HUMAN MITOCHONDRIA AND MITOCHONDRIAL GENOME FUNCTION AS A SINGLE DYNAMIC CELLULAR-UNIT

Rho0 HeLa cells entirely lacking mitochondrial DNA (mtDNA) and mitochondrial transfection techniques were used to examine intermitochondrial interactions between mitochondria with and without mtDNA, and also between those with wild-type (wt) and mutant-type mtDNA in living human cells. First, unambiguous evidence was obtained that the DNA-binding dyes ethidium bromide (EtBr) and 4',6-diamidino-2-phenylindole (DAPI) exclusively stained mitochondria containing mtDNA in living human cells. Then, using EtBr or DAPI fluorescence as a probe, mtDNA was shown to spread rapidly to all rho0 HeLa mitochondria when EtBr- or DAPI-stained HeLa mitochondria were introduced into rho0 HeLa cells. Moreover, coexisting wt-mtDNA and mutant mtDNA with a large deletion (DELTA-mtDNA) were shown to mix homogeneously throughout mitochondria, not to remain segregated by use of electron microscopic analysis of cytochrome c oxidase activities of individual mitochondria as a probe to identify mitochondria with predominantly wt- or DELTA-mtDNA in single cells. This rapid diffusion of mtDNA and the resultant homogeneous distribution of the heteroplasmic wt- and DELTA-mtDNA molecules throughout mitochondria in a cell suggest that the mitochondria in living human cells have lost their individuality. Thus, the actual number of mitochondria per cell is not of crucial importance, and mitochondria in a cell should be considered as a virtually single dynamic unit.