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.

Keywords: STED; cryo-ET; mitochondria; nanoscopy; nucleoids.

Fig. 1.

Electron microscopy reveals that TFAM packages single mtDNA molecules and that cross-strand TFAM binding is necessary for compaction of mtDNA. (A) Electron micrograph showing a DNA U-turn with TFAM bound (white arrowhead). (Scale bar: 20 nm.) Crystal structure of TFAM (blue) and DNA (orange/green) [Protein Data Bank ID code 3TMM (16)]. (B) Electron micrographs of spread DNA incubated with increasing concentrations of TFAM. TFAM molecules are indicated by white arrowheads. White asterisks mark unbound TFAM molecules. (Scale bar: 100 nm.) (C) Diameters of DNA incubated with increasing concentrations of recombinant TFAM protein by the mean of the long and short axes. Data are represented as mean ± SD, n = 134. (D) TFAM binds to DNA in two different ways. TFAM binds single DNA duplexes as beads on a string inducing bending of DNA (Left) or bridges two DNA duplexes resulting in loops (Right). TFAM molecules are indicated by white arrowheads. (E) TFAM binding to DNA as beads on a string (red) or by bridging two DNA duplexes (dark red). Subsequent binding preferentially occurs at sites already occupied by TFAM as can be observed by an increase in particle size. (Scale bars: 50 nm.)

Fig. S1.

(A) EMSA of DNA with increasing concentrations of recombinant TFAM. The stability of the formed complex was investigated with or without ammonium acetate in the buffer. Constant amounts of DNA templates were incubated with increasing amounts of recombinant TFAM protein in standard and ammonium acetate buffer. A clear shift of the DNA fragments was visible in the EMSA, indicating that the ammonium acetate required for EM does not interfere with TFAM–DNA binding. (B) Representative electron micrographs of in vitro-generated nucleoids with TFAM concentrations >1 TFAM/6 bp. (Scale bar: 100 nm.)

Fig. 2.

Model for packaging mtDNA into the mitochondrial nucleoid. (A) Outline of the naked mtDNA duplex (gray). (B) TFAM molecules (red) bind to mtDNA and induce bending. (C) TFAM bridges neighboring mtDNA duplexes (arrows) by cross-strand binding. (D) A combination of mtDNA duplex bending and cross-strand binding by TFAM compacts mtDNA. (E) Further compaction of mtDNA by cooperative TFAM binding into patches. (F) The final tightly packaged mtDNA in the mitochondrial nucleoid. The green triangle illustrates increasing concentration of TFAM protein per base pair mtDNA.

Fig. S2.

(A) Electron micrographs of spread DNA incubated with increasing concentrations of TFAM dimer mutant (DM). (Scale bar: 100 nm.) (B) Quantification of the diameters of DNA incubated with increasing concentrations of TFAM DM protein by the mean of the long and short axis. Data are represented as mean ± SD; n = 82. (C and D) Electron micrographs showing that TFAM DM binds to DNA in two different ways. TFAM binds single DNA duplexes as beads on a string inducing bending of DNA (C) or bridges two DNA duplexes resulting in loops (D). (Scale bar: 20 nm.)

Fig. 3.

Mitochondrial nucleoids frequently contain a single mtDNA molecule, their intracellular abundance depends on total mtDNA copy number, and most mitochondrial nucleoids have a slightly ellipsoid shape. (A) mtDNA (green, DNA antibodies) is localized in nucleoids in the tubular mitochondria (red, anti-TOM20) of MEFs. DAPI staining of the nucleus is in blue. (Scale bar: 25 µm.) (B) Number of mtDNA molecules per cell in MEFs by qPCR. (C) Comparison of confocal microscopy with superresolution STED microscopy of mitochondrial nucleoids visualized by DNA antibodies (deconvolved data). Shown are MEFs from wild-type (wt) and TFAM overexpressor (TFAM OE) mice. (Scale bar: 1 µm.) (D) Mean diameter of nucleoids labeled with DNA antibodies as determined by STED imaging; n = 70,638 nucleoids. (E) Nucleoids per cell area; n = 717 cells. (F) Ratio of the number of nucleoids detected by STED microscopy versus the number of nucleoids detected by confocal microscopy; n = 28,712 nucleoids. (G) Number of nucleoids per cell; n = 717 cells. (H) Ratio of long to short axis of nucleoids in MEFs imaged by STED microscopy; n = 70,638 nucleoids. (I) A variety of nucleoid shapes visualized by confocal and STED microscopy using DNA antibodies (deconvolved data). Asterisks mark the structures measured (in nm). The ratio is the ratio of long to short axis of the nucleoids. (Scale bar: 200 nm.) (J) Histogram of the relative frequency of binned ratios of long to short axis of nucleoids in wild-type and TFAM OE MEFs. Ellipsoids below the graph depict the resulting ellipses. We used six independent TFAM OE MEF lines and four independent wild-type lines for the analyses. wt: wild-type; TFAM OE: TFAM overexpressor. Data are represented as mean ± SD.

Fig. S3.

(A) Southern blot analysis of the mtDNA content in independent MEF lines. (B) Diameters of mitochondrial nucleoids imaged by STED microscopy for four independent wild-type (wt) and six independent TFAM OE MEF cell lines. (C) Quantification of the long and short axis length of nucleoids in MEFs imaged by STED microscopy. (D) Cell size of MEFs. n = 717 cells. wt: wild-type; TFAM OE: TFAM overexpressor. Notched boxplots: the boxed intervals contain the data points from the first to third quartiles of the distribution. With a size of 1.5 times the interquartile range, the whiskers encompass ∼99% of all data. The notches describe a confidence interval around the median, which equals the 95% confidence interval.

Fig. 4.

Mitochondrial nucleoids observed by cryo-electron tomography in situ are ellipsoid. (A) Tomographic slice through a whole bovine heart mitochondrion showing mitochondrial nucleoids (boxed areas). White blobs are gas bubbles, caused by the interaction of the electron beam with the biological material (28). (Scale bars: 100 nm.) (B) Segmented surface representation of A showing position of mitochondrial nucleoids (green) in a bovine heart mitochondrion. Green: nucleoids; gray: outer membrane; gray-blue: cristae. (C) Dimensions and long to short axis ratio of 13 nucleoids from four tomographic volumes of isolated bovine heart mitochondria (mitoch.) (n = 13). See also Movie S1.

Fig. S4.

(A and B) Projection images #52 and #78 from a tomographic tilt series of a bovine heart mitochondrion showing mitochondrial nucleoids (boxed areas). The total number of projection images in the tilt series is 86. Total accumulated electron dose for the series was 130 electrons per Ångström squared (e⁻/Ų). The total accumulated electron dose for each image is displayed at the bottom right corner of the image. (C) Tomographic slice through a whole bovine heart mitochondrion showing mitochondrial nucleoids (boxed areas). (Scale bar: 100 nm.)

Fig. S5.

(A and D) Tomographic slice through a whole bovine heart mitochondrion showing mitochondrial nucleoids (boxed areas). (Scale bar: 100 nm.) (B and E) Segmented surface representation of A with D showing position of mitochondrial nucleoids (green) in a bovine heart mitochondrion. Green: nucleoids; gray: outer membrane; gray-blue: cristae. (C and F) Crista extracted from B and E showing crista morphology. (Scale bar: 50 nm.)