G. Gasset

Plant cell proliferation and growth are altered by microgravity conditions in spaceflight.

Seeds of Arabidopsis thaliana were sent to space and germinated in orbit. Seedlings grew for 4d and were then fixed in-flight with paraformaldehyde. The experiment was replicated on the ground in a Random Positioning Machine, an effective simulator of microgravity. In addition, samples from a different space experiment, processed in a similar way but fixed in glutaraldehyde, including a control flight experiment in a 1g centrifuge, were also used. In all cases, comparisons were performed with ground controls at 1g. Seedlings grown in microgravity were significantly longer than the ground 1g controls. The cortical root meristematic cells were analyzed to investigate the alterations in cell proliferation and cell growth. Proliferation rate was quantified by counting the number of cells per millimeter in the specific cell files, and was found to be higher in microgravity-grown samples than in the control 1g. Cell growth was appraised through the rate of ribosome biogenesis, assessed by morphological and morphometrical parameters of the nucleolus and by the levels of the nucleolar protein nucleolin. All these parameters showed a depletion of the rate of ribosome production in microgravity-grown samples versus samples grown at 1g. The results show that growth in microgravity induces alterations in essential cellular functions. Cell growth and proliferation, which are strictly associated functions under normal ground conditions, appeared divergent after gravity modification; proliferation was enhanced, whereas growth was depleted. We suggest that the cause of these changes could be an alteration in the cell cycle regulation, at the levels of checkpoints regulating cell cycle progression, leading to a shortened G2 period.

Growth and division of Escherichia coli under microgravity conditions.

The growth rate in glucose minimal medium and time of entry into the stationary phase in pepton cultures were determined during the STS 42 mission of the space shuttle Discovery. Cells were cultured in plastic bags and growth was stopped at six different time points by lowering the temperature to 5 degrees C, and at a single time point, by formaldehyde fixation. Based on cell number determination, the doubling time calculated for the flight samples of glucose cells was shorter (46 min) than for the ground samples (59 min). However, a larger cell size expected for more rapidly growing cells was not observed by volume measurements with the electronic particle counter, nor by electron microscopic measurement of cell dimensions. Only for cells fixed in flight was a larger cell length and percentage of constricted cells found. An optical density increase in the peptone cultures showed an earlier entry into the stationary phase in flight samples, but this could not be confirmed by viability counts. The single sample with cells fixed in flight showed properties indicative of growth stimulation. However, taking all observations together, we conclude that microgravity has no effect on the growth rate of exponentially growing Escherichia coli cells.

Weightlessness acts on human breast cancer cell line MCF-7

Because cells are sensitive to mechanical forces, weightlessness might act on stress-dependent cell changes. Human breast cancer cells MCF-7, flown in space in a Photon capsule, were fixed after 1.5, 22 and 48 h in orbit. Cells subjected to weightlessness were compared to 1 g in-flight and ground controls. Post-flight, fluorescent labeling was performed to visualize cell proliferation (Ki-67), three cytoskeleton components and chromatin structure. Confocal microscopy and image analysis were used to quantify cycling cells and mitosis, modifications of the cytokeratin network and chromatin structure. Several main phenomena were observed in weightlessness: The perinuclear cytokeratin network and chromatin structure were looser; More cells were cycling and mitosis was prolonged. Finally, cell proliferation was reduced as a consequence of a cell-cycle blockade; Microtubules were altered in many cells. The results reported in the first point are in agreement with basic predictions of cellular tensegrity. The prolongation of mitosis can be explained by an alteration of microtubules. We discuss here the different mechanisms involved in weightlessness alteration of microtubules: i) alteration of their self-organization by reaction-diffusion processes, and a mathematical model is proposed, ii) activation or deactivation of microtubules stabilizing proteins, acting on both microtubule and microfilament networks in cell cortex.

Preliminary results of Cytos 2 experiment.

Cytos 2 experiment, carried out during the French-Soviet manned flight (July 1982), has studied the antibiotics sensitivity of bacteria cultivated in vitro during the orbital flight. The results show an increase of the antibiotics resistance and a larger thickness of the cellular envelope for the inflight cells. The increase of antibiotics resistance can be related to a stimulating effect of space on the cell growth rate or to changes of the cellular envelope structure.

