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Visualizing, quantifying, and manipulating mitochondrial DNA in vivo

Open AccessPublished:December 18, 2020DOI:https://doi.org/10.1074/jbc.REV120.015101
      Mitochondrial DNA (mtDNA) encodes proteins and RNAs that support the functions of mitochondria and thereby numerous physiological processes. Mutations of mtDNA can cause mitochondrial diseases and are implicated in aging. The mtDNA within cells is organized into nucleoids within the mitochondrial matrix, but how mtDNA nucleoids are formed and regulated within cells remains incompletely resolved. Visualization of mtDNA within cells is a powerful means by which mechanistic insight can be gained. Manipulation of the amount and sequence of mtDNA within cells is important experimentally and for developing therapeutic interventions to treat mitochondrial disease. This review details recent developments and opportunities for improvements in the experimental tools and techniques that can be used to visualize, quantify, and manipulate the properties of mtDNA within cells.
      Mitochondrial DNA (mtDNA) encodes a variety of proteins, peptides, transfer RNAs, and ribosomal RNAs that support the functions of mitochondria and is thereby central to numerous physiological and pathophysiological processes, including development, disease, and aging (
      • Park C.B.
      • Larsson N.G.
      Mitochondrial DNA mutations in disease and aging.
      ). Both the number of mtDNAs in a cell (mtDNA copy number) and the sequences of mtDNAs are important phenotypic determinants (
      • Aryaman J.
      • Bowles C.
      • Jones N.S.
      • Johnston I.G.
      Mitochondrial network state scales mtDNA genetic dynamics.
      ). Mutations of mtDNA can cause a spectrum of mitochondrial diseases where the clinical expression depends on the specific mutation and the degree of heteroplasmy (the proportion of mutated mtDNA in a single cell) between WT and mutant mtDNA (
      • Stewart J.B.
      • Chinnery P.F.
      The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease.
      ,
      • Hahn A.
      • Zuryn S.
      The cellular mitochondrial genome landscape in disease.
      ). The copy number of mtDNA is controlled by poorly understood mechanisms but varies between tissues, during aging, and in cancer (
      • Wachsmuth M.
      • Hubner A.
      • Li M.
      • Madea B.
      • Stoneking M.
      Age-related and heteroplasmy-related variation in human mtDNA copy number.
      ,
      • Chen J.
      • Zheng Q.
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      • Rosenberg A.Z.
      • Levi M.
      • Wang X.X.
      • Ozbek B.
      • Baena-Del Valle J.
      • Yegnasubramanian S.
      • De Marzo A.M.
      An in situ atlas of mitochondrial DNA in mammalian tissues reveals high content in stem/progenitor cells.
      ,
      • Yuan Y.
      • Ju Y.S.
      • Kim Y.
      • Li J.
      • Wang Y.
      • Yoon C.J.
      • Yang Y.
      • Martincorena I.
      • Creighton C.J.
      • Weinstein J.N.
      • Xu Y.
      • Han L.
      • Kim H.L.
      • Nakagawa H.
      • Park K.
      • et al.
      Comprehensive molecular characterization of mitochondrial genomes in human cancers.
      ). The mtDNA within mitochondria is present within nucleoids (
      • Gilkerson R.
      • Bravo L.
      • Garcia I.
      • Gaytan N.
      • Herrera A.
      • Maldonado A.
      • Quintanilla B.
      The mitochondrial nucleoid: integrating mitochondrial DNA into cellular homeostasis.
      ). Nucleoids contain complexes of mtDNA with proteins and other factors that comprise the machinery required for regulated transcription (
      • Gilkerson R.
      • Bravo L.
      • Garcia I.
      • Gaytan N.
      • Herrera A.
      • Maldonado A.
      • Quintanilla B.
      The mitochondrial nucleoid: integrating mitochondrial DNA into cellular homeostasis.
      ,
      • Garrido N.
      • Griparic L.
      • Jokitalo E.
      • Wartiovaara J.
      • van der Bliek A.M.
      • Spelbrink J.N.
      Composition and dynamics of human mitochondrial nucleoids.
      ,
      • Legros F.
      • Malka F.
      • Frachon P.
      • Lombès A.
      • Rojo M.
      Organization and dynamics of human mitochondrial DNA.
      ). The abundance and sequence of mtDNA can affect mitochondrial function, whereas the mitochondrial network, which is regulated by dynamic fission and fusion events (
      • Giacomello M.
      • Pyakurel A.
      • Glytsou C.
      • Scorrano L.
      The cell biology of mitochondrial membrane dynamics.
      ), can impact the turnover and copy number of mtDNA (
      • Aryaman J.
      • Bowles C.
      • Jones N.S.
      • Johnston I.G.
      Mitochondrial network state scales mtDNA genetic dynamics.
      ,
      • Lieber T.
      • Jeedigunta S.P.
      • Palozzi J.M.
      • Lehmann R.
      • Hurd T.R.
      Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline.
      ).
      Single-cell studies have shown that mtDNA content and heteroplasmy are dynamic throughout life, with marked heterogeneity (
      • Stewart J.B.
      • Chinnery P.F.
      The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease.
      ,
      • Lawless C.
      • Greaves L.
      • Reeve A.K.
      • Turnbull D.M.
      • Vincent A.E.
      The rise and rise of mitochondrial DNA mutations.
      ,
      • van den Ameele J.
      • Li A.Y.Z.
      • Ma H.
      • Chinnery P.F.
      Mitochondrial heteroplasmy beyond the oocyte bottleneck.
      ,
      • Burgstaller J.P.
      • Kolbe T.
      • Havlicek V.
      • Hembach S.
      • Poulton J.
      • Piálek J.
      • Steinborn R.
      • Rülicke T.
      • Brem G.
      • Jones N.S.
      • Johnston I.G.
      Large-scale genetic analysis reveals mammalian mtDNA heteroplasmy dynamics and variance increase through lifetimes and generations.
      ). Although vegetative segregation and relaxed replication of mtDNA appear to be important, it remains unclear how and when these processes are involved in different tissues because most methods for quantifying mtDNA variants are in vitro biochemical assays that are destructive to cells and preclude measurements of mtDNA over time (
      • Stewart J.B.
      • Chinnery P.F.
      The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease.
      ,
      • Lawless C.
      • Greaves L.
      • Reeve A.K.
      • Turnbull D.M.
      • Vincent A.E.
      The rise and rise of mitochondrial DNA mutations.
      ,
      • Longchamps R.J.
      • Castellani C.A.
      • Yang S.Y.
      • Newcomb C.E.
      • Sumpter J.A.
      • Lane J.
      • Grove M.L.
      • Guallar E.
      • Pankratz N.
      • Taylor K.D.
      • Rotter J.I.
      • Boerwinkle E.
      • Arking D.E.
      Evaluation of mitochondrial DNA copy number estimation techniques.
      ). Direct visualization of mtDNA in vivo can thus offer further mechanistic insight. Visualization of the mtDNA copy number has revealed that mtDNA increases its population during S-phase in the cell cycle (
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
      ), that mtDNA copy number differs between tissues and can decline during aging (
      • Chen J.
      • Zheng Q.
      • Peiffer L.B.
      • Hicks J.L.
      • Haffner M.C.
      • Rosenberg A.Z.
      • Levi M.
      • Wang X.X.
      • Ozbek B.
      • Baena-Del Valle J.
      • Yegnasubramanian S.
      • De Marzo A.M.
      An in situ atlas of mitochondrial DNA in mammalian tissues reveals high content in stem/progenitor cells.
      ), and that mtDNA copy number is reduced in some cancers such as glioma (
      • Shen H.
      • Yu M.
      • Tsoli M.
      • Chang C.
      • Joshi S.
      • Liu J.
      • Ryall S.
      • Chornenkyy Y.
      • Siddaway R.
      • Hawkins C.
      • Ziegler D.S.
      Targeting reduced mitochondrial DNA quantity as a therapeutic approach in pediatric high-grade gliomas.
      ). Visualization of mtDNA in yeast has shown that segregation of mtDNA during cell division preserves the density of mtDNA in daughter cells, in part via the semi-regular spacing of nucleoids within mitochondria (
      • Jajoo R.
      • Jung Y.
      • Huh D.
      • Viana M.P.
      • Rafelski S.M.
      • Springer M.
      • Paulsson J.
      Accurate concentration control of mitochondria and nucleoids.
      ,
      • Aretz I.
      • Jakubke C.
      • Osman C.
      Power to the daughters—mitochondrial and mtDNA transmission during cell division.
      ). Visualization of replicating mtDNA nucleoids has revealed that they coincide with endoplasmic reticulum–mitochondria contact sites, mitochondrial fission, and actin (
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      ,
      • Friedman J.R.
      • Lackner L.L.
      • West M.
      • DiBenedetto J.R.
      • Nunnari J.
      • Voeltz G.K.
      ER tubules mark sites of mitochondrial division.
      ,
      • Ban-Ishihara R.
      • Ishihara T.
      • Sasaki N.
      • Mihara K.
      • Ishihara N.
      Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c.
      ). High-resolution and superresolution microscopy (SRM) imaging has revealed that there are relatively small numbers of mtDNAs per nucleoid (mean ∼1.4, and often only one), that nucleoids have a relatively uniform size of ∼100-nm diameter (
      • Ban-Ishihara R.
      • Ishihara T.
      • Sasaki N.
      • Mihara K.
      • Ishihara N.
      Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c.
      ,
      • Brown T.A.
      • Tkachuk A.N.
      • Shtengel G.
      • Kopek B.G.
      • Bogenhagen D.F.
      • Hess H.F.
      • Clayton D.A.
      Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.
      ,
      • Kukat C.
      • Wurm C.A.
      • Spåhr H.
      • Falkenberg M.
      • Larsson N.G.
      • Jakobs S.
      Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.
      ), that there are relatively small numbers (∼1–15) of nucleoids per mitochondrion (
      • Satoh M.
      • Kuroiwa T.
      Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell.
      ), and that mtDNA resides in voids between mitochondrial cristae (
      • Stephan T.
      • Roesch A.
      • Riedel D.
      • Jakobs S.
      Live-cell STED nanoscopy of mitochondrial cristae.
      ). Fluorescence in situ hybridization has shown (in a manner consistent with the low number of mtDNAs per nucleoid) that individual mtDNA nucleoids maintain their genetic autonomy rather than freely exchanging mtDNA between nucleoids (
      • Gilkerson R.W.
      • Schon E.A.
      • Hernandez E.
      • Davidson M.M.
      Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation.
      ) and that removal of deleterious mutant mtDNA from the germline may occur after mitochondrial fragmentation (
      • Lieber T.
      • Jeedigunta S.P.
      • Palozzi J.M.
      • Lehmann R.
      • Hurd T.R.
      Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline.
      ).
      Despite considerable advances in our understanding of mtDNA biology, fundamental questions remain, such as how mtDNA nucleoids are formed and distributed within cells, how mtDNA copy number is controlled, and how mtDNA heteroplasmy is determined in different cells and tissues.
      This review aims to assemble the existing suite of experimental tools and techniques that can be used to visualize, quantify, and manipulate mtDNA within cells; it places a particular emphasis on visualization. In the first section, we discuss methods for labeling mtDNA nucleoids in cells. The next section provides details of imaging methods for visualizing mtDNA in cells. Next, we discuss the manipulation of mtDNA in cells. Finally, we discuss some of the future challenges and new approaches in the field that may enable a greater understanding of the roles and regulation of mtDNA in cells. Tools used to probe more general mitochondrial physiology are reviewed elsewhere (
      • Glancy B.
      Visualizing mitochondrial form and function within the cell.
      ,
      • Gökerküçük E.B.
      • Tramier M.
      • Bertolin G.
      Imaging mitochondrial functions: from fluorescent dyes to genetically-encoded sensors.
      ).

