Comparison of the effects of selected chalcones, dihydrochalcones and some cyclic flavonoids on mitochondrial outer membrane determined by fluorescence spectroscopy

The effect on mitochondrial outer membrane of 4-hydroxychalcone (1), the cyclic chalcone analogues E-2-(4'-hydroxybenzylidene)-1-indanone (2a) and E-2-(4'-hydroxybenzylidene)-1-tetralone (2b), the dihydrochalcones phloretin (3a) and phloridzin (3b), the flavanones naringenin (4a) and naringin (4b), and the flavonol quercetin (5) was investigated by fluorescence spectroscopy. Excitation and emission fluorescence spectra of each flavonoid and synthetic analogue were recorded in respiration medium containing 1 mM succinate. Initial interaction of the compounds with the outer mitochondrial membrane was investigated by recording their fluorescence polarization in the presence of rat liver mitochondria. Most of the compounds displayed an elevated fluorescence polarization on mixing with mitochondria at the zero time point. During the investigated 20 min period the initial fluorescence polarization values remained constant (1, 2a), or a gradual depression of the measured polarization values could be observed (2b, 3a, 4b, 5). In the case of naringenin (4a), however, similar to the previously investigated seven-membered cyclic chalcone analogue E-2-(4 -methoxybenzylidene)-1-benzosuberone, a slight, continuous increase of fluorescence polarization could be detected during the 20 min experiment. Phloridzin (3b) showed an increased fluorescence polarization in first 10 min, which was slightly depressed by the 20 min time point.     

Assessment of the effect of cyclic chalcone analogues on mitochondrial membrane and DNA

The cytotoxic and protective effects of selected synthetic chalcone analogues have been shown in previous studies. We studied their cytotoxic effect on the modification of mitochondrial membrane potential and on DNA. The first spectral information about the methoxy group as well as the dimethylamino substituent in E-2-arylmethylene-1-benzosuberones molecule was obtained by absorption and emission spectra. The cytotoxic effect of both cyclic chalcone analogues on DNA were detected by alkaline single-cell gel electrophoresis. Better fluorescent chalcone analogue E-2-(4′-dimethylamino-benzylidene)-1-benzosuberone was studied further in fresh isolated mitochondria. The decrease of rat liver mitochondria membrane potential (Δψ) was observed by fluorescence emission spectra. For the collapsing of mitochondrial potentials and as the negative control of mitochondrial function the CCCP uncoupler was used. The absorption maximum of the methoxy group was found at a shorter wavelength (λ = 335 nm) than that of the dimethylamino group (λ = 406 nm). The excitation spectra were very similar to the absorption spectra for both molecules but the emission spectra showed a better fluorescence for dimethylamino derivative. After the addition of E-2-(4′-dimethylamino-benzylidene)-1-benzosuberone to the intact mitochondria the decrease of mitochondrial membrane potential Δψ was observed by emisssion fluorescence spectra. Both cyclic chalcone analogues induced DNA damage, which was detected by alkaline comet assay. Mainly the apoptotic cells were detected, but necrotic cells were also present. Similarities in the percentages of DNA migration from the head were observed in both treatment groups. Both benzosuberones, with dimethylamino- and methoxy- substituent, were very active biologically, as shown by DNA results of the comet assay. Due to its better fluorescence properties, only the fluorophore with dimethylamino substituent was selected for further study of the function of rat liver mitochondria. Decline of mitochondrial function as well as mitochondrial DNA damage were evident between experimental and control groups.     

Phosphate is required to maintain the outer membrane integrity and membrane potential of respiring yeast mitochondria.

The buffer requirements to maintain mitochondrial intactness and membrane potential in in vitro studies were investigated, using gradient purified yeast mitochondria. It was found that the presence of phosphate is crucial for generation of a stable membrane potential and for preserving the intactness of the outer membrane, as assessed by probing the accessibility of Tom40p to trypsin and the leakage of cytochrome b2 from the intermembrane space. Upon addition of respiratory substrate in the absence of phosphate, mitochondria generate a membrane potential that collapses within 1 min. Under the same conditions, the mitochondrial outer membrane is disrupted. The presence of phosphate prevents both phenomena. The DeltapH component of the proton motive force appears to be responsible for the compromised outer membrane integrity. The collapse of the membrane potential is reversible to a limited extent. Only when phosphate is added soon enough after the addition of exogenous respiratory substrate can a stable membrane potential be obtained again. Within a few minutes, this capacity is lost. The presence of Mg(2+) prevents rupture of the outer membrane, but does not prevent rapid dissipation of the membrane potential. Similar results were obtained for mitochondria isolated and stored in the presence of dextran or bovine serum albumin.     

