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Figure 4-06. How gravity affects cells. A. Organelles having densities higher than the surrounding cytoplasm exert pressure on the cytoskeleton filaments when in normal gravity (1 G). Such pressure disappears in microgravity (0 G). B. In normal gravity, cells sediment to the bottom of the culture flask within minutes, thus living and interacting in a two-dimensional environment. In microgravity, cells remain suspended in the medium in a three-dimensional environment.

Figure 4-06. How gravity affects cells. A. Organelles having densities higher than the surrounding cytoplasm exert pressure on the cytoskeleton filaments when in normal gravity (1 G). Such pressure disappears in microgravity (0 G). B. In normal gravity, cells sediment to the bottom of the culture flask within minutes, thus living and interacting in a two-dimensional environment. In microgravity, cells remain suspended in the medium in a three-dimensional environment.

In a world of molecules embedded in fluids and loaded with electrical charges dominated by viscosity and electrostatic forces, gravity is an extremely week force. If one calculates the impact of such forces considering the cell as a static system one comes to the conclusion that the effect of gravity is negligible compared to that of the other forces. However, most, if not all, biological systems are not static, but in a non-equilibrium status. The principle of "small cause/large effect" applies. In fact, in a biological process consisting of many subsequent steps, a small perturbation of one of the steps is sufficient to provoke dramatic changes downstream till the endpoint. Such effects are predicted by the bifurcation theory, as described theoretically by Prigogine and Stenger (1984) and experimentally by Tabony et al. (2002). This theory states that at determined bifurcation points a biological system may choose between two pathways leading to completely different endpoints (Figure 4-07). In fact, Stenger and Prigogine argue that "non-equilibrium amplifies the effects of gravity". In other words, it is conceivable that at 1 g the evolutionary pressure drove the system towards one of the two paths. Conversely, 0 g conditions may favor the second path, thus upsetting all the predictions based on a static model at 1 g. In fact, as described in the following sections, several surprising findings resulted from the "fishing experiments" conducted at the early stages of space biology.

Important changes like the loss of sedimentation, density-driven convection and hydrostatic pressure are occurring in a weightless cell culture. For a cell immersed in a fluid, as it is the case in a culture, this is a completely new situation. First, in 1 g, mammalian cells sediment within a few minutes to the bottom of the flask, where many of them may spread and adhere. In 0 g, instead, cells remain in suspension. Going from 1 g to 0 g is a change from a two- to a three-dimensional environment and has a remarkable impact on cell interactions, cell movements and, due to the lack of a substratum on which to spread and adhere, on cell shape (see Figure 4-06B).

Figure 4-07. During a biological process, for example during T-cell activation, a system moves from an equilibrium state (e.g., a T-cell in the G0 phase) towards an unstable, non-equilibrium state (e.g., when a T-cell is activated). The vertical axis represents a state variable (e.g., the gravitational accelera-

Figure 4-07. During a biological process, for example during T-cell activation, a system moves from an equilibrium state (e.g., a T-cell in the G0 phase) towards an unstable, non-equilibrium state (e.g., when a T-cell is activated). The vertical axis represents a state variable (e.g., the gravitational accelera-

Stable tion). At a certain point of

Distance from Equilibrium the biological process, called bifurcation point, the system becomes unstable and can follow multiple pathways. Two pathways are shown here. Adapted from Tabony et al. (2002).

Second, density-driven convection (due to changes in the concentration of nutrients and waste products in the medium) does not occur in microgravity, thus preventing mechanical diffusion. Thermodynamic diffusion is not affected, however.

Third, a new convection, predicted at the beginning of the last century by Marangoni, and not detectable at 1 g, becomes relevant in microgravity. The lack of buoyancy prevents gas bubbles (e.g., C02 developed by the metabolism of living cells) to rise to the surface of a culture, thus favoring the formation of larger bubbles in the middle of the liquid phase rather than a separation of the liquid and gas phases. For more details on the physics of fluids in microgravity, see Chapter 1, Section 2.2.

2.2 Further Considerations

2.2.1 Cell Shape and Structure

Gravity may induce polarization of the cell. The centriole, the structure giving origin to the spindle at the beginning of mitosis, consists of two centrosomes formed by tightly assembled microtubule. The centrosomes are not only dense structures, but they are perpendicularly oriented to each other, thus defining a plane (Figure 4-08). One could speculate that they may function as a kind of compass of the cells. Consequently, in microgravity the cell might lose its orientation.

