Fig 2: Rab14 resides on late endosomes and lysosomes containing endocytic Env in CEM-A T cells. (A and B) GFP-Rab14 knock-in (KI) or GFP-Rab11A KI CEM-A T cells were infected with HIV-1 virus and pulsed with anti-Env Fab b12-AF647 for 15 min at 4°C and then chased at 37°C for 1 h, with LysoTracker Red DND-99 added in the final 50 min. The cells were fixed and imaged by confocal fluorescence microscopy. Rab14 partially overlapped with LysoTracker, while Rab11 did not. (C and D) CEM-A cells stably overexpressing low levels of TagRFP-Rab14 were infected with HIV-1 virus possessing either LAMP1-Emerald or CTLA4-GFP reporters. The cells were pulsed with anti-Env Fab b12-AF647 for 15 min at 4°C, then chased at 37°C for 1 h, fixed, and imaged by confocal fluorescence microscopy. Rab14 colocalized with LAMP1 and with CTLA4, which also possessed Env staining in many instances. White arrows indicate examples of colocalization, while red arrows indicate examples of noncolocalization. Scale bars are 5 µm.

Fig 3: EGFR continuously recycles through the RE.(A) Co-localization of EGFR and Rab11. Left panel, first row: Immunostaining of endogenous EGFR (left), Rab11 (middle) and corresponding green/magenta overlay (right) in fixed COS-7 cells. Left panel, second row: Fluorescence images of EGFR-mCitrine (left), BFP-Rab11a (middle), and corresponding green/magenta overlay (right) in living COS-7 cells. Right panel: Quantification of co-localization between EGFR and Rab11 in A (n=15 cells), B (n=12) by Pearson’s correlation coefficient. (B) Fluorescence redistribution after photoactivation of EGFR-paGFP on the recycling endosome. First row: EGFR-paGFP fluorescence at the indicated time in seconds (photoactivation area: black rectangle in the pre-activation image), second row: corresponding EGFR-mCherry fluorescence and image in third row: BFP-Rab11a fluorescence. Right graphs: Loss of EGFR-paGFP fluorescence at the RE normalized to EGFR-mCherry fluorescence (left) and corresponding gain of EGFR-paGFP fluorescence at the PM (right), normalized to EGFR-mCherry fluorescence. Average trace (black ± SEM.; n=6 cells) was fitted to an exponential function (red) to retrieve time constants (t) of EGFR fluorescence intensity lossat the RE and its gain at the PM. (C) Fluorescence redistribution of TAMRA-labeled-EGFR in HEK293 cells. Representative fluorescence images of EGFR-(128TCOK)-GFP (first row), TAMRA-labeled EGFR-(128TCOK)-GFP (second row), and BFP-Rab11a (third row, left panel) or LAMP1-BFP (third row, right panel) for the indicated time (min) in the respective columns (see 'Materials and methods'). (D) Co-localization of ligand-activated EGFR and Rab5 in COS-7 cells. Representative fluorescence images of EGFR-mCitrine (first column), Rab5-mCherry (second column), and green/magenta overlay of selected ROIs (third column), pre- and 15-min post- stimulation with EGF (100 ng/ml). (E) Co-localization of spontaneously activated EGFR and Rab5 in COS-7 cells. Representative fluorescence images of EGFR-mCitrine (first column), Rab5-mCherry (second column), and green/magenta overlay of selected ROIs (third column), pre- and 15-min post- treatment with 0.33 mM PV. (F) Quantification of co-localization between EGFR-mCitrine and Rab5-mCherry with Pearson’s correlation coefficient upon EGF stimulation (100 ng/ml, n=5 cells) or PV treatment (0.33 mM, n=7 cells) for the indicated time points. All scale bars: 10 µm. EGFR, epidermal growth factor receptor; PM, plasma membrane; PV, pervanadate; RE, recycling endosome; SEM, standard error of the mean.DOI: http://dx.doi.org/10.7554/eLife.12223.010

