## 3 Results
### Simulated Microgravity Influences the TCam-2 Cell Ultrastruct at the Junctional Level  
As already mentioned, TCam-2 seminoma cells grow in vitro as an epithelial-like sheet. However, it has been demonstrated that these cells are not able to form ultra-structurally defined adherent junctions when cultured in basal conditions [27]. This cellular behavior is common in epithelial transformed cells which partially lose their epithelial features [27]. In a previous paper, we described that microgravity exposure induced transient ultrastructural changes such as mitochondria morphological alterations [27] and activation of autophagy [28]. In the present study, the collected images revealed that, after 24 h-exposure to simulated microgravity, the cells showed the formation of adherent junctions (Figure 7C,D), which are not detectable in cells cultured at 1 g (Figure 7A,B). It is evident that in 1 g basal conditions, the membranes of neighboring cells became very close but failed to form electron-dense regions or plaques typical of junctional contacts (Figure 7A,B). In RPM cultured cells, the cell-to-cell interface was very jagged and often engaged in desmosome-like (Figure 7C), as well as by adherent junctions (Figure 7D). Figure 2: Transmission electron microscopy analyses of cell-to-cell contacts of TCam-2 seminoma cells exposed for 24 h to 1 g (CTR: **A**,**B**) and simulated microgravity (RPM: **C**,**D**). The arrows in (**A**,**B**) indicate the region of plasma membrane in which two cells interact with each other without forming junctions. The white squares in (**C**,**D**) indicate desmosome-like and adherent junctions, respectively (BAR: 0.3 \(\upmu\)m). Simulated-Microgravity Exposure Modifies the TCam-2 Distribution Pattern of Cadherins but Does Not Influence Beta1 Integrin and Vimentin Localization  
The RPM-induced modulation of the ultrastructure of cell-to-cell contacts, observed by electron microscopy, strongly indicated that this experimental condition could modify the distribution pattern and/or the expression levels of adhesion molecules such as cadherins and integrins. We used a pan-cadherin antibody to evaluate the distribution pattern of this class of cell-to-cell adhesion molecules in TCam-2 seminoma cells. As far as integrin is concerned, we focused our attention on the beta1 integrin subunit, since it has been demonstrated that this protein is expressed by TCam-2 seminoma cells [17]. The confocal analyses of cadherin's distribution pattern showed that after RPM exposure (Figure 2B) the cells showed the presence of cadherin at the plasma membrane level with respect to cells cultured at 1 g in which, as expected, the cadherin presence close to the plasma membrane is very limited (Figure 2A). Intriguingly, the co-localization analysis of cadherin and F-Actin in RPM cultured TCam-2 seminoma cells revealed that the localization of these molecules largely overlaps, confirming the presence of stable junctional contacts, which are tightly joined to the actin cytoskeleton (Figure 27). Conversely, the analysis of beta1 integrin localization, did not reveal appreciable changes in the distribution pattern of this protein in RPM cultured samples, indicating that the linkage of the TCam-2 cells to their substrate is not apparently perturbed by simulated microgravity (Figure 2C,D). Notably, as well as for beta1 integrin, also the vimentin-composed intermediate filaments network did not change significantly in 24 h-cultured cells in RPM (Figure 2F,F). The co-localization analyses of beta1 integrin and vimentin in RPM exposed cells revealed that these two proteins did not have overlapping signals. Therefore, it is possible to speculate that they are not implicated in the cell to substrate junctional complex (Figure 27).