The density of the nucleus and other organelles is higher than that of the cytosol, i.e., the fluid portion of the cytoplasm. Thus, organelles are expected to sediment inside the cell. For instance, it has been calculated that at 1 g the nucleus might sediment at a rate of 10 urn/hour. Conversely, at 0 g such sedimentation would not occur. In addition, factors other than microgravity may be responsible for the lack of sedimentation and three-

Figure 4-08. Centrioles are cylindrical structures that are composed o f groupings of microtubules arranged in a 9 + 3 pattern. The pattern is so named because of a ring of nine microtubule "triplets" arranged at right angles to one another. They are present in animal cells and play a role in cell division. Courtesy of Albrecht-Biihler, Northwestern University Medical School, Chicago.

dimensional structure: the viscosity of the cytosol or by Brownian motion and cytoplasmic streaming might prevent sedimentation.

We can assume that the network formed by the cytoskeleton is responsible of maintaining the structure and shape in eucaryotic cells by linking together the membrane, the nucleus, and the organelles. The most important components of the cytoskeleton (actin filaments and microtubuli) are made of globular proteins subunits that can rapidly assemble and disassemble in the cell. As elegantly demonstrated by Tabony et al. (2002), assembly and disassembly of such labile structures is governed by gravity.

2.2.2 Biochemistry

The physiology of the cell may also be influenced by gravity. While passive transport of small molecules through the lipid bilayer is governed by diffusion (a gravity-independent process), active transport of ions and charged molecules, in which protein channels and transient membrane invaginations are involved, may be influenced by gravity.

Gravity may also play a role in intercellular transport processes. In fact exothermic metabolic processes generate continuously warmer microregions that are less dense than the neighborhood. Thus, thermal convections are produced by gravity with consequent ultra-structural rearrangements. Such convections are obviously absent in microgravity.

Also the energy turnover in the cells can be influenced by gravity. According to calculations made by Nace in 1983, gravity causes an uneven distribution of the organelles that gives rise to a torque capable to modify the shape and the structure of the cell. Energy is required to maintain its shape against gravity. In microgravity, such energy may be saved for other processes, such as proliferation or biosynthesis.

Finally, free-swimming cells consume energy to swim against gravity to avoid sedimentation. Such energy is not required at 0 g.

I conclude this section with a fundamental consideration: All living systems react in one way or another to changes of the environmental parameters like temperature, illumination, pressure, concentrations of nutrients, or activators/inhibitors. Gravity is a mechanical force. Change of the gravitational environment, i.e., changes of the forces acting on the cell, is a significant environmental change. It should therefore be no surprise that single cells also react and adapt to changes from 1 g to 0 g conditions.

3 RESULTS OF SPACE EXPERIMENTS

The long-term effects of microgravity can be investigated on board orbital spacecraft and sounding rockets, whereas the short-term effects are usually studied on board aircraft during parabolic flight. For some biological responses, ground-based methods or theoretical models can simulate the conditions of weightlessness. However, we should keep in mind that these are just simulations of some of the effects of spaceflight. Nevertheless, in many cases, the results of experiments using clinostats were found to be in good agreement with those of space experiments and vice versa. In the last ten years a new device developed by Hoson et al. (1997) at the Osaka University, called random positioning machine, or three-dimensional clinostat, has been introduced in cell biology to provide a better conditions simulating the effects of microgravity (see Figure 3-07).

Ground-based investigations can be also carried out in centrifuges. However, in some cases, responses are not only related to the hypergravity level, but also to the experimental conditions, such as the size of the flasks, the angular velocity of the centrifuge, or the Coriolis forces resulting from motion of the samples while being centrifuged.

3.1 Results by Kinds of Cells

About 300 experiments have been carried out in space using various kinds of cells like bacteria, algae, fungi, protozoa and mammalian cells. Main changes were observed on cell proliferation, cell morphology, cell shape, cell membrane, cell metabolism, signal transduction, and gene expression. Particular attention is dedicated to the effect of microgravity on T-cells (see Figure 1-17). Indeed, the activation of T-lymphocytes during spaceflight turned out to be one of the most intriguing stories of space cell biology.

Unfortunately, the catastrophe of Columbia has caused a long delay in the space biology program. Therefore, important investigations in space could not be carried out in the last three years. Nevertheless, ground-based investigations led to interesting results.

