Plasma Membrane Of Single Cells Research Articles

Electrophysiology of fluoride channels in the yeasts Saccharomyces cerevisiae and Candida albicans.

Tight regulation of molecules moving through the cell membrane is particularly important for free-living microorganisms because of their small cell volumes and frequent changes in the chemical composition of the extracellular environment. This is true for nutrients, but even more so for toxic molecules. Traditionally, the transport of these diverse molecules in microorganisms has been studied on cell populations rather than on single cells, mainly because of technical difficulties. The goal of this chapter is to make available a detailed method to prepare yeast spheroplasts to study the movement of fluoride ions across the plasma membrane of single cells by the patch-clamp technique. In this procedure, three steps are critical to achieve high resistance (GΩ) seals between the membrane and the glass electrode: (1) appropriate removal of the cell wall by enzymatic treatment; (2) balance between the osmotic strength of sealing solutions and cell membrane turgor; and (3) meticulous morphological inspection of spheroplasts suitable for gigaseal formation. We show now that this method, originally developed for Saccharomyces cerevisiae, can also be applied to Candida albicans, an opportunistic human pathogen.

Highly specific and non-invasive imaging of Piezo1-dependent activity across scales using GenEPi

Mechanosensing is a ubiquitous process to translate external mechanical stimuli into biological responses. Piezo1 ion channels are directly gated by mechanical forces and play an essential role in cellular mechanotransduction. However, readouts of Piezo1 activity are mainly examined by invasive or indirect techniques, such as electrophysiological analyses and cytosolic calcium imaging. Here, we introduce GenEPi, a genetically-encoded fluorescent reporter for non-invasive optical monitoring of Piezo1-dependent activity. We demonstrate that GenEPi has high spatiotemporal resolution for Piezo1-dependent stimuli from the single-cell level to that of the entire organism. GenEPi reveals transient, local mechanical stimuli in the plasma membrane of single cells, resolves repetitive contraction-triggered stimulation of beating cardiomyocytes within microtissues, and allows for robust and reliable monitoring of Piezo1-dependent activity in vivo. GenEPi will enable non-invasive optical monitoring of Piezo1 activity in mechanochemical feedback loops during development, homeostatic regulation, and disease.

Unfolding and identification of membrane proteins in situ.

Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscopy (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, which involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from mass spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing four constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment.

High spatial resolution observation of Temporin A at cell membranes using scanning ion conductive microscopy

The electrochemical visualization of molecules binding at the plasma membrane of single cells is important, but challenging. Here, the binding of Temporin A at the plasma membrane of a single living A549 cell is imaged using high spatial resolution scanning ion conductive microscopy (SICM). A relatively steep approach curve is observed at the binding region, compared with that at the non-binding cellular membrane. This difference is ascribed to the neutralization of the negative charges at the cellular membrane by the positively charged Temporin A through electrostatic binding. The pit-like structure of the plasma membrane is visualized in the SICM image, exhibiting unhomogeneous binding of Temporin A at the cell, and providing direct evidence about the interaction of Temporin A with certain regions of the membrane. The information will facilitate a deeper understanding of the cytotoxicity of Temporin A for the treatment of lung cancer. Moreover, the successful visualization of this process is an example of an electrochemical strategy that could be used to study other molecular interactions at cellular membranes.

Effect of magnetic and electric fields on plasma membrane of single cells: A computational approach

AbstractCell membrane is a lipid bilayer that allows the flow of ions through their ionic pumping proteins. The ionic flow can be stimulated with external stimuli to activate specific signaling pathways intracellularly. Although studies have applied electric and magnetic stimuli to modify the cell function, the parameters to stimulate the cell membrane are unknown. Accordingly, a computational model to simulate the effect of electric and magnetic fields on the cell membrane was developed. Cells were stimulated with electric fields from 45 × 103 V/m to 12.6 × 105 V/m and magnetic fields of 2 mT, at frequencies of 60 kHz, 10 MHz, and 1 GHz. Results showed that the electric fields applied to the cell membrane tend to increase according to the frequency used, while magnetic fields do not have any effect on it. It was observed that electric fields generate a high voltage concentrator in the cell membrane of ellipsoidal cells when a frequency window from 1 kHz to 1 GHz was applied. These findings demonstrate that depending on the intensity of the field and frequency, it was possible to stimulate different cell membrane zones. This model is a promising tool to establish the adequate parameters to stimulate cells, and accurately predict if the stimulation modifies the cell membrane potential.