      Labeling mtDNA nucleoids in cells

      Desirable properties for tools to label and visualize mtDNA

      The experimental tools and techniques that can currently be used to label, visualize, and quantitatively describe the characteristics of mtDNA include those summarized in Table 1. The ideal tool for labeling and visualizing mtDNA would enable the most challenging experimental approaches to investigate mtDNA physiology. These include long-term time-lapse microscopy to monitor mtDNA throughout the life of a cell or organism, superresolution microscopy to determine the architecture of nucleoids and their relationship to mitochondria, and selective visualization of different variants of mtDNA within cells and tissues to reveal the dynamics of each mtDNA variant and their effects on the mitochondria and cells in which they reside. To achieve these aims, the tools for labeling mtDNA would have the following nine challenging but desirable properties. 1) It should selectively label mtDNA rather than nuclear DNA, in both live and fixed cells. 2) It should be nontoxic and nonperturbing, thus allowing visualization over time. 3) It should be photostable for extended periods of video imaging and particle tracking. 4) It should be flexible with respect to spectral characteristics, to enable multicolor imaging with other labels directed to other targets and to enable pulse-chase experiments. 5) It should also be flexible with respect to binding affinity for mtDNA, so that stable or reversible binding can be employed. 6) It should be capable of specifically detecting replicating mtDNA. 7) It should be compatible with SRM to achieve images with the highest spatial resolution. 8) It should be applicable to intravital imaging of tissues and organisms in vivo. 9) It should label sequence variants of mtDNA selectively, including single-nucleotide variants, to help understand the pathophysiology of mtDNA heteroplasmy. All of the tools that are currently available have limitations with respect to these desirable properties (Table 1).
      Table 1Tools and techniques for visualizing mtDNA in cells
      Tool/TechniqueAdvantagesDisadvantagesReferences
      Microscopy techniques
       Confocal microscopyEase, speed, 3DResolution restricted to optical diffraction limit
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
       Superresolution microscopyHigh resolutionLow number of colors possible (one to three); sometimes restricted modality (e.g. TIRFM)
      • Brown T.A.
      • Tkachuk A.N.
      • Shtengel G.
      • Kopek B.G.
      • Bogenhagen D.F.
      • Hess H.F.
      • Clayton D.A.
      Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.
      ,
      • Kukat C.
      • Wurm C.A.
      • Spåhr H.
      • Falkenberg M.
      • Larsson N.G.
      • Jakobs S.
      Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.
      ,
      • Stephan T.
      • Roesch A.
      • Riedel D.
      • Jakobs S.
      Live-cell STED nanoscopy of mitochondrial cristae.
       Light-sheet microscopySpeed for large samples, low phototoxicityCost of hardware; not maximum resolution
      • McArthur K.
      • Whitehead L.W.
      • Heddleston J.M.
      • Li L.
      • Padman B.S.
      • Oorschot V.
      • Geoghegan N.D.
      • Chappaz S.
      • Davidson S.
      • San Chin H.
      • Lane R.M.
      • Dramicanin M.
      • Saunders T.L.
      • Sugiana C.
      • Lessene R.
      • et al.
      BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.
       EMHighest resolutionFixation, heavy metal staining, and sectioning may be necessary
      • McArthur K.
      • Whitehead L.W.
      • Heddleston J.M.
      • Li L.
      • Padman B.S.
      • Oorschot V.
      • Geoghegan N.D.
      • Chappaz S.
      • Davidson S.
      • San Chin H.
      • Lane R.M.
      • Dramicanin M.
      • Saunders T.L.
      • Sugiana C.
      • Lessene R.
      • et al.
      BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.
       CryotomographyHighest resolution; no fixation, staining or sectioning requiredRapid freezing required
       CLEMCombination of EM and fluorescence enables correlation of proteins and structuresExpense, technical expertise, laborious
      • McArthur K.
      • Whitehead L.W.
      • Heddleston J.M.
      • Li L.
      • Padman B.S.
      • Oorschot V.
      • Geoghegan N.D.
      • Chappaz S.
      • Davidson S.
      • San Chin H.
      • Lane R.M.
      • Dramicanin M.
      • Saunders T.L.
      • Sugiana C.
      • Lessene R.
      • et al.
      BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.
      ,
      • Kopek B.G.
      • Shtengel G.
      • Xu C.S.
      • Clayton D.A.
      • Hess H.F.
      Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes.
      ,
      • Okayama S.
      • Ohta K.
      • Higashi R.
      • Nakamura K.
      Correlative light and electron microscopic observation of mitochondrial DNA in mammalian cells by using focused-ion beam scanning electron microscopy.
      DNA-binding dyes
       SYBR GreenLive cellsFixed λ, fixation, and permeabilization disrupt labeling, toxic in some cells, bleaches relatively rapidly
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
       SYBR GoldLive cellsFixed λ
      • Jevtic V.
      • Kindle P.
      • Avilov S.V.
      SYBR Gold dye enables preferential labelling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy.
       PicoGreenLive cellsFixed λ, fixation, and permeabilization disrupt labeling, toxic in some cells, bleaches relatively rapidly
      • Ashley N.
      • Harris D.
      • Poulton J.
      Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining.
       EtBrLive cells in some casesFixed UV λ, inhibits mtDNA replication, low membrane permeability, toxic, may require fixation and permeabilization
      • Villa A.M.
      • Doglia S.M.
      Ethidium bromide as a vital probe of mitochondrial DNA in carcinoma cells.
       DAPILive cellsFixed UV λ
      • Dellinger M.
      • Gèze M.
      Detection of mitochondrial DNA in living animal cells with fluorescence microscopy.
       SiR-HoechstLive cells, far-redFixed λ
      • Lukinavičius G.
      • Blaukopf C.
      • Pershagen E.
      • Schena A.
      • Reymond L.
      • Derivery E.
      • Gonzalez-Gaitan M.
      • D'Este E.
      • Hell S.W.
      • Wolfram Gerlich D.
      • Johnsson K.
      SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy.
      Nucleotide analogues
       EdU-clickVariable λFixation and permeabilization necessary
      • Davis A.F.
      • Clayton D.A.
      In situ localization of mitochondrial DNA replication in intact mammalian cells.
       EdU-click with amplificationVariable λ

      Increased signal/noise ratio
      Fixation and permeabilization necessary
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
       BrdUVariable λFixation and permeabilization necessary, harsh denaturing step
      • Ligasová A.
      • Strunin D.
      • Liboska R.
      • Rosenberg I.
      • Koberna K.
      Atomic scissors: a new method of tracking the 5-bromo-2′-deoxyuridine-labeled DNA in situ.
      Fluorescence-tagged proteins
       TFAM-FPVariable λPossible perturbing effect of FP tag, overexpression artifacts
      • Alam T.I.
      • Kanki T.
      • Muta T.
      • Ukaji K.
      • Abe Y.
      • Nakayama H.
      • Takio K.
      • Hamasaki N.
      • Kang D.
      Human mitochondrial DNA is packaged with TFAM.
       POLG2-FPVariable λ, selective for replicating mtDNAPossible perturbing effect of FP tag, overexpression artifacts
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      ,
      • Young M.J.
      • Humble M.M.
      • DeBalsi K.L.
      • Sun K.Y.
      • Copeland W.C.
      POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity.
      Antibodies
       Ab-TFAMVariable λFixation and permeabilization necessary
      • Alam T.I.
      • Kanki T.
      • Muta T.
      • Ukaji K.
      • Abe Y.
      • Nakayama H.
      • Takio K.
      • Hamasaki N.
      • Kang D.
      Human mitochondrial DNA is packaged with TFAM.
       Ab-DNAVariable λFixation and permeabilization necessary
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      In situ hybridization
       FISHVariable λ, sequence-specificFixation and permeabilization necessary
      • Lieber T.
      • Jeedigunta S.P.
      • Palozzi J.M.
      • Lehmann R.
      • Hurd T.R.
      Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline.
      ,
      • Gilkerson R.W.
      • Schon E.A.
      • Hernandez E.
      • Davidson M.M.
      Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation.
       Padlock probesVariable λ, sequence-specificFixation and permeabilization necessary
      • Larsson C.
      • Koch J.
      • Nygren A.
      • Janssen G.
      • Raap A.K.
      • Landegren U.
      • Nilsson M.
      In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes.
      ,
      • Yaroslavsky A.I.
      • Smolina I.V.
      Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly.
      Targeted nucleases
       CasPLAVariable λ, sequence-specificFixation and permeabilization required, few NGG PAM sites in mtDNA
      • Zhang K.
      • Deng R.
      • Teng X.
      • Li Y.
      • Sun Y.
      • Ren X.
      • Li J.
      Direct visualization of single-nucleotide variation in mtDNA using a CRISPR/Cas9-mediated proximity ligation assay.

      Fluorescent DNA-binding dyes

      A wide variety of DNA-binding dyes exist that can label DNA in cells (
      • Lakadamyali M.
      • Cosma M.P.
      Advanced microscopy methods for visualizing chromatin structure.
      ,
      • Kaur G.
      • Lewis J.S.
      • van Oijen A.M.
      Shining a spotlight on DNA: single-molecule methods to visualise DNA.
      ), but these have been used mainly to label nuclear DNA. Only a few of these have been demonstrated to label mtDNA within cells. These include SYBR Green I (
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
      ,
      • Ban-Ishihara R.
      • Ishihara T.
      • Sasaki N.
      • Mihara K.
      • Ishihara N.
      Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c.
      ), SYBR Gold (
      • Jevtic V.
      • Kindle P.
      • Avilov S.V.
      SYBR Gold dye enables preferential labelling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy.
      ), PicoGreen (
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      ,
      • Ashley N.
      • Harris D.
      • Poulton J.
      Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining.
      ,
      • He J.
      • Mao C.C.
      • Reyes A.
      • Sembongi H.
      • Di Re M.
      • Granycome C.
      • Clippingdale A.B.
      • Fearnley I.M.
      • Harbour M.
      • Robinson A.J.
      • Reichelt S.
      • Spelbrink J.N.
      • Walker J.E.
      • Holt I.J.
      The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization.
      ,
      • Benke A.
      • Manley S.
      Live-cell dSTORM of cellular DNA based on direct DNA labeling.
      ), DAPI (
      • Dellinger M.
      • Gèze M.
      Detection of mitochondrial DNA in living animal cells with fluorescence microscopy.
      ), and ethidium bromide (
      • Villa A.M.
      • Doglia S.M.
      Ethidium bromide as a vital probe of mitochondrial DNA in carcinoma cells.
      ). Red fluorescent DNA-binding dyes that label mtDNA have been described recently (
      • Uno K.
      • Sugimoto N.
      • Sato Y.
      N-Aryl pyrido cyanine derivatives: nuclear and organelle DNA markers for two-photon and super-resolution imaging.
      ). DNA-binding organic dyes can be relatively bright and photostable and can bind mtDNA stably over periods of days within cells (
      • Jevtic V.
      • Kindle P.
      • Avilov S.V.
      SYBR Gold dye enables preferential labelling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy.
      ). An example of live mammalian cells in which mtDNA nucleoids have been labeled with SYBR Green is shown in Fig. 1 (
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
      ).
      Figure thumbnail gr1
      Figure 1Labeling mtDNA with SYBR Green I. Shown is staining of HeLa cells with different concentrations of SYBR Green I. Dilutions of SYBR Green I are shown above the images. DNA was immunostained using anti-DNA antibodies in cells without staining with SYBR Green I (far right panel). Scale bars, 10 μm. The images are taken from previously published data (
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
      ); the figure panel has not been changed; and the images are covered by a license (http://creativecommons.org/licenses/by/4.0/).
      DNA-binding dyes must traverse the plasma membrane and mitochondrial outer and inner membranes in live cells to gain access to mtDNA in the mitochondrial matrix. The physicochemical properties of dyes have therefore been used to rationalize the choice of dyes that might accumulate in mitochondria and bind to mtDNA, for example yielding SYBR Gold as a suitable dye (
      • Jevtic V.
      • Kindle P.
      • Avilov S.V.
      SYBR Gold dye enables preferential labelling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy.
      ). Relevant properties of the dye include delocalized positive charge to favor uptake across the negative membrane potential of the mitochondrial membrane; lipophilicity, to penetrate the plasma membrane and mitochondrial membranes; high-affinity binding to DNA; and preferably fluorogenicity (i.e. a fluorescence enhancement upon binding to mtDNA).
      A DNA-binding dye consisting of SYBR Green linked to a cationic rhodamine-B moiety to enable mitochondrial targeting yields green fluorescence in mitochondria but red fluorescence in lysosomes after mitophagy (
      • Zou X.
      • Shi Y.
      • Zhu R.
      • Han J.
      • Han S.
      Organelle-redirected chameleon sensor-enabled live cell imaging of mitochondrial DNA.
      ). This type of DNA-binding dye/sensor may have utility in revealing mechanisms involved in mtDNA turnover during mitophagy. A ruthenium(II)-peptide conjugate is reported to target mtDNA in live cells and enable the induction of targeted phototoxicity in selected cells (
      • Burke C.S.
      • Byrne A.
      • Keyes T.E.
      Highly selective mitochondrial targeting by a ruthenium(II) peptide conjugate: imaging and photoinduced damage of mitochondrial DNA.
      ). The mtDNA can be a target of oxidative attack by hydrogen peroxide and reactive oxygen species. To study this process, a fluorescent mtDNA-tethered peroxide sensor has been reported, comprised of a DNA-binding peptide coupled to a green fluorescent peroxide sensor and a charged red fluorescent dye (
      • Wen Y.
      • Liu K.
      • Yang H.
      • Liu Y.
      • Chen L.
      • Liu Z.
      • Huang C.
      • Yi T.
      Mitochondria-directed fluorescent probe for the detection of hydrogen peroxide near mitochondrial DNA.
      ).
      The existing organic mtDNA-binding dyes are limited to certain wavelengths of excitation and emission light. SYBR Green, SYBR Gold, and PicoGreen all emit green fluorescence and require illumination in the 488-nm region. EtBr and DAPI require shorter-wavelength illumination in the UV region, which can be biologically damaging. Red fluorescent DNA-binding dyes that label mtDNA have been described recently (
      • Uno K.
      • Sugimoto N.
      • Sato Y.
      N-Aryl pyrido cyanine derivatives: nuclear and organelle DNA markers for two-photon and super-resolution imaging.
      ). The lower toxicity of longer-wavelength illumination, together with the expanded spectral flexibility these red dyes provide, may be useful for time-lapse imaging and combination with other fluorescent labels (e.g. to identify replicating mtDNA and total mtDNA simultaneously). They may also be suitable for SRM to determine the architecture of nucleoids (
      • Uno K.
      • Sugimoto N.
      • Sato Y.
      N-Aryl pyrido cyanine derivatives: nuclear and organelle DNA markers for two-photon and super-resolution imaging.
      ). Other DNA-binding dyes based on Hoechst (
      • Lukinavičius G.
      • Blaukopf C.
      • Pershagen E.
      • Schena A.
      • Reymond L.
      • Derivery E.
      • Gonzalez-Gaitan M.
      • D'Este E.
      • Hell S.W.
      • Wolfram Gerlich D.
      • Johnsson K.
      SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy.
      ) or rhodamine derivatives (
      • Wang L.
      • Tran M.
      • D'Este E.
      • Roberti J.
      • Koch B.
      • Xue L.
      • Johnsson K.
      A general strategy to develop cell permeable and fluorogenic probes for multicolour nanoscopy.
      ), that are red fluorescent or far-red fluorescent, have been described recently, but it remains to be determined whether these dyes can be used for visualizing mtDNA.
      In addition to labeling mtDNA with a fluorescent DNA-binding dye, it is often experimentally desirable to label other cellular components with antibodies or membrane-impermeant probes, which necessitates fixation and permeabilization of cells. Fixation with paraformaldehyde and detergent permeabilization can reduce staining of cellular mtDNA with DNA-binding dyes (
      • Jevtic V.
      • Kindle P.
      • Avilov S.V.
      SYBR Gold dye enables preferential labelling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy.
      ). Another limitation of current DNA-binding dyes for visualizing mtDNA is their binding to nuclear DNA within the same cells. Extreme dilution of the DNA-binding dye has been demonstrated to minimize this issue by aiding the selective labeling of mitochondrial nucleoids by SYBR Gold (
      • Jevtic V.
      • Kindle P.
      • Avilov S.V.
      SYBR Gold dye enables preferential labelling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy.
      ) and SYBR Green (
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
      ) for reasons that remain unclear. DNA-binding dyes also intercalate between the two strands of dsDNA. This may alter the ability of DNA strands to dissociate during replication of both nuclear DNA and mtDNA and may thereby alter cell division and be toxic in some cell types.

