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Cross-Strand Binding of TFAM to a Single Mtdna Molecule Forms the Mitochondrial Nucleoid

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Cross-Strand Binding of TFAM to a Single Mtdna Molecule Forms the Mitochondrial Nucleoid Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid Christian Kukata,b,1, Karen M. Daviesc,1, Christian A. Wurmd,1, Henrik Spåhra, Nina A. Bonekampa, Inge Kühla, Friederike Joosc, Paola Loguercio Polosae, Chan Bae Parka,f, Viktor Posseg, Maria Falkenbergg, Stefan Jakobsd,h, Werner Kühlbrandtc, and Nils-Göran Larssona,i,2 aDepartment of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany; bFACS & Imaging Core Facility, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany; cDepartment of Structural Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany; dDepartment of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; eDepartment of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, 70125 Bari, Italy; fInstitute for Medical Sciences, Ajou University School of Medicine, Suwon 443-721, Korea; gDepartment of Medical Biochemistry and Cell Biology, University of Gothenburg, 405 30 Gothenburg, Sweden; hDepartment of Neurology, University of Göttingen Medical School, 37073 Göttingen, Germany; and iDepartment of Laboratory Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden Edited by F. Ulrich Hartl, Max Planck Institute of Biochemistry, Martinsried, Germany, and approved August 4, 2015 (received for review June 23, 2015) Mammalian mitochondrial DNA (mtDNA) is packaged by mito- To date, mitochondrial transcription factor A (TFAM) is the chondrial transcription factor A (TFAM) into mitochondrial nucle- only protein that fulfills a stringent definition of a structural oids that are of key importance in controlling the transmission and component that packages mtDNA in the mammalian nucleoid expression of mtDNA. Nucleoid ultrastructure is poorly defined, (8). TFAM is very abundant, fully coats mtDNA (11, 12), and and therefore we used a combination of biochemistry, superreso- is essential for mtDNA maintenance (1). In addition to its lution microscopy, and electron microscopy to show that mito- function in mtDNA compaction, TFAM is also an essential chondrial nucleoids have an irregular ellipsoidal shape and typically component of the mammalian mtDNA transcription initiation contain a single copy of mtDNA. Rotary shadowing electron complex (13). It specifically binds the mitochondrial promoters microscopy revealed that nucleoid formation in vitro is a multistep to recruit the mitochondrial RNA polymerase (POLRMT), process initiated by TFAM aggregation and cross-strand binding. which, in turn, recruits mitochondrial transcription factor B2 (14, Superresolution microscopy of cultivated cells showed that in- 15). Crystallographic studies have revealed that TFAM induces a creased mtDNA copy number increases nucleoid numbers without 180° U-turn when bound to promoters or unspecific mtDNA altering their sizes. Electron cryo-tomography visualized nucleoids sequences (16–18). TFAM contains two high mobility group at high resolution in isolated mammalian mitochondria and con- (HMG)-box domains (HMG-box A and B) that each intercalate firmed the sizes observed by superresolution microscopy of cell into the minor groove of a half-site of a DNA duplex. Between lines. We conclude that the fundamental organizational unit of the two HMG-box domains is a helical linker with a positively the mitochondrial nucleoid is a single copy of mtDNA compacted charged surface that interacts with the negatively charged by TFAM, and we suggest a packaging mechanism. backbone of the DNA (16, 17). TFAM has the capacity to in- crease the flexibility of DNA to promote compaction (19, 20). nucleoids | mitochondria | cryo-ET | STED | nanoscopy However, the mechanism of mtDNA compaction by TFAM is currently under debate. Apart from imposing U-turns, TFAM he thousands of copies of mitochondrial DNA (mtDNA) in Tmammalian cells encode 13 essential subunits of the mito- Significance chondrial oxidative phosphorylation system, along with 2 rRNAs and 22 tRNAs (1). Despite its few genes, the mtDNA of ∼16 kb is essential for biogenesis of the oxidative phosphorylation sys- Altered expression of mitochondrial DNA (mtDNA) is heavily tem, which produces the energy currency ATP used for a variety implicated in human disease and aging, but the basic organiza- of metabolic reactions (1). Mutations in mtDNA are an impor- tional unit of mtDNA, the mitochondrial nucleoid, is poorly un- tant cause of human disease and are heavily implicated in the derstood. Here, we have used a combination of biochemistry, aging process (2, 3). After the discovery of mtDNA by electron superresolution microscopy, and electron microscopy to show that mammalian