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Seminars in Cell & Developmental Biology Mar 2024Membrane structural integrity is essential for optimal mitochondrial function. These organelles produce the energy needed for all vital processes, provided their outer... (Review)
Review
Membrane structural integrity is essential for optimal mitochondrial function. These organelles produce the energy needed for all vital processes, provided their outer and inner membranes are intact. This prevents the release of mitochondrial apoptogenic factors into the cytosol and ensures intact mitochondrial membrane potential (ΔΨ) to sustain ATP production. Cell death by apoptosis is generally triggered by outer mitochondrial membrane permeabilization (MOMP), tightly coupled with loss of ΔΨ . As these two processes are essential for both mitochondrial function and cell death, researchers have devised various techniques to assess them. Here, we discuss current methods and biosensors available for detecting MOMP and measuring ΔΨ , focusing on their advantages and limitations and discuss what new imaging tools are needed to improve our knowledge of mitochondrial function.
Topics: Mitochondrial Membranes; Membrane Potentials; Mitochondria; Apoptosis; Biosensing Techniques
PubMed: 37438211
DOI: 10.1016/j.semcdb.2023.07.003 -
ELife Jan 2024Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite...
Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in . These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.
Topics: Animals; Membrane Potential, Mitochondrial; Phosphates; Saccharomyces cerevisiae; Adenosine Triphosphate; Respiration; Mammals
PubMed: 38251707
DOI: 10.7554/eLife.84282 -
Life Science Alliance Dec 2023Mitochondria are central to numerous metabolic pathways whereby mitochondrial dysfunction has a profound impact and can manifest in disease. The consequences of...
Mitochondria are central to numerous metabolic pathways whereby mitochondrial dysfunction has a profound impact and can manifest in disease. The consequences of mitochondrial dysfunction can be ameliorated by adaptive responses that rely on crosstalk from the mitochondria to the rest of the cell. Such mito-cellular signalling slows cell cycle progression in mitochondrial DNA-deficient (ρ) cells, but the initial trigger of the response has not been thoroughly studied. Here, we show that decreased mitochondrial membrane potential (ΔΨm) acts as the initial signal of mitochondrial stress that delays G1-to-S phase transition in both ρ and control cells containing mtDNA. Accordingly, experimentally increasing ΔΨm was sufficient to restore timely cell cycle progression in ρ cells. In contrast, cellular levels of oxidative stress did not correlate with the G1-to-S delay. Restored G1-to-S transition in ρ cells with a recovered ΔΨm is likely attributable to larger cell size, whereas the timing of G1/S transcription remained delayed. The identification of ΔΨm as a regulator of cell cycle progression may have implications for disease states involving mitochondrial dysfunction.
Topics: Membrane Potential, Mitochondrial; Cell Division; Mitochondria; Cell Size; Cross Reactions; DNA, Mitochondrial
PubMed: 37696576
DOI: 10.26508/lsa.202302091 -
Cardiovascular Research Nov 2023Cardiotoxicity is one major reason why drugs do not enter or are withdrawn from the market. Thus, approaches are required to predict cardiotoxicity with high specificity...
AIMS
Cardiotoxicity is one major reason why drugs do not enter or are withdrawn from the market. Thus, approaches are required to predict cardiotoxicity with high specificity and sensitivity. Ideally, such methods should be performed within intact cardiac tissue with high relevance for humans and detect acute and chronic side effects on electrophysiological behaviour, contractility, and tissue structure in an unbiased manner. Herein, we evaluate healthy pig myocardial slices and biomimetic cultivation setups (BMCS) as a new cardiotoxicity screening approach.
METHODS AND RESULTS
Pig left ventricular samples were cut into slices and spanned into BMCS with continuous electrical pacing and online force recording. Automated stimulation protocols were established to determine the force-frequency relationship (FFR), frequency dependence of contraction duration, effective refractory period (ERP), and pacing threshold. Slices generated 1.3 ± 0.14 mN/mm2 force at 0.5 Hz electrical pacing and showed a positive FFR and a shortening of contraction duration with increasing pacing rates. Approximately 62% of slices were able to contract for at least 6 days while showing stable ERP, contraction duration-frequency relationship, and preserved cardiac structure confirmed by confocal imaging and X-ray diffraction analysis. We used specific blockers of the most important cardiac ion channels to determine which analysis parameters are influenced. To validate our approach, we tested five drug candidates selected from the Comprehensive in vitro Proarrhythmia Assay list as well as acetylsalicylic acid and DMSO as controls in a blinded manner in three independent laboratories. We were able to detect all arrhythmic drugs and their respective mode of action on cardiac tissue including inhibition of Na+, Ca2+, and hERG channels as well as Na+/Ca2+ exchanger.