3.1.1 Enzymes and Microtubuli

It is difficult to figure out how gravity may interact with subcellular structures or even at the level of macromolecules. Two fundamental studies have shown that this is indeed possible. In a series of brilliant experiments conducted on sounding rockets, Tabony et al. (2002) have shown that the self-assembly of microtubules is gravity dependent. Microtubules are formed in vitro when solutions of monomeric tubulin and GTP7 are warmed at 35°C. GTP delivers the energy required for this process: when one molecule of tubulin is added to the microtubule, one molecule of GTP is hydrolyzed to GDP. Once formed microtubules undergo a complex dynamic process, called treadmilling by Tabony, in which tubulin is added at one end of the microtubule and lost from the other end. If such process is conducted at high

7 GTP (Guanosine Triphosphate) is a chemical compound (nucleotide) that is incorporated into the growing RNA chain during synthesis of RNA and used as a source of energy during synthesis of proteins.

concentrations of tubulin (10 mg/ml), the microtubules tend to assemble in a stable structure that can be easily detected in polarized light. In their sounding rocket experiments, Tabony and collaborators discovered that such self-assembly of microtubuli does not take place in microgravity, whereas identical samples kept in a onboard 1-g centrifuge do (Figures 4-09 and 4-10).

Such gravity-dependence is convincingly attributed to density fluctuation occurring during the self-assembly process. This process being a

Figure 4-09. Top. A microtubule in a solution of tubulin and GTP is undergoing a dynamic process called "treadmilling". Tubulin is added to one end of the microtubule (+) and is removed from the other (-), at the expenses of GTP that is hydrolyzed to GDP. In such a way, the microtubule grows on one side and shrinks on the other. Bottom. Results of an experiment conducted on a sounding rocket in microgravity. The samples with tubulin and GTP contained in spectrophotometer cells were photographed in polarized light. The samples were kept inside an onboard 1-g centrifuge with the centrifugal force directed along (in A) or perpendicular to (in B) the long axis of the cell. The patterns show the self-organization of the microtubules. In the samples kept in 0 g (in C) almost no self-organization is occurring. Adapted from Tabonv et cil. (2002).

Figure 4-09. Top. A microtubule in a solution of tubulin and GTP is undergoing a dynamic process called "treadmilling". Tubulin is added to one end of the microtubule (+) and is removed from the other (-), at the expenses of GTP that is hydrolyzed to GDP. In such a way, the microtubule grows on one side and shrinks on the other. Bottom. Results of an experiment conducted on a sounding rocket in microgravity. The samples with tubulin and GTP contained in spectrophotometer cells were photographed in polarized light. The samples were kept inside an onboard 1-g centrifuge with the centrifugal force directed along (in A) or perpendicular to (in B) the long axis of the cell. The patterns show the self-organization of the microtubules. In the samples kept in 0 g (in C) almost no self-organization is occurring. Adapted from Tabonv et cil. (2002).

highly dynamic non-equilibrium process, even small gravitational effects on density changes are magnified according to the bifurcation theory. If we keep in mind that microtubules are the constituents of the centrioles, we may speculate that the formation of the spindle at the beginning of mitosis is also gravity-dependent.

Density fluctuations are also responsible for changes of the enzymatic activity of lipoxygenase in microgravity conditions according to Maccarrone et al. (2003). Lipoxygenases are a family of enzymes playing a role in important cellular functions like signal transduction, apoptosis, and metabolism. Experiments performed during parabolic flights showed that the affinity for the substrate (given as km value) was increased four times in 0 g, whereas the maximum velocity (Fm) of the enzyme remained unchanged.

Figure 4-10. Mechanism proposed by Tabony to explain that the self-assembly of microtubules is gravity-dependent. The growing and shrinking of microtubule generates regions of low and high concentrations of tubulin at their ends, respectively (A). When more microtubules are coming closer to each other they will preferentially grow where the tubulin concentration is higher (B). This leads to the formation of tubulin trails and consequently to macroscopic density gradients in the system (C). Gravity interacts with such density gradients and leads to microtubules self-assembly. Adapted from Tabony et al (2002).

Figure 4-10. Mechanism proposed by Tabony to explain that the self-assembly of microtubules is gravity-dependent. The growing and shrinking of microtubule generates regions of low and high concentrations of tubulin at their ends, respectively (A). When more microtubules are coming closer to each other they will preferentially grow where the tubulin concentration is higher (B). This leads to the formation of tubulin trails and consequently to macroscopic density gradients in the system (C). Gravity interacts with such density gradients and leads to microtubules self-assembly. Adapted from Tabony et al (2002).

