Figure 6.
mtDNA-containing EVs promote endosomal trafficking and invasiveness. (a) Protocol to study the influence of EVs collected from glutamine-replete (+Gln) donor cells on the invasive behavior of Gln-starved (−Glu) recipient cells. (b) Donor MDA-MB-231 cells were incubated in glutamine-containing medium in the absence (Ctrl) and presence of LY95 0.3 µM for 48 h, and EVs were purified from this using differential centrifugation. Glutamine-starved recipient cells were incubated in the absence (−EVs) or presence of EVs from the indicated donor cells (+EVs [Ctrl]; +EVs [LY95]) for 3 d and then transfected with mCherry-MT1-MMP and imaged using TIRF microscopy. Time-lapse videos were recorded with frames being collected every 1.5 s for 5 min. Kymographs of representative videos are displayed. The rate of appearance of mCherry-positive structures in the TIRF field was calculated using the ImageJ plugin TrackMate. Values represent mean ± SEM from three independent experiments. **, P < 0.002; ***, P < 0.001; two-way ANOVA with Dunn’s multiple comparison test. (c) Glutamine-starved recipient cells were treated for 3 d with EVs from LY95- or vehicle-treated (Ctrl) donor cells (as for b) or donor cells that had been transfected with siRNAs targeting PINK1 (siPINK1), Rab27a/b (siRab27), or nontargeting control (siNT). Recipient cells were then replated onto dishes coated with fluorescently conjugated gelatin (FITC-gelatin) in the absence of glutamine, and imaging was performed using confocal microscopy. Bar, 20 µm; bar in zoom, 2 µm. Gelatin degradation was determined using ImageJ. Values are mean ± SEM; n = 2 independent experiments, n = 4 technical repeats with a minimum of 45 cells being quantified for each condition. **, P < 0.002; ***, P < 0.001; two-way ANOVA with Dunn’s multiple comparison test. (d) Glutamine-starved recipient cells were pretreated for 3 d with EVs from LY95- or vehicle-treated (Ctrl) donor cells (as in b) or donor cells that had been transfected with siRNAs targeting PINK1 (siPINK1), Rab27a/b (siRab27), CD63 (siCD63), or nontargeting control (siNT). Recipient cells were plated into fibroblast-derived ECM and imaged using time-lapse video microscopy. The length of protrusions extending in the direction of migration (distance from the nucleus to cell front) was determined using ImageJ. Bar, 30 µm. Whiskers are 10th–90th percentiles; + represents the mean. ***, P < 0.001; two-way ANOVA with Dunn’s multiple comparison test.

mtDNA-containing EVs promote endosomal trafficking and invasiveness. (a) Protocol to study the influence of EVs collected from glutamine-replete (+Gln) donor cells on the invasive behavior of Gln-starved (−Glu) recipient cells. (b) Donor MDA-MB-231 cells were incubated in glutamine-containing medium in the absence (Ctrl) and presence of LY95 0.3 µM for 48 h, and EVs were purified from this using differential centrifugation. Glutamine-starved recipient cells were incubated in the absence (−EVs) or presence of EVs from the indicated donor cells (+EVs [Ctrl]; +EVs [LY95]) for 3 d and then transfected with mCherry-MT1-MMP and imaged using TIRF microscopy. Time-lapse videos were recorded with frames being collected every 1.5 s for 5 min. Kymographs of representative videos are displayed. The rate of appearance of mCherry-positive structures in the TIRF field was calculated using the ImageJ plugin TrackMate. Values represent mean ± SEM from three independent experiments. **, P < 0.002; ***, P < 0.001; two-way ANOVA with Dunn’s multiple comparison test. (c) Glutamine-starved recipient cells were treated for 3 d with EVs from LY95- or vehicle-treated (Ctrl) donor cells (as for b) or donor cells that had been transfected with siRNAs targeting PINK1 (siPINK1), Rab27a/b (siRab27), or nontargeting control (siNT). Recipient cells were then replated onto dishes coated with fluorescently conjugated gelatin (FITC-gelatin) in the absence of glutamine, and imaging was performed using confocal microscopy. Bar, 20 µm; bar in zoom, 2 µm. Gelatin degradation was determined using ImageJ. Values are mean ± SEM; n = 2 independent experiments, n = 4 technical repeats with a minimum of 45 cells being quantified for each condition. **, P < 0.002; ***, P < 0.001; two-way ANOVA with Dunn’s multiple comparison test. (d) Glutamine-starved recipient cells were pretreated for 3 d with EVs from LY95- or vehicle-treated (Ctrl) donor cells (as in b) or donor cells that had been transfected with siRNAs targeting PINK1 (siPINK1), Rab27a/b (siRab27), CD63 (siCD63), or nontargeting control (siNT). Recipient cells were plated into fibroblast-derived ECM and imaged using time-lapse video microscopy. The length of protrusions extending in the direction of migration (distance from the nucleus to cell front) was determined using ImageJ. Bar, 30 µm. Whiskers are 10th–90th percentiles; + represents the mean. ***, P < 0.001; two-way ANOVA with Dunn’s multiple comparison test.

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