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Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a polyglutamine (polyQ) tract near the N-terminus of the huntingtin (htt) protein. Expanded polyQ tracts are prone to aggregate into oligomers and insoluble fibrils. Mutant htt (mhtt) localizes to variety of organelles, including mitochondria. Specifically, mitochondrial defects, morphological alteration, and dysfunction are observed in HD. Mitochondrial lipids, cardiolipin (CL) in particular, are essential in mitochondria function and have the potential to directly interact with htt, altering its aggregation. Here, the impact of mitochondrial membranes on htt aggregation was investigated using a combination of mitochondrial membrane mimics and tissue-derived mitochondrial-enriched fractions. The impact of exposure of outer and inner mitochondrial membrane mimics (OMM and IMM respectively) to mhtt was explored. OMM and IMM reduced mhtt fibrillization, with IMM having a larger effect. The role of CL in mhtt aggregation was investigated using a simple PC system with varying molar ratios of CL. Lower molar ratios of CL (<5%) promoted fibrillization; however, increased CL content retarded fibrillization. As revealed by in situ AFM, mhtt aggregation and associated membrane morphological changes at the surface of OMM mimics was markedly different compared to IMM mimics. While globular deposits of mhtt with few fibrillar aggregates were observed on OMM, plateau-like domains were observed on IMM. A similar impact on htt aggregation was observed with exposure to purified mitochondrial-enriched fractions. Collectively, these observations suggest mitochondrial membranes heavily influence htt aggregation with implication for HD.
The molecular mechanisms of hepatotoxicity after propofol anaesthesia have not been fully elucidated, although there is a relation with mitochondrial dysfunction. The action of propofol on mitochondrial hepatic functions in a rat model was evaluated by infusion for 4h with 25 and 62.5mg/kg/h propofol or 3.125ml/kg/h (vehicle). Liver mitochondrial respiratory rates were evaluated as well as mitochondrial transmembrane potential (ΔΨ), calcium fluxes, mitochondrial enzymatic activities (Complex I-V) and oxidative stress biomarkers (superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase, lipid peroxidation and the oxidised/reduced glutathione ratio). Biophysical interactions with membrane models were also performed. The mitochondrial transmembrane potential was decreased and the opening time of the mitochondrial permeability transition pore was slightly reduced for the highest dose. The activity of complex II was stimulated by propofol, which also causes fluctuations on some respiratory parameters, whereas the antioxidant system was affected in a nonspecific manner. Fluorescence quenching studies suggested that propofol is preferably located in deeper regions of the bilayer and has a high affinity to mitochondrial membranes. It is suggested that propofol interacts with liver mitochondrial membranes with mild modification in mitochondrial function.
Recent reports have indicated the presence of P-glycoprotein in crude mitochondrial membrane fractions, leading to the assumption that P-glycoprotein is present in mitochondrial membranes, and may be involved in transport across these membranes. To determine the validity of this claim, two cell lines overexpressing endogenous P-glycoprotein were investigated. Using various centrifugation steps, mitochondria were purified from these cells and analyzed by Western blot reaction with the anti-P-glycoprotein antibody C219 and organelle-specific antibodies. While P-glycoprotein is present in crude mitochondrial fractions, these fractions are contaminated with plasma membranes. Further purification of the mitochondria to remove plasma membranes revealed that P-glycoprotein is not expressed in mitochondria of the KB-V1 (vinblastine-resistant KB-3-1 cells) or MCF-7(ADR) (adriamycin-resistant MCF-7 cells) cell lines. To further substantiate these findings, we used confocal microscopy and the anti-P-glycoprotein antibody 17F9. This demonstrated that in intact cells, P-glycoprotein is not present in mitochondria and is primarily localized to the plasma membrane. These findings are consistent with the role of P-glycoprotein in conferring multidrug resistance by decreasing cellular drug accumulation. Therefore, contrary to previous speculation, P-glycoprotein does not confer cellular protection by residing in mitochondrial membranes.
Mitochondrial-associated membranes (MAMs) are known to modulate organellar and cellular functions and can subsequently affect pathophysiology including myocardial ischemia-reperfusion (IR) injury. Thus, identifying molecular targets in MAMs that regulate the outcome of IR injury will hold a key to efficient therapeutics. Here, we found chloride intracellular channel protein (CLIC4) presence in MAMs of cardiomyocytes and demonstrate its role in modulating ER and mitochondrial calcium homeostasis under physiological and pathological conditions. In a murine model, loss of CLIC4 increased myocardial infarction and substantially reduced cardiac function after IR injury. CLIC4 null cardiomyocytes showed increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation injury in comparison to wild-type cardiomyocytes. Overall, our results indicate that MAM-CLIC4 is a key mediator of cellular response to IR injury and therefore may have a potential implication on other pathophysiological processes.