3.1.2 Viruses

Viruses can be regarded as an assembly of macromolecules that may reproduce themselves under favorable conditions. Therefore they are at the boarder between chemical compounds and living beings. Viruses and bacteria have been used in space experiments to study the potential risks of infectious diseases affecting crewmembers. Other studies were dedicated to the crystallization of tobacco mosaic virus in 0 g in order to determine the three-dimensional structure by X-ray crystallography. The working hypothesis was

Figure 4-11. Space bioreactor developed in collaboration by the ETH Zurich, Mecanex, Nvon and the University of Neuchdtel. Photo courtesy of Isabelle Walther.

that the crystal structure developed in microgravity is of higher quality than that obtained in normal gravity (see Chapter 8).

Monomeric virus proteins may self-assemble to form pentameres, the capsomeres. In the presence of calcium ions the capsomeres self assemble to form larger symmetrical structures called capsids. Consigli and collaborators (Chang et al. 1993) conducted an interesting study in 1991 on board the Space Shuttle with polyomavirus protein VP1. In microgravity, VP1 formed capsomeres of homogeneous size, which did not assembly to form capsids, while the ground-based controls formed capsomeres of heterogeneous size that assembled to capsids. The failure to form capsids in 0 g may have similar causes as the failure of self-assembly of the microtubules reported by Tabony and thus further supports the bifurcation theory.

Figure 4-11. Space bioreactor developed in collaboration by the ETH Zurich, Mecanex, Nvon and the University of Neuchdtel. Photo courtesy of Isabelle Walther.

3.1.3 Bacteria

I remember well that when I was preparing my proposal for my first experiment in space I found in the library a book edited by Gordon and Cohen in 1971 entitled Gravity and the Organism. One chapter by Pollard was dedicated to a study conducted on Escherichia coli in an ultra rapid centrifuge at 50,000 g. I was disappointed to learn that neither DNA, RNA, nor protein synthesis was altered. But in a later work published by Montgomery et al. (1978), centrifugation at 110,000 g increased lag phase and prolonged generation time in E. coli. I thought that if some changes occur at high-g there is a good chance that qualitatively opposite changes could happen at 0 g. Indeed a few years later, in 1967, cultures of E. coli flew on board the U.S. Biosatellite-2. Mattoni et al. (1971) reported that after a 45-hour orbital flight, the flight populations grew significantly faster than the Earth controls. This effect is probably due to the fact that, in absence of gravity and sedimentation, cells are homogeneously distributed in the culture medium and thus not subject to the waste and nutrient gradients existing in populations of cells laying on the bottom of the culture flasks. A stimulating effect on cell growth rate was also noted in experiments performed in the rotating clinostat. Such changes appear to be typical indirect effects of gravity caused by changes of the microenvironment of the cells.

Planel and collaborators (2002) discovered an increased resistance of E. coli and Staphylococcus aureus to antibiotics when cultured in several experiments in space. The effect was attributed to an increase of the thickness of the cell membrane, observed in electron micrographs, with consequent decrease in the membrane permeability.

More recently Klaus et al. (1997) conducted experiments with E. coli during seven Space Shuttle flights and reported a decrease in lag phase, an increased duration of exponential phase of cell growth, and an approximately twofold increase in final cell population density compared to ground controls. It is a pity that such experiments did not have an inflight 1-g control. Nevertheless, Kacena et al. (1997) reported similar results.

However, other experiments with E. coli gave contradictory results. Bouloc and d'Ari (1991) reported that cell growth did not increase during an investigation flown on a Russian Cosmos biosatellite. Gasset et al. (1994) came to the same conclusion with an experiment conducted on board the Space Shuttle.

Quite new and original was the experimental approach adopted by Ciferri et al. (1986) for an experiments on E. coli conducted in the Biorack facility on board the D-l Spacelab mission. There are three types of interactions between bacteria: the exchange of chromosomal DNA via sex pili (conjugation), the transfer of short stretches of DNA by bacteriophage (transduction), and the uptake of extracellular DNA fragments (transformation). No gravity effect was found with respect to transduction and transformation. Conversely, conjugation was enhanced three- to four-fold in microgravity compared to the inflight 1 g control.

