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Due to their exceptional physicochemical features, green synthesized silver nanoparticles (AgNPs) have been of considerable interest in cancer treatment. In the present study, for the first time, we aimed to green synthesize AgNPs from Euphorbia retusa and explore their anticancer potential on human breast cancer (MCF-7) cells. First, the green synthesized AgNPs (EU-AgNPs) were well characterized by UV-visible spectroscopy, Fourier transmission infrared (FTIR) spectrum, XRD, scanning and transmission electron microscopy (SEM and TEM), and EDX techniques. The characterization data exhibited that EU-AgNPs were spherical in shape and crystalline in nature with an average size of 17.8 nm. FTIR results established the presence of active metabolites in EU-AgNPs. Second, the anticancer effect of EU-AgNPs was evaluated against MCF-7 cells by MTT and neutral red uptake (NRU) assays. Moreover, morphological changes, ROS production, MMP, and apoptotic marker genes were also studied upon exposure to cytotoxic doses of EU-AgNPs. Our results showed that EU-AgNPs induce cytotoxicity in a concentration-dependent manner, with an IC 50 value of 40 μg/mL. Morphological changes in MCF-7 cells exposed to EU-AgNPs also confirm their cytotoxic effects. Increased ROS and decreased MMP levels revealed that EU-AgNPs induced oxidative stress and mitochondrial membrane dysfunction. Moreover, ROS-mediated apoptosis was confirmed by elevated levels of proapoptotic marker genes (p53, Bax, caspase-3, and caspase-9) and reduced levels of an antiapoptotic gene (Bcl-2). Altogether, these findings suggested that EU-AgNPs could induce potential anticancer effects through ROS-mediated apoptosis in MCF-7 cells., (© 2024 John Wiley & Sons Ltd.)

Plasma membrane large-conductance calcium-activated potassium (BK Ca ) channels are important players in various physiological processes, including those mediated by epithelia. Like other cell types, human bronchial epithelial (HBE) cells also express BK Ca in the inner mitochondrial membrane (mitoBK Ca ). The genetic relationships between these mitochondrial and plasma membrane channels and the precise role of mitoBK Ca in epithelium physiology are still unclear. Here, we tested the hypothesis that the mitoBK Ca channel is encoded by the same gene as the plasma membrane BK Ca channel in HBE cells. We also examined the impact of channel loss on the basic function of HBE cells, which is to create a tight barrier. For this purpose, we used CRISPR/Cas9 technology in 16HBE14o- cells to disrupt the KCNMA1 gene, which encodes the α-subunit responsible for forming the pore of the plasma membrane BK Ca channel. Electrophysiological experiments demonstrated that the disruption of the KCNMA1 gene resulted in the loss of BK Ca -type channels in the plasma membrane and mitochondria. We have also shown that HBE ΔαBK Ca cells exhibited a significant decrease in transepithelial electrical resistance which indicates a loss of tightness of the barrier created by these cells. We have also observed a decrease in mitochondrial respiration, which indicates a significant impairment of these organelles. In conclusion, our findings indicate that a single gene encodes both populations of the channel in HBE cells. Furthermore, this channel is critical for maintaining the proper function of epithelial cells as a cellular barrier., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Mutations in ATP13A2 cause Kufor-Rakeb Syndrome (KRS), a juvenile form of Parkinson's Disease (PD). The gene product belongs to a diverse family of ion pumps and mediates polyamine influx from lysosomal lumen. While the biochemical and structural studies highlight its unique mechanics, how PD pathology is linked to ATP13A2 function remains unclear. Here we report that localization of overexpressed TOM20, a mitochondrial outer-membrane protein, is significantly altered upon ATP13A2 expression to partially merge with lysosome. Using Halo-fused version of ATP13A2, ATP13A2 was identified in lysosome and autophagosome. Upon ATP13A2 co-expression, overexpressed TOM20 was found not only in mitochondria but also within ATP13A2-containing autolysosome. This modification of TOM20 localization was inhibited by adding 1-methyl-4-phenylpyridinium (MPP+) and not accompanied with mitophagy induction. We suggest that ATP13A2 may participate in the control of overexpressed proteins targeted to mitochondrial outer-membrane., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Hatori et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Regulated cell death is a vital and dynamic process in multicellular organisms that maintains tissue homeostasis and eliminates potentially dangerous cells. Apoptosis, one of the better-known forms of regulated cell death, is activated when cell-surface death receptors like Fas are engaged by their ligands (the extrinsic pathway) or when BCL-2-family pro-apoptotic proteins cause the permeabilization of the mitochondrial outer membrane (the intrinsic pathway). Both the intrinsic and extrinsic pathways of apoptosis lead to the activation of a family of proteases, the caspases, which are responsible for the final cell demise in the so-called execution phase of apoptosis. In this review, I will first discuss the most common types of regulated cell death on a morphological basis. I will then consider in detail the molecular pathways of intrinsic and extrinsic apoptosis, discussing how they are activated in response to specific stimuli and are sometimes overlapping. In-depth knowledge of the cellular mechanisms of apoptosis is becoming more and more important not only in the field of cellular and molecular biology but also for its translational potential in several pathologies, including neurodegeneration and cancer., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The outer mitochondrial membrane (OMM) is essential for cellular homeostasis. Yet little is known of the mechanisms that remodel it during natural stresses. We found that large “SPOTs” (structures positive for OMM) emerge during Toxoplasma gondii infection in mammalian cells. SPOTs mediated the depletion of the OMM proteins mitofusin 1 and 2, which restrict parasite growth. The formation of SPOTs depended on the parasite effector TgMAF1 and the host mitochondrial import receptor TOM70, which is required for optimal parasite proliferation. TOM70 enabled TgMAF1 to interact with the host OMM translocase SAM50. The ablation of SAM50 or the overexpression of an OMM-targeted protein promoted OMM remodeling independently of infection. Thus, Toxoplasma hijacks the formation of SPOTs, a cellular response to OMM stress, to promote its growth.

Mitochondrial dysfunctions are implicated in several pathologies, such as metabolic, cardiovascular, respiratory, and neurological diseases, as well as in cancer and aging. These metabolic alterations are usually assessed in human or murine samples by mitochondrial respiratory chain enzymatic assays, by measuring the oxygen consumption of intact mitochondria isolated from tissues, or from cells obtained after physical or enzymatic disruption of the tissues. However, these methodologies do not maintain tissue multicellular organization and cell-cell interactions, known to influence mitochondrial metabolism. Here, we develop an optimal model to measure mitochondrial oxygen consumption in heart and lung tissue samples using the XF24 Extracellular Flux Analyzer (Seahorse) and discuss the advantages and limitations of this technological approach. Our results demonstrate that tissue organization, as well as mitochondrial ultrastructure and respiratory function, are preserved in heart and lung tissues freshly processed or after overnight conservation at 4 °C. Using this method, we confirmed the repeatedly reported obesity-associated mitochondrial dysfunction in the heart and extended it to the lungs. We set up and validated a new strategy to optimally assess mitochondrial function in murine tissues. As such, this method is of great potential interest for monitoring mitochondrial function in cohort samples.

