Nucleoid clusters
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Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators. We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (>48h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity.
- MeSH
- buňky Hep G2 MeSH
- DNA vazebné proteiny metabolismus MeSH
- doxorubicin MeSH
- ethidium MeSH
- GTP-fosfohydrolasy metabolismus MeSH
- jaterní mitochondrie metabolismus MeSH
- lidé MeSH
- mitochondriální DNA metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- poškození DNA MeSH
- proteiny asociované s mikrotubuly metabolismus MeSH
- transkripční faktory metabolismus MeSH
- transportní proteiny mitochondriální membrány metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The mitochondrion owns an autonomous genome. Double-stranded circular mitochondrial DNA (mtDNA) is organized in complexes with a packing/stabilizing transcription factor TFAM, having multiple roles, and proteins of gene expression machinery in structures called nucleoids. From hundreds to thousands nucleoids exist distributed in the matrix of mitochondrial reticulum network. A single mtDNA molecule contained within the single nucleoid is a currently preferred but questioned model. Nevertheless, mtDNA replication should lead transiently to its doubling within a nucleoid. However, nucleoid division has not yet been documented in detail. A 3D superresolution microscopy is required to resolve nucleoid biology occurring in ∼100 nm space, having an advantage over electron microscopy tomography in resolving the particular protein components. We discuss stochastic vs. stimulated emission depletion microscopy yielding wide vs. narrow nucleoid size distribution, respectively. Nucleoid clustering into spheroids fragmented from the continuous mitochondrial network, likewise possible nucleoid attachment to the inner membrane is reviewed.
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
Mitochondrial DNA (mtDNA) is organized in nucleoids in complex with accessory proteins, proteins of mtDNA replication and gene expression machinery. A robust mtDNA genome is represented by hundreds to thousands of nucleoids in cell mitochondrion. Detailed information is lacking about the dynamics of nucleoid distribution within the mitochondrial network upon physiological and pathological events. Therefore, we used confocal microscopy to study mitochondrial nucleoid redistribution upon mitochondrial fission and following reintegration of the mitochondrial network. Fission was induced by oxidative stress at respiration inhibition by rotenone or upon elimination of the protonmotive force by uncoupling or upon canceling its electrical component, ΔΨ(m), by valinomycin; and by silencing of mitofusin MFN2. Agent withdrawal resulted in concomitant mitochondrial network reintegration. We found two major principal morphological states: (i) a tubular state of the mitochondrial network with equidistant nucleoid spacing, 1.10±0.2 nucleoids per μm, and (ii) a fragmented state of solitary spheroid objects in which several nucleoids were clustered. We rarely observed singular mitochondrial fragments with a single nucleoid inside and very seldom we observed empty fragments. Reintegration of fragments into the mitochondrial network re-established the tubular state with equidistant nucleoid spacing. The two major morphological states coexisted at intermediate stages. These observations suggest that both mitochondrial network fission and reconnection of the disintegrated network are nucleoid-centric, i.e., fission and new mitochondrial tubule formation are initiated around nucleoids. Analyses of combinations of these morphological icons thus provide a basis for a future mitochondrial morphology diagnostics.
- MeSH
- buňky Hep G2 MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- mitochondriální DNA metabolismus ultrastruktura MeSH
- mitochondriální dynamika genetika fyziologie MeSH
- mitochondriální proteiny genetika metabolismus ultrastruktura MeSH
- mitochondrie ultrastruktura MeSH
- replikace DNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
... DNA codes for few proteins 83 -- The chloroplast genome codes for -100 proteins and RNAs 84 -- 4 Clusters ... ... and repeats 89 -- Gene clusters are formed by duplication and divergence 90 -- Sequence divergence is ... ... on specific sites 262 -- More subunits for RNA polymerase 267 -- 10 Theoperon -- Structural gene clusters ... ... 312 -- 11 Phage strategies 319 -- Lytic development is controlled by a cascade 321 -- Functional clustering ... ... 18 Chromosomes 545 -- Condensing viral genomes into their coats 546 -- The bacterial genome is a nucleoid ...
First published xvii, 990 stran : ilustrace, tabulky ; 28 cm
- MeSH
- DNA MeSH
- genetika MeSH
- geny MeSH
- molekulární biologie MeSH
- Publikační typ
- monografie MeSH
- Konspekt
- Obecná genetika. Obecná cytogenetika. Evoluce
- NLK Obory
- genetika, lékařská genetika
- biologie
... 297 -- 302 -- 305 -- 309 -- 312 -- 317 -- 320 -- 323 -- 329 -- 12: The operon -- Structural gene clusters ... ... 387 -- 13: Phage strategies 395 -- Lytic development is controlled by a cascade 397 -- Functional clustering ... ... Gene numbers 687 -- Essential genes and total gene number 689 -- Globin genes are organized in two clusters ... ... 692 -- Unequal crossing-over rearranges gene clusters 694 -- Gene clusters suffer continual reorganization ... ... 26: Chromosomes 743 -- Condensing viral genomes into their coats 744 -- The bacterial genome is a nucleoid ...
xviii, 1260 stran : ilustrace ; 28 cm
... 1 Expressed Gene Number Can Be Measured En Masse 93 iJEfci Summary 94 -- 6 Clusters and Repeats 98 -- ... ... Globin Clusters Are Formed by Duplication and Divergence 101 -- IMI Sequence Divergence Is the Basis ... ... * Unequal Crossing-over Rearranges Gene Clusters 109 1??? Genes for rRNA Form Tandem Repeats 112 ?? ... ... Structural Gene Clusters Are Coordinately Controlled 304 -- ??? -- ??? -- ????? -- 12.7 -- ??? ... ... 39 The T7 and T4 Genomes Show Functional Clustering 355 ESQ Lambda Immediate Early and Delayed Early ...
xvii, 892 s. : il.
- MeSH
- DNA genetika MeSH
- genetické jevy MeSH
- genom MeSH
- geny fyziologie MeSH
- proteiny genetika MeSH
- RNA genetika MeSH
- Publikační typ
- monografie MeSH
- Konspekt
- Obecná genetika. Obecná cytogenetika. Evoluce
- NLK Obory
- genetika, lékařská genetika
