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Classification of gravity effects on “free” cells
Adv. Space Res. Vol. 6, No. 12, pp. 15—19, 1986 Printed in Great Britain. All rights reserved.
0273—1177/86 $0.00 + .50 Copyright © COSPAR
CLASSIFICATION OF GRAVITY EFFECTS ON “FREE” CELLS W. Briegleb and I. Block DFVLR-Institute for Aerospace Medicine, Linder Höhe, D-5000 Cologne 90, F.R.G.
ABSTRACT
When cell physiologists detect gravity related reactions of their objects it is often difficult to decide where the receptors for the observed effects are located. Answering this question is necessary for any further analysis of a detected gravity effect on cells. In previous papers we have discussed direct and indirect gravity effects in relation to the ~uallest functional units where the primary receptor, which interacts with gravity, is positioned inside and outside of such a unit, respectively. So, in a first approximation we can conclude that in a multicellular aquatic organism, which changes its metabolism in weightlessness, the primary receptors of gravity are located inside the cells of that organism. A special approach is necessary when free living cells, the density of which may be higher than the one of the (liquid) medium, or even cells living on a free surface are observed. In these two cases also indirect effects have to be taken into account, which will be demonstrated with the aid of the slime mold Physarum polycephalum. Additionally the environment of the organisms can be changed directly and indirectly by gravity. INTRODUCTION Today a growing number of biologists is interested in whether cell function is directly affected by gravity. For most types of cells direct gravity effects are merely an assumption. In these cases two essential premises can be postulated. Firstly, physically the cell does interact with gravity; we only do not know if the cell reacts passively or even actively (see below). Secondly, with some restrictions of lower significance, gravity is the only stimulus on earth the cell cannot escape from for a time longer than about 1 second (free fall). In case biologists could prove the sensation of gravity in any part of a randomly selected cell, then the second premise leads to the conclusion that gravity has influenced the evolution of cells and consequently via direct effects also the evolution of multicellular organisms /1,2/. DIRECT VERSUS INDIRECT GRAVITY EFFECTS In previous papers /1,2,3/ we found it useful to make an ecophysiologic classification of gravity effects. Using the definition of SMALLEST FUNCTIONAL UNITS (SFU), which explain the function of the morphologic characters of the organisms, and by correlating the SFUs with possible g—effects, we are able to make the following classification (see figure 1): If the interacting mass, which functions as a primary receptor for an acceleration stimulus, is spatially arranged inside a SFU, we define it as a system of direct or primary interaction (SF13 I). In this case a special density and size of the primary receptor is necessary. If the interacting mass is located in the neighborhood or far away from the SF13, we define it as a system of indirect or secondary (even tertiary) interaction (SFU II). Here, in a certain range, no special size and density of the primary receptor is necessary for its function. The ecophysiologic value of explanation of this classification is evident: In a first approach direct interactions between gravity and the SFU5 I exist in the cells and in gravity receptors of both water and land organisms, and indirect interactions between gravity and the SFUs II exist only in land organisms. Here it is presumed that water organisms have a similar density like water. Land animals have developed morphologic characters which 15
16
W. Briegleb and I. Block
compensate the indirect effects. These are musculo—skeletal and the circulatory systems.
the
antigravity parts
of
the
GRAVITY PERCEPTION BY SMALLEST FUNCTIONAL UNITS (SFU)
TBEHAVb0~1
SFUI
~
/WODYMASSOR
1::: \
[
EXCITATION AND ITS CONDUCTION
/7~CELLS
fLITHS,CELL ORGANELLES)\
L
BEHAVIOR (ORIENTATION)
\\
Functional
ENVIRONMENT ___________
//
-
GEN~ ENVIRONMENT (ECOTOP)
DIRECT PRIMARY INFLUENCE ON SFU I (DENSITY DIFFERENCES PRESUMED) INDIRECT SECONDARY INFLUENCE ON SFU 11 (EVEN WITHOUT DENSITY DIFFERENCES!) Fig. 1. Scheme of Smallest interaction with gravity.
