Link to Pubmed [PMID] – 23239030
Link to DOI – 10.1242/jcs.114322
J Cell Sci 2013 Feb; 126(Pt 4): 914-26
Mitochondrial DNA (mtDNA) replication and transcription are crucial for cell function, but these processes are poorly understood at the single-cell level. We describe a novel fluorescence in situ hybridization protocol, called mTRIP (mitochondrial transcription and replication imaging protocol), that reveals simultaneously mtDNA and RNA, and that can also be coupled to immunofluorescence for in situ protein examination. mTRIP reveals mitochondrial structures engaged in initiation of DNA replication by identification of a specific sequence in the regulatory D-loop, as well as unique transcription profiles in single human cells. We observe and quantify at least three classes of mitochondrial structures: (i) replication initiation active and transcript-positive (Ia-Tp); (ii) replication initiation silent and transcript-positive (Is-Tp); and (iii) replication initiation silent and transcript-negative (Is-Tn). Thus, individual mitochondria are dramatically heterogeneous within the same cell. Moreover, mTRIP exposes a mosaic of distinct nucleic acid patterns in the D-loop, including H-strand versus L-strand transcripts, and uncoupled rRNA transcription and mtDNA initiation of replication, which might have functional consequences in the regulation of the mtDNA. Finally, mTRIP identifies altered mtDNA processing in cells with unbalanced mtDNA content and function, including in human mitochondrial disorders. Thus, mTRIP reveals qualitative and quantitative alterations that provide additional tools for elucidating the dynamics of mtDNA processing in single cells and mitochondrial dysfunction in diseases.