GEEC Endocytosis in Drosophila Figure 5. Role of actin and cholesterol in S2R+ endocytic pathways. (A) Panels shows fluorescent micrographs of filipin staining in: untreated S2R+ cells (control); cells adapted to growth in delipidated, cholesterol-free medium (delip; see Materials and Methods); and control cells treated with 10 mM MbCD for 30 min. Bar graph shows quantification of the total filipin intensity across the different treatments (mean6s.e.m., .90 cells for each treatment from 2 experiments; asterix denote P,0.05 by Student’s t-test). (B) Normalized Fdex and TfR uptake in: control S2R+ cell line, cholesterol- free adapted line (delip), cholesterol-depleted lines repleted with 100 mg/ml cholesterol for 12 hrs (delip+100c) and control line grown in 100 mg/ml cholesterol for 12 hrs (control+100c). Mean6s.e.m. values are derived from 2 independent experiments with n.200 cells in each condition. Asterixs denote P,0.05 by Student’s t-test. (C) Normalized uptake of Fdex and TfR in S2R+ cells over different pulse times (in minutes) in medium containing either carrier dimethyl sulphoxide (dmso); 1 mM Latrunculin A (Lat) or 4 mM Jasplakinolide (Jas). Values are represented as mean6s.e.m. at each time point/treatment, .200 cells per treatment in 2 independent experiments. (D) S2R+ cells were prepulsed for 2 hrs with Fdex and then chased in medium without probe either with Latrunculin A (Lat) or carrier alone (dmso) for increasing lengths of time in minutes. At the end of each chase time, cells were fixed and the total remaining cell-associated Fdex intensity was quantitated and normalized to control levels. Each point on the graph is represents mean6s.e.m. from 2 independent experiments with at least 100 cells each. Bar in A = 5 mm. doi:10.1371/journal.pone.0006768.g005 presence of Arf1 dsRNA, and this inhibition was not affected by the presence or absence of shibire dsRNA (Figure 6C). Conserved role of garz and its mammalian counterpart GBF1 in Arf1-mediated endocytic uptake After ascertaining the presence of an Arf1-mediated pinocytic pathway in Drosophila cells and establishing the necessary tools to assay endocytosis, we sought to identify conserved components of this pathway using functional genomic RNAi analysis. To find candidate regulators of Arf1, we screened several orthologous members of the family of Arf GEFs and GAPs in Drosophila for defects in quantitative fluid phase and Tf uptake (Figure 7A). While there were several interesting molecules whose depletion affected both Tf and Fdex uptake, we chose to focus on the Drosophila Arf GEF gartenzwerg (garz) because it was a candidate GTPase regulator which appeared to be exclusively required for Fdex uptake (Figure 7A). Sequence alignment and domain analysis indicates that Drosophila garz is the likely ortholog of human GBF1, with over 60% amino acid conservation over the entire length of the protein products (Figure 7B). We used the well-characterized GTPase exchange mutant E794K to perturb GBF1 function [46,47]. The glutamic acid to lysine substitution within the catalytic Sec7 domain abolishes the nucleotide exchange activity, such that this mutant GEF binds Arf–GDP, but does not catalyze GDP displacement [46]. The overexpression of GBF1E794K in CHO cells caused a specific inhibition of Rdex uptake, whereas internalization of Tf was unaffected (Figure 7C). Dynamics of GBF1 reveals a functional association with Arf1 GFPGBF1, which reflects the native GBF1 distribution in cells [46], is primarily cytosolic and cycles rapidly on and off PLoS ONE | www.plosone.org 9 August 2009 | Volume 4 | Issue 8 | e6768