GEEC Endocytosis in Drosophila steady state pool of Rab5 GFP were examined over different chase times, most Rdex and A647Tf containing endosomes were found to lack Rab5 during the short pulse (30 s–60 s), and then acquire Rab5 steadily over a chase time of upto 30 min (Figure 2A). Interestingly, mixed endosomes overlapped with Rab5 punctae in a biphasic pattern, rising rapidly to encompass over 60% of Rab5 punctae at 2 min post probe entry and falling to pre-pulse levels with longer chase times (Figure 2A). This time course is not influenced by overexpression of Rab5-GFP protein, as mixing kinetics of Fdex and Tf in lines with or without Rab5 GFP was similar (Figure S2A). Furthermore, heterotypic mixing between Fdex and TfR probes in wild type cells also rises rapidly to a peak in a 2 min pulse (Figure 2B), a process that in mammalian cells has been shown to require Rab5 function [38]. A similar time course of mixing was found when examining homotypic fusion between fluid-containing endosomes, which always occurred in Rab5 marked structures (Figure 2C). We next examined the kinetics of Tf binding and recycling in Drosophila cells in detail because it is unclear whether the canonical Tf pathway operates in insect lines [39]. The binding and uptake of TfR was kinetically analyzed over 2 hrs with saturating levels of A568Tf (Figure 3A) in an approach to steady-state assay (Materials and Methods; [32]). At each time point, total binding of Tf at room temperature was normalized to levels of Tf bound out to steady state (upto 2 hrs). Total binding at 4uC was assumed to be the initial surface receptor occupancy. Uptake at room temper- ature exceeded binding at 4uC by 2.5-fold and saturated after 15– 20 min, and similar values were obtained when the assay was carried out with cyclohexamide (to block synthesis of new TfR; Figure S2B). The kinetics of approach to steady state reflect the rate of export of the TfR to the cell surface with a rate constant of 0.216/min (t1/2,3 min) if we assume a single first order process. This value agrees with the time course of quantitative TfR uptake in S2R+ cells after surface receptor normalization with Okt9 (not Figure 2. Fluid phase and TfR pathways intersect in Rab5-positive compartments and subsequently follow expected fates. (A) TMR- dextran (Rdex) was co-pulsed with A647Tf (TfR) in cells expressing Rab5 GFP (Rab5 GFP) for 30 s (left pane) or the pulse was chased for 4 min in complete medium (right pane). Graph shows quantified colocalization indices over different pulse (30 s or 60 s) and pulse and chase times (1 min pulse and 1, 2, 3 or 4 min chase; and a 3 min pulse and 30 min chase) of: Tf-labeled endosomes co-localized with Rab5 GFP (blue symbols and line); Rdex endosomes with Rab5 GFP (red symbols and line); and Rab5 GFP-labelled endosomes that contained both Rdex and Tf (green symbols and line). Data points represent mean6s.e.m pooled from 2 independent experiments with at least 20 cells for each point. (B) Time course of mixing of Fdex, mBSA and TfR probes in S2R+ cells. Colocalization values for different probes and pulse times (0.5 min, 1 min, 2 min, 5 min and 10 min; or a 15 min chase following a 1 min pulse) were obtained as described earlier (Figure 1A). The fraction of TfR or mBSA endosomes that also contain Fdex is relatively low at all time points [TfR-Fdex (brown symbols and line) and mBSA-Fdex (red symbols and line), respectively]). Conversely, the fraction of Fdex endosomes that contain TfR (Fdex-TfR; blue symbols and line) or mBSA (Fdex-mBSA; green symbols and line) is initially low, and increases with longer pulse times. (C) S2R+ cells expressing Rab5 GFP were pulsed with Rdex, washed and then pulsed with A647dex to examine homotypic fusion of newly formed fluid endosomes (see schema at the top of image). Rab5 GFP was not found on endosomes labeled with either the first or the second short pulse of dextran (left panel). When the two staggered pulses were chased for 4 min (right panel), endosomes containing the first pulse acquired Rab5 GFP (empty arrowheads) and a fraction of these also contained the second pulse (filled arrowheads). There was little or no mixing between the two pulses outside Rab5 GFP labeled structures. Bars in A, C = 5 mm, insets magnified 2x. doi:10.1371/journal.pone.0006768.g002 PLoS ONE | www.plosone.org 5 August 2009 | Volume 4 | Issue 8 | e6768