Hypoxia causes mitochondrial cristae widening, enabled by the ~20% degradation of Mic60/mitofilin, with concomitant clustering of the MICOS complex, reflecting the widening of crista junctions (outlets) (Plecitá-Hlavatá et al. FASEB J., 2016 30:1941-1957). Attempting to accelerate metabolism by the addition of membrane-permeant dimethyl-2-oxoglutarate (dm2OG) to HepG2 cells pre-adapted to hypoxia, we found cristae narrowing by transmission electron microscopy. Glycolytic HepG2 cells, which downregulate hypoxic respiration, instantly increased respiration with dm2OG. Changes in intracristal space (ICS) morphology were also revealed by 3D super-resolution microscopy using Eos-conjugated ICS-located lactamase-β. Cristae topology was resolved in detail by focused-ion beam/scanning electron microscopy (FIB/SEM). The spatial relocations of key cristae-shaping proteins were indicated by immunocytochemical stochastic 3D super-resolution microscopy (dSTORM), while analyzing inter-antibody-distance histograms: i) ATP-synthase dimers exhibited a higher fraction of shorter inter-distances between bound F1-α primary Alexa-Fluor-647-conjugated antibodies, indicating cristae narrowing. ii) Mic60/mitofilin clusters (established upon hypoxia) decayed, restoring isotropic random Mic60/mitofilin distribution (a signature of normoxia). iii) outer membrane SAMM50 formed more focused clusters. Less abundant fractions of higher ATP-synthase oligomers of hypoxic samples on blue-native electrophoresis became more abundant fractions at the high dm2OG load and at normoxia. This indicates more labile ATP-synthase dimeric rows established at crista rims upon hypoxia, strengthened at normoxia or dm2OG-substrate load. Hypothetically, the increased Krebs substrate load stimulates the cross-linking/strengthening of rows of ATP-synthase dimers at the crista rims, making them sharper. Crista narrowing ensures a more efficient coupling of proton pumping to ATP synthesis. We demonstrated that cristae morphology changes even within minutes.
- MeSH
- buněčné dýchání MeSH
- buňky Hep G2 MeSH
- dimerizace MeSH
- hypoxie MeSH
- kyseliny ketoglutarové farmakologie MeSH
- lidé MeSH
- mitochondriální membrány účinky léků ultrastruktura MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondriální protonové ATPasy metabolismus MeSH
- mitochondrie ultrastruktura MeSH
- transmisní elektronová mikroskopie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
3D super-resolution microscopy based on the direct stochastic optical reconstruction microscopy (dSTORM) with primary Alexa-Fluor-647-conjugated antibodies is a powerful method for accessing changes of objects that could be normally resolved only by electron microscopy. Despite the fact that mitochondrial cristae yet to become resolved, we have indicated changes in cristae width and/or morphology by dSTORM of ATP-synthase F1 subunit α (F1α). Obtained 3D images were analyzed with the help of Ripley's K-function modeling spatial patterns or transferring them into distance distribution function. Resulting histograms of distances frequency distribution provide most frequent distances (MFD) between the localized single antibody molecules. In fasting state of model pancreatic β-cells, INS-1E, MFD between F1α were ~80 nm at 0 and 3 mM glucose, whereas decreased to 61 nm and 57 nm upon glucose-stimulated insulin secretion (GSIS) at 11 mM and 20 mM glucose, respectively. Shorter F1α interdistances reflected cristae width decrease upon GSIS, since such repositioning of F1α correlated to average 20 nm and 15 nm cristae width at 0 and 3 mM glucose, and 9 nm or 8 nm after higher glucose simulating GSIS (11, 20 mM glucose, respectively). Also, submitochondrial entities such as nucleoids of mtDNA were resolved e.g. after bromo-deoxyuridine (BrDU) pretreatment using anti-BrDU dSTORM. MFD in distances distribution histograms reflected an average nucleoid diameter (<100 nm) and average distances between nucleoids (~1000 nm). Double channel PALM/dSTORM with Eos-lactamase-β plus anti-TFAM dSTORM confirmed the latter average inter-nucleoid distance. In conclusion, 3D single molecule (dSTORM) microscopy is a reasonable tool for studying mitochondrion.