AND SOCIAL
“~
..:
[~ATI~~
Units
of
organisms
and
their
If a rat is sent into space and a change in certain hormone or enzyme concentrations is found, it is impossible to discriminate between a direct or indirect zero—g effect. If a comparable hormone complex of a fish is measured in space and a similar reaction to the first one is found, then this will be a strong evidence for a direct interaction on the cellular level somewhere in the organism. FREE CELLS I½ND GRAVITY
The consequent scientific use of SFUs I and II can also be brought to an extreme. If an entire organism, such as a human being, is defined as a SFU the classification must be improved. An autonomous organism always consists of a number of functional units. If the organism as a whole is influenced by gravity, and if there is any active reaction at all, only sub—SFUs (ordinary SFUs in figure 2) start to work. So, an autonomous organism as a whole can only be regarded as a SUPER SF13 (SSFU) (see figure 2). This holds also true for a single cell. It can be subdivided also into INFRA SFUs (ISFU). In a free living cell, which is somewhat flattenend by gravity, its ISFUs II will react indirectly against gravity, too. In other words, a cell, which is flattened by its own weight, is physiologically in the same situation as a tissue cell, which is flattened by the weight of other cells of the (respective) whole organism. Without destroying our first classification we are now able to define a single or a free cell as a SSFU, which consists of many ISFUs I and II. ISFU5 I, which can directly interact with gravity, may be the nucleolus together with the nucleus, mitochondria together with parts of the endoplasmic reticulum and the cytoskeleton, or the MUller’s vesicles in certain protozoa. ISFUs II for indirect interaction with gravity may include parts of the cell’s fibrils and parts of the cytoskeleton. A free cell in suspension may settle to the bottom of the vessel. Approximately 10 to 20% of its weight forces the cell to the bottom. A flattening of the cell is presumably an active process (a sort of haptotaxis). Thus, the resulting effects cannot be classified as direct gravity effects. A special case are free living cells on a wet surface. Depending on their size gravity may flatten those cells to a small extent. In this case ISFUs 11 will interact with gravity indirectly (see below).
Gravity Effects on Cells
Super SFU (autonomous organisms)
/ I
_
cells
multicellular organisms
I
infra SFU I and II
ordinary SFU I and II
(consisting of portions of celLs)
(consisting of cells or parts of organs)
Fig. 2. Hierarchy of Smallest Functional Units, which are the objects for direct or indirect interactions of organisms with gravity (or other accelerations). Compare also Figure 1 and text! GRAVITY AND THE ENVIRONMENT The influence of gravity on the environment of multicellular organisms and free cells may also be put into a classification system. For this classifi1so the terms direct (primary) and indirect cation we are going to use a . Direct changes of the environment by gravity (secondary and tertiary)effects are convections in the media air or water caused by the metabolisms of the organisms. A resulting altered ion exchange within cell membranes will represent a direct gravity effect. Indirect effects of gravity such as stratification of the medium or hydrostatic pressure, may be called indirect environmental effects. Tertiary environmental effects may be geophysical factors like the direction of light, tides, geophysical rhythms and so on. We are able to contribute an observation gained during our experimentation with cells, especially animal eggs, on the fast running clinostat as well as in space. Large eggs seem to be at the limit of a sufficient ogygen supply. After exposing insect eggs (Tribolium) contained in oat meal, on the fast running clinostat to simulated weightlessness a lot of teratogenic anomalies were generated. When raising the oxygen content in the air of the experiment container to 40% the rate of anomalies nearly dropped to the normal value (from above 50% down to about 5%). We relate our success in cultivating Xenopus eggs in space, where we did not observe teratogenic effects, also to the oxygen enriched atmosphere (40%) /4/. Under these conditions, the lack of mass convection in the vicinity of eggs exposed to clinostat or to space conditions may be counterbalanced by diffusion. We do not expect or believe in anomalies or in increased mortality in the embryonic development of organisms raised under zero—g conditions (see also /5/!). GRAVITY AND THE POLAR CELL STRUCTURE OF PHYSARUM POLYCEPHALUM The authors are preparing a space experiment in the European Biorack with the unicellular slime mold Physarum polycephalum to be flown in the International Microgravity Laboratory (IML). One of the three questions, which will be investigated in this IML experiment, is the analysis of the to date unexplained morphologic polarity of this giant cell which is possibly induced by gravity. Physarum is typically differentiated into tubelike strands forming a network which can grow up to several square meters. The cross sections of the strands are rather circular and their diameters range from 0.1 to 1 millimeter. The walls (ectoplasm) of the strands are of a complicated structure built up of a gel containing many invaginations of the cell membrane (see figure 3) which are accompanied by longitudinal, radial and circular actomyosin fibrils. These fibrils contract and dilatate in a regulated manner inducing a peristaltic—like pumping. This pumping mechanism moves the sol—like endoplasm inside the strands force and back = shuttle streaming. In a cross section of a strand the physically lower side of the wall is thinner than the lateral and upper sides (see figure 3).
