Abstract
BackgroundWe analyzed the organization and function of mitochondrial DNA in a stable human cell line (ECV304, which is also known as T-24) containing mitochondria tagged with the yellow fluorescent protein.ResultsMitochondrial DNA is organized in ~475 discrete foci containing 6–10 genomes. These foci (nucleoids) are tethered directly or indirectly through mitochondrial membranes to kinesin, marked by KIF5B, and microtubules in the surrounding cytoplasm. In living cells, foci have an apparent diffusion constant of 1.1 × 10-3 μm2/s, and mitochondria always split next to a focus to distribute all DNA to one daughter. The kinetics of replication and transcription (monitored by immunolabelling after incorporating bromodeoxyuridine or bromouridine) reveal that each genome replicates independently of others in a focus, and that newly-made RNA remains in a focus (residence half-time ~43 min) long after it has been made. This mitochondrial RNA colocalizes with components of the cytoplasmic machinery that makes and imports nuclear-encoded proteins – that is, a ribosomal protein (S6), a nascent peptide associated protein (NAC), and the translocase in the outer membrane (Tom22).ConclusionsThe results suggest that clusters of mitochondrial genomes organize the translation machineries on both sides of the mitochondrial membranes. Then, proteins encoded by the nuclear genome and destined for the mitochondria will be made close to mitochondrial-encoded proteins so that they can be assembled efficiently into mitochondrial complexes.
Highlights
We analyzed the organization and function of mitochondrial DNA in a stable human cell line (ECV304, which is known as T-24) containing mitochondria tagged with the yellow fluorescent protein
A stable cell line with YFP-tagged mitochondria We first derived a stable cell line containing mitochondria tagged with the yellow fluorescent protein (YFP)
The kinesin motor implicated in moving mitochondria along microtubules – KIF5B [25] – often colocalized with individual mitochondrial DNA (mtDNA) foci (Figure 3E,3F,3G), and a close association was confirmed by the peak in Pearson's coefficient at a ∆x of zero (Figure 3H)
Summary
We analyzed the organization and function of mitochondrial DNA in a stable human cell line (ECV304, which is known as T-24) containing mitochondria tagged with the yellow fluorescent protein. Mitochondria are the centre of energy production in the cell They are usually numerous and polymorphic, and their overall shape depends on a balance between the fusion and fission of individual mitochondria [1]. These processes are controlled by several proteins [2], including dynamin-related protein 1 (Drp1) [3,4], and the mitofusins (for example, Mfn 1 and 2) [5]. A noncoding regulatory region harbours an origin of replication plus two promoters, one on each of the two strands Transcription from these promoters generates polycistronic transcripts that are processed to produce mature rRNAs, tRNAs, and mRNAs; a transcript generated from one of the promoters primes mitochondrial DNA (mtDNA) replication. Most mitochondrial proteins are encoded by the nucleus; they are made by cytoplasmic ribosomes and imported through specialized pores into mitochondria where they combine with those encoded by the mitochondrial genome [11,12]
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