Point-localized, site-specific membrane potential optical recording by single fluorescent nanodiscs.
Ontology highlight
ABSTRACT:
SUBMITTER: Grupi A
PROVIDER: S-EPMC8448295 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The study of electrical activity in single cells and local circuits of excitable cells, such as neurons, requires an easy-to-use, high-throughput methodology that allows for the measurement of membrane potential. Investigating the electrical properties in specific subcompartments of neurons, or in a specific type of neurons, introduces additional complexity. An optical voltage-imaging technique that allows high spatial and temporal resolution could be an ideal solution. However, most valid voltage-imaging techniques are nonspecific. Those that are more site-directed require a lot of preliminary work and specific adaptations, among other drawbacks. Here, we explore a new method for membrane voltage imaging, based on Förster resonance energy transfer between fluorescent polystyrene (FPS) beads and dipicrylamine. Not only has it been shown that fluorescence intensity correlates with membrane potential, but more importantly, the membrane potential from individual particles can be detected. Among other advantages, FPS beads can be synthesized with surface functional groups and can be targeted to specific proteins by conjugation of recognition molecules. Therefore, in the presence of dipicrylamine, FPS beads represent single-particle detectors of membrane potential that can be localized to specific membrane compartments. This new and easily accessible platform for targeted optical voltage imaging can further elucidate the mechanisms of neuronal electrical activity.
| S-EPMC8651512 | biostudies-literature
Project description:In order to determine the sequence of cellular processes in glutamate toxicity, we simultaneously recorded O(2) consumption, cytosolic Ca(2+) concentration ([Ca(2+)](i)), and mitochondrial membrane potential (mDeltapsi) in single cortical neurons. Oxygen consumption was measured using an amperometric self-referencing platinum electrode adjacent to neurons in which [Ca(2+)](i) and mDeltapsi were monitored with Fluo-4 and TMRE(+), respectively, using a spinning disk laser confocal microscope. Excitotoxic doses of glutamate caused an elevation of [Ca(2+)](i) followed seconds afterwards by an increase in O(2) consumption which reached a maximum level within 1-5 min. A modest increase in mDeltapsi occurred during this time period, and then, shortly before maximal O(2) consumption was reached, the mDeltapsi, as indicated by TMRE(+) fluorescence, dissipated. Maximal O(2) consumption lasted up to 5 min and then declined together with mDeltapsi and ATP levels, while [Ca(2+)](i) further increased. mDeltapsi and [Ca(2+)](i) returned to baseline levels when neurons were treated with an NMDA receptor antagonist shortly after the [Ca(2+)](i) increased. Our unprecedented spatial and time resolution revealed that this sequence of events is identical in all neurons, albeit with considerable variability in magnitude and kinetics of changes in O(2) consumption, [Ca(2+)](i), and mDeltapsi. The data obtained using this new method are consistent with a model where Ca(2+) influx causes ATP depletion, despite maximal mitochondrial respiration, minutes after glutamate receptor activation.
| S-EPMC2805059 | biostudies-literature
Project description:Electrophysiological field potential dynamics are of fundamental interest in basic and clinical neuroscience, but how specific cell types shape these dynamics in the live brain is poorly understood. To empower mechanistic studies, we created an optical technique, TEMPO, that records the aggregate trans-membrane voltage dynamics of genetically specified neurons in freely behaving mice. TEMPO has >10-fold greater sensitivity than prior fiber-optic techniques and attains the noise minimum set by quantum mechanical photon shot noise. After validating TEMPO's capacity to track established oscillations in the delta, theta, and gamma frequency bands, we compared the D1- and D2-dopamine-receptor-expressing striatal medium spiny neurons (MSNs), which are interspersed and electrically indistinguishable. Unexpectedly, MSN population dynamics exhibited two distinct coherent states that were commonly indiscernible in electrical recordings and involved synchronized hyperpolarizations across both MSN subtypes. Overall, TEMPO allows the deconstruction of normal and pathologic neurophysiological states into trans-membrane voltage activity patterns of specific cell types.