—
18
W. Briegleb and I. Block
>~
H— ~
1’ _ ‘I,
_____
I
_ _______________________
I
Fig. 3. Cross section of a strand of Physarum polycephalum growing on agar. The outer area of the strand (ec = ectoplasm) contains actomyosin fibrils and invaginations of the plasmalemma, the inner portion of the strand (en = endoplasm) is free of these structures. The arrows indicate the varying thickness of the ectoplasmic wall at different parts of the strand: Note the thin ectoplasmic wall at the physically lower side of the strand. From /6/. This is even more pronounced if the strands “hang” on an inversed horizontal surface. We try to explain this phenomenon in two ways: Firstly, the endoplasm may be of a higher density than the ectoplasm. This is possible if slime (polysaccharids) , which is contained in the outer invagina— tions of the ectoplasm, is of lower density than the endoplasm. In this case the polarity of the strand would be a primary effect of gravity with (active interaction ) or without (passive interaction) a respective sensation for the cell. Such a phenomenon of polarity should not disappear if the strand becomes submerged. Secondly, the polarity of the strand may be caused by its total weight. In this case the ISFUs II of the cell will interact indirectly with gravity. For example, in the laying position it would be unefficient for the strand to contract within the area where the strand adheres to the substratum. As a consequence the contractile fibrils would be reduced and the wall would become thinner. This would be an active regulation of ISFU II. In the hanging position, with the substratum upside—down, the contact of the strand to the substratum would be loosened by the strands’ contractions. As a consequence, the cell would have to provide a thick wall in the area of contact, e.i. a sort of a plate containing less contractile fibrils. The adhesion of such a plate would be strong enough to bear the weight of the strand. The construction of such a plate would need a regulated cycle of the ISFU II type. To discriminate between these different possible mechanisms many experiments are necessary, some of which preferably have to be performed in space. REFERENCES 1.
W. Briegleb, Acceleration Reactions of Cells and Tissues Phylogenic Implications, Adv. Space Res. 4, 12, 5 (1984)
2.
w. Briegleb, Some Remarks on Gravitational Biology, in: Gravitational Biology in the Federal Republic of Germany, Proceedings and a Prograrrine Draft, European Space Agency Technical Translation, ESA—TT—988, 1986, p.26.
3.
w. Brieg].eb, Proceedings of New Buildings Porz, 10 March 857, 1984, p.
—
Their Genetic—
Are Living Cells Generally Sensitive to Gravity?, in: a Scientific Meeting on the Occasion of the Inauguration of for the DFVLR—Institute for Aerospace Medicine in Cologne 1982, European Space Agency, Technical Translation, ESA—TT— 92 (87).
Gravity Effects on Cells
19
4.
W. Briegleb, J. Neubert A. Schatz, T. Klein, and B. Kruse (Technical Assistance), Survey of the Vest ibulum, and Behavior of Xenopus Laevis Larvae Developed During a 7—Days Spaceflight, Adv. Space Res. in press (1986)
5.
Y. Gaubin, B. Pianezzi, G. Gasset, H. Planel, and E.E. Kovalev, Stimulating Effect of Space Flight Factors on Artemia Cysts: Comparison with Irradiation by ~ Rays, Aviat., Space, and Env. Med. 57, 583—590 (1986)
6.