Fig 4: Ubiquitin-mediated switch in ligand-activated EGFR trafficking.(A) Time-lapse of EGFR and Rab11a co-localization after EGF stimulation of COS-7 cells with Alexa647-labeled EGF (5 ng/ml). Representative fluorescence images of EGFR-mCitrine (first row), c-Cbl-mCherry (second row), and BFP-Rab11a merge with Alexa647-labeled EGF (third row) at the indicated time in minutes. (B) Change in spatial distribution of EGFR-mCitrine upon EGF stimulation calculated as the difference between each time point and the one acquired prior to it, indicates areas with increased (cyan) or decreased (magenta) fluorescence. (C) Plot shows the normalized average fraction ± SEM of EGFR-mCitrine at the PM (PM-EGFR over total EGFR) in the presence (red, n=10 cells) or absence (black, n=6 cells) of c-Cbl-mCherry over time (see 'Materials and methods'). (D) Fraction of EGFR-mCitrine fluorescence at the RE with (n=10 cells) and without (n=6 cells) ectopic expression of c-Cbl-mCherry upon EGF stimulation for the indicated time. (E) Differential ubiquitination of ligand- and autonomously-activated EGFR. COS–7 lysates immunoprecipitated with anti-EGFR (left panel) or blotted for total proteins (middle panel). IP was probed with anti-HA (HA-ubiquitin) and anti-GFP (EGFR-mCitrine) and total lysates were probed with anti-GFP (EGFR-mCitrine), anti-Cbl (c-Cbl-mCherry), anti-tubulin (Tubulin), and anti-pY1045. Right panel: Quantification shows relative EGFR ubiquitination (Ub over EGFR) in the upper graph and degradation (EGFR over Tubulin) in the lower graph upon EGF stimulation (100 ng/ml) or PV treatment (0.33 mM) for the indicated time in minutes (see Figure 5—figure supplement 1A,B). (F) Fraction of EGFR-mCitrine fluorescence at the RE upon stimulation with 100 ng/ml EGF (n=12 cells) or 0.33 mM PV (n=14 cells) for the indicated time (see Figure 5—figure supplement 2). (G) Time-lapse of EGFR-Y1045F-mCitrine and BFP-Rab11a co-localization after EGF stimulation of COS-7 cells with Alexa647-labeled EGF (5 ng/ml). Representative fluorescence images of EGFR-Y1045F-mCitrine (first row), c-Cbl-mCherry (second row), and BFP-Rab11a merge with Alexa647-labeled EGF (third row) at the indicated time in minutes. (H) Fraction of EGFR-Y1045F-mCitrine (n=9 cells) or EGFR-Y1045/1068/1086F-mCitrine (n=9 cells) fluorescence at the RE upon EGF stimulation for the indicated time (min) in the presence of ectopically expressed c-Cbl-mCherry (see Figure 5—figure supplement 3A). (I) Sustained phosphorylation of EGFR-Y1045F-mCitrine and EGFR-Y1045/1068/1086F-mCitrine as compared to EGFR-mCitrine upon EGF stimulation. COS-7 lysates were probed with generic phosphotyrosine (PY72) and anti-GFP (EGFR-mCitrine) and bar diagram shows relative EGFR phosphorylation (PY72/EGFR) upon EGF stimulation (100 ng/ml) for the indicated time (see Figure 5—figure supplement 3B). All blots are n=3 (mean ± SEM). All scale bars: 10 µm. EGFR, epidermal growth factor receptor; SEM, standard error of the mean.DOI: http://dx.doi.org/10.7554/eLife.12223.017

Fig 5: Endocytosed HIV-1 Env traffics through Rab5+ early endosomes and into Rab14+ compartments. (A to E) HIV-1-infected CEM-A T cells were pulsed with probes for Env and transferrin (Tfn) for 15 min at 4°C, then chased at 37°C, fixed, and imaged by confocal fluorescence microscopy. Representative confocal slices are shown. All scale bars 5 µm. White arrows indicate examples of colocalization, while red arrows indicate examples of noncolocalization. (A) mRuby3-Rab5A KI CEM-A cells were infected, pulse-labeled with anti-Env Fab b12-AF647 and Tfn-AF488, and chased for 1 h. Env and Tfn partially colocalized with Rab5A. (B) Wild-type (WT) CEM-A cells were infected with HIV-1 virus bearing a Rab5A(Q79L) expression cassette in replacement of the nef open reading frame, expressing GFP-Rab5A(Q79L) in place of HIV-1 Nef protein. The cells were pulse-labeled with anti-Env Fab b12-Atto565 and Tfn-AF647, and chased for 20 min. Both Env and Tfn accumulated in Rab5A(Q79L)-induced swollen EEs. (C) HIV-1-infected GFP-Rab11A KI CEM-A cells were pulse-labeled with anti-Env Fab b12-AF647 and Tfn-AF555, and chased for 1 h. Env showed little overlap with Rab11, and Tfn overlapped partially with Rab11, but Tfn colocalized more so with Env. (D) CEM-A cells stably overexpressing high levels of TagRFP-Rab11A were treated as in panel C. Tfn accumulated in the swollen ERC induced by Rab11 overexpression, while Env did not. (E) GFP-Rab14 KI CEM-A cells were treated as in (C). Env and Tfn colocalized significantly with Rab14. (F) Fractional overlap between probes in the assays represented in panels A, C, and E; n = 40 infected cells per sample. “Env w/Rab5” as example denotes the percentage of Env-positive pixels that were also positive for Rab5. Bars represent means, error bars represent SE, and points represent the values for individual cells. Statistical significance was assessed by Brown-Forsythe and Welch ANOVA tests and Dunnett’s T3 multiple-comparison test. n.s., P > 0.05, *, P = 0.05, **, P = 0.01, ***, P = 0.001, ****, P = 0.0001.