Significance: Mitochondrial (mt) reticulum network in the cell possesses amazing ultramorphology of parallel lamellar cristae, formed by the invaginated inner mitochondrial membrane. Its non-invaginated part, the inner boundary membrane (IBM) forms a cylindrical sandwich with the outer mitochondrial membrane (OMM). Crista membranes (CMs) meet IBM at crista junctions (CJs) of mt cristae organizing system (MICOS) complexes connected to OMM sorting and assembly machinery (SAM). Cristae dimensions, shape, and CJs have characteristic patterns for different metabolic regimes, physiological and pathological situations. Recent Advances: Cristae-shaping proteins were characterized, namely rows of ATP-synthase dimers forming the crista lamella edges, MICOS subunits, optic atrophy 1 (OPA1) isoforms and mitochondrial genome maintenance 1 (MGM1) filaments, prohibitins, and others. Detailed cristae ultramorphology changes were imaged by focused-ion beam/scanning electron microscopy. Dynamics of crista lamellae and mobile CJs were demonstrated by nanoscopy in living cells. With tBID-induced apoptosis a single entirely fused cristae reticulum was observed in a mitochondrial spheroid. Critical Issues: The mobility and composition of MICOS, OPA1, and ATP-synthase dimeric rows regulated by post-translational modifications might be exclusively responsible for cristae morphology changes, but ion fluxes across CM and resulting osmotic forces might be also involved. Inevitably, cristae ultramorphology should reflect also mitochondrial redox homeostasis, but details are unknown. Disordered cristae typically reflect higher superoxide formation. Future Directions: To link redox homeostasis to cristae ultramorphology and define markers, recent progress will help in uncovering mechanisms involved in proton-coupled electron transfer via the respiratory chain and in regulation of cristae architecture, leading to structural determination of superoxide formation sites and cristae ultramorphology changes in diseases. Antioxid. Redox Signal. 39, 635-683.

• Klíčová slova
• ATP-synthase dimeric rows, MICOS, OPA1, mitochondrial cristae, mitochondrial superoxide formation, respiratory chain supercomplexes,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• homeostáza MeSH
• mitochondriální membrány * metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• oxidace-redukce MeSH
• superoxidy * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• adenosintrifosfát MeSH
• mitochondriální proteiny MeSH
• superoxidy * MeSH

Previously, a number of ~ 1.4 of mitochondrial DNA (mtDNA) molecules in a single nucleoid was reported, which would reflect a minimum nucleoid division. We applied 3D-double-color direct stochastic optical reconstruction microscopy (dSTORM), i.e. nanoscopy with ~ 25-40 nm x,y-resolution, together with our novel method of Delaunay segmentation of 3D data to identify unbiased 3D-overlaps. Noncoding D-loops were recognized in HeLa cells by mtDNA fluorescence in situ hybridization (mtFISH) 7S-DNA 250-bp probe, containing biotin, visualized by anti-biotin/Cy3B-conjugated antibodies. Other mtFISH probes with biotin or Alexa Fluor 647 (A647) against ATP6-COX3 gene overlaps (1,100 bp) were also used. Nucleoids were imaged by anti-DNA/(A647-)-Cy3B-conjugated antibodies. Resulting histograms counting mtFISH-loci/nucleoid overlaps demonstrated that 45% to 70% of visualized nucleoids contained two or more D-loops or ATP6-COX3-loci, indicating two or more mtDNA molecules per nucleoid. With increasing number of mtDNA per nucleoid, diameters were larger and their distribution histograms peaked at ~ 300 nm. A wide nucleoid diameter distribution was obtained also using 2D-STED for their imaging by anti-DNA/A647. At unchanged mtDNA copy number in osteosarcoma 143B cells, TFAM expression increased nucleoid spatial density 1.67-fold, indicating expansion of existing mtDNA and its redistribution into more nucleoids upon the higher TFAM/mtDNA stoichiometry. Validation of nucleoid imaging was also done with two TFAM mutants unable to bend or dimerize, respectively, which reduced both copy number and nucleoid spatial density by 80%. We conclude that frequently more than one mtDNA molecule exists within a single nucleoid in HeLa cells and that mitochondrial nucleoids do exist in a non-uniform size range.

• MeSH
• DNA vazebné proteiny * genetika   metabolismus   MeSH
• HeLa buňky MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• mitochondriální DNA * genetika   metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny * MeSH
• mitochondriální DNA * MeSH
• mitochondriální proteiny MeSH

Mammalian genes were long thought to be constrained within somatic cells in most cell types. This concept was challenged recently when cellular organelles including mitochondria were shown to move between mammalian cells in culture via cytoplasmic bridges. Recent research in animals indicates transfer of mitochondria in cancer and during lung injury in vivo, with considerable functional consequences. Since these pioneering discoveries, many studies have confirmed horizontal mitochondrial transfer (HMT) in vivo, and its functional characteristics and consequences have been described. Additional support for this phenomenon has come from phylogenetic studies. Apparently, mitochondrial trafficking between cells occurs more frequently than previously thought and contributes to diverse processes including bioenergetic crosstalk and homeostasis, disease treatment and recovery, and development of resistance to cancer therapy. Here we highlight current knowledge of HMT between cells, focusing primarily on in vivo systems, and contend that this process is not only (patho)physiologically relevant, but also can be exploited for the design of novel therapeutic approaches.

