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

Pancreatic cancer is one of the deadliest forms of cancer, which is attributed to lack of effective treatment options and drug resistance. Mitochondrial inhibitors have emerged as a promising class of anticancer drugs, and several inhibitors of the electron transport chain (ETC) are being clinically evaluated. We hypothesized that resistance to ETC inhibitors from the biguanide class could be induced by inactivation of SMAD4, an important tumor suppressor involved in transforming growth factor β (TGFβ) signaling, and associated with altered mitochondrial activity. Here we show that, paradoxically, both TGFβ-treatment and the loss of SMAD4, a downstream member of TGFβ signaling cascade, induce resistance to biguanides, decrease mitochondrial respiration, and fragment the mitochondrial network. Mechanistically, the resistance of SMAD4-deficient cells is mediated by increased mitophagic flux driven by MAPK/ERK signaling, whereas TGFβ-induced resistance is autophagy-independent and linked to epithelial-to-mesenchymal transition (EMT). Interestingly, mitochondria-targeted tamoxifen, a complex I inhibitor under clinical trial, overcomes resistance mediated by SMAD4-deficiency or TGFβ signaling. Our data point to differential mechanisms underlying the resistance to treatment in PDAC arising from TGFβ signaling and SMAD4 loss, respectively. The findings will help the development of mitochondria-targeted therapy for pancreatic cancer patients with SMAD4 as a plausible predictive marker.

• MeSH
• lidé MeSH
• mitofagie MeSH
• nádory slinivky břišní genetika   metabolismus   patologie   MeSH
• protein Smad4 metabolismus   MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein Smad4 MeSH
• SMAD4 protein, human MeSH Prohlížeč

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

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

Mitochondrial nucleoids are confined sites of mitochondrial DNA existing in complex clusters with the DNA-compacting mitochondrial (mt) transcription factor A (TFAM) and other accessory proteins and gene expression machinery proteins, such as a mt single-stranded-DNA-binding protein (mtSSB). To visualize nucleoid distribution within the mt reticular network, we have employed three-dimensional (3D) double-color 4Pi microscopy. The mt network was visualized in hepatocellular carcinoma HepG2 cells via mt-matrix-addressed GFP, while 3D immunocytochemistry of mtSSB was performed. Optimization of iso-surface computation threshold for nucleoid 4Pi images to 30 led to an average nucleoid diameter of 219 ± 110 and 224 ± 100 nm in glucose- and galactose-cultivated HepG2 cells (the latter with obligatory oxidative phosphorylation). We have positioned mtDNA nucleoids within the mt reticulum network and refined our model for nucleoid redistribution within the fragmented network--clustering of up to ten nucleoids in 2 μm diameter mitochondrial spheroids of a fragmented mt network, arising from an original 10 μm mt tubule of a 400 nm diameter. However, the theoretically fragmented bulk parts were observed most frequently as being reintegrated into the continuous mt network in 4Pi images. Since the predicted nucleoid counts within the bulk parts corresponded to the model, we conclude that fragmentation/reintegration cycles are not accompanied by mtDNA degradation or that mtDNA degradation is equally balanced by mtDNA replication.

• MeSH
• buněčné kultury MeSH
• buňky Hep G2 MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• fluorescenční mikroskopie metody   MeSH
• imunohistochemie MeSH
• konfokální mikroskopie metody   MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• mitochondriální DNA genetika   metabolismus   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• molekulární modely * MeSH
• počítačové zpracování obrazu MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zelené fluorescenční 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
• TFAM protein, human MeSH Prohlížeč
• transkripční faktory MeSH
• zelené fluorescenční proteiny MeSH

Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5'-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor(®) 488 or 647 dye, we obtained the fluorescent "L-ND5 probe" containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5'-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5'-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import.

• MeSH
• genetická transkripce MeSH
• lidé MeSH
• mitochondriální DNA chemie   genetika   MeSH
• nukleové kyseliny chemie   genetika   MeSH
• oligonukleotidy chemie   genetika   MeSH
• RNA ribozomální chemie   genetika   MeSH
• RNA chemie   genetika   MeSH
• sekvenční homologie nukleových kyselin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA MeSH
• nukleové kyseliny MeSH
• oligonukleotidy MeSH
• RNA ribozomální MeSH
• RNA MeSH