CONCLUSION
We systematically evaluate this approach for cardiotoxicity screening, which is of high relevance for humans and can be upscaled to medium-throughput screening. Thus, our approach will improve the predictive value and efficiency of preclinical cardiotoxicity screening.
Topics: Humans; Swine; Animals; Cardiotoxicity; Calcium; Myocardial Contraction; Heart Ventricles; Heart; Myocytes, Cardiac; Action Potentials
PubMed: 37934066
DOI: 10.1093/cvr/cvad141 -
Cells Aug 2023Optical mapping is a powerful imaging technique widely adopted to measure membrane potential changes and intracellular Ca variations in excitable tissues using... (Review)
Review
Optical mapping is a powerful imaging technique widely adopted to measure membrane potential changes and intracellular Ca variations in excitable tissues using voltage-sensitive dyes and Ca indicators, respectively. This powerful tool has rapidly become indispensable in the field of cardiac electrophysiology for studying depolarization wave propagation, estimating the conduction velocity of electrical impulses, and measuring Ca dynamics in cardiac cells and tissues. In addition, mapping these electrophysiological parameters is important for understanding cardiac arrhythmia mechanisms. In this review, we delve into the fundamentals of cardiac optical mapping technology and its applications when applied to hiPSC-derived cardiomyocytes and discuss related advantages and challenges. We also provide a detailed description of the processing and analysis of optical mapping data, which is a crucial step in the study of cardiac diseases and arrhythmia mechanisms for extracting and comparing relevant electrophysiological parameters.
Topics: Humans; Myocytes, Cardiac; Induced Pluripotent Stem Cells; Heart Diseases; Cardiac Electrophysiology; Coloring Agents
PubMed: 37681899
DOI: 10.3390/cells12172168 -
Journal of Investigative Surgery : the... Dec 2023Cerebral ischemia-reperfusion (I/R) injury (CI/RI) is a severe problem in patients with cerebral ischemia. The current study explored the influences of circular...
Cerebral ischemia-reperfusion (I/R) injury (CI/RI) is a severe problem in patients with cerebral ischemia. The current study explored the influences of circular (circ)-Gucy1a2 on neuronal apoptosis and mitochondrial membrane potential (MMP) in the brain tissue of CI/RI mice. Forty-eight mice were randomized into the sham group, transient middle cerebral artery occlusion (tMCAO) group, lentivirus negative control (LV-NC) group, and LV-Gucy1a2 group. Mice were first injected with lentivirus loaded with LV-Gucy1a2 or LV-NC via lateral ventricle, followed by the establishment of CI/RI models 2 weeks later. Twenty-four hours after CI/RI, the neurological impairment of mice was assessed using a 6-point scoring system. The cerebral infarct volume and brain histopathological changes were determined in CI/RI mice through histological staining. , pcDNA3.1-NC and pcDNA3.1-Gucy1a2 were transfected into mouse primary cortical neurons for 48 hours, followed by the establishment of oxygen-glucose deprivation/reoxygenation (OGD/R) models. The levels of circ-Gucy1a2 in mouse brain tissues and neurons were examined using RT-qPCR. Neuronal proliferation and apoptosis, MMP loss, and oxidative stress (OS)-related indexes in neurons were detected using CCK-8 assay, flow cytometry, JC-1 staining, and H2DFFDA staining. CI/RI mouse models and OGD/R cell models were successfully established. After CI/RI, neurons in mice were impaired and the cerebral infarction volume was increased. circ-Gucy1a2 was poorly expressed in CI/RI mouse brain tissues. Overexpression of circ-Gucy1a2 increased OGD/R-induced neuronal proliferation and mitigated apoptosis, MMP loss, and OS. Overall, circ-Gucy1a2 was down-regulated in brain tissues of CI/RI mice, and overexpression of circ-Gucy1a2 can protect mice from CI/RI.
Topics: Mice; Animals; Membrane Potential, Mitochondrial; Brain Ischemia; Apoptosis; Brain; Reperfusion Injury; Glucose; MicroRNAs
PubMed: 37277119
DOI: 10.1080/08941939.2022.2152509 -
The Journal of Physiology Aug 2023For the past seven decades, the Hodgkin-Huxley (HH) formalism has been an invaluable tool in the arsenal of neuroscientists, allowing for robust and reproducible... (Review)
Review
For the past seven decades, the Hodgkin-Huxley (HH) formalism has been an invaluable tool in the arsenal of neuroscientists, allowing for robust and reproducible modelling of ionic conductances and the electrophysiological phenomena they underlie. Despite its apparent age, its role as a cornerstone of computational neuroscience has not waned. The discovery of dendritic regenerative events mediated by ionic and synaptic conductances has solidified the importance of HH-based models further, yielding new predictions concerning dendritic integration, synaptic plasticity and neuronal computation. These predictions are often validated through in vivo and in vitro experiments, advancing our understanding of the neuron as a biological system and emphasizing the importance of HH-based detailed computational models as an instrument of dendritic research. In this article, we discuss recent studies in which the HH formalism is used to shed new light on dendritic function and its role in neuronal phenomena.
