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Correspondence: D. Schmitt, MSM-GS, European Space Agency/ESTEC, Keplerlaan 1, 2200 AG Noordwijk, The Netherlands.
The two questions discussed below were used to guide workshop discussions.
1. WHAT COULD BE THE MECHANISMS OF SINGLE CELL GRAVISENSITIVITY? CAN GRAVITY BE A STIMULUS?
From the various investigations that have been done in this field, we have to distinguish between what one could call "professional gravisensing cells" and "non-professional gravisensing cells." In cells like Euglena and Paramecium, a specific gravireceptor organelles' statoliths has been identified. This is also the case for statocytes in plant root tips. For all other cell types, today we can only formulate hypotheses on gravisensing mechanisms.
The cytoskeleton may be a good candidate for gravisensing. It has been suggested, for example, that it can serve as a mechanical filter or a force-gathering antenna, as in the concept of tensegrity (1). If this is a possibility, then a time constraint for a relaxation mechanism must exist (milliseconds, seconds, minutes?). At the level of the plasma membrane, receptors, channels, or adhesion molecules, gravity may also be sensed. Indeed, the membrane is very flexible and stretching/unstretching of the membrane due to changes in gravity (at 1 G nonadhering cells are lying on a surface and not in microgravity) may lead to a different cell response. These changes may trigger stretch-activated receptors in the membrane solely or in synergy with stretch changes in the cytoskeleton connected to the membrane. The presence or absence of hydrostatic pressure caused by changes in gravity does not seem to be a possible mechanism for sensing gravity via stretch-activated receptors.
Changes in energy levels or changes in the state of equilibrium may also be potential mechanisms for sensing gravity. Near the threshold where channels can be in an open or closed state, small changes may have larger effects than one could expect.
Sensing of gravity is certainly not an on/off response. There is probably a proportional–or even a linear–response as shown in some experiments with professional gravisensing cells (2). Even when thresholds are observed (at about 0.1 G), the question remains as to whether this might be due to a technical limitation in detection.
2. AT WHICH CELLULAR LEVEL(S) ARE THE EFFECTS OF MICROGRAVITY POSSIBLE?
Effects of gravity (and therefore the absence of gravity) are likely to occur at a macromolecular level. The probability of the opening of some ion channels, for example, seems to be modified not only by stretch but also by the absence of gravity. The assembly of macromolecules is also changed by the orientation of the gravity vector as has been shown with in vitro experiments involving the auto-assembly of microtubules. At the level of single cells, it is obvious that some cells, such as swimming protists, are gravisensing cells.
The specific organelles responsible in these cells for the graviperception have been identified; however, the exact biochemical mechanisms involved have still to be understood. For other isolated eukaryotic cells in culture, several effects have been observed in microgravity compared to Earth gravity. Despite all these findings, it is still not clear whether these effects are a direct consequence of the absence of gravity or related to an indirect effect via changes in convection of the surrounding culture medium, for example. Some experiments conducted at the tissue level have shown differences during spaceflight compared to ground controls, but the question also remains as to whether this is due to a direct or an indirect effect of the absence of gravity. At the tissue level, as in single adherent cells, tension may play a role as well.
RECOMMENDATIONS
Research on the specific biochemical mechanisms of gravisensing/gravitaxis in professional gravisensing cells such as protists should be pursued.
Experiments using other cell types (non-professional gravisensing cells) should be performed more carefully. Indeed, many studies up until now in this field are not conclusive as to the direct or indirect effect of gravity/microgravity on single, isolated cells in culture. This is true for adhering as well as for nonadhering cells.
Unmasking the effects of gravity on the cellular machinery must run parallel with the identification of potential artifacts, which could modify cellular responses in spaceflight experiments. Therefore, specific recommendations in the following areas should be observed.
Ground preparatory research
Cell-cell contacts and cell-culture hardware
contacts as a consequence of sedimentation must be more carefully
studied. This type of research can be performed in clinostats or random
positioning machines or other facilities using density or viscosity as
means to prevent sedimentation.
Studies using
hypergravity in the range of 1-10 G by using centrifuges
should always be a prerequisite for a spaceflight
experiment.
Action of shear forces on cells with
specifically developed hardware should be investigated. In this field,
much should be learned from fluid physics in microgravity as compared
to 1 G. The question is, are shear forces and
stress-activated signals part of gravisensing?
New
fields of investigation should be developed. For example, stirring can
be considered in order to study the effects of local heating of cell
clusters and the distribution of nutrients. Indeed, these phenomena are
altered in the absence of gravity-driven
convection.
Hardware for ground research needs new
developments and access to the existing hardware or facilities must be
promoted. The more this hardware is available, the more complementary
experiments can be performed among different laboratories. The access
to ground hardware should also be internationalized. A flight-identical
hardware must be made available in the laboratories at least 2 years
before the flight itself in order to perform extensive
testing.
Centrifuge effects, mainly vibration, must be
studied when comparing microgravity experiments with 1-G
in-flight centrifuge controls.
Vibrations and
hypergravity encountered during launch of an experiment on a spacecraft
need to be investigated before any flight experiment is carried
out.
Flight experiments
The microgravity levels required could not be well
specified at the present time, but G levels of
10-3/10-4 seem sufficient. More importantly,
the microgravity level must be uninterrupted during the
experiments.
In certain cases, transitions from 1
G to microgravity and vice versa should be used, because
studying gravity transitions may be more important than just looking at
the difference in effects between 1 G and microgravity. The
gravity level must be measured by accelerometers placed close to the
experiment.
Access to short-duration microgravity must
be fully used by means of drop towers, parabolic plane flights, and
balloon-borne drop capsules.
To study gravity changes and
thresholds, in-flight hypergravity controls should also be performed
(up to 3 G).
Real-time environment controls
(partial gas pressures, temperature, pH, etc.) must be accurately
measured in the cell culture hardware.
Optical controls
should be available in order to visualize whether cells are in
clusters, stay sedimented, or are in contact with surfaces while in
microgravity.
Enough replicates and storage
capabilities in-flight must be made available in the design of hardware
and during a given flight.
WHAT DOES MICROGRAVITY BIOLOGICAL RESEARCH FINALLY BRING?
Unlike what has been seen in physiology, concepts in cell biology have not been changed in the last 20 years by research in microgravity. Nevertheless, studies using professional gravisensing cells have clearly shown that graviorientation in plants and swimming protists, which is a matter of survival, is abolished in microgravity and that this type of research will help clarify the fine mechanisms involved. Space experiments have contributed markedly to the elucidation of the signal transduction chain in gravitaxis (and also gravitropism of higher plants). The effects of altered gravity on non-professional gravisensing cells, such as eukaryotic cells in culture, are not as easy to study. The increasing research in this field in the last 10 years has not yet unequivocally shown whether or not the effects that have been demonstrated were a direct or an indirect consequence of microgravity. However, whether direct or not, gravity is one of the parameters that cells in culture take into account, just as they do for temperature or pH. The study of the alterations in the environment, such as by changing gravity (hypergravity or microgravity), will undoubtedly lead to a better overall understanding of the cellular machinery.
CONCLUSION
The cost effectiveness of research using the space environment, mainly the quasi-absence of gravity, has not been extensively discussed in the workshop because spaceflights are often planned for reasons other than just biological science. Nevertheless, spaceflight experiments should only be considered if they have a sound ground-based program and then should be funded accordingly.
Research on cell gravisensing may have important implications for understanding fundamental cell biology. The ground research that is linked to space research has still to be promoted and might prove productive in various fields of cell biology.
REFERENCES
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