Distribution and dynamics of mitochondrial nucleoids in animal cells in culture

Mitochondrial (mt) nucleoids were visualized in living cells in culture by staining with the fluorochrome picoGreen. The cell types included a line derived from Xenopus heart endothelial cells (XTH-2), 3T3 cells, SV40-transformed 3T3 cells and primary cultures of Xenopus tadpole epidermis cells. In the permanent cell lines 6-60% of the mitochondria were found to be devoid of DNA. The peaks of the frequency distribution of mtDNA content, as revealed by microfluorometry, were not very distinct, indicating the presence of a high amount of aneuploid mt nucleoids. The maximum size of nucleoids (as derived from fluorescence intensity) was 10-12 times that of the minimum peak value in proliferating cell cultures. A linear ratio was found between the volume of the nucleoids and their DNA content, which is interpreted as a uniform package density. In terminally differentiating tadpole epidermis cells mitochondria form large bodies containing giant nucleoids, while in mitotic cells the mt nucleoids are small and of uniform size. Fusion and fission of the nucleoids were observed to occur either for no visible reason or in connection with fusion and fission events of the mitochondria.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Organization, dynamics and transmission of mitochondrial DNA: focus on vertebrate nucleoids

Eukaryotic cells contain numerous copies of the mitochondrial genome (from 50 to 100 copies in the budding yeast to some thousands in humans) that localize to numerous intramitochondrial nucleoprotein complexes called nucleoids. The transmission of mitochondrial DNA differs significantly from that of nuclear genomes and depends on the number, molecular composition and dynamic properties of nucleoids and on the organization and dynamics of the mitochondrial compartment. While the localization, dynamics and protein composition of mitochondrial DNA nucleoids begin to be described, we are far from knowing all mechanisms and molecules mediating and/or regulating these processes. Here, we review our current knowledge on vertebrate nucleoids and discuss similarities and differences to nucleoids of other eukaryots.     

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.     

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.     

The yeast mitochondrial succinylome: Implications for regulation of mitochondrial nucleoids

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.     

Isolation of giant mitochondrial nucleoids from the yeastSaccharomyces cerevisiae

Summary The yeast cellsSaccharomyces cerevisiae grown up to stationary phase under either anaerobic conditions, or aerobic conditions in the presence of a respiratory inhibitor, antimycin A, had distinctive giant mitochondrial nucleoids (mt-nucleoids) (apparent diameter 0.6–0.9 m) in contrast with the small mt-nucleoids (apparent diameter 0.2–0.4 m) in respiratory-sufficient cells grown aerobically, as revealed by DAPI-fluorescence microscopy. The cytoplasmic respiratory-deficient cells (rho^– cells), which were induced by treatment of wild-type cells with ethidium bromide, showed both giant and small mt-nucleoids of irregular size. In order to examine the structural and functional differences between giant and small mt-nucleoids, the former were successfully isolated from spheroplasts of three different cells by differential centrifugation and centrifugation on a discontinuous sucrose gradient. The isolated giant mt-nucleoids were intact in the morphology and were free of significant contamination by nuclear chromatin. The number of protein components involved in each of three different giant mt-nucleoids was similar to the number in small mt-nucleoids from aerobically grown cells, though a few noticeable differences were also recognized. DNA-binding proteins with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa, 26 kDa, and 20 kDa were the main components of small mt-nucleoids from aerobically grown cells as detected by chromatography on native DNA-cellulose. In contrast, the 67 kDa and 52 kDa proteins were hardly detected in corresponding fractions of giant mt-nucleoids from anaerobically grown cells and from rho^– cells grown aerobically. On the other hand, mt-nucleoids from aerobically grown cells in the presence of antimycin A seemed to lack the 67 kDa protein but to have a small amount of the 52 kDa protein. This is the first demonstration of the variance of protein species involved in yeast mt-nucleoids according to the respiratory activity of mitochondria.     

Polymer-mediated compaction and internal dynamics of isolated Escherichia coli nucleoids

Nucleoids of Escherichia coli were isolated by osmotic shock under conditions of low salt in the absence of added polyamines or Mg(2+). As determined by fluorescence microscopy, the isolated nucleoids in 0.2 M NaCl are expanded structures with an estimated volume of about 27 microm(3) according to a procedure based on a 50% threshold for the fluorescence intensity. The nucleoid volume is measured as a function of the concentration of added polyethylene glycol. The collapse is a continuous process, so that a coil-globule transition is not witnessed. The Helmholtz free energy of the nucleoids is determined via the depletion interaction between the DNA helix and the polyethylene glycol chains. The resulting compaction relation is discussed in terms of the current theory of branched DNA supercoils and it is concluded that the in vitro nucleoid is crosslinked in a physical sense. Despite the congested and crosslinked state of the nucleoid, the relaxation rate of its superhelical segments, as monitored by dynamic light scattering, turns out to be purely diffusional. At small scales, the nucleoid behaves as a fluid.     

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.     

Human prohibitin 1 maintains the organization and stability of the mitochondrial nucleoids

Mitochondrial prohibitin (PHB) proteins have diverse functions, such as the regulation of apoptosis and the maintenance of mitochondrial morphology. In this study, we clarified a novel mitochondrial function of PHB1 that regulates the organization and maintenance of mitochondrial DNA (mtDNA). In PHB1-knockdown cells, we found that mtDNA is not stained by fluorescent dyes, such as ethidium bromide and PicoGreen, although the mitochondrial membrane potential still maintains. We also demonstrated that mtDNA, which is predominantly found in the NP-40-insoluble fraction when isolated from normal mitochondria, is partially released into the soluble fraction when isolated from PHB1-knockdown cells, indicating that the organization of the mitochondrial nucleoids has been altered. Furthermore, we found that PHB1 regulates copy number of mtDNA by stabilizing TFAM protein, a known protein component of the mitochondrial nucleoids. However, TFAM does not affect the organization of mtDNA as observed in PHB1-knockdown cells. Taken together, these results demonstrate that PHB1 maintains the organization and copy number of the mtDNA through both TFAM-independent and -dependent pathways.     

