Fig. 6: Characterization of mitochondrial fate after delivery using NLuc complementation.

A Schematic representation of three possible outcomes following cytosolic delivery of extracellular mitochondria: Hypothesis I: Individual donor mitochondria are delivered into the cytosol and remain isolated, with none too moderate complementation with outermembrane LgBit-protein or cytosolic LgBit. Hypothesis II: Donor mitochondria cluster with acceptor cell mitochondria. In this case, outer membrane (OM) protein complementation is expected to be positive, while inner membrane (IM) proteins should remain negative. Hypothesis III: Donor mitochondria fully integrate into the acceptor cell’s mitochondrial network. Both OM and IM proteins are expected to show positive complementation in during homotypic exchange. A summary table outlines the expected outcomes for each donor/acceptor combination under these three models. B extracellular mitochondria from donor cells expressing either HiBiT-OMP25 or HiBiT-COX8 were incubated for 24 hours with acceptor cells expressing either cytosolic LgBiT, LgBiT-OMP25, or LgBiT-COX8. NLuc activity was measured to assess complementation. Three independent experiments were performed, each including three technical replicates. Each dot represents a replicate. Error bars indicate standard deviation, center represents the means. *Statistical significance was assessed by unpaired, two-tailed t-test comparing homotypic combinations (OMP25/OMP25 and COX8/COX8) to the heterotypic OMP25/LgBiT control; p < 0.01.