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Featured Application: Studies on the effects of microgravity on cell–cell and cell–matrix interaction may help increase the safety and improve the health of astronauts in space. Changes in cell–matrix and cell-to-cell adhesion patterns are dramatically fostered by the microgravity exposure of living cells. The modification of adhesion properties could promote the emergence of a migrating and invasive phenotype. We previously demonstrated that short exposure to the simulated microgravity of human keratinocytes (HaCaT) promotes an early epithelial–mesenchymal transition (EMT). Herein, we developed this investigation to verify if the cells maintain the acquired invasive phenotype after an extended period of weightlessness exposure. We also evaluated cells' capability in recovering epithelial characteristics when seeded again into a normal gravitational field after short microgravity exposure. We evaluated the ultra-structural junctional features of HaCaT cells by Transmission Electron Microscopy and the distribution pattern of vinculin and E-cadherin by confocal microscopy, observing a rearrangement in cell–cell and cell–matrix interactions. These results are mirrored by data provided by migration and invasion biological assay. Overall, our studies demonstrate that after extended periods of microgravity, HaCaT cells recover an epithelial phenotype by re-establishing E-cadherin-based junctions and cytoskeleton remodeling, both being instrumental in promoting a mesenchymal–epithelial transition (MET). Those findings suggest that cytoskeletal changes noticed during the first weightlessness period have a transitory character, given that they are later reversed and followed by adaptive modifications through which cells miss the acquired mesenchymal phenotype. [ABSTRACT FROM AUTHOR]

One of the hallmarks of microgravity-induced alterations in several cell models is an alteration in oxidative balance. Notably, male germ cells, sensitive to oxidative stress, have also been shown susceptibility to changes in gravitational force. To gain more insights into the mechanisms of male germ cells' response to altered gravity, a 3D cell culture model was established from TCam-2 cells, a seminoma cell line and the only available in vitro model to study mitotically active human male germ cells. TCam-2 spheroids were cultured for 24 hours under unitary gravity (UG) or simulated microgravity conditions (SM), which was achieved using a random positioning machine (RPM). Apoptosis and necrosis analyses performed on the UG- and SM exposed samples revealed no significant differences in all of the cell death markers. Notably, the Mitosox assay revealed significant oxidation of mitochondria, after microgravity exposure, at least at this culture time. In the SM-treated samples, gene expression levels (evaluated by real-time PCR) of the main enzymes of the antioxidant barrier, GPX1 and NCF1, were reduced, indicating an influence of SM on mitochondrial function. Notably, the expression of HMOX, involved in the heme catabolism of mitochondrial cytochromes, was increased. The SOD, XDH, CYBA, NCF-2, TXN, and TXNRD genes were not affected. The ultrastructural analysis by transmission electron microscopy revealed that SM significantly altered TCam-2 spheroid mitochondria, which appeared swollen and, in some cases, disrupted. Indeed, mitophagy, or mitochondrial autophagy, appears to be more represented in the samples exposed to simulated microgravity. This result seems to be in line with the increase, mediated by the simulated microgravity, in the enzyme HMOX. All together, these preliminary data demonstrate TCam-2 spheroids' sensitivity to acute SM exposure, strongly indicating a microgravity-dependent modulation of mitochondrial morphology and activity and encouraging us to perform further investigations on the chronical exposure to SM of TCam-2 spheroids. [ABSTRACT FROM AUTHOR]

The past few decades have seen an increasing number of both space travels and studies aimed at investigating the effects induced by space flights and the environment on humans. One of the main features of these conditions is the presence of altered gravity, mostly represented by microgravity experienced by astronauts. Microgravity is well known to induce deleterious effects at cellular, organ and systemic levels, including alterations in the male and female reproductive systems. In the present study, we investigated the effect of simulated microgravity on the metabolic activity of male germ cells using TCam-2 line as a cell model. These cells were cultured in the Random Positioning Machine that simulated microgravity conditions, and were grown as 2D monolayers or 3D spheroids to assay the effects on single cells or on organ-like structures. After a 24 hour-exposure to simulated microgravity, TCam-2 monolayers showed: (1) a decreased proliferation rate and a delay in cell cycle progression; (2) increased anaerobic metabolism; (3) increased levels of reactive oxygen species and superoxide anion; (4) modifications in mitochondrial morphology. After the same 24 hour-exposure, TCam-2 spheroids showed: (1) an increased anaerobic and aerobic activity in 40% and 26% of samples, respectively; (2) alterations in the redox balance with a decrease in catalase activity in about 65% of cell samples, and therefore, a deficit in the cellular antioxidant capacity; (3) increases in oxidative damage to proteins and lipids in more than 50% of cell samples. In conclusion, these data demonstrated a clear inference of simulated microgravity on the metabolic activity of TCam-2 cells, which is expressed through the activation of an oxidative stress state, that, if not compensated for, could be deleted over time. [ABSTRACT FROM AUTHOR]

One of the most important hazards of the space environment is microgravity, which causes an alteration in the physiology of different systems, including the reproductive one. It is widely accepted that cytoskeleton is the microgravity-sensitive apparatus of the cells, and that cytoskeletal modifications are responsible for microgravity-triggered cell alterations. We established a 3D free-floating culture system from TCam-2 cell, a human seminoma cell line, and then exposed the obtained TCam-2 spheroids for 24 h at unitary gravity (UG), or under a simulated microgravity condition (SM), using the random position machine (RPM). We tested the cytoskeletal and junctional features of these samples using Western blot and confocal microscopy analysis to elucidate the impact of microgravity on the adherent and occluding junctions of TCam-2 spheroids. The junctional ultrastructure was studied using transmission electron microscopy (TEM). TEM analysis revealed the presence of occluding junctions both in UG or SM samples. Even if Western blot revealed no quantitative difference in actin and occludin proteins both in UG and SM exposed samples, fluorescence colocalization analysis showed a significative increase in the colocalization area of occludin and actin proteins in the superficial layer of TCam-2 spheroids grown in RPM conditions. This result let us speculate that tight junction functionality is different in UG and SM exposed spheroids. As far as adherent junctions are concerned, TEM analysis revealed adherent junctions both in UG or SM samples. Moreover, we observed by Western blot a trend in terms of the increase in the vimentin expression in SM exposed spheroids. Confocal microscopy analyses confirmed this significant increase. All together, these data suggest that simulated microgravity conditions in TCam-2 spheroids alter the tight junction assembly, while the increase in the intermediate filament's structures can in part be associated with an enrichment in the adherent junctions. A functional investigation is needed to more deeply clarify this hypothesis. [ABSTRACT FROM AUTHOR]