but may not inhibit all GBF1 activity in vivo. Equally, it is possible that GBF1 sensitivity to BFA is modulated by its membrane receptors, and the membrane recruitment of GBF1 at the ER- Golgi route is more sensitive to BFA than membrane recruitment at the plasma membrane. A third non-mutually exclusive possibility is that the presence of excess Arf1 that has been released from the ER-Golgi pool upon BFA addition stimulates GBF1 activity at the plasma membrane, which compensates for GBF inhibition. Identification and characterization of the receptor(s) and the membrane recruitment mechanism for GBF1 would help to distinguish between these possibilities. A role for GBF1 in endocytosis is not without precedent. In Arabidopsis, the GBF1-ortholog GNL1 has a conserved role at the Golgi but is also required for BFA-insensitive endocytosis at the plasma membrane [55]. Moreover, the Drosophila GBF1 ortholog garz has been identified as a candidate in genomic screens which tested the entry and infection of the pathogens Listeria monocytogenes [56] and Candida albicans [57,58]. While direct involvement of candidate genes in pathogen entry was not tested in these screens, our finding that garz affects Arf1-mediated endocytosis in Drosophila implicate this pathway as a mode of entry for these pathogens. There is growing evidence that Arf1, GBF1 and part of the COPI machinery can be recruited not only onto transitional ER and the Golgi, but also onto other membranes where they can modulate trafficking or modify membranes (replication complexes during picornaviral infection - [59,60]; lipid bodies – [61,62]. While both replication complexes and lipid bodies are probably ER-derived membranes [63,64], our results suggest that Arf1 and GBF1 may also act together during endocytic events at the plasma membrane. In this regard, it would be interesting to test the involvement of components of the COPI machinery in Arf1-mediated endocytosis. Conclusion The availability of heterologous gene expression and functional genomic RNAi in the S2R+ line, combined with high-resolution imaging promises to be a powerful approach to study endocytosis in Drosophila. These methods have allowed us to systematically identify and define the role of a novel molecule in GEEC endocytosis. The demonstration of conserved pathways in Drosophila indicates that these findings can be broadly applicable. Supporting Information Figure S1 Automated quantitation of uptake assay measure- ments. (A) High penetrance of a dsRNA induced phenotype in S2R+ cells cultured on dishes. Images taken from a single field of S2R+ cells treated with control dsRNA or dsRNA against pavarotti, which encodes a kinesin like protein required for cytokinesis[1]. Note the accumulation of abnormally large multinucleate cells with pavarotti dsRNA as shown in the brightfield/Hoechst-stained nuclear channels. (B) Stepwise proce- dure for performing automated cell identification using the MATLAB image processing toolbox. The nuclear image (Hoechst) is thresholded using Otsu’s method [2] and an empirically- determined correction factor to create a binary mask (nuclear binary mask). This binary mask is subjected to a Euclidean distance transform followed by a watershed transform, generating References GEEC Endocytosis in Drosophila the first crude stage of cell segmentation (nuclear distance transform, ndt). A Sobel edge-detection filter is applied to the Okt9 surface label image (Okt9) to enhance cell boundaries. The watershed and the nuclear mask are superimposed upon the enhanced Okt9 image as local minima (local minima of ndt+Okt9). A final watershed transform is applied to this combined image to generate cell outlines which are then filled in (surface mask). (C) Scatterplot comparing the performance of automated intensity/cell quantitation vs manual quantitation. 24 dishes were pulsed with Fdex for different times to generate a ,10fold range of fluorescence intensity values across cells. Each point represents the mean6s.e.m. intensity/cell in a dish (with measurements from .100 cells from each dish) calculated manually or via MATLAB routines. The two methods show linear correlation over a wide range of cell intensities. Bar in A = 20 mm, B = 5 mm. References: 1.Adams, R.R., et al., pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev, 1998. 12(10): p. 1483-94. 2. Otsu, N., A Threshold Selection Method from Gray-Level Histograms. IEEE Transactions on Systems, Man, and Cybernetics 1979. 9: p. 62–66. Found at: doi:10.1371/journal.pone.0006768.s001 (3.45 MB TIF) Figure S2 Controls for trafficking assays. (A) Direct comparison of the mixing of Fdex and TfR probes at various pulse and chase times in S2R+ cells expressing TfR alone or S2R+ cells expressing TfR and Rab5 GFP. Fdex was co-pulsed with A568Tf (TfR) in wild-type S2R+ cells or S2R+ cells expressing Rab5 GFP for 30 s and 60 s, or pulsed for 60 s and chased for 1 min or 4 min in complete medium. The histogram shows quantified colocalization indices of: the fraction of Fdex-labeled endosomes that co-localized with Tf. Bars represent mean6s.e.m pooled from 2 independent experiments with .20 cells each. (B) Approach to steady state assay of TfR in cells treated with cyclohexamide. S2R+ cells expressing TfR were pretreated with 75 uM cyclohexamide (Chx) for 2 hrs to block protein synthesis. They were then pulsed with A647Tf for different times according to the ‘approach to steady state’ assay (see Methods and Figure 2A) in the presence of Chx. Data for control S2R+ cells expressing TfR, but without Chx pretreatment was taken from Figure 3A and is plotted for comparison. Each data point represents the mean6s.e.m. of a replicate experiment from at least 50 cells/replicate. Points have been fit to y = 0.381+0.666(1 - e-0.216t), R2 = 0.922 for TfR and y = 0.332+0.698(1 - e-0.18t), R2 = 0.994 for Tfr with Chx. Found at: doi:10.1371/journal.pone.0006768.s002 (0.62 MB TIF) Acknowledgments We would like to thank Abhijit Kale, Rahul Chadda and Neha Vyas for sharing reagents, and Rajesh M and Brojo for preliminary work. Author Contributions Conceived and designed the experiments: GDG SMG Sk. Performed the experiments: GDG SMG Sk RL. Analyzed the data: GDG SMG Sk RL GD. Contributed reagents/materials/analysis tools: GDG. Wrote the paper: GDG. Guided the work: SM. 1. Conner SD, Schmid SL (2003) Regulated portals of entry into the cell. Nature 422: 37–44. 2. Benmerah A, Lamaze C (2007) Clathrin-coated pits: vive la difference? Traffic 8: 970–982. 3. Mayor S, Pagano RE (2007) Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol 8: 603–612. 4. Sabharanjak S, Sharma P, Parton RG, Mayor S (2002) GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin- independent pinocytic pathway. Dev Cell 2: 411–423. 5. Kirkham M, Fujita A, Chadda R, Nixon SJ, Kurzchalia TV, et al. (2005) Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles. J Cell Biol 168: 465–476. PLoS ONE | www.plosone.org 14 August 2009 | Volume 4 | Issue 8 | e6768