Enzyme-modified microelectrodes for electrochemical detection of sphingomyelin in the plasma membranes of single cells

In this communication, sphingomyelin (SM) in the plasma membrane of single cells is detected electrochemically using an enzyme-modified Pt microelectrode for the first time. The enzymes immobilized at the electrode surface, which include sphingomyelinase (SMase), alkaline phosphatase (ALP), and choline oxidase, react with SM in a series of reactions to generate hydrogen peroxide. The electrochemical oxidation of hydrogen peroxide induces an elevation in the current, which offers a quantitative determination of SM in a range from 140 to 560 µM. Further contact of the microelectrode with one oocyte cell results in the reaction of SM in the plasma membrane with the enzymes at the electrode. The resulting current increase is correlated with the content of SM in the plasma membrane, confirming the electrochemical detection of membrane SM in single cells. This success in single cell electrochemical analysis of membrane SM expands the application of microelectrodes in single cell studies, which will help in the elucidation of cellular heterogeneity in lipid trafficking.

Codetermination of Sphingomyelin and Cholesterol in Cellular Plasma Membrane in Sphingomyelin-Depletion-Induced Cholesterol Efflux.

Quantification of multiple lipids with different contents in plasma membrane in single cells is significant, but challenging, for investigating lipid interactions and the role of dominant protein transporters. In this paper, comonitoring the alteration of low-content sphingomyelin (SM) and high-content cholesterol in plasma membrane of one living cell is realized by use of luminol electrochemiluminescence (ECL) for the first time. Concentrations of SM as low as 0.5 μM are detected, which permits the measurement of low-content membrane SM in single cells. More membrane cholesterol is observed in individual cells after depletion of membrane SM, providing direct evidence about SM-depletion-induced cholesterol efflux. The upregulation of ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) in SM-depleted cells induces a further increase in membrane cholesterol. These results imply that higher expression of ABCA1/G1 promotes cholesterol trafficking, which offers additional information to solve the debate about ABC transporters in cholesterol efflux. Moreover, the established approach offers a special strategy to investigate the correlation of membrane lipids and the role of transporters in cholesterol trafficking.

Surface-Confined Electrochemiluminescence Microscopy of Cell Membranes

Herein is reported a surface-confined microscopy based on electrochemiluminescence (ECL) that allows to image the plasma membrane of single cells at the interface with an electrode. By analyzing photoluminescence (PL), ECL and AFM images of mammalian CHO cells, we demonstrate that, in contrast to the wide-field fluorescence, ECL emission is confined to the immediate vicinity of the electrode surface and only the basal membrane of the cell becomes luminescent. The resulting ECL microscopy reveals details that are not resolved by classic fluorescence microscopy, without any light irradiation and specific setup. The thickness of the ECL-emitting regions is ∼500 nm due to the unique ECL mechanism that involves short-lifetime electrogenerated radicals. In addition, the reported ECL microscopy is a dynamic technique that reflects the transport properties through the cell membranes and not only the specific labeling of the membranes. Finally, disposable transparent carbon nanotube (CNT)-based electrodes inkjet-printed on classic microscope glass coverslips were used to image cells in both reflection and transmission configurations. Therefore, our approach opens new avenues for ECL as a surface-confined microscopy to develop single cell assays and to image the dynamics of biological entities in cells or in membranes.

Determining the Composition of Active Cholesterol in the Plasma Membrane of Single Cells by using Electrochemiluminescence

AbstractActive and inactive cholesterol in the plasma membrane of one cell were co‐measured by using luminol electrochemiluminescence, so that the composition of active membrane cholesterol was determined at single cells. The cellular plasma membrane remained intact after the removal of active membrane cholesterol and, thus, inactive cholesterol was activated for reaction with cholesterol oxidase. By utilizing the reactions of active and inactive cholesterol with oxidase in series, both of them generated hydrogen peroxide, inducing the corresponding electrochemiluminescence for the respective quantitation of these two states of membrane cholesterol. The successful determination of active and inactive membrane cholesterol at one cell provides information about the composition of active membrane cholesterol, which is significant for the study of cellular cholesterol trafficking.