F. Achenbach, W. Naib—Majani, and K.E. Wohlfarth—Bottermann, Plasmalemma Invaginations of Physarum Dependent on the Nutritional Content of the Plasmodial Environment, J. Cell Sci. 36, 355—359 (1979)
0273—1177/86 $0.00 + .50 Copyright © COSPAR
CLASSIFICATION OF GRAVITY EFFECTS ON “FREE” CELLS W. Briegleb and I. Block DFVLR-Institute for Aerospace Medicine, Linder Höhe, D-5000 Cologne 90, F.R.G.
ABSTRACT
When cell physiologists detect gravity related reactions of their objects it is often difficult to decide where the receptors for the observed effects are located. Answering this question is necessary for any further analysis of a detected gravity effect on cells. In previous papers we have discussed direct and indirect gravity effects in relation to the ~uallest functional units where the primary receptor, which interacts with gravity, is positioned inside and outside of such a unit, respectively. So, in a first approximation we can conclude that in a multicellular aquatic organism, which changes its metabolism in weightlessness, the primary receptors of gravity are located inside the cells of that organism. A special approach is necessary when free living cells, the density of which may be higher than the one of the (liquid) medium, or even cells living on a free surface are observed. In these two cases also indirect effects have to be taken into account, which will be demonstrated with the aid of the slime mold Physarum polycephalum. Additionally the environment of the organisms can be changed directly and indirectly by gravity. INTRODUCTION Today a growing number of biologists is interested in whether cell function is directly affected by gravity. For most types of cells direct gravity effects are merely an assumption. In these cases two essential premises can be postulated. Firstly, physically the cell does interact with gravity; we only do not know if the cell reacts passively or even actively (see below). Secondly, with some restrictions of lower significance, gravity is the only stimulus on earth the cell cannot escape from for a time longer than about 1 second (free fall). In case biologists could prove the sensation of gravity in any part of a randomly selected cell, then the second premise leads to the conclusion that gravity has influenced the evolution of cells and consequently via direct effects also the evolution of multicellular organisms /1,2/. DIRECT VERSUS INDIRECT GRAVITY EFFECTS In previous papers /1,2,3/ we found it useful to make an ecophysiologic classification of gravity effects. Using the definition of SMALLEST FUNCTIONAL UNITS (SFU), which explain the function of the morphologic characters of the organisms, and by correlating the SFUs with possible g—effects, we are able to make the following classification (see figure 1): If the interacting mass, which functions as a primary receptor for an acceleration stimulus, is spatially arranged inside a SFU, we define it as a system of direct or primary interaction (SF13 I). In this case a special density and size of the primary receptor is necessary. If the interacting mass is located in the neighborhood or far away from the SF13, we define it as a system of indirect or secondary (even tertiary) interaction (SFU II). Here, in a certain range, no special size and density of the primary receptor is necessary for its function. The ecophysiologic value of explanation of this classification is evident: In a first approach direct interactions between gravity and the SFU5 I exist in the cells and in gravity receptors of both water and land organisms, and indirect interactions between gravity and the SFUs II exist only in land organisms. Here it is presumed that water organisms have a similar density like water. Land animals have developed morphologic characters which 15
16
W. Briegleb and I. Block
compensate the indirect effects. These are musculo—skeletal and the circulatory systems.
the
antigravity parts
of
the
GRAVITY PERCEPTION BY SMALLEST FUNCTIONAL UNITS (SFU)
TBEHAVb0~1
SFUI
~
/WODYMASSOR
1::: \
[
EXCITATION AND ITS CONDUCTION
/7~CELLS
fLITHS,CELL ORGANELLES)\
L
BEHAVIOR (ORIENTATION)
\\
Functional
ENVIRONMENT ___________
//
-
GEN~ ENVIRONMENT (ECOTOP)
DIRECT PRIMARY INFLUENCE ON SFU I (DENSITY DIFFERENCES PRESUMED) INDIRECT SECONDARY INFLUENCE ON SFU 11 (EVEN WITHOUT DENSITY DIFFERENCES!) Fig. 1. Scheme of Smallest interaction with gravity.