The phosphatidylcholine-transfer protein catalyzed import of phosphatidylcholine into isolated rat liver mitochondria

In order to study the individual steps involved in the import of phosphatidylcholine (PC) into rat liver mitochondria, a number of PC analogues were introduced into the outer membrane of isolated mitochondria. Two fluorescent PC species, i.e. 1-palmitoyl-2-(16-bimanylthio)hexadecanoyl-PC (bimane-PC) and 1-palmitoyl-2-(10-pyrene)decanoyl-PC (pyrene-PC), and one radiolabeled PC species, i.e. 1-palmitoyl-2-[1-14C]oleoyl-PC (14C-POPC), were studied. The PC analogues were introduced from small unilamellar vesicles with the use of PC-specific transfer protein. The amount of PC imported was quantified by reisolation of the mitochondria. Import of the fluorescent PC species was monitored by on-line fluorescence spectroscopy. The distribution of the newly inserted PC between the outer and the inner membrane was assessed by separation of the two membranes using digitonin treatment. All analogues tested remained exclusively localized in the outer membrane thereby suggesting that additional (extramitochondrial) factors are required to initiate transfer of PC to the inner membrane.     

Effects of chronic ethanol ingestion on the activity of rat liver mitochondrial 2',3'-cyclic nucleotide 3'-phosphohydrolase

Chronic ethanol ingestion induced a 47% increase in the specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (nucleoside-2':3'-cyclic-phosphate 2'-nucleotidohydrolase, EC 3.1.4.37) in whole mitochondria. Both inner and outer mitochondrial membranes showed increased (cyclic nucleotide)phosphohydrolase activity, but the inner was increased 94% compared to 67% for the outer. Techniques which disrupt membrane structure increased (cyclic nucleotide)phosphohydrolase activity. After these treatments, whole mitochondria from ethanol-treated animals still showed a 50% increase in activity. This increase may be related either to an inherent increase in the resistance of (cyclic nucleotide)phosphohydrolase to protein degradation or turnover, or to ethanol-induced membrane changes. An increase in (cyclic nucleotide)phosphohydrolase reaction medium pH was observed when freshly isolated, highly-coupled mitochondria were used. The total increase in pH was about 2-fold greater in the controls compared to the ethanol-treated mitochondria. It is suggested that the smaller initial increase in pH and the greater activity of (cyclic nucleotide)phosphohydrolase in the mitochondria from the ethanol-treated animals relate to previously observed changes in the lipid and protein composition of the mitochondrial membranes. In addition, (cyclic nucleotide)phosphohydrolase may represent an excellent marker for membrane integrity.     

2',3'-cyclic nucleotide 3'-phosphohydrolase in rat liver mitochondrial membranes

2',3'-Cyclic nucleotide 3'-phosphohydrolase (nucleoside-2':3'-cyclic-phosphate 2'-nucleotidohydrolase, EC 3.1.4.37) activity has been demonstrated in rat liver mitochondria. The enzyme was localized in both the outer and inner mitochondrial membranes but was absent from the intermembrane space and matrix. The mitochondrial (cyclic nucleotide) phosphohydrolase was activated by freezing and thawing and by treatment with digitonin or detergents. It is suggested that (cyclic nucleotide) phosphohydrolase is an integral membrane protein which is buried to a significant degree within the membrane. Atractyloside was found to be a noncompetitive inhibitor of the enzyme both in intact mitochondria and in preparations of the mitochondrial membranes. The enzyme substrate, 2',3'-cyclic adenosine monophosphate, had no effect on the oxidation of exogenous beta-hydroxybutyrate or succinate by intact mitochondria. These findings suggest that 2',3'-cyclic nucleotide 3'phosphohydrolase is more widely distributed than was previously thought and that the enzyme may play a fundamental role in membranes, independent of their specialized structure or functions.     

