Mitochondrial fusion remains a largely unknown process despite its observation by live microscopy and the identification of few implicated proteins. Using green and red fluorescent proteins targeted to the mitochondrial matrix, we show that mitochondrial fusion in human cells is efficient and achieves complete mixing of matrix contents within 12 h. This process is maintained in the absence of a functional respiratory chain, despite disruption of microtubules or after significant reduction of cellular ATP levels. In contrast, mitochondrial fusion is completely inhibited by protonophores that dissipate the inner membrane potential. This inhibition, which results in rapid fragmentation of mitochondrial filaments, is reversible: small and punctate mitochondria fuse to reform elongated and interconnected ones upon withdrawal of protonophores. Expression of wild-type or dominant-negative dynamin-related protein 1 showed that fragmentation is due to dynamin-related protein 1-mediated mitochondrial division. On the other hand, expression of mitofusin 1 (Mfn1), one of the human Fzo homologues, increased mitochondrial length and interconnectivity. This process, but not Mfn1 targeting, was dependent on the inner membrane potential, indicating that overexpressed Mfn1 stimulates fusion. These results show that human mitochondria represent a single cellular compartment whose exchanges and interconnectivity are dynamically regulated by the balance between continuous fusion and fission reactions. INTRODUCTIONThe morphology and distribution of mitochondria differ significantly between the cells of different species and tissues. In addition, mitochondrial volume and morphology vary in function of cellular metabolism, are modulated during cell cycle and development, and during apoptosis (Stevens, 1981;Tzagoloff, 1982;Bereiter-Hahn and Voth, 1994;Church and Poyton, 1998;Diaz et al., 1999;Frank et al., 2001). Live microscopy has revealed that mitochondrial morphology is continuously remodeled by fission and fusion (Nunnari et al., 1997;Rizzuto et al., 1998). In yeast, selective inhibition of either process significantly modifies mitochondrial size and interconnectivity (Bleazard et al., 1999;Sesaki and Jensen, 1999). Among the best known proteins involved in mitochondrial dynamics are Fzo/ mitofusin, a transmembrane GTPase involved in fusion (Hales and Fuller, 1997;Hermann et al., 1998;Santel and Fuller, 2001;Rojo et al., 2002), and Dnm1p/dynamin-related protein 1 (Drp1), a dynamin-related protein involved in fission (Bleazard et al., 1999;Pitts et al., 1999;Smirnova et al., 2001).In yeast, mitochondrial fusion has been demonstrated by the diffusion and/or mixing of different matrix proteins during mating of haploid cells (Azpiroz and Butow, 1993;Nunnari et al., 1997). In contrast, mitochondrial fusion has not been studied with similar assays in human cells, and it is not known to what extent the apparent fusion events observed by live microscopy correspond to the formation of intermitochondrial junctions (Bakeeva et al., 1978;Amche...

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.