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Methods in Enzymology 2020Voltage imaging in living cells offers the tantalizing possibility of combining the temporal resolution of electrode-based methods with the spatial resolution of imaging...
Voltage imaging in living cells offers the tantalizing possibility of combining the temporal resolution of electrode-based methods with the spatial resolution of imaging techniques. Our lab has been developing voltage-sensitive fluorophores, or VoltageFluors, that respond to changes in cellular and neuronal membrane potential via a photoinduced electron transfer (PeT)-based mechanism. This unique mechanism enables both the fast response kinetics and high sensitivity required to record action potentials in single trials, across multiple cells without the need for stimuli-triggered averaging. In this chapter, we present a methodology for imaging membrane potential dynamics from dozens of neurons simultaneously in vitro. Using simple, commercially available cameras, illumination sources, and microscope optics in combination with the far-red synthetic voltage-sensitive fluorophore BeRST-1 (Berkeley Red Sensor of Transmembrane potential) provides a readily applied method for monitoring neuronal activity in cultured neurons. We discuss different types of voltage-sensitive dyes, considerations for selecting imaging modalities, and outline procedures for the culture of rat hippocampal neurons and performing voltage imaging experiments with these samples. Finally, we provide an example of how changes to the metabolic input to cultured hippocampal neurons can alter their activity profile.
Topics: Action Potentials; Animals; Fluorescent Dyes; Hippocampus; Membrane Potentials; Neurons; Rats
PubMed: 32560798
DOI: 10.1016/bs.mie.2020.04.028 -
Antimicrobial Agents and Chemotherapy May 2023The treatment of bacterial infections is becoming increasingly challenging with the emergence of antimicrobial resistance. Thus, the development of antimicrobials with...
The treatment of bacterial infections is becoming increasingly challenging with the emergence of antimicrobial resistance. Thus, the development of antimicrobials with novel mechanisms of action is much needed. Previously, we designed several cationic main-chain imidazolium compounds and identified the polyimidazolium PIM1 as a potent antibacterial against a wide panel of multidrug-resistant nosocomial pathogens, and it had relatively low toxicity against mammalian epithelial cells. However, little is known about the mechanism of action of PIM1. Using an oligomeric version of PIM1 with precisely six repeating units (OIM1-6) to control for consistency, we showed that OIM1-6 relies on an intact membrane potential for entry into the bacterial cytoplasm, as resistant mutants to OIM1-6 have mutations in their electron transport chains. These mutants demonstrate reduced uptake of the compound, which can be circumvented through the addition of a sub-MIC dose of colistin. Once taken up intracellularly, OIM1-6 exerts double-stranded DNA breaks. Its potency and ability to kill represents a promising class of drugs that can be combined with membrane-penetrating drugs to potentiate activity and hedge against the rise of resistant mutants. In summary, we discovered that cationic antimicrobial OIM1-6 exhibits an antimicrobial property that is dissimilar to the conventional cationic antimicrobial compounds. Its killing mechanism does not involve membrane disruption but instead depends on the membrane potential for uptake into bacterial cells so that it can exert its antibacterial effect intracellularly.
Topics: Animals; DNA, Bacterial; Membrane Potentials; Antimicrobial Cationic Peptides; Anti-Bacterial Agents; Anti-Infective Agents; Bacteria; Microbial Sensitivity Tests; Mammals
PubMed: 37125913
DOI: 10.1128/aac.00355-23 -
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 -
The Journal of Experimental Biology Jan 2023Mammalian sperm capacitation involves biochemical and physiological changes, such as an increase in intracellular calcium ion concentration ([Ca2+]i), hyperpolarization...
Mammalian sperm capacitation involves biochemical and physiological changes, such as an increase in intracellular calcium ion concentration ([Ca2+]i), hyperpolarization of the plasma membrane potential and sperm hyperactivation, among others. These changes provide sperm with the ability to fertilize. In the bat Corynorhinus mexicanus, there is an asynchrony between spermatogenesis and sperm storage in the male with the receptivity of the female. For instance, in C. mexicanus, spermatogenesis occurs before the reproductive season. During the reproductive period, sperm are stored in the epididymis for a few months and the testis undergoes a regression, indicating low or almost null sperm production. Therefore, it is unclear whether the elements necessary for sperm fertilization success undergo maturation or preparation during epididymis storage. Here, we characterized pH-sensitive motility hyperactivation and Ca2+ influx in sperm, regulated by alkalinization and progesterone. In addition, by electrophysiological recordings, we registered currents that were stimulated by alkalinization and inhibited by RU1968 (a CatSper-specific inhibitor), strongly suggesting that these currents were evoked via CatSper, a sperm Ca2+-specific channel indispensable for mammalian fertilization. We also found hyperpolarization of the membrane potential, such as in other mammalian species, which increased according to the month of capture, reaching the biggest hyperpolarization during the mating season. In conclusion, our results suggest that C. mexicanus sperm have functional CatSper and undergo a capacitation-like process such as in other mammals, particularly Ca2+ influx and membrane potential hyperpolarization.