• MeSH
• energetický metabolismus MeSH
• fylogeneze MeSH
• mitochondrie * metabolismus   MeSH
• nádory * genetika   metabolismus   MeSH
• savci MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• komentáře MeSH
• práce podpořená grantem MeSH

Mitochondrial DNA copy number has been previously shown to be elevated with severe and chronic stress, as well as stress-related pathology like Major Depressive Disorder (MDD) and post-traumatic stress disorder (PTSD). While experimental data point to likely recovery of mtDNA copy number changes after the stressful event, time needed for full recovery and whether it can be achieved are still unknown. Further, while it has been shown that stress-related mtDNA elevation affects multiple tissues, its specific consequences for oogenesis and maternal inheritance of mtDNA has never been explored. In this study, we used qPCR to quantify mtDNA copy number in 15 Holocaust survivors and 102 of their second- and third-generation descendants from the Czech Republic, many of whom suffer from PTSD, and compared them to controls in the respective generations. We found no significant difference in mtDNA copy number in the Holocaust survivors compared to controls, whether they have PTSD or not, and no significant elevation in descendants of female Holocaust survivors as compared to descendants of male survivors or controls. Our results showed no evidence of persistence or inheritance of mtDNA changes in Holocaust survivors, though that does not rule out effects in other tissues or mitigating mechanism for such changes.

• Klíčová slova
• Holocaust-psychic trauma, copy number variation, mitochondrial DNA, posttraumatic stress disorder, quantitative PCR,
• Publikační typ
• časopisecké články MeSH

Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents.

• Klíčová slova
• OXPHOS, cancer, coenzyme Q, dihydroorotate dehydrogenase, mitochondria, pyrimidine biosynthesis, respiration,
• MeSH
• buněčné dýchání MeSH
• dihydroorotátdehydrogenasa MeSH
• lidé MeSH
• mitochondriální DNA metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory metabolismus   MeSH
• oxidativní fosforylace MeSH
• oxidoreduktasy působící na CH-CH vazby fyziologie   MeSH
• pyrimidiny metabolismus   MeSH
• ubichinon metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• dihydroorotátdehydrogenasa MeSH
• mitochondriální DNA MeSH
• oxidoreduktasy působící na CH-CH vazby MeSH
• pyrimidine MeSH Prohlížeč
• pyrimidiny MeSH
• ubichinon MeSH

Hypertrophic pancreatic islets (PI) of Goto Kakizaki (GK) diabetic rats contain a lower number of β-cells vs. non-diabetic Wistar rat PI. Remaining β-cells contain reduced mitochondrial (mt) DNA per nucleus (copy number), probably due to declining mtDNA replication machinery, decreased mt biogenesis or enhanced mitophagy. We confirmed mtDNA copy number decrease down to <30% in PI of one-year-old GK rats. Studying relations to mt nucleoids sizes, we employed 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with lentivirally transduced Eos conjugate of mt single-stranded-DNA-binding protein (mtSSB) or transcription factor TFAM; or by 3D immunocytochemistry. mtSSB (binding transcription or replication nucleoids) contoured "nucleoids" which were smaller by 25% (less diameters >150 nm) in GK β-cells. Eos-TFAM-visualized nucleoids, composed of 72% localized TFAM, were smaller by 10% (immunochemically by 3%). A theoretical ~70% decrease in cell nucleoid number (spatial density) was not observed, rejecting model of single mtDNA per nucleoid. The β-cell maintenance factor Nkx6.1 mRNA and protein were declining with age (>12-fold, 10 months) and decreasing with fasting hyperglycemia in GK rats, probably predetermining the impaired mtDNA replication (copy number decrease), while spatial expansion of mtDNA kept nucleoids with only smaller sizes than those containing much higher mtDNA in non-diabetic β-cells.