Topics: Action Potentials; Models, Neurological; Neurons; Electrophysiological Phenomena; Neuronal Plasticity
PubMed: 36218068
DOI: 10.1113/JP282756 -
Pflugers Archiv : European Journal of... Jun 2024Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane...
Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane potential dynamics during the initial phase of neutrophil activation and its relation to migration and production of reactive oxygen species (ROS). We performed ROS production measurements of neutrophils from healthy C57BL/6J mice after TNFα-priming and/or C5a stimulation. The actin cytoskeleton was visualized with fluorescence microscopy. Furthermore, we combined migration assays and measurements of membrane potential dynamics after stimulating unprimed and/or TNFα-primed neutrophils with C5a. We show that C5a has a concentration-dependent effect on ROS production and chemokinetic migration. Chemokinetic migration and chemotaxis are impaired at C5a concentrations that induce ROS production. The actin cytoskeleton of unstimulated and of ROS-producing neutrophils is not distributed in a polarized way. Inhibition of the phagocytic NADPH oxidase NOX2 with diphenyleneiodonium (DPI) leads to a polarized distribution of the actin cytoskeleton and rescues chemokinetic migration of primed and C5a-stimulated neutrophils. Moreover, C5a evokes a pronounced depolarization of the cell membrane potential by 86.6 ± 4.2 mV starting from a resting membrane potential of -74.3 ± 0.7 mV. The C5a-induced depolarization occurs almost instantaneously (within less than one minute) in contrast to the more gradually developing depolarization induced by PMA (lag time of 3-4 min). This initial depolarization is accompanied by a decrease of the migration velocity. Collectively, our results show that stimulation with C5a evokes parallel changes in membrane potential dynamics, neutrophil ROS production and motility. Notably, the amplitude of membrane potential dynamics is comparable to that of excitable cells.
Topics: Animals; Neutrophils; Complement C5a; Reactive Oxygen Species; Mice, Inbred C57BL; Mice; Membrane Potentials; NADPH Oxidases; Actin Cytoskeleton; Tumor Necrosis Factor-alpha; Cell Movement; Neutrophil Activation; NADPH Oxidase 2
PubMed: 38613695
DOI: 10.1007/s00424-024-02947-8 -
Nature Jan 2024Prokaryotic type III CRISPR-Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors. Recognition of...
Prokaryotic type III CRISPR-Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors. Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain. Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF-transmembrane helix fusion proteins facilitate the type III CRISPR-Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses.
Topics: Bacteriophages; CRISPR-Associated Proteins; CRISPR-Cas Systems; Membrane Potentials; Nucleotides, Cyclic; RNA, Guide, CRISPR-Cas Systems; Second Messenger Systems; Staphylococcus aureus
PubMed: 38200316
DOI: 10.1038/s41586-023-06902-y -
ACS Nano Jan 2024We present super-resolution microscopy of isolated functional mitochondria, enabling real-time studies of structure and function (voltages) in response to...
We present super-resolution microscopy of isolated functional mitochondria, enabling real-time studies of structure and function (voltages) in response to pharmacological manipulation. Changes in mitochondrial membrane potential as a function of time and position can be imaged in different metabolic states (not possible in whole cells), created by the addition of substrates and inhibitors of the electron transport chain, enabled by the isolation of vital mitochondria. By careful analysis of structure dyes and voltage dyes (lipophilic cations), we demonstrate that most of the fluorescent signal seen from voltage dyes is due to membrane bound dyes, and develop a model for the membrane potential dependence of the fluorescence contrast for the case of super-resolution imaging, and how it relates to membrane potential. This permits direct analysis of mitochondrial structure and function (voltage) of isolated, individual mitochondria as well as submitochondrial structures in the functional, intact state, a major advance in super-resolution studies of living organelles.
Topics: Mitochondria; Organelles; Microscopy; Membrane Potentials; Coloring Agents; Fluorescent Dyes
PubMed: 37289571
DOI: 10.1021/acsnano.3c02768