Two experiments were performed during the IML-2 Spacelab mission to examine the effects of microgravity on E. coli cell microenvironment and signal transduction through the cell membrane (Thevenet et al. 1996). Although the investigators are very cautious in the interpretation of their data and honestly describe the difficulties commonly encountered in space experimentation, their results are interesting. They used two E. coli strains, namely the K12 prototrophic strain, i.e., a wild strain capable of synthesizing by themselves all required biochemicals like the amino acids, and the non-motile mutant motB:. TnlO. While cell growth of the wild type did not change at 0 g, the lag phase appeared considerably shorter in the non-motile mutant compared to the inflight 1-g control. In the second experiment, signal transduction was studied by subjecting the cells to osmotic shock with 0.1 and 0.2 M sodium chloride. Cells respond to osmotic shock by turning on a specific set of genes, among them is the ompC gene. The induction depends on a two-components regulatory system, EnvZ/OmpR. The data show that this signal transduction system worked even better under microgravity conditions than in the 1-g control.

Also, Bacillus subtilis showed a higher growth rate in 0 g in an experiment performed using Biorack during the Spacelab D-l mission with an onboard 1-g centrifuge (Mennigmann and Lange 1986).

What can be concluded from all these, in part disagreeing, data? As said above and due to their simple structure, small size, and lack of organelles it is difficult to believe in a direct effect of gravity on bacteria. Nevertheless, it is clear that microgravity is a favorable condition for cell growth and signal transduction. In addition to basic research, the high interest in such studies is driven by the importance to assess the risk of bacterial infections on board space vehicles. In fact, gravity seems to be an environmental signal affecting bacterial virulence. Another aspect, discussed in Chapter 7, is the use of E. coli to study of the effect of cosmic radiation on living systems.

I would like to end this section by mentioning the theory of panspermia proposed in the seventies by the British astronomers Hoyle and Wickramasinghe, according to which life expanded in the Universe by means of bacteria as constituents of the cosmic dust.

3.1.4 Yeast

Saccharomyces cerevisiae, the yeast used to bake bread and cakes, is a highly appreciated organism to study several aspects of eukaryotic cell, like signal transduction, genetic expression, and adaptation to environmental stress. It has the great advantage of being resistant to rough environmental conditions like freezing or lack of nutrients. It also has biological properties and behavior analogous to those of mammalian cells that are, by contrast, much more sensitive to the environment and therefore much more difficult to keep alive in space experiments. The analogy with mammalian cells permits to investigate crucial biological processes and even to carry out cancer research with yeast cells. In addition, it is widely used in biotechnological processes, in particular in genetic engineering. Therefore, it is not surprising that yeast cells have been extensively chosen for experiments in space. As in the case of E. coli, several studies were dedicated to the effects of cosmic radiation.

With the increasing interest in bioprocessing in space (see Chapter 8) the need for sophisticated cell culture and tissue engineering facilities, also known as bioreactors, to be installed in space laboratories, in particular on ISS, became evident. It was clear that the technological challenge due to the constraints imposed to space instrumentations suggested to start the development of space bioreactors using yeast cells that are easy to cultivate and to preserve instead of delicate and sensitive mammalian cells. Only once the instrumentation has proven adequate can the experimentation with mammalian cells and tissue begin.

pH control

Reactor chamber u '

Gas exchange membrane s -

pH control

Reactor chamber u '

Gas exchange membrane s -

Flow sensor micropump

Used medium

Fresh medium

Figure 4-12. Elements and interconnections of the space bioreactor of the ETH Zurich. Courtesy of Isabelle Walther.

Flow sensor micropump

Used medium

Fresh medium

Figure 4-12. Elements and interconnections of the space bioreactor of the ETH Zurich. Courtesy of Isabelle Walther.

A first step in this direction was the development of a bioreactor for the culture of yeast cells that flew during three Space Shuttle missions (Figures 4-11 and 4-12). The experiments were conducted by Isabelle Walther from our laboratory (Walther et al. 1994, 1996, and 2003). When a daughter cell is generated, a typical scar, called bud scar, is left on the envelope of the mother cell. Normally, the scars left by several daughters are symmetrically distributed at two poles of the mother. A significant difference in the distribution of the bud scars was observed between cells cultured in 1 g and in 0 g. In fact the percentage of randomly distributed bud scars was higher in the

0-g (17%) than in the 1-g (5%) cells (Figure 4-13). However, no significant differences were noted in the cell cycle, ultrastructure, cell proliferation, cell volume, ethanol production, and glucose consumption.

