Composition and dynamics of human mitochondrial nucleoids.
The organization of multiple mitochondrial DNA (mtDNA) molecules in discrete protein-DNA complexes called nucleoids is well studied in Saccharomyces cerevisiae. Similar structures have recently been observed in human cells by the colocalization of a Twinkle-GFP fusion protein with mtDNA. However, nucleoids in mammalian cells are poorly characterized and are often thought of as relatively simple structures, despite the yeast paradigm. In this article we have used immunocytochemistry and biochemical isolation procedures to characterize the composition of human mitochondrial nucleoids. The results show that both the mitochondrial transcription factor TFAM and mitochondrial single-stranded DNA-binding protein colocalize with Twinkle in intramitochondrial foci defined as nucleoids by the specific incorporation of bromodeoxyuridine. Furthermore, mtDNA polymerase POLG and various other as yet unidentified proteins copurify with mtDNA nucleoids using a biochemical isolation procedure, as does TFAM. The results demonstrated that mtDNA in mammalian cells is organized in discrete protein-rich structures within the mitochondrial network. In vivo time-lapse imaging of nucleoids show they are dynamic structures able to divide and redistribute in the mitochondrial network and suggest that nucleoids are the mitochondrial units of inheritance. Nucleoids did not colocalize with dynamin-related protein 1, Drp1, a protein of the mitochondrial fission machinery.
Garrido
Nuria
N
Institute of Medical Technology and Tampere University Hospital, Lenkkeilijänkatu 6, 33014 University of Tampere, Tampere, Finland.
Griparic
Lorena
L
Jokitalo
Eija
E
Wartiovaara
Jorma
J
van der Bliek
Alexander M
AM
Spelbrink
Johannes N
JN
D001483:Base Sequence; D002460:Cell Line; D004265:DNA Helicases; D000074002:DNA Polymerase gamma; D019915:DNA Primase / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013367:Submitochondrial Particles / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C499631:TFAM protein, human; D014157:Transcription Factors; C422562:mitochondrial transcription factor A; D049452:Green Fluorescent Proteins; D019915:DNA Primase; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C413027:POLG protein, human; D004265:DNA Helicases; C431576:TWNK protein, human