2′,3′-Cyclic nucleotide 3′-phosphohydrolase in rat liver mitochondrial membranes

2′,3′-Cyclic nucleotide 3′-phosphohydrolase (nucleoside-2′:3′-cyclic-phosphate 2′-nucleotidohydrolase, EC 3.1.4.37) activity has been demonstrated in rat liver mitochondria. The enzyme was localized in both the outer and inner mitochondrial membranes but was absent from the intermembrane space and matrix. The mitochondrial (cyclic nucleotide) phosphohydrolase was activated by freezing and thawing and by treatment with digitonin or detergents. It is suggested that (cyclic nucleotide) phosphohydrolase is an integral membrane protein which is buried to a significant degree within the membrane. Atractyloside was found to be a noncompetitive inhibitor of the enzyme both in intact mitochondria and in preparations of the mitochondrial membranes. The enzyme substrate, 2′,3′-cyclic adenosine monophosphate, had no effect on the oxidation of exogenous β-hydroxybutyrate or succinate by intact mitochondria. These findings suggest that 2′,3′-cyclic nucleotide 3′phosphohydrolase is more widely distributed than was previously thought and that the enzyme may play a fundamental role in membranes, independent of their specialized structure or functions.     

Use of Human Cancer Cell Lines Mitochondria to Explore the Mechanisms of BH3 Peptides and ABT-737-Induced Mitochondrial Membrane Permeabilization

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.     

Activation of oxidative stress response by hydroxyl substituted chalcones and cyclic chalcone analogues in mitochondria

We investigated the effect of hydroxyl substituted chalcone (1a) and some chalcone analogues (1b-d) on isolated rat liver mitochondria to gain new insights into the cytotoxic mechanism of these compounds. We observed an inhibitory effect on phosphorylation and the partial uncoupling of compounds 1a and 1d. Increased radical generation and possible covalent interaction of the compounds with cellular thiols resulted in glutathione (GSH) depletion and modulation of the investigated mitochondrial activities. Disruption of interconnected mechanisms as electron transport chain and energetic metabolism, ROS production and insufficiency of antioxidant defensive system could lead to induction of cell death.     

Nitric oxide and mitochondrial status in noise-induced hearing loss

The study investigated the distribution of nitric oxide (NO) within isolated outer hair cells (OHCs) from the cochlea, its relationship to mitochondria and its modulation of mitochondrial function. Using two fluorescent dyes--4,5-diamino-fluorescein diacetate (DAF-2DA), which detects NO, and tetramethyl rhodamine methyl ester (TMRM+), a mitochondrial membrane potential dye--it was found that a relatively greater amount of the DAF fluorescence in OHCs co-localized with mitochondria in comparison to DAF fluorescence in the cytosole. This study also observed reduced mitochondrial membrane potential of OHCs and increased DAF fluorescence following exposure of the cells to noise (120 dB SPL for 4 h) and to an exogenous NO donor, NOC-7 (>350 mm). Antibody label for nitrotyrosine was also increased, indicating NO-related formation of peroxynitrite in both mitochondria and the cytosol. The results suggest that NO may play an important physiological role in regulating OHC energy status and act as a potential agent in OHC pathology.     

A high-throughput assay for mitochondrial membrane potential in permeabilized yeast cells

A fluorometric assay for mitochondrial membrane potential in permeabilized yeast cells has been developed. This method involves permeabilizing the plasma membrane and measuring the distribution of a mitochondrial membrane potential sensitive probe 3,3'-dipropylthiadicarbocyanine iodide (DiSC(3)(5); DiSC(3)). In permeabilized cells, DiSC(3) fluorescence decreased when introduced into energized mitochondria and increased three- to sixfold when the mitochondrial membrane potential was dissipated by the chemical uncoupler carbonylcyanide m-chlorophenyl hydrazone. Plasma membrane potential was abolished by permeabilization, as shown by a lack of polarization of the plasma membrane induced by K(+) and glucose. Uncoupling protein 1 (UCP1), a mitochondrial H(+) transporter, was used as a model for method validation. The fluorescence intensity responded vigorously to specific modulators in UCP1-expressing cells. This method has been adapted as a high-throughput assay to screen for modulators of mitochondrial membrane potential.     