mitochondrial nucleoids have an irregular ellipsoidal microscopy (EM) of vertebrate mitochondria in the early 1960s, shape and typically contain a single copy of mtDNA. Furthermore, it soon became evident that this circular molecule with a contour we show that the nucleoid ultrastructure is independent of cel- length of ∼5 μm must be compacted to fit within a mitochondrion, lular mtDNA copy number and that the core nucleoid structure is which typically has a width of ∼0.5 μm (4). Subsequent light mi- formed by cross-strand binding of mitochondrial transcription croscopy studies showed that mtDNA is present in cells as hun- factor A (TFAM) to a single copy of mtDNA. The clarification of dreds of discrete condensed spots, denoted as mitochondrial the ultrastructure of the mammalian mitochondrial nucleoid nucleoids, each with a diameter of a few hundred nanometers (5–7). provides the fundamental basis for the understanding of regu- The existence of nucleoids in mammalian cells has been docu- lation of mtDNA maintenance and expression in mammals. mented in detail with a variety of light microscopy approaches (8), but their true size has been much debated as conventional Author contributions: C.K., K.M.D., C.A.W., H.S., S.J., W.K., and N.-G.L. designed research; light microscopy cannot resolve structures smaller than ∼250 nm C.K., K.M.D., C.A.W., H.S., N.A.B., F.J., P.L.P., C.B.P., V.P., and M.F. performed research; C.K., (9). It came as a surprise when superresolution microscopy [stim- K.M.D., and C.A.W. analyzed data; and C.K., K.M.D., C.A.W., I.K., S.J., W.K., and N.-G.L. ulated emission depletion (STED) nanoscopy] showed that nu- wrote the paper. cleoids in a variety of mammalian species had a mean diameter The authors declare no conflict of interest. below ∼100 nm, which was much smaller than anticipated (6). An This article is a PNAS Direct Submission. independent approach using different forms of superresolution Freely available online through the PNAS open access option. microscopy [photoactivatable localization microscopy (PALM), 1C.K., K.M.D., and C.A.W. contributed equally to this work. interferometric PALM (iPALM), and direct stochastic optical 2To whom correspondence should be addressed. Email: [email protected]. reconstruction microscopy (dSTORM)] confirmed that the mean This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. diameter of nucleoids is ∼110 nm in human cells (10). 1073/pnas.1512131112/-/DCSupplemental. 11288–11293 | PNAS | September 8, 2015 | vol. 112 | no. 36 www.pnas.org/cgi/doi/10.1073/pnas.1512131112 Downloaded by guest on October 2, 2021 can also bind to nonspecific DNA sequences in a cooperative manner to form stable protein patches (19). Beyond DNA bending, dimerization of TFAM has been reported to be necessary for full compaction of mtDNA into nucleoids (18, 20). Although important biophysical principles for TFAM-mediated mtDNA compaction are beginning to be understood, a number of properties of the mammalian nucleoids, such as their mor- phology and mtDNA content, are subject to considerable debate. Estimates of the mtDNA copy number per nucleoid, performed in the pre-superresolution microscopy era, had yielded values of 2–10 copies of mtDNA per nucleoid (5, 7, 21, 22). With STED microscopy a previously unrecognized clustering of nucle- oids was discovered and the cell was found to contain ∼60% more nucleoids than previously estimated (6). As a consequence, the number of mtDNA molecules per nucleoid in human fibroblasts wasreportedtobeaslowas∼1.4 in human cells (6). However, this number was questioned by another study that reported approxi- mately three mtDNA molecules per nucleoid using confocal mi- croscopy (10). Also, the shape of the mitochondrial nucleoid as determined by superresolution microscopy is disputed. Ellipsoid contours of nucleoids have been reported (10, 23), whereas we have documented a similar mean nucleoid diameter in a variety of mammalian species (6). The organization of mtDNA into nucleoids is of fundamental interest as it will impact our understanding of mtDNA segregation in the germline and somatic tissues. Furthermore, replication and transcription of mtDNA is inhibited if the nucleoid is highly com- pacted (24). We have used biochemistry, STED superresolution microscopy, EM, and electron cryo-tomography (cryo-ET) of mammalian cells and nucleoids reconstituted in vitro to charac- terize the ultrastructure of the mammalian mitochondrial nucle- oid. Here we report that the organizational unit of the mammalian mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM aggregation and cross-strand binding. Furthermore, we report that most nucleoids have a slightly ellipsoidal shape. Our Fig. 1. Electron microscopy reveals that TFAM packages single mtDNA molecules and
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