Mitochondrial DNA nucleoids determine mitochondrial genetics and dysfunction

Mitochondrial DNA plays a crucial role in cellular homeostasis; however, the molecular mechanisms underlying mitochondrial DNA inheritance and propagation are only beginning to be understood. To ensure the distribution and propagation of the mitochondrial genome, mitochondrial DNA is packaged into macromolecular assemblies called nucleoids, composed of one or more copies of mitochondrial DNA and associated proteins. We review current research on the mitochondrial nucleoid, including nucleoid-associated proteins, nucleoid dynamics within the cell, potential mechanisms to ensure proper distribution of nucleoids, and the impact of nucleoid organization on mitochondrial dysfunction. The nucleoid is the molecular organizing unit of mitochondrial genetics, and is the site of interactions that ultimately determine the bioenergetic state of the cell as a whole. Current and future research will provide essential insights into the molecular and cellular interactions that cause bioenergetic crisis, and yield clues for therapeutic rescue of mitochondrial dysfunction.     

TFAM knockdown-triggered mtDNA-nucleoid aggregation and a decrease in mtDNA copy number induce the reorganization of nucleoid populations and mitochondria-associated ER-membrane contacts

The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution.     

Proteins associated with mitochondrial DNA protect it against the action of X-rays and hydrogen peroxide

It has been suggested in a number of investigations that the high vulnerability of mitochondrial DNA to reactive oxygen species and other damaging agents is due to the absence in mitochondria of histones complexed with DNA. In the present study it was shown that DNA-binding proteins of mitochondrial nucleoids were able to shield mitochondrial DNA from X-ray radiation and hydrogen peroxide, as nuclear histones did. Mitochondria, mitochondrial nucleoid proteins, and histones were isolated from mouse liver cells. The degree of damage to or protection of mitochondrial DNA was assessed from the yield of its PCR amplification product. The in vitro experiments demonstrated that mouse mitochondrial DNA, when in complex with mitochondrial nucleoids or nuclear histones, was damaged much less by radiation and/or hydrogen peroxide than in the absence of these proteins and histones. No significant difference between mitochondrial nucleoid proteins and nuclear histones was revealed in their efficiency to protect mitochondrial DNA from the damaging effect of radiation and hydrogen peroxide. It is likely that the nucleoid proteins in the mitochondria shield mitochondrial DNA against the attack of reactive oxygen species, thus significantly decreasing the level of the oxidative damage to mitochondrial DNA.     

线粒体拟核结构及相关疾病研究进展

线粒体DNA(mitochondrial DNA,mtDNA)与一系列蛋白质相互作用形成核蛋白复合体,并包装折叠成类似原核生物拟核的结构,称为线粒体拟核(mitochondrial nucleoid)。参与线粒体拟核组成的相关蛋白包括线粒体转录因子、线粒体单链DNA结合蛋白以及多种参与线粒体中代谢途径的多功能蛋白。线粒体拟核结构的阐明对于进一步研究线粒体形态与功能以及mtDNA的遗传模式、基因表达调控具有重要意义。本文综述了线粒体拟核结构的最新研究进展,着重介绍组成拟核结构的重要蛋白,以及这些蛋白如何将mtDNA与柠檬酸循环等线粒体重要代谢途径相联系。同时,拟核相关蛋白(nucleoid-associated protein)的异常涉及多种人类疾病,这为研究线粒体相关疾病提供了新的思路。     

Characterization of the structure and DNA complexity of mung bean mitochondrial nucleoids

Electron microscopic images of mitochondrial nucleoids isolated from mung bean seedlings revealed a relatively homogeneous population of particles, each consisting of a chromatin-like structure associated with a membrane component. Association of F-actin with mitochondrial nucleoids was also observed. The mitochondrial nucleoid structure identified in situ showed heterogeneous genomic organization. After pulsed-field gel electrophoresis (PFGE), a large proportion of the mitochondrial nucleoid DNA remained in the well, whereas the rest migrated as a 50–200 kb smear zone. This PFGE migration pattern was not affected by high salt, topoisomerase I or latrunculin B treatments; however, the mobility of a fraction of the fastmoving DNA decreased conspicuously following an in-gel ethidium-enhanced UV-irradiation treatment, suggesting that molecules with intricately compact structures were present in the 50-200 kb region. Approximately 70% of the mitochondrial nucleoid DNA molecules examined via electron microscopy were open circles, supercoils, complex forms, and linear molecules with interspersed sigma-shaped structures and/or loops. Increased sensitivity of mtDNA to DNase I was found after mitochondrial nucleoids were pretreated with high salt. This result indicates that some loosely bound or peripheral DNA binding proteins protected the mtDNA from DNase I degradation.     

Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation

Mitochondrial DNA (mtDNA) is packaged into DNA-protein assemblies called nucleoids, but the mode of mtDNA propagation via the nucleoid remains controversial. Two mechanisms have been proposed: nucleoids may consistently maintain their mtDNA content faithfully, or nucleoids may exchange mtDNAs dynamically. To test these models directly, two cell lines were fused, each homoplasmic for a partially deleted mtDNA in which the deletions were nonoverlapping and each deficient in mitochondrial protein synthesis, thus allowing the first unequivocal visualization of two mtDNAs at the nucleoid level. The two mtDNAs transcomplemented to restore mitochondrial protein synthesis but were consistently maintained in discrete nucleoids that did not intermix stably. These results indicate that mitochondrial nucleoids tightly regulate their genetic content rather than freely exchanging mtDNAs. This genetic autonomy provides a molecular mechanism to explain patterns of mitochondrial genetic inheritance, in addition to facilitating therapeutic methods to eliminate deleterious mtDNA mutations.     

Evolutionary tinkering with mitochondrial nucleoids

Mitochondrial DNA (mtDNA) is organized in nucleoprotein particles called nucleoids. Each nucleoid, which is considered a heritable unit of mtDNA, might contain several copies of the mitochondrial genome and several different proteins. Some nucleoid-associated proteins, such as the high mobility group (HMG) box family, have well defined functions in mtDNA maintenance and packaging; others, such as Aco1 and IIv5, are bifunctional, fulfilling their roles in nucleoids in addition to well established metabolic functions. The fact that the HMG box mtDNA packaging proteins are of eukaryotic rather than bacterial origin and also that every organism seems to have a unique set of nucleoid-associated proteins suggests that evolutionary tinkering occurred to reinvent mitochondrial nucleoprotein during the evolution of mitochondrial genomes.     

The mitochondrial genome. The nucleoid

Mitochondrial DNA (mtDNA) in cells is organized in nucleoids containing DNA and various proteins. This review discusses questions of organization and structural dynamics of nucleoids as well as their protein components. The structures of mt-nucleoid from different organisms are compared. The currently accepted model of nucleoid organization is described and questions needing answers for better understanding of the fine mechanisms of the mitochondrial genetic apparatus functioning are discussed.     

Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.     

No sex please, we're mitochondria: a hypothesis on the somatic unit of inheritance of mammalian mtDNA

In this article we develop a model for the organization and maintenance of mitochondrial DNA (mtDNA) in mammalian somatic cells, based on the idea that the unit of genetic function comprises a group of mtDNA molecules that are semi-permanently associated as a mitochondrial nucleoid. Different mtDNA molecules within a nucleoid need not be genetically identical. We propose that nucleoids replicate faithfully via a kind of mitochondrial mitosis, generating daughter nucleoids that are identical copies of each other, but which can themselves segregate freely. This model can account for the very slow rates of mitotic segregation observed in cultured, heteroplasmic cell-lines, and also for the apparently poor complementation observed between different mutant mtDNAs co-introduced into rho(0) cells (cells that lack endogenous mtDNA). It also provides a potential system for maintaining the mitochondrial genetic fitness of stem cells in the face of a presumed high somatic mutation rate of mtDNA and many rounds of cell division in the absence of phenotypic selection. BioEssays 22:564-572, 2000.     

The numbers of individual mitochondrial DNA molecules and mitochondrial DNA nucleoids in yeast are co-regulated by the general amino acid control pathway

Mitochondrial DNA (mtDNA) is inherited as a protein-DNA complex (the nucleoid). We show that activation of the general amino acid response pathway in rho(+) and rho(-) petite cells results in an increased number of nucleoids without an increase in mtDNA copy number. In rho(-) cells, activation of the general amino acid response pathway results in increased intramolecular recombination between tandemly repeated sequences of rho(-) mtDNA to produce small, circular oligomers that are packaged into individual nucleoids, resulting in an approximately 10-fold increase in nucleoid number. The parsing of mtDNA into nucleoids due to general amino acid control requires Ilv5p, a mitochondrial protein that also functions in branched chain amino acid biosynthesis, and one or more factors required for mtDNA recombination. Two additional proteins known to function in mtDNA recombination, Abf2p and Mgt1p, are also required for parsing mtDNA into a larger number of nucleoids, although expression of these proteins is not under general amino acid control. Increased nucleoid number leads to increased mtDNA transmission, suggesting a mechanism to enhance mtDNA inheritance under amino acid starvation conditions.     

Proteins associated with mitochondrial DNA protect it against X-rays and hydrogen peroxide

The high susceptibility of mitochondrial DNA to reactive oxygen species and other damaging agents has been supposed to result from the absence of histones. Here we show that DNA-binding proteins of mitochondrial nucleoids can shield mtDNA from X-ray radiation and hydrogen peroxide just as nuclear histones do. Mitochondria, mitochondrial nucleoid proteins, and histones were isolated from mouse liver and assessed for mtDNA protection by the yield of PCR products. In vitro, mtDNA in complex either with nucleoid proteins or with nuclear histones proved to be much less damaged than naked mtDNA, with little difference in protective efficacy. Most probably, in mitochondria the nucleoid proteins also protect mtDNA against reactive oxygen species and thus attenuate the oxidative damage.     

Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction

A fundamental objective in molecular biology is to understand how DNA is organized in concert with various proteins, RNA, and biological membranes. Mitochondria maintain and express their own DNA (mtDNA), which is arranged within structures called nucleoids. Their functions, dimensions, composition, and precise locations relative to other mitochondrial structures are poorly defined. Superresolution fluorescence microscopy techniques that exceed the previous limits of imaging within the small and highly compartmentalized mitochondria have been recently developed. We have improved and employed both two- and three-dimensional applications of photoactivated localization microscopy (PALM and iPALM, respectively) to visualize the core dimensions and relative locations of mitochondrial nucleoids at an unprecedented resolution. PALM reveals that nucleoids differ greatly in size and shape. Three-dimensional volumetric analysis indicates that, on average, the mtDNA within ellipsoidal nucleoids is extraordinarily condensed. Two-color PALM shows that the freely diffusible mitochondrial matrix protein is largely excluded from the nucleoid. In contrast, nucleoids are closely associated with the inner membrane and often appear to be wrapped around cristae or crista-like inner membrane invaginations. Determinations revealing high packing density, separation from the matrix, and tight association with the inner membrane underscore the role of mechanisms that regulate access to mtDNA and that remain largely unknown.     

Effects of Fcj1-Mos1 and mitochondrial division on aggregation of mitochondrial DNA nucleoids and organelle morphology

Mitochondrial DNA (mtDNA) is packaged into DNA–protein complexes called nucleoids, which are distributed as many small foci in mitochondria. Nucleoids are crucial for the biogenesis and function of mtDNA. Here, using a yeast genetic screen for components that control nucleoid distribution and size, we identify Fcj1 and Mos1, two evolutionarily conserved mitochondrial proteins that maintain the connection between the cristae and boundary membranes. These two proteins are also important for establishing tubular morphology of mitochondria, as mitochondria lacking Fcj1 and Mos1 form lamellar sheets. We find that nucleoids aggregate, increase in size, and decrease in number in fcj1∆ and mos1∆ cells. In addition, Fcj1 form punctate structures and localized adjacent to nucleoids. Moreover, connecting mitochondria by deleting the DNM1 gene required for organelle division enhances aggregation of mtDNA nucleoids in fcj1∆ and mos1∆ cells, whereas single deletion of DNM1 does not affect nucleoids. Conversely, deleting F1Fo-ATP synthase dimerization factors generates concentric ring-like cristae, restores tubular mitochondrial morphology, and suppresses nucleoid aggregation in these mutants. Our findings suggest an unexpected role of Fcj1-Mos1 and organelle division in maintaining the distribution and size of mtDNA nucleoids.     

Whole Cell Formaldehyde Cross-Linking Simplifies Purification of Mitochondrial Nucleoids and Associated Proteins Involved in Mitochondrial Gene Expression

Mitochondrial DNA/protein complexes (nucleoids) appear as discrete entities inside the mitochondrial network when observed by live-cell imaging and immunofluorescence. This somewhat trivial observation in recent years has spurred research towards isolation of these complexes and the identification of nucleoid-associated proteins. Here we show that whole cell formaldehyde crosslinking combined with affinity purification and tandem mass-spectrometry provides a simple and reproducible method to identify potential nucleoid associated proteins. The method avoids spurious mitochondrial isolation and subsequent multifarious nucleoid enrichment protocols and can be implemented to allow for label-free quantification (LFQ) by mass-spectrometry. Using expression of a Flag-tagged Twinkle helicase and appropriate controls we show that this method identifies many previously identified nucleoid associated proteins. Using LFQ to compare HEK293 cells with and without mtDNA, but both expressing Twinkle-FLAG, identifies many proteins that are reduced or absent in the absence of mtDNA. This set not only includes established mtDNA maintenance proteins but also many proteins involved in mitochondrial RNA metabolism and translation and therefore represents what can be considered an mtDNA gene expression proteome. Our data provides a very valuable resource for both basic mitochondrial researchers as well as clinical geneticists working to identify novel disease genes on the basis of exome sequence data.     

Circular nucleoids isolated from chloroplasts in a brown alga Ectocarpus siliculosus

Circular nucleoids have been isolated from the chloroplasts of a brown alga, Ectocarpus indicus, by Nonidet P-40 treatment. Enzymatic treatments of the isolated nucleoids reveal that the nucleoid is a circle composed of bead-like particles interconnected by DNA strands. The beads contain predominantly DNA and proteins.