- MeSH
- beta-buňky cytologie metabolismus MeSH
- buňky Hep G2 MeSH
- DNA vazebné proteiny metabolismus MeSH
- fluorescenční mikroskopie přístrojové vybavení MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- mitochondriální DNA chemie metabolismus MeSH
- mitochondriální membrány metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- potkani Wistar MeSH
- zobrazování trojrozměrné metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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
Insulin production in pancreatic beta-cells is critically linked to mitochondrial oxidative phosphorylation. Increased ATP production triggered by blood glucose represents the beta-cells' glucose sensor. Type-2 diabetes mellitus results from insulin resistance in peripheral tissues and impaired insulin secretion. Pathology of diabetic beta-cells might be reflected by the altered morphology of mitochondrial network. Its characterization is however hampered by the complexity and density of the three-dimensional (3D) mitochondrial tubular networks in these cell types. Conventional confocal microscopy does not provide sufficient axial resolution to reveal the required details; electron tomography reconstruction of these dense networks is still difficult and time consuming. However, mitochondrial network morphology in fixed cells can also be studied by 4Pi microscopy, a laser scanning microscopy technique which provides an approximately 7-fold improved axial resolution (approximately 100 nm) over conventional confocal microscopy. Here we present a quantitative study of these networks in insulinoma INS-1E cells and primary beta-cells in Langerhans islets. The former were a stably-transfected cell line while the latter were transfected with lentivirus, both expressing mitochondrial matrix targeted redox-sensitive GFP. The mitochondrial networks and their partial disintegration and fragmentation are revealed by carefully created iso-surface plots and their quantitative analysis. We demonstrate that beta-cells within the Langerhans islets from diabetic Goto Kakizaki rats exhibited a more disintegrated mitochondrial network compared to those from control Wistar rats and model insulinoma INS-1E cells. Standardization of these patterns may lead to development of morphological diagnostics for Langerhans islets, for the assessment of beta-cell condition, before their transplantations. Copyright 2010 Elsevier B.V. All rights reserved.
- MeSH
- beta-buňky * patologie ultrastruktura MeSH
- diabetes mellitus 2. typu * patologie MeSH
- inzulinom patologie MeSH
- konfokální mikroskopie * metody MeSH
- krysa rodu rattus MeSH
- mitochondrie * patologie ultrastruktura MeSH
- modely nemocí na zvířatech MeSH
- nádorové buněčné linie MeSH
- nádory slinivky břišní patologie MeSH
- potkani Wistar MeSH
- rekombinantní fúzní proteiny genetika MeSH
- techniky in vitro MeSH
- transfekce MeSH
- zelené fluorescenční proteiny genetika MeSH
- zobrazování trojrozměrné MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Complex I, i.e. proton-pumping NADH:quinone oxidoreductase, is an essential component of the mitochondrial respiratory chain but produces superoxide as a side-reaction. However, conditions for maximum superoxide production or its attenuation are not well understood. Unlike for Complex III, it has not been clear whether a Complex I-derived superoxide generation at forward electron transport is sensitive to membrane potential or protonmotive force. In order to investigate this, we used Amplex Red for H(2)O(2) monitoring, assessing the total mitochondrial superoxide production in isolated rat liver mitochondria respiring at state 4 as well as at state 3, namely with exclusive Complex I substrates or with Complex I substrates plus succinate. We have shown for the first time, that uncoupling diminishes rotenone-induced H(2)O(2) production also in state 3, while similar attenuation was observed in state 4. Moreover, we have found that 5-(N-ethyl-N-isopropyl) amiloride is a real inhibitor of Complex I H(+) pumping (IC(50) of 27 microM) without affecting respiration. It also partially prevented suppression by FCCP of rotenone-induced H(2)O(2) production with Complex I substrates alone (glutamate and malate), but nearly completely with Complexes I and II substrates. Sole 5-(N-ethyl-N-isopropyl) amiloride alone suppressed 20% and 30% of total H(2)O(2) production, respectively, under these conditions. Our data suggest that Complex I mitochondrial superoxide production can be attenuated by uncoupling, which means by acceleration of Complex I H(+) pumping due to the respiratory control. However, when this acceleration is prevented by 5-(N-ethyl-N-isopropyl) amiloride inhibition, no attenuation of superoxide production takes place.
