Figure 1. Rab conversion and completion of endolysosomal stages of endosome maturation can be observed in enlarged endosomes, induced with short nigericin treatment.

Nigericin was added to HeLa cells at 10 μM for 20 min and washed away, and cells were imaged by time-lapse microscopy. Recovery times are specified relative to removal of nigericin. (A,C,D) Cells stably expressing mApple-Rab5 and GFP-Rab7. (A) Images to show enlarged Rab5- (white arrows) and Rab7- (magenta arrows) positive compartments and return to normal morphology by 20 hr. (B) Lysotracker Red (LTR) was added to cells before, during and following nigericin treatment. Images show rapid re-accumulation of Lysotracker in treated cells (bottom row). Untreated cells were tracked in parallel (upper row). (C) The enlarged endosome was selected to show Rab5 recruitment, Rab conversion and endolysosomal maturation. (D) Example of homotypic fusion of two Rab5-positive endosome and subsequent Rab5 removal/weak Rab7 recruitment. (E) Cells stably expressing mApple-Rab7 were imaged in the presence of Lysotracker Green. An enlarged Rab7-positive endosome was selected to show accumulation of Lysotracker concomitant with the loss of spherical shape and a reduction in size of the maturing endolysome. (C–E) Scale bar = 2 μm. Time-lapse videos of the endosomes in (C–E) at 1 min interval are available in Figure 1—video 1, Figure 1—video 2, Figure 1—video 3, respectively.

Figure 1—figure supplement 1. Enlarged compartments interact with early endosomes and acquire intraluminal content from the endolysosomal pathway.

Nigericin was added to HeLa cells, stably expressing mApple-Rab5 and GFP-Rab7 (A,B) or mApple-Rab5 alone (C), for 20 min and washed away, and cells were imaged by time-lapse microscopy. Recovery times are specified relative to removal of nigericin. (A) Example to show asynchronous nature of the transient Rab5-positive endosomes together with the closeup of a Rab5-positive endosome undergoing Rab conversion shortly after nigericin washout. (B) Example to show an enlarged compartment devoid of Rab5 fusing with Rab5-positive endosome to subsequently undergo Rab conversion. (C) Example to show an enlarged Rab5-positive endosome later acquiring Dextran-AF488, which had been pulse-chased into the endolysosomal pathway (4 hr pulse, 1.5 hr chase) prior to nigericin treatment. Recovery times are specified relative to removal of nigericin.

Figure 1—figure supplement 2. Enlarged endosomes that undergo Rab conversion can be induced by different treatments.

HeLa cells stably expressing mApple-Rab5 and GFP-Rab7 were treated for 4 hr with ammonium chloride (A) or for 20 min with monensin (B), washed and imaged at specified times after the wash. Representative images of endosomes to show Rab conversion. Homotypic fusion of two Rab5-positive endosomes is evident in (B).

Figure 1—figure supplement 3. Enlarged endosomes that undergo Rab conversion can be induced in different cell types.

Cells were transiently transfected with mApple-Rab5 and GFP-Rab7, and treated 20 min with nigericin followed by recovery and time-lapse microscopy. (A) HEK293 cells, (B) Neuro2A cells, (C) Cos-1 cells. Images were selected to show Rab conversion in the enlarged endosomes.

Figure 1—video 1. Rab5 recruitment, Rab conversion and endolysosomal maturation.

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Figure 1—video 2. Homotypic fusion of two Rab5-positive endosome and subsequent Rab5 removal/weak Rab7 recruitment.

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Figure 1—video 3. An enlarged Rab7-positive endosome was selected to show accumulation of Lysotracker concomitant with the loss of spherical shape and a reduction in size of the maturing endolysosome.

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