Analysis of Endocytic Pathways in Drosophila Cells Reveals a Conserved Role for GBF1 in Internalization via GEECs Gagan D. Gupta¤*, Swetha M. G., Sudha Kumari, Ramya Lakshminarayan, Gautam Dey, Satyajit Mayor* National Centre for Biological Sciences, Bangalore, India Abstract In mammalian cells, endocytosis of the fluid phase and glycosylphosphatidylinositol-anchored proteins (GPI-APs) forms GEECs (GPI-AP enriched early endosomal compartments) via an Arf1- and Cdc42-mediated, dynamin independent mechanism. Here we use four different fluorescently labeled probes and several markers in combination with quantitative kinetic assays, RNA interference and high resolution imaging to delineate major endocytic routes in Drosophila cultured cells. We find that the hallmarks of the pinocytic GEEC pathway are conserved in Drosophila and identify garz, the fly ortholog of the GTP exchange factor GBF1, as a novel component of this pathway. Live confocal and TIRF imaging reveals that a fraction of GBF1 GFP dynamically associates with ABD RFP (a sensor for activated Arf1 present on nascent pinosomes). Correspondingly, a GTP exchange mutant of GBF1 has altered ABD RFP localization in the evanescent field and is impaired in fluid phase uptake. Furthermore, GBF1 activation is required for the GEEC pathway even in the presence of Brefeldin A, implying that, like Arf1, it has a role in endocytosis that is separable from its role in secretion. Citation: Gupta GD, MG S, Kumari S, Lakshminarayan R, Dey G, et al. (2009) Analysis of Endocytic Pathways in Drosophila Cells Reveals a Conserved Role for GBF1 in Internalization via GEECs. PLoS ONE 4(8): e6768. doi:10.1371/journal.pone.0006768 Editor: Jean Gruenberg, University of Geveva, Switzerland Received May 14, 2009; Accepted July 28, 2009; Published August 26, 2009 Copyright: 2009 Gupta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded in part by a grant from the Nanotechnology mission [Government of India, (GOI)]. G.D.G was supported by fellowships from NSERC (Canada) and NCBS. SM is a JC Bose Fellow (Department of Science and Technology, GOI), and SMG and SK acknowledge pre-doctoral fellowships from the CSIR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gagan@ncbs.res.in (GDG); mayor@ncbs.res.in (SM) ¤ Current address: Chromous Biotech Pvt. Ltd., Sahakar Bhavan, Sahakarnagar, Bangalore, India Introduction Internalization of cargo at the cell surface takes place via multiple mechanisms. For instance, the well-studied transferrin receptor (TfR) is endocytosed via the dynamin-mediated pinching of clathrin coated vesicles from the plasma membrane [1,2]. Other pathways that do not require clathrin or dynamin for internali- zation are less well characterized, but are also prevalent - the GEEC pathway is one such example [3]. This pathway was originally identified on the basis of the selective internalization of various GPI-APs such as the folate receptor [4], but has also been shown to facilitate the entry of: cholera toxin bound to its ganglioside receptor GM1[5]; the vacuolating toxins aerolysin and VacA[6]; and bulk fluid phase (pinocytosis) in diverse mammalian cells [4]. At the cell surface, a fraction of GPI-APs are organized in nanoclusters which are sensitive to the extraction of cholesterol [7]. The cholesterol-dependent organization of GPI-APs, as well as their endocytosis via the GEEC pathway, requires cortical actin polymerization [8,9]. Recent evidence links local actin polymer- ization to GEEC endosome formation via a regulatory circuit of two small GTPases - Cdc42 and Arf1[8,10]. Here, Cdc42 cycles on or off the plasma membrane according to its active (GTP- bound) or inactive (GDP-bound) state respectively, where it promotes actin polymerization [8]. Cdc42 cycling is modulated by ARHGAP10, a Rho GTPase- activating-protein (GAP) that also harbours an Arf-binding domain which is specific for Arf-GTP (ABD; [10,11]). Thus Cdc42–GAP activity, and actin-driven GEEC endocytosis is coupled to Arf1 activation [10]. However, it is not known how Arf1 activation occurs during GEEC endocytosis. Arf1 activation is facilitated by guanine-nucleotide exchange factors (GEFs; [12]), which contain a conserved Sec7 domain responsible for GDP-GTP exchange [13,14]. Two well charac- terized Arf-GEF subfamilies, GBF1 and BIGs, appear to regulate Arf activation for coat recruitment and vesicular traffic at the Golgi complex [15,16]. It follows that these Arf-GEFs are predicted in vivo targets for the fungal metabolite Brefeldin A (BFA), an established inhibitor of the secretory pathway which binds at the interface between the Sec7 domain and Arf-GDP in vitro [17,18]. Since the GEEC pathway is upregulated in the presence of BFA, it has been proposed that the effector GEF(s) required to activate Arf1 for GEEC endocytosis may be BFA- insensitive [10]. The significance of the GEEC pathway is broadening as it is identified in more mammalian cell types and additional cargo is found [3]. Furthermore, evidence from Drosophila cells indicates that the GEEC pathway may be conserved across phyla. In hemocytes derived from shibire (Drosophila dynamin) temperature- sensitive mutants, GPI-APs and the fluid phase continue to be internalized at the restrictive temperature [19]. However, the roles of key molecules such as Arf1, Cdc42, cholesterol and actin with PLoS ONE | www.plosone.org 1 August 2009 | Volume 4 | Issue 8 | e6768