AND SOCIAL
“~
..:
[~ATI~~
Units
of
organisms
and
their
If a rat is sent into space and a change in certain hormone or enzyme concentrations is found, it is impossible to discriminate between a direct or indirect zero—g effect. If a comparable hormone complex of a fish is measured in space and a similar reaction to the first one is found, then this will be a strong evidence for a direct interaction on the cellular level somewhere in the organism. FREE CELLS I½ND GRAVITY
The consequent scientific use of SFUs I and II can also be brought to an extreme. If an entire organism, such as a human being, is defined as a SFU the classification must be improved. An autonomous organism always consists of a number of functional units. If the organism as a whole is influenced by gravity, and if there is any active reaction at all, only sub—SFUs (ordinary SFUs in figure 2) start to work. So, an autonomous organism as a whole can only be regarded as a SUPER SF13 (SSFU) (see figure 2). This holds also true for a single cell. It can be subdivided also into INFRA SFUs (ISFU). In a free living cell, which is somewhat flattenend by gravity, its ISFUs II will react indirectly against gravity, too. In other words, a cell, which is flattened by its own weight, is physiologically in the same situation as a tissue cell, which is flattened by the weight of other cells of the (respective) whole organism. Without destroying our first classification we are now able to define a single or a free cell as a SSFU, which consists of many ISFUs I and II. ISFU5 I, which can directly interact with gravity, may be the nucleolus together with the nucleus, mitochondria together with parts of the endoplasmic reticulum and the cytoskeleton, or the MUller’s vesicles in certain protozoa. ISFUs II for indirect interaction with gravity may include parts of the cell’s fibrils and parts of the cytoskeleton. A free cell in suspension may settle to the bottom of the vessel. Approximately 10 to 20% of its weight forces the cell to the bottom. A flattening of the cell is presumably an active process (a sort of haptotaxis). Thus, the resulting effects cannot be classified as direct gravity effects. A special case are free living cells on a wet surface. Depending on their size gravity may flatten those cells to a small extent. In this case ISFUs 11 will interact with gravity indirectly (see below).
Gravity Effects on Cells
Super SFU (autonomous organisms)
/ I
_
cells
multicellular organisms
I
infra SFU I and II
ordinary SFU I and II
(consisting of portions of celLs)
(consisting of cells or parts of organs)
Fig. 2. Hierarchy of Smallest Functional Units, which are the objects for direct or indirect interactions of organisms with gravity (or other accelerations). Compare also Figure 1 and text! GRAVITY AND THE ENVIRONMENT The influence of gravity on the environment of multicellular organisms and free cells may also be put into a classification system. For this classifi1so the terms direct (primary) and indirect cation we are going to use a . Direct changes of the environment by gravity (secondary and tertiary)effects are convections in the media air or water caused by the metabolisms of the organisms. A resulting altered ion exchange within cell membranes will represent a direct gravity effect. Indirect effects of gravity such as stratification of the medium or hydrostatic pressure, may be called indirect environmental effects. Tertiary environmental effects may be geophysical factors like the direction of light, tides, geophysical rhythms and so on. We are able to contribute an observation gained during our experimentation with cells, especially animal eggs, on the fast running clinostat as well as in space. Large eggs seem to be at the limit of a sufficient ogygen supply. After exposing insect eggs (Tribolium) contained in oat meal, on the fast running clinostat to simulated weightlessness a lot of teratogenic anomalies were generated. When raising the oxygen content in the air of the experiment container to 40% the rate of anomalies nearly dropped to the normal value (from above 50% down to about 5%). We relate our success in cultivating Xenopus eggs in space, where we did not observe teratogenic effects, also to the oxygen enriched atmosphere (40%) /4/. Under these conditions, the lack of mass convection in the vicinity of eggs exposed to clinostat or to space conditions may be counterbalanced by diffusion. We do not expect or believe in anomalies or in increased mortality in the embryonic development of organisms raised under zero—g conditions (see also /5/!). GRAVITY AND THE POLAR CELL STRUCTURE OF PHYSARUM POLYCEPHALUM The authors are preparing a space experiment in the European Biorack with the unicellular slime mold Physarum polycephalum to be flown in the International Microgravity Laboratory (IML). One of the three questions, which will be investigated in this IML experiment, is the analysis of the to date unexplained morphologic polarity of this giant cell which is possibly induced by gravity. Physarum is typically differentiated into tubelike strands forming a network which can grow up to several square meters. The cross sections of the strands are rather circular and their diameters range from 0.1 to 1 millimeter. The walls (ectoplasm) of the strands are of a complicated structure built up of a gel containing many invaginations of the cell membrane (see figure 3) which are accompanied by longitudinal, radial and circular actomyosin fibrils. These fibrils contract and dilatate in a regulated manner inducing a peristaltic—like pumping. This pumping mechanism moves the sol—like endoplasm inside the strands force and back = shuttle streaming. In a cross section of a strand the physically lower side of the wall is thinner than the lateral and upper sides (see figure 3).
—
18
W. Briegleb and I. Block
>~
H— ~
1’ _ ‘I,
_____
I
_ _______________________
I
Fig. 3. Cross section of a strand of Physarum polycephalum growing on agar. The outer area of the strand (ec = ectoplasm) contains actomyosin fibrils and invaginations of the plasmalemma, the inner portion of the strand (en = endoplasm) is free of these structures. The arrows indicate the varying thickness of the ectoplasmic wall at different parts of the strand: Note the thin ectoplasmic wall at the physically lower side of the strand. From /6/. This is even more pronounced if the strands “hang” on an inversed horizontal surface. We try to explain this phenomenon in two ways: Firstly, the endoplasm may be of a higher density than the ectoplasm. This is possible if slime (polysaccharids) , which is contained in the outer invagina— tions of the ectoplasm, is of lower density than the endoplasm. In this case the polarity of the strand would be a primary effect of gravity with (active interaction ) or without (passive interaction) a respective sensation for the cell. Such a phenomenon of polarity should not disappear if the strand becomes submerged. Secondly, the polarity of the strand may be caused by its total weight. In this case the ISFUs II of the cell will interact indirectly with gravity. For example, in the laying position it would be unefficient for the strand to contract within the area where the strand adheres to the substratum. As a consequence the contractile fibrils would be reduced and the wall would become thinner. This would be an active regulation of ISFU II. In the hanging position, with the substratum upside—down, the contact of the strand to the substratum would be loosened by the strands’ contractions. As a consequence, the cell would have to provide a thick wall in the area of contact, e.i. a sort of a plate containing less contractile fibrils. The adhesion of such a plate would be strong enough to bear the weight of the strand. The construction of such a plate would need a regulated cycle of the ISFU II type. To discriminate between these different possible mechanisms many experiments are necessary, some of which preferably have to be performed in space. REFERENCES 1.
W. Briegleb, Acceleration Reactions of Cells and Tissues Phylogenic Implications, Adv. Space Res. 4, 12, 5 (1984)
2.
w. Briegleb, Some Remarks on Gravitational Biology, in: Gravitational Biology in the Federal Republic of Germany, Proceedings and a Prograrrine Draft, European Space Agency Technical Translation, ESA—TT—988, 1986, p.26.
3.
w. Brieg].eb, Proceedings of New Buildings Porz, 10 March 857, 1984, p.
—
Their Genetic—
Are Living Cells Generally Sensitive to Gravity?, in: a Scientific Meeting on the Occasion of the Inauguration of for the DFVLR—Institute for Aerospace Medicine in Cologne 1982, European Space Agency, Technical Translation, ESA—TT— 92 (87).
Gravity Effects on Cells
19
4.
W. Briegleb, J. Neubert A. Schatz, T. Klein, and B. Kruse (Technical Assistance), Survey of the Vest ibulum, and Behavior of Xenopus Laevis Larvae Developed During a 7—Days Spaceflight, Adv. Space Res. in press (1986)
5.
Y. Gaubin, B. Pianezzi, G. Gasset, H. Planel, and E.E. Kovalev, Stimulating Effect of Space Flight Factors on Artemia Cysts: Comparison with Irradiation by ~ Rays, Aviat., Space, and Env. Med. 57, 583—590 (1986)
6.
F. Achenbach, W. Naib—Majani, and K.E. Wohlfarth—Bottermann, Plasmalemma Invaginations of Physarum Dependent on the Nutritional Content of the Plasmodial Environment, J. Cell Sci. 36, 355—359 (1979)