Background

Mitochondria, the organelles best known for their orchestration of cellular bioenergetics, operate semi-autonomously within eukaryotic cells and are key signaling hubs for intracellular communication (Friedman and Nunnari, 2014). Each mitochondrion harbors multiple copies of its ~16 kb circular genome, which encodes essential subunits of the electron transport chain as well as a set of transfer and ribosomal RNAs (Falkenberg et al., 2007). Mitochondrial DNA (mtDNA) is replicated separately from nuclear DNA (nDNA): the mitochondrial genome is replicated by its organelle-specific replisome, comprised of DNA polymerase POLγ and other nuclear-encoded proteins (Holt and Reyes, 2012; Gustafsson et al., 2016; Bailey and Doherty, 2017). mtDNA replication can occur at any cell cycle stage. Because of its proximity to events during oxidative phosphorylation, mtDNA is constantly exposed to reactive oxygen species, rendering it highly susceptible to oxidative DNA damage (Kang and Hamasaki, 2002). Failure to repair damage of the mitochondrial genome additionally results in instability of mtDNA and mitochondrial dysfunction, which is associated with premature aging, tumorigenesis, and neurological disorders (Trifunovic et al., 2004; Chatterjee et al., 2006; Ishikawa et al., 2008; Park and Larsson, 2011). Apart from mtDNA repair pathways, mtDNA stability is also regulated at the level of mtDNA replication with the exonuclease activity of POLγ polymerase and MGME1 (mitochondrial genome maintenance exonuclease 1) degrading damaged mtDNA and generating new mtDNA (Torregrosa-Munumer et al., 2019).

To date, detailed mechanisms of mtDNA replication linking to stability are incompletely understood. A comprehensive understanding of these mechanisms is imperative to allow the development of effective and specific agents targeting disease outcomes. For this purpose, efficient tools to specifically monitor and quantifiably measure nascent mtDNA replication are needed. mtDNA is replicated primarily by POLγ within one to two hours, yet methods for labeling mtDNA have historically relied on prolonged exposures (up to 24 h) of 5′-bromo-2′-deoxyuridine (BrdU) or 5′-ethynyl-2′-deoxyuridine (EdU) (Clayton, 1982; Korhonen et al., 2004). Incorporation of these nucleoside analogs for labeling times sufficiently short to monitor nascent mtDNA replication before completion of the entire mtDNA genome, e.g. less than two hours, does not produce signals suited for efficient quantitative analysis (Luzwick et al., 2021). The assay system described here, termed Mitochondrial replication assay (MIRA), utilizes proximity ligation assay (PLA) as a means of signal amplification of the mtDNA-incorporated nucleoside analogs to overcome this limitation. Specifically, mtDNA is labeled with the thymidine analog EdU, which is then biotinylated using Click-IT chemistry (Moses and Moorhouse, 2007). The PLA technology was developed by Soderberg et al. (2008) for single-molecule protein–protein interaction studies, utilizing antibodies with oligonucleotide conjugates that can be ligated into a circular DNA molecule when two antibodies are in close proximity to each other (<40 nm). Once ligated, the DNA circle can be amplified by rolling circle DNA polymerase. After annealing of fluorescent DNA probes that are complementary to the DNA circle, this procedure results in a highly amplified fluorescent signal, readily detectable by conventional immunofluorescence (IF) microscopy. In MIRA, PLA is adapted to signal-amplify the nascent mtDNA signals by utilizing complementary antibodies against biotin (detecting biotinylated EdU). As with conventional EdU and BrdU labeling, MIRA also detects and visualizes nDNA. Nonetheless, only cytoplasmic MIRA signals are considered for analysis, which are present irrespective of the cell cycle state and do not form without EdU or in mtDNA-depleted Rho zero cells (Luzwick et al., 2021). With amplification, the nascent mtDNA can be readily detected and quantified with a 1 h EdU pulse, which is less time than needed to replicate the entire genome. This assays system enables the monitorization of ongoing mtDNA replication prior to completion of mtDNA genome replication. It therefore differs from most common mtDNA label schemes, which use prolonged nucleoside analog incorporation for extended time over multiple hours. Since the mtDNA is replicated within one to two hours, prolonged labeling will predominantly result in post-replicative signals. Moreover, as an adaptation from our single-cell assay for in situ protein Interactions with nascent DNA Replication Forks (SIRF) protocol (Roy et al., 2018; Roy and Schlacher, 2019), MIRA can be combined with a primary antibody against a protein of interest to detect protein mtDNA interactions (mitoSIRF). The MIRA and mitoSIRF assay systems allowed the discovery of a new mitochondrial stability pathway, mtDNA fork protection (Luzwick et al., 2021). Specifically, BRCA and FANC tumor-suppressor proteins protect nascent mtDNA from instability during ongoing mtDNA replication; this would not have been detectable by previous assay systems that visualize post-replicative events. The instability is caused by MRE11-dependent nuclease degradation, which results in cGAS-mediated inflammatory signaling (Luzwick et al., 2021).

The MIRA method requires a minimal number of cells (~100–1,000), preserves the single-cell resolution as seen with IF, which provides valuable information regarding cell morphology, and can be readily and accurately quantified. The MIRA assay can furthermore be combined with conventional IF for multi-parameter biomarker analysis, including cell cycle stage or cell identity. If combined with other biomarkers, primary and secondary antibody staining for conventional IF is performed following the Click-IT and PLA reaction to avoid interference of PLA secondary antibodies from cross-reacting with IF primary antibodies.