Mouse anti-c-myc (clone 9E10, MA1-980) was from Invitrogen

Mouse anti-c-myc (clone 9E10, MA1-980) was from Invitrogen. altered the morphology of actin recruitments to EA invasion sites. Additionally, EA internalization was increased in cells overexpressing CA-Rac1 but inhibited in cells overexpressing CA-RhoA. WT-Cdc42 expression increased EA internalization, but curiously, CA-Cdc42 inhibited it. Altogether, these results corroborate the hypothesis of EA internalization in non-phagocytic cells by a phagocytosis-like mechanism and present Rac1 as the important Rho-family GTPase in this process. is a protozoan parasite that causes Chagas’ disease and affects approximately 6C7 million people worldwide, mostly in Latin America (WHO., 2017). Classically, contamination begins by metacyclic trypomastigote forms released in the feces of triatomine vectors. Unlike the metacyclic or bloodstream trypomastigote forms, host cell invasion by extracellular amastigotes (EAs) is usually highly dependent on the actin cytoskeleton of host cell (Mortara et al., 2005; Ferreira et al., 2012). During host cell invasion, EAs induce recruitment and colocalization with actin of diverse host cell molecules, such as integrins, extracellular matrix components and actin binding proteins, in a cup-like structure (Procpio et al., 1999). EAs also promote the sequential and coordinated formation of phosphoinositides at their access site around the plasma membrane of HeLa cells, suggesting that they induce a phagocytosis-like process in non-phagocytic cells (Fernandes et al., 2013). Recently, our group also showed that EAs induce selective phosphorylation of cortactin by ERK, which is abolished if heat-killed parasites or non-infective epimastigote forms are used (Bonfim-Melo et al., 2015). These studies demonstrate the importance of the actin cytoskeleton and its regulatory proteins during EA invasion of non-phagocytic cells. Cdc42, Rac1, and RhoA, the key regulators of actin cytoskeleton signaling, have been evaluated during invasion of intracellular bacteria, viruses and protozoa (Krause-Gruszczynska et al., 2011; Reed et al., 2012; Van den Broeke et al., 2014). Cdc42 and Rac1 induce actin polymerization by the Arp2/3 complex through binding to and activation of their effector proteins, N-WASP and WAVE-2, respectively (Spiering and Hodgson, 2011). In canonic phagocytosis, actin polymerization is usually mediated by these proteins during their translocation to the plasma membrane after formation of phosphatidylinositol bi (4,5) or tri (3,4,5) phosphate (PIP2 or PIP3) at the inner leaflet of the plasma membrane (Takenawa and Suetsugu, 2007; Spiering and Hodgson, 2011). During actin remodeling, signaling of Rho GTPases can cooperate or inhibit each other’s activity (Guilluy et al., 2011). For instance, RhoA effector protein ROCK is able to activate FilGAP, a Rac1 inhibitory protein (Ohta et al., 2006). Rac1 activity can also be inhibited after the recruitment of PBR (polybasic region) containing GAPs (GTPase Activating Proteins) by PIP3 generated after activation of PI3k by Cdc42 (Campa et al., 2015). N-WASP and WAVE2 pathways can cooperate or not ABT-492 (Delafloxacin) during invasion of ssp. depending on the host cell (Bierne et ABT-492 (Delafloxacin) al., 2005). Using MDCK cells stably expressing Rho GTPase constructs, our group showed that Rac1 is usually involved in G strain EAs invasion but not invasions by other parasite strains or forms (Fernandes and Mortara, 2004). Despite these initial results, their precise role during internalization remains poorly characterized. Considering actin involvement in EA internalization and the importance of Rho-family GTPases in actin dynamics, the aim of this ABT-492 (Delafloxacin) study was to ABT-492 (Delafloxacin) evaluate the role of Rho GTPases and their effector proteins, N-WASP and WAVE-2, in microfilament modulation during host cell invasion by EAs. Using cells depleted of or overexpressing these proteins and microcopy techniques, we found that Rac1 is the important Rho GTPase in this process, possibly acting together with WAVE2, whereas Cdc42 displays a minor role in parallel with N-WASP participation; RhoA had a negative role in the regulation of Rabbit Polyclonal to 5-HT-3A actin dynamics involved in EA entry. Materials and methods Antibodies, reagents and plasmids Mouse anti-Rac1 (clone 23A8, #05-389) and rabbit anti-Cdc42 (#07-1466) were purchased from Millipore. Rabbit anti-RhoA (R9404) and mouse anti-WAVE2 (clone 8E7, WH0010163 M2) were from Sigma-Aldrich. Rabbit anti-N-WASP (clone 30D10, #4848), mouse -actin (clone 8H10D10, #3700) and rabbit anti-GAPDH (clone D16H11, #5174) were from Cell Signaling. Mouse anti-c-myc (clone 9E10, MA1-980) was from Invitrogen. Secondary antibody goat anti-rabbit Alexa Fluor 488 (A11008) was from Invitrogen. Goat anti-rabbit IgG-peroxidase (A6154), goat anti-mouse IgG-peroxidase (A4416) and goat anti-human Alexa Flour 488 (A11013) were from Sigma-Aldrich. Anti-was.