      Fluorescent nucleotide analogues

      5-bromo-2-deoxyuridine (BrdU) is a nucleotide analog that can incorporate into replicating mtDNA (
      • Kukat C.
      • Wurm C.A.
      • Spåhr H.
      • Falkenberg M.
      • Larsson N.G.
      • Jakobs S.
      Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.
      ,
      • Ligasová A.
      • Strunin D.
      • Liboska R.
      • Rosenberg I.
      • Koberna K.
      Atomic scissors: a new method of tracking the 5-bromo-2′-deoxyuridine-labeled DNA in situ.
      ). Once incorporated into DNA, the BrdU is inaccessible to antibodies and fluorescent labeling until relatively harsh chemical or enzymatic cleavage of the DNA is used to uncover the BrdU epitope and facilitate binding of labeled antibodies (
      • Ligasová A.
      • Strunin D.
      • Liboska R.
      • Rosenberg I.
      • Koberna K.
      Atomic scissors: a new method of tracking the 5-bromo-2′-deoxyuridine-labeled DNA in situ.
      ).
      5-Ethynyl-2′-deoxyuridine (EdU) is another nucleotide analog that can incorporate specifically into replicating mtDNA (
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
      ,
      • Davis A.F.
      • Clayton D.A.
      In situ localization of mitochondrial DNA replication in intact mammalian cells.
      ,
      • Iborra F.J.
      • Kimura H.
      • Cook P.R.
      The functional organization of mitochondrial genomes in human cells.
      ). EdU can be fluorescently labeled using a copper(I)-catalyzed click reaction to form a covalent attachment between the alkyne group of EdU and an azide group on an appropriately conjugated fluorescent dye. Labeling of EdU does not require the denaturation step involved in BrdU labeling, and EdU can be visualized directly (e.g. using an azide-Alexa Fluor label) (Fig. 2) (
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      ). Alternatively, a signal amplification step can increase signal/noise ratio by using a fluorescence-conjugated antibody directed against the click-conjugated fluorescent dye to introduce further fluorescent dye molecules (
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
      ,
      • Davis A.F.
      • Clayton D.A.
      In situ localization of mitochondrial DNA replication in intact mammalian cells.
      ,
      • Iborra F.J.
      • Kimura H.
      • Cook P.R.
      The functional organization of mitochondrial genomes in human cells.
      ). For example, an antibody against Oregon Green 488 can amplify the signal from EdU labeled with Oregon Green 488-azide (
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
      ).
      Figure thumbnail gr2
      Figure 2Labeling replicating mtDNA with EdU and POLG2-GFP. Shown is a representative U2OS cell expressing POLG2-GFP and labeled with EdU (50 μm), fixed and stained with DAPI (DNA, blue), MitoTracker (mitochondria, red), anti-GFP–Alexa Fluor 488 conjugate antibody (POLG2-GFP, green), and Click-iT EdU-Alexa Fluor 647 (nascent DNA, magenta). Arrowheads, colocalization. Scale bar, 10 μm. The images are reproduced from previously published data (
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      ), reprinted with permission from the American Association for the Advancement of Science.
      A wide variety of azide-conjugated fluorescent dyes exist, and the resulting spectral flexibility provides an advantage of EdU and BrdU techniques over currently available organic DNA-binding dyes. Furthermore, a combination of BrdU and EdU can be utilized in sequential pulse-chase experiments, to monitor mtDNA replication (
      • Lentz S.I.
      • Edwards J.L.
      • Backus C.
      • McLean L.L.
      • Haines K.M.
      • Feldman E.L.
      Mitochondrial DNA (mtDNA) biogenesis: visualization and duel incorporation of BrdU and EdU into newly synthesized mtDNA in vitro.
      ).
      BrdU and EdU incorporate into replicating strands of both nuclear DNA and mtDNA. Specific visualization of mtDNA using these nucleotide analogues may, therefore, require pharmacological inhibition of nuclear DNA replication by drugs, such as aphidicolin (
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
      ), or restricted use in postmitotic or quiescent cells. Other limitations are the incorporation of these nucleotides during repair of DNA and the reported cytotoxicity of EdU in some cells (
      • Diermeier-Daucher S.
      • Clarke S.T.
      • Hill D.
      • Vollmann-Zwerenz A.
      • Bradford J.A.
      • Brockhoff G.
      Cell type specific applicability of 5-ethynyl-2′-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry.
      ,
      • Kohlmeier F.
      • Maya-Mendoza A.
      • Jackson D.A.
      EdU induces DNA damage response and cell death in mESC in culture.
      ). Finally, labeling of incorporated EdU and BrdU for most applications requires fixation and permeabilization, as most of the reactive probes are membrane-impermeant; a strategy for labeling EdU and BrdU, or other similar probes, in live cells would be of substantial utility to detect replicating mtDNA. Labeling EdU with a membrane-permeant tetramethylrhodamine azide, together with copper(I) generated in situ, has been employed without fixation, but this use has not been developed significantly, in part due to toxicity of the probes used (
      • Salic A.
      • Mitchison T.J.
      A chemical method for fast and sensitive detection of DNA synthesis in vivo.
      ).
      Halogenated thymidine analogues have also been used to label replicating mtDNA, in a technique termed mitochondrial single-molecule analysis of replicating DNA (
      • Phillips A.F.
      • Millet A.R.
      • Tigano M.
      • Dubois S.M.
      • Crimmins H.
      • Babin L.
      • Charpentier M.
      • Piganeau M.
      • Brunet E.
      • Sfeir A.
      Single-molecule analysis of mtDNA replication uncovers the basis of the common deletion.
      ). Relatively short pulses of incubation with the halogenated thymidine derivatives 5-iodo-2′-deoxyuridine or 5-chloro-2′-deoxyuridine can lead to incorporation of these analogues into replicating mtDNA within cells over defined time periods of time (
      • Phillips A.F.
      • Millet A.R.
      • Tigano M.
      • Dubois S.M.
      • Crimmins H.
      • Babin L.
      • Charpentier M.
      • Piganeau M.
      • Brunet E.
      • Sfeir A.
      Single-molecule analysis of mtDNA replication uncovers the basis of the common deletion.
      ). Specific antibodies directed against either 5-iodo-2′-deoxyuridine or 5-chloro-2′-deoxyuridine can then be used to visualize the locations of each incorporated analog within single mtDNA genomes, via SRM (
      • Phillips A.F.
      • Millet A.R.
      • Tigano M.
      • Dubois S.M.
      • Crimmins H.
      • Babin L.
      • Charpentier M.
      • Piganeau M.
      • Brunet E.
      • Sfeir A.
      Single-molecule analysis of mtDNA replication uncovers the basis of the common deletion.
      ). This technique has revealed mechanisms underlying the replication of mtDNA and the generation of common mtDNA deletion mutants (
      • Phillips A.F.
      • Millet A.R.
      • Tigano M.
      • Dubois S.M.
      • Crimmins H.
      • Babin L.
      • Charpentier M.
      • Piganeau M.
      • Brunet E.
      • Sfeir A.
      Single-molecule analysis of mtDNA replication uncovers the basis of the common deletion.
      ).

      Antibodies to DNA and proteins

      Antibodies raised against DNA can detect mtDNA in fixed, permeabilized cells (
      • Legros F.
      • Malka F.
      • Frachon P.
      • Lombès A.
      • Rojo M.
      Organization and dynamics of human mitochondrial DNA.
      ,
      • Ban-Ishihara R.
      • Ishihara T.
      • Sasaki N.
      • Mihara K.
      • Ishihara N.
      Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c.
      ,
      • Kukat C.
      • Wurm C.A.
      • Spåhr H.
      • Falkenberg M.
      • Larsson N.G.
      • Jakobs S.
      Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.
      ,
      • Iborra F.J.
      • Kimura H.
      • Cook P.R.
      The functional organization of mitochondrial genomes in human cells.
      ) (Fig. 3). These antibodies do not currently distinguish between nuclear DNA and mtDNA. Antibodies to protein constituents of mitochondrial nucleoids are commonly used to identify mtDNA. A critical component of the mtDNA transcription machinery, and a commonly used marker for nucleoids, is the mitochondrial transcription factor A (TFAM) protein (
      • Alam T.I.
      • Kanki T.
      • Muta T.
      • Ukaji K.
      • Abe Y.
      • Nakayama H.
      • Takio K.
      • Hamasaki N.
      • Kang D.
      Human mitochondrial DNA is packaged with TFAM.
      ,
      • Holt I.J.
      • He J.
      • Mao C.C.
      • Boyd-Kirkup J.D.
      • Martinsson P.
      • Sembongi H.
      • Reyes A.
      • Spelbrink J.N.
      Mammalian mitochondrial nucleoids: organizing an independently minded genome.
      ,
      • Kaufman B.A.
      • Durisic N.
      • Mativetsky J.M.
      • Costantino S.
      • Hancock M.A.
      • Grutter P.
      • Shoubridge E.A.
      The mitochondrial transcription factor TFAM coordinates the assembly of multiple DNA molecules into nucleoid-like structures.
      ). An example of fixed, permeabilized cells in which mtDNA nucleoids have been labeled with an antibody directed against TFAM is shown in Fig. 3 (
      • Kukat C.
      • Wurm C.A.
      • Spåhr H.
      • Falkenberg M.
      • Larsson N.G.
      • Jakobs S.
      Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.
      ). Other protein targets include DNA polymerase subunit γ (POLG) and the Twinkle helicase (
      • Garrido N.
      • Griparic L.
      • Jokitalo E.
      • Wartiovaara J.
      • van der Bliek A.M.
      • Spelbrink J.N.
      Composition and dynamics of human mitochondrial nucleoids.
      ). Protein constituents of nucleoids may be present at different levels, depending on the structure and functional state of the mtDNA. For example, POLG induces replication of mtDNA and thereby labels replicating mtDNA preferentially (
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      ), whereas methylation of mtDNA may reduce binding of TFAM (
      • Hao Z.
      • Wu T.
      • Cui X.
      • Zhu P.
      • Tan C.
      • Dou X.
      • Hsu K.W.
      • Lin Y.T.
      • Peng P.H.
      • Zhang L.S.
      • Gao Y.
      • Hu L.
      • Sun H.L.
      • Zhu A.
      • Liu J.
      • et al.
      N6-deoxyadenosine methylation in mammalian mitochondrial DNA.
      ).
      Figure thumbnail gr3
      Figure 3Labeling mtDNA with antibodies directed against DNA and TFAM. A, human fibroblasts showing mtDNA (green, DNA antibodies) localized in nucleoids in the mitochondrial network (red, anti-TOM20) with DAPI staining of nucleus in blue. B, confocal microscopy image of nucleoids labeled with antiserum against DNA. C, STED microscopy image of the same nucleoids shown in B. D, sizes of nucleoids labeled with a DNA antibody as measured using confocal and STED imaging. E, TFAM (green, anti-TFAM) located in nucleoids within the mitochondrial network (red, anti-TOM20) of human fibroblasts. Nuclear DAPI staining is shown in blue. F, confocal microscopy image of nucleoids labeled with antiserum against TFAM. G, STED microscopy image of the same nucleoids shown in F. H, quantification of sizes of nucleoids labeled with TFAM antibodies, determined by confocal and STED. Error bars, S.E. Scale bars, 20 μm in A and E and 0.5 μm in B, C, F, and G. The images and figure are reproduced from previously published data (
      • Kukat C.
      • Wurm C.A.
      • Spåhr H.
      • Falkenberg M.
      • Larsson N.G.
      • Jakobs S.
      Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.
      ), reprinted with permission from the United States National Academy of Sciences.

