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

• Publikační typ
• abstrakty 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

Existing controversies led us to analyze absolute mRNA levels of mitochondrial uncoupling proteins (UCP1-UCP5). Individual UCP isoform mRNA levels varied by up to four orders of magnitude in rat and mouse tissues. UCP2 mRNA content was relatively high (0.4 to 0.8 pg per 10 ng of total mRNA) in rat spleen, rat and mouse lung, and rat heart. Levels of the same order of magnitude were found for UCP3 mRNA in rat and mouse skeletal muscle, for UCP4 and UCP5 mRNA in mouse brain, and for UCP2 and UCP5 mRNA in mouse white adipose tissue. Significant differences in pattern were found for rat vs. mouse tissues, such as the dominance of UCP3/UCP5 vs. UCP2 transcript in mouse heart and vice versa in rat heart; or UCP2 (UCP5) dominance in rat brain contrary to 10-fold higher UCP4 and UCP5 dominance in mouse brain. We predict high antioxidant/antiapoptotic UCP function in tissues with higher UCP mRNA content.

• MeSH
• DNA primery genetika   MeSH
• druhová specificita MeSH
• iontové kanály metabolismus   MeSH
• krysa rodu rattus MeSH
• membránové transportní proteiny metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mozek metabolismus   MeSH
• myokard metabolismus   MeSH
• myši MeSH
• plíce metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteiny nervové tkáně metabolismus   MeSH
• slezina metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH
• srovnávací studie MeSH

• Publikační typ
• abstrakty MeSH

Cell death-inducing DFF[DNA fragmentation factor]-like effector-a (CIDEa), may initiate apoptosis by disrupting a complex consisting of 40-kDa caspase-3-activated nuclease (DFF40/CAD) and its 45-kDa inhibitor (DFF45/ICAD). CIDEa, however, was found to be localized in mitochondria. We have performed immunodetection of CIDEa in whole cells and subcellular fractions of HeLa cells adapted for a tetracycline-inducible CIDEa expression. Using immunocytochemistry we observed redistribution, enhanced upon treatment with camptothecin or valinomycin, of CIDEa to nucleus. Similarly, CIDEa content increased in the nuclear fraction but decreased in cytosolic fraction in cells treated to initiate apoptosis. We hypothesize that CIDEa is sequestered in mitochondria while transfer of this potentially dangerous protein from mitochondria into nucleus intensifies or even initiates apoptosis.

• MeSH
• apoptóza MeSH
• buněčné jádro metabolismus   MeSH
• financování organizované MeSH
• HeLa buňky MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• proteiny regulující apoptózu biosyntéza   metabolismus   MeSH
• transport proteinů MeSH
• Check Tag
• lidé MeSH

Mitochondria in numerous cell types, especially in cultured cells, form a reticular network undergoing constant fusion and fission. The three dimensional (3D) morphology of these networks however has not been studied in detail to our knowledge. We have investigated insulinoma INS-1E and hepatocellular carcinoma HEP-G2 cells transfected with mitochondria-addressed GFP. Using 4Pi microscopy, 3D morphology changes responding to decreased oxidative phosphorylation and/or energetic status could be observed in these cells at an unprecedented 100 nm level of detail. In INS-1E cells cultivated at 11 mM glucose, the mitoreticulum appears predominantly as one interconnected mitochondrion with a nearly constant 262+/-26 nm tubule diameter. If cultured at 5 mM glucose, INS-1E cells show 311+/-36 nm tubules coexisting with numerous flat cisternae. Similar interconnected 284+/-38 nm and 417+/-110 nm tubules were found in HEP-G2 cells cultivated at 5 mM and hyperglycaemic 25 mM glucose, respectively. With rotenone inhibiting respiration to approximately 10%, disintegration into several reticula and numerous approximately 300 nm spheres or short tubules was observed. De-energization by uncoupling additionally led to formation of rings and bulky cisternae of 1.4+/-0.4 microm diameter. Rotenone and uncoupler acted synergically in INS-1E cells and increased fusion (ongoing with fission) forming bowl-like shapes. In HEP-G2 cells fission partially ceased with FCCP plus rotenone. Thus we have revealed previously undescribed details for shapes upon mitochondrial disintegration and clearly demonstrate that high resolution 3D microscopy is required for visualization of mitochondrial network. We recommend 4Pi microscopy as a new standard.

• MeSH
• energetický metabolismus MeSH
• financování organizované MeSH
• hepatocelulární karcinom MeSH
• inzulinom MeSH
• konfokální mikroskopie MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• mitochondrie metabolismus   patologie   ultrastruktura   MeSH
• nádorové buněčné linie MeSH
• nádory jater MeSH
• nádory slinivky břišní MeSH
• oxidativní fosforylace MeSH
• počítačové zpracování obrazu MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH

Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Although specific bioenergetics might be involved, parallel studies of cell respiration and mitochondrial membrane potential (DeltaPsi(m)) during GSIS are lacking. Using high resolution respirometry and parallel DeltaPsi(m) monitoring by two distinct fluorescence probes we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model to study in vitro insulin secretion. Upon glucose addition to glucose-depleted cells we demonstrated a simultaneous increase in respiration and DeltaPsi(m) during GSIS and showed that the endogenous state 3/state 4 respiratory ratio hyperbolically increased with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the "toxic" effect of fatty acids on insulin secretion, GSIS was studied after linoleic acid addition, which diminished respiration increase, DeltaPsi(m) jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose. Its effects were due to protonophoric function, i.e. uncoupling, since without glucose, linoleic acid accelerated both state 3 and state 4 respiration by similar extent. In turn, state 3 respiration increased marginally with linoleic acid at 10-20mM glucose. We conclude that upon glucose addition in physiological range, the INS-1E cells are able to regulate the oxidative phosphorylation rate from nearly zero to maximum and that the impairment of GSIS by linoleic acid is caused by mitochondrial uncoupling. These findings may be relevant to the pathogenesis of type 2 diabetes.

• MeSH
• adenosindifosfát metabolismus   MeSH
• adenosintrifosfát metabolismus   MeSH
• financování organizované MeSH
• glukosa farmakologie   MeSH
• inzulinom sekrece   MeSH
• krysa rodu rattus MeSH
• kyselina linolová farmakologie   MeSH
• Langerhansovy ostrůvky sekrece   účinky léků   MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• mitochondrie metabolismus   účinky léků   MeSH
• nádorové buňky kultivované MeSH
• nádory slinivky břišní sekrece   MeSH
• potkani Wistar MeSH
• spotřeba kyslíku účinky léků   MeSH
• transmisní elektronová mikroskopie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata 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