How Mitochondria Protect Themselves from Mutations
by Ross Anderson, PhD
Mitochondria are very interesting and unique organelles. All eukaryotic cells possess them. They are indispensable in a number of cell activities beyond their well-known role of synthesizing the bulk of ATP used to power cells. Mitochondria reproduce at rates that are, for the most part, independent of the cell division cycle. However, they always manage to generate about twice the number just before the time of mitosis and cytokinesis so that each daughter cell receives approximately equal numbers of mitochondria. How this is regulated and coordinated is unknown.
The Mitochondrial Genome
Mitochondria have their own genomes separate from that in the nucleus. Each mitochondrion may have 5 to 10 copies of the genome organized into clusters called nucleoids, and a cell may have a thousand mitochondria. This means that each cell may have 5,000 to 10,000 copies of the mitochondrial genome! At conception it is estimated that mammals inherit 100,000 to 500,000 mitochondrial genomes via the egg!
The Problem of Mutational Degradation
Due to mutations, it is possible for a single mitochondrion to have “healthy” copies as well as defective/mutated copies. Such cells with two or more distinct varieties of mitochondrial DNA (mtDNA) are said to be heteroplasmic. Mitochondrial DNA encodes various protein subunits used for ATP production, their own transfer RNAs (tRNA), and ribosomal RNAs (rRNA). Most of the proteins used in the mitochondria, however, are encoded in nuclear genes.
Like nuclear DNA, mtDNA mutates, but at a much greater rate; 5 to 10 times greater than the genes in the nucleus. This may possibly be due to lower fidelity in DNA replication, lower efficiency in DNA repair mechanisms, or both. There are a number of disease states that are the result of too many mutated mitochondrial genes.
Mitochondria of mammalian cells and fruit flies, indeed most eukaryotes, are inherited from the mother’s egg cells. These diseases then are inherited through the mother. Consequently, it is imperative that those mitochondria be as “healthy” genetically as possible. If a mitochondrion generates more mutated copies than can be tolerated, there must be a mechanism for recognizing these defective mitochondria and destroying them so that only “healthy” mitochondria are maintained in the egg and passed on to the next generation. The high rates of mutation would suggest that most cells would exhibit heteroplasmy, but this is not the case. Most cells are homoplastic; i.e., they have mitochondria with identical genomes. Thus, there must be a way that cells are able to identify mitochondria with defective DNA, keep them from reproducing and eliminate them. The cells, in effect, create a bottleneck through which only “healthy” mitochondria are permitted to pass.
Forcing Mitochondria Through a Bottleneck Inspection
The authors of a letter in Nature by Lieber et al. performed a series of ingenious experiments investigating factors that may be involved in identifying defective mitochondria; i.e., creating the bottleneck. They transplanted wild-type mitochondria from one species of fruit fly, D. yakuba, into another species of fruit fly, D. melanogaster, which already had mitochondrial genomes with a temperature-sensitive point mutation in the cytochrome c oxidase subunit I gene. Flies grown at the permissive temperature (18○C) exhibited little effect, while those grown at the restrictive temperature (29○C) exhibited reduced cytochrome c oxidase activity.
When grown at the restrictive temperature, they found there was a marked increase in the proportion of wild-type mitochondrial DNA from D. yakuba relative to the defective mtDNA, but only in the germline cells—not somatic cells. This indicated that the defective mtDNA was somehow selectively detected and destroyed.
Whether from an evolutionary or a design perspective, this makes perfect sense. Somatic cells can tolerate defective mitochondria more than germline cells because only that cell would be affected; it would not necessarily impact the next generation. Germline cells, though, would carry the defect into the next generation. Not surprisingly, it was also found that detection and destruction of defective mtDNA was limited to female germline cells, not male germline cells.
Selection for destruction appeared to initiate in the very early stages of egg development; after the stem cell stage and during the cyst formation stage. Further experiments revealed that selection was at the level of whole mitochondria—not just defective DNA. Thus, it was the defective mitochondria that were being detected and destroyed.
How Fragmentation Rescues Healthy mtDNA
The morphology of the mitochondrion changes from the stem cell stage to the cyst stage. The authors postulate that this change is due to fragmentation of the mitochondria, such that defective mitochondria are more easily detected. Additionally, from the 2-to-8-cell stage in the cyst, mtDNA is not replicated. This reduces the number of genomes per mitochondrion and decreases the possibility of both defective and wild-type genomes residing in the same mitochondrion. This strategy leads to improved selection. Mitochondria in the stem cell stage (which occurs prior to the cyst stage) were shown to share their contents easily. Fragmentation in the cyst cell stage, however, decreases the possibility of defective and wild-type mitochondria from sharing their contents.