Crucial role of sulfhydryl groups in the mitochondrial inner membrane structure

The mitochondrial inner membrane lost its selectivity for the transport of solutes after reaction of hydrophobic sulfhydryl groups with alkylating agents (maleimide derivatives). The nature of the thiol reagent-induced membrane perturbations was investigated. Modifications of the interactions between membrane components after treatment with thiol reagents were assessed by measuring the binding parameters of 1-anilinonaphtalene-8-sulfonate. An enhancement (about 50%) of the fluorescence intensity, a weak increase of the number of binding sites, and a decrease of the apparent dissociation constant were observed. However, no significant modification of the net surface charge was detected. The osmotic behavior of mitochondria in hypotonic solutions of sucrose was altered after thiol modification. The outer membrane did not seem to influence the matricial volume expansion when thiols were alkylated. After swelling in an isotonic solution of permeant ions, N-butylmaleimide-treated mitochondrial lost one-half of their malate dehydrogenase content, whereas fumarase and glutamate dehydrogenase did not leave the matrix space. Addition of polyethylene glycol of molecular weight below 6000 to swollen mitochondria induced a rapid but transient shrinkage. In swollen mitochondria, the above results indicate a possible holes formation in the membrane structure. The size of these holes was estimated to be about 3 nm. This process which required the presence of the outer membrane, was favored by increasing the temperature and was antagonized by specific effectors of the adenine nucleotide translocator.     

Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader

Opening of permeability transition (PT) pores in the mitochondrial inner membrane causes the mitochondrial permeability transition (MPT) and leads to mitochondrial swelling, membrane depolarization, and release of intramitochondrial solutes. Here, our aim was to develop high-throughput assays using a fluorescence plate reader to screen potential inducers and blockers of the MPT. Isolated rat liver mitochondria (0.5 mg/ml) were incubated in multiwell plates with tetramethylrhodamine methyl ester (TMRM, 1 microM), a potential-indicating fluorophore, and Fluo-5N (1 microM), a low-affinity Ca(2+) indicator. Incubation led to mitochondrial polarization, as indicated by uncoupler-sensitive quenching of the red TMRM fluorescence. CaCl(2) (100 microM) addition led to ruthenium red-sensitive mitochondrial Ca(2+) uptake, as indicated by green Fluo-5N fluorescence. After Ca(2+) accumulation, mitochondria depolarized, released Ca(2+) into the medium, and began to swell. This swelling was monitored as a decrease in light absorbance at 620 nm. Swelling, depolarization, and Ca(2+) release were prevented by cyclosporin A (1 microM), confirming that these events represented the MPT. Measurements of Ca(2+), mitochondrial membrane potential, and swelling could be made independently from the same wells without cross interference, and all three signals could be read from every well of a 48-well plate in about 1 min. In other experiments, mitochondria were ester-loaded with carboxydichlorofluorescein (carboxy-DCF) during the isolation procedure. Release of carboxy-DCF after PT pore opening led to an unquenching of green carboxy-DCF fluorescence occurring simultaneously with swelling. By combining measurements of carboxy-DCF release, Ca(2+) uptake, membrane potential, and swelling, MPT inducers and blockers can be distinguished from uncouplers, respiratory inhibitors, and blockers of Ca(2+) uptake. This high-throughput multiwell assay is amenable for screening panels of compounds for their ability to promote or block the MPT.     

The vectorial orientation of human monoamine oxidase in the mitochondrial outer membrane.

1. The localization of monoamine oxidase in the mitochondrial outer membrane was studied in preparations of human liver mitochondrial and brain-cortex non-synaptosomal and synaptosomal mitochondria. 2. Immunochemical accessibility in iso-osmotic and hypo-osmotic mitochondrial preparations was used to localize the enzyme. 3. It was shown that the immunochemically accessible tyramine-oxidizing activity was distributed approximately equally on both surfaces of the membrane in human liver and brain-cortex non-synaptosomal mitochondria. However, the immunochemically accessible beta-phenethylamine-oxidizing activity was situated predominantly on the outer surface, and the immunochemically accessible 5-hydroxytryptamine-oxidizing activity was situated predominantly on the inner surface of the mitochondrial outer membrane in liver and brain-cortex non-synaptosomal mitochondrial preparations. 4. Considerable variation in the distribution of the enzyme in preparations of synaptosomal mitochondria was seen. 5. The simplest model consistent with our observations is that, in liver and brain-cortex non-synaptosomal mitochondria, the tyramine-oxidizing activity is distributed on both sides of the mitochondrial outer membrane, the beta-phenethylamine-oxidizing activity is located on the outer surface of the outer membrane and the 5-hydroxytryptamine-oxidizing activity is located on the inner surface of the mitochondria outer membrane.     

