Phylum Euglenozoa comprises three groups of eukaryotic microbes (kinetoplastids, diplonemids, and euglenids), the mitochondrial (mt) genomes of which exhibit radically different modes of organization and expression. Gene fragmentation is a striking feature of both euglenid and diplonemid mtDNAs. To rationalize the emergence of these highly divergent mtDNA types and the existence of insertion/deletion RNA editing (in kinetoplastids) and trans-splicing (in diplonemids), we propose that in the mitochondrion of the common evolutionary ancestor of Euglenozoa, small expressed gene fragments promoted a rampant neutral evolutionary pathway. Interactions between small antisense transcripts of these gene fragments and full-length transcripts, assisted by RNA-processing enzymes, permitted the emergence of RNA editing and/or trans-splicing activities, allowing the system to tolerate indel mutations and further gene fragmentation, respectively, and leading to accumulation of additional mutations. In this way, dramatically different mitochondrial genome structures and RNA-processing machineries were able to evolve. The paradigm of constructive neutral evolution acting on the widely different mitochondrial genetic systems in Euglenozoa posits the accretion of initially neutral molecular interactions by genetic drift, leading inevitably to the observed 'irremediable complexity'.

• MeSH
• editace RNA MeSH
• Euglenozoa genetika   MeSH
• genom mitochondriální MeSH
• modely genetické MeSH
• molekulární evoluce MeSH
• replikace DNA MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy 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

Diplonemids are highly abundant heterotrophic marine protists. Previous studies showed that their strikingly bloated mitochondrial genome is unique because of systematic gene fragmentation and manifold RNA editing. Here we report a comparative study of mitochondrial genome architecture, gene structure and RNA editing of six recently isolated, phylogenetically diverse diplonemid species. Mitochondrial gene fragmentation and modes of RNA editing, which include cytidine-to-uridine (C-to-U) and adenosine-to-inosine (A-to-I) substitutions and 3' uridine additions (U-appendage), are conserved across diplonemids. Yet as we show here, all these features have been pushed to their extremes in the Hemistasiidae lineage. For example, Namystynia karyoxenos has its genes fragmented into more than twice as many modules than other diplonemids, with modules as short as four nucleotides. Furthermore, we detected in this group multiple A-appendage and guanosine-to-adenosine (G-to-A) substitution editing events not observed before in diplonemids and found very rarely elsewhere. With >1,000 sites, C-to-U and A-to-I editing in Namystynia is nearly 10 times more frequent than in other diplonemids. The editing density of 12% in coding regions makes Namystynia's the most extensively edited transcriptome described so far. Diplonemid mitochondrial genome architecture, gene structure and post-transcriptional processes display such high complexity that they challenge all other currently known systems.

• MeSH
• chromozomy genetika   MeSH
• editace RNA genetika   MeSH
• Euglenozoa genetika   MeSH
• fylogeneze MeSH
• genom mitochondriální * MeSH
• geny * MeSH
• konzervovaná sekvence MeSH
• mitochondriální DNA genetika   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tamoxifen resistance remains a clinical obstacle in the treatment of hormone sensitive breast cancer. It has been reported that tamoxifen is able to target respiratory complex I within mitochondria. Therefore, we established two tamoxifen-resistant cell lines, MCF7 Tam5R and T47D Tam5R resistant to 5 μM tamoxifen and investigated whether tamoxifen-resistant cells exhibit mitochondrial changes which could help them survive the treatment. The function of mitochondria in this experimental model was evaluated in detail by studying i) the composition and activity of mitochondrial respiratory complexes; ii) respiration and glycolytic status; iii) mitochondrial distribution, dynamics and reactive oxygen species production. We show that Tam5R cells exhibit a significant decrease in mitochondrial respiration, low abundance of assembled mitochondrial respiratory supercomplexes, a more fragmented mitochondrial network connected with DRP1 Ser637 phosphorylation, higher glycolysis and sensitivity to 2-deoxyglucose. Tam5R cells also produce significantly higher levels of mitochondrial superoxide but at the same time increase their antioxidant defense (CAT, SOD2) through upregulation of SIRT3 and show phosphorylation of AMPK at Ser 485/491. Importantly, MCF7 ρ0 cells lacking functional mitochondria exhibit a markedly higher resistance to tamoxifen, supporting the role of mitochondria in tamoxifen resistance. We propose that reduced mitochondrial function and higher level of reactive oxygen species within mitochondria in concert with metabolic adaptations contribute to the phenotype of tamoxifen resistance.