• MeSH
• beta-buňky metabolismus   patologie   MeSH
• DNA vazebné proteiny genetika   MeSH
• experimentální diabetes mellitus genetika   metabolismus   patologie   MeSH
• homeodoménové proteiny genetika   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• mitochondriální DNA genetika   MeSH
• mitochondrie genetika   patologie   MeSH
• mitofagie genetika   MeSH
• pankreas exokrinní metabolismus   MeSH
• potkani Wistar MeSH
• replikace DNA genetika   MeSH
• transkripční faktory genetika   MeSH
• variabilita počtu kopií segmentů DNA genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• homeodoménové proteiny MeSH
• mitochondriální DNA MeSH
• Nkx6-1 protein, rat MeSH Prohlížeč
• Tfam protein, rat MeSH Prohlížeč
• transkripční faktory MeSH

Mitochondrial nucleoids consist of several different groups of proteins, many of which are involved in essential cellular processes such as the replication, repair and transcription of the mitochondrial genome. The eukaryotic, ATP-dependent protease Lon is found within the central nucleoid region, though little is presently known about its role there. Aside from its association with mitochondrial nucleoids, human Lon also specifically interacts with RNA. Recently, Lon was shown to regulate TFAM, the most abundant mtDNA structural factor in human mitochondria. To determine whether Lon also regulates other mitochondrial nucleoid- or ribosome-associated proteins, we examined the in vitro digestion profiles of the Saccharomyces cerevisiae TFAM functional homologue Abf2, the yeast mtDNA maintenance protein Mgm101, and two human mitochondrial proteins, Twinkle helicase and the large ribosomal subunit protein MrpL32. Degradation of Mgm101 was also verified in vivo in yeast mitochondria. These experiments revealed that all four proteins are actively degraded by Lon, but that three of them are protected from it when bound to a nucleic acid; the Twinkle helicase is not. Such a regulatory mechanism might facilitate dynamic changes to the mitochondrial nucleoid, which are crucial for conducting mitochondrial functions and maintaining mitochondrial homeostasis.

• MeSH
• aktivace enzymů MeSH
• DNA vazebné proteiny metabolismus   MeSH
• lidé MeSH
• mitochondriální DNA metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• proteasa La metabolismus   MeSH
• proteolýza MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• substrátová specifita MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• mitochondriální DNA MeSH
• mitochondriální proteiny MeSH
• proteasa La MeSH

Data segmentation and object rendering is required for localization super-resolution microscopy, fluorescent photoactivation localization microscopy (FPALM), and direct stochastic optical reconstruction microscopy (dSTORM). We developed and validated methods for segmenting objects based on Delaunay triangulation in 3D space, followed by facet culling. We applied them to visualize mitochondrial nucleoids, which confine DNA in complexes with mitochondrial (mt) transcription factor A (TFAM) and gene expression machinery proteins, such as mt single-stranded-DNA-binding protein (mtSSB). Eos2-conjugated TFAM visualized nucleoids in HepG2 cells, which was compared with dSTORM 3D-immunocytochemistry of TFAM, mtSSB, or DNA. The localized fluorophores of FPALM/dSTORM data were segmented using Delaunay triangulation into polyhedron models and by principal component analysis (PCA) into general PCA ellipsoids. The PCA ellipsoids were normalized to the smoothed volume of polyhedrons or by the net unsmoothed Delaunay volume and remodeled into rotational ellipsoids to obtain models, termed DVRE. The most frequent size of ellipsoid nucleoid model imaged via TFAM was 35 × 45 × 95 nm; or 35 × 45 × 75 nm for mtDNA cores; and 25 × 45 × 100 nm for nucleoids imaged via mtSSB. Nucleoids encompassed different point density and wide size ranges, speculatively due to different activity stemming from different TFAM/mtDNA stoichiometry/density. Considering twofold lower axial vs. lateral resolution, only bulky DVRE models with an aspect ratio >3 and tilted toward the xy-plane were considered as two proximal nucleoids, suspicious occurring after division following mtDNA replication. The existence of proximal nucleoids in mtDNA-dSTORM 3D images of mtDNA "doubling"-supported possible direct observations of mt nucleoid division after mtDNA replication.

• Klíčová slova
• 3D object segmentation, 3D super-resolution microscopy, Delaunay algorithm, Mitochondrial DNA replication, Nucleoids, Principal component analysis,
• MeSH
• algoritmy * MeSH
• analýza hlavních komponent * MeSH
• buňky Hep G2 MeSH
• DNA vazebné proteiny metabolismus   MeSH
• fluorescenční mikroskopie * MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• mitochondriální DNA chemie   metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• molekulární modely MeSH
• zobrazování trojrozměrné * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• mitochondriální DNA MeSH
• mitochondriální proteiny MeSH
• NABP2 protein, human MeSH Prohlížeč

Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species.

• Klíčová slova
• DNA compaction, DNA-binding protein, HMG-box containing protein, Holliday junction, mitochondrial DNA (mtDNA), mitochondrial nucleoid,
• MeSH
• Candida genetika   metabolismus   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• molekulární evoluce * MeSH
• proteiny s vysokou pohyblivostí genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Yarrowia genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• proteiny s vysokou pohyblivostí MeSH
• Saccharomyces cerevisiae - proteiny MeSH