## 4 Discussion  
HaCaT cells exposed to simulated microgravity show a biphasic response, displaying increased motility and invasiveness during the early period (24 h), which was followed by the recovering of a noninvasive morphological phenotype that is partly observed after 48 h and fully acquired at 72 h. Similar results were obtained by seeding again into a normal gravitational field those invasive HaCaT cells previously exposed to simulated microgravity for 24 h. Overall, these findings suggest that changes occurring at the morphological and functional level in cells exposed to microgravity for short periods have a transitory character and then are followed by adaptive modifications through which cells strive in recovering their native phenotype. Both E-cadherin and vinculin distribution patterns are likely to play a key role during those changes. The actin ability to generate/transduce forces through cytoskeleton remodeling is a pivotal tool that enables cells in mechanosensing the environmental imbalance of physical forces. The transduction of mechanical stresses involves the clustering of transmembrane receptors that are instrumental in the successive assembly of focal contacts and adhesions that physically connect the extracellular matrix to the cell cytoskeleton [23]. Binding en  
Figure 8: Representative images of Matrigel invasion by HaCaT after 24 h. HaCaT cells (red) were cultured on GFR Matrigel in normal gravitational condition ((**A–C**), scale bar 7.5 \(\upmu\)m) and microgravity conditions ((**D–F**), scale bar 150 \(\upmu\)m) for 24 h; (**A,D**) represent the confocal maximum projection; (**B,E**) represent 3D projection; (**C,F**) shows the depth analysis. As shown in this figure, HaCaT cells maintained at unit gravity invade matrix up to a depth of about 44.56 \(\upmu\)m (**C**), while cells maintained in the microgravity condition are able to invade to a depth of about 138.56 \(\upmu\)m (**F**). The table indicates the depth along the zeta axis measured in the three experiments performed (1g vs. RPM \(p\) < 0.001). hances the bundling of F-actin at the cell-substratum adhesion sites with the concomitant maturation of both the focal adhesions and the actin stress fibers [24], which represent mandatory steps to trigger the required tension for ensuring cell adherence and motility capability. It has been reported that exposure to microgravity reduces the number and total area of focal adhesions per cell [25], thus significantly reducing adherence to the substratum and migratory capability [26], albeit some contradictory results have been reported [27]. These changes are mandatory for the emergence of floating populations in cell cultures exposed to weightlessness, i.e., an organoid-like structure in which cells aggregate themselves after detaching from the substrate [28]. Therefore, our results are apparently at odds with those reported for other kind of cells. Differences in the experimental model, namely those involving different times of observation, can probably help in explaining this conundrum. Indeed, we observed increased motility only within the limited window of the first 24 h of microgravity exposure, given that after that time, HaCaT cells progressively recovered the native phenotype, losing the invasive/motile phenotype. Moreover, effects of weightlessness on F-actin cytoskeleton are still a matter of debate. While a number of studies have reported that microgravity decreases F-actin expression, leading to disorganization of the cytoskeleton [29], other reports have reported opposite findings, with increased F-actin and stress fiber formation with the emergence of lamellipodia protrusions [30]. It is well known that vinculin has several effects on actin polymerization in newly formed adhesions. Vinculin can cross-link and bundle actin filaments, and it can recruit actin modifiers such as the Arp2/3 complex, which is a potent nucleator of actin polymerization. By contrast, vinculin and F-actin polymerization at cell-cell adhesions is less understood [18].