Heteroplasmic mutations of mitochondrial DNA (mtDNA) are an important source of human diseases. The mechanisms governing transmission, segregation and complementation of heteroplasmic mtDNA-mutations are unknown but depend on the nature and dynamics of the mitochondrial compartment as well as on the intramitochondrial organization and mobility of mtDNA. We show that mtDNA of human primary and immortal cells is organized in several hundreds of nucleoids that contain a mean of 2-8 mtDNA-molecules each. Nucleoids are enriched in mitochondrial transcription factor A and distributed throughout the entire mitochondrial compartment. Using cell fusion experiments, we demonstrate that nucleoids and respiratory complexes are mobile and diffuse efficiently into mitochondria previously devoid of mtDNA. In contrast, nucleoid-mobility was lower within mitochondria of mtDNA-containing cells, as differently labeled mtDNA-molecules remained spatially segregated in a significant fraction (37%) of the polykaryons. These results show that fusion-mediated exchange and intramitochondrial mobility of endogenous mitochondrial components are not rate-limiting for intermitochondrial complementation but can contribute to the segregation of mtDNA molecules and of mtDNA mutations during cell growth and division.

Enlarged or giant mitochondria have often been documented in aged tissues although their role and underlying mechanism remain unclear. We report here how highly elongated giant mitochondria are formed in and related to the senescent arrest. The mitochondrial morphology was progressively changed to a highly elongated form during deferoxamine (DFO)-induced senescent arrest of Chang cells, accompanied by increase of intracellular ROS level and decrease of mtDNA content. Interestingly, under exposure to subcytotoxic doses of H 2 O 2 (200 mM), about 65% of Chang cells harbored elongated mitochondria with senescent phenotypes whereas ethidium bromide (EtBr) (50 ng/ml) only reformed the cristae structure. Elongated giant mitochondria were also observed in TGF b1-or H 2 O 2 -induced senescent Mv1Lu cells and in old human diploid fibroblasts (HDFs). In all senescent progresses employed in this study Fis1 protein, a mitochondrial fission modulator, was commonly downexpressed. Overexpression of YFP-Fis1 reversed both mitochondrial elongation and appearance of senescent phenotypes induced by DFO, implying its critical involvement in the arrest. Finally, we found that direct induction of mitochondrial elongation by blocking mitochondrial fission process with Fis1-DTM or Drp1-K38A was sufficient to develop senescent phenotypes with increased ROS production. These data suggest that mitochondrial elongation may play an important role as a mediator in stress-induced premature senescence.