Mitochondrial functional integrity depends on protein and lipid homeostasis in the mitochondrial membranes and disturbances in their accumulation can cause disease. AGK , a mitochondrial acylglycerol kinase, is not only involved in lipid signaling but is also a component of the TIM22 complex in the inner mitochondrial membrane, which mediates the import of a subset of membrane proteins. AGK mutations can alter both phospholipid metabolism and mitochondrial protein biogenesis, contributing to the pathogenesis of Sengers syndrome. We describe the case of an infant carrying a novel homozygous AGK variant, c.518+1G>A, who was born with congenital cataracts, pielic ectasia, critical congenital dilated myocardiopathy, and hyperlactacidemia and died 20 h after birth. Using the patient's DNA, we performed targeted sequencing of 314 nuclear genes encoding respiratory chain complex subunits and proteins implicated in mitochondrial oxidative phosphorylation (OXPHOS). A decrease of 96-bp in the length of the AGK cDNA sequence was detected. Decreases in the oxygen consumption rate (OCR) and the OCR:ECAR (extracellular acidification rate) ratio in the patient's fibroblasts indicated reduced electron flow through the respiratory chain, and spectrophotometry revealed decreased activity of OXPHOS complexes I and V. We demonstrate a clear defect in mitochondrial function in the patient's fibroblasts and describe the possible molecular mechanism underlying the pathogenicity of this novel AGK variant. Experimental validation using in vitro analysis allowed an accurate characterization of the disease-causing variant.

An extended biophysical model was obtained by upgrading the previously reported one (Khmelinskii and Makarov, 2021). The upgraded model accommodates variations of solute transport rates through the inner mitochondrial membrane (IMM) within the mitochondrial population, described by a Gaussian distribution. However, the model may be used for any functional form of the distribution. The dynamics of system parameters as predicted by the current model differed from that predicted by the previous model in the same initial conditions (Khmelinskii and Makarov, 2021). The amount of change varied from one parameter to the other, remaining in the 1-38% range. The upgraded model fitted the available experimental data with a better accuracy (R = 0.993) compared to the previous model (R = 0.978) using the same experimental data (Khmelinskii and Makarov, 2021). The fitting procedure also estimated the Gaussian distribution parameters. The new model requires much larger computational resources, but given its higher accuracy, it may be used for better analysis of experimental data and for better prediction of MS dynamics in different initial conditions. Note that activities of individual mitochondria in mitochondrial populations should vary within biological tissues. Thus, the currently upgraded model is a better tool for biological and bio-medical applications. We believe that this model is much better adapted to the analysis of MS dynamics in vivo., (Published by Elsevier B.V.)

Aims: To overcome radioresistant cancer cells, clinically relevant radioresistant (CRR) cells were established. To maintain their radioresistance, CRR cells were exposed 2 Gy/day of X-rays daily (maintenance irradiation: MI). To understand whether the radioresistance induced by X-rays was reversible or irreversible, the difference between CRR cells and those without MI for a year (CRR-NoIR cells) was investigated by the mitochondrial function as an index., Main Methods: Radiation sensitivity was determined by modified high density survival assay. Mitochondrial membrane potential (Δψm) was determined by 5,5',6,6'-tetrachloro-1,1', tetraethylbenzimidazolocarbo-cyanine iodide (JC-1) staining. Rapid Glucose-Galactose assay was performed to determine the shift in their energy metabolism from aerobic glycolysis to oxidative phosphorylation in CRR cells. Involvement of prohibitin-1 (PHB1) in Δψm was evaluated by knockdown of PHB1 gene followed by real-time PCR., Key Findings: CRR cells that exhibited resistant to 2 Gy/day X-ray lost their radioresistance after more than one year of culture without MI for a year. In addition, CRR cells lost their radioresistance when the mitochondria were activated by galactose. Furthermore, Δψm were increased and PHB1 expression was down-regulated, in the process of losing their radioresistance., Significance: Our finding reveled that tune regulation of mitochondrial function is implicated in radioresistance phenotype of cancer cells. Moreover, as our findings indicate, though further studies are required to clarify the precise mechanisms underlying cancer cell radioresistance, radioresistant cells induced by irradiation and cancer stem cells that are originally radioresistant should be considered separately, the radioresistance of CRR cells is reversible., (Copyright © 2021 Elsevier Inc. All rights reserved.)

ER lipid raft-associated protein 1 (ERLIN1) and 2 (ERLIN2) are 40 kDa transmembrane glycoproteins belonging to the family of prohibitins, containing a PHB domain. They are generally localized in the endoplasmic reticulum (ER), where ERLIN1 forms a heteroligomeric complex with its closely related ERLIN2. Well-defined functions of ERLINS are promotion of ER-associated protein degradation, mediation of inositol 1,4,5-trisphosphate (IP 3 ) receptors, processing and regulation of lipid metabolism. Until now, ERLINs have been exclusively considered protein markers of ER lipid raft-like microdomains. However, under pathophysiological conditions, they have been described within mitochondria-associated endoplasmic reticulum membranes (MAMs), tethering sites between ER and mitochondria, characterized by the presence of specialized raft-like subdomains enriched in cholesterol and gangliosides, which play a key role in the membrane scrambling and function. In this context, it is emerging that ER lipid raft-like microdomains proteins, i.e., ERLINs, may drive mitochondria-ER crosstalk under both physiological and pathological conditions by association with MAMs, regulating the two main processes underlined, survival and death. In this review, we describe the role of ERLINs in determining cell fate by controlling the "interchange" between apoptosis and autophagy pathways, considering that their alteration has a significant impact on the pathogenesis of several human diseases.

Mitochondria possess transport mechanisms for import of RNA and DNA. Based on import into isolated Solanum tuberosum mitochondria in the presence of competitors, inhibitors or effectors, we show that DNA fragments of different size classes are taken up into plant organelles through distinct channels. Alternative channels can also be activated according to the amount of DNA substrate of a given size class. Analyses of Arabidopsis thaliana knockout lines pointed out a differential involvement of individual voltage-dependent anion channel (VDAC) isoforms in the formation of alternative channels. We propose several outer and inner membrane proteins as VDAC partners in these pathways., (Copyright © 2021 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Mitochondrial activity determines aging rate and the onset of chronic diseases. The mitochondrial permeability transition pore (mPTP) is a pathological pore in the inner mitochondrial membrane thought to be composed of the F-ATP synthase (complex V). OSCP, a subunit of F-ATP synthase, helps protect against mPTP formation. How the destabilization of OSCP may contribute to aging, however, is unclear. We have found that loss OSCP in the nematode Caenorhabditis elegans initiates the mPTP and shortens lifespan specifically during adulthood, in part via initiation of the mitochondrial unfolded protein response (UPR mt ). Pharmacological or genetic inhibition of the mPTP inhibits the UPR mt and restores normal lifespan. Loss of the putative pore-forming component of F-ATP synthase extends adult lifespan, suggesting that the mPTP normally promotes aging. Our findings reveal how an mPTP/UPR mt nexus may contribute to aging and age-related diseases and how inhibition of the UPR mt may be protective under certain conditions., Competing Interests: SA, AF, MC, TP, AG, AS, JA, GL No competing interests declared, (© 2021, Angeli et al.)

