Figure 11. GalT-pHlemon sensor detects endosomal acidification, which correlates with Rab conversion.
HeLa cells, transiently expressing mApple-Rab5 (A–C) or stably expressing mApple-Rab7 (D–F) and transiently transfected with GalT-pHlemon, were treated for 20 min with nigericin, washed and imaged over 3 hr, as described in Figure 4A. (A,D) Time-lapse images of a representative endosome to show association of Rab conversion with acidification, as detected by the decrease in the YFP signal and relatively constant CFP at the rim. Time-lapse videos of the endosomes at 85 s interval are available in Figure 11—videos 1 and 2. (B,E) Corresponding graphs of normalised MFI of Rab5 (B) or Rab7 (E) at the rim and lumenal YFP/CFP ratio of GalT-pHlemon signal of the endosomes in (A) or (D), respectively, during and around the time of Rab conversion. (C,F) Averaged Rab5 (C) or Rab7 (F) and GalT-pH kinetics of 19 and 18 endosomes, respectively. Error bars represent standard deviation. Pooled data from two independent experiments. Numerical data for all analysed endosomes is available in Figure 11—source data 1 and Figure 11—source data 2.
