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

Rho0 HeLa cells entirely lacking mitochondrial DNA (mtDNA) and mitocho ndrial transfection techniques were used to examine intermitochondrial interactions between mitochondria with and without mtDNA, and also be tween those with wild-type (wt) and mutant-type mtDNA in living human cells. First, unambiguous evidence was obtained that the DNA-binding d yes ethidium bromide (EtBr) and 4',6-diamidino-2-phenylindole (DAPI) e xclusively 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-stain ed HeLa mitochondria were introduced into rho0 HeLa cells. Moreover, c oexisting wt-mtDNA and mutant mtDNA with a large deletion (DELTA-mtDNA ) were shown to mix homogeneously throughout mitochondria, not to rema in segregated by use of electron microscopic analysis of cytochrome c oxidase activities of individual mitochondria as a probe to identify m itochondria with predominantly wt- or DELTA-mtDNA in single cells. Thi s rapid diffusion of mtDNA and the resultant homogeneous distribution of the heteroplasmic wt- and DELTA-mtDNA molecules throughout mitochon dria in a cell suggest that the mitochondria in living human cells hav e lost their individuality. Thus, the actual number of mitochondria pe r cell is not of crucial importance, and mitochondria in a cell should be considered as a virtually single dynamic unit.