Mitochondria associated membranes (MAM), which are the contact sites between endoplasmic reticulum (ER) and mitochondria, have emerged as an important hub for signaling molecules to integrate the cellular and organelle homeostasis, thus facilitating the adaptation of energy metabolism to nutrient status. This review explores the dynamic structural and functional features of the MAM and summarizes the various abnormalities leading to the impaired insulin sensitivity and metabolic diseases.

Transmission electron microscopy (TEM) is widely used as an imaging modality to provide high-resolution details of subcellular components within cells and tissues. Mitochondria and endoplasmic reticulum (ER) are organelles of particular interest to those investigating metabolic disorders. A straightforward method for quantifying and characterizing particular aspects of these organelles would be a useful tool. In this protocol, we outline how to accurately assess the morphology of these important subcellular structures using open source software ImageJ , originally developed by the National Institutes of Health (NIH). Specifically, we detail how to obtain mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology and measuring mito/endoplasmic reticulum (ER) interactions. These procedures provide useful tools for quantifying and characterizing key features of sub-cellular morphology, leading to accurate and reproducible measurements and visualizations of mitochondria and ER.

Mitochondrial alternative oxidase (AOX) is predicted to be present in mitochondria of several invertebrate taxa including tardigrades. Independently of the reason concerning the enzyme occurrence in animal mitochondria, expression of AOX in human mitochondria is regarded as a potential therapeutic strategy. Till now, relevant data were obtained due to heterologous AOX expression in cells and animals without natively expressed AOX. Application of animals natively expressing AOX could importantly contribute to the research. Thus, we decided to investigate AOX activity in intact specimens of the tardigrade Hypsibius exemplaris. We observed that H. exemplaris specimens' tolerance to the blockage of the mitochondrial respiratory chain (MRC) cytochrome pathway was diminished in the presence of AOX inhibitor and the inhibitor-sensitive respiration enabled the tardigrade respiration under condition of the blockage. Importantly, these observations correlated with relevant changes of the mitochondrial inner membrane potential (Δψ) detected in intact animals. Moreover, detection of AOX at protein level required the MRC cytochrome pathway blockage. Overall, we demonstrated that AOX activity in tardigrades can be monitored by the animals' behavior observation as well as by measurement of intact specimens' whole-body respiration and Δψ. Furthermore, it is also possible to check the impact of the MRC cytochrome pathway blockage on AOX level as well as AOX inhibition in the absence of the blockage on animal functioning. Thus, H. exemplaris could be consider as a whole-animal model suitable to study AOX., Competing Interests: The authors have declared that no competing interests exist.

Adenine nucleotide translocase 1 (ANT1) transfers ATP and ADP over the mitochondrial inner membrane and thus supplies the cell with energy. This study analyzed the role of ANT1 in the immune response of ischemic heart tissue. Ischemic ANT1 overexpressing hearts experienced a shift toward an anti-inflammatory immune response. The shift was characterized by low interleukin (IL)-1β expression and M1 macrophage infiltration, whereas M2 macrophage infiltration and levels of IL-10, IL-4, and transforming growth factor (TGFβ) were increased. The modulated immune response correlated with high mitochondrial integrity, reduced oxidative stress, low left ventricular end-diastolic heart pressure, and a high survival rate. Isolated ANT1-transgenic (ANT1-TG) cardiomyocytes expressed low levels of pro-inflammatory cytokines such as IL-1α, tumor necrosis factor α, and TGFβ. However, they showed increased expression and cellular release of anti-inflammatory immunomodulators such as vascular endothelial growth factor. The secretome from ANT1-TG cardiomyocytes initiated stress resistance when applied to ischemic wild-type cardiomyocytes and endothelial cells. It additionally prevented macrophages from expressing pro-inflammatory cytokines. Additionally, ANT1 expression correlated with genes that are related to cytokine and growth factor pathways in hearts of patients with ischemic cardiomyopathy. In conclusion, ANT1-TG cardiomyocytes secrete soluble factors that influence ischemic cardiac cells and initiate an anti-inflammatory immune response in ischemic hearts.

Recently published work suggests that highly permeable low-molecular-weight (LMW) acidic drugs are transported by organic anion transporter 2 (OAT2). However, an asymmetric distribution of ionizable drugs in subcellular organelles where pH gradients are significant may occur in the presence of an inhibitor relative to its absence (e.g., lysosomal trapping). In the present study, OAT2-mediated transport of highly permeable LMW anions could not be demonstrated using OAT2 transfected cells, despite robust transport of the OAT2 substrate penciclovir. Moreover, a rifamycin SV (RifSV)-dependent reduction in the accumulation of highly permeable LMW anions previously observed in hepatocytes could be qualitatively reproduced using HepG2 cells and also in Madin-Darby canine kidney (MDCK) cells, which lack expression of OAT2. Neither HepG2 nor MDCK cells demonstrated meaningful penciclovir transport, nor was the cellular accumulation of the highly permeable LMW anions sensitive to competitive inhibition by the neutral OAT2 substrate penciclovir. Both cell lines, however, demonstrated sensitivity to the mitochondrial uncoupler p -trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) in a manner similar to RifSV. Furthermore, the transepithelial MDCK permeability of the highly permeable LMW anions was measured in the absence and presence of RifSV and FCCP at concentrations that reduced the cellular accumulation of anions. Neither inhibitor, nor the OAT2 inhibitor ketoprofen, reduced the transepithelial flux of the anions as would be anticipated for transported substrate inhibition. The findings presented here are aligned with cellular accumulation of highly permeable LMW anions being significantly determined by ion trapping sensitive to mitochondrial uncoupling, rather than the result of OAT2-mediated transport. SIGNIFICANCE STATEMENT: The manuscript illustrates that passive influx and ion trapping are more relevant to the cellular accumulation of highly permeable low-molecular-weight acidic drugs than is the previously proposed mechanism of OAT2-mediated transport. The outcome illustrated here highlights a rare, and perhaps previously not reported, observation of anionic drug trapping in a compartment sensitive to mitochondrial uncoupling (e.g., the mitochondrial matrix) that may be confused for transporter-mediated uptake., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Transmembrane electrostatically localized protons (TELP) theory has been recently recognized as an important addition over the classic Mitchell's chemiosmosis; thus, the proton motive force (pmf) is largely contributed from TELP near the membrane. As an extension to this theory, a novel phenomenon of mitochondrial thermotrophic function is now characterized by biophysical analyses of pmf in relation to the TELP concentrations at the liquid-membrane interface. This leads to the conclusion that the oxidative phosphorylation also utilizes environmental heat energy associated with the thermal kinetic energy (k B T) of TELP in mitochondria. The local pmf is now calculated to be in a range from 300 to 340 mV while the classic pmf (which underestimates the total pmf) is in a range from 60 to 210 mV in relation to a range of membrane potentials from 50 to 200 mV. Depending on TELP concentrations in mitochondria, this thermotrophic function raises pmf significantly by a factor of 2.6 to sixfold over the classic pmf. Therefore, mitochondria are capable of effectively utilizing the environmental heat energy with TELP for the synthesis of ATP, i.e., it can lock heat energy into the chemical form of energy for cellular functions., (© 2021. The Author(s).)