Cardiolipin is an anionic lipid found in the mitochondrial membranes of eukaryotes ranging from unicellular microorganisms to metazoans. This unique lipid contributes to various mitochondrial functions, including metabolism, mitochondrial membrane fusion and/or fission dynamics, and apoptosis. However, differences in cardiolipin content between the two mitochondrial membranes, as well as dynamic fluctuations in cardiolipin content in response to stimuli and cellular signaling events, raise questions about how cardiolipin concentration affects mitochondrial membrane structure and dynamics. Although cardiolipin's structural and dynamic roles have been extensively studied in binary mixtures with other phospholipids, the biophysical properties of cardiolipin in higher number lipid mixtures are still not well resolved. Here, we used molecular dynamics simulations to investigate the cardiolipin-dependent properties of ternary lipid bilayer systems that mimic the major components of mitochondrial membranes. We found that changes to cardiolipin concentration only resulted in minor changes to bilayer structural features but that the lipid diffusion was significantly affected by those alterations. We also found that cardiolipin position along the bilayer surfaces correlated to negative curvature deflections, consistent with the induction of negative curvature stress in the membrane monolayers. This work contributes to a foundational understanding of the role of cardiolipin in altering the properties in ternary lipid mixtures composed of the major mitochondrial phospholipids, providing much-needed insights to help understand how cardiolipin concentration modulates the biophysical properties of mitochondrial membranes.
Mitochondrial morphology is controlled by two opposing processes: fusion and fission. Drp1 (dynamin-related protein 1) and hFis1 are two key players of mitochondrial fission, but how Drp1 is recruited to mitochondria and how Drp1-mediated mitochondrial fission is regulated in mammals is poorly understood. Here, we identify the vertebrate-specific protein MIEF1 (mitochondrial elongation factor 1; independently identified as MiD51), which is anchored to the outer mitochondrial membrane. Elevated MIEF1 levels induce extensive mitochondrial fusion, whereas depletion of MIEF1 causes mitochondrial fragmentation. MIEF1 interacts with and recruits Drp1 to mitochondria in a manner independent of hFis1, Mff (mitochondrial fission factor) and Mfn2 (mitofusin 2), but inhibits Drp1 activity, thus executing a negative effect on mitochondrial fission. MIEF1 also interacts with hFis1 and elevated hFis1 levels partially reverse the MIEF1-induced fusion phenotype. In addition to inhibiting Drp1, MIEF1 also actively promotes fusion, but in a manner distinct from mitofusins. In conclusion, our findings uncover a novel mechanism which controls the mitochondrial fusion-fission machinery in vertebrates. As MIEF1 is vertebrate-specific, these data also reveal important differences between yeast and vertebrates in the regulation of mitochondrial dynamics.
Mitochondria are the principal producers of energy in cells of higher organisms. It was recently reported that mutations and defects in mitochondrial DNA (mtDNA) are associated with various mitochondrial diseases including a variety of neurodegenerative and neuromuscular diseases. Therefore, an effective mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve this, therapeutic agents need to be delivered into the innermost mitochondrial space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the development of MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. In this study, we provide a demonstration of mitochondrial matrix delivery and the visualization of mitochondrial genes (mtDNA) in living cells using the MITO-Porter. We first prepared MITO-Porter containing encapsulated propidium iodide (PI), a fluorescent dye used to stain nucleic acids to detect mtDNA. We then confirmed the emission of red-fluorescence from PI by conjugation with mtDNA, when the carriers were incubated in the presence of isolated rat liver mitochondria. Finally, intracellular observation by confocal laser scanning microscopy clearly verified that the MITO-Porter delivered PI to the mitochondrial matrix.
Mitochondria, the centers of energy production, are highly organized with inner membranes, cristae and outer membranes. The mitochondrial architecture determines their functions in all cellular processes. Changes in the mitochondrial ultrastructure are tightly related to a wide variety of diseases. MGARP, a mitochondria-localized protein, was predicted by bioinformatics and confirmed by cellular and biochemical methods to be located in mitochondria, but there is no direct and clear evidence for its precise location. This report demonstrates the precise ultrastructural location of MGARP within mitochondria by the ascorbate peroxidase 2 (APEX2) system in combination with electron microscopy (EM). EM revealed that more MGARP is located in the inner/cristae membranes, with its C-terminus at the inner faces of the intramembrane spaces, than in the outer membranes. MGARP overexpression caused both mitochondrial remodeling and cristae shaping, leading to the collapse of the mitochondrial network. The mitochondrial morphologies in MGARP-overexpressing cells were diverse; the cells became round or short, and their cristae were deformed and became discontinuous or circular. An engineered MGARP mutant deficient in its transmembrane domain no longer localized to the mitochondria and lost its effects on mitochondrial structure, confirming that the localization of MGARP in the mitochondria depends on its structural integrity. Collectively, our findings define the location of MGARP within the mitochondria, which is associated with its functional implications for the architecture and organization of mitochondria.
The GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial division, but the mechanisms remain poorly understood. Much of what is attributed to Drp1's mechanism of action in mitochondrial membrane fission parallels that of prototypical dynamin in endocytic vesicle scission. Unlike the case for dynamin, however, no lipid target for Drp1 activation at the mitochondria has been identified. In addition, the oligomerization properties of Drp1 have not been well established. We show that the mitochondria-specific lipid cardiolipin is a potent stimulator of Drp1 GTPase activity, as well as of membrane tubulation. We establish further that under physiological conditions, Drp1 coexists as two morphologically distinct polymeric species, one nucleotide bound in solution and the other membrane associated, which equilibrate via a dimeric assembly intermediate. With two mutations, C300A and C505A, that shift Drp1 polymerization equilibria in opposite directions, we demonstrate that dimers, and not multimers, potentiate the reassembly and reorganization of Drp1 for mitochondrial membrane remodeling both in vitro and in vivo.
This paper demonstrates the membranotropic effect of modified levopimaric acid diene adducts on liver mitochondria and lecithin liposomes. We found that the derivatives dose-dependently reduced the efficiency of oxidative phosphorylation of mitochondria due to inhibition of the activity of complexes III and IV of the respiratory chain and protonophore action. This was accompanied by a decrease in the membrane potential in the case of organelle energization both by glutamate/malate (complex I substrates) and succinate (complex II substrate). Compounds 1 and 2 reduced the generation of H2O2 by mitochondria, while compound 3 exhibited a pronounced antioxidant effect on glutamate/malate-driven respiration and, on the other hand, caused ROS overproduction when organelles are energized with succinate. All tested compounds exhibited surface-active properties, reducing the fluidity of mitochondrial membranes and contributing to nonspecific permeabilization of the lipid bilayer of mitochondrial membranes and swelling of the organelles. Modified levopimaric acid diene adducts also induced nonspecific permeabilization of unilamellar lecithin liposomes, which confirmed their membranotropic properties. We discuss the mechanisms of action of the tested compounds on the mitochondrial OXPHOS system and the state of the lipid bilayer of membranes, as well as the prospects for the use of new modified levopimaric acid diene adducts in medicine.
Proteins destined for the mitochondrial matrix are imported by the translocase of the outer membrane--the TOM complex--and the presequence translocase of the inner membrane--the TIM23 complex. At present, there is no structural information on components of the presequence translocase. Tim21, a subunit of the presequence translocase consisting of a membrane anchor and a carboxy-terminal domain exposed to the intermembrane space, directly connects the TOM and TIM23 complexes by binding to the intermembrane space domain of the Tom22 receptor. We crystallized the binding domain of Tim21 of Saccharomyces cerevisiae and determined its structure at 1.6 A resolution. The Tim21 structure represents a new alpha/beta-mixed protein fold with two alpha-helices flanked by an extended eight-stranded beta-sheet. We also identified a core sequence of Tom22 that binds to Tim21. Furthermore, negatively charged amino-acid residues of Tom22 are important for binding to Tim21. Here we suggest a mechanism for the TOM-TIM interaction.
Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle.
Studies on the amyloidogenic N-terminal domain of the E. coli HypF protein (HypF-N) have contributed significantly to a detailed understanding of the pathogenic mechanisms in neurodegenerative diseases characterised by the formation of misfolded oligomers, by proteins such as amyloid-β, α-synuclein and tau. Given that both cell membranes and mitochondria are increasingly recognised as key targets of oligomer toxicity, we investigated the damaging effects of aggregates of HypF-N on mitochondrial membranes. Essentially, we found that HypF-N oligomers characterised by high surface hydrophobicity (type A) were able to trigger a robust permeabilisation of mito-mimetic liposomes possessing cardiolipin-rich membranes and dysfunction of isolated mitochondria, as demonstrated by a combination of mitochondrial shrinking, lowering of mitochondrial membrane potential and cytochrome c release. Furthermore, using single-channel electrophysiology recordings we obtained evidence that the type A aggregates induced currents reflecting formation of ion-conducting pores in mito-mimetic planar phospholipid bilayers, with multi-level conductances ranging in the hundreds of pS at negative membrane voltages. Conversely, HypF-N oligomers with low surface hydrophobicity (type B) could not permeabilise or porate mitochondrial membranes. These results suggest an inherent toxicity of membrane-active aggregates of amyloid-forming proteins to mitochondria, and that targeting of oligomer-mitochondrial membrane interactions might therefore afford protection against such damage.
