Integrity of the yeast mitochondrial genome, but not its distribution and inheritance, relies on mitochondrial fission and fusion

Abstract

Mitochondrial DNA (mtDNA) is essential for mitochondrial and cellular function. In Saccharomyces cerevisiae, mtDNA is organized in nucleoprotein structures termed nucleoids, which are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle. How the cell distributes and inherits mtDNA is incompletely understood although an involvement of mitochondrial fission and fusion has been suggested. We developed a LacO-LacI system to noninvasively image mtDNA dynamics in living cells. Using this system, we found that nucleoids are nonrandomly spaced within the mitochondrial network and observed the spatiotemporal events involved in mtDNA inheritance. Surprisingly, cells deficient in mitochondrial fusion and fission distributed and inherited mtDNA normally, pointing to alternative pathways involved in these processes. We identified such a mechanism, where we observed fission-independent, but F-actin-dependent, tip generation that was linked to the positioning of mtDNA to the newly generated tip. Although mitochondrial fusion and fission were dispensable for mtDNA distribution and inheritance, we show through a combination of genetics and next-generation sequencing that their absence leads to an accumulation of mitochondrial genomes harboring deleterious structural variations that cluster at the origins of mtDNA replication, thus revealing crucial roles for mitochondrial fusion and fission in maintaining the integrity of the mitochondrial genome.

Keywords: Dnm1; Fzo1; mitochondria; mtDNA; yeast.

Fig. 1.

The mt-LacO-LacI system. (A) Histogram showing the petite frequency of cells grown for 6 h in the absence or presence of indicated amounts of DAPI in the growth medium. (B) Representative image of the petite frequency assay. White colonies indicate respiratory deficiency. (C) Cells grown overnight in the presence of DAPI (1µg/mL) were examined by light microscopy. (D) Schematic representation of the mt-LacO-LacI system. The length of individual genetic elements is drawn to scale. (E) Maximum intensity Z-projections of WT cells or cells harboring the mt-LacO repeats, each expressing mt-3xGFP-LacI and mt-dsRed. (F) Maximum intensity Z-projection of an mt-LacO-LacI cell treated with DAPI (1 µg/mL). Close-ups of mitochondrial segments are shown that illustrate colocalization of the mt-3xGFP-LacI and the DAPI signal. (G) The petite frequency was determined for WT, mt-LacO-LacI, and ∆mmr1 cells. (H) qPCR analysis of total levels of mtDNA relative to nuclear DNA in WT and mt-LacO-LacI cells. (I) Serial dilutions of WT and mt-LacO-LacI cells were spotted on YPD or YPEG plates containing a fermentable or nonfermentable carbon source, respectively.

Fig. 2.

mtDNA distribution in WT cells. (A) Maximum intensity Z-projection of a representative WT mt-LacO-LacI cell expressing mt-dsRed (Left). Image of the same cell with skeletonized mitochondria and spheres at positions of mtDNA (Middle). Illustration of the distance measurements between nucleoids or between mitochondrial tips and nucleoids (Right) (Bottom Right image is rotated by 90°; compare Movie S1). (B) The number of nucleoids is plotted against the network length of the mitochondria. Datasets from mother, daughter, or both cells are distinguished; the data for small buds are indicated. (C) Distances between each nucleoid and the closest neighboring nucleoids along the mitochondrial filament (see A) of experimentally determined or randomly distributed nucleoids were determined for 23 cells, binned and plotted in a histogram. The dashed line indicates the mean distance between nucleoids. Note that the mean distances are similar for the real and the simulated dataset. The x axis was limited to 3.5 µm; less frequently observed longer distances are thus omitted from the figure. (D) Distances between mitochondrial tips and the closest nucleoid (see A) were determined for the real and the simulated dataset, binned, and plotted in a histogram. Distances smaller than 500 nm are highlighted. (E) Time-lapse microscopy of a representative WT mt-LacO-LacI cell expressing mt-dsRed shows the spatiotemporal relationship of mitochondrial and mtDNA inheritance.

Fig. 3.

mtDNA distribution in ∆dnm1∆fzo1 cells. (A) Maximum intensity Z-projection of a representative ∆dnm1∆fzo1 mt-LacO-LacI cell expressing mt-dsRed is shown (Left). Image of the same cell with skeletonized mitochondria and spheres at positions of mtDNA (Middle). Illustration of the distance measurements between nucleoids or between mitochondrial tips and nucleoids (Right) (Bottom Right image is rotated by 90°; compare Movie S3). (B) Correlation between nucleoid number and network length. (C) Distribution of internucleoid distances. (D) Distribution of nucleoid-mitochondrial-tip distances. (E) Time-lapse microscopy of a representative WT mt-LacO-LacI cell expressing mt-dsRed shows the spatiotemporal relationship of mitochondrial and mtDNA inheritance.

Fig. 4.

Fission-independent mitochondrial tip generation. (A and B) Single frames from time-lapse microscopy of (A) ∆dnm1∆fzo1 or (B) WT cells harboring the mt-LacO-LacI system and expressing mt-dsRed. Arrows indicate the start of fission-independent tubule generation. (C) ∆dnm1∆fzo1 cells expressing mt-dsRed were treated with LatA or not, and Z-stacks of complete cells were acquired every 4 s for a total of 160 s, and fission-independent generation of mitochondrial tips was quantified in 40 cells in each sample (compare Movie S5 with Movie S6).

Fig. 5.

∆dnm1∆fzo1 accumulate ρ⁻ genomes. (A, Top) Table reporting the ρ⁰ and petite frequencies for WT and ∆dnm1∆fzo1 cells that were determined by DAPI staining and the petite frequency assay, respectively. Representative images of DAPI-stained ∆dnm1∆fzo1 cells with the corresponding brightfield (Middle) and the petite frequency assay (Bottom) are shown. (B) mtDNA complementation assay and PCR analysis of petite ∆dnm1∆fzo1 cells. Seven individual ∆dnm1∆fzo1 petite colonies were crossed against tester strains containing either a small deletion in the COX3 gene or a compete deletion of the COB gene, and crosses were replicated onto YPEG plates. Cell growth indicates complementation of the genetic defects of mtDNA in the parental strains. Loci in the mtDNA (COB, COX3, ATP9) and nuclear DNA (ACT1) were amplified by PCR in the parental ∆dnm1∆fzo1 petite strains used for the complementation analysis. (C) Analysis of small mtDNA variations (SMVs) by next-generation sequencing. Total number of SMVs (Left) and number of nonsynonymous SMVs detected in protein coding regions (Right) of WT and ∆dnm1∆fzo1 cells. The amount of SMVs was normalized to the number of reads that aligned to the mtDNA in each sample. (D) The mutational landscapes of mtDNA in WT and ∆dnm1∆fzo1 cells. The outer ring represents the mitochondrial genome, and features are indicated. The histograms in the inner rings show the percentage of detected SMV’s binned into 100-bp segments for WT (blue) and ∆dnm1∆fzo1 (orange). The arcs in the middle represent structural variations (compare Table S2) for WT (light orange) and ∆dnm1∆fzo1 (shades of blue). Each end of the arc points to the region of mtDNA to which each of the paired reads aligned. These regions are fused in the mutant mtDNA that gave rise to the respective sequencing reads. For ∆dnm1∆fzo1, a darker shade of blue indicates that these variations were detected more often (compare Table S2). A region of the diagram is enlarged to illustrate SMVs and structural variations at two of the origins of replication. Promoter elements associated with the origins are indicated by a black bar.