Abnormal function of suborganelles such as mitochondria and endoplasmic reticulum often leads to abnormal function of cardiomyocytes or vascular endothelial cells and cardiovascular disease (CVD). Mitochondria-associated membrane (MAM) is involved in several important cellular functions. Increasing evidence shows that MAM is involved in the pathogenesis of CVD. MAM mediates multiple cellular processes, including calcium homeostasis regulation, lipid metabolism, unfolded protein response, ROS, mitochondrial dynamics, autophagy, apoptosis, and inflammation, which are key risk factors for CVD. In this review, we discuss the structure of MAM and MAM-associated proteins, their role in CVD progression, and the potential use of MAM as the therapeutic targets for CVD treatment., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Yi Luan et al.)

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that contributes to protein biogenesis as a subunit of the TIM22 complex at the inner mitochondrial membrane. Mutations in AGK cause Sengers syndrome, an autosomal recessive condition characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis. We mapped the proteomic changes in Sengers patient fibroblasts and AGK KO cell lines to understand the effects of AGK dysfunction on mitochondria. This uncovered down-regulation of a number of proteins at the inner mitochondrial membrane, including many SLC25 carrier family proteins, which are predicted substrates of the complex. We also observed down-regulation of SFXN proteins, which contain five transmembrane domains, and show that they represent a novel class of TIM22 complex substrate. Perturbed biogenesis of SFXN proteins in cells lacking AGK reduces the proliferative capabilities of these cells in the absence of exogenous serine, suggesting that dysregulation of one-carbon metabolism is a molecular feature in the biology of Sengers syndrome.

In the last decades, the communication between the Endoplasmic reticulum (ER) and mitochondria has obtained great attention: mitochondria-associated membranes (MAMs), which represent the contact sites between the two organelles, have indeed emerged as central hub involved in different fundamental cell processes, such as calcium signalling, apoptosis, autophagy and lipid biosynthesis. Consistently, dysregulation of ER-mitochondria crosstalk has been associated with different pathological conditions, ranging from diabetes to cancer and neurodegenerative diseases. In this review, we will try to summarize the current knowledge on MAMs' structure and functions in health and their relevance for human diseases., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ectopic fat deposition (EFD) in the kidney plays a key role in the development of diabetic nephropathy (DN). Mitochondria-associated ER membranes (MAMs) are structures that connect to the endoplasmic reticulum (ER) and are involved in lipid metabolism. However, there are few studies on MAMs in the field of kidney disease, and the relationship between EFD and MAMs in DN is still unclear. In this study, increased EFD in the kidneys of DN patients was observed, and analysis showed that the degree of EFD was positively correlated with renal damage. Then, the MAMs were quantified by an in situ proximity ligation assay (PLA). The MAMs in the kidneys were found to gradually decrease through the different stages of DN, while the expression of ADRP (a marker of lipid droplets) and tubulointerstitial damage increased. Moreover, correlation analysis showed that the MAMs were negatively correlated with serum lipid levels, the EFD in the kidney and renal damage. Finally, we observed decreased expression of MAM-control proteins (DsbA-L, PACS-2, and MFN-2) in different stages of DN and they were associated with lipid deposition and renal damage. These data showed that the destruction of MAMs in DN might be the cause of EFD and interstitial damage in the kidney., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yang, Han, Luo, Xiong, Zhu, Zhao, Jiang, Xiao, Wei, Li, Yang and Sun.)

Among the phenolic acids tested on the K562 cell line, a model of chronic myeloid leukemia (CML), caffeic acid (CA) was biologically active on sensitive and imatinib (IM)-resistant cells at micro-molar concentration, either in terms of reduction of cell proliferation or triggering of apoptosis. The CA treatment provoked mitochondrial membrane depolarization, genomic DNA fragmentation and phosphatidylserine exposure, hallmarks of apoptosis. Cell cycle analysis following the treatment with comparable cytotoxic concentrations of IM or CA showed marked differences in the distribution profiles. The reduction of cell proliferation by CA administration was associated with increased expression of two cell cycle repressor genes, CDKN1A and CHES1 , while IM at a cytotoxic concentration increased the CHES1 but not the CDKN1A expression. In addition, CA treatment affected the proliferation and triggered the apoptosis in IM-resistant cells. Taken together, these data suggested that CA induced the anti-proliferative effect and triggered apoptosis of CML cells by a different mechanism than IM. Finally, the combined administration of IM and CA at suboptimal concentrations evidenced a synergy of action in determining the anti-proliferative effect and triggering apoptosis. The ability of CA to potentiate the anti-leukemic effect of IM highlighted the nutraceutical potential of CA in CML.

Mitochondria form networks that continually remodel and adapt to carry out their cellular function. The mitochondrial network is remodeled through changes in mitochondrial morphology, number, and distribution within the cell. Mitochondrial dynamics depend directly on fission, fusion, shape transition, and transport or tethering along the cytoskeleton. Over the past several years, many of the mechanisms underlying these processes have been uncovered. It has become clear that each process is precisely and contextually regulated within the cell. Here, we discuss the mechanisms regulating each aspect of mitochondrial dynamics, which together shape the network as a whole., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Deficiencies in mitochondrial import cause the toxic accumulation of non-imported mitochondrial precursor proteins. Numerous fates for non-imported mitochondrial precursors have been identified in budding yeast, including proteasomal destruction, deposition into protein aggregates, and mistargeting to other organelles. Amongst organelles, the ER has emerged as a key destination for a subset of non-imported mitochondrial proteins. However, how ER targeting of various types of mitochondrial proteins is achieved remains incompletely understood. Here, we show that the ER delivery of endogenous mitochondrial transmembrane proteins, especially those belonging to the SLC25A mitochondrial carrier family, is dependent on the guided entry of tail-anchored proteins (GET) complex. Without a functional GET pathway, non-imported mitochondrial proteins destined for the ER are alternatively sequestered into Hsp42-dependent protein foci. Loss of the GET pathway is detrimental to yeast cells experiencing mitochondrial import failure and prevents re-import of mitochondrial proteins from the ER via the ER-SURF pathway. Overall, this study outlines an important role for the GET complex in ER targeting of non-imported mitochondrial carrier proteins., (© 2021 Xiao et al.)