Topics: Animals; Male; Female; Calcium; Chiroptera; Membrane Potentials; Semen; Spermatozoa; Sperm Motility
PubMed: 36541225
DOI: 10.1242/jeb.244878 -
Cells Aug 2022All living cells maintain a charge distribution across their cell membrane (membrane potential) by carefully controlled ion fluxes. These bioelectric signals regulate...
All living cells maintain a charge distribution across their cell membrane (membrane potential) by carefully controlled ion fluxes. These bioelectric signals regulate cell behavior (such as migration, proliferation, differentiation) as well as higher-level tissue and organ patterning. Thus, voltage gradients represent an important parameter for diagnostics as well as a promising target for therapeutic interventions in birth defects, injury, and cancer. However, despite much progress in cell and molecular biology, little is known about bioelectric states in human stem cells. Here, we present simple methods to simultaneously track ion dynamics, membrane voltage, cell morphology, and cell activity (pH and ROS), using fluorescent reporter dyes in living human neurons derived from induced neural stem cells (hiNSC). We developed and tested functional protocols for manipulating ion fluxes, membrane potential, and cell activity, and tracking neural responses to injury and reinnervation in vitro. Finally, using morphology sensor, we tested and quantified the ability of physiological actuators (neurotransmitters and pH) to manipulate nerve repair and reinnervation. These methods are not specific to a particular cell type and should be broadly applicable to the study of bioelectrical controls across a wide range of combinations of models and endpoints.
Topics: Humans; Induced Pluripotent Stem Cells; Membrane Potentials; Neural Stem Cells; Neuronal Outgrowth; Neurons
PubMed: 36010547
DOI: 10.3390/cells11162470 -
The Journal of Physiology May 2016Activation of neurons not only changes their membrane potential and firing rate but as a secondary action reduces membrane resistance. This loss of resistance, or... (Review)
Review
Activation of neurons not only changes their membrane potential and firing rate but as a secondary action reduces membrane resistance. This loss of resistance, or increase of conductance, may be of central importance in non-invasive magnetic or electric stimulation of the human brain since electrical fields cause larger changes in transmembrane voltage in resting neurons with low membrane conductances than in active neurons with high conductance. This may explain why both the immediate effects and after-effects of brain stimulation are smaller or even reversed during voluntary activity compared with rest. Membrane conductance is also increased during shunting inhibition, which accompanies the classic GABAA IPSP. This short-circuits nearby EPSPs and is suggested here to contribute to the magnitude and time course of short-interval intracortical inhibition and intracortical facilitation.
Topics: Biophysical Phenomena; Excitatory Postsynaptic Potentials; Humans; Inhibitory Postsynaptic Potentials; Membrane Potentials; Neural Inhibition; Synapses
PubMed: 26940751
DOI: 10.1113/JP271452 -
Experimental Brain Research Aug 2020I-waves represent high-frequency (~ 600 Hz) repetitive discharge of corticospinal fibers elicited by single-pulse stimulation of motor cortex. First detected and...
I-waves represent high-frequency (~ 600 Hz) repetitive discharge of corticospinal fibers elicited by single-pulse stimulation of motor cortex. First detected and examined in animal preparations, this multiple discharge can also be recorded in humans from the corticospinal tract with epidural spinal electrodes. The exact underpinning neurophysiology of I-waves is still unclear, but there is converging evidence that they originate at the cortical level through synaptic input from specific excitatory interneuronal circuitries onto corticomotoneuronal cells, controlled by GABAAergic interneurons. In contrast, there is at present no supportive evidence for the alternative hypothesis that I-waves are generated by high-frequency oscillations of the membrane potential of corticomotoneuronal cells upon initial strong depolarization. Understanding I-wave physiology is essential for understanding how TMS activates the motor cortex.
Topics: Animals; Evoked Potentials, Motor; Humans; Interneurons; Membrane Potentials; Motor Cortex; Pyramidal Tracts; Transcranial Magnetic Stimulation
PubMed: 32185405
DOI: 10.1007/s00221-020-05764-4 -
Methods in Enzymology 2021Membrane potential is a fundamental biophysical parameter common to all of cellular life. Traditional methods to measure membrane potential rely on electrodes, which are...