Results on Artemia cysts, lettuce and tobacco seeds in the biobloc 4 experiment flown aboard the Soviet biosatellite Cosmos 1129

Artemia cysts, lettuce and tobacco seeds were flown aboard the Cosmos 1129 for 19 days. A correlative method was used in order to determine the passage of cosmic heavy ions (HZE particles) through the biological test objects. This space flight resulted in a decrease on hatchability, nucleic acid and protein synthesis in hydrated Artemia cysts. HZE particle effects on plant cellular chromosomes are confirmed. In tobacco seeds, a stimulating effect on germination rate and a higher frequency of abnormalities were observed. Dormant biological objects are a very suitable material to study cosmic ray effects: these objects can be arranged in monolayers and sandwiched between visual track detectors in order to determine the passage of the cosmic heavy ions (HZE particles). On the other hand this method allows us to study effects of microgravity and those of the protonic component of cosmic rays in the objects not hit by the HZE articles.

Effects of gravity and cosmic rays on cell proliferation kinetics in Paramecium tetraurelia

Space flights resulted in a stimulating effect on kinetics of proliferation in Paramecium tetraurelia. Additional experiments were performed in order to determine the origin of this phenomena. Paramecia were cultivated in balloon flights or in a slow clinostat, or were exposed to different levels of hypergravity. The results suggest that changes in cell proliferation rate are related to cosmic rays and to a direct effect of microgravity.

Changes in developmental capacity of artemia cyst and chromosomal aberrations in lettuce seeds flown aboard Salyut-7 (Biobloc III experiment)

This paper gives the results of investigations performed on the first container (A) of the Biobloc III experiment, flown aboard the orbital station Salyut 7 for 40 days. The space flight resulted in a decreased developmental capacity of Arterlia cysts, hit or not hit by the HZE particles. No effect was observed in cysts in bulk. A synergetic effect of microgravity and gamma pre irradiation is described. The germination of in-flight lettuce seeds was decreased. The space flight resulted also in a higher percentage of cells with chromosomal aberrations. Relations between biological response, TEL and location of HZE particles are discussed.

Space environmental factors affecting responses to radiation at the cellular level.

Previous space experiments suggest a high value for the RBE of cosmic radiation. A possible explanation could be a change in cell radiosensitivity due to a combined effect of radiation and other factors related to the space environment and to the space flight. Results of the EXOBLOC II experiment support this assumption. On earth, vibrations or accelerations applied before or after irradiation can change the responses to radiation. Microgravity could be the main factor affecting the radiosensitivity and DNA repair but this hypothesis must be confirmed by additional experiments.

The "Root" experiment of the "Cervantes" Spanish Soyuz Mission: cell proliferation and nucleolar activity alterations in Arabidopsis roots germinated in real or simulated microgravity

In an experiment conducted in the “Cervantes” Spanish Soyuz Mission, a 10-day flight to the International Space Station, Arabidopsis seeds were germinated, seedlings grew for 4 days at 22ºC, and they were fixed in flight with paraformaldehyde. A ground 1 g control experiment was replicated, and an additional experiment in simulated microgravity, using a Random Positioning Machine, was performed in the same conditions. Structural, morphometric and immunocytochemical data were compared. Glutaraldehyde-fixed seedlings similarly grown in the Space Shuttle (STS-84 Mission) were also used for ultrastructural and morphometric studies. Seedlings grown for 4 days in real or simulated weightlessness showed a longer size than the ground 1 g control. Root meristematic cells showed an enhanced proliferating rate, but ribosome biogenesis was reduced, as inferred from the nucleolar size and from the levels of the nucleolar protein nucleolin. This could be the consequence of the acceleration of the cell cycle, with shortening of its phases. Weightlessness induces stress in the plant, influencing cellular processes decisive for development and morphogenesis. This stress may endanger the plant and would require the action of compensating specialized defence mechanisms.

The "Gene" Experiment in the Spanish Soyuz Mission to the ISS. Effects of the cold transportation step

If exploration of outer space is going to be a major human enterprise in the future, it is important to establish the nature of the biological response to the space environment. In one of the recent Soyuz missions to serve the ISS, the Spanish Soyuz Mission in October 2003, we sent a group of Drosophila pupae that underwent almost complete development there. Microarray analyses of the RNAs extracted from flies fixed in the ISS revealed that a relatively large set of genes (15% of the total number assayed) suffered a significant expression change in these conditions. Furthermore, the samples had to be transported to the launch site and it was necessary to slow down their development by exposing them to a lower temperature, fully compatible with pupal development. Such a pre- exposure had an effect by itself on the pattern of gene expression observed after pupal development at normal temperature, but the two environmental factors seemed to act synergistically together with the containment in the type I container. These findings indicate the importance of maintaining a vigorous scientific program in the ISS to understand the consequences of the modified environment in outer space on living organisms.