TIM29 is a mitochondrial inner membrane protein that interacts with the protein import complex TIM22. TIM29 was shown to stabilize the TIM22 complex but its biological function remains largely unknown. Until recently, it was classified as one of the Domain of Unknown Function (DUF) genes, with a conserved protein domain DUF2366 of unclear function. Since characterizing DUF genes can provide novel biological insight, we used previously established transcriptional profiles of the germline and stem cells of the flatworm Macrostomum lignano to probe conserved DUFs for their potential role in germline biology, stem cell function, regeneration, and development. Here, we demonstrate that DUF2366/TIM29 knockdown in M. lignano has very limited effect during the normal homeostatic condition but prevents worms from adapting to a highly proliferative state required for regeneration.

Mitochondria have a remarkable ability to uptake and store massive amounts of calcium. However, the consequences of massive calcium accumulation remain enigmatic. In the present study, we analyzed a series of time-course experiments to identify the sequence of events that occur in a population of guinea pig cardiac mitochondria exposed to excessive calcium overload that cause mitochondrial permeability transition (MPT). By analyzing coincident structural and functional data, we determined that excessive calcium overload is associated with large calcium phosphate granules and inner membrane fragmentation, which explains the extent of mitochondrial dysfunction. This data also reveals a novel mechanism for cyclosporin A, an inhibitor of MPT, in which it preserves cristae despite the presence of massive calcium phosphate granules in the matrix. Overall, these findings establish a mechanism of calcium-induced mitochondrial dysfunction and the impact of calcium regulation on mitochondrial structure and function.

We studied respiratory activity and microviscosity of mitochondrial membranes from the left ventricular myocardium of 2-month-old (n=8) and 15-month-old (n=8) rats. The respiratory control (RC) during substrate phosphorylation was calculated as the ratio of oxygen absorption rates in the presence of ADP and after its utilization. In 2-month-old rats, RC was 4.66 (4.56; 4.71); in 15-month-old animals, it was significantly (р<0.05) lower: 3.57 (3.50; 3.62). Pyrene probe eximerization indices for regions of protein-lipid and lipid-lipid interactions in mitochondria of 2-month-old animals were significantly lower than in the group of 15-month-old rats, which indicated reduced microviscosity of the lipid environment of proteins. Thus, the decrease in RC of mitochondria from the left ventricle of 15-month-old animals and the increase in the microviscosity coefficients of their membranes indicate age-related changes in the structural and functional activity of mitochondria.

Ethnopharmacological Relevance: Shikonin, one of the main active ingredients of Chinese herbal medicine Lithospermum erythrorhizon, has been widely used to treat various disease including virus infection and inflammation in clinical. Its anti-tumor activity has been recorded in "Chinese herbal medicine". Recently, some studies about its anti-glioma effects have been reported. However, little is known about the molecular pharmacological activity of Shikonin in glioma., Aim: This study aimed to systematically uncover and validate the pharmacological mechanism of Shikonin against glioma., Material and Methods: Network pharmacology approach, survival analysis, and Pearson co-expression analysis were performed to uncover and test the pharmacological mechanisms of Shikonin in glioma. Apoptosis assay, Caspase-3 activity assay and immunoblot analysis were practiced to validate the mechanisms., Results: Network pharmacology results suggested, anti-glioma effect of Shikonin by interfering endoplasmic reticulum (ER) stress-mediated tumor apoptosis targeting Caspase-3, and Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP) triggering cancer cell apoptosis. Survival analysis suggested the association of CASP3 with glioma (P < 0.05). Pearson correlation analysis indicated possible interaction of CASP3 with PERK through positive feedback regulation. Shikonin or in combination with 14G2a induced cell apoptosis in oligodendroglioma Hs683 cells in a dose-dependent manner with at a maximum apoptosis rate of 33%-37.5%, and 73%-77% respectively. Immunoblot analysis showed that Shikonin increased Caspase-3 activity to about 4.29 times, and increased 9 times when it combined with 14G2a. Shikonin increased also the expression levels of the proteins PERK and CHOP by about 4.4 and 5.6 folds, respectively, when it combined with 14G2a., Conclusions: This study highlights the pharmacological mechanisms of Shikonin in the induction of tumor apoptosis in glioma cells., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The synthesis of ATP, life's "universal energy currency," is the most prevalent chemical reaction in biological systems and is responsible for fueling nearly all cellular processes, from nerve impulse propagation to DNA synthesis. ATP synthases, the family of enzymes that carry out this endless task, are nearly as ubiquitous as the energy-laden molecule they are responsible for making. The F-type ATP synthase (F-ATPase) is found in every domain of life and has facilitated the survival of organisms in a wide range of habitats, ranging from the deep-sea thermal vents to the human intestine. Accordingly, there has been a large amount of work dedicated toward understanding the structural and functional details of ATP synthases in a wide range of species. Less attention, however, has been paid toward integrating these advances in ATP synthase molecular biology within the context of its evolutionary history. In this review, we present an overview of several structural and functional features of the F-type ATPases that vary across taxa and are purported to be adaptive or otherwise evolutionarily significant: ion channel selectivity, rotor ring size and stoichiometry, ATPase dimeric structure and localization in the mitochondrial inner membrane, and interactions with membrane lipids. We emphasize the importance of studying these features within the context of the enzyme's particular lipid environment. Just as the interactions between an organism and its physical environment shape its evolutionary trajectory, ATPases are impacted by the membranes within which they reside. We argue that a comprehensive understanding of the structure, function, and evolution of membrane proteins-including ATP synthase-requires such an integrative approach., (© 2020 Nirody et al.)

Neutrophils participate in innate immunity as the first line of host defence against microorganisms. However, persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues; this problem occurs in diverse inflammatory diseases, including asthma-affected horses. Previous studies in horses with acute lung inflammation indicated that treatment with tamoxifen (TX), a selective oestrogen receptor modulator, produces a significant decrease in bronchoalveolar lavage fluid (BALF) neutrophil content. The aim of this study was to investigate the effect of tamoxifen and its metabolites (N-desmethyltamoxifen and endoxifen) on the mitochondrial membrane potential assay by flow cytometry, and the activation of effector caspase-3 through immunoblotting, in peripheral blood neutrophils obtained from healthy horses (n = 5). Results show that tamoxifen, N-desmethyltamoxifen and endoxifen depolarize the mitochondrial membrane and activate caspase-3 in healthy equine neutrophils in vitro. These findings suggest that tamoxifen and its metabolites may activate the intrinsic apoptotic pathway in equine neutrophils. However, more studies are necessary to further explore the signalling pathways of these drugs in the induction of apoptosis., (© 2020 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

The large-conductance voltage- and Ca2+-activated K+ channels (BK) are encoded in humans by the Kcnma1 gene. Nevertheless, BK channel isoforms in different locations can exhibit functional heterogeneity mainly due to the alternative splicing during the Kcnma1 gene transcription. Here, we would like to examine the existence of dynamic diversity of BK channels from the inner mitochondrial and cellular membrane from human glioblastoma (U-87 MG). Not only the standard characteristics of the spontaneous switching between the functional states of the channel is discussed, but we put a special emphasis on the presence and strength of correlations within the signal describing the single-channel activity. The considered short- and long-range memory effects are here analyzed as they can be interpreted in terms of the complexity of the switching mechanism between stable conformational states of the channel. We calculate the dependencies of mean dwell-times of (conducting/non-conducting) states on the duration of the previous state, Hurst exponents by the rescaled range R/S method and detrended fluctuation analysis (DFA), and use the multifractal extension of the DFA (MFDFA) for the series describing single-channel activity. The obtained results unraveled statistically significant diversity in gating machinery between the mitochondrial and cellular BK channels.

Mitochondria contain the genetic information and expression machinery to produce essential respiratory chain proteins. Within the mitochondrial matrix, newly synthesized RNA, RNA processing proteins and mitoribosome assembly factors form punctate sub-compartments referred to as mitochondrial RNA granules (MRGs) 1-3 . Despite their proposed importance in regulating gene expression, the structural and dynamic properties of MRGs remain largely unknown. We investigated the internal architecture of MRGs using fluorescence super-resolution localization microscopy and correlative electron microscopy, and found that the MRG ultrastructure consists of compacted RNA embedded within a protein cloud. Using live-cell super-resolution structured illumination microscopy and fluorescence recovery after photobleaching, we reveal that MRGs rapidly exchange components and can undergo fusion, characteristic properties of fluid condensates 4 . Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with mitochondrial remodelling. Inhibition of mitochondrial fission or fusion leads to an aberrant accumulation of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. Together, our findings reveal that MRGs are nanoscale fluid compartments, which are dispersed along mitochondria via membrane dynamics.

Ca 2+ uptake by mitochondria regulates bioenergetics, apoptosis, and Ca 2+ signaling. The primary pathway for mitochondrial Ca 2+ uptake is the mitochondrial calcium uniporter (MCU), a Ca 2+ -selective ion channel in the inner mitochondrial membrane. MCU-mediated Ca 2+ uptake is driven by the sizable inner-membrane potential generated by the electron-transport chain. Despite the large thermodynamic driving force, mitochondrial Ca 2+ uptake is tightly regulated to maintain low matrix [Ca 2+ ] and prevent opening of the permeability transition pore and cell death, while meeting dynamic cellular energy demands. How this is accomplished is controversial. Here we define a regulatory mechanism of MCU-channel activity in which cytoplasmic Ca 2+ regulation of intermembrane space-localized MICU1/2 is controlled by Ca 2+ -regulatory mechanisms localized across the membrane in the mitochondrial matrix. Ca 2+ that permeates through the channel pore regulates Ca 2+ affinities of coupled inhibitory and activating sensors in the matrix. Ca 2+ binding to the inhibitory sensor within the MCU amino terminus closes the channel despite Ca 2+ binding to MICU1/2. Conversely, disruption of the interaction of MICU1/2 with the MCU complex disables matrix Ca 2+ regulation of channel activity. Our results demonstrate how Ca 2+ influx into mitochondria is tuned by coupled Ca 2+ -regulatory mechanisms on both sides of the inner mitochondrial membrane., Competing Interests: The authors declare no competing interest.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present the characterisation of an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery, represents a robust biomarker for both USP30 loss and inhibition. A proteomics analysis, on a SHSY5Y neuroblastoma cell line model, directly compares the effects of genetic loss of USP30 with chemical inhibition. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 ubiquitin proceeds more rapidly in cells either lacking USP30 or subject to USP30 inhibition., (© 2020 Rusilowicz-Jones et al.)

The concept of "electric cables" involved in bioenergetic processes in a living cell was proposed half a century ago [Skulachev, V. P. (1971) Curr. Top. Bioenerg., Elsevier, pp. 127-190]. Membrane structures of a cell were considered as probable pathways for transferring transmembrane electrochemical potential. Further studies have shown that coupling membranes (inner mitochondrial membrane or bacterial cell membrane), i.e., those involved in the generation of membrane potential, can also serve for its transfer. A wide range of organisms from almost all major taxa have been discovered to employ the energy-transmitting function of coupling membranes. Macroscopic (millimeter or even centimeter in length) cable-like structures have been found, the most striking examples of which are giant mitochondria of some unicellular organisms (algae, fungi, protozoa) and animal tissues, filamentous mitochondria, mitochondrial reticulum in animal muscle tissue, and trichomes of cyanobacteria. The importance of such "electric cables" in cells or multicellular structures is determined by their ability to provide rapid energy exchange between metabolic counterparts, energy producers and energy consumers, as the diffusive transport of soluble macroergic molecules (ATP, etc.) requires much longer time. However, in the last 10-15 years, a new type of bacterial "electric cables" of presumably proteinaceous nature has been discovered, which serve a quite different purpose in cell bioenergetics. The molecular structure and functions of these cables will be discussed in the second part of the review ("Electric cables of living cells. II. Bacterial electron conductors").

Recent breakthroughs in live-cell imaging have enabled visualization of cristae, making it feasible to investigate the structure-function relationship of cristae in real time. However, quantifying live-cell images of cristae in an unbiased way remains challenging. Here, we present a novel, semi-automated approach to quantify cristae, using the machine-learning Trainable Weka Segmentation tool. Compared with standard techniques, our approach not only avoids the bias associated with manual thresholding but more efficiently segments cristae from Airyscan and structured illumination microscopy images. Using a cardiolipin-deficient cell line, as well as FCCP, we show that our approach is sufficiently sensitive to detect perturbations in cristae density, size, and shape. This approach, moreover, reveals that cristae are not uniformly distributed within the mitochondrion, and sites of mitochondrial fission are localized to areas of decreased cristae density. After a fusion event, individual cristae from the two mitochondria, at the site of fusion, merge into one object with distinct architectural values. Overall, our study shows that machine learning represents a compelling new strategy for quantifying cristae in living cells., (© 2020 Segawa et al.)

Background: For decades, semiquinone intermediates have been suggested to play an essential role in catalysis by one of the most enigmatic proton-pumping enzymes, respiratory complex I, and different mechanisms have been proposed on their basis. However, the difficulty in investigating complex I semiquinones, due to the many different enzymes embedded in the inner mitochondrial membrane, has resulted in an ambiguous picture and no consensus., Results: In this paper, we re-examine the highly debated origin of semiquinone species in mitochondrial membranes using a novel approach. Our combination of a semi-artificial chimeric respiratory chain with pulse EPR spectroscopy (HYSCORE) has enabled us to conclude, unambiguously and for the first time, that the majority of the semiquinones observed in mitochondrial membranes originate from complex III. We also identify a minor contribution from complex II., Conclusions: We are unable to attribute any semiquinone signals unambiguously to complex I and, reconciling our observations with much of the previous literature, conclude that they are likely to have been misattributed to it. We note that, for this earlier work, the tools we have relied on here to deconvolute overlapping EPR signals were not available. Proposals for the mechanism of complex I based on the EPR signals of semiquinone species observed in mitochondrial membranes should thus be treated with caution until future work has succeeded in isolating any complex I semiquinone EPR spectroscopic signatures present.

