title: Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid.
abstract: Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.
authors: Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany; FACS & Imaging Core Facility, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany;
authors: Christian
authors: C
authors: Kukat
authors: Department of Structural Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
authors: Karen M
authors: KM
authors: Davies
authors: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany;
authors: Christian A
authors: CA
authors: Wurm
authors: Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany;
authors: Henrik
authors: H
authors: Spåhr
authors: Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany;
authors: Nina A
authors: NA
authors: Bonekamp
authors: Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany;
authors: Inge
authors: I
authors: Kühl
authors: Department of Structural Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
authors: Friederike
authors: F
authors: Joos
authors: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, 70125 Bari, Italy;
authors: Paola Loguercio
authors: PL
authors: Polosa
authors: Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany; Institute for Medical Sciences, Ajou University School of Medicine, Suwon 443-721, Korea;
authors: Chan Bae
authors: CB
authors: Park
authors: Department of Medical Biochemistry and Cell Biology, University of Gothenburg, 405 30 Gothenburg, Sweden;
authors: Viktor
authors: V
authors: Posse
authors: Department of Medical Biochemistry and Cell Biology, University of Gothenburg, 405 30 Gothenburg, Sweden;
authors: Maria
authors: M
authors: Falkenberg
authors: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; Department of Neurology, University of Göttingen Medical School, 37073 Göttingen, Germany;
authors: Stefan
authors: S
authors: Jakobs
authors: Department of Structural Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany;
authors: Werner
authors: W
authors: Kühlbrandt
authors: Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany; Department of Laboratory Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden larsson@age.mpg.de.
authors: Nils-Göran
authors: NG
authors: Larsson
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D020285:Cryoelectron Microscopy; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D055032:Electron Microscope Tomography; D054629:Genome, Mitochondrial / Q000235:genetics; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D009154:Mutation; D009698:Nucleoproteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding
chemical_list: D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D009698:Nucleoproteins; C081930:Tfam protein, mouse
keywords: STED; cryo-ET; mitochondria; nanoscopy; nucleoids