Periodic Reporting for period 4 - IlluMitoDNA (Illuminating the mechanisms of mitochondrial DNA quality control and inheritance)
Periodo di rendicontazione: 2022-02-01 al 2023-07-31
Mitochondria, often referred to as the powerhouses of eukaryotic cells, play a pivotal role in energy production through a remarkable process known as oxidative phosphorylation (OXPHOS), which relies on several large protein complexes that eventually produce ATP. While numerous OXPHOS proteins originate from the nuclear genome and are subsequently transported into the mitochondria, key subunits of the OXPHOS complexes are encoded within the mitochondrial genome (mtDNA). Remarkably, mtDNA exists in multiple copies, dispersed throughout a tubular and reticulated mitochondrial network (as illustrated for S.cerevisiae in Fig. 1).
Given the indispensability of mtDNA to OXPHOS, it becomes imperative for cells to uphold an adequate number of mtDNA copies and ensure their equitable distribution within the mitochondrial network. This organisation is vital to guarantee a consistent supply of mitochondrial subregions with mtDNA-encoded OXPHOS components. Equally critical is the necessity to thwart the accumulation of mutant mtDNA copies, which can severely compromise cellular energy supply. The gravity of this matter is underscored by the connection between mtDNA mutations and mitochondrial diseases and the aging process.
However, intriguingly, fundamental questions pertaining to the distribution, control of mtDNA copy number, and the preservation of its integrity across generations remain enigmatic. What precisely are the molecular mechanisms governing mtDNA copy number? How does mtDNA withstand the relentless assault of reactive oxygen species, abundant within mitochondria, for generations?
To tackle these mysteries, we harnessed the unique advantages of the budding yeast, S. cerevisiae, as our model organism. This choice allowed us to exploit its genetic tractability, facilitating the manipulation of mtDNA.
We firmly believe that the insights gleaned from unraveling the mechanisms underpinning mtDNA copy number, distribution, and preservation across generations in yeast will not only expand our understanding of this fundamental biological process but also pave the way for a deeper comprehension of mitochondrial disorders in humans where these processes go awry.
Given the indispensability of mtDNA to OXPHOS, it becomes imperative for cells to uphold an adequate number of mtDNA copies and ensure their equitable distribution within the mitochondrial network. This organisation is vital to guarantee a consistent supply of mitochondrial subregions with mtDNA-encoded OXPHOS components. Equally critical is the necessity to thwart the accumulation of mutant mtDNA copies, which can severely compromise cellular energy supply. The gravity of this matter is underscored by the connection between mtDNA mutations and mitochondrial diseases and the aging process.
However, intriguingly, fundamental questions pertaining to the distribution, control of mtDNA copy number, and the preservation of its integrity across generations remain enigmatic. What precisely are the molecular mechanisms governing mtDNA copy number? How does mtDNA withstand the relentless assault of reactive oxygen species, abundant within mitochondria, for generations?
To tackle these mysteries, we harnessed the unique advantages of the budding yeast, S. cerevisiae, as our model organism. This choice allowed us to exploit its genetic tractability, facilitating the manipulation of mtDNA.
We firmly believe that the insights gleaned from unraveling the mechanisms underpinning mtDNA copy number, distribution, and preservation across generations in yeast will not only expand our understanding of this fundamental biological process but also pave the way for a deeper comprehension of mitochondrial disorders in humans where these processes go awry.
In the first part of our project, we delved into the intricate process of maintaining mitochondrial DNA (mtDNA) across cell generations, focusing on a critical but poorly understood aspect—regulating the correct number of mtDNA copies within cells. We embarked on a systematic analysis of approximately 5,000 mutant yeast strains to pinpoint the genes that are essential for maintaining normal cellular mtDNA levels. This comprehensive screen unveiled several mutants with elevated levels of mtDNA. Among them, we narrowed our focus to two enigmatic genes: MRX6 and CIM1.
Our investigation into MRX6-deficient cells unveiled clusters of mtDNA copies, indicating a deficiency in segregating replicated mtDNA copies. We found that Mrx6 interacts physically with the evolutionarily conserved protease Pim1, a central player in mitochondrial protein quality control. Depleting Pim1 mirrored the effects of MRX6 deficiency on mtDNA levels, and further depletion of MRX6 did not exacerbate the situation. These findings support a model in which MRX6 regulates mtDNA levels by modulating Pim1-mediated protein degradation.
Our attention then shifted to the CIM1 gene, identified in our systematic analysis as pivotal in regulating mtDNA copy number. Surprisingly, we discovered that Cim1 requires two HMG boxes for its function, akin to the well-characterized mammalian mtDNA packaging factor TFAM and its yeast counterpart, Abf2. Surprisingly, we found that Abf2 and Cim1 form an antagonistic relationship in which Abf2 promotes and Cim1 reduces mtDNA copy number. We observed that Pim1-mediated regulation keeps Cim1 levels low within cells, presumably to prevent toxic effects on cellular mtDNA maintenance. Taken together, our findings underscore the pivotal role of the conserved Lon protease, Pim1, in mtDNA copy number regulation.
In a second part of this project, we set out to unravel how S. cerevisiae cells maintain pristine mtDNA. We introduced innovative single-cell assays and unearthed a fascinating capability of yeast cells to detect mutant mtDNA and support the generation of progeny predominantly harboring intact mtDNA. This discovery raised the intriguing question of how cells differentiate between the quality of various mtDNA copies within the same mitochondrial network. To address this question, we engineered yeast strains expressing fluorescently-tagged mtDNA-encoded subunits and mated them with cells expressing untagged counterparts. This approach revealed the strikingly limited mobility of fluorescent mtDNA-encoded proteins within mitochondria, which depended on mitochondrial cristae, invaginations of the inner mitochondrial membrane. Based on these experimental findings, we formulated "The Sphere of Influence Model" model (Fig. 2). We propose that mtDNA-encoded proteins remain in close proximity to the copy by which they are encoded in a cristae-dependent manner, creating a spatial genotype-phenotype link. Dysfunctional mtDNA-encoded subunits will thus result in local OXPHOS complexes defects, which can be detected by cellular quality control pathways. Our study provides a foundational insight into the mechanisms facilitating the detection of low-functioning mitochondria, a compelling avenue for future research.
Our investigation into MRX6-deficient cells unveiled clusters of mtDNA copies, indicating a deficiency in segregating replicated mtDNA copies. We found that Mrx6 interacts physically with the evolutionarily conserved protease Pim1, a central player in mitochondrial protein quality control. Depleting Pim1 mirrored the effects of MRX6 deficiency on mtDNA levels, and further depletion of MRX6 did not exacerbate the situation. These findings support a model in which MRX6 regulates mtDNA levels by modulating Pim1-mediated protein degradation.
Our attention then shifted to the CIM1 gene, identified in our systematic analysis as pivotal in regulating mtDNA copy number. Surprisingly, we discovered that Cim1 requires two HMG boxes for its function, akin to the well-characterized mammalian mtDNA packaging factor TFAM and its yeast counterpart, Abf2. Surprisingly, we found that Abf2 and Cim1 form an antagonistic relationship in which Abf2 promotes and Cim1 reduces mtDNA copy number. We observed that Pim1-mediated regulation keeps Cim1 levels low within cells, presumably to prevent toxic effects on cellular mtDNA maintenance. Taken together, our findings underscore the pivotal role of the conserved Lon protease, Pim1, in mtDNA copy number regulation.
In a second part of this project, we set out to unravel how S. cerevisiae cells maintain pristine mtDNA. We introduced innovative single-cell assays and unearthed a fascinating capability of yeast cells to detect mutant mtDNA and support the generation of progeny predominantly harboring intact mtDNA. This discovery raised the intriguing question of how cells differentiate between the quality of various mtDNA copies within the same mitochondrial network. To address this question, we engineered yeast strains expressing fluorescently-tagged mtDNA-encoded subunits and mated them with cells expressing untagged counterparts. This approach revealed the strikingly limited mobility of fluorescent mtDNA-encoded proteins within mitochondria, which depended on mitochondrial cristae, invaginations of the inner mitochondrial membrane. Based on these experimental findings, we formulated "The Sphere of Influence Model" model (Fig. 2). We propose that mtDNA-encoded proteins remain in close proximity to the copy by which they are encoded in a cristae-dependent manner, creating a spatial genotype-phenotype link. Dysfunctional mtDNA-encoded subunits will thus result in local OXPHOS complexes defects, which can be detected by cellular quality control pathways. Our study provides a foundational insight into the mechanisms facilitating the detection of low-functioning mitochondria, a compelling avenue for future research.
Recent years have seen limited progress in unraveling the mechanistic intricacies of mtDNA copy number control, largely due to the scarcity of knowledge concerning the key players in this intricate process. Our systematic screening in S. cerevisiae has successfully surmounted this barrier by unearthing a plethora of proteins that may play pivotal roles in mtDNA copy number regulation. Notably, our analysis of Mrx6 and Cim1 has already demonstrated that the screen has led us to components intricately connected to mtDNA copy number control, rendering it a valuable resource for future investigations.
The discovery of Cim1 is particularly noteworthy as it challenges the long-held assumption that mitochondrial HMG box-containing proteins like TFAM (in mammals) and Abf2 (in yeast) primarily govern mtDNA copy number regulation.
Our studies on Mrx6 are equally significant for two reasons. Firstly, they establish a connection between the highly conserved Lon protease, Pim1, and mtDNA copy number regulation, shedding light on an important regulatory mechanism. Secondly, these findings may serve as a catalyst for future research investigating how Lon proteases are regulated to target specific substrates. Mrx6, belonging to the Pet20 protein family, raises the intriguing possibility that all these proteins might modulate Pim1 according to cellular demand, which is a promising avenue for further study.
Our experimental validation of the "Sphere of Influence Model" and its role in mtDNA quality control marks a substantial leap forward in our comprehension of how cells uphold the integrity of mtDNA. This breakthrough will inspire future research aimed at testing the impact of tightening or loosening the "Sphere of Influence" on mtDNA quality maintenance. Given the critical importance of intact mtDNA for both cellular and organismal health, these findings have the potential to catalyze experiments exploring ways to enhance mtDNA quality control, with the promise of positive outcomes for organisms.
The discovery of Cim1 is particularly noteworthy as it challenges the long-held assumption that mitochondrial HMG box-containing proteins like TFAM (in mammals) and Abf2 (in yeast) primarily govern mtDNA copy number regulation.
Our studies on Mrx6 are equally significant for two reasons. Firstly, they establish a connection between the highly conserved Lon protease, Pim1, and mtDNA copy number regulation, shedding light on an important regulatory mechanism. Secondly, these findings may serve as a catalyst for future research investigating how Lon proteases are regulated to target specific substrates. Mrx6, belonging to the Pet20 protein family, raises the intriguing possibility that all these proteins might modulate Pim1 according to cellular demand, which is a promising avenue for further study.
Our experimental validation of the "Sphere of Influence Model" and its role in mtDNA quality control marks a substantial leap forward in our comprehension of how cells uphold the integrity of mtDNA. This breakthrough will inspire future research aimed at testing the impact of tightening or loosening the "Sphere of Influence" on mtDNA quality maintenance. Given the critical importance of intact mtDNA for both cellular and organismal health, these findings have the potential to catalyze experiments exploring ways to enhance mtDNA quality control, with the promise of positive outcomes for organisms.