GEEC Endocytosis in Drosophila Figure 3. Characteristics of endosomal progression in S2R+ cells. (A) A568Tf (TfR) was pulsed for 15 min and cells were processed for immunocytochemistry with anti-Rab11 (aRab11), and subsequently imaged at high resolution. Plot shows kinetics of Tf and aGFP uptake in S2R+ cells transfected with the either the human TfR or GFP-GPI in an approach to steady state assay (see Materials and Methods). Each data point represents the mean6s.e.m. of a replicate experiment from at least 200 cells/replicate. Points have been fit to y = 0.381+0.666(1 – e-0.216t), R2 = 0.922 for TfR and y = 0.339+0.698(1 - e-0.029t), R2 = 0.966 for GFP GPI. (B) TMR-dextran was co-pulsed with A647Tf for 15 min in cells expressing Rab7 YFP, and cells were imaged with a spinning disk confocal. A single (median) plane from a stack is shown. (C) Cells expressing corresponding receptors were pulsed with TfR, mBSA or aGFP for 2 hrs and then the probes were chased in complete medium for an additional 2 hrs in the presence (+PI) or absence (-PI) of protease inhibitors. The integrated intensity of each probe in cells was then quantified and normalized to controls. Values are represented as mean6s.e.m. from at least 100 cells per treatment in 2 independent experiments. Asterix denote P,0.05 by Student’s t-test. (D) Endosomal pH measurements. S2R+ cells were pulsed for 30 min with Fdex and Rdex, washed and mildly fixed, and subsequently equilibriated with a range of different buffer pHs to obtain a linear calibration of the ratio of Fdex/Rdex fluorescence against pH (dotted line through empty circles; R2.0.95). To label different populations of endosomes, S2R+ cells were either pulsed for 30 s with Fdex and Rdex or pulsed and then chased for 2 min, 4 min, 10 min and 45 min (shown on the graph as squares filled in descending grayscale). The ratio of Fdex/Rdex intensities was measured and compared to equilibriated values to obtain pH values. 30 s fluid endosomes were mildly acidic with a pH of 6.560.3 (mean6s.e.m. of 2 independent experiments, .30 cells for each time point) whereas the pH of more mature fluid endosomes was progressively more acidic, with 45 min endosomes having a pH of 4.460.1, close to values reported for lysosomes. Bars in A = 2.5 mm, B = 5 mm, insets magnified 2x. doi:10.1371/journal.pone.0006768.g003 shown). Also, this externalization rate is 3 fold higher than in CHO cells (0.069/min, t1/2,10 min; Johnson et al., 1993), and ,10 fold higher than that of GFP-GPI in S2R+ cells (0.02883/ min, t1/2,25 min; Figure 3A). Consistent with a recycling step, a significant pool of internalized TfR after a 20 min pulse was found in large endosomes that also labeled with antibodies to the recycling marker Rab11 (Figure 3A) and were devoid of the lysosomally directed fluid phase (Figure 3B), and unlikely to enter a degradative pathway. Consistent with this, Tf levels inside cells were not sensitive to the addition of protease inhibitors (PI; Figure 3C). After a 20 min pulse, the majority of internalized endosomes that accumulated Fdex were marked by YFP-Rab7 (Figure 3B), and co-localized with Cy3-mBSA (not shown; [19]). Internalized Cy3mBSA, a marker that traverses the lysosomal route (Sriram et al., 2003), is protected from degradation in the presence of PI (Figure 3C). We also monitored the change in pH accessed by fluid-phase markers, upon increasing chase time in the endocytic pathway. Consistent with Rab5 acquisition in sorting endosomes, the pH of fluid-containing endosomes 2 min after internalization, as measured by a ratiometric assay[38], is ,6.0 (Figure 3D; [40]). This value drops to pH 4.5 once the probes reach deeper in the endocytic pathway (Figure 3D). Thus, S2R+ cells also have an endocytic system that resembles that of a typical mammalian cell (early endosomal [41]; late endosomal transport [42]). Here, endocytic trafficking involves a hetero and homo-typic mixing of PLoS ONE | www.plosone.org 6 August 2009 | Volume 4 | Issue 8 | e6768