Owing to their ability to efficiently generate ATP required to sustain normal cell function, mitochondria are often considered the 'powerhouses of the cell'. However, our understanding of the role of mitochondria in cell biology recently expanded when we recognized that they are key platforms for a plethora of cell signalling cascades. This functional versatility is tightly coupled to constant reshaping of the cellular mitochondrial network in a series of processes, collectively referred to as mitochondrial membrane dynamics and involving organelle fusion and fission (division) as well as ultrastructural remodelling of the membrane. Accordingly, mitochondrial dynamics influence and often orchestrate not only metabolism but also complex cell signalling events, such as those involved in regulating cell pluripotency, division, differentiation, senescence and death. Reciprocally, mitochondrial membrane dynamics are extensively regulated by post-translational modifications of its machinery and by the formation of membrane contact sites between mitochondria and other organelles, both of which have the capacity to integrate inputs from various pathways. Here, we discuss mitochondrial membrane dynamics and their regulation and describe how bioenergetics and cellular signalling are linked to these dynamic changes of mitochondrial morphology.

The mitochondria of various tissues from mice, naked mole rats (NMRs), and bats possess two mechanistically similar systems to prevent the generation of mitochondrial reactive oxygen species (mROS): hexokinases I and II and creatine kinase bound to mitochondrial membranes. Both systems operate in a manner such that one of the kinase substrates (mitochondrial ATP) is electrophoretically transported by the ATP/ADP antiporter to the catalytic site of bound hexokinase or bound creatine kinase without ATP dilution in the cytosol. One of the kinase reaction products, ADP, is transported back to the mitochondrial matrix via the antiporter, again through an electrophoretic process without cytosol dilution. The system in question continuously supports H + -ATP synthase with ADP until glucose or creatine is available. Under these conditions, the membrane potential, ∆ψ, is maintained at a lower than maximal level (i.e., mild depolarization of mitochondria). This ∆ψ decrease is sufficient to completely inhibit mROS generation. In 2.5-y-old mice, mild depolarization disappears in the skeletal muscles, diaphragm, heart, spleen, and brain and partially in the lung and kidney. This age-dependent decrease in the levels of bound kinases is not observed in NMRs and bats for many years. As a result, ROS-mediated protein damage, which is substantial during the aging of short-lived mice, is stabilized at low levels during the aging of long-lived NMRs and bats. It is suggested that this mitochondrial mild depolarization is a crucial component of the mitochondrial anti-aging system., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Optic atrophy type 1 protein (OPA1), a dynamin-related GTPase, that, in addition to mitochondrial fusion, plays an important role in maintaining the structural organization and integrity of the inner mitochondrial membrane (IMM). OPA1 exists in two forms: IMM-bound long-OPA1 (L-OPA1) and soluble short-OPA1 (S-OPA1), a product of L-OPA1 proteolytic cleavage localized in the intermembrane space. In addition to OPA1, the structural and functional integrity of IMM can be regulated by changes in the matrix volume due to the opening/closure of permeability transition pores (PTP). Herein, we investigated the crosstalk between the PTP and OPA1 to clarify whether PTP opening is involved in OPA1-mediated regulation of respiratory chain supercomplexes (RCS) assembly using cardiac mitochondria and cell line. We found that: 1) Proteolytic cleavage of L-OPA1 is stimulated by PTP-induced mitochondrial swelling, 2) OPA1 knockdown reduces PTP-induced mitochondrial swelling but enhances ROS production, 3) OPA1 deficiency impairs the RCS assembly associated with diminished ETC activity and oxidative phosphorylation, 4) OPA1 has no physical interaction with phospholipid scramblase 3 although OPA1 downregulation increases expression of the scramblase. Thus, this study demonstrates that L-OPA1 cleavage depends on the PTP-induced mitochondrial swelling suggesting a regulatory role of the PTP-OPA1 axis in RCS assembly and mitochondrial bioenergetics., (Copyright © 2019 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Through their many and varied metabolic functions, mitochondria power life. Paradoxically, mitochondria also have a central role in apoptotic cell death. Upon induction of mitochondrial apoptosis, mitochondrial outer membrane permeabilization (MOMP) usually commits a cell to die. Apoptotic signalling downstream of MOMP involves cytochrome c release from mitochondria and subsequent caspase activation. As such, targeting MOMP in order to manipulate cell death holds tremendous therapeutic potential across different diseases, including neurodegenerative diseases, autoimmune disorders and cancer. In this Review, we discuss new insights into how mitochondria regulate apoptotic cell death. Surprisingly, recent data demonstrate that besides eliciting caspase activation, MOMP engages various pro-inflammatory signalling functions. As we highlight, together with new findings demonstrating cell survival following MOMP, this pro-inflammatory role suggests that mitochondria-derived signalling downstream of pro-apoptotic cues may also have non-lethal functions. Finally, we discuss the importance and roles of mitochondria in other forms of regulated cell death, including necroptosis, ferroptosis and pyroptosis. Collectively, these new findings offer exciting, unexplored opportunities to target mitochondrial regulation of cell death for clinical benefit.

Mitochondrial membrane dynamics is a cellular rheostat that relates metabolic function and organelle morphology. Using an in vitro reconstitution system, we describe a mechanism for how mitochondrial inner-membrane fusion is regulated by the ratio of two forms of Opa1. We found that the long-form of Opa1 (l-Opa1) is sufficient for membrane docking, hemifusion and low levels of content release. However, stoichiometric levels of the processed, short form of Opa1 (s-Opa1) work together with l-Opa1 to mediate efficient and fast membrane pore opening. Additionally, we found that excess levels of s-Opa1 inhibit fusion activity, as seen under conditions of altered proteostasis. These observations describe a mechanism for gating membrane fusion., Competing Interests: YG, XS, SB, JM, AS, LC No competing interests declared, (© 2020, Ge et al.)

