Vimentin as a Marker of Early Differentiating, Highly Motile Corneal Epithelial Cells FEDERICO CASTRO-MU ~ NOZLEDO, 1 * DIANA G. MEZA-AGUILAR, 1 ROCÍO DOMÍNGUEZ-CASTILLO, 2 VEREMUNDO HERN  ANDEZ-ZEQUINELY, 1 AND ERIKA S  ANCHEZ-GUZM  AN 1 1 Department of Cell Biology, Centro de Investigaci  on y de Estudios Avanzados del IPN, M exico City, Mexico 2 Department of Molecular Biomedicine, Centro de Investigaci  on y de Estudios Avanzados del IPN, M exico City, Mexico Vimentin (Vim), a cytoskeletal intermediate filament, is part of a naturally occurring reversible program, the Epithelial-Mesenchymal Transition (EMT), which converts epithelial cells into mesenchymal-like derivatives. Based on previous results showing that epithelial cells co-express Vim and keratin (Krt) as part of a cytoskeletal network which confers them a highly motile phenotype, we explored the role of Vim in rabbit corneal epithelial cells or RCE1(5T5) cells, an established model of corneal epithelial differentiation. Vim and keratin filaments were co-expressed in cells localized at the proliferative/migratory rim of the growing colonies, but not in basal cells from the center of the colonies nor at suprabasal cell layers. Flow cytometry and qPCR demonstrated that there was a decrease in Krt þ /Vim þ cell number and DNp63a expression when cells reached confluence and formed a 4–5 layered epithelium, while there was a concomitant increase of both Pax-6 expression and Krt þ /Vim  cells. Inhibition of cell proliferation with mitomycin C did not modify cell motility nor the expression of Vim. We studied the distribution and expression of a6 integrin, a protein also involved in cell migration. The results demonstrated that a6 integrin had a distribution which was, in part, co-linear with Vim at the proliferative/migratory rim of cell colonies, suggesting an indirect interaction between these proteins. Immunoprecipitation and immunostaining assays indicated that plectin might be mediating such interaction. These data suggest that Vim expression in corneal epithelium is found in a cell population composed of highly motile cells with a Vim þ /Krt þ /DNp63a þ /Pax-6 low /a6 integrin þ phenotype. J. Cell. Physiol. 9999: 1–13, 2016. ß 2016 Wiley Periodicals, Inc. Vimentin (Vim), a cytoskeletal protein that belongs to the type III intermediate filament family (reviewed by Fuchs and Weber, 1994) was long considered a specific marker of mesenchymal cells, forming a unique and distinctive type of intermediate filaments (IFs) in fibroblasts and other related cells (Franke et al., 1979a; Herrmann and Aebi, 2000). Nevertheless, Vim was also identified in epithelial cells and neurons during cell migration and morphogenetic movements in embryos (Bignami et al., 1982; Lane et al., 1983; Kasper et al., 1989), being regarded as an element associated to early development, and opening the possibility that Vim could be required for assembly of the cytoskeletal network associated to the differentiated phenotype (Schnitzer et al., 1981; Wikstr€ om et al., 1988; Oudega and Marani, 1991). Further investigations showed that Vim is also associated to epithelial pathologies, contributing to the migratory and aggressive behavior of tumor cells (Moll et al., 1982; Ramaekers et al., 1983). Based on its expression during epithelial- mesenchymal transition observed in metastatic tumors, Vim was described as a marker of oncogenic progression and invasiveness (Summerhayes et al., 1981; Miettinen et al., 1982; Raymond and Leong, 1989; Dou et al., 2014; Yi et al., 2015). On the other hand, the presence of Vim in adult epithelial cells has been mainly reported in cultured cells and consequently its expression was assumed to result from in vitro conditions (Franke et al., 1979a,b; Biddle and Spandau, 1996). However, we recently identified in cell culture, an epidermal keratinocyte subpopulation that shows a p63 þ / a5b1 bright phenotype and displays Vim IFs alongside an extensive keratin (Krt) network (Castro-Mu~ nozledo et al., 2015). These Vim/Krt-positive cells are basal cells mostly located at the proliferative/migratory rim of the growing colonies; but they are also found in confluent stratified epithelia, forming scarce and scattered small groups of basal cells (Castro-Mu~ nozledo et al., 2015). At present, evidence suggests that Vim filaments play a dynamic role during cell motility, spreading and signaling (reviewed by Clarke and Allan, 2002; Ivaska et al., 2007). Interestingly, Vim expression has been observed in epithelial cells located at the edges of healing wounds in corneal epithelium (SundarRaj et al., 1992); and in an specific cell population at limbal epithelium (Lauweryns et al., 1993a,b; Schl€ otzer-Schrehardt and Kruse, 2005; Schl € otzer-Schrehardt et al., 2007; Utheim et al., 2009; Merjava et al., 2011), which is the anatomic site viewed as the stem cell or early progenitor reservoir of the corneal epithelium (Schermer et al., 1986; Cotsarelis et al., 1989; reviewed by Castro-Mu~ nozledo, 2013). These results open the possibility that Vim could be an important part of the migratory/proliferative phenotype Conflicts of interest: The authors have no conflict of interest. Contract grant sponsor: Institute of Science and Technology from Mexico City (ICyTDF); Contract grant number: 138/2012. Contract grant sponsor: Consejo Nacional de Ciencia y Tecnología (CONACyT); Contract grant number: 219601. *Correspondence to: Federico Castro-Mu~ nozledo, PhD, Department of Cell Biology, Centro de Investigaci  on y de Estudios Avanzados del IPN, Apdo. Postal 14–740, M exico City 07000, Mexico. E-mail: fcastro@cell.cinvestav.mx Manuscript Received: 11 February 2016 Manuscript Accepted: 11 July 2016 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 00 Month 2015. DOI: 10.1002/jcp.25487 ORIGINAL RESEARCH ARTICLE 1 Journal of Journal of Cellular Physiology Cellular Physiology © 2016 WILEY PERIODICALS, INC.