Nitric oxide and mitochondrial status in noise-induced hearing loss

This study investigated the distribution of nitric oxide (NO) within isolated outer hair cells (OHCs) from the cochlea, its relationship to mitochondria and its modulation of mitochondrial function. Using two fluorescent dyes—4,5-diaminofluorescein diacetate (DAF-2DA), which detects NO, and tetramethyl rhodamine methyl ester (TMRM⁺), a mitochondrial membrane potential dye—it was found that a relatively greater amount of the DAF fluorescence in OHCs co-localized with mitochondria in comparison to DAF fluorescence in the cytosole. This study also observed reduced mitochondrial membrane potential of OHCs and increased DAF fluorescence following exposure of the cells to noise (120 dB SPL for 4 h) and to an exogenous NO donor, NOC-7 (>350 nm). Antibody label for nitrotyrosine was also increased, indicating NO-related formation of peroxynitrite in both mitochrondria and the cytosol. The results suggest that NO may play an important physiological role in regulating OHC energy status and act as a potential agent in OHC pathology.     

Calix[4]arenes C-136 and C-137 hyperpolarize myometrium mitochondria membranes

Calixarenes are supramolecular compounds interacting with bioactive molecules and ions, causing changes in biochemical and biophysical processes. The aim of this work was to study the effects of calix[4]arenes C-136, C-137, and C-138 at the level of polarization of the rat myometrium mitochondria membrane. The structure of synthesized calix[4]arene molecules was confirmed by the methods of ¹H NMR and infrared spectroscopy. Calix[4]arenes C-136 and C-137 each possess two chalcone amide moieties at the lower rim, while calix[4]arene C-138, only one. Calix[4]arenes C-136 and C-137 differ by the presence of ether or hydroxyl groups, respectively, at the lower rim of calix[4]arene skeleton, as well as the length of alkyl spacer between chalcone amide group and the macrocycle. It was shown that calix[4]arenes C-136, C-137, and C-138 form micelles in aqueous medium and in dimethylformamide (DMF). Irradiation of micelles with an argon laser on the flow cytometer results in the rise of autofluorescence. In an aqueous medium, calix[4]arene micelles interact with a positively charged voltage-sensitive fluorescent probe TMRM, which can testify to the presence of negative charge in these structures. However, calix[4]arene micelles do not interact with TMRM in DMF solution. The mitochondrial membrane potential was measured using fluorescent dyes MTG and TMRM with confocal microscopy and fluorescent dye TMRM with flow cytometry. Experiments were conducted on myometrium cells in culture and on suspension of digitonin-permeabilized uterus myocytes. It was shown that the fluorescent signal was stable during the time of experiment. Calix[4]arenes C-136 and C-137 (10 μM) hyperpolarize mitochondria membranes. At maximum, the effect was 173% relative to the control. At the same time, calix[4]arene C-138 did not influence the mitochondria membrane potential. The relationship between the structural organization of investigated calix[4]arene molecules and their effect on polarization of the mitochondria membrane is discussed.     

The concise synthesis of chalcone, indanone and indenone analogues of combretastatin A4

A series of aryl- and aroyl-substituted chalcone analogues of the tubulin binding agent combretastatin A4 (1) were prepared, using a recently introduced one-pot palladium-mediated hydrostannylation-coupling reaction sequence. These chalcones were converted to indanones by Nazarov cyclisation, followed by oxidation to give the corresponding indenones. Indenones were also prepared using a palladium-mediated formal [3+2]-cycloaddition process between ortho-halobenzaldehydes and diarylpropynones. All compounds were assessed as inhibitors of tubulin polymerisation, but only E-31 had activity similar to that of 1. However, compound E-31 did not exhibit antiproliferative activity against the MCF-7 cell line.     