In this respect the stem cells are more like somatic cells. The reasoning is that in somatic cells mitochondria with defective DNA can fuse and share their contents, thus a “healthy” mitochondrion can complement and rescue a mitochondrion with defective DNA. Fusion cannot be permitted in germline cells, because the defective mtDNA would be masked by the “healthy” DNA, allowing mutations to accrue and to be passed on to each subsequent generation. The authors suggest that fragmentation, in some unknown way, aids in distinguishing defective from wild-type mitochondria.
The authors were able to show that either overexpression of the protein Mitofusin (a protein involved in the fusion of mitochondria, and suppression of fragmentation), or the reduced expression of DrpI (a protein involved in mitochondrial fission; promotes fragmentation) led to loss of selection. This, again, suggested that a time of sustained fragmentation is necessary for selection. It was shown that mitochondrial fragmentation is not only necessary, but also sufficient for effective selection against defective mtDNA.
Interestingly, experimentally reduced expression of Mitofusin in somatic cells also led to sustained fragmentation and subsequent selection against defective mtDNA. However, a reduction in Mitofusin expression (both the protein and its mRNA) is normally found only in the germline cells at the cyst cell stage; i.e., this reduction in Mitofusin is observed selectively in germline cells, not somatic cells.
Design in the Timing, Too
This observation strongly suggests a design feature for the purpose of generating a period of sustained fragmentation early in egg development so that selection against defective mtDNA can be more effective. Further investigation is needed to ascertain just how defective mtDNA is identified. However, the authors were able to determine that reduction in ATP production is sufficient to induce selection. This makes sense considering that most of the protein-coding genes of mitochondria are for subunits of the ATP synthase rotary engines or for the other complex machinery of the electron transport chain.
Another protein, BNIP3, was also found to be selectively upregulated in cyst cells. This protein is located in the mitochondrial outer membrane. It plays an important role in mitophagy [mitochondrial recycling] in somatic cells as when maturing red blood cells need to rid themselves of all their organelles including mitochondria.
So, the selective reduction of Mitofusin expression and increased expression of DrpI and BNIP3 at the same time and in the same select group of cells (cyst cells) smacks of design! We must also keep in mind that these proteins do not work alone, but rather in coordinated complexes with a number of other proteins designed to carry out a specific function at a specific time and place. If that’s not enough, these authors also mention reports that another protein, Pink1, may recognize and inhibit replication of the defective mtDNA, which in some unknown way stimulates replication of the wild-type DNA so that the “healthy” DNA can dominate.
Design in the Cleanup
Once mitochondria carrying defective DNA have been selected, they are destroyed by a process known as mitophagy, a type of autophagy whereby certain organelles are selectively destroyed, and their contents recycled by the cell. Pink1 in conjunction with another protein, Parkin, initiate mitophagy. In healthy mitochondria, Pink1 is imported into the matrix of the mitochondrion, but is not imported by defective mitochondria. Instead it accumulates on the outer surface of the outer membrane, recruits Parkin which, in turn, adds ubiquitin tags to outer membrane proteins targeting them for destruction.
It appears that the ability of a developing egg cell to create the genetic bottleneck leading to homoplastic cells is its ability to initiate sustained fragmentation whereby mitochondria containing defective DNA are selectively eliminated. Taken in toto with other information not in the article, it seems to me that the design inference is strong.
This article, like virtually all others of its kind, addresses the scientific questions of ‘What is it? What does it do?’ And ‘How does it do it?’ While we slowly learn more and more answers to these questions, and someday may be able to describe in intimate detail the inner workings of cells, we will never be able to accurately answer the question of ‘How it came to be?’ short of invoking the genius of an all-powerful, creative designer.
The authors, unfortunately, felt the need to attribute the process of selective identification and destruction of aberrant mtDNA to the all-powerful deity, Evolution, and thus gave their token bow. I find it simply amazing how so many intelligent researchers can report many of the detailed features of living creatures yet still attribute it to chance and state that any inference to design is only an illusion. All researchers need to take a few steps back and look at the forest, and not be so focused on the trees.
Ross Anderson (PhD biochemistry) is professor of biochemistry at The Master’s University in southern California. Dr Anderson’s expertise is in the area of biochemistry and molecular biology. He has taught Biochemistry and helped to direct research projects of graduate and medical students at Baylor College of Medicine, Houston, TX. Dr. Anderson was a post-doctoral researcher in the Molecular Genetics Division of the Department of Ophthalmology at the Houston Neurosensory Center.
Dr Anderson was a member of both the undergraduate and graduate faculty at Lamar University, Beaumont, TX. There he taught and directed the research activities of undergraduates and Masters of Science degree candidates in Biology. Currently he is professor of biochemistry at The Master’s University in southern California.
Dr Anderson’s research interests include structure-function studies of DNA polymerizing enzymes and the synthesis and expression of synthetic human genes in bacterial hosts. He has authored or co-authored several publications in major, peer-reviewed journals. He is a member of the American Chemical Society and Sigma Xi Research Society.