- MeSH
- amilorid analogy a deriváty farmakologie MeSH
- biologické modely MeSH
- buněčné dýchání účinky léků MeSH
- financování organizované MeSH
- jaterní mitochondrie enzymologie účinky léků MeSH
- krysa rodu rattus MeSH
- kyselina glutamová farmakologie MeSH
- kyselina jantarová farmakologie MeSH
- maláty farmakologie MeSH
- peroxid vodíku metabolismus MeSH
- potkani Wistar MeSH
- protonové pumpy metabolismus MeSH
- respirační komplex I metabolismus MeSH
- rozpřahující látky farmakologie MeSH
- superoxidy metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
Vulnerability of mitochondrial Complex I to oxidative stress determines an organism's lifespan, pace of aging, susceptibility to numerous diseases originating from oxidative stress and certain mitopathies. The mechanisms involved, however, are largely unknown. We used confocal microscopy and fluorescent probe MitoSOX to monitor superoxide production due to retarded forward electron transport in HEPG2 cell mitochondrial Complex I in situ. Matrix-released superoxide production, the un-dismuted surplus (J(m)) was low in glucose-cultivated cells, where an uncoupler (FCCP) reduced it to half. Rotenone caused a 5-fold J(m) increase (AC(50) 2 microM), which was attenuated by uncoupling, membrane potential (DeltaPsi(m)), and DeltapH-collapse, since addition of FCCP (IC(50) 55 nM), valinomycin, and nigericin prevented this increase. J(m) doubled after cultivation with galactose/glutamine (i.e. at obligatory oxidative phosphorylation). A hydrophobic amiloride that acts on the ND5 subunit and inhibits Complex I H(+) pumping enhanced J(m) and even countered the FCCP effect (AC(50) 0.3 microM). Consequently, we have revealed a new principle predicting that Complex I produces maximum superoxide only when both electron transport and H(+) pumping are retarded. H(+) pumping may be attenuated by high protonmotive force or inhibited by oxidative stress-related mutations of ND5 (ND2, ND4) subunit. We predict that in a vicious cycle, when oxidative stress leads to higher fraction of, e.g. mutated ND5 subunits, it will be accelerated more and more. Thus, inhibition of Complex I H(+) pumping, which leads to oxidative stress, appears to be a missing link in the theory of mitochondrial aging and in the etiology of diseases related to oxidative stress.
- MeSH
- financování organizované MeSH
- fluorescenční barviva metabolismus MeSH
- fosforylace účinky léků MeSH
- glukosa chemie MeSH
- intracelulární prostor metabolismus účinky léků MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- mitochondriální DNA genetika MeSH
- mitochondrie metabolismus účinky léků MeSH
- mutace MeSH
- nádorové buněčné linie MeSH
- nemoc MeSH
- oxidační stres účinky léků MeSH
- respirační komplex I antagonisté a inhibitory genetika metabolismus MeSH
- rotenon toxicita MeSH
- rozpřahující látky toxicita MeSH
- stárnutí MeSH
- superoxidy metabolismus MeSH
- transport elektronů MeSH
- Check Tag
- lidé MeSH
Thermogenic uncoupling has been proven only for UCP1 in brown adipose tissue. All other isoforms of UCPs are potentially acting in suppression of mitochondrial reactive oxygen species (ROS) production. In this contribution we show that BAT mitochondria can be uncoupled by lauric acid in the range of approximately 100 nM when endogenous fatty acids are combusted by carnitine cycle and beta-oxidation is properly separated from the uncoupling effect. Respiration increased up to 3 times when related to the lowest fatty acid content (BSA present plus carnitine cycle). We also illustrated that any effect leading to more coupled states leads to enhanced H2O2 generation and any effect resulting in uncoupling gives reduced H2O2 generation in BAT mitochondria. Finally, we report doubling of plant UCP transcript in cells as well as amount of protein detected by 3H-GTP-binding sites in mitochondria of shoots and roots of maize seedlings subjected to the salt stress.
- MeSH
- buněčné dýchání MeSH
- financování organizované MeSH
- guanosintrifosfát metabolismus MeSH
- hnědá tuková tkáň metabolismus MeSH
- iontové kanály MeSH
- karnitin metabolismus MeSH
- kořeny rostlin metabolismus MeSH
- kukuřice setá metabolismus MeSH
- kyseliny laurové farmakologie metabolismus MeSH
- mastné kyseliny metabolismus MeSH
- membránové proteiny fyziologie MeSH
- mitochondriální proteiny MeSH
- mitochondrie metabolismus MeSH
- oxidační stres MeSH
- peroxid vodíku metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- rostlinné proteiny fyziologie MeSH
- rozpřahující látky farmakologie metabolismus MeSH
- savci MeSH
- techniky in vitro MeSH
- transportní proteiny fyziologie MeSH
- vazebná místa MeSH
- výhonky rostlin metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- přehledy MeSH