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. 2017 Oct 30;114(46):E9883–E9892. doi: 10.1073/pnas.1713524114

Fig. 6.

Fig. 6.

Interference with endosomal trafficking by diverting endosomes to mitochondria via fusion with targeted liposomes. (A) Overview over the experimental design. LUVs were prepared that contain both 4-EE-SNAREs and HaloTag ligand. These liposomes are then injected into HeLa cells expressing HaloTag-GFP targeted to the outer mitochondrial membrane as shown in Fig. 2. (B) In vitro recruitment of endosomes incubated with 4-EE-SNARE LUVs containing HaloTag ligand to mitochondria. Postnuclear supernatants (PNSs) were used as source for endosomes and mitochondria that were obtained from HeLa cells expressing either GFP-MAO or GFP-MAO fused to HaloTag, and incubation was carried out for 30 min with the liposomes. Mitochondria were then immunoisolated using an anti-GFP antibody. Note that transferrin receptor (TfnR) showed significant coprecipitation with mitochondria when 4-EE-SNARE + HaloTag ligand proteoliposomes (PL), but not when HaloTag ligand liposomes (Lipo) were used for microinjection. (C) HaloTag-GFP-MAO–expressing HeLa cells were injected with HaloTag ligand LUVs containing 4-EE-SNARE proteins (Top) or with HaloTag ligand LUVs devoid of 4-EE-SNAREs (Bottom). Fifteen minutes after injection of the LUVs, Alexa 633-Tfn was internalized for 5 min and the cells were chased for 30 min. Note that Alexa 633-positive endosomes were associated with mitochondria and showed colocalization with liposomes only when the liposomes contained both 4-EE-SNARE and HaloTag ligand. (Scale bar, 2.5 µm.) The panels on the Right show intensity plots of the line scans (a purple line) in the pictures on the Left. (D) Alexa 633-Tfn (green) trafficking in 4-EE-SNARE + HaloTag ligand LUV (red)-injected cells expressing HaloTag-GFP-MAO (blue). Fifteen minutes after injection of the LUVs, Alexa 633-Tfn was internalized for 5 min and the cells were chased for 3 or 15 min. Asterisks indicate the HaloTag-GFP-MAO–expressing cells injected with 4-EE-SNARE LUVs containing HaloTag ligand. (Scale bar, 20 µm.) (E) Fluorescence intensity ratio of internalized Alexa 633-Tfn between HaloTag-GFP-MAO–expressing HeLa cells injected with 4-EE-SNARE + HaloTag ligand LUVs and noninjected cells. Microinjection of 4-EE-SNARE + HaloTag ligand LUVs resulted in retention of the fluorescence signal that was significantly higher than in control cells. (F) Quantification of the distribution of Alexa 633-Tfn–positive endosomes between the peripheral region and the perinuclear region after 15-min chasing. The perinuclear index (see Methods for details) decreased in HaloTag-GFP-MAO–expressing cells after microinjection of 4-EE-SNARE + HaloTag ligand LUVs, suggesting that Alexa 633-Tfn–positive endosomes were scattered. For all plots in this figure, error bars indicate SEM, *P < 0.05, **P < 0.01, determined by unpaired t test.