Human uterine epithelial RL95-2 cells reorganize their cytoplasmic architecture with respect to Rho protein and F-actin in response to trophoblast binding

Abstract

Embryo implantation is initiated by interaction of trophoblast with uterine epithelium via the apical cell poles of both partners. Using spheroids of human trophoblastoid JAR cells and monolayers of human uterine epithelial RL95-2 cells to simulate this initial interaction, we previously demonstrated that formation of stable cell-to-cell bonds depends on actin cytoskeleton (F-actin) and small GTPases of the Rho family, most likely RhoA. In this study, we determined the apical as well as the basal distribution of these proteins by fluorescence confocal microscopy before and after binding of JAR spheroids. We focussed on changes in cytoplasmic organization with respect to apicobasal polarity of RL95-2 cells. Before binding of spheroids, significantly higher fluorescence signals of RhoA [37 +/- 6 grey scale values (gsv)] and of F-actin (41 +/- 3 gsv) were found in the basal region of RL95-2 cells as compared to the apical pole (RhoA: 24 +/- 3 gsv, F-actin: 28 +/- 2 gsv). After binding of JAR spheroids, this apicobasal asymmetry was inverted (RhoA: 55 +/- 10 gsv apical vs. 25 +/- 3 gsv basal; F-actin: 108 +/- 17 gsv apical vs. 57 +/- 7 gsv basal). Inactivation of Rho GTPases in RL95-2 cells by Clostridium difficile toxin A leads to a loss of their apical adhesion competence, as previously published. Here, we observed a uniform distribution of RhoA and F-actin between apical and basal region rather than an asymmetric one in toxin A-treated cells. These data suggest that activation of Rho GTPases and coordinated rearrangement of F-actin within uterine epithelial cells in response to trophoblast binding are part of a generalized structural and functional reorganization of the cytoplasm. This involves not only the immediate contact zone (apical) but also the opposite (basal) cell pole and may be a critical element of uterine epithelial reactions during transition between trophoblast adhesion and transmigration.