Fast Serial Analysis of Active Cholesterol at the Plasma Membrane in Single Cells

Previously, our group has utilized the luminol electrochemiluminescence to analyze the active cholesterol at the plasma membrane in single cells by the exposure of one cell to a photomultiplier tube (PMT) through a pinhole. In this paper, fast analysis of active cholesterol at the plasma membrane in single cells was achieved by a multimicroelectrode array without the pinhole. Single cells were directly located on the microelectrodes using cell-sized microwell traps. A cycle of voltage was applied on the microelectrodes sequentially to induce a peak of luminescence from each microelectrode for the serial measurement of active membrane cholesterol. A minimal time of 1.60 s was determined for the analysis of one cell. The simulation and the experimental data exhibited a semisteady-state distribution of hydrogen peroxide on the microelectrode after the reaction of cholesterol oxidase with the membrane cholesterol, which supported the relative accuracy of the serial analysis. An eight-microelectrode array was demonstrated to analyze eight single cells in 22 s serially, including the channel switching time. The results from 64 single cells either activated by low ion strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT) revealed that most of the cells analyzed had the similar active membrane cholesterol, while few cells had more active cholesterol resulting in the cellular heterogeneity. The fast single-cell analysis platform developed will be potentially useful for the analysis of more molecules in single cells using proper oxidases.

Anisotropy of Crumbs and aPKC Drives Myosin Cable Assembly during Tube Formation

SummaryThe formation of tubular structures from epithelial sheets is a key process of organ formation in all animals, but the cytoskeletal rearrangements that cause the cell shape changes that drive tubulogenesis are not well understood. Using live imaging and super-resolution microscopy to analyze the tubulogenesis of the Drosophila salivary glands, I find that an anisotropic plasma membrane distribution of the protein Crumbs, mediated by its large extracellular domain, determines the subcellular localization of a supracellular actomyosin cable in the cells at the placode border, with myosin II accumulating at edges where Crumbs is lowest. Laser ablation shows that the cable is under increased tension, implying an active involvement in the invagination process. Crumbs anisotropy leads to anisotropic distribution of aPKC, which in turn can negatively regulate Rok, thus preventing the formation of a cable where Crumbs and aPKC are localized.

Mechanical Activation of Cells Induces Chromatin Remodeling Preceding MKL Nuclear Transport

For cells to adapt to different tissues and changes in tissue mechanics, they must be able to respond to mechanical cues by changing their gene expression patterns. Biochemical signaling pathways for these responses have been elucidated, and recent evidence points to the involvement of force-induced deformation of the nucleus. However, it is still unclear how physical cues received at the plasma membrane (PM) spatiotemporally integrate to the functional chromatin organization of the cell nucleus. To investigate this issue, we applied mechanical forces through magnetic particles adhered to the PM of single cells and mapped the accompanying changes in actin polymerization, nuclear morphology, chromatin remodeling, and nuclear transport of soluble signaling intermediates using high-resolution fluorescence anisotropy imaging. Using this approach, we show the timescales associated with force-induced polymerization of actin and changes in the F/G actin ratio resulting in nuclear translocation of the G-actin-associated transcriptional cofactor, megakaryoblastic acute leukemia factor-1 (MKL). Further, this method of measuring nuclear organization at high spatiotemporal resolution with simultaneous force application revealed the physical propagation of forces to the nucleus, resulting in changes to chromatin organization, followed by nuclear deformation. We also describe a quantitative model that incorporates active stresses and chemical kinetics to evaluate the observed timescales. Our work suggests that mechanical activation of cells is accompanied by distinct timescales involved in the reorganization of actin and chromatin assembly, followed by translocation of transcription cofactors from the cytoplasm to the nucleus.