Renal cysteine conjugate beta-lyase. Bioactivation of nephrotoxic cysteine S-conjugates in mitochondrial outer membrane

Cysteine conjugate beta-lyase activity from rat kidney cortex was found in the cystosolic and mitochondrial fractions. With 2 mM S-(2-benzothiazolyl)-L-cysteine as the substrate, approximately two-thirds of the total beta-lyase activity was present in the cytosolic fraction. The kinetics of beta-lyase activity with three cysteine S-conjugates were different in the cytosolic and mitochondrial fractions, and the mitochondrial beta-lyase was much more sensitive to inhibition by aminooxyacetic acid than was the cytosolic activity. These results indicate that the beta-lyase activities in the two subcellular fractions are catalyzed by distinct enzymes. Nephrotoxic cysteine S-conjugates of halogenated hydrocarbons that require bioactivation by cysteine conjugate beta-lyase (S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, CTFC) were potent inhibitors of state 3 respiration in rat kidney mitochondria. Fractionation of mitochondria by digitonin treatment and comparison with marker enzyme distributions showed that the mitochondrial beta-lyase activity is localized in the outer mitochondrial membrane. Inhibition of the beta-lyase prevented the mitochondrial toxicity of DCVC and CTFC, and nonmetabolizable, alpha-methyl analogues of DCVC and CTFC were not toxic. Neither DCVC nor CTFC was toxic to mitoplasts, indicating that activation by the beta-lyase occurs on the outer membrane and may be essential for the expression of toxicity; in contrast, the direct acting nephrotoxin S-(2-chloroethyl)-DL-cysteine was toxic to both mitochondria and mitoplasts. Thus, the suborganelle localization of DCVC and CTFC bioactivation correlates with the observed pattern of toxicity.     

Mitochondrial clustering induced by overexpression of the mitochondrial fusion protein Mfn2 causes mitochondrial dysfunction and cell death

Mitochondria change their shapes dynamically mainly through fission and fusion. Dynamin-related GTPases have been shown to mediate remodeling of mitochondrial membranes during these processes. One of these GTPases, mitofusin, is anchored at the outer mitochondrial membrane and mediates fusion of the outer membrane. We found that overexpression of a mitofusin isoform, Mfn2, drastically changes mitochondrial morphology, forming mitochondrial clusters. High-resolution microscopic examination indicated that the mitochondrial clusters consisted of small fragmented mitochondria. Inhibiting mitochondrial fission prevented the cluster formation, supporting the notion that mitochondrial clusters are formed by fission-mediated mitochondrial fragmentation and aggregation. Mitochondrial clusters displayed a decreased inner membrane potential and mitochondrial function, suggesting a functional compromise of small fragmented mitochondria produced by Mfn2 overexpression; however, mitochondrial clusters still retained mitochondrial DNA. We found that cells containing clustered mitochondria lost cytochrome c from mitochondria and underwent caspase-mediated apoptosis. These results demonstrate that mitochondrial deformation impairs mitochondrial function, leading to apoptotic cell death and suggest the presence of an intricate form-function relationship in mitochondria.     

Phosphorylation/dephosphorylation of maize mitochondrial proteins with different localization

Phosphorylation and dephosphorylation of the proteins residing in the outer mitochondrial membrane, mitoplasts and whole mitochondria of maize (Zea mays L.) were investigated in order to reveal the possible participation of these processes in mitochondrial signaling. A mitochondrial protein of around 57 kD was identified by immunocytochemistry as α-subunit of the F₁-ATPase complex. In isolated mitochondria of maize, phosphorylation of this protein could be visualized only after treating mitochondria with endotholl, an inhibitor of the PP1a and PP2A protein phosphatases. A phosphorylated protein of 46.6 kD was identified as β-subunit of the F₁-ATPase complex. Ca²⁺ is the most common second messenger participating in mitochondrial signaling. We conclude that the transmission of the Ca²⁺ signal to the plant mitochondria occurs via proteins of the outer mitochondrial membrane. The systems perceiving this signal could include the protein phosphatases residing in the outer mitochondrial membrane, which preferentially dephosphorylate the proteins in the inner membrane.