Mitochondria have been widely accepted as the main source of ATP in the cell. The inner mitochondrial membrane (IMM) is important for the maintenance of ATP production and other functions of mitochondria. The electron transport chain (ETC) generates an electrochemical gradient of protons known as the proton-motive force across the IMM and thus produces the mitochondrial membrane potential that is critical to ATP synthesis. One of the main factors regulating the structural and functional integrity of the IMM is the changes in the matrix volume. Mild (reversible) swelling regulates mitochondrial metabolism and function; however, excessive (irreversible) swelling causes mitochondrial dysfunction and cell death. The central mechanism of mitochondrial swelling includes the opening of non-selective channels known as permeability transition pores (PTPs) in the IMM by high mitochondrial Ca 2+ and reactive oxygen species (ROS). The mechanisms of reversible and irreversible mitochondrial swelling and transition between these two states are still unknown. The present study elucidates an upgraded biophysical model of reversible and irreversible mitochondrial swelling dynamics. The model provides a description of the PTP regulation dynamics using an additional differential equation. The rigidity tensor was used in numerical simulations of the mitochondrial parameter dynamics with different initial conditions defined by Ca 2+ concentration in the sarco/endoplasmic reticulum. We were able to estimate the values of the IMM rigidity tensor components by fitting the model to the previously reported experimental data. Overall, the model provides a better description of the reversible and irreversible mitochondrial swelling dynamics., (Copyright © 2019 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

The proton electrochemical gradient generated by respiratory chain activity accounts for over 90% of all available ATP and, as such, its evaluation and accurate measurements regarding its total values and fluctuations is an invaluable component in the understanding of mitochondrial functions. Consequently, alterations in electric potential across the inner mitochondrial membrane generated by differential protonic accumulations and transport are known as the mitochondrial membrane potential, or Δψ, and are reflective of the functional metabolic status of mitochondria. There are several experimental approaches to measure Δψ, ranging from fluorometric evaluations to electrochemical probes. Here we discuss the advantages and disadvantages of several of these methods, ranging from one that is dependent on the movement of a particular ion (tetraphenylphosphonium (TPP + ) with a selective electrode) to the selection of a fluorescent dye from various types to achieve the same goal. The evaluation of the accumulation and movements of TPP + across the inner mitochondrial membrane, or the fluorescence of accumulated dye particles, is a sensitive and accurate method of evaluating the Δψ in respiring mitochondria (either isolated or still inside the cell).

Background: Chondrogenesis represents a highly dynamic cellular process that leads to the establishment of various types of cartilage. However, when stress-related injuries occur, a rapid and efficient regeneration of the tissues is necessary to maintain cartilage integrity. Mesenchymal stem cells (MSCs) are known to exhibit high capacity for self-renewal and pluripotency effects, and thus play a pivotal role in the repair and regeneration of damaged cartilage. On the other hand, the influence of certain pathological conditions such as metabolic disorders on MSCs can seriously impair their regenerative properties and thus reduce their therapeutic potential., Objectives: In this investigation, we attempted to improve and potentiate the in vitro chondrogenic ability of adipose-derived mesenchymal stromal stem cells (ASCs) isolated from horses suffering from metabolic syndrome., Methods: Cultured cells in chondrogenic-inductive medium supplemented with Cladophora glomerata methanolic extract were experimented for expression of the main genes and microRNAs involved in the differentiation process using RT-PCR, for their morphological changes through confocal and scanning electron microscopy and for their physiological homeostasis., Results: The different added concentrations of C. glomerata extract to the basic chondrogenic inductive culture medium promoted the proliferation of equine metabolic syndrome ASCs (ASCs EMS ) and resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II, aggrecan, cartilage oligomeric matrix protein, and Sox9 among others. The results reveal an obvious inhibitory effect of hypertrophy and a strong repression of miR-145-5p, miR-146-3p, and miR-34a and miR-449a largely involved in cartilage degradation. Treated cells additionally exhibited significant reduced apoptosis and oxidative stress, as well as promoted viability and mitochondrial potentiation., Conclusion: Chondrogenesis in EqASCs EMS was found to be prominent after chondrogenic induction in conditions containing C. glomerata extract, suggesting that the macroalgae could be considered for the enhancement of ASC cultures and their reparative properties.

A biochemical reaction model clarifies for the first time how cold atmospheric plasmas (CAPs) affect mitochondrial redox homeostasis and energy metabolism. Fundamental mitochondrial functions in pyruvic acid oxidation, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation involving the respiratory chain (RC), adenosine triphosphate/adenosine diphosphate (ATP/ADP) synthesis machinery and reactive oxygen species/reactive nitrogen species (ROS/RNS)-mediated mechanisms are numerically simulated. The effects of CAP irradiation are modelled as 1) the influx of hydrogen peroxide (H[Formula: see text]O[Formula: see text]) to an ROS regulation system and 2) the change in mitochondrial transmembrane potential induced by RNS on membrane permeability. The CAP-induced stress modifies the dynamics of intramitochondrial H[Formula: see text]O[Formula: see text] and superoxide anions, i.e., the rhythm and shape of ROS oscillation are disturbed by H[Formula: see text]O[Formula: see text] infusion. Furthermore, CAPs control the ROS oscillatory behaviour, nicotinamide adenine dinucleotide redox state and ATP/ADP conversion through the reaction mixture over the RC, the TCA cycle and ROS regulation system. CAPs even induce a homeostatic or irreversible state transition in cell metabolism. The present computational model demonstrates that CAPs crucially affect essential mitochondrial functions, which in turn affect redox signalling, metabolic cooporation and cell fate decision of survival or death.

Mitochondria are highly dynamic organelle that undergo frequent fusion and division, and the balance of these opposing processes regulates mitochondrial morphology, distribution, and function. Mitochondrial fission facilitates the replication and distribution of mitochondria during cell division, whereas the fusion process including inner and outer mitochondrial membrane fusion allows the exchange of intramitochondrial material between adjacent mitochondria. Despite several GTPase family proteins have been implicated as key modulators of mitochondrial dynamics, the mechanisms by which these proteins regulate mitochondrial homeostasis and function remain not clearly understood. Neuronal function and survival are closely related to mitochondria dynamics, and disturbed mitochondrial fission/fusion may influence neurotransmission, synaptic maintenance, neuronal survival and function. Recent studies have shown that mitochondrial dysfunction caused by aberrant mitochondrial dynamics plays an essential role in the pathogenesis of both sporadic and familial Parkinson's disease (PD). Collectively, we review the molecular mechanism of known GTPase proteins in regulating mitochondrial fission and fusion, but also highlight the causal role for mitochondrial dynamics in PD pathogenesis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The endoplasmic reticulum (ER) and mitochondria are two important organelles in cells. Mitochondria-associated membranes (MAMs) are lipid raft-like domains formed in the ER membranes that are in close apposition to mitochondria. They play an important role in signal transmission between these two essential organelles. When cells are exposed to internal or external stressful stimuli, the ER will activate an adaptive response called the ER stress response, which has a significant effect on mitochondrial function. Mitochondrial quality control is an important mechanism to ensure the functional integrity of mitochondria and the effect of ER stress on mitochondrial quality control through MAMs is of great significance. Therefore, in this review, we introduce ER stress and mitochondrial quality control, and discuss how ER stress signals are transmitted to mitochondria through MAMs. We then review the important roles of MAMs in mitochondrial quality control under ER stress., (Copyright © 2019 Elsevier Inc. All rights reserved.)