Free mitochondria enter cells by macropinocytosis, and a small fraction escapes endosomes

Mitochondrial transfer between cells has been reported from yeast to humans, and mitochondrial transplantation is being explored as a therapy. Yet the form of the transported material, how efficiently it moves, and what recipient cells do with it remain unclear. Earlier work has often relied on diffusible fluorescent dyes, which can produce artifacts. Clarifying these basics matters for any future mitochondria-based treatment aimed at restoring cellular energy function.

The team, from Université Paris Cité/INSERM, engineered HeLa donor cells to express NanoLuciferase (NLuc)-tagged mitochondrial proteins: OMP25 on the outer membrane, facing the cytosol, and COX8a in the inner membrane, facing the intermembrane space. They separated conditioned media by size exclusion chromatography and compared it with mitochondria isolated directly from donor cells. They characterized the fractions by nanoparticle tracking, immunoblotting, and proteinase K protection assays. They then measured uptake in unlabeled acceptor cells by luminescence and used confocal imaging to follow intracellular fate.

Only a small amount of the tagged signal in conditioned media was in the large-particle fraction (F4), about 0.13-0.18%, versus roughly 17-21% in the equivalent fraction of isolated mitochondria. Particles were 100-500 nm, and ER markers were absent, arguing against cell lysis as the source. In protease protection assays, OMP25 was protease-sensitive while COX8a was protected, the pattern expected for free mitochondria rather than mitochondria enclosed in extracellular vesicles. Acceptor cells took up about 1-2% of F4 and isolated mitochondria over 24 hours, with uptake rising with time and dose, while the small-particle fraction was essentially not taken up. Plasmid DNA was not detected, mitochondrial DNA was, and a stable donor line gave consistent uptake in HeLa, A431, and SKOV3 cells.

Uptake was virtually undetectable at 4 °C, arguing against a high-affinity receptor, though the authors note heparan sulfate-mediated electrostatic docking is compatible with the data. Internalized mitochondria colocalized with early endosome (39%) and lysosome (30%) markers and strongly with fluid-phase dextran, supporting macropinocytosis followed by endo-lysosomal trafficking. Using an adapted split-NLuc (HiBiT/LgBiT) complementation assay, the authors report that fewer than 10% of internalized mitochondria escape endosomes into the cytosol, where they may integrate into the host mitochondrial network. The provided text ends before the details of this assay, so those results are taken from the abstract and introduction.

The findings suggest that free mitochondria transfer is a low-efficiency process, which tempers expectations for mitochondrial transplantation and highlights endosomal escape as a bottleneck to improve. Caveats include the use of immortalized cell lines, tagged reporter proteins, and transient overexpression in donor cells. The wording of the conclusions is also tentative: uptake is "primarily" fluid-phase, and integration is something mitochondria "may" do. Functional benefit to recipient cells and in vivo relevance were not established in the text available.