      Fluorescently tagged mtDNA-binding proteins

      TFAM tagged with a fluorescent protein is a commonly used marker of mitochondrial nucleoids (
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
      ,
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      ,
      • Jevtic V.
      • Kindle P.
      • Avilov S.V.
      SYBR Gold dye enables preferential labelling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy.
      ). Potential limitations of this method are that overexpression of exogenous TFAM can increase the number of nucleoids per cell (
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      ) and that fluorescent proteins may be less bright and photostable relative to some fluorescent dyes.
      A fluorescently tagged processivity subunit of POLG (POLG2-GFP) can be used to specifically label replicating mtDNA within cells (
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      ,
      • Young M.J.
      • Humble M.M.
      • DeBalsi K.L.
      • Sun K.Y.
      • Copeland W.C.
      POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity.
      ) (Fig. 2), and together with the simultaneous labeling of all mtDNA nucleoids, this can reveal subcellular heterogeneity in the replicative status of mtDNA (
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      ,
      • Young M.J.
      • Humble M.M.
      • DeBalsi K.L.
      • Sun K.Y.
      • Copeland W.C.
      POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity.
      ). Future advances in this field may include animal models or cell lines with endogenously tagged TFAM, POLG, and other proteins associated with mtDNA nucleoids, whereby overexpression artifacts are avoided.

      In situ hybridization probes

      In situ hybridization probes can detect mtDNA and have been used in many applications, including the comparison of mtDNA copy numbers in different tissues (
      • Chen J.
      • Zheng Q.
      • Peiffer L.B.
      • Hicks J.L.
      • Haffner M.C.
      • Rosenberg A.Z.
      • Levi M.
      • Wang X.X.
      • Ozbek B.
      • Baena-Del Valle J.
      • Yegnasubramanian S.
      • De Marzo A.M.
      An in situ atlas of mitochondrial DNA in mammalian tissues reveals high content in stem/progenitor cells.
      ), visualizing the removal of deleterious mtDNA from the germline after mitochondrial fragmentation (
      • Lieber T.
      • Jeedigunta S.P.
      • Palozzi J.M.
      • Lehmann R.
      • Hurd T.R.
      Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline.
      ), and visualization of mtDNA association with particular nucleoids and the resulting genetic autonomy of nucleoids (
      • Gilkerson R.W.
      • Schon E.A.
      • Hernandez E.
      • Davidson M.M.
      Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation.
      ).

      Labeling specific mtDNA variants in cells

      Many of the aforementioned methods for visualizing mtDNA do not reliably distinguish between sequence variants of mtDNA. Successful labeling of specific variants of mtDNA and heteroplasmy within single cells would greatly enhance the understanding of the mechanisms involved in heteroplasmic mitochondrial disease (e.g. by helping to resolve whether mtDNA variants are degraded or segregated differentially in cells and during cell division). Progress has been made toward this using several techniques, but each currently has drawbacks that limit their application. Only a single copy of any specific region of an mtDNA allele is present within each strand of the double-stranded mtDNA. This poses a challenge for achieving adequate signal/noise ratios for stoichiometric probes, and it may be advantageous to employ an amplification step to increase the signal/noise ratio. For in vitro assays of mtDNA variants, PCR-based amplification is often used, but this is prone to PCR-derived point mutations due to the imperfect fidelity of the DNA polymerases. Duplex sequencing can be used to control for the occurrence of these mutations and as a sensitive method to detect rare sequence variants of mtDNA in vitro (
      • Kennedy S.R.
      • Schmitt M.W.
      • Fox E.J.
      • Kohrn B.F.
      • Salk J.J.
      • Ahn E.H.
      • Prindle M.J.
      • Kuong K.J.
      • Shen J.C.
      • Risques R.A.
      • Loeb L.A.
      Detecting ultralow-frequency mutations by duplex sequencing.
      ,
      • Schmitt M.W.
      • Kennedy S.R.
      • Salk J.J.
      • Fox E.J.
      • Hiatt J.B.
      • Loeb L.A.
      Detection of ultra-rare mutations by next-generation sequencing.
      ).
      Fluorescence in situ hybridization can selectively label specific alleles or specific mtDNA variants in situ, but often only with partial selectivity that may be dependent on context (
      • Lieber T.
      • Jeedigunta S.P.
      • Palozzi J.M.
      • Lehmann R.
      • Hurd T.R.
      Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline.
      ,
      • Gilkerson R.W.
      • Schon E.A.
      • Hernandez E.
      • Davidson M.M.
      Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation.
      ,
      • Kempfer R.
      • Pombo A.
      Methods for mapping 3D chromosome architecture.
      ). This is also limited to fixed, permeabilized cells, and the specificity of the hybridization probes is often limited to the differential labeling of mtDNA variants containing large deletions, rather than single-nucleotide variations, and may provide limited sensitivity (
      • Kempfer R.
      • Pombo A.
      Methods for mapping 3D chromosome architecture.
      ).
      Padlock probes (
      • Larsson C.
      • Koch J.
      • Nygren A.
      • Janssen G.
      • Raap A.K.
      • Landegren U.
      • Nilsson M.
      In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes.
      ) can be used to achieve specificity for single-nucleotide variants of mtDNA in situ (
      • Yaroslavsky A.I.
      • Smolina I.V.
      Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly.
      ). This technique employs a peptide nucleic acid probe to locally open a target DNA site, allowing a “padlock” DNA probe to access the site and become ligated. A rolling circle amplification then generates thousands of single-stranded copies of the target sequence that can be visualized with fluorescent in situ hybridization (
      • Larsson C.
      • Koch J.
      • Nygren A.
      • Janssen G.
      • Raap A.K.
      • Landegren U.
      • Nilsson M.
      In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes.
      ,
      • Yaroslavsky A.I.
      • Smolina I.V.
      Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly.
      ). Detection efficiency for padlock probes at target sites can be in the region of 90% (
      • Yaroslavsky A.I.
      • Smolina I.V.
      Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly.
      ). This technique is limited to fixed, permeabilized cells and often confers only limited specificity for single-nucleotide variants due to substantial binding of the probe to the other variants (
      • Larsson C.
      • Koch J.
      • Nygren A.
      • Janssen G.
      • Raap A.K.
      • Landegren U.
      • Nilsson M.
      In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes.
      ,
      • Yaroslavsky A.I.
      • Smolina I.V.
      Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly.
      ).
      Labeling of mtDNA containing single-nucleotide variations has also been achieved in situ using a technique termed CasPLA (
      • Zhang K.
      • Deng R.
      • Teng X.
      • Li Y.
      • Sun Y.
      • Ren X.
      • Li J.
      Direct visualization of single-nucleotide variation in mtDNA using a CRISPR/Cas9-mediated proximity ligation assay.
      ). In this technique, Cas9/guide RNA (gRNA) is directed to a specific sequence on mtDNA, and local amplification of a fluorescence signal is achieved via a proximity ligation assay. This can enable visualization of individual nucleoids containing the specific mtDNA sequence of interest (
      • Zhang K.
      • Deng R.
      • Teng X.
      • Li Y.
      • Sun Y.
      • Ren X.
      • Li J.
      Direct visualization of single-nucleotide variation in mtDNA using a CRISPR/Cas9-mediated proximity ligation assay.
      ). The low copy number of mtDNAs in nucleoids (
      • Kukat C.
      • Wurm C.A.
      • Spåhr H.
      • Falkenberg M.
      • Larsson N.G.
      • Jakobs S.
      Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.
      ) confirms that CasPLA can achieve high sensitivities at the level of single molecules. Fixed, permeabilized cells are required for the method, to enable access of the probes to the mtDNA; there is currently no reported method for visualizing single-nucleotide mtDNA variants within live cells (
      • Zhang K.
      • Deng R.
      • Teng X.
      • Li Y.
      • Sun Y.
      • Ren X.
      • Li J.
      Direct visualization of single-nucleotide variation in mtDNA using a CRISPR/Cas9-mediated proximity ligation assay.
      ).

      Imaging methods for visualizing mtDNA in cells

      Superresolution techniques and tools.

      SRM comprises an array of techniques, such as photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion (STED) microscopy, that overcome the diffraction barrier and can achieve a resolution <10 nm and thereby generate novel biological insights (
      • Baddeley D.
      • Bewersdorf J.
      Biological insight from super-resolution microscopy: what we can learn from localization-based images.
      ,
      • Sigal Y.M.
      • Zhou R.
      • Zhuang X.
      Visualizing and discovering cellular structures with super-resolution microscopy.
      ,
      • Lakadamyali M.
      • Cosma M.P.
      Visualizing the genome in high resolution challenges our textbook understanding.
      ,
      • Jacquemet G.
      • Carisey A.F.
      • Hamidi H.
      • Henriques R.
      • Leterrier C.
      The cell biologist's guide to super-resolution microscopy.
      ).
      SRM has enabled insights into the structure and dynamics of submitochondrial components, including mtDNA and nucleoids. For example, it has shown that mitochondrial cristae undergo remodeling on a submitochondrial level (
      • Kondadi A.K.
      • Anand R.
      • Hänsch S.
      • Urbach J.
      • Zobel T.
      • Wolf D.M.
      • Segawa M.
      • Liesa M.
      • Shirihai O.S.
      • Weidtkamp-Peters S.
      • Reichert A.S.
      Cristae undergo continuous cycles of membrane remodelling in a MICOS-dependent manner.
      ); that the organization of cristae may be spatially and functionally linked to the mitochondrial transport machinery at endoplasmic reticulum–mitochondria contact sites (
      • Modi S.
      • López-Doménech G.
      • Halff E.F.
      • Covill-Cooke C.
      • Ivankovic D.
      • Melandri D.
      • Arancibia-Cárcamo I.L.
      • Burden J.J.
      • Lowe A.R.
      • Kittler J.T.
      Miro clusters regulate ER-mitochondria contact sites and link cristae organization to the mitochondrial transport machinery.
      ); that mitochondrial nucleoids occupy voids between cristae (
      • Stephan T.
      • Roesch A.
      • Riedel D.
      • Jakobs S.
      Live-cell STED nanoscopy of mitochondrial cristae.
      ); and that individual cristae show remarkable functional independence, including different membrane potentials (
      • Kondadi A.K.
      • Anand R.
      • Hänsch S.
      • Urbach J.
      • Zobel T.
      • Wolf D.M.
      • Segawa M.
      • Liesa M.
      • Shirihai O.S.
      • Weidtkamp-Peters S.
      • Reichert A.S.
      Cristae undergo continuous cycles of membrane remodelling in a MICOS-dependent manner.
      ). These techniques achieve resolutions capable of determining the structure and composition of individual nucleoids and nucleoid clusters (
      • Brown T.A.
      • Tkachuk A.N.
      • Shtengel G.
      • Kopek B.G.
      • Bogenhagen D.F.
      • Hess H.F.
      • Clayton D.A.
      Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.
      ,
      • Kukat C.
      • Wurm C.A.
      • Spåhr H.
      • Falkenberg M.
      • Larsson N.G.
      • Jakobs S.
      Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.
      ).
      SRM entails unique requirements for the fluorescent labels that are used to visualize mtDNA or other structures, as well as suitable cameras and analysis software (
      • Jacquemet G.
      • Carisey A.F.
      • Hamidi H.
      • Henriques R.
      • Leterrier C.
      The cell biologist's guide to super-resolution microscopy.
      ). For example, STORM requires fluorophores, such as Alexa Fluor 647, that alternate between dark and emitting states, or “blink”, upon exposure to illumination. SRM imaging of mtDNA by PALM has been achieved using an overexpressed TFAM tagged with a photoconvertible fluorescent protein, mEos2 (
      • Brown T.A.
      • Tkachuk A.N.
      • Shtengel G.
      • Kopek B.G.
      • Bogenhagen D.F.
      • Hess H.F.
      • Clayton D.A.
      Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.
      ). SRM imaging of mtDNA has also been achieved with STED using antibodies directed against DNA and TFAM (Fig. 3) (
      • Kukat C.
      • Wurm C.A.
      • Spåhr H.
      • Falkenberg M.
      • Larsson N.G.
      • Jakobs S.
      Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.
      ). Newer red fluorescent DNA-binding dyes that label mtDNA have been described recently and may prove useful for SRM applications (
      • Uno K.
      • Sugimoto N.
      • Sato Y.
      N-Aryl pyrido cyanine derivatives: nuclear and organelle DNA markers for two-photon and super-resolution imaging.
      ,
      • Lukinavičius G.
      • Blaukopf C.
      • Pershagen E.
      • Schena A.
      • Reymond L.
      • Derivery E.
      • Gonzalez-Gaitan M.
      • D'Este E.
      • Hell S.W.
      • Wolfram Gerlich D.
      • Johnsson K.
      SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy.
      ,
      • Wang L.
      • Tran M.
      • D'Este E.
      • Roberti J.
      • Koch B.
      • Xue L.
      • Johnsson K.
      A general strategy to develop cell permeable and fluorogenic probes for multicolour nanoscopy.
      ). Most SRM methods are constrained to imaging only one or two different labels with superresolution in any given sample, but some allow several labels to be imaged at superresolution. These include 4Pi single-molecule switching (4Pi-SMS) microscopy, which is reported to enable imaging of three labels in three dimensions, at 5–10 nm resolution, and has been applied to the imaging of mtDNA (
      • Zhang Y.
      • Schroeder L.K.
      • Lessard M.D.
      • Kidd P.
      • Chung J.
      • Song Y.
      • Benedetti L.
      • Li Y.
      • Ries J.
      • Grimm J.B.
      • Lavis L.D.
      • De Camilli P.
      • Rothman J.E.
      • Baddeley D.
      • Bewersdorf J.
      Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging.
      ).

