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.

Demonstration of Ubiquitin Thiolester Formation of UBE2Q2 (UBCi), a Novel Ubiquitin-Conjugating Enzyme with Implantation Site-Specific Expression1

“…The two cell populations differ in the expression of genes, with the luminal epithelial cells interacting directly with the trophoblast and the glandular epithelial cells expressing secretory proteins for conceptus recognition, survival, and development. During implantation, luminal epithelial cells also undergo progressive differentiation with reorganization of the cytoskeletal networks as invasion of the trophoblast occurs [4,[45][46][47]. Some of the peri-implantation cytoskeletal changes have been shown to involve an upregulated expression of cytokeratin 13, specifically in the luminal epithelial cells of both rabbits and humans [47], as well as changes in the localization and expression of other intermediate filament proteins [46,48,49].…”

Human uterine epithelial RL95-2 and HEC-1A cell-line adhesiveness: the role of plexin B1

“…This process prepares the contact of apical cell pole of uterine cells with the trophoblast (25). In this study, we performed attachment assays with spheroids of the human trophoblast JAR cell line, a wellestablished model for adhesion assays, based on human EEC lines (16,26,27). The human EEC line RL95-2 demonstrated increased adhesiveness for JAR spheroids (71% spheroid attachment) compared with another completely polarized human EEC line, HEC-1A (23% spheroid attachment).…”

“…Fluorescence study localization techniques have shown an almost nonpolarized organization of the actin cytoskeleton in RL95-2 cells. In these cells, peripheral actin cortex was observed without microvilli and actin stress fibers at their basal membrane (16,26). Whether the effect of plexin-B1 is via F-actin reorganization and GTPase distribution requires further investigation.…”

Epithelial–mesenchymal transition in rhesus monkey embryonic stem cell colonies: the role of culturing conditions

“…Confocal microscopy was performed using a Zeiss Axiovert 100M microscope attached to a confocal laser-scanning unit (model LSM 510; Carl Zeiss) as described previously (Heneweer et al 2002(Heneweer et al , 2003. Two helium-neon lasers with output at 543 and 633 nm were used as excitation sources.…”