| S-EPMC5382987 | biostudies-literature
Project description:Graphene oxides (GOs) have emerged as precursors offering the potential of a cost-effective and large-scale production of graphene-based materials. Despite that their intrinsic fluorescence property has already brought interest of researchers for optical applications, to date, refractive-index modulation as one of the fundamental aspects of optical properties of GOs has received less attention. Here we reported on a giant refractive-index modulation on the order of 10(-2) to 10(-1), accompanied by a fluorescence intensity change, through the two-photon reduction of GOs. These features enabled a mechanism for multimode optical recording with the fluorescence contrast and the hologram-encoded refractive-index modulation in GO-dispersed polymers for security-enhanced high-capacity information technologies. Our results show that GO-polymer composites may provide a new material platform enabling flexible micro-/nano-photonic information devices.
| S-EPMC3788367 | biostudies-literature
Project description:In Mitchell's chemiosmotic theory, membrane potential Δψ was given as the electric potential difference across the membrane. However, its physical origin for membrane potential Δψ was not well explained. Using the Lee proton electrostatic localization model with a newly formulated equation for protonic motive force (pmf) that takes electrostatically localized protons into account, membrane potential has now been better understood as the voltage difference contributed by the localized surface charge density ([HL+]+∑i=1n[MLi+]) at the liquid-membrane interface as in an electrostatically localized protons/cations-membrane-anions capacitor. That is, the origin of membrane potential Δψ is now better understood as the electrostatic formation of the localized surface charge density that is the sum of the electrostatically localized proton concentration [HL+] and the localized non-proton cations density ∑i=1n[MLi+] at the liquid membrane interface. The total localized surface charge density equals to the ideal localized proton population density [HL+]0 before the cation-proton exchange process; since the cation-proton exchange process does not change the total localized charges density, neither does it change to the membrane potential Δψ . The localized proton concentration [HL+] represents the dominant component, which accounts about 78% of the total localized surface charge density at the cation-proton exchange equilibrium state in animal mitochondria. Liquid water as a protonic conductor may play a significant role in the biological activities of membrane potential formation and utilization.
| S-EPMC6646885 | biostudies-literature
Project description:Skeletal muscle fibers have been used to examine a variety of cellular functions and pathologies. Among other parameters, skeletal muscle action potential (AP) propagation has been measured to assess the integrity and function of skeletal muscle. In this work, we utilize 1-(3-sulfonatopropyl)-4[?[2-(Di-n-octylamino)-6-naphtyl]vinyl]pyridinium betaine, a potentiometric dye, and mag-fluo-4, a low-affinity intracellular Ca2+indicator, to noninvasively and reliably measure AP conduction velocity in skeletal muscle. We used remote extracellular bipolar electrodes to generate an alternating polarity electric field that initiates an AP at either end of the fiber. Using enzymatically dissociated flexor digitorum brevis (FDB) fibers and high-speed line scans, we determine the conduction velocity to be ?0.4 m/s. We applied these methodologies to FDB fibers under elevated extracellular potassium conditions and confirmed that the conduction velocity is significantly reduced in elevated [K+]o. Because our recorded velocities for FDB fibers were much slower than previously reported for other muscle groups, we compared the conduction velocity in FDB fibers to that of extensor digitorum longus (EDL) fibers and measured a significantly faster velocity in EDL fibers than FDB fibers. As a basis for this difference in conduction velocity, we found a similarly higher level of expression of Na+ channels in EDL than in FDB fibers. In addition to measuring the conduction velocity, we can also measure the passive electrotonic potentials elicited by pulses by applying tetrodotoxin and have constructed a circuit model of a skeletal muscle fiber to predict passive polarization of the fiber by the field stimuli. Our predictions from the model fiber closely resemble the recordings acquired from in vitro assays. With these techniques, we can examine how various pathologies and mutations affect skeletal muscle AP propagation. Our work demonstrates the utility of using 1-(3-sulfonatopropyl)-4[?[2-(Di-n-octylamino)-6-naphtyl]vinyl]pyridinium betaine or mag-fluo-4 to noninvasively measure AP initiation and conduction.