      Other methods for visualizing mtDNA in situ

      Light-sheet microscopy can enable rapid and high-resolution imaging of relatively large samples, such as embryos (
      • Chen B.C.
      • Legant W.R.
      • Wang K.
      • Shao L.
      • Milkie D.E.
      • Davidson M.W.
      • Janetopoulos C.
      • Wu X.S.
      • Hammer 3rd, J.A.
      • Liu Z.
      • English B.P.
      • Mimori-Kiyosue Y.
      • Romero D.P.
      • Ritter A.T.
      • Lippincott-Schwartz J.
      • et al.
      Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.
      ,
      • Peng X.
      • Huang X.
      • Du K.
      • Liu H.
      • Chen L.
      High spatiotemporal resolution and low photo-toxicity fluorescence imaging in live cells and in vivo.
      ), and has been used to visualize the escape of mtDNA from mitochondria during apoptosis (
      • McArthur K.
      • Whitehead L.W.
      • Heddleston J.M.
      • Li L.
      • Padman B.S.
      • Oorschot V.
      • Geoghegan N.D.
      • Chappaz S.
      • Davidson S.
      • San Chin H.
      • Lane R.M.
      • Dramicanin M.
      • Saunders T.L.
      • Sugiana C.
      • Lessene R.
      • et al.
      BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.
      ).
      EM has been used to demonstrate the compaction of mtDNA by TFAM in vitro (
      • Kukat C.
      • Davies K.M.
      • Wurm C.A.
      • Spåhr H.
      • Bonekamp N.A.
      • Kühl I.
      • Joos F.
      • Polosa P.L.
      • Park C.B.
      • Posse V.
      • Falkenberg M.
      • Jakobs S.
      • Kühlbrandt W.
      • Larsson N.G.
      Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid.
      ) and the mitochondrial herniation of mtDNA in situ (
      • McArthur K.
      • Whitehead L.W.
      • Heddleston J.M.
      • Li L.
      • Padman B.S.
      • Oorschot V.
      • Geoghegan N.D.
      • Chappaz S.
      • Davidson S.
      • San Chin H.
      • Lane R.M.
      • Dramicanin M.
      • Saunders T.L.
      • Sugiana C.
      • Lessene R.
      • et al.
      BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.
      ). Visualization of mtDNA by EM may typically be achieved by negative staining and platinum shadowing in vitro (
      • Kukat C.
      • Davies K.M.
      • Wurm C.A.
      • Spåhr H.
      • Bonekamp N.A.
      • Kühl I.
      • Joos F.
      • Polosa P.L.
      • Park C.B.
      • Posse V.
      • Falkenberg M.
      • Jakobs S.
      • Kühlbrandt W.
      • Larsson N.G.
      Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid.
      ) or immunogold labeling of TFAM in situ (
      • McArthur K.
      • Whitehead L.W.
      • Heddleston J.M.
      • Li L.
      • Padman B.S.
      • Oorschot V.
      • Geoghegan N.D.
      • Chappaz S.
      • Davidson S.
      • San Chin H.
      • Lane R.M.
      • Dramicanin M.
      • Saunders T.L.
      • Sugiana C.
      • Lessene R.
      • et al.
      BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.
      ). Electron cryotomography has been used to visualize mtDNA nucleoids at high resolution within isolated mitochondria after rapid freezing (
      • Kukat C.
      • Davies K.M.
      • Wurm C.A.
      • Spåhr H.
      • Bonekamp N.A.
      • Kühl I.
      • Joos F.
      • Polosa P.L.
      • Park C.B.
      • Posse V.
      • Falkenberg M.
      • Jakobs S.
      • Kühlbrandt W.
      • Larsson N.G.
      Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid.
      ) (Fig. 4), and this method can obviate the need for chemical fixation, dehydration, heavy metal staining, and sectioning (
      • Wagner J.
      • Schaffer M.
      • Fernández-Busnadiego R.
      Cryo-electron tomography—the cell biology that came in from the cold.
      ). Correlative light and EM images the same sample with both EM and fluorescence microscopy. This technique can visualize mtDNA and correlate it with other structural features within cells labeled with fluorescent probes (
      • McArthur K.
      • Whitehead L.W.
      • Heddleston J.M.
      • Li L.
      • Padman B.S.
      • Oorschot V.
      • Geoghegan N.D.
      • Chappaz S.
      • Davidson S.
      • San Chin H.
      • Lane R.M.
      • Dramicanin M.
      • Saunders T.L.
      • Sugiana C.
      • Lessene R.
      • et al.
      BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.
      ,
      • Kopek B.G.
      • Shtengel G.
      • Xu C.S.
      • Clayton D.A.
      • Hess H.F.
      Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes.
      ,
      • Okayama S.
      • Ohta K.
      • Higashi R.
      • Nakamura K.
      Correlative light and electron microscopic observation of mitochondrial DNA in mammalian cells by using focused-ion beam scanning electron microscopy.
      ).
      Figure thumbnail gr4
      Figure 4Mitochondrial nucleoids observed by cryo-electron tomography in situ. A segmented surface representation shows the position of mitochondrial nucleoids (green) in a bovine heart mitochondrion. Green, nucleoids; gray, outer membrane; gray-blue, cristae. The images are reproduced from previously published data (
      • Kukat C.
      • Davies K.M.
      • Wurm C.A.
      • Spåhr H.
      • Bonekamp N.A.
      • Kühl I.
      • Joos F.
      • Polosa P.L.
      • Park C.B.
      • Posse V.
      • Falkenberg M.
      • Jakobs S.
      • Kühlbrandt W.
      • Larsson N.G.
      Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid.
      ), reprinted with permission from the United States National Academy of Sciences.
      Although they are beyond the scope of this review and not currently possible in situ, biochemical and sequence-based methods can be used to measure the mtDNA copy number in vitro. These include quantitative PCR, duplex sequencing (
      • Kennedy S.R.
      • Schmitt M.W.
      • Fox E.J.
      • Kohrn B.F.
      • Salk J.J.
      • Ahn E.H.
      • Prindle M.J.
      • Kuong K.J.
      • Shen J.C.
      • Risques R.A.
      • Loeb L.A.
      Detecting ultralow-frequency mutations by duplex sequencing.
      ,
      • Schmitt M.W.
      • Kennedy S.R.
      • Salk J.J.
      • Fox E.J.
      • Hiatt J.B.
      • Loeb L.A.
      Detection of ultra-rare mutations by next-generation sequencing.
      ,
      • Arbeithuber B.
      • Hester J.
      • Cremona M.A.
      • Stoler N.
      • Zaidi A.
      • Higgins B.
      • Anthony K.
      • Chiaromonte F.
      • Diaz F.J.
      • Makova K.D.
      Age-related accumulation of de novo mitochondrial mutations in mammalian oocytes and somatic tissues.
      ), microarrays, and DNA-sequencing read counts (
      • Longchamps R.J.
      • Castellani C.A.
      • Yang S.Y.
      • Newcomb C.E.
      • Sumpter J.A.
      • Lane J.
      • Grove M.L.
      • Guallar E.
      • Pankratz N.
      • Taylor K.D.
      • Rotter J.I.
      • Boerwinkle E.
      • Arking D.E.
      Evaluation of mitochondrial DNA copy number estimation techniques.
      ).

      Quantification of mtDNA images

      Images of mtDNA and mitochondria within cells can be analyzed quantitatively using an array of packages (
      • Bros H.
      • Hauser A.
      • Paul F.
      • Niesner R.
      • Infante-Duarte C.
      Assessing mitochondrial movement within neurons: manual versus automated tracking methods.
      ). The most common methods use plugins for the open-source ImageJ software. For example, the plugins TrackMate (
      • Tinevez J.Y.
      • Perry N.
      • Schindelin J.
      • Hoopes G.M.
      • Reynolds G.D.
      • Laplantine E.
      • Bednarek S.Y.
      • Shorte S.L.
      • Eliceiri K.W.
      TrackMate: an open and extensible platform for single-particle tracking.
      ,
      • Thillaiappan N.B.
      • Chavda A.P.
      • Tovey S.C.
      • Prole D.L.
      • Taylor C.W.
      Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.
      ) and PunctaSpeck (
      • Shah S.I.
      • Ong H.L.
      • Demuro A.
      • Ullah G.
      PunctaSpecks: a tool for automated detection, tracking, and analysis of multiple types of fluorescently labeled biomolecules.
      ) can track particles such as mtDNA nucleoids within cells and measure their numbers and intensities over time.
      The biological properties of mtDNA nucleoids may affect the choice of analysis tools. The diameter of a single nucleoid is ∼100 nm (
      • Ban-Ishihara R.
      • Ishihara T.
      • Sasaki N.
      • Mihara K.
      • Ishihara N.
      Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c.
      ,
      • Brown T.A.
      • Tkachuk A.N.
      • Shtengel G.
      • Kopek B.G.
      • Bogenhagen D.F.
      • Hess H.F.
      • Clayton D.A.
      Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.
      ,
      • Kukat C.
      • Wurm C.A.
      • Spåhr H.
      • Falkenberg M.
      • Larsson N.G.
      • Jakobs S.
      Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.
      ), and SRM may therefore be required for adequate resolution. Analysis of experiments involving incorporation and labeling of nucleotide analogues in replicating mtDNA (
      • Ligasová A.
      • Strunin D.
      • Liboska R.
      • Rosenberg I.
      • Koberna K.
      Atomic scissors: a new method of tracking the 5-bromo-2′-deoxyuridine-labeled DNA in situ.
      ,
      • Davis A.F.
      • Clayton D.A.
      In situ localization of mitochondrial DNA replication in intact mammalian cells.
      ,
      • Phillips A.F.
      • Millet A.R.
      • Tigano M.
      • Dubois S.M.
      • Crimmins H.
      • Babin L.
      • Charpentier M.
      • Piganeau M.
      • Brunet E.
      • Sfeir A.
      Single-molecule analysis of mtDNA replication uncovers the basis of the common deletion.
      ) may involve time-lapse images acquired over several hours or days in live cells, because >1 h is required for a single mtDNA genome to replicate completely (
      • Clayton D.A.
      Replication of animal mitochondrial DNA.
      ,
      • Korhonen J.A.
      • Pham X.H.
      • Pellegrini M.
      • Falkenberg M.
      Reconstitution of a minimal mtDNA replisome in vitro.
      ). The fluorescence intensity of labeled mtDNA nucleoids may not be linearly related to the amount of mtDNA because factors including the replicative state and epigenetic modifications of mtDNA may alter the binding of dyes or TFAM (
      • Hao Z.
      • Wu T.
      • Cui X.
      • Zhu P.
      • Tan C.
      • Dou X.
      • Hsu K.W.
      • Lin Y.T.
      • Peng P.H.
      • Zhang L.S.
      • Gao Y.
      • Hu L.
      • Sun H.L.
      • Zhu A.
      • Liu J.
      • et al.
      N6-deoxyadenosine methylation in mammalian mitochondrial DNA.
      ).
      The Mitochondrial Network Analysis (MiNA) plugin can analyze the morphology of mitochondrial networks in 3D stacks, to estimate network volumes and lengths of individual structures (
      • Valente A.J.
      • Maddalena L.A.
      • Robb E.L.
      • Moradi F.
      • Stuart J.A.
      A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture.
      ). The open-source Mitograph has also been used to measure the characteristics of the mitochondrial network in cells (
      • Jajoo R.
      • Jung Y.
      • Huh D.
      • Viana M.P.
      • Rafelski S.M.
      • Springer M.
      • Paulsson J.
      Accurate concentration control of mitochondria and nucleoids.
      ,
      • Harwig M.C.
      • Viana M.P.
      • Egner J.M.
      • Harwig J.J.
      • Widlansky M.E.
      • Rafelski S.M.
      • Hill R.B.
      Methods for imaging mammalian mitochondrial morphology: a prospective on MitoGraph.
      ). Commercial packages, such as Imaris (
      • Chevrollier A.
      • Cassereau J.
      • Ferré M.
      • Alban J.
      • Desquiret-Dumas V.
      • Gueguen N.
      • Amati-Bonneau P.
      • Procaccio V.
      • Bonneau D.
      • Reynier P.
      Standardized mitochondrial analysis gives new insights into mitochondrial dynamics and OPA1 function.
      ) and Volocity (
      • Higuchi-Sanabria R.
      • Charalel J.K.
      • Viana M.P.
      • Garcia E.J.
      • Sing C.N.
      • Koenigsberg A.
      • Swayne T.C.
      • Vevea J.D.
      • Boldogh I.R.
      • Rafelski S.M.
      • Pon L.A.
      Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae.
      ), have also been used to analyze mitochondrial characteristics, such as movement and network distribution, and can track the intensities of mtDNA nucleoids (
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
      ). An array of software also exists for analyzing SRM images, such as the open-source SR-Tesseler software for analysis of two-dimensional localization-based superresolution microscopy data (
      • Levet F.
      • Hosy E.
      • Kechkar A.
      • Butler C.
      • Beghin A.
      • Choquet D.
      • Sibarita J.B.
      SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data.
      ).