Probing the Functional Integration of Mechanical Signals to Cell Nucleus

For cells to adapt to different tissues and changes in tissue mechanics, they must be able to respond to mechanical cues by changing gene expression patterns. These responses potentially involve major changes in nuclear organization and structure to reflect epigenetic changes in the nucleus. However, it is unclear how physical cues received at the plasma membrane integrate to the functional architecture of the cell nucleus. To probe this, we applied mechanical forces through magnetic particles adhered to the plasma membrane of single cells and mapped accompanying changes in cytoskeletal reorganization, soluble signalling intermediates, nuclear morphology and chromatin remodelling using high resolution fluorescence anisotropy imaging. Application of force on the plasma membrane resulted in spatio-temporal reorganization of actin cytoskeleton and chromatin assembly and the translocation of transcription co-factor Megakaryoblastic acute leukemia factor (MKL) from the cytoplasm to the nucleus. Taken together our results evidence a strong architectural coupling between physico-chemical networks and spatial organization of the chromosomes within the nucleus facilitating mechanotransduction.

A microfluidic platform for probing single cell plasma membranes using optically trapped Smart Droplet Microtools (SDMs)

We recently introduced a novel platform based upon optically trapped lipid coated oil droplets (Smart Droplet Microtools-SDMs) that were able to form membrane tethers upon fusion with the plasma membrane of single cells. Material transfer from the plasma membrane to the droplet via the tether was seen to occur. Here we present a customised version of the SDM approach based upon detergent coated droplets deployed within a microfluidic format. These droplets are able to differentially solubilise the plasma membrane of single cells with spatial selectivity and without forming membrane tethers. The microfluidic format facilitates separation of the target cells from the bulk SDM population and from downstream analysis modules. Material transfer from the cell to the SDM was monitored by tracking membrane localized EGFP.

Spatially selective sampling of single cells using optically trapped fusogenic emulsion droplets: a new single-cell proteomic tool

We present a platform for the spatially selective sampling of the plasma membrane of single cells. Optically trapped lipid-coated oil droplets (smart droplet microtools, SDMs), typically 0.5-5 microm in size, composed of a hexadecane hydrocarbon core and fusogenic lipid outer coating (mixture of 1,2-dioleoyl-phosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) were brought into controlled contact with target colon cancer cells leading to the formation of connecting membrane tethers. Material transfer from the cell to the SDM across the membrane tether was monitored by tracking membrane-localized enhanced green fluorescent protein.

Gating of cx46 gap junction hemichannels by calcium and voltage.

Connexin 46 (cx46), when expressed in Xenopus oocytes, not only forms typical gap junction channels between paired cells but also forms open gap junction hemichannels in the plasma membrane of single cells. The gap junction hemichannels share properties with complete gap junction channels in terms of permeability and gating. Here we characterize the gate that closes hemichannels in response to increased calcium concentration with whole-cell and single-channel records. The channels close within a narrow range of extracellular calcium concentrations (1-2 mM) which includes the calcium concentration prevailing in the primary site of cx46 expression, the lens. The effect of calcium on the channels is determined by voltage. A cysteine mutant of cx46, cx46L35C, was used to determine the localization of the gate. Experimental evidence suggests that position 35 is pore lining. The localization protocol tests the accessibility of position 35 for thiol reagents applied extra- or intracellularly to the channel closed by calcium. Channel closure by calcium excluded the thiol reagent from the outside but not from the inside. Consequently, the gate results in a regional closure of the pore and it is located extracellular to the position 35 of cx46. The present data also suggest that the cx46 gap junction hemichannel may exert a physiological function in the lens. Considering the association of calcium with cataract formation, it is feasible that misregulation of cx46 gap junction hemichannels could be a cause for cataract.

Translational diffusion in the plasma membrane of single cells as studied by fluorescence microphotolysis

Translational diffusion in the plasma membrane of single cells as studied by fluorescence microphotolysis

Type B and Type C RNA Tumor Virus Replication in Single Cells. Electron Microscopy With Tannic Acid2

Simultaneous replication of murine mammary tumor virus (type B) and murine leukemia virus (type C) was demonstrated electron microscopically along continuous stretches of the plasma membrane of single cells in cultures ofthe Mm5mt/c1 cell line. Types B and C virus buds were discriminated in thin sections with the aid of a tannic acid fixative that revealed the type B surface spikes as a homogeneous band of intermediate density and constant width on the surfaces of some buds (type B), whereas others (type C) remained with relatively smooth envelopes. Both types B and C buds may contain morphologically identical horseshoe-shaped nucleoids. Therefore, their identify (type B or C) could be ascertained in thin sections only on the basis of recognition of surface spikes.