Mitochondria play key roles in the life and death of cells. We investigated whether mitochondria represent morphologically continuous and functionally homogenous entities within single intact cells. Physical continuity of mitochondria was determined by three-dimensional reconstruction of fluorescence from mitochondrially targeted DsRed1 (red fluorescent protein from Discosoma sp.; obtained from Clontech). The mitochondria of HeLa and HUVEC cells displayed heterogeneous distributions and were of varying sizes. In general, there was a denser aggregation of mitochondria in perinuclear positions than in the cell periphery where individual isolated mitochondria could clearly be seen. DsRed1 was highly mobile within the lumen of individual mitochondria, equilibrating across a 22 µm long mitochondrion in the periphery of the cell within 30 s. However, a lack of fluorescence recovery after photobleaching of DsRed1 for at least 20 min after bleaching demonstrated lumenal discontinuity between mitochondria in the perinuclear aggregation. Mitochondria were not electrically coupled, since only individual mitochondria were observed to depolarise following irradiation of tetramethyl rhodamine ethyl ester-loaded cells.

Heterogeneity in membrane potential was observed with a predominance of highly energised mitochondria in the periphery of the cell. In 5,5fi6,6fi tetrachloro 1,1fi3,3fi tetraethylbenzimidazol carbocyanine iodide (JC1)-loaded cells, mitochondria fluoresce red at high and green at low membrane potentials. In the periphery of JC1-loaded cells, 68 ± 5 % of mitochondria emitted red fluorescence compared with 28 ± 4% in the perinuclear region (S.E.M., n = 25 cells, P < 0.005, paired t test). There were also differences in sequestration of hormonally evoked cytosolic calcium signals and the timing of permeability transition pore opening in response to tert-butyl hydroperoxide. Our data indicate that the mitochondria within individual cells are morphologically heterogeneous and unconnected, thus allowing them to have distinct functional properties.