrechtBuehler /l/ states that when suspended cells are neither stirred nor sedimenting, extracellular metabolites such as glucose are depleted from an unstirred boundary layer su~ounding the cell. This leads to a lower steady-state metabolite ~oncen~ation available to the cell. In addition, the quiescent, unstirred boundary layer ‘allows byproducts from the cell to accumulate. ‘Ibis situation is depicted diagrammatically in Figure 3 (left). If glucose at the cell boundary is depleted to a steady-state level around 0.2 g/l /27/ there should be negligible change in growth rate (a possible slight reduction) and a two-fold increase in final biomass (Figure 3, growth curve A). If glucose at the cell boundary is depleted to below about 0.05 g/l there should be a reduction in growth rate according to p =

PmaxES1/(KS + Fl)

where [S] is substrate (glucose concentration). shown in Figure 3 (right).

The resulting possible growth curves are

In recent experiments bacteria grown in orbital space flight with initial glucose concentration of 5 g/l and their matched ground controls, which were allowed to sediment 7.5 cm over a 4-day period, produced growth curves like those in Figure 3 (B) and (C), respectively /29/. It is tentatively concluded that increases in cell biomass routinely seen as a consequence of bacterial growth in low gravity are consistent with the extracellular transport model. Elaborating briefly on this model, Albrecht-Buehler also commented that rno~~tio~ in solute concentration in the extracellular unstirred layer might lead to regulatory responses by the cell. This elaboration is consistent with the regulatory effect of steady-state glucose concentration on acetate (or other toxic metabolite) production and subsequently on final biomass concentration.

0

50 Di8t8nC6, m&On8

100 Time, hours

Figure 3. Left: proposed concentration profile of nutrients (solid curve) and metabolic products (dashed curve) in the unstirred layer around a bacterial cell in low gravity. Right: Predicted growth curves with glucose present at 0.2 g/l at steady state (growth curve A), 0.02 g/l at steady state (growth curve B) and 5 g/l (growth curve C). JASR 11:1/T-8

P. Todd and D. M. Klaus

SUMMARY The above taxonomic categorization of cellular responses to gravity and its application to only two basic principles is summarized in Figure 4. Under each of the two principles, three physical models are identified, and under each of these models a few examples of cellular responses are cited. The diagram is not intended to be complete, and it is hoped that additions to the lists will be made as research progresses in gravitational cell biology.

Symmetry

Signal

Brsakina

Transduction

Figure 4. A proposed aviation of cellular responses to inertial acceleration. Two major principles are suggested, each enveloping a small number of “models” (three each, in this diagram, as elaborated in the text), and representative cases of each model can be identified. ACKNOWLEDGMENTS Portions of this research were supported by the NASA Specialized Center on Research and Training in Space Environmental Health, the NASA Center for the Commercial Development of Space/BioServe Space Technologies, and the Colorado Institute for Research in Biotechnology. This paper is dedicated to the late Marvin W. Luttges, Director of Bioserve Space Technologies and a nation~ly-~0~ leader in experimental lactations biology and bioastronautics. REFERENCES 1. G. Albrecht-Buehler, Possible mechanisms of indirect gravity sensing by cells, Amer, Sot. Gravitational and Space Biol. Bull 4(2), 25-34 (1991). 2. J. P. Ding and B. G. Pickard, Mech~ose~o~ epidermal cells, The Plant Journal 3, 83 (1993).

mourn-sele~ive

cation channels in

3. A. Sievers, Gravity sensing mechanisms in plant cells, Amer. Sot. Gmvitational und Space Biol. Balk 4(2), 43-50 (1991). 4. J. 0. Kessler, The dynamics of uniceIlular swimming organisms. Amer. Sot. Gravitational and Space Biol Bull. 4(2), 97-106 (1991).

Theories of Ceils in Space

a-09

5. D. Ingber, The riddle of morphogenesis: A question of solution chemistry or molecular cell engineering? Cell 75, 1249-1252 (1993). 6. P. Todd, Mechanical analysis of statolith action in roots and rhizoids, A&. Space Res. 14, #8, 121-124 (1994). 7. D. K Kondepudi, State selection dynamics in seem-bre~g transitions. In Noise in ~o~Z~~e~ ~~~cu~ Systems, F. Moss and P. V. E. McClintock, Eds., Cambridge University Press, Vol. 2, 1989, pp. 251-267. D. K. Kondepudi, Detection of gravity through non-equilibrium mechanisms, Amer. Sot. Gravitutiontzl and Space l3ioZ.BuZL 4(2), 119-124 (1991). D. A. M. Mesland, Mech~s~ of gravity effects on cellx are there gravitysensitive windows? Advances in Space Biology and Medicine 2, 211-228 JAI Press, Inc. (1992). 8. M. Desrosiers, Cellular responses to endogenous morphological development, Adv. Space Res. this issue.

