Cross-strand Binding of TFAM to a Single mtDNA Molecule Forms the Mitochondrial Nucleoid

Fundamental insights into the nanoworld of cellular power plants gained by scientists of three Max Planck Institutes

November 17, 2015

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.

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Kukat C, Davies KM, Wurm CA, Spar H, Bonekamp NA, Kühle I, Joos F, Polosa PL, Park CB, Posse V, Falkenberg M, Jakobs S, Kühlbrandt W, and Larsson NG

Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid

PNAS 112 (36):11288–11293 (2015); doi: 10.1073/pnas.1512131112

Scientists from the Max Planck Institute for Biology of Ageing (MPI AGE) in Cologne, the Max Planck Institute of Biophysics (MPI BP) in Frankfurt a.M., and the Max Planck Institute for Biophysical Chemistry (MPI BPC) in Göttingen have gained fundamental insights into the organization of mitochondrial DNA (mtDNA). The researchers observed in high-resolution images gained with nobel prize-winning microscopy techniques that single copies of mtDNA are packaged by a specialized protein into slightly elongated structures of circa 100 nm in length.

Mitochondria are described as cellular power plants, because they produce energy in nearly every cell in the (human) body. They harbor their own genome, the mitochondrial DNA or mtDNA. The mtDNA is organized together with specific proteins in complexes inside mitochondria, the so called mitochondrial nucleoids. “The structure of these nucleoids is poorly understood. We can visualize them in the microscope, but we quickly reach the limits of conventional light microscopy when we want to look at finer structures”, says Christian Kukat, researcher at the Max Planck Institute for Biology of Ageing in Cologne. “Therefore we asked for help from other Max Planck Institutes.”

Scientists from the Max Planck Institute in Göttingen and Frankfurt use the latest microscopy techniques, which can bypass the resolution limit of the conventional light microscopy. With the help of super-resolution STED microscopy, electron microscopy (EM) and electron cryo-tomography (cryo-ET) they characterized the ultrastructure of the mam malian mitochondrial nucleoid. “We saw that nucleoids have an irregular, ellipsoidal shape and contain typically just a single copy of mitochondrial DNA. Even if the cell has more mtDNA molecules, the nucleoid ultrastructure remains the same”, says Kukat. The other mtDNA molecules are packaged into additional nucleoids.

These insights of the researchers from three Max Planck Institutes establish a new basis for the investigation of diseases that are inherited mitochondrially, from mothers to the offspring. Christian Kukat: “In future we will continue our studies on the connection of mitochondria and the processes of ageing.”

Image above: 3D model/tomographic volume of a bovine heart mitochondrion. Segmented surface representation showing position of mitochondrial nucleoids (green) in a bovine heart mitochondrion. Green: nucleoids, gray: outer membrane, grayblue: cristae. Credit: MPI BP Frankfurt a.M. / Karen Davies and Proceedings of the National Academy of Sciences of the United States of America (PNAS).

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