A new tool reveals the distribution and dynamics of mitochondrial DNA in living cells [with video]

Mitochondrial DNA (mtDNA) encodes essential subunits of respiratory complexes, which are responsible for the generation of ATP through oxidative phosphorylation in mitochondria. Copies of mtDNA are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle, processes previously believed to require mitochondrial dynamics driven by fusion and fission of the organelle. We have developed a novel tool in Saccharomyces cerevisiae that allows us to watch mtDNA dynamics in living cells and to characterize its distribution and inheritance. We show that, surprisingly, mitochondrial fusion and fission are dispensable for both processes. The absence of fusion and fission events, however, leads to the accumulation of rearranged and dysfunctional mitochondrial genomes. These results reveal crucial roles of mitochondrial fusion and fission in maintaining the quality and integrity of the mitochondrial genome.

HFSP Long-Term Fellow Christof Osman and colleagues
authored on Thu, 02 April 2015

Mitochondria contain their own genome, which is present in multiple copies in each cell. These copies are distributed in the form of nucleoprotein complexes, termed nucleoids, throughout the mitochondrial network that adopts a tubular and reticulated morphology in most cells. The mitochondrial genome encodes several subunits of the respiratory chain and it is therefore essential for mitochondria to fulfill their most prominent role - the generation of ATP through oxidative phosphorylation. Accordingly, mutations within mitochondrial DNA can lead to defective respiratory chains, which in turn has pleiotropic effects on cell physiology. Thus, it is not surprising that such mutations have been linked to a variety of human diseases and ageing. Quite astonishingly, given the fundamental importance of mtDNA for cell physiology, it is poorly understood how the integrity of the mitochondrial genome is maintained, how it is distributed throughout the mitochondrial network and how it is faithfully inherited by daughter cells during cell division. Part of the reason for our poor understanding of these processes is that good methods to follow the dynamics of mtDNA in living cells have been lacking to date.

Figure caption: (A) Schematic representation of the mt-LacO-LacI system. mtDNA is shown as a circle. Genes encoding proteins or RNA are indicated in blue or red, respectively. Fission-independent (B) and fission-dependent (C) generation of mtDNA loaded mitochondrial tips. The mitochondrial matrix is shown in red and the mitochondrial intermembrane space is shown in yellow.

To eliminate this methodological shortcoming and to characterize the dynamics associated with mtDNA, we have developed a novel tool in the model organism S. cerevisiae that allows us to non-invasively follow mtDNA by live-cell microscopy. This system is based on a non-recombinable array of LacO-repeats, which we have integrated into the mitochondrial genome. These repeats can be bound by mitochondrially targeted 3xGFP-LacI proteins, which leads to accumulation of GFP at the LacO arrays that can be observed as clearly discernible foci by fluorescent microscopy (see Figure A and video). We used this system to quantitatively describe the distribution of mtDNA within the mitochondrial network and find that nucleoids are non-randomly distributed throughout the mitochondrial network (see video), indicating that mechanisms are in place that appropriately space nucleoids. Furthermore, we were able, for the first time, to follow the inheritance of mtDNA in living cells. We find that mtDNA localizes to the leading tip of the first mitochondrial tubule, which invades the daughter cell. These findings suggest that transport of mitochondria is coupled to the presence of mtDNA, which ensures faithful inheritance of mtDNA.

The tubular mitochondrial network is highly dynamic and is constantly remodeled by fusion and fission events, which has been proposed to be important for the distribution and inheritance of mtDNA. mtDNA localizes to sites of mitochondrial fission and is segregated to both of the resulting mitochondrial tips (Figure C). It has been hypothesized that mtDNA-loaded tips are transported to distant places within the cell and re-fusion with the mitochondrial network drives distribution of mtDNA. We directly examined the importance of mitochondrial fusion and fission for the distribution of mtDNA by utilizing our mt-LacO-LacI system in cells lacking the key components required for mitochondrial fusion and fission, Fzo1 and Dnm1, respectively. Surprisingly, we find that these cells displayed a normal distribution of mtDNA (see video) and that the inheritance of mtDNA occurred in the tip of a mitochondrial tubule, indistinguishable from WT cells. Live-cell microscopy revealed that cells possess a fission-independent mechanism for mitochondrial tip generation, where tips are pulled out of existing tubules in an actin-dependent manner. This form of mitochondrial tip generation is similarly linked to localization of mtDNA to the resulting mitochondrial tips (Figure B). These results indicate that fission-independent tip generation is sufficient for cells to maintain a normal distribution and inheritance of mtDNA in the absence of mitochondrial fusion and fission.

While the apparent distribution and inheritance of mtDNA in fusion and fission deficient cells was unaffected, we uncovered through a combination of yeast genetics and next generation sequencing that these cell accumulated compromised mitochondrial genomes that were characterized by rearrangements of the mtDNA. Taken together, our work shows that mitochondrial fusion and fission are dispensable for the distribution and inheritance of the mitochondrial genome, but that they are required for maintaining the integrity of the mitochondrial genome. Based on these findings, we propose that fusion and fission are involved in the quality control of the mitochondrial genome, which may involve selective removal of mitochondrial fragments containing dysfunctional mitochondrial genomes or their retention in the mother cell.

In conclusion, the development of the mt-LacO-LacI system facilitated important novel insights into the dynamics of mtDNA, which pave the way for the elucidation of the molecular mechanisms that underlie mtDNA distribution, inheritance and the maintenance of its integrity. Given the importance of these features of mtDNA for cellular function, such analyses promise to shed light onto disease states, where these processes may be dysregulated.

Video: The distribution of mtDNA in wildtype and fusion and fission deficient cells (∆dnm1fzo1) yeast cells. 3D reconstructions of microscopic images are shown. mtDNA as visualized with the mt-LacO-LacI system is shown in green and mitochondria as visualized with mitochondrially targeted dsRed is shown in red.


Integrity of the yeast mitochondrial genome, but not its distribution and inheritance, relies on mitochondrial fission and fusion. Osman, C., Noriega, T. R., Okreglak, V., Fung, J. C., & Walter, P. (2015). Proceedings of the National Academy of Sciences of the United States of America, 112(9), E947–956. doi:10.1073/pnas.1501737112.

Link to PNAS (open access)

Link to Pubmed