• MeSH
• apoptóza MeSH
• buněčný cyklus MeSH
• chemorezistence * MeSH
• fenotyp MeSH
• glykolýza * MeSH
• hormonální protinádorové látky farmakologie   MeSH
• lidé MeSH
• mitochondrie metabolismus   patologie   MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• nádory prsu farmakoterapie   metabolismus   patologie   MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• respirační komplex I metabolismus   MeSH
• superoxidy metabolismus   MeSH
• tamoxifen farmakologie   MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mitochondrial morphology was studied in cultivated myoblasts obtained from patients with mitochondrial disorders, including CPEO, MELAS and TMEM70 deficiency. Mitochondrial networks and ultrastructure were visualized by fluorescence microscopy and transmission electron microscopy, respectively. A heterogeneous picture of abnormally sized and shaped mitochondria with fragmentation, shortening, and aberrant cristae, lower density of mitochondria and an increased number of "megamitochondria" were found in patient myoblasts. Morphometric Fiji analyses revealed different mitochondrial network properties in myoblasts from patients and controls. The small number of cultivated myoblasts required for semiautomatic morphometric image analysis makes this tool useful for estimating mitochondrial disturbances in patients with mitochondrial disorders.

• MeSH
• dítě MeSH
• fluorescenční mikroskopie MeSH
• kojenec MeSH
• lidé MeSH
• mitochondriální nemoci patologie   MeSH
• mitochondrie ultrastruktura   MeSH
• myoblasty ultrastruktura   MeSH
• transmisní elektronová mikroskopie MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Booklice in the genus Liposcelis (Psocodea: Liposcelididae) are essential storage pests worldwide. Fragmented mt genomes have been identified in the Liposcelis species together with the typical mitochondrial (mt) genome, which is a single circular chromosome with 37 genes. Gene rearrangement, pseudogenes, and repeat regions (RRs) are very common among fragmented mt genomes. We sequenced the mt genome of the booklouse L. brunnea, the type species of the genus Liposcelis. We identified 37 genes in the mt genome of L. brunnea, which was fragmented into three chromosomes. The chromosomes I, II, III were 7.3 kb, 5.5 kb, and 5.3 kb in size with 9, 19, and 15 genes, respectively. In addition, 16 pseudogenes and four repeat regions were present in three chromosomes. Gene rearrangement in the mt genome of L. brunnea was obvious compared to that in other mt genomes in the genus Liposcelis. We found a possible correlation among mt genome rearrangement, the morphological classification standard, and phylogenetic relationships. In summary, a three-chromosome mt genome in an insect was identified for the first time, which may aid in understanding mt genome fragmentation, gene rearrangement, and evolution.