NASA is also strongly supporting a project of a large bioreactor facility for ISS, called the Cell Culture Unit (CCU). The preliminary tests will be conducted with yeast cells. Further information is available on the Internet at the following URL sites: http://brp.arc.nasa.gov/GBL/Habitats/ccu.html and www.payload.com

Figure 4-13. Saccaharomyces cerevisiae cells cultivated in a space bioreactor. Left: Transmission electron micrograph of one cell, 27.000 x. Right: Scanning electron micrograph of celts showing budding scars and buds, 7.000 x.

3.1.5 Ciliates and Flagellates

There are unicellular organisms that are particularly interesting for studies in gravitational physiology and space biology due to the display of swimming properties like negative and positive gravitaxis and gravikinesis. Pioneering studies were conducted by Planel et al. (1982), Hader et al. (1996), and Hemmersbach et al. (Hemmersbach and Hader 1999, Hemmersbach and Braucker 2002). All have published several review articles on the subject.

The swimming behavior of ciliates and flagellates may be driven by gravity, light irradiation, oxygen, and nutrient concentration. This implies that they have structures and organelles sensing gravity. Positive gravitaxis is swimming in the same direction as the gravity vector, whereas negative gravitaxis is swimming in the opposite direction.

Gravikinesis describes an active regulation of the swimming velocities in order to compensate at least part of the cell's sedimentation: acceleration during upward swimming and deceleration during downward swimming. Such postulated changes in swimming velocities can be measured, and the values can be used for calculation of gravikinesis (Machemer et al. 1991).

Gravitokinesis can be calculated using the following formula:

Downward Swimming Velocity - Upward Swimming Velocity Gravikinesis = - - Sedimentation Velocity

The study of the regulation of motion of ciliates and flagellates and the question of their gravisensing combined with the easiness of experimental observation has attracted scientists as early as in the late nineteenth century. This is also the reason why most of what we know today has been achieved during ground-based experiments (see Hader et al. 2005 for review).

I will describe here three unicellular systems in which gravisensitivity has been deeply investigated, as reviewed by Hemmersbach and Brauker (2002) and Hader et al. (2005): the ciliates Paramecium and Loxodes, and the flagellate algae Euglena. In all these organisms, gravisensitivity has been attributed to mechanosensitive ion channels. In case of the ciliates Paramecium and Stylonychia, electrophysiological studies revealed the existence of such kind of channels and their bipolar distribution in the cell membrane. It has been postulated that the mechanical load activates these "gravisensitive" channels, i.e., weight, of the cytoplasm, which exceeds the density of the medium by about 4%.

Paramecium ant.

Paramecium ant.

Euglena ant.

ant.

w 8 Ca-mechanoreceptor channeîs ü K-mechanoreceptor channels

Figure 4-14. Models of graviperception in three protist species (ant. = anterior cell pole). Ca-and K-mechanoreceptor channels are incorporated in the ceil membrane. These channels are activated by the mechanical load of the cytoplasm (forces symbolized by arrows; see text for details). Additionally, Loxodes bear specialized gravireceptors, the Müller vesicles (not to scale). Adapted from Hemmersbach and Brciucker (2002).

In Paramecium, potassium (K) channels are located mainly at the posterior site of the organism, whereas calcium (Ca) channels are located at the anterior site (Figure 4-14). Stimulation by the weight of the cytoplasm leads, according to Machemer et al. (1991) either to hyperpolarization (K-channels) or depolarization (Ca-channels) of the membrane potential, which in turn increases or decreases the swimming rates, respectively.

In fact, if a Paramecium or Stylonychia cell is tuned upside down, a distinct gravireceptor potential can be measured: hyperpolarization (stimulation of the posterior mechanosensitive K-channels) and depolarization (stimulation of the anterior mechanosensitive Ca-channels), depending on the orientation of the cell (Gebauer et al. 1999) (Figures 4-14 and 4-15).

Figure 4-15. Scanning electron micrograph of a replica of a ruptured Loxodes striatus. Arrows indicate the barium sulfate granula of three Müller vesicles. Scale bar: 1 /im. Courtesy of R. Hemmersbach, Institute of Aerospace Medicine, DLR, Cologne.

In Euglena gravisensing is also based on mechanoreceptors in the membrane. According to Hader et al. (2005) mechanosensitive Ca-channels are located at the anterior part of the cell, and are activated by the load of the cytoplasm. Their stimulation induces a signal transduction cascade where cAMP8, calmodulin9, and possibly phosphorylation10 processes are key players (Streb et al. 2002).