DNA synthesis in isolated mitochondrial nucleoids from plasmodia ofPhysarum polycephalum

Summary A mitochondrion contains multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial nucleoid (mt-nucleoid, synonym for mitochondrial nuclei). Replicaton of mtDNA in the mtnucleoids appears to be regulated within groups of adjacent mtDNA molecules, known as mitochondrial replicon clusters (MRCs). In this study, we isolated structurally intact mt-nucleoids from the plasmodia ofPhysarum polycephalum and characterized DNA synthesis in the isolated mt-nucleoids. The mt-nucleoids were isolated by dissolving the membranes of highly purified mitochondria with 0.5% Nonidet P-40. The structural integrity of the isolated mt-nucleoid was determined by observing the rod shape of the mt-nucleoid and the structure of the MRC. The isolated mt-nucleoids required four deoxyribonucleoside triphosphates and MgCl₂ for DNA synthesis. The DNA synthesis was resistant to aphidicolin and showed only low sensitivity to N-ethylmaleimide and to ddTTP, suggesting that the DNA synthesis is catalyzed by plant-type mitochondrial DNA polymerase. The capacity for DNA synthesis in the isolated mt-nucleoids was similar to that in the isolated mitochondria, despite removal of most of the mitochondrial matrix and membrane. Furthermore, visualization of sites of DNA synthesis in vitro revealed that DNA synthesis in the isolated mt-nucleoids occurred in each MRC. These results suggest that the isolated mt-nucleoids are capable of efficient and systematic DNA synthesis in vitro. Therefore, the use of isolated mt-nucleoids should permit in vitro characterization of the molecular mechanism of mtDNA replication in the MRC.Abbreviations BrdU 5-bromodeoxyuridine - BrdUTP 5-bromo-deoxyuridine triphosphate - DAPI 4,6-diamidino-2-phenylindole - dNTP deoxyribonucleoside triphosphate - ddCTP dideoxycytidine triphosphate - NEM N-ethylmaleimide - MRC mitochondrial replicon cluster; mt mitochondrial - NP-40 Nonidet P-40 - PBS phosphatebuffered saline - PMSF phenylmethanesulfonyl fluoride - rNTP ribonucleoside triphosphate - VIMPCS video-intensified microscope photon-counting system     

Comparative characterization of mitochondrial nucleoids and of nuclear chromatin of sea urchin embryos

Summary Chromatin and mitochondrial nucleoids of sea urchin embryos were found to have different isopycnic densities in metrizamide gradients. The banding density of nucleoids was constant during development, while there appeared to be alterations in the density profiles of chromatin banded at low ionic strength.     

Studies on mitochondrial structure and function in Physarum polycephalum. V. Behaviour of mitochondrial nucleoids throughout mitochondrial division cycle

The fine structure of mitochondria and mitochondrial nucleoids in exponentially growing Physarum polycephalum was studied at various periods throughout the mitochondrial division cycle by light and electron microscopy. The mitochondrial nucleoid elongates lingitudinally while the mitochondrion increases in size. When the nucleoid reaches a length of approximately 1.5 mum the mitochondrial membrane invaginates at the center of the mitochondrion and separates the mitochondrial contents. However, the nucleoid does not divide even when the mitochondrial sections are connected by a very narrow bridge. Just before division of the mitochondrion, the nucleoid divides by constriction of the limiting membrane of the dividing mitochondrion. After division, one end of the nucleoid appears to be associated with the inner mitochondrial membrane. The nucleoid then again becomes situated in the center of the mitochondrion before repeating these same processes.     

Ecm10p localizes in yeast mitochondrial nucleoids and its overexpression induces extensive mitochondrial DNA aggregations

Ecm10p was initially identified as a cell wall synthesis-related gene product [Genetics 147 (1997) 435] and also reported as a mitochondrial protein which was partially capable of compensating the phenotypic defect by SSC1 gene mutation [FEBS Lett. 487 (2000) 307]. Here we report that ecm10p is localized in mitochondrial nucleoids as its major component and the targeting signal resides between amino acid residues 161 and 240. Overexpression of ecm10p induces extensive mitochondrial DNA aggregations, which might be due to aberrant mitochondrial DNA cleavages through an altered endonuclease activity in mitochondrial nucleoids.     

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.     

Effects of ethidium bromide and chloramphenicol on the mitochondrial nucleoids in Euglena gracilis

The effects of ethidium bromide (EB) at 0.13 m M and of chloramphenicol (CAP) at 46 m M on the mitochondria and mitochondrial nucleoids in Euglena gracilis . Z strain, were examined by fluorescence microscopy and by electron microscopy. Ethidium bromide stopped the multiplication of cells and decreased their respiratory activity by 55% after treatment for 10 days. Most of the mitochondria became slender with few cristae and some became cup-shaped with stacked cristac. Mitochondrial nucleoids decreased markedly in number after treatment with EB for more than 2 days. After treatment for 3 days with EB, mitochondrial nucleoids could not be detected in about half of all cells examined. Treatment with CAP for 10 days reduced the respiratory activity by 47%. Chloramphenicol did not decrease the number of mitochondrial nucleoids but it increased the number of cristae and the volume of mitochondria.