## 4 Discussion
In our study, through analyzing the fluorescence intensity (SUM (I)) of actin and vinculin in cells exposed to simulated microgravity, we obtained a positive correlation between the two markers only at the level of newly formed FAs in cells cultured in the microgravity condition for 24 h. This correlation persists but decreases in membrane protrusions observed at 48 h of simulated microgravity, while at 72 h, it disappears and reaches values overlapping those observed in cells cultured in 1g. On the basis of these results, we can hypothesize that in the FAs that we observed and analyzed, vinculin is able to recruit actin filament within the FAs, while this phenomenon does not occur in cell-cell contact or in membrane protrusions of cells deprived of their migratory capability. Furthermore, in HaCaT cells cultured in RPM for 24 h and then transferred into a normal gravitational field for a further 24 h (recovery conditions), both F-actin filament and vinculin recovered almost completely the distribution and correlation pattern that we observed at 1g. All these data demonstrated that the emergence of FAs and consequently the F-actin remodeling occurring during the first 24 h should be considered a transient and adaptive phenomenon. Noticeably, these data outline that a critical factor in the emergence of the migrating/invasive phenotype is represented by the concomitant down-regulation of E-cadherin in cells exposed to microgravity for limited periods. Indeed, reduction in the expression pattern of E-cadherin during weightlessness indicated a transitory commitment toward an invasive phenotype, which is a critical feature that is also recorded in thyroid cancer cells exposed to short-term microgravity [30]. It is worth noting that the concomitant reduction in vinculin expression, which is witnessed by confocal studies during the first 24 h of microgravity exposure, can ultimately contribute in decreasing E-cadherin values, given that vinculin exerts a positive regulatory influence upon E-cadherin levels [31]. We hypothesize that the loosening of cell-to-cell adhesion, which is ascribed to reduced E-cadherin levels, plays a key role in favoring the emergence of a migratory/invasive phenotype despite the fact that the concomitant changes occurring in the cytoskeleton--i.e., the disorganization of F-Actin network and weakening of FA--would impair the migrating capability of HaCaT cells, at least in principle. This conclusion deserves to be emphasized, as it pinpoints E-cadherin as the pivotal factor that can switch cells toward a motile, invasive phenotype. Given that the metastatic potential of tumors is highly dependent on such capabilities, it is worth noting that the critical target of the treatment should be the restoration of proper E-cadherin values, as already underlined [32]. Loss of cell-to-cell adhesions is instrumental in enabling the disassembly of the cell cluster and in promoting their migratory capability. When and if such a condition persists, then organoid-like structures--in which cells are detached from the substratum and are recognizable as floating aggregates in the supernatant--appear [33]. In many microgravity-based models, the emergence of these organoid bodies is associated with the persistence of adherent cells, so it can be assumed that microgravity enacts the partitioning of living cells into two different populations, which is characterized by different morphological and functional properties [34]. However, by using the HaCaT model, we did not observe the formation of any organoid-like structure. On the contrary, these cells were shown to be able to counteract microgravity effects on both E-cadherin and F-actin, thus re-establishing a native, non-motile phenotype within 48-72 h of adaptation in simulated microgravity. These results suggest that caution should be kept when considering data from weightlessness-based experiments, given that different exposure times can generate different biological behaviors.