Hundreds of mitochondrial proteins with N-terminal presequences are translocated across the outer and inner mitochondrial membranes via the TOM and TIM23 complexes, respectively. How translocation of proteins across two mitochondrial membranes is coordinated is largely unknown. Here, we show that the two domains of Tim50 in the intermembrane space, named core and PBD, both have essential roles in this process. Building upon the surprising observation that the two domains of Tim50 can complement each other in trans, we establish that the core domain contains the main presequence-binding site and serves as the main recruitment point to the TIM23 complex. On the other hand, the PBD plays, directly or indirectly, a critical role in cooperation of the TOM and TIM23 complexes and supports the receptor function of Tim50. Thus, the two domains of Tim50 both have essential but distinct roles and together coordinate translocation of proteins across two mitochondrial membranes.
The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.
Mitochondria in neurons contribute to energy supply, the regulation of synaptic transmission, Ca2+ homeostasis, neuronal excitability, and stress adaptation. In recent years, several studies have highlighted that the neurotransmitter serotonin (5-HT) plays an important role in mitochondrial biogenesis in cortical neurons, and regulates mitochondrial activity and cellular function in cardiomyocytes. 5-HT exerts its diverse actions by binding to cell surface receptors that are classified into seven distinct families (5-HT1 to 5-HT7). Recently, it was shown that 5-HT3 and 5-HT4 receptors are located on the mitochondrial membrane and participate in the regulation of mitochondrial function. Furthermore, it was observed that activation of brain 5-HT7 receptors rescued mitochondrial dysfunction in female mice from two models of Rett syndrome, a rare neurodevelopmental disorder characterized by severe behavioral and physiological symptoms. Our Western blot analyses performed on cell-lysate and purified mitochondria isolated from neuronal cell line SH-SY5Y showed that 5-HT7 receptors are also expressed into mitochondria. Maximal binding capacity (Bmax) obtained by Scatchard analysis on purified mitochondrial membranes was 0.081 pmol/mg of 5-HT7 receptor protein. Lastly, we evaluated the effect of selective 5-HT7 receptor agonist LP-211 and antagonist (inverse agonist) SB-269970 on mitochondrial respiratory chain (MRC) cytochrome c oxidase activity on mitochondria from SH-SY5Y cells. Our findings provide the first evidence that 5-HT7 receptor is also expressed in mitochondria.
Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼ 100-160 nm in diameter after 60-90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10-500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress.
Fusaricidins and related LI-F compounds are effective bactericides and fungicides. Recently, we have found that they are highly toxic to mammalian cells. Here, we studied the effect of fusaricidin-type compounds (FTCs) on the membranes of mammalian cells. Ethanol extracts from Paenibacillus polymyxa strains, RS10 and I/Sim, were fractionated and analyzed by HPLC and mass spectrometry. The effects of FTCs on mitochondrial functions and integrity were studied by standard methods: measurements of swelling, membrane potential (ΔΨm), respiration rate, cytochrome c release, and pore sizes. Superoxide flashes were registered by 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA). Plasma membrane permeability was assessed by propidium iodide (PI) staining and ATP release. FTCs caused the permeabilization of the inner mitochondria membrane (IMM) to ions and low-molecular-weight (~750 Da) solutes. The permeabilization did not depend on the permeability transition pore (mPTP) but was strongly dependent on ΔΨm. Fusaricidins A plus B, LI-F05a, and LI-F05b-LI-F07b permeabilized IMM with comparable efficiency. They created pores and affected mitochondrial functions and integrity similarly to mPTP opening. They permeabilized the sperm cell plasma membrane to ATP and PI. Thus, the formation of pores in polarized membranes underlays the toxicity of FTCs to mammals. Besides, FTCs appeared to be superior reference compounds for mPTP studies.
The paper assesses the membranotropic action of the natural antibiotic fusidic acid (FA) and its derivatives. It was found that a FA analogue with ethylenediamine moiety (derivative 2), in contrast to native FA and 3,11-dioxime analogue (derivative 1), is able to increase the mobility of the lipid bilayer in the zone of lipid headgroups, as well as to induce permeabilization of lecithin liposome membranes. A similar effect of derivative 2 is also observed in the case of rat liver mitochondrial membranes. We noted a decrease in the microviscosity of the mitochondrial membrane and nonspecific permeabilization of organelle membranes in the presence of this agent, which was accompanied by a decrease in mitochondrial Δψ and OXPHOS efficiency. This led to a reduction in mitochondrial calcium retention capacity. The derivatives also reduced the production of H2O2 by mitochondria. The paper considers the relationship between the structure of the tested compounds and the observed effects.
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