## 3 Results
### Cytoskeleton Modifications of HaCaT Exposed to Simulated Microgravity
#### 3.2.2 F-Actin and Vinculin Organization at 48-72 h of Simulated Microgravity  
To verify if the acquired phenotype at 24 h is stably maintained by cells exposed to microgravity, we extended the exposure times to 48 and 72 h. Surprisingly, after 48 h of simulated microgravity, lamellipodia and ruffles markedly decreased when compared to 24 h RPM cultured cells. After 72 h of microgravity exposition, the cytoskeleton architecture observed in the microgravity condition recovered almost completely the cytoskeleton organization usually observed in 1g control.  
Specifically, by confocal analysis, we observed that after 48 h of microgravity, the exposition of vinculin at the cell periphery was appreciably diminished when compared to cells exposed for 24 h in weightlessness. Colocalization analyses demonstrated little or no colocalization (Figure 5C), although a good correlation with actin (r = 0.92; \(p>\) 0.05) can be recorded in few points. At cell-to-cell contact, colocalization with F-actin was observed (Figure 5E) and, as described in 1g treated samples, there is no significant positive relationship between the two markers (r = 0.21 \(p>\) 0.05).  
Figure 4: Representative images of colocalization analysis of vinculin (green) and F-actin (red) in HaCaT cells cultured in normal gravitational condition (1g) and microgravity conditions (RPM) for 24 h. In A, vinculin distribution is shown. Vinculin is distributed in cell–cell contact in control condition (**A**), (Scale bars 37.5 μm), while it is mostly displaced at the free front of cell membrane in RPM-treated cells (**B**) Scale bars 30 μm). (**C–F**) show the colocalization analysis of vinculin and F-actin. In the X/Y axes, at the level of the dotted lines, the colocalization (green and red picks) is shown. Colocalization is evident at the level of membrane protrusion in RPM cultured cells ((**D**), dotted circle) and in cell–cell contact in 1g cultured cells ((**E**) dotted circle). Contrarily, no colocalization is observed at the level of membrane protrusion in on 1g cultured cells ((**C**), dotted circle) and in cell–cell contact in RPM cultured cells ((**F**), dotted circles).  
Figure 5: Representative images of colocalization analysis of vinculin (green) and F-actin (red) in HaCaT cells cultured in RPM for 48 and 72 h. In A, B vinculin distribution is shown; vinculin distribution is appreciably reduced at the cell periphery, while it is increased at the cell-cell contact both at 48 h (**A**) and 72 h (**B**). (**C**–**F**) show colocalization analysis of F-actin and vinculin. In the X/Y axes, at the level of the dotted lines, the colocalization (green and red picks) is shown. Colocalization after 48 h is still evident in some parts of the cellular periphery ((**C**), dotted circle), albeit reduced compared to RPM at 24 h, and it appears in cell-cell contacts ((**E**), dotted circle). Contrarily, at 72 h, no colocalization is observed at the level of periphery ((**D**), dotted circle), while being recognizable in cell–cell contact areas ((**F**), dotted circles). Scale bars 20.6 μm.  
After 72 h of microgravity exposition, the distribution of vinculin is almost concentrated within the areas of cell-to-cell contact, in which it shows a striking colocalization with F-actin bundles (Figure 5D,F), while little or no colocalization was observed in cell membranes at the leading edge of culture. Correlation analyses of vinculin with actin at the leading edge of the cell culture and in cell-cell contact showed no significant positive relationships (r = 0.173 \(p\) > 0.05; r = 0.57 \(p\) > 0.05 respectively), as we observed in the control condition.  
Overall, these findings witness that after longer periods of microgravity exposure, cells recover their native phenotype, losing their migrating/invasive properties.