      Manipulating mtDNA in cells

      Tools and techniques for perturbing mtDNA copy number

      The mtDNA copy number varies between cells (
      • Chen J.
      • Zheng Q.
      • Peiffer L.B.
      • Hicks J.L.
      • Haffner M.C.
      • Rosenberg A.Z.
      • Levi M.
      • Wang X.X.
      • Ozbek B.
      • Baena-Del Valle J.
      • Yegnasubramanian S.
      • De Marzo A.M.
      An in situ atlas of mitochondrial DNA in mammalian tissues reveals high content in stem/progenitor cells.
      ,
      • Aryaman J.
      • Johnston I.G.
      • Jones N.S.
      Mitochondrial heterogeneity.
      ) but is often roughly several hundred to several thousand mtDNAs per mammalian somatic cell (
      • Sasaki T.
      • Sato Y.
      • Higashiyama T.
      • Sasaki N.
      Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells.
      ). Manipulation of the mtDNA copy number can be useful to determine the mechanisms involved in mtDNA replication and turnover, to understand the etiology of disease, and to develop novel therapeutics. The mtDNA copy number is altered in many primary human cancers (
      • Shen H.
      • Yu M.
      • Tsoli M.
      • Chang C.
      • Joshi S.
      • Liu J.
      • Ryall S.
      • Chornenkyy Y.
      • Siddaway R.
      • Hawkins C.
      • Ziegler D.S.
      Targeting reduced mitochondrial DNA quantity as a therapeutic approach in pediatric high-grade gliomas.
      ,
      • Yu M.
      Generation, function and diagnostic value of mitochondrial DNA copy number alterations in human cancers.
      ). Lowering the mtDNA copy number of some cancer cells leads to increased susceptibility of these cells to anti-cancer drugs (
      • Mei H.
      • Sun S.
      • Bai Y.
      • Chen Y.
      • Chai R.
      • Li H.
      Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs.
      ), whereas decreasing mtDNA copy number in pancreatic cancer cells leads to autophagy-dependent ferroptotic cell death of these cells (
      • Li C.
      • Zhang Y.
      • Liu J.
      • Kang R.
      • Klionsky D.J.
      • Tang D.
      Mitochondrial DNA stress triggers autophagy-dependent ferroptotic death.
      ). The copy number of mtDNA also changes with aging in some human tissues. Decreases have been reported in skeletal muscle (
      • Wachsmuth M.
      • Hubner A.
      • Li M.
      • Madea B.
      • Stoneking M.
      Age-related and heteroplasmy-related variation in human mtDNA copy number.
      ), blood mononuclear cells (
      • Wachsmuth M.
      • Hubner A.
      • Li M.
      • Madea B.
      • Stoneking M.
      Age-related and heteroplasmy-related variation in human mtDNA copy number.
      ,
      • Kim J.H.
      • Kim H.K.
      • Ko J.H.
      • Bang H.
      • Lee D.C.
      The relationship between leukocyte mitochondrial DNA copy number and telomere length in community-dwelling elderly women.
      ,
      • O'Hara R.
      • Tedone E.
      • Ludlow A.
      • Huang E.
      • Arosio B.
      • Mari D.
      • Shay J.W.
      Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution.
      ), and kidney (
      • Chen J.
      • Zheng Q.
      • Peiffer L.B.
      • Hicks J.L.
      • Haffner M.C.
      • Rosenberg A.Z.
      • Levi M.
      • Wang X.X.
      • Ozbek B.
      • Baena-Del Valle J.
      • Yegnasubramanian S.
      • De Marzo A.M.
      An in situ atlas of mitochondrial DNA in mammalian tissues reveals high content in stem/progenitor cells.
      ), whereas an increase was reported in liver (
      • Wachsmuth M.
      • Hubner A.
      • Li M.
      • Madea B.
      • Stoneking M.
      Age-related and heteroplasmy-related variation in human mtDNA copy number.
      ) and some controversy remains for other tissues (
      • Kazachkova N.
      • Ramos A.
      • Santos C.
      • Lima M.
      Mitochondrial DNA damage patterns and aging: revising the evidences for humans and mice.
      ).
      Depletion of mtDNA within cells can be achieved via diverse methods, including chemically, enzymatically, or via manipulation of regulatory proteins or cellular organelles. Cells in which mtDNA is absent are termed rho0 cells and can be generated by a variety of methods, including treatment of cells with ethidium bromide or targeted nucleases (
      • Kukat A.
      • Kukat C.
      • Brocher J.
      • Schäfer I.
      • Krohne G.
      • Trounce I.A.
      • Villani G.
      • Seibel P.
      Generation of rho0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses.
      ,
      • Spadafora D.
      • Kozhukhar N.
      • Chouljenko V.N.
      • Kousoulas K.G.
      • Alexeyev M.F.
      Methods for efficient elimination of mitochondrial DNA from cultured cells.
      ,
      • King M.P.
      • Attardi G.
      Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation.
      ). Introduction of new mtDNA into rho0 cells results in cybrid cells (
      • Wallace D.C.
      • Pollack Y.
      • Bunn C.L.
      • Eisenstadt J.M.
      Cytoplasmic inheritance in mammalian tissue culture cells.
      ,
      • Seibel P.
      • Di Nunno C.
      • Kukat C.
      • Schäfer I.
      • Del Bo R.
      • Bordoni A.
      • Comi G.P.
      • Schön A.
      • Capuano F.
      • Latorre D.
      • Villani G.
      Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids.
      ), and this technique can yield insights into the function of mtDNA variants. Techniques for directly manipulating mtDNA within cells are summarized in Table 2.
      Table 2Manipulating mtDNA in cells
      Tool/TechniqueMechanism of actionNotesReferences
      Chemical treatments
       ddCInhibition of POLG, mtDNA chain terminationMost potent nucleotide analog tested, cellular toxicity in some contexts
      • Chen C.H.
      • Vazquez-Padua M.
      • Cheng Y.C.
      Effect of anti-human immunodeficiency virus nucleoside analogs on mitochondrial DNA and its implication for delayed toxicity.
      ,
      • Wallace K.B.
      Mitochondrial off targets of drug therapy.
      ,
      • Martin J.L.
      • Brown C.E.
      • Matthews-Davis N.
      • Reardon J.E.
      Effects of antiviral nucleoside analogs on human DNA polymerases and mitochondrial DNA synthesis.
       AzidothymidineInhibition of POLG, mtDNA chain terminationCellular toxicity in some contexts
      • Chen C.H.
      • Vazquez-Padua M.
      • Cheng Y.C.
      Effect of anti-human immunodeficiency virus nucleoside analogs on mitochondrial DNA and its implication for delayed toxicity.
      ,
      • Wallace K.B.
      Mitochondrial off targets of drug therapy.
      ,
      • Martin J.L.
      • Brown C.E.
      • Matthews-Davis N.
      • Reardon J.E.
      Effects of antiviral nucleoside analogs on human DNA polymerases and mitochondrial DNA synthesis.
       DideoxyinosineInhibition of POLG, mtDNA chain terminationCellular toxicity in some contexts
      • Chen C.H.
      • Vazquez-Padua M.
      • Cheng Y.C.
      Effect of anti-human immunodeficiency virus nucleoside analogs on mitochondrial DNA and its implication for delayed toxicity.
      ,
      • Wallace K.B.
      Mitochondrial off targets of drug therapy.
      ,
      • Martin J.L.
      • Brown C.E.
      • Matthews-Davis N.
      • Reardon J.E.
      Effects of antiviral nucleoside analogs on human DNA polymerases and mitochondrial DNA synthesis.
       DideoxydidehydrothymidineInhibition of POLG, mtDNA chain terminationCellular toxicity in some contexts
      • Chen C.H.
      • Vazquez-Padua M.
      • Cheng Y.C.
      Effect of anti-human immunodeficiency virus nucleoside analogs on mitochondrial DNA and its implication for delayed toxicity.
      ,
      • Wallace K.B.
      Mitochondrial off targets of drug therapy.
      ,
      • Martin J.L.
      • Brown C.E.
      • Matthews-Davis N.
      • Reardon J.E.
      Effects of antiviral nucleoside analogs on human DNA polymerases and mitochondrial DNA synthesis.
       DideoxydidehydrocytidineInhibition of POLG, mtDNA chain terminationCellular toxicity in some contexts
      • Chen C.H.
      • Vazquez-Padua M.
      • Cheng Y.C.
      Effect of anti-human immunodeficiency virus nucleoside analogs on mitochondrial DNA and its implication for delayed toxicity.
      ,
      • Wallace K.B.
      Mitochondrial off targets of drug therapy.
      ,
      • Martin J.L.
      • Brown C.E.
      • Matthews-Davis N.
      • Reardon J.E.
      Effects of antiviral nucleoside analogs on human DNA polymerases and mitochondrial DNA synthesis.
       TFAM RNAi or knockoutDestabilizes mtDNASlow action, temporary, specific
      • Larsson N.G.
      • Wang J.
      • Wilhelmsson H.
      • Oldfors A.
      • Rustin P.
      • Lewandoski M.
      • Barsh G.S.
      • Clayton D.A.
      Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice.
      ,
      • Ekstrand M.I.
      • Falkenberg M.
      • Rantanen A.
      • Park C.B.
      • Gaspari M.
      • Hultenby K.
      • Rustin P.
      • Gustafsson C.M.
      • Larsson N.G.
      Mitochondrial transcription factor A regulates mtDNA copy number in mammals.
       TFAM overexpressionIncreases mtDNA copy numberSlow action, temporary
      • Ekstrand M.I.
      • Falkenberg M.
      • Rantanen A.
      • Park C.B.
      • Gaspari M.
      • Hultenby K.
      • Rustin P.
      • Gustafsson C.M.
      • Larsson N.G.
      Mitochondrial transcription factor A regulates mtDNA copy number in mammals.
      ,
      • Ikeda M.
      • Ide T.
      • Fujino T.
      • Arai S.
      • Saku K.
      • Kakino T.
      • Tyynismaa H.
      • Yamasaki T.
      • Yamada K.
      • Kang D.
      • Suomalainen A.
      • Sunagawa K.
      Overexpression of TFAM or Twinkle increases mtDNA copy number and facilitates cardioprotection associated with limited mitochondrial oxidative stress.
       Twinkle overexpressionIncreases mtDNA copy numberSlow action, temporary
      • Ikeda M.
      • Ide T.
      • Fujino T.
      • Arai S.
      • Saku K.
      • Kakino T.
      • Tyynismaa H.
      • Yamasaki T.
      • Yamada K.
      • Kang D.
      • Suomalainen A.
      • Sunagawa K.
      Overexpression of TFAM or Twinkle increases mtDNA copy number and facilitates cardioprotection associated with limited mitochondrial oxidative stress.
      ,
      • Tyynismaa H.
      • Sembongi H.
      • Bokori-Brown M.
