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. 2015 Feb 3;16(3):297–311. doi: 10.15252/embr.201439464

Figure 4.

Figure 4

RAB21 and Sbf/MTMR13 regulate dVamp7/VAMP8 trafficking to lysosomes

  • A-D GFP:Vamp7 in Drosophila starved larval fat body. (A) Control. Vamp7 trafficking is blocked with (B) Rab21RNAi or (C) SbfRNAi. (D) Number of GFP:Vamp7 punctae normalized to fat body area; SEM.
  • E-H RAB21 and VAMP8 co-localize at EEA1 early endosomes. (E) GFP:RAB21, (F) VAMP8:3xHA and (G) EEA1 co-localize (arrowheads) in starved HeLa cells, merged in (H).
  • I Per cell average Pearson Correlation of GFP:RAB21 with VAMP8:3xHA, GFP:RAB21 with EEA1, VAMP8:3xHA with EEA1 and GFP:RAB7 with mCherry:VAMP8 in response to starvation over time; SEM. Only VAMP8 co-localization with RAB7 was enhanced by starvation.
  • J-L RAB7 and VAMP8 co-localize at late endosomes. (J) GFP:RAB7 and (K) mCherry:VAMP8 co-localize (arrowheads) in starved HeLa cells, merged in (L).
  • M-P Starvation-enhanced co-localization between endogenous RAB7 and GFP:VAMP8 as in (M) scramble control requires (N) RAB21 and (O) MTMR13 function. (M', N' and O') Zooms of boxed regions shown above. (P) Per cell average Pearson Correlation between endogenous RAB7 and GFP:VAMP8 in siRNA-treated cells; SEM.
  • Q VAMP8 exit from early endosomes is delayed in RAB21 or MTMR13 siRNA-depleted cells. Per cell average object co-localization between internalized VAMP8 and EEA1 in chloroquine-treated cells; SEM. See Supplementary Fig S4Q–S.
  • R-U VAMP8 delivery to lysosomes is blocked in RAB21- or MTMR13-depleted cells. Antibody-uptake assay of VAMP8:3xHA (internalized anti-HA, red) with LAMP1 (green) and DAPI nuclei. (R) Scramble siRNA, (S) RAB21 siRNA and (T) MTMR13 siRNA-treated cells. (R', S' and T') Zooms of boxed regions shown above. (U) Per cell average object co-localization of internalized VAMP8:3xHA with LAMP1 over time; SEM. See also Supplementary Figs S3 and S4.