Figure 9. Selective cargo recycling takes place in enlarged Rab5-positive endosomes.

HeLa cells, stably expressing mApple-Rab5 and transiently transfected with the cargos TfR-GFP (A,D,G), GFP-CDMPR (B,E,H) or GalT-GFP (C,F,I), were treated for 20 min with nigericin, washed and imaged over 3 hr, as described in Figure 4A. (A–C) Time-lapse images of representative endosomes to show cargo acquisition and removal relative to Rab5 recruitment. Time-lapse videos of the endosomes in (A,D,G) at 1 min interval are available in Figure 9—video 1, Figure 9—video 2, and Figure 9—video 3, respectively. (D–F) Corresponding graphs of MFI of Rab5 and specified cargo at the endosome in (A,B,C), respectively, over the time the endosome was detectable, to show prompt removal of TfR and CDMPR, and GalT remaining unchanged. (G–I) Averaged Rab5 and specified cargo acquisition and removal, representing 24, 24, and 17 endosomes, respectively. Error bars represent standard deviation. Three independent experiments were performed. Numerical data for all analyzed endosomes is available in Figure 9—source data 1, Figure 9—source data 2, and Figure 9—source data 3.

Figure 9—figure supplement 1. CDMPR can be acquired by Rab5-positive endosomes.

HeLa cells, stably expressing mApple-Rab5 and transiently transfected with CDMPR-GFP, were treated for 20 min with nigericin, washed and imaged over 3 hr, as described in Figure 4A. Time-lapse images of a representative endosome to show CDMPR acquisition at Rab5-positive enlarged endosome.

Figure 9—video 1. TfR acquisition and removal relative to Rab5 recruitment.

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Figure 9—video 2. CDMPR acquisition and removal relative to Rab5 recruitment.

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Figure 9—video 3. GalT acquisition and removal relative to Rab5 recruitment.

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