ele~roche~cal

gradients

in

9. R. S. Bandurski, A. Schulze, A. and W. Domagalski, Possible effects of organelle charge density on cell metabolism, Adv. Space Res. 6, #12, 47-54 (1986). 10. P. Todd, Essay: Gravity-dependent physical processes at the dimensions of the single Gravitutio~ and Space BioZogvBull. 2, 101-129 (1989). cell. American Society for 11. J. Tabony and D. Job, Gravitational symmetry breaking in microtubular structures, froc. Natl Acud. Sci USA 89, 6948-6952 (1992).

dissipative

12. K. Weber and M. Osborne, The molecules of the cell matrix, Scien@c Ame&xm 253, 110-120 (1985). 13. F. Gittes, B. Mickey, J. Nettleton and J. Howard, Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape, .T. Celf Biol. 120, 923-934 (1993). 14. M. Ataka, M. Asai, Systematic studies on the ~t~~~tion of lysozyme. Determination and use of phase diagrams, J, CkystaZGrowth 90, 86-93 (1988). 15. M. L. Grant and D. A. Saville, The role of transport phenomena growth. J. Crystal Greats 108, 8-18 (1991).

in protein crystal

16. A. J. Hunt and J. Howard, Kinesin swivels to permit microtubule direction. Proc. NutZ.Acad. Sci USA 90, 11653-11657 (1993).

movement

in any

17. L. Hou, F. Larmi and K. Luby-Phelps, Tracer diffusion in F-actin in Ficoll mixtures, Biophys. J. 58, 31-43 (1990).

18. E. L. Bearer, J. A. DeGeorgis, R. A. Bodner, A. W. Kao and T. S. Reese, Evidence for myosin motors on organelles in squid axoplasm. Proc. NutZ. Acad. Sci. USA 90, 1125211256 (1993). 19, X. Xiang, S. M. Be&with and N. R. Morris, Cytoplasmic dynein is involved in nuclear migration in Aspe@Zus nidulans, Proc. NatZ.Acad. Sci. USA 91, 2100-2104 (1994). 20. J. Guikema, Effects of cytochalasin D and weightlessness on the structure of plant roottip cells, Amer. Sot. Gr~~t~‘o~~Z and Space BioL BUZZ.5 (1992).

(6/7)10

P. T&d and D. M. Kkw

21. A. Sievers, M. Braun and B. Buchen, Statoliths cause tension of microfilaments: proven during parabolic flights of sounding rockets. Adv. Space Res. 14, #8, 116-120 (1994). 22. C. E. Morris, and W. J. Sigurdson, W. J., Stretch-ina~ivated stretch-activated ion channels. Science 243, 807-809 (1989).

ion channels coexist with

23.0. Ciferi, 0. Tiboni, A. M. Oriandoni and M. L. Marchesi, The effects of microgravity on genetic recombination in E~che~chja cofi, ~~~~e~cha~en 73, 418421 (1986). 24. L. Lapchine, N. Moatti, G. Richoilley, G. Templier, G. Gasset and R. Tixador, The antibio experiment, Biorack on Spacelab Dl (ESA SP-1091), pp. 45-51 (1988). 25. H.-D. Mermigman and M. Heise, Response of growing bacteria to reduction in gravity, Fifth European Symposium on Life Sciences and Space Research, Conference Proceedings, 1993. 26. R. Tixador, G. Richoilley, G. Gasset, J. Ternplier, J. C. Bes, N. Moatti, and L. Lapchine, Study of minimal inhibitory concentration of antibiotics on bacteria cultivated in vitro in space (CYTOS 2 Experiment). Aviaf. SpruceE~v~o* M&i. 56, 748-751 (1985). 27. M. J. Frude, A. Read and L. D. Kennedy. Scale-up production of a recombinant Mycobacterium Zepraeantigen. Ann. NYAcad. Sci. 721, 100-104 (1994). 28. J. E. Bailey and D. F. Ollis, Biochemical Engineering Fundamentals, McGraw-Hill Publishing Company, New York, 1986. 30. D. M. Klaus, M. W. Luttges and L. S. Stodieck, rnvestigation of space flight effects on SAE Technical Paper Series 941260, SAE International, Escherichiu coli growth, Warrendale, PA, 1994.