Abstract: The acquisition of cell polarity, which includes the establishment of the tight junction barrier, the polarized assembly of the cytoskeleton and the appropriate organization of membrane traffic, requires external cues, that in epithelial cells are represented by the interaction of cells with their neighbors and with the extracellular matrix. The Rho family of small GTPases, whose prototypes are RhoA, Rac1 and Cdc42, regulates many biological processes including cell cycle progression, apoptosis, migration and intercellular adhesion. Rho-GTPases and their effectors are also key regulators of microfilament and microtubule dynamics and, consequently, are crucially involved in polarity signaling. Unraveling how signaling transduced via Rac1 are translated into oriented distribution of molecules in epithelial cells is a central issue to fully understand the processes of acquisition/maintenance of cell polarity. In order to contribute to the understanding of the molecular basis of this complex issue in the present study we have focused our attention on the analysis of the role of Rac1 protein in the acquisition and mantainance of the polarized phenotype in the FRT, rat thyroid epithelial cell line. The FRT cells were chosen as a model system since they exhibit a fully polarized epithelial phenotype, manifest high transepithelial electrical resistance and express apical and basolateral marker proteins. They also form organized tridimensional follicular structures in suspension culture. The Rac1 subcellular distribution in FRT cells was initially investigated. Subsequentely, to analyze the role of Rac1 in the control of cell polarization in the FRT cells, the cultures were treated with NSC23766, a molecule that does not allow Rac1 specific GEFs, such as Tiam1 and Trio, to bind to Rac1 and therefore acts as an inibitor of Rac1 activation. Several aspects including directional migration, TER acquisition, cell aggregation and formation of polarized follicles were investigated and found to be affected by the pharmacological inhibition of Rac1 activity. Moreover in this type of experiment, changes in subcellular localization of the Rac1 molecule were analyzed in parallel with those regarding E-cadherin. This experimental appoach allowed us to establish that Rac1 is a major regulator of the polarization process in FRT cells. The data presented also suggest that, in addition to the control of cell-cell adhesion, Rac1 may be involved also in the control of the Golgi apparatus integrity and therefore in the polarized intracellular traffic of proteins. The role of integrin signaling in the acquisition/maintenance of cell polarity has been studied to some extent in polarized epithelial cells in culture. For example in the FRT-β1B cells, derived from FRT parental cells after transfection of the dominant-negative β1B integrin, an impairment of the polarized phenotype have been described. The observation that these cells manifest properties similar to those evidenced in FRT cells where Rac1 is inhibited prompted us to test the hypothesis that this may be due to a reduced Rac1 activity. Subclones derived from FRT-β1B cells were obtained that stably express a Rac1 construct that is constitutively activated upon 4-OH-tamoxifen treatment. However it was not possibile to rescue the normal phenotype in these cells by this experimental approach. Furthermore also FRT parental cells expressing the same construct appeared to be hampered in the expression of certain properties of the polarized epithelium, indicating that sustained but not regulated acivation of Rac1 impairs the acquisition of cell polarity. In conclusion this report combines the interest in defining the role of Rac1 in a model of thyroid derived-epithelium, in which the functional properties of this small Rho GTPase heve not been investigated yet, to novel experimental approaches, such as the use of the Rac1-GEFs-interaction inhibitor and a novel conditional expression vector of Rac1 protein
