Panel A1 shows time-lapse images of transferred import-competent mitochondria at 6, 18, and 30 hours. Panel A2 shows time-lapse images of transferred import-defective mitochondria at 6, 18, and 30 hours. Panel B shows normalized transferred mitochondrial intensity over time for import-competent and import-defective mitochondria. Panel C shows fluorescence images highlighting lysosomes and colocalization of transferred mitochondria with lysosomes. Panel D contains bar graphs quantifying mitochondrial colocalization with lysosome-associated membrane protein 1 using Manders’ coefficient. Panel E presents a schematic model of tunneling nanotube-mediated mitochondrial exchange and degradation pathways.
Transferred mitochondria are ultimately degraded. (Ai) Stills from a representative movie of transferred import-defective mitochondria (su9-mTagBFP2-DHFR +100 nM MTX) in a cell containing import-competent mitochondria (su9-mScarlet) at the indicated time points after sorting. (Aii) Stills from a representative movie of transferred import-competent mitochondria (su9-mTagBFP2) in a cell containing import-defective mitochondria (su9-mScarlet-DHFR +100 nM MTX) at the indicated time points after sorting. (B) Quantification of intensity of transferred import-defective mitochondria (su9-mTagBFP2-DHFR +100 nM MTX) normalized to intensity at 6 h after sorting (n = 11 cells), and quantification of intensity of transferred import-competent mitochondria (su9-mTagBFP2) normalized to intensity at 6 h after sorting (n = 7 cells). Lines plotted from mean normalized intensity for each time point, and area shading shows standard deviation. (C) Representative images of LAMP1-stained double-positive cells from a 48 h co-culture of su9-mTagBFP2 and su9-mScarlet-DHFR cell lines in the presence of MTX. Scale bars 20 μm. (D) Histogram of Mander’s coefficient for mitochondrial co-localization with LAMP1. n = 47 cells from N = 3 technical replicates. (E) Schematic of proposed model.