ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion Claudio Vieira da Silva a,b , Erika Alves da Silva b , Mário Costa Cruz b , Philippe Chavrier c , Renato Arruda Mortara b, * a Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Av. Pará, 1720, Campus Umuarama, Uberlândia, 38400-902, MG, Brazil b Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, 862, 6° andar, 04023-062 São Paulo, SP, Brazil c UMR144 CNRS, Institut Curie, 26 rue d’Ulm, F-75248 Paris, France article info Article history: Received 20 November 2008 Available online 4 December 2008 Keywords: Toxoplasma gondii Host cell invasion Parasitophorous vacuole ARF6 PI3-kinase Actin cytoskeleton abstract Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endo- cytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrange- ments at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP 2 and PIP 3 to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion. Ó 2008 Elsevier Inc. All rights reserved. The ADP-ribosylation factor-6 (ARF6) belongs to the ARF fam- ily of small GTP-binding proteins. ARF6 regulates membrane trafficking and the actin cytoskeleton at the plasma membrane [1]. It is involved in membrane trafficking during receptor-med- iated endocytosis, endosomal recycling and exocytosis of secre- tory granules [2–5]. It is also implicated in the formation of actin-rich membrane protrusions and ruffles [6]. Trafficking of rafts seems to be a major regulatory pathway by which ARF6 controls Rac1 activation and cell spreading [7]. Like all small GTP-binding proteins, ARF6 cycles between an inactive GDP- bound state and an active form when bound to GTP. ARF6 is activated following agonist stimulation of phosphatidylinositol 3-kinase (PI3-kinase) [8]. GTP-ARF6 acts through the activation of downstream effectors, including lipid-modifying enzymes such as phospholipase D (PLD) and phosphatidylinositol 4-phosphate 5-kinase (PIP5-kinase) [9,10]. PIP5-kinase, which is responsible for generating phosphatidylinositol 4,5-biphosphate (PIP 2 ), has been implicated in the phagocytosis of IgG-coated erythrocytes, probably through the regulation of actin cytoskeleton by PIP 2 [11]. Altogether, changes in membrane lipid composition and structure may mediate ARF6 alterations of the cortical actin cytoskeleton and regulation of membrane traffic and signal transduction [12]. Interestingly, it was shown that a PIP 2 -depen- dent pathway regulated by ARF6 is associated with Yersinia pseu- dotuberculosis internalization [13] and also, ARF6 undergoes a rapid and transient activation after infection by Chlamydia caviae and plays a critical role in bacterial uptake [12]. We have also observed that ARF6 is recruited to the parasitophorous vacuole of invading Trypanosoma cruzi amastigotes and that knockdown on endogenous ARF6 expression reduced the internalization of the parasites (Silva et al., unpublished data). Taking together, these results suggested that ARF6 controls actin polymerization at the site of entry of these actin polymerization-dependent pathogens. On the other hand, in the case of phagocytosis of red blood cells by macrophages, ARF6 was shown to control membrane recruitment at the site of phagocytosis rather than actin polymerization [14]. Toxoplasma gondii is a widespread, obligate intracellular para- site capable of infecting virtually all types of nucleated mamma- lian and avian cells. T. gondii is a member of a large phylum of related parasites, called the Apicomplexa that are unified by a common set of apical structures that are involved in cell inva- sion [15]. Unlike phagocytic uptake, host cell invasion by T. gon- dii occurs by active penetration of the host cell [16]. The process of invasion is accompanied by the sequential discharge of three separate sets of secretory organelles termed micronemes, rhop- tries and dense granules [17]. Following invasion, T. gondii re- 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.108 * Corresponding author. Fax: +55 11 5571 1095. E-mail address: ramortara@unifesp.br (R.A. Mortara). Biochemical and Biophysical Research Communications 378 (2009) 656–661 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc