Raquel Moreno-Loshuertos

Supercomplex Assembly Determines Electron Flux in the Mitochondrial Electron Transport Chain

Respiration Refined Cells derive energy from redox reactions mediated by mitochondrial enzymes that form the electron transport chain. The enzymes can form large complexes, known as supercomplexes, whose function has been controversial. Lapuente-Brun et al. (p. 1567) discovered that a mouse protein, supercomplex assembly factor I (SCAFI), specifically modulates assembly of respiratory complexes into supercomplexes. Formation of the supercomplexes appears to cause electrons to be processed differently, depending on the substrate from which they are derived. Ordered formation of supercomplexes of respiratory enzymes influences metabolic efficiency in response to food supply. The textbook description of mitochondrial respiratory complexes (RCs) views them as free-moving entities linked by the mobile carriers coenzyme Q (CoQ) and cytochrome c (cyt c). This model (known as the fluid model) is challenged by the proposal that all RCs except complex II can associate in supercomplexes (SCs). The proposed SCs are the respirasome (complexes I, III, and IV), complexes I and III, and complexes III and IV. The role of SCs is unclear, and their existence is debated. By genetic modulation of interactions between complexes I and III and III and IV, we show that these associations define dedicated CoQ and cyt c pools and that SC assembly is dynamic and organizes electron flux to optimize the use of available substrates.

Respiratory Complex III Is Required to Maintain Complex I in Mammalian Mitochondria

A puzzling observation in patients with oxidative phosphorylation (OXPHOS) deficiencies is the presence of combined enzyme complex defects associated with a genetic alteration in only one protein-coding gene. In particular, mutations in the mtDNA encoded cytochrome b gene are associated either with combined complex I+III deficiency or with only complex III deficiency. We have reproduced the combined complex I+III defect in mouse and human cultured cell models harboring cytochrome b mutations. In both, complex III assembly is impeded and causes a severe reduction in the amount of complex I, not observed when complex III activity was pharmacologically inhibited. Metabolic labeling in mouse cells revealed that complex I was assembled, although its stability was severely hampered. Conversely, complex III stability was not influenced by the absence of complex I. This structural dependence among complexes I and III was confirmed in a muscle biopsy of a patient harboring a nonsense cytochrome b mutation.

Differences in reactive oxygen species production explain the phenotypes associated with common mouse mitochondrial DNA variants

Common mitochondrial DNA (mtDNA) haplotypes in humans and mice have been associated with various phenotypes, including learning performance and disease penetrance. Notably, no influence of mtDNA haplotype in cell respiration has been demonstrated. Here, using cell lines carrying four different common mouse mtDNA haplotypes in an identical nuclear background, we show that the similar level of respiration among the cell lines is only apparent and is a consequence of compensatory mechanisms triggered by different production of reactive oxygen species. We observe that the respiration capacity per molecule of mtDNA in cells with the NIH3T3 or NZB mtDNA is lower than in those with the C57BL/6J, CBA/J or BALB/cJ mtDNA. In addition, we have determined the genetic element underlying these differences. Our data provide insight into the molecular basis of the complex phenotypes associated with common mtDNA variants and anticipate a relevant contribution of mtDNA single nucleotide polymorphisms to phenotypic variability in humans.

Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing

Human mitochondrial DNA (mtDNA) shows extensive within-population sequence variability. Many studies suggest that mtDNA variants may be associated with ageing or diseases, although mechanistic evidence at the molecular level is lacking. Mitochondrial replacement has the potential to prevent transmission of disease-causing oocyte mtDNA. However, extension of this technology requires a comprehensive understanding of the physiological relevance of mtDNA sequence variability and its match with the nuclear-encoded mitochondrial genes. Studies in conplastic animals allow comparison of individuals with the same nuclear genome but different mtDNA variants, and have provided both supporting and refuting evidence that mtDNA variation influences organismal physiology. However, most of these studies did not confirm the conplastic status, focused on younger animals, and did not investigate the full range of physiological and phenotypic variability likely to be influenced by mitochondria. Here we systematically characterized conplastic mice throughout their lifespan using transcriptomic, proteomic, metabolomic, biochemical, physiological and phenotyping studies. We show that mtDNA haplotype profoundly influences mitochondrial proteostasis and reactive oxygen species generation, insulin signalling, obesity, and ageing parameters including telomere shortening and mitochondrial dysfunction, resulting in profound differences in health longevity between conplastic strains.

Five Entry Points of the Mitochondrially Encoded Subunits in Mammalian Complex I Assembly

ABSTRACT Complex I (CI) is the largest enzyme of the mammalian mitochondrial respiratory chain. The biogenesis of the complex is a very complex process due to its large size and number of subunits (45 subunits). The situation is further complicated due to the fact that its subunits have a double genomic origin, as seven of them are encoded by the mitochondrial DNA. Understanding of the assembly process and characterization of the involved factors has advanced very much in the last years. However, until now, a key part of the process, that is, how and at which step the mitochondrially encoded CI subunits (ND subunits) are incorporated in the CI assembly process, was not known. Analyses of several mouse cell lines mutated for three ND subunits allowed us to determine the importance of each one for complex assembly/stability and that there are five different steps within the assembly pathway in which some mitochondrially encoded CI subunit is incorporated.

Evolution Meets Disease: Penetrance and Functional Epistasis of Mitochondrial tRNA Mutations

About half of the mitochondrial DNA (mtDNA) mutations causing diseases in humans occur in tRNA genes. Particularly intriguing are those pathogenic tRNA mutations than can reach homoplasmy and yet show very different penetrance among patients. These mutations are scarce and, in addition to their obvious interest for understanding human pathology, they can be excellent experimental examples to model evolution and fixation of mitochondrial tRNA mutations. To date, the only source of this type of mutations is human patients. We report here the generation and characterization of the first mitochondrial tRNA pathological mutation in mouse cells, an m.3739G>A transition in the mitochondrial mt-Ti gene. This mutation recapitulates the molecular hallmarks of a disease-causing mutation described in humans, an m.4290T>C transition affecting also the human mt-Ti gene. We could determine that the pathogenic molecular mechanism, induced by both the mouse and the human mutations, is a high frequency of abnormal folding of the tRNAIle that cannot be charged with isoleucine. We demonstrate that the cells harboring the mouse or human mutant tRNA have exacerbated mitochondrial biogenesis triggered by an increase in mitochondrial ROS production as a compensatory response. We propose that both the nature of the pathogenic mechanism combined with the existence of a compensatory mechanism can explain the penetrance pattern of this mutation. This particular behavior can allow a scenario for the evolution of mitochondrial tRNAs in which the fixation of two alleles that are individually deleterious can proceed in two steps and not require the simultaneous mutation of both.

Respiratory supercomplexes and the functional segmentation of the CoQ pool

The evidence accumulated during the last fifteen years on the existence of respiratory supercomplexes and their proposed functional implications has changed our understanding of the OXPHOS system complexity and regulation. The plasticity model is a point of encounter accounting for the apparently contradictory experimental observations claimed to support either the solid or the fluid models. It allows the explanation of previous observations such as the dependence between respiratory complexes, supercomplex assembly dynamics or the existence of different functional ubiquinone pools. With the general acceptation of respiratory supercomplexes as true entities, this review evaluates the supporting evidences in favor or against the existence of different ubiquinone pools and the relationship between supercomplexes, ROS production and pathology.

Length variation in the mouse mitochondrial tRNAArg DHU loop size promotes oxidative phosphorylation functional differences

The efficiency of the cellular oxidative phosphorylation system was recently shown to be modulated by common mitochondrial tRNAArg haplotypes. The molecular mechanism by which some mt‐Tr haplotypes induce these functional differences remains undetermined. Common polymorphisms in mouse mt‐Tr genes affect the size of the dihydrouridine loop in the mature tRNA, producing loops of between five and seven nucleotides, the largest being a rare variant among mammals. Here, we analyzed a new mt‐Tr variant identified in C3H mice, and found that it is mitochondrial tRNA loop size, but not the specific sequence, that is responsible for the observed differences in cellular respiration. We further found that the sensitivity of mitochondrial protein synthesis to specific inhibitors is dependent on the mt‐Tr gene haplotype, and confirmed that the differences in oxidative phosphorylation performance are masked by a reactive oxygen species‐induced compensatory increase in mitochondrial biogenesis.

Corrigendum: Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing

This corrects the article DOI: 10.1038/nature18618

Structural biology response of a collagen hydrogel synthetic extracellular matrix with embedded human fibroblast: computational and experimental analysis

Adherent cells exert contractile forces which play an important role in the spatial organization of the extracellular matrix (ECM). Due to these forces, the substrate experiments a volume reduction leading to a characteristic shape. ECM contraction is a key process in many biological processes such as embryogenesis, morphogenesis and wound healing. However, little is known about the specific parameters that control this process. With this aim, we present a 3D computational model able to predict the contraction process of a hydrogel matrix due to cell–substrate mechanical interaction. It considers cell-generated forces, substrate deformation, ECM density, cellular migration and proliferation. The model also predicts the cellular spatial distribution and concentration needed to reproduce the contraction process and confirms the minimum value of cellular concentration necessary to initiate the process observed experimentally. The obtained continuum formulation has been implemented in a finite element framework. In parallel, in vitro experiments have been performed to obtain the main model parameters and to validate it. The results demonstrate that cellular forces, migration and proliferation are acting simultaneously to display the ECM contraction.