respect to endocytosis are unexplored in Drosophila, so the presence of a canonical GEEC pathway in this organism is still an open question. In this report, we develop quantitative endocytic assays in Drosophila S2R+ cultured cells using a variety of fluorescent probes, reporters and high resolution microscopy. We then exploit the facility of long double-stranded RNA (dsRNA) mediated RNA interference in this cell line (RNAi; [20]) to establish the presence of an Arf1-mediated pinocytic pathway that bears a striking resemblance to the GEEC pathway. With the added benefit of lower gene redundancy in the Drosophila genome [21], we utilize functional RNAi genomics to identify the Arf GEF, garz, as a candidate Arf1 effector during pinocytosis in S2R+ cells. We validate this strategy by examining the role of the mammalian ortholog of garz, GBF1, and find that it is a novel regulator of the GEEC pathway. Materials and Methods Materials Apart from the fluorochromes Cy3, Cy5 (AP Biotech, UK), Alexa488/568/647, FITC and Latrunculin A (Molecular Probes, OR), all chemicals were obtained from Sigma-Aldrich, MO. Hybridoma cultures containing anti-GFP monoclonal (1B3A8) and anti-hTFr monoclonal (OKT9) were obtained from S. Sundaresan (Bangalore Genei, India) and American Type Culture Collection (ATCC,VA) respectively, and purified with IgG Fast Flow columns (Amersham,UK). Probes were labeled with Alexa or Cy3/Cy5 fluorophores according to manufacturers’ instructions. Anti-Drosophila dynamin antibodies were from BD Biosciences, CA. Secondary antibodies were from Jackson Laboratories, ME. PI- PLC was purified from PI-PLC-expressing bacterial strains [22]. Plasmids and Constructs pUAST hTFr encoding the human transferrin (Tf) receptor (TfR), and pCasper YFP-Rab7 [23] were kind gifts from S.Cohen (EMBL, Heidelberg) and S.Eaton (MPI Cell Biology, Dresden) respectively. pBSPURO was obtained from M. Wilm (EMBL, Heidelberg). pUAST-GFPRab5 and pUAST-GFPRab11 were cloned by ligation of PCR generated fragments of GFP and Drosophila Rab5 or Rab11 cDNAs into the pUAST vector [24], and subsequently sequenced. pUAST-GFPGPI [25] was obtained from the Drosophila Genome Resource Center (DGRC, IN). Mammalian GFPGBF1 and GFPGBF1E794K constructs were a kind gift from E. Sztul (U. Alabama, Birmingham). Cell culture, Fly stocks, RNAi and stable lines Drosophila S2R+ cells were obtained from S. Yanagawa [26], split every 6–8 days and grown at room temperature (21–24uC). They were maintained in Schneider’s Drosophila Insect Medium (SDM; Invitrogen) supplemented with 7.5% heat-inactivated fetal bovine serum (FBS; Gibco-BRL) and 150 mg/ml penicillin, 250 mg/ml streptomycin, and 750 mg/ml glutamine. RNAi was performed according to the procedure of Clemens [20]. Transfections were performed with Cellfectin (Invitrogen, CA) or Effectene (Qiagen, Germany), according to the manufacturer’s instructions. For the generation of stable lines, the PBSPURO plasmid bearing puromycin N-acetyltransferase was co-transfected at a 1:30 ratio with the desired constructs. Stable lines were selected over 2–3 weeks by incubation in medium containing 1 ug/ml puromycin. Chinese Hamster Ovary (CHO) cells stably expressing FR-GPI and human TfR (IA2.2 cells) were used for endocytic assays. CHO cells were grown in HF-12 (HiMEDIA, Mumbai, India) containing NaHCO3 and penicillin, streptomycin GEEC Endocytosis in Drosophila (100 mg/ml) and supplemented with 10% FBS (GibcoBRL, Rockville, MD). CHOs were transfected with different DNA constructs using FuGENE6 (Roche Diagnostics, Germany) according to standard protocol, and assayed 18–20 hrs after transfection [10]. Probes for endocytosis and immunodetection Since both the S2 line and primary cultures of Drosophila hemocytes have been previously shown to exhibit scavenger receptor-endocytosis [19,27], we tested for the presence of this pathway in S2R+ cells using a polyanionic ligand (malelyated BSA; mBSA; [19,28]). The 10 kDa fluid phase tracer FITC- dextran was made as described previously (Fdex; [4]) and 10 kDa tetramethyl rhodamine-dextran (Rdex) was purchased from Molecular Probes, OR. Human apo-transferrin was iron-loaded and conjugated to Alexa 568 or Alexa 647 dyes [29,30]. Anti-GFP hybridoma was purified (see Materials) and conjugated to Alexa 647 to yield A647aGFP. Immunofluorescence was carried out as described [31]. For Tf and GPI-AP probes, stable S2R+ lines constitutively expressing the well characterized human transferrin (Tf) receptor (TfR) and/or GFP-GPI were generated. Cy3 conjugated mBSA (Cy3mBSA; [19]) or Alexa568/647 conjugated Tf (A568Tf/A647Tf) was used to probe receptor-mediated pathways whereas FITC or TMR conjugated to Dextran (FDex and RDex respectively) were used as fluid-phase tracers. The receptor probes Cy3mBSA, Cy5mBSA and biotinylated Cy3m- BSA (Cy3bmBSA; [19]) were used at 10–30 mg/ml in S2R+ cells. At these concentrations, the binding and internalization of labeled mBSA was completely competed by 100 fold excess unlabeled mBSA. Fdex and Rdex, which are markers for bulk fluid uptake, were used at 1–2 mg/ml, unless otherwise specified. Tf and aGFP probes were used at 10 ug/ml and 5 ug/ml respectively. In the corresponding cells, Cy3mBSA, A568/A647 Tf and aGFP ligands show saturable receptor labeling, which is competed by 100 fold excess unconjugated ligand (not shown). Uptake assays For S2R+ uptake experiments, cells were incubated with endocytic probes in M1 buffer (150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, pH 6.9; modified from [29]) supplemented with BSA (1.5 mg/ml) and D-glucose (2 mg/ml) at room temperature (21–24uC), unless otherwise indicated, and extensively washed in the same medium. To visualize the specific endocytic uptake of GFP-GPI or TfR, cells were incubated with fluorescent conjugates of aGFP or Tf (along with fluid phase markers if needed) at room temperature for different pulse times and then washed in M1 and placed on ice. Where necessary, PI-PLC (0.3 mg/ml; 45 min on ice) or ascorbate buffer with 50 ug/ml desferroxamine mesylate (pH 4.5; 10 min on ice) treatment was carried out to remove cell-surface GFP-GPI or Tf respectively. Surface pools of receptors were labeled by further incubation on ice with antibodies to GFP (aGFP with a different fluorochrome) or anti-hTfR (Okt9) for 30 min. Subsequently, cells were washed, fixed (2.5% paraformaldehyde in M1) and imaged. When required, endosomal pH was neutralized by the addition of ammonium chloride (30 mM in M1) post-fixation so as to dequench pH-sensitive fluorochromes. Assays for surface accessibility of mBSA were carried out as described previously [19]. Mammalian uptake experiments were carried out exactly as described previously [10]. For Brefeldin A (BFA) experiments, IA2.2 cells were treated with 20 mg/ml BFA for 60 min and pulsed with TMR-Dex during the last 10 min before fixation and imaging. For determining externalization rates in S2R+ lines (see [32]), cells were incubated in the presence of a saturating concentration PLoS ONE | www.plosone.org 2 August 2009 | Volume 4 | Issue 8 | e6768