• MeSH
• fylogeneze MeSH
• genom mitochondriální genetika   MeSH
• hmyz genetika   MeSH
• hmyzí geny genetika   MeSH
• molekulární evoluce MeSH
• multigenová rodina genetika   MeSH
• pořadí genů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Mitochondrial protein quality control is crucial for the maintenance of correct mitochondrial homeostasis. It is ensured by several specific mitochondrial proteases located across the various mitochondrial subcompartments. Here, we focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 (AFG3 Like Matrix AAA Peptidase Subunit 2) and YME1L (YME1 like ATPase) of mitochondrial inner membrane AAA (ATPases Associated with diverse cellular Activities) complexes in the maintenance of mitochondrial structure and respiratory chain integrity. We demonstrate that loss of AFG3L2 and YME1L, both alone and in combination, results in diminished cell proliferation, fragmentation of mitochondrial reticulum, altered cristae morphogenesis, and defective respiratory chain biogenesis. The double AFG3L2/YME1L knockdown cells showed marked upregulation of OPA1 protein forms, with the most prominent increase in short OPA1 (optic atrophy 1). Loss of either protease led to marked elevation in OMA1 (OMA1 zinc metallopeptidase) (60 kDa) and severe reduction in the SPG7 (paraplegin) subunit of the m-AAA complex. Loss of the YME1L subunit led to an increased Drp1 level in mitochondrial fractions. While loss of YME1L impaired biogenesis and function of complex I, knockdown of AFG3L2 mainly affected the assembly and function of complex IV. Our results suggest cooperative and partly redundant functions of AFG3L2 and YME1L in the maintenance of mitochondrial structure and respiratory chain biogenesis and stress the importance of correct proteostasis for mitochondrial integrity.

• MeSH
• ATPázy spojené s různými buněčnými aktivitami genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• metaloendopeptidasy genetika   metabolismus   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie metabolismus   ultrastruktura   MeSH
• proliferace buněk genetika   fyziologie   MeSH
• proteasy závislé na ATP genetika   metabolismus   MeSH
• transmisní elektronová mikroskopie MeSH
• western blotting 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.

• MeSH
• fluorescenční mikroskopie metody   MeSH
• konfokální mikroskopie metody   MeSH
• lidé MeSH
• mitochondriální DNA metabolismus   MeSH
• mitochondrie metabolismus   ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Arguably, the most bizarre mitochondrial DNA (mtDNA) is that of the euglenozoan eukaryote Diplonema papillatum. The genome consists of numerous small circular chromosomes none of which appears to encode a complete gene. For instance, the cox1 coding sequence is spread out over nine different chromosomes in non-overlapping pieces (modules), which are transcribed separately and joined to a contiguous mRNA by trans-splicing. Here, we examine how many genes are encoded by Diplonema mtDNA and whether all are fragmented and their transcripts trans-spliced. Module identification is challenging due to the sequence divergence of Diplonema mitochondrial genes. By employing most sensitive protein profile search algorithms and comparing genomic with cDNA sequence, we recognize a total of 11 typical mitochondrial genes. The 10 protein-coding genes are systematically chopped up into three to 12 modules of 60-350 bp length. The corresponding mRNAs are all trans-spliced. Identification of ribosomal RNAs is most difficult. So far, we only detect the 3'-module of the large subunit ribosomal RNA (rRNA); it does not trans-splice with other pieces. The small subunit rRNA gene remains elusive. Our results open new intriguing questions about the biochemistry and evolution of mitochondrial trans-splicing in Diplonema.

• MeSH
• chromozomy chemie   MeSH
• Euglenozoa genetika   MeSH
• genetická transkripce MeSH
• genom mitochondriální MeSH
• mitochondriální DNA chemie   MeSH
• mitochondriální geny MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• sekvenční analýza DNA MeSH
• trans-splicing MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The methods of the detection of (1) non-labeled and (2) BrdU-labeled mitochondrial DNA (mtDNA) are described. They are based on the production of singlet oxygen by monovalent copper ions and the subsequent induction of DNA gaps. The ends of interrupted DNA serve as origins for the labeling of mtDNA by DNA polymerase I or they are utilized by exonuclease that degrades DNA strands, unmasking BrdU in BrdU-labeled DNA. Both methods are sensitive approaches without the need of additional enhancement of the signal or the use of highly sensitive optical systems.

• MeSH
• barvení a značení metody   MeSH
• biotin chemie   MeSH
• bromodeoxyuridin chemie   MeSH
• DNA-polymerasa I metabolismus   MeSH
• indoly chemie   MeSH
• kultivované buňky MeSH
• kyslík chemie   MeSH
• lidé MeSH
• měď chemie   MeSH
• mitochondriální DNA genetika   MeSH
• mitochondrie genetika   MeSH
• replikace DNA MeSH
• zelené fluorescenční proteiny chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH