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. 2023 May 20;13(10):e4680.
doi: 10.21769/BioProtoc.4680.

Mitochondrial Replication Assay (MIRA) for Efficient in situ Quantification of Nascent mtDNA and Protein Interactions with Nascent mtDNA (mitoSIRF)

Macy Lozen  1 , Yue Chen  1 , Rebecca A Boisvert  1 , Katharina Schlacher  1
Affiliations

Mitochondrial Replication Assay (MIRA) for Efficient in situ Quantification of Nascent mtDNA and Protein Interactions with Nascent mtDNA (mitoSIRF)

Macy Lozen et al. Bio Protoc. .

Abstract

Mitochondria play decisive roles in bioenergetics and intracellular communication. These organelles contain a circular mitochondrial DNA (mtDNA) genome that is duplicated within one to two hours by a mitochondrial replisome, independently from the nuclear replisome. mtDNA stability is regulated in part at the level of mtDNA replication. Consequently, mutations in mitochondrial replisome components result in mtDNA instability and are associated with diverse disease phenotypes, including premature aging, aberrant cellular energetics, and developmental defects. The mechanisms ensuring mtDNA replication stability are not completely understood. Thus, there remains a need to develop tools to specifically and quantifiably examine mtDNA replication. To date, methods for labeling mtDNA have relied on prolonged exposures of 5'-bromo-2'-deoxyuridine (BrdU) or 5'-ethynyl-2'-deoxyuridine (EdU). However, labeling with these nucleoside analogs for a sufficiently short time in order to monitor nascent mtDNA replication, such as under two hours, does not produce signals suited for efficient or accurate quantitative analysis. The assay system described here, termed Mitochondrial Replication Assay (MIRA), utilizes proximity ligation assay (PLA) combined with EdU-coupled Click-IT chemistry to address this limitation, thereby enabling sensitive and quantitative analysis of nascent in situ mtDNA replication with single-cell resolution. This method can be further paired with conventional immunofluorescence (IF) for multi-parameter cell analysis. By enabling monitoring nascent mtDNA prior to the complete replication of the entire mtDNA genome, this new assay system allowed the discovery of a new mitochondrial stability pathway, mtDNA fork protection. Moreover, a modification in primary antibodies application allows the adaptation of our previously described in situ protein Interactions with nascent DNA Replication Forks (SIRF) for the detection of proteins of interest to nascent mtDNA replication forks on a single molecule level (mitoSIRF). Graphical overview Schematic overview of Mitochondrial Replication Assay (MIRA). 5'-ethynyl-2'-deoxyuridine (EdU; green) incorporated in DNA is tagged with biotin (blue) using Click-IT chemistry. Subsequent proximity ligation assay (PLA, pink circles) using antibodies against biotin allows the fluorescent tagging of the nascent EdU and amplification of the signal sufficient for visualization by standard immunofluorescence. PLA signals outside the nucleus denote mitochondrial DNA (mtDNA) signals. Ab, antibody. In in situ protein interactions with nascent DNA replication forks (mitoSIRF), one of the primary antibodies is directed against a protein of interest, while the other detects nascent biotinylated EdU, thus enabling in situ protein interactions with nascent mtDNA.

Keywords: BRCA2; Fanconi anemia; MIRA; Mitochondria; Mitochondrial DNA; Proximity ligation assay; mtDNA instability; mtDNA replication.

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Conflict of interest statement

Competing interestsThe authors have no competing interests.

Figures

Figure 1.
Figure 1.. Silicone gasket removal. Removal of silicone gasket using fine curved forceps.
Take caution to avoid scratching slide with the forceps.
Figure 2.
Figure 2.. Humidified slide chamber.
A. Preparation of a humidified chamber by placing folded paper towels or Kimwipes wetted with distilled water or PBS in an empty slide box. B. Slides are laid flat, facing up during incubations, and are carefully covered with plastic coverslips. Air bubbles can be avoided by applying the coverslip one end at a time.
Figure 3.
Figure 3.. Representative image to distinguish MIRA signals at various stages of the cell cycle and controls.
A. Example of MIRA in UWB1.289 cells treated with 20 μM EdU for 1 h. Note that the green nuclear signal for Alexa 488 azide co-click enables distinction of S-phase from non-S-phase cells [no green nuclear signal, DAPI signal (blue) only]. PLA signal (red) is detected inside the nucleus during S-phase and outside the nucleus in mitochondria during all cell cycle phases. Scale bars = 20 μm. B. Representative images of MIRA assay (red) in Rho zero SH2038 cells that are depleted of mtDNA as seen by PicoGreen DNA stain (green), with and without EdU as negative control. Scale bars = 10 μm.
See this image and copyright information in PMC

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