TcRho1, the Trypanosoma cruzi Rho homologue, regulates cell-adhesion properties: Evidence for a conserved function Luiz Dione Barbosa De Melo 1 , Nicole Eisele 1 , Jose ´ Luciano Nepomuceno-Silva, Ulisses Gazos Lopes * Laborato ´ rio de Parasitologia Molecular, Instituto de Biofı ´sica Carlos Chagas Filho, CCS, UFRJ, Rio de Janeiro, Brazil Received 12 April 2006 Available online 27 April 2006 Abstract Rho proteins are members of the Ras superfamily of small GTPases. In higher eukaryotes these proteins play pivotal role in cell movement, phagocytosis, intracellular transport, cell-adhesion, and maintenance of cell morphology, mainly through the regulation of actin microfilaments. The GTPase TcRho1 is the only member of the Rho family described in human protozoan parasite Trypanosoma cruzi. We previously demonstrated that TcRho1 is actually required for differentiation of epimastigote to trypomastigote forms during the parasite cell cycle. In the present work, we describe cellular phenotypes induced by TcRho1 heterologous expression in NIH 3T3 fibroblasts. The NIH-3T3 lineages expressing the TcRho1-G15V and TcRho1-Q76L mutants displayed decreased levels of migration compared to the control lineage NIH-3T3 pcDNA3.1, a phenotype probably due to distinct cell-substrate adhesion properties expressed by the mutant cell lines. Accordingly, cell-substrate adhesion assays revealed that the mutant cell lines of NIH-3T3 expressing TcRho1- positive dominants constructions present enhanced substrate-adhesion phenotype. Furthermore, similar experiments with T. cruzi expressing TcRho1 mutants also revealed an enhancement of cell attachment. These results suggest that TcRho1 plays a conserved reg- ulatory role in cell-substrate adhesion in both NIH-3T3 fibroblasts and T. cruzi epimastigotes. Taken together, our data corroborate the notion that TcRho1 may regulate the substrate-adhesion in T. cruzi, a critical step for successful progression of the parasite life cycle. Ó 2006 Elsevier Inc. All rights reserved. Keywords: GTPase; Rho; TcRho1; Actin; Cytoskeleton; Trypanosoma cruzi; NIH-3T3 Rho GTPases comprise a family of the Ras superfamily of small GTPases which also includes the families: Ras, Rab, Arf, Ran, RGK, and RJL [1,2]. As other small GTPases, the Rho GTPases switch between an active, GTP-bound state and an inactive, GDP-bound state. This activity is regulated by guanine nucleotide factors (GEFs), which activate the GTPases, promoting the exchange of GDP for GTP, while GTPase-activating proteins (GAPs) increase the intrinsic rate of hydrolysis of bound GTP to GDP [3]. In addition, GDP dissociation inhibitors (GDIs) can sequester Rho GTPases in an inactive state on cyto- plasm [3]. The Rho family of small GTPases plays pivotal roles in the maintenance of mammalian cell morphology, control- ling the dynamics of the actin cytoskeleton, cell-matrix adhesion, and cell–cell-adhesion [4,5]. Other cellular pro- cesses under Rho family control are signaling pathways, which lead to activation of some transcription factors [6], the control of cell cycle progression [7], and the activation of the NADPH oxidase complex [8]. Concerning to cyto- skeletal organization and migration behavior at a single- cell level: RhoA, Rac1, and Cdc42 are spatiotemporally regulated. On early stage of adhesion and spreading, new focal adhesions and actin-rich protrusion structures are induced by the activation of Cdc42 and Rac1, which lead, respectively, to formation of finger-like membranes (filopodia) and protrusions at the leading edge (lamellipodia). The later stage leads to RhoA activation, 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.04.075 * Corresponding author. Fax: +55 21 2280 8193. E-mail address: lopesu@biof.ufrj.br (U.G. Lopes). 1 These authors contributed equally to this work. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 345 (2006) 617–622 BBRC