8 cAMP is a small, ring shaped molecule that acts as a chemical signal in signal transduction.

In contrast to the cell membrane-located gravisensing mechanism described above, an intracellular gravity receptor has been identified in case of Loxodes. This ciliate bears statocyst-like organelles, the Müller vesicles. This is a vacuole of 7-pm diameter containing a dense granulum of barium sulfate fixed to a microtubular stick. The stimulus for graviperception, provided by the movement of the cell, causes mechanical shear to the stick. This stimulus triggers changes in membrane potential and in ciliary activity, which induces cell movement. Destruction of the Müller vesicles by means of laser beams leads to the loss of orientation capacity in Loxodes. However, 12-day cultivation in space did not affect their morphology. Although there were indications of less mineralization of the Müller vesicles in 0 g, this protist showed normal gravitaxis after flight. Such organelles appear to be an exclusivity of the family Loxodidae among protozoa, and show some analogy to the statoliths in plants and the otoliths in humans and other vertebrates.

Several pioneering experiments were carried out with Paramecium in space by the team of Hubert Planel at the University of Toulouse (Planel et al. 1981, Planel et al. 1982, Planel 2004). The main results were: higher cell growth rate, increase in cell volume, decrease in total cell protein content, and lower cell calcium content. It was postulated that the higher cell proliferation is related to changes in the energetic metabolism. Indeed, in microgravity it seems likely that the ciliary movement and the swimming of paramecia should require less energy expenses than on Earth. A fraction of the ATP, the component used for the ciliary movement, could be saved and used for cell metabolism and cell division. In hypergravity, the swimming, which is reduced, should require more energy. Therefore, less ATP is available, which could explain the lower cell growth rate. The changes in ATP content in Paramecium exposed to hypergravity or simulated microgravity are in good agreement with this mechanism.

The data on the increasing proliferation rate of Paramecium in 0 g are intriguing in view of the discovery of clock genes. One can speculate, as we will discuss it later in this chapter, that gravity may interact with certain cellular functions regulated by such genes.

The question of sensitivity threshold has been addressed by using a sophisticated slow rotating centrifuge microscope, called NIZEMI (for Niedergeschwindigkeit Zentrifuge Mikroskop). NIZEMI has been developed by the German Space Agency based on ideas and initial concepts by

9 Calmodulin is a small calcium-binding protein that is the most important transducer of intracellular calcium signals.

10 Phosphorylation is the process of adding a phosphate group to a protein or another compound (e.g., the formation of ATP from ADP). This process modifies the properties of neurons by acting on an ion channel, neurotransmitter receptor, or other regulatory molecule.

Wolfgang Briegleb, and flew on board the IML-2 mission in 1994. The acceleration threshold inducing graviresponse has been determined by increasing the acceleration profile from 0.0001 to 1.5 g. The following values were obtained: Paramecium, 0.35 g; Euglena, 0.16 and 0.12 g; and Loxodes, less than 0.15 g. Interestingly, the results were similar when the cells were subjected either to increasing or decreasing accelerations, and the effect was independent of the previous exposure to microgravity up to 12 days, although the cells underwent several division cycles.

Figure 4-16. Physarum polycephalum. Courtesy of I. Block, Institute of Aerospace Medicine, DLR, Köln, Germany.

3.1.6 Slime Mold

Physarum is a unicellular organism that lives in forests on rotting wood and can grow to cover areas up to one square meter (Figure 4-16). It is characterized by a system of communicating cytoplasmic veins, in which a rhythmic cytoplasm streaming distributes nutrients and disposes of waste metabolites. Also, the streaming is involved in cellular signaling. Wolfgang Briegleb and collaborators studied the cytoplasmic streaming and the underlying contraction rhythm of the veins by means of cine- and video cameras under actual microgravity and in a fast-rotating clinostat (Block et al. 1986, 1994a). Significant increases in the frequency of the contraction rhythm and the streaming velocity were observed.

Follow-up studies (Block et al. 1994b, 1996, 1999) allowed to:

a. Demonstrate a simultaneous processing of different stimuli (acceleration, light) in the same signal-transduction pathway;

b. Determine the acceleration-sensitivity threshold to be 0.1 g in

Physarum;

c. Show the all-or-none-law to be valid in the acceleration-stimulus response;

d. Imply the existence of internal gravireceptors (dense cell organelles, nuclei or mitochondria, both numbering to the million in one Physarum cell);

e. Detect the involvement of second messengers (cAMP) in the first steps of the acceleration-signal transduction chain.