| S-EPMC6289662 | biostudies-literature
Project description:Protein-lipid interactions are vital for numerous transmembrane signaling pathways. However, simple tools to characterize these interactions remain scarce and are much needed to advance our understanding of signal transduction across lipid bilayers. To tackle this challenge, we herein engineer nanodisc as a robust fluorescent sensor for reporting membrane biochemical reactions. We circularize nanodiscs via split GFP and thereby create an intensity-based fluorescent sensor (isenND) for detecting membrane binding and remodeling events. We show that isenND responds robustly and specifically to the action of a diverse array of membrane-interacting proteins and peptides, ranging from synaptotagmin and synuclein involved in neurotransmission to viral fusion peptides of HIV-1 and SARS-CoV-2. Together, isenND can serve as a versatile biochemical reagent useful for basic and translational research of membrane biology.
| S-EPMC9135701 | biostudies-literature
Project description:Recent studies have shown that bacterial membrane potential is dynamic and plays signaling roles. Yet, little is still known about the mechanisms of membrane potential dynamics regulation-owing to a scarcity of appropriate research tools. Optical modulation of bacterial membrane potential could fill this gap and provide a new approach for studying and controlling bacterial physiology and electrical signaling. Here, the authors show that a membrane-targeted azobenzene (Ziapin2) can be used to photo-modulate the membrane potential in cells of the Gram-positive bacterium Bacillus subtilis. It is found that upon exposure to blue-green light (λ = 470 nm), isomerization of Ziapin2 in the bacteria membrane induces hyperpolarization of the potential. To investigate the origin of this phenomenon, ion-channel-deletion strains and ion channel blockers are examined. The authors found that in presence of the chloride channel blocker idanyloxyacetic acid-94 (IAA-94) or in absence of KtrAB potassium transporter, the hyperpolarization response is attenuated. These results reveal that the Ziapin2 isomerization can induce ion channel opening in the bacterial membrane and suggest that Ziapin2 can be used for studying and controlling bacterial electrical signaling. This new optical tool could contribute to better understand various microbial phenomena, such as biofilm electric signaling and antimicrobial resistance.
| S-EPMC10015841 | biostudies-literature
Project description:Studying cellular and developmental processes in complex multicellular organisms can require the non-destructive observation of thousands to billions of cells deep within an animal. DNA recorders address the staggering difficulty of this task by converting transient cellular experiences into mutations at defined genomic sites that can be sequenced later in high throughput. However, existing recorders act primarily by erasing DNA. This is problematic because, in the limit of progressive erasure, no record remains. We present a DNA recorder called CHYRON (Cell History Recording by Ordered Insertion) that acts primarily by writing new DNA through the repeated insertion of random nucleotides at a single locus in temporal order. To achieve in vivo DNA writing, CHYRON combines Cas9, a homing guide RNA and the template-independent DNA polymerase terminal deoxynucleotidyl transferase. We successfully applied CHYRON as an evolving lineage tracer and as a recorder of user-selected cellular stimuli.
| S-EPMC8891441 | biostudies-literature
Project description:Recent studies have shown that the small GTPase KRAS adopts multiple orientations with respect to the plane of anionic model membranes, whereby either the three C-terminal helices or the three N-terminal β-strands of the catalytic domain face the membrane. This has functional implications because, in the latter, the membrane occludes the effector-interacting surface. However, it remained unclear how membrane reorientation occurs and, critically, whether it occurs in the cell in which KRAS operates as a molecular switch in signaling pathways. Herein, using data from a 20 μs-long atomistic molecular dynamics simulation of the oncogenic G12V-KRAS mutant in a phosphatidylcholine/phosphatidylserine bilayer, we first show that internal conformational fluctuations of flexible regions in KRAS result in three distinct membrane orientations. We then show, using single-molecule fluorescence resonance energy transfer measurements in native lipid nanodiscs derived from baby hamster kidney cells, that G12V-KRAS samples three conformational states that correspond to the predicted orientations. The combined results suggest that relatively small energy barriers separate orientation states and that signaling-competent conformations dominate the overall population.
| S-EPMC6350008 | biostudies-literature