      • Granycome C.
      • Ashley N.
      • Poulton J.
      • Jalanko A.
      • Spelbrink J.N.
      • Holt I.J.
      • Suomalainen A.
      Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number.
       Twinkle RNAi or knockoutDestabilizes mtDNASlow action, temporary, specific
      • Tyynismaa H.
      • Sembongi H.
      • Bokori-Brown M.
      • Granycome C.
      • Ashley N.
      • Poulton J.
      • Jalanko A.
      • Spelbrink J.N.
      • Holt I.J.
      • Suomalainen A.
      Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number.
       Ethidium bromideIntercalation; prevents replicationCellular toxicity in some contexts
      • King M.P.
      • Attardi G.
      Injection of mitochondria into human cells leads to a rapid replacement of the endogenous mitochondrial DNA.
      Targeted nucleases
       Mito-CRISPR/Cas9Cuts DNA locally at CRISPR/Cas9-binding site, targeting with guide RNALocus-specific, requires PAM sites, requires guide RNA access to mitochondrial matrix
      • Jo A.
      • Ham S.
      • Lee G.H.
      • Lee Y.I.
      • Kim S.
      • Lee Y.S.
      • Shin J.H.
      • Lee Y.
      Efficient Mitochondrial Genome Editing by CRISPR/Cas9.
       Mito-TALEN-FokITargets and cuts DNA locally at TALEN-binding siteLocus-specific, no guide RNA required
      • Bacman S.R.
      • Williams S.L.
      • Pinto M.
      • Peralta S.
      • Moraes C.T.
      Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs.
       Mito-ZFN-FokITargets and cuts DNA locally at ZFN-binding siteLocus-specific, no guide RNA required
      • Gammage P.A.
      • Rorbach J.
      • Vincent A.I.
      • Rebar E.J.
      • Minczuk M.
      Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations.
       Mito-PstICuts DNA at CTGCAGMultiple sites on mtDNA, not targetable
      • Bacman S.R.
      • Williams S.L.
      • Pinto M.
      • Moraes C.T.
      The use of mitochondria-targeted endonucleases to manipulate mtDNA.
      ,
      • Fukui H.
      • Moraes C.T.
      Mechanisms of formation and accumulation of mitochondrial DNA deletions in aging neurons.
       Mito-XhoICuts DNA at CTCGAGMultiple sites on mtDNA, not targetable
      • Bacman S.R.
      • Williams S.L.
      • Pinto M.
      • Moraes C.T.
      The use of mitochondria-targeted endonucleases to manipulate mtDNA.
       Mito-ScaICuts DNA at AGTACTMultiple sites on mtDNA, not targetable
      • Nissanka N.
      • Bacman S.R.
      • Plastini M.J.
      • Moraes C.T.
      The mitochondrial DNA polymerase γ degrades linear DNA fragments precluding the formation of deletions.
       Mito-EGFP-EcoRICuts DNA at GAATTCMultiple sites on mtDNA, not targetable
      • Kukat A.
      • Kukat C.
      • Brocher J.
      • Schäfer I.
      • Krohne G.
      • Trounce I.A.
      • Villani G.
      • Seibel P.
      Generation of rho0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses.
      Other
       DddA cytidine deaminase-TALEBase editor targeted to mtDNA sequence via fused TALELocus-specific, no guide RNA required, no PAM sites required
      • Mok B.Y.
      • de Moraes M.H.
      • Zeng J.
      • Bosch D.E.
      • Kotrys A.V.
      • Raguram A.
      • Hsu F.
      • Radey M.C.
      • Peterson S.B.
      • Mootha V.K.
      • Mougous J.D.
      • Liu D.R.
      A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.
      The antiretroviral drug zalcitabine is a nucleoside analog (ddC) that inhibits the mitochondrial POLG and causes a reduction in mtDNA copy number (
      • Starnes M.C.
      • Cheng Y.C.
      Cellular metabolism of 2′,3′-dideoxycytidine, a compound active against human immunodeficiency virus in vitro.
      ). This reduction in mtDNA by zalcitabine can lead to the death of some cancer cells (
      • Li C.
      • Zhang Y.
      • Liu J.
      • Kang R.
      • Klionsky D.J.
      • Tang D.
      Mitochondrial DNA stress triggers autophagy-dependent ferroptotic death.
      ). Other nucleoside analogues have been used and have similar effects to zalcitabine, although they are less potent (
      • Chen C.H.
      • Vazquez-Padua M.
      • Cheng Y.C.
      Effect of anti-human immunodeficiency virus nucleoside analogs on mitochondrial DNA and its implication for delayed toxicity.
      ).
      Targeted cleavage of mtDNA can lead to its degradation in cells (
      • Nissanka N.
      • Minczuk M.
      • Moraes C.T.
      Mechanisms of mitochondrial DNA deletion formation.
      ), in part via proteins that form part of the mtDNA replication machinery (
      • Nissanka N.
      • Minczuk M.
      • Moraes C.T.
      Mechanisms of mitochondrial DNA deletion formation.
      ). Cleavage and breakdown of mtDNA has been achieved using mitochondrially targeted transcription activator–like effector nucleases (TALENs) (
      • Bacman S.R.
      • Williams S.L.
      • Pinto M.
      • Peralta S.
      • Moraes C.T.
      Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs.
      ,
      • Hashimoto M.
      • Bacman S.R.
      • Peralta S.
      • Falk M.J.
      • Chomyn A.
      • Chan D.C.
      • Williams S.L.
      • Moraes C.T.
      MitoTALEN: a general approach to reduce mutant mtDNA loads and restore oxidative phosphorylation function in mitochondrial diseases.
      ,
      • Reddy P.
      • Ocampo A.
      • Suzuki K.
      • Luo J.
      • Bacman S.R.
      • Williams S.L.
      • Sugawara A.
      • Okamura D.
      • Tsunekawa Y.
      • Wu J.
      • Lam D.
      • Xiong X.
      • Montserrat N.
      • Esteban C.R.
      • Liu G.H.
      • et al.
      Selective elimination of mitochondrial mutations in the germline by genome editing.
      ,
      • Yahata N.
      • Matsumoto Y.
      • Omi M.
      • Yamamoto N.
      • Hata R.
      TALEN-mediated shift of mitochondrial DNA heteroplasmy in MELAS-iPSCs with m.13513G>A mutation.
      ,
      • Bacman S.R.
      • Kauppila J.H.K.
      • Pereira C.V.
      • Nissanka N.
      • Miranda M.
      • Pinto M.
      • Williams S.L.
      • Larsson N.G.
      • Stewart J.B.
      • Moraes C.T.
      MitoTALEN reduces mutant mtDNA load and restores tRNA(Ala) levels in a mouse model of heteroplasmic mtDNA mutation.
      ,
      • Yang Y.
      • Wu H.
      • Kang X.
      • Liang Y.
      • Lan T.
      • Li T.
      • Tan T.
      • Peng J.
      • Zhang Q.
      • An G.
      • Liu Y.
      • Yu Q.
      • Ma Z.
      • Lian Y.
      • Soh B.S.
      • et al.
      Targeted elimination of mutant mitochondrial DNA in MELAS-iPSCs by mitoTALENs.
      ) and zinc finger nucleases (ZFNs) (
      • Gammage P.A.
      • Rorbach J.
      • Vincent A.I.
      • Rebar E.J.
      • Minczuk M.
      Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations.
      ,
      • Gammage P.A.
      • Gaude E.
      • Van Haute L.
      • Rebelo-Guiomar P.
      • Jackson C.B.
      • Rorbach J.
      • Pekalski M.L.
      • Robinson A.J.
      • Charpentier M.
      • Concordet J.P.
      • Frezza C.
      • Minczuk M.
      Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs.
      ).
      Targeting of restriction enzymes, such as EcoRI, PstI, or XhoI, to mitochondria can also be used to decrease or eliminate mtDNA in cells (
      • Kukat A.
      • Kukat C.
      • Brocher J.
      • Schäfer I.
      • Krohne G.
      • Trounce I.A.
      • Villani G.
      • Seibel P.
      Generation of rho0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses.
      ,
      • Bacman S.R.
      • Williams S.L.
      • Pinto M.
      • Moraes C.T.
      The use of mitochondria-targeted endonucleases to manipulate mtDNA.
      ). These enzymes can be placed under tissue-specific or inducible control and can be used to create cell or animal models and define mechanisms of mtDNA copy number control (
      • Bacman S.R.
      • Williams S.L.
      • Pinto M.
      • Moraes C.T.
      The use of mitochondria-targeted endonucleases to manipulate mtDNA.
      ).
      Manipulation of mtDNA copy number can also be achieved by alteration of the mtDNA replication machinery, and mutation of the key components of the replication machinery can cause a spectrum of heritable diseases (
      • Young M.J.
      • Copeland W.C.
      Human mitochondrial DNA replication machinery and disease.
      ). TFAM overexpression increases mtDNA copy number (
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      ), whereas knockdown or deletion of TFAM reduces or eliminates mtDNA (
      • Larsson N.G.
      • Wang J.
      • Wilhelmsson H.
      • Oldfors A.
      • Rustin P.
      • Lewandoski M.
      • Barsh G.S.
      • Clayton D.A.
      Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice.
      ,
      • Desdín-Micó G.
      • Soto-Heredero G.
      • Aranda J.F.
      • Oller J.
      • Carrasco E.
      • Gabandé-Rodríguez E.
      • Blanco E.M.
      • Alfranca A.
      • Cussó L.
      • Desco M.
      • Ibañez B.
      • Gortazar A.R.
      • Fernández-Marcos P.
      • Navarro M.N.
      • Hernaez B.
      • et al.
      T cells with dysfunctional mitochondria induce multimorbidity and premature senescence.
      ). Perturbing other components of the mtDNA replication machinery may also be expected to alter mtDNA copy number. These components may include POLG, the helicase Twinkle, topoisomerase, mitochondrial RNA polymerase, RNase H1, mitochondrial ssDNA-binding protein, and mitochondrial ligase III (
      • Young M.J.
      • Copeland W.C.
      Human mitochondrial DNA replication machinery and disease.
      ).
      Mitochondrial network fragmentation is intimately involved in the mechanisms of mitophagy and may also enhance the breakdown of mtDNA (
      • Aryaman J.
      • Bowles C.
      • Jones N.S.
      • Johnston I.G.
      Mitochondrial network state scales mtDNA genetic dynamics.
      ,
      • Lieber T.
      • Jeedigunta S.P.
      • Palozzi J.M.
      • Lehmann R.
      • Hurd T.R.
      Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline.
      ). Altering the levels of the endogenous regulators of mitochondrial network architecture, including Drp1, Opa1, and Mfn1/2 (
      • Giacomello M.
      • Pyakurel A.
      • Glytsou C.
      • Scorrano L.
      The cell biology of mitochondrial membrane dynamics.
      ), may therefore also impose changes to the mtDNA copy number.