Membrane potential is a fundamental biophysical parameter common to all of cellular life. Traditional methods to measure membrane potential rely on electrodes, which are invasive and low-throughput. Optical methods to measure membrane potential are attractive because they have the potential to be less invasive and higher throughput than classic electrode based techniques. However, most optical measurements rely on changes in fluorescence intensity to detect changes in membrane potential. In this chapter, we discuss the use of fluorescence lifetime imaging microscopy (FLIM) and voltage-sensitive fluorophores (VoltageFluors, or VF dyes) to estimate the millivolt value of membrane potentials in living cells. We discuss theory, application, protocols, and shortcomings of this approach.
Topics: Fluorescent Dyes; Membrane Potentials; Microscopy, Fluorescence; Optical Imaging
PubMed: 34099175
DOI: 10.1016/bs.mie.2021.02.009 -
Biochimica Et Biophysica Acta.... May 2018Particularly in Asia medicinal plants with antimicrobial activity are used for therapeutic purpose. One such plant-derived antibiotic is rhodomyrtone (Rom) isolated from...
Particularly in Asia medicinal plants with antimicrobial activity are used for therapeutic purpose. One such plant-derived antibiotic is rhodomyrtone (Rom) isolated from Rhodomyrtus tomentosa leaves. Rom shows high antibacterial activity against a wide range of Gram-positive bacteria, however, its mode of action is still unclear. Reporter gene assays and proteomic profiling experiments in Bacillus subtilis indicate that Rom does not address classical antibiotic targets like translation, transcription or DNA replication, but acts at the cytoplasmic membrane. In Staphylococcus aureus, Rom decreases the membrane potential within seconds and at low doses, causes release of ATP and even the excretion of cytoplasmic proteins (ECP), but does not induce pore-formation as for example nisin. Lipid staining revealed that Rom induces local membrane damage. Rom's antimicrobial activity can be antagonized in the presence of a very narrow spectrum of saturated fatty acids (C15:0, C16:0, or C18:0) that most likely contribute to counteract the membrane damage. Gram-negative bacteria are resistant to Rom, presumably due to reduced penetration through the outer membrane and its neutralization by LPS. Rom is cytotoxic for many eukaryotic cells and studies with human erythrocytes showed that Rom induces eryptosis accompanied by erythrocyte shrinkage, cell membrane blebbing, and membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Rom's distinctive interaction with the cytoplasmic membrane reminds on the amphipathic, alpha-helical peptides, the phenol-soluble modulins (PSMs), and renders Rom an important tool for the investigation of membrane physiology.
Topics: Animals; Anti-Infective Agents; BALB 3T3 Cells; Bacillus subtilis; Cells, Cultured; HeLa Cells; Hemolysis; Humans; Membrane Potentials; Membranes; Mice; Microbial Sensitivity Tests; Staphylococcus aureus; Xanthones
PubMed: 29317198
DOI: 10.1016/j.bbamem.2018.01.011 -
Communications Biology Jul 2022Mitochondrial ultrastructure represents a pinnacle of form and function, with the inner mitochondrial membrane (IMM) forming isolated pockets of cristae membrane (CM),...
Mitochondrial ultrastructure represents a pinnacle of form and function, with the inner mitochondrial membrane (IMM) forming isolated pockets of cristae membrane (CM), separated from the inner-boundary membrane (IBM) by cristae junctions (CJ). Applying structured illumination and electron microscopy, a novel and fundamental function of MICU1 in mediating Ca control over spatial membrane potential gradients (SMPGs) between CM and IMS was identified. We unveiled alterations of SMPGs by transient CJ openings when Ca binds to MICU1 resulting in spatial cristae depolarization. This Ca/MICU1-mediated plasticity of the CJ further provides the mechanistic bedrock of the biphasic mitochondrial Ca uptake kinetics via the mitochondrial Ca uniporter (MCU) during intracellular Ca release: Initially, high Ca opens CJ via Ca/MICU1 and allows instant Ca uptake across the CM through constantly active MCU. Second, MCU disseminates into the IBM, thus establishing Ca uptake across the IBM that circumvents the CM. Under the condition of MICU1 methylation by PRMT1 in aging or cancer, UCP2 that binds to methylated MICU1 destabilizes CJ, disrupts SMPGs, and facilitates fast Ca uptake via the CM.
Topics: Biological Transport; Membrane Potentials; Mitochondria; Mitochondrial Membranes
PubMed: 35778442
DOI: 10.1038/s42003-022-03606-3