## 1 Introduction  
Microgravity has noticeable effects at the cellular level, producing morphological and functional alterations in a large number of different cell types [1; 2; 3; 4]. It is believed that cellular modifications induced by weightlessness play a fundamental role in the development of diseases caused by microgravity in astronauts, so in the last decades, it has become crucial to study the effects of simulated microgravity in various experimental models that would mimic different aspects of human physiology [1]. Simulated microgravity can be obtained through the use of various devices. The random positioning machine (RPM) is considered and recognized as a good tool for studying the effects of weightlessness on cell cultures [5]. Studies performed on human epidermal keratinocytes have shown that simulated microgravity exposure induced the modification of expression profiles of several genes in a time-dependent manner and during recovery [6], and it influenced the circadian oscillations of clock genes without affecting either cell proliferation or apoptosis rates [7]. These results underline the existence of an integrated mechanical and chemical signals that influence gene regulation and transcription [8]. In addition, long-term exposure to microgravity conditions is able to affect mouse skin homeostasis [9; 10]. In a previous study [11], we have demonstrated that, after exposure to simulated microgravity in random positioning machine (RPM) for 24 h, human immortalized keratinocytes (HaCaT) cells underwent an epithelial-mesenchymal transition (EMT) through the expression of the typical EMT transcription factors and markers. During cutaneous wound healing, the EMT process is necessary to restore the epidermal barrier through re-epithelialization. Keratinocytes surrounding the wound lose their adherent epithelial phenotype and acquire a mesenchymal phenotype [12; 13]. Wound re-epithelialization is generally considered a partial and reversible form of EMT [14; 15]. In fact, under normal conditions, keratinocytes regain epithelial characteristics when the wound is closed [13]. However, in some pathological conditions, an uncontrolled continued EMT from keratinocytes to myofibroblasts can result in fibrosis [12]. Cutaneous fibrosis, characterized by the accumulation of extracellular matrix components, normally occurs during scar formation in wound repair, but when this process is deregulated and/or accentuated, it can lead to the formation of hypertrophic scars or keloids [16; 17]. Therefore, it was extremely important to determine whether the observed effects of simulated microgravity on keratinocytes [11] were constant over time and whether these effects were reversible. These issues are usually left aside by most published studies. Herein, we extend our previous investigation by culturing HaCaT cells in simulated microgravity for up to 72 h to assess their adaptive capability. To evaluate the phenomenon of reversibility, cells subjected to 24 h of microgravity were then cultured under a normal gravitational field (1g) for an additional 24 h. We specifically focused on the maintenance of the EMT phenotype. The migratory phenotype is characterized by cytoskeletal rearrangement and involves new focal adhesion (FA) formation and cell-cell contact disassembling. Vinculin is a key molecule highly expressed in focal adhesion, where it colocalizes with the actin binding layer to facilitate the recruitment of proteins involved in FA dynamics and in the regulation of cell migration. Vinculin is also recruited at cadherin, containing adheren junctions [18], and its activation is sensitive to mechanical stresses, given that focal adhesions (FAs) respond to mechanical forces by inducing conformational changes of the protein [18; 19; 20; 21]. Therefore, in order to fully address the study of HaCaT cell behavior under simulated microgravity exposure, in this study, we performed confocal and electron microscopy analysis of the junctional features dynamics followed by in vitro migration and invasion assay.