      Repair and elimination of pathogenic mutant mtDNA

      Mutant mtDNA can be inherited or acquired (e.g. during aging and cellular redox dyshomeostasis) (
      • Kowalska M.
      • Piekut T.
      • Prendecki M.
      • Sodel A.
      • Kozubski W.
      • Dorszewska J.
      Mitochondrial and nuclear DNA oxidative damage in physiological and pathological aging.
      ). Mutant mtDNA can be pathogenic, and a therapeutic strategy is to shift mtDNA heteroplasmy toward the WT mtDNA species (
      • Stewart J.B.
      • Chinnery P.F.
      The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease.
      ,
      • Hahn A.
      • Zuryn S.
      The cellular mitochondrial genome landscape in disease.
      ,
      • Nissanka N.
      • Moraes C.T.
      Mitochondrial DNA heteroplasmy in disease and targeted nuclease-based therapeutic approaches.
      ) using mitochondrially targeted ZFNs (
      • Gammage P.A.
      • Viscomi C.
      • Simard M.L.
      • Costa A.S.H.
      • Gaude E.
      • Powell C.A.
      • Van Haute L.
      • McCann B.J.
      • Rebelo-Guiomar P.
      • Cerutti R.
      • Zhang L.
      • Rebar E.J.
      • Zeviani M.
      • Frezza C.
      • Stewart J.B.
      • et al.
      Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo.
      ) or TALENs (
      • Pereira C.V.
      • Bacman S.R.
      • Arguello T.
      • Zekonyte U.
      • Williams S.L.
      • Edgell D.R.
      • Moraes C.T.
      mitoTev-TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels.
      ). Concerns exist that the gRNA necessary for CRISPR/Cas9 editing may have limited access to the interior of mitochondria (
      • Gammage P.A.
      • Viscomi C.
      • Simard M.L.
      • Costa A.S.H.
      • Gaude E.
      • Powell C.A.
      • Van Haute L.
      • McCann B.J.
      • Rebelo-Guiomar P.
      • Cerutti R.
      • Zhang L.
      • Rebar E.J.
      • Zeviani M.
      • Frezza C.
      • Stewart J.B.
      • et al.
      Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo.
      ,
      • Slone J.
      • Huang T.
      The special considerations of gene therapy for mitochondrial diseases.
      ,
      • Gammage P.A.
      • Moraes C.T.
      • Minczuk M.
      Mitochondrial genome engineering: the revolution may not be CRISPR-Ized.
      ). Another approach to enhance mitochondrial delivery uses microprojectile transformation of mitochondria with plasmid encoding gRNA, Cas9, and DNA repair template (
      • Yoo B.C.
      • Yadav N.S.
      • Orozco Jr., E.M.
      • Sakai H.
      Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts.
      ).
      The most widely used and conventional variants of Cas9, such as the SpCas9 variant from Streptococcus pyogenes used for cleavage of DNA and gene editing, require protospacer adjacent motif (PAM) sites with a sequence NGG to be present within the target DNA, to enable binding of the Cas9. This presents an additional barrier to mtDNA cleavage and editing because NGG PAM sites, although numerous within nuclear DNA, are scarce in mtDNA (
      • Jackson C.B.
      • Turnbull D.M.
      • Minczuk M.
      • Gammage P.A.
      Therapeutic manipulation of mtDNA heteroplasmy: a shifting perspective.
      ). Development of Cas9 variants with a less stringent dependence on specific PAM sites (
      • Miller S.M.
      • Wang T.
      • Randolph P.B.
      • Arbab M.
      • Shen M.W.
      • Huang T.P.
      • Matuszek Z.
      • Newby G.A.
      • Rees H.A.
      • Liu D.R.
      Continuous evolution of SpCas9 variants compatible with non-G PAMs.
      ,
      • Walton R.T.
      • Christie K.A.
      • Whittaker M.N.
      • Kleinstiver B.P.
      Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.
      ,
      • Legut M.
      • Daniloski Z.
      • Xue X.
      • McKenzie D.
      • Guo X.
      • Wessels H.H.
      • Sanjana N.E.
      High-throughput screens of PAM-flexible Cas9 variants for gene knockout and transcriptional modulation.
      ) may now enable their adoption for mtDNA manipulation.
      A recently described CRISPR-free method for precise editing of mtDNA at the level of single nucleotides using a bacterial cytidine deaminase may overcome some of the difficulties associated with the use of CRISPR-based methods (
      • Mok B.Y.
      • de Moraes M.H.
      • Zeng J.
      • Bosch D.E.
      • Kotrys A.V.
      • Raguram A.
      • Hsu F.
      • Radey M.C.
      • Peterson S.B.
      • Mootha V.K.
      • Mougous J.D.
      • Liu D.R.
      A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.
      ).
      Mitophagy also plays a role in the regulation of pathophysiological mtDNA heteroplasmy. A mouse engineered to express the mitophagy sensor, mito-QC, has proved an invaluable resource to further our understanding of mitophagy, heteroplasmy, and mitochondrial architecture (
      • McWilliams T.G.
      • Prescott A.R.
      • Allen G.F.
      • Tamjar J.
      • Munson M.J.
      • Thomson C.
      • Muqit M.M.
      • Ganley I.G.
      mito-QC illuminates mitophagy and mitochondrial architecture in vivo.
      ). Mito-QC is a GFP-mCherry fluorescent reporter targeted to the outer mitochondrial membrane (
      • McWilliams T.G.
      • Prescott A.R.
      • Allen G.F.
      • Tamjar J.
      • Munson M.J.
      • Thomson C.
      • Muqit M.M.
      • Ganley I.G.
      mito-QC illuminates mitophagy and mitochondrial architecture in vivo.
      ). When mitophagy occurs, the fluorescence of green fluorescent GFP, but not red fluorescent mCherry, is quenched, leading to a change in the measured fluorescence ratio. Another recently described mitophagy sensor is mito-SRAI, which may have improved properties (
      • Katayama H.
      • Hama H.
      • Nagasawa K.
      • Kurokawa H.
      • Sugiyama M.
      • Ando R.
      • Funata M.
      • Yoshida N.
      • Homma M.
      • Nishimura T.
      • Takahashi M.
      • Ishida Y.
      • Hioki H.
      • Tsujihata Y.
      • Miyawaki A.
      Visualizing and modulating mitophagy for therapeutic studies of neurodegeneration.
      ).
      Modulation of mitophagy may comprise a therapeutic strategy in mitochondrial diseases (
      • Dombi E.
      • Mortiboys H.
      • Poulton J.
      Modulating mitophagy in mitochondrial disease.
      ,
      • Alsina D.
      • Lytovchenko O.
      • Schab A.
      • Atanassov I.
      • Schober F.A.
      • Jiang M.
      • Koolmeister C.
      • Wedell A.
      • Taylor R.W.
      • Wredenberg A.
      • Larsson N.-G.
      FBXL4 deficiency increases mitochondrial removal by autophagy.
      ). Mitophagy can be stimulated by drugs such as the antibiotic actinonin (
      • Sun N.
      • Yun J.
      • Liu J.
      • Malide D.
      • Liu C.
      • Rovira I.I.
      • Holmström K.M.
      • Fergusson M.M.
      • Yoo Y.H.
      • Combs C.A.
      • Finkel T.
      Measuring in vivo mitophagy.
      ) or the anti-diabetic metformin (
      • Barzilai N.
      • Crandall J.P.
      • Kritchevsky S.B.
      • Espeland M.A.
      Metformin as a tool to target aging.
      ). Stimulation of mitophagy reverses memory impairment in animal models of Alzheimer's disease (
      • Fang E.F.
      • Hou Y.
      • Palikaras K.
      • Adriaanse B.A.
      • Kerr J.S.
      • Yang B.
      • Lautrup S.
      • Hasan-Olive M.M.
      • Caponio D.
      • Dan X.
      • Rocktäschel P.
      • Croteau D.L.
      • Akbari M.
      • Greig N.H.
      • Fladby T.
      • et al.
      Mitophagy inhibits amyloid-β and tau pathology and reverses cognitive deficits in models of Alzheimer's disease.
      ) and delays age-related morbidities. Caloric restriction may extend lifespan in part by effects on mitophagy (
      • Diot A.
      • Morten K.
      • Poulton J.
      Mitophagy plays a central role in mitochondrial ageing.
      ). Conversely, increased mitophagy may also be involved in mitochondrial diseases (
      • Alsina D.
      • Lytovchenko O.
      • Schab A.
      • Atanassov I.
      • Schober F.A.
      • Jiang M.
      • Koolmeister C.
      • Wedell A.
      • Taylor R.W.
      • Wredenberg A.
      • Larsson N.-G.
      FBXL4 deficiency increases mitochondrial removal by autophagy.
      ). Reactive oxygen species may also affect mitophagy and impact many diseases and the aging process (
      • Diot A.
      • Morten K.
      • Poulton J.
      Mitophagy plays a central role in mitochondrial ageing.
      ).

      Future challenges and new approaches

      Significant challenges remain to enable improved visualization of mtDNA within cells and to reveal the functions and regulation of mtDNA within cells. Many advances have been made, but the existing tools for labeling mtDNA have limitations (Table 1) and do not fulfill the ideal requirements listed earlier. For example, currently used organic DNA-binding dyes bind to both nuclear DNA and mtDNA, they are spectrally limited to 488-nm excitation light, and they bleach relatively rapidly; current strategies for labeling EdU and BrdU require fixation and permeabilization and the use of toxic azides and copper(I); and antibody labeling requires fixation and permeabilization (Table 1). Currently, the best choice for many applications may be the expression of TFAM tagged with a fluorescent protein. This strategy provides selective labeling of mtDNA nucleoids that is relatively bright and photostable to enable video imaging (Fig. 1), although overexpression of fluorescently tagged TFAM may increase mtDNA copy number (
      • Lewis S.C.
      • Uchiyama L.F.
      • Nunnari J.
      ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.
      ). Development of new tools may help to overcome the existing limitations. For example, live-cell and time-lapse imaging would benefit from improved nonperturbative and photostable mtDNA labels with a range of spectral characteristics and nontoxic labeling of replicating mtDNA. Useful future approaches may include the fluorescent tagging of native proteins such as TFAM or POLG2 via gene editing (
      • Thillaiappan N.B.
      • Chavda A.P.
      • Tovey S.C.
      • Prole D.L.
      • Taylor C.W.
      Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.
      ) to obviate the need for overexpression or the use of fluorescently labeled single-domain antibodies (chromobodies) to bind to target proteins in live cells (
      • Traenkle B.
      • Rothbauer U.
      Under the microscope: single-domain antibodies for live-cell imaging and super-resolution microscopy.
      ).
      Visualization of the mtDNA epigenetics and post-translational modification of nucleoids would enhance our understanding of mtDNA physiology and disease. One example is DNA methylation, which is widespread and functionally important (
      • Jones P.A.
      • Takai D.
      The role of DNA methylation in mammalian epigenetics.
      ). 5-Methylcytosine is common in nuclear DNA but is considered to occur less frequently in mtDNA (
      • Hao Z.
      • Wu T.
      • Cui X.
      • Zhu P.
      • Tan C.
      • Dou X.
      • Hsu K.W.
      • Lin Y.T.
      • Peng P.H.
      • Zhang L.S.
      • Gao Y.
      • Hu L.
      • Sun H.L.
      • Zhu A.
      • Liu J.
      • et al.
      N6-deoxyadenosine methylation in mammalian mitochondrial DNA.
      ). In contrast, N6-methyldeoxyadenosine (6mA) methylation is reported to occur at 1,300-fold higher levels in mtDNA relative to nuclear DNA and reduces transcription of mtDNA (
      • Hao Z.
      • Wu T.
      • Cui X.
      • Zhu P.
      • Tan C.
      • Dou X.
      • Hsu K.W.
      • Lin Y.T.
      • Peng P.H.
      • Zhang L.S.
      • Gao Y.
      • Hu L.
      • Sun H.L.
      • Zhu A.
      • Liu J.
      • et al.
      N6-deoxyadenosine methylation in mammalian mitochondrial DNA.
      ). This characteristic methylation signature of mtDNA may play important physiological roles and may also provide an avenue for selective visualization and manipulation of 6mA-methylated mtDNA. An antibody against 6mA stains mitochondria in normal cells but not rho0 cells (
      • Hao Z.
      • Wu T.
      • Cui X.
      • Zhu P.
      • Tan C.
      • Dou X.
      • Hsu K.W.
      • Lin Y.T.
      • Peng P.H.
      • Zhang L.S.
      • Gao Y.
      • Hu L.
      • Sun H.L.
      • Zhu A.
      • Liu J.
      • et al.
      N6-deoxyadenosine methylation in mammalian mitochondrial DNA.
      ), suggesting that similar antibodies might identify 6mA-methylated mtDNA selectively. A limitation of the 6mA-antibody is cross-reactivity with RNA containing N6-methyladenosine and the consequent need for stringent RNase treatment to selectively visualize mtDNA (
      • Hao Z.
      • Wu T.
      • Cui X.
      • Zhu P.
      • Tan C.
      • Dou X.
      • Hsu K.W.
      • Lin Y.T.
      • Peng P.H.
      • Zhang L.S.
      • Gao Y.
      • Hu L.
      • Sun H.L.
      • Zhu A.
      • Liu J.
      • et al.
      N6-deoxyadenosine methylation in mammalian mitochondrial DNA.
      ).
      An exciting avenue for development is the visualization of mtDNA breakdown. Indirect methods such as PCR have been utilized to determine some of the proteins and mechanisms involved (
      • Nissanka N.
      • Minczuk M.
      • Moraes C.T.
      Mechanisms of mitochondrial DNA deletion formation.
      ), but a direct method for visualizing breakdown within live cells has not yet been described. Mitophagy is known to turn over entire mitochondria (
      • McWilliams T.G.
      • Prescott A.R.
      • Allen G.F.
      • Tamjar J.
      • Munson M.J.
      • Thomson C.
      • Muqit M.M.
      • Ganley I.G.
      mito-QC illuminates mitophagy and mitochondrial architecture in vivo.
      ), but where the mtDNA is broken down and disposed of during this process remains unclear. It remains possible that mtDNA is metabolized to some extent within mitochondria, in the absence of mitophagy or preceding it. Mitochondria can contain lysosome-like organelles (
      • Höglinger D.
      • Burgoyne T.
      • Sanchez-Heras E.
      • Hartwig P.
      • Colaco A.
      • Newton J.
      • Futter C.E.
      • Spiegel S.
      • Platt F.M.
      • Eden E.R.
      NPC1 regulates ER contacts with endocytic organelles to mediate cholesterol egress.
      ) and vesicles containing cytosolic components (
      • Chen P.L.
      • Huang K.T.
      • Cheng C.Y.
      • Li J.C.
      • Chan H.Y.
      • Lin T.Y.
      • Su M.P.
      • Yang W.Y.
      • Chang H.C.
      • Wang H.D.
      • Chen C.H.
      Vesicular transport mediates the uptake of cytoplasmic proteins into mitochondria in Drosophila melanogaster.
      ). Whether these intramitochondrial organelles or other factors within mitochondria play roles in mtDNA metabolism remains to be resolved.
      Last, a significant challenge is to develop a technique that is capable of visualizing specific single-nucleotide mtDNA variants within live cells. This may be via techniques analogous to the CasPLA (
      • Zhang K.
      • Deng R.
      • Teng X.
      • Li Y.
      • Sun Y.
      • Ren X.
      • Li J.
      Direct visualization of single-nucleotide variation in mtDNA using a CRISPR/Cas9-mediated proximity ligation assay.
      ) or padlock probes (
      • Larsson C.
      • Koch J.
      • Nygren A.
      • Janssen G.
      • Raap A.K.
      • Landegren U.
      • Nilsson M.
      In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes.
      ,
      • Yaroslavsky A.I.
      • Smolina I.V.
      Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly.
      ) used to label DNA variants in fixed and permeabilized cells or via improvements to fluorescence in situ hybridization methods (
      • Goh J.J.L.
      • Chou N.
      • Seow W.Y.
      • Ha N.
      • Cheng C.P.P.
      • Chang Y.C.
      • Zhao Z.W.
      • Chen K.H.
      Highly specific multiplexed RNA imaging in tissues with split-FISH.
      ). The development of new variants of Cas9 may also assist (
      • Miller S.M.
      • Wang T.
      • Randolph P.B.
      • Arbab M.
      • Shen M.W.
      • Huang T.P.
      • Matuszek Z.
      • Newby G.A.
      • Rees H.A.
      • Liu D.R.
      Continuous evolution of SpCas9 variants compatible with non-G PAMs.
      ,
      • Walton R.T.
      • Christie K.A.
      • Whittaker M.N.
      • Kleinstiver B.P.
      Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.