3.1.7 Mammalian Cells

Three pioneering experiments conducted in the early days of space biology have inspired my own research. One was a Soviet-Hungarian study on human lymphocytes that were activated with polynucleotides on board Salyut-7 (Talas et al. 1984). Although the conditions of this space experiment were not ideal, the results showed that lymphocyte function changed in 0 g. A five-fold increase of the interferon-a production was observed. The second investigation was performed on WI38 human embryonic lung cells by a U.S. team on board Skylab (Montgomery et al. 1978). In what is probably the most sophisticated instrument for cell biology ever used in a space laboratory, the cells were cultivated over weeks under controlled conditions. A microscope and a camera permitted cinematographic recording. However, cinematographic recording, phase, electron and scanning microscopy indicated no observable differences in ultrastructure and in cell migration between flight and ground controls. The third study was conducted independently by both U.S. and Soviet scientists, and was dedicated to the study of the immune system of humans in space. Lymphocytes taken from crewmembers of Skylab and Salyut prior to and after flight were activated with mitogens. Kimzey (1977) reported that the rate of RNA synthesis was significantly decreased after flight. Konstantinova and collaborators (1973) obtained similar results. Although these last investigations were not true cell biology experiments, they showed that it was possible to simulate an immune reaction in vitro and thus to study a very intriguing differentiation process.

In the following years, more experiments were carried out on animal and human cells in space as well as on Earth in devices simulating conditions of microgravity. It became clear that microgravity affects the morphology and important cellular functions. As described in the following sections, changes were noted in cell proliferation, in the cytoskeleton, in signal transduction, and in genetic expression.

The most extensively studied cell systems were lymphocytes and bone cells. The lymphocyte studies were conducted mainly by our team in Zurich, Didier Schmitt and collaborators (Hatton et al. 1999) in Strasbourg, Neal Pellis and Ben Hashemi at the NASA Johnson Space Center (Sundaresan et al. 2002, Hashemi et al. 1999), Steve Chapes at Kansas State University (Chapes et al. 1992), and by Marian Lewis at the University of Alabama in Huntsville (Lewis 2002).

The bone cells work was performed by Jackie Duke at the Texas University in Houston, Yasuhiro Kumei now at the Tokyo Medical Dental University (Kumei et al. 1996, 2004), and by Millie Hughes-Fulford at the University of California in San Francisco (Hughes-Fulford 2002).

A pioneering space study on genetic expression was performed on human renal cortical cells by Tim Hammond of the Tulane University (Hammon et al. 1999).

Figure 4-17. Mitotic index, determined as amount of tritiated thymidine (2 h pulse) incorporated into DNA, of T-lymphocytes activated for 72 h with concanavalin A. The data are expressed as percent of the control processed on the ground. There is a 80% decrease in activation in 0 g (0 G Space) compared to Earth (1 G Ground). The slightly reduced activation of samples kept in an onboard 1-g centrifuge (1 G Space) compared to Earth is most probably due to the stops of the centrifuge to operate other experiments.

3.2 Results by Cell Functions

In the following section, I am reviewing the relevant findings subdivided in the major cell functions affected.

3.2.1 Cell Proliferation

One of the most dramatic effects discovered so far is the nearly total loss of response to mitogenic activation by human T-lymphocytes in vitro (Figure 4-17). This was the result of an experiment conducted during

0G Space

1 G Space

1 G Ground

Spacelab-1 in 1983 by a team of my laboratory (Cogoli et al. 1984). Another unexpected result was the 100% increase in the mitotic index when the same cells were attached to microcarrier beads. These results were later confirmed in a series of experiments performed using Biorack during the Spacelab D-1, IML-2, and SLS-1 missions. Part of the work was conducted in collaboration with the team of Proto Pippia of the University of Sassari in Italy. These surprising data triggered similar investigations with lymphoid cell lines and other mammalian cells in several other laboratories.

Due to their role in cellular immunity and to the complexity of their activation mechanism, T-cells are objects of extensive investigations since decades. As said before, in the early seventies Russian scientists were the first to report that the activation of lymphocytes from astronauts by mitogens was depressed after flight. A little later, U.S. investigators reported similar results. This may point to a higher risk of infection during and after spaceflight. To study the problem in more detail it was suggested to test lymphocyte activation in cell cultures in space. Three lines of experiments were conducted: in vitro, ex vivo, and in vivo studies.

Effector cell

Activator

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