Panel A shows schematic illustrations and flow cytometry dot plots identifying EGFP-positive and mScarlet-positive cell populations under different culture conditions. Panel B shows a line graph with x-axis Hours post coculture plus 100 nanomolar MTX and y-axis Percentage of coculture double-positive cells. Panel C shows a schematic workflow illustrating coculture, MTX treatment, fluorescence-activated cell sorting, and selection of double-positive cells. Panels D, E, and F show fluorescence microscopy images of import-competent and import-defective mitochondria under DHFR-MTX, DHFR, and MTX conditions. Panel G shows scatter plots with x-axis Experimental condition and y-axis Mean size of transferred mitochondria per cell (square micrometers) or Number of transferred mitochondria per cell. Panel H shows scatter plots with x-axis Experimental condition and y-axis Mean size of transferred mitochondria per cell (square micrometers) or Number of transferred mitochondria per cell.
Transferred import-competent and import-defective mitochondria have distinct fates. (A) Four-way gating system for identification of EGFP+/mScarlet+ cells. (B) Time course of the percentage population of co-culture that is double-positive for EGFP and mScarlet in a co-cultivation of su9-EGFP line (import-competent) with su9-mScarlet-DHFR line in the presence of MTX (import-defective). N = 4 technical replicates. Error bars represent standard deviation. (C) Schematic of workflow. (D) Representative images of double-positive cells from a 48 h co-culture of su9-mTagBFP2 and su9-mScarlet-DHFR cell lines in the presence of MTX. (E) Representative images of double-positive cells from a 48 h co-culture of su9-mTagBFP2 and su9-mScarlet-DHFR cell lines. (F) Representative images of double-positive cells from a 48 h co-culture of su9-mTagBFP2 and su9-mScarlet cell lines in the presence of MTX. Scale bars 20 μm. (G) Quantification of (i) mean size of transferred import-defective mitochondria per cell; (ii) number of transferred import-defective mitochondria per cell; (iii) mean size of transferred import-competent mitochondria per cell. For transferred import-competent mitochondria localized in MDBs, individual mitochondria could not be distinguished, so the total MDB area was quantified; (iv) number of transferred import-competent mitochondria per cell. All quantification was done on max. projected images. n = 62 cells from N = 3 technical replicates. P values from ordinary one-way ANOVA, multiple comparisons corrected for by the Dunnett test. Data distribution was assumed to be normal, but this was not formally tested. Mean lines shown on the graph. (H) Quantification of double-positive cells from a 48 h co-culture of su9-mTagBFP2 line and a su9-mScarlet line, in which the su9-mScarlet line has been pre-treated with siRNA against TIM23, TOM40, or a non-targeting control. (i) mean size of transferred import-defective mitochondria per cell; (ii) number of transferred import-defective mitochondria per cell; (iii) mean size of transferred import-competent mitochondria per cell. For transferred import-competent mitochondria localized in MDBs, individual mitochondria could not be distinguished, so the total MDB area was quantified; (iv) number of transferred import-competent mitochondria per cell. All quantification was done on max. projected images. n = 45 cells from N = 3 technical replicates. P values from ordinary one-way ANOVA, multiple comparisons corrected for by the Dunnett test. Data distribution was assumed to be normal, but this was not formally tested. Mean lines shown on the graph.