of A568 Tf for varying times until they achieved a maximum, steady-state value of cell-associated label. Since Tf binding to surface TfR is rapid, and since saturating levels of Tf were used in this assay, the rate of accumulation of Tf is mainly dependent upon the rate at which unoccupied intracellular TfR are externalized to the plasma membrane to acquire Tf. The exocytic rate constant, ke can be quantitated once the parameters Tfs and Tfss are estimated from a best fit to the function: Tft ~ Tfsz Tfss h 1 { expð{ke tÞi where Tft is the total amount of Tf bound to TfR at time t; Tfs is the amount of Tf bound to surface TfR (constant in this assay); and Tfss is the Tf bound to internal TfR at steady state. The values for ke are determined from the best least-squares fit to this function [29]. As a control, to evaluate the contribution of the biosynthetic pool of TfR during the course of the assay, cells were pretreated with 75 mM cyclohexaminde for 2 hrs and the assay was carried out in the presence of cyclohexamide (Figure S2B). Cholesterol measurements and serum delipidation FBS was delipidated by organic extraction with diethyl ether according to Cham and Knowles [33], and dialysed extensively in PBS. This extraction reduced total cholesterol content by over 99% as measured by the Red Amplex Cholesterol Assay Kit (,10 ug/ml sterol; Molecular Probes, OR), while leaving overall protein levels in serum unchanged. S2R+ cells were adapted to growth in delipidated medium containing 10% lipid-extracted FBS in SDM. This was achieved by serial passage in 150 ml flasks with linearly increasing levels of lipid-extracted FBS over 5–8 weeks. Filipin staining and methl-b-cyclodextin (MbCD) treat- ments were as described previously [8] except that the incubation temperature was reduced to 22uC to match optimum growth conditions for Drosophila cells. Fluorescence imaging, quantification and processing Wide field imaging was carried out on a Nikon TE300 inverted fluorescence microscope and images captured with a CASCADE II camera (Photometrics, AZ). Fluorescence quantification was carried out with a low magnification objective (20x, 0.75NA) to obtain 30–100 cells per field, while higher resolution images for visualization purposes were obtained with a 60X, 1.4NA objective. Confocal microscopy was carried out on an Andor Spinning Disc confocal imaging system (60x 1.42 or 100x 1.4 NA, with Ixon EMCCD; Andor Technologies, Ireland) using appropriate factory- set filters and dichroics for different fluorophores. Care was taken to avoid fluorophore saturation during acquisition. Total internal reflection fluorescence (TIRF) microscopy imaging was done on a custom-built setup around a Nikon Eclipse TE 2000U inverted microscope (described in detail in [8]). Images were collected using appropriate filters onto cooled EMCCD-based Cascade 512B cameras (Roper Scientific, AZ) with ‘on-chip multiplication gain’ feature for providing single-molecule detection sensitivity in solutions and cells. For two-colour TIRF microscopy, cells were illuminated sequentially by 488 or 543 nm lasers and fluorescence emission was collected using corresponding emission filters mounted on a shutter wheel (Sutter Instruments, CA) operating coordinately with the illuminating lasers. Background subtraction and fluorescence quantitation was carried out as described previously [29] using MetaMorph or custom routines written in MATLAB (described in Figure S1). Statistical methods were as described previously [10]. Images were pseudo-coloured using Adobe Photoshop and composites were assembled using the same GEEC Endocytosis in Drosophila software. Quantification of colocalization was performed as described [4,34]. All processing including determination of colocalization was performed using similar parameters regardless of the type of endocytic tracer used. Colocalization fraction was calculated as the number of endosomes per cell whose surface area overlapped at least 30% in each cell. The maximum extent of colocalization obtained by this method is 80% for cointernalized A647-TfR and A568-TfR in the same cell [4]. RT-PCR and Western blotting For each condition or dsRNA treatment, about 10 ug total RNA was extracted from ,106 S2R+ cells with Trizol reagent (Sigma). First strand cDNA synthesis was performed with Superscript III Reverse Transcriptase (Invitrogen, CA) and a polyT22 primer according to the manufacturer’s instructions. The reaction product was then treated with DNAse-free RNAse (Ambion, CA) to remove RNA template. Gene-specific primers spanning introns were designed to confirm the absence of genomic DNA contamination during PCR for each transcript. PCR was performed for 20 cycles and 30 cycles for semi-quantitative comparison of amplification. Western blotting was performed according to standard protocols. Results Pathways of endocytosis in Drosophila S2R+cells The S2R+ cell line, which expresses a wingless pathway receptor [26] is believed to be a derivative of the embryonic S2 line [35]. The morphology of these cells is heterogeneous, but the majority of cells spread out, exhibiting broad and thin lamellapodia [36]. These characteristics allow for high resolution wide field imaging of early endosomes. To visualize early steps of internalization, we co-pulsed S2R+ cells with fluid phase (Fdex/Rdex) and receptor (Cy3mBSA and/or A647Tf) probes for very short times (30–60 s) and fixed them before imaging (see Materials and Methods for details of probes). Post-pulse most of the Fdex and Cy3mBSA probes were found in separate, peripheral structures (Figure 1A). By contrast, Cy3mBSA was largely found together with internal- ized Cy5Tf in the same period (Figure 1A). Quantification of colocalization of these structures indicated that Tf puncta were ,5 fold enriched in mBSA when compared to Fdex (graph in Figure 1A). Conversely, only a small fraction of Fdex punctae also contained Tf and mBSA when all three probes were co-pulsed (Figure 1A). To follow the endocytic route of GPI- anchored proteins (GPI- APs), S2R+ cells stably expressing GFP-GPI were pulsed with Alexa 647-coupled antibodies to GFP (A647aGFP) and Rdex (Figure 1A). Internalized Rdex labeled structures contained significantly more GFP-GPI as marked by A647aGFP, compared to mBSA or Tf (graph in Figure 1A). To examine nascent endosomes present close to the plasma membrane at higher resolution, we pulsed cells expressing clathrin light chain GFP with Rdex and A647Tf for 40 s and imaged them with TIRF microscopy. In the evanescent field, the fraction of clathrin GFP punctae that colocalized with Tf at early times was over 10 fold greater than those that contained Rdex (Figure 1B). Sequence of endosome mixing and separation, and the fate of endocytic cargo To study endosomal dynamics, we generated a stable S2R+ line expressing moderate levels of Rab5 GFP, an early endosomal marker which participates in homo- and hetero-typic endosome fusion and sorting steps [37], and performed pulse-chase assays with Rdex and A647Tf. When levels of colocalization with the PLoS ONE | www.plosone.org 3 August 2009 | Volume 4 | Issue 8 | e6768