Membranes Of Viable Cells Research Articles

Exogenous polyserine fibrils change membrane properties of phosphatidylcholine-liposome and red blood cells

The causative genes for neurodegenerative polyglutamine (polyQ) diseases produce homopolymeric polyglutamine (polyQ), polyserine (polyS), polyalanine (polyA), polycysteine (polyC), and polyleucine (polyL) sequences by repeat-associated non-AUG (RAN) translation. The cytotoxicity of the intracellular polyQ and RAN products has been extensively investigated. However, little is known about the toxicity of the extracellular polyQ and RAN products on the membranes of viable cells. Because polyQ aggregates induce a deflated morphology of a model membrane, we hypothesized that extracellular polyQ and RAN products might affect the membrane properties of viable cells. In this study, we demonstrated that exogenous polyS fibrils but not polyS or polyQ non-fibril aggregates altered the thermal phase transition behavior of a model membrane composed of a phosphatidylcholine bilayer using differential scanning calorimetry. PolyS fibrils induced morphological changes in viable red blood cells (RBCs). However, both polyS and polyQ non-fibril aggregates had no effects on RBCs. These results highlight the possibility that extracellular fibrils generated from RAN products may alter the properties of neuronal cell membranes, which may contribute to changes in the brain pathology.

Assessment of bacterial membrane fluidity by flow cytometry

It can be interesting to evaluate the cytoplasmic membrane fluidity of bacteria in order to understand the impacts of stresses during processing. Traditionally, membrane fluidity was assessed by fluorescence anisotropy measured by spectrofluorimetry, but this method does not make it possible to reveal the heterogeneity of bacterial populations. Flow cytometry, as opposed to spectrofluorimetry, has the ability to simultaneously detect different sub-populations. We developed a new method to measure the fluorescence anisotropy of bacterial membranes using flow cytometry. This technique, coupled with viability/mortality/fluidity co-staining, now allows us to assess the membrane fluidity of viable, damaged and dead cells. Then, we show that membrane of viable bacterial cells becomes more and more rigid during batch culture, culminating in the death of the cells. This new flow cytometric method therefore opens new perspectives to study changes and adaptations in bacterial membranes of viable cells depending on the micro-environment during fermentation or stress applications.

TNT-Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of Pro-Phagocytic Signals From Apoptotic to Viable Cells.

The exposure of phosphatidylserine (PS) on the surface membrane of apoptotic cells triggers the recruitment of phagocytic receptors and subsequently results in uptake by phagocytes. Here we describe how apoptotic cells can use intercellular membrane nanotubes to transfer exposed PS to neighboring viable cells, and thus deposit an “eat‐me” tag on the viable cells. Tunneling nanotubes (TNTs) connected UV‐treated apoptotic rat pheochromocytoma PC12 cells with neighboring untreated cells. These TNTs were composed of PS‐exposed plasma membrane and facilitated the transfer of the membrane from apoptotic to viable cells. Other pro‐phagocytic signals, such as oxidized phospholipids and calreticulin, were also transferred to viable cells. In addition, anti‐phagocytic signal CD47 presenting on the plasma membrane of viable cells was masked by the transferred PS‐membrane. Confocal imaging revealed an increase of phagocytosis of viable PC12 cells by murine RAW264.7 macrophages when the viable PC12 cells were cocultured with UV‐treated PC12 cells. Treatment with 50 nM cytochalasin D would abolish TNTs and correspondingly inhibit this phagocytosis of the viable cells. Our study indicates that exposed‐PS membrane is delivered from apoptotic to viable cells through TNTs. This transferred membrane may act as a pro‐phagocytic signal for macrophages to induce phagocytosis of viable cells in a situation where they are in the vicinity of apoptotic cells. J. Cell. Physiol. 232: 2271–2279, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.

Quantification and viability analyses of Pseudokirchneriella subcapitata algal cells using image-based cytometry

This work aims to evaluate the feasibility of using image-based cytometry (IBC) in the analysis of algal cell quantification and viability, using Pseudokirchneriella subcapitata as a cell model. Cell concentration was determined by IBC to be in a linear range between 1 × 105 and 8 × 106 cells mL−1. Algal viability was defined on the basis that the intact membrane of viable cells excludes the SYTOX Green (SG) probe. The disruption of membrane integrity represents irreversible damage and consequently results in cell death. Using IBC, we were able to successfully discriminate between live (SG-negative cells) and dead algal cells (heat-treated at 65 °C for 60 min; SG-positive cells). The observed viability of algal populations containing different proportions of killed cells was well correlated (R 2 = 0.994) with the theoretical viability. The validation of the use of this technology was carried out by exposing algal cells of P. subcapitata to a copper stress test for 96 h. IBC allowed us to follow the evolution of cell concentration and the viability of copper-exposed algal populations. This technology overcomes several main drawbacks usually associated with microscopy counting, such as labour-intensive experiments, tedious work and lack of the representativeness of the cell counting. In conclusion, IBC allowed a fast and automated determination of the total number of algal cells and allowed us to analyse viability. This technology can provide a useful tool for a wide variety of fields that utilise microalgae, such as the aquatic toxicology and biotechnology fields.

Why we get a blue stain

Sir, We wish to add our comments to the recent review article describing the use of trypan blue (TB) in anterior segment surgery.1 To understand its potential uses, we need some knowledge of its biochemistry. TB (C34H23N6O14S4Na4) is a negatively charged, water soluble bis-axo dye that is used in cell biology to confirm cell viability. The selectively permeable cell membrane of viable cells does not allow this ‘foreign' chemical to enter and hence appear colourless while the uptake in dead cells results in a blue discolouration. Similarly, TB will delineate collagen type IV-rich membranes. Both these properties are utilized in ophthalmology, the former in highlighting viable cells after conjunctival brush cytology, the latter in staining the anterior lens capsule (as well as ILM and ERM tissue).2 Permanent discolouration is avoided by dilution with balanced salt solution. TB also stains poorly hydrated tissues better than well-hydrated ones explaining the contrast seen between the acellular lens capsule and the water-rich cells of the cortical lens. Unmentioned risks include potential teratogenicity (has been associated with encephalocoele formation in animal models) and carcinogenicity (lymphoma in the rat model); both models, however, used subcutaneous injections of TB. Similarly, ocular abnormalities have been described in the pregnant murine model.3 As a cautionary measure, the risk benefit ratio should be properly assessed before proceeding with TB-assisted ophthalmic surgery in pregnant or nursing women.

Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide

Flow cytometric monitoring of propidium iodide (PI) uptake is a well-established and rapid method for monitoring cell death and is used on the basis that the intact membrane of viable cells excludes the propidium ion and that loss of this permeability barrier represents irreparable damage and thus cell death. These assumptions are typically based on analysis of live and killed cells. Here we have identified stress levels that lead to a loss of viability of a proportion of Saccharomyces cerevisiae cells and under these conditions we show that there is a subpopulation of cells that can take up PI during and immediately following exposure to stress but that a short incubation allows repair of the membrane damage such that subsequent exposure to PI does not result in staining. Irrespective of the stress applied, approximately 7% of cells exhibited the ability to repair. These results indicate that the level of damage that the yeast cell membrane can sustain and yet retain the ability to repair is greater than previously recognized and care must therefore be taken in using the terms 'PI-positive' and 'dead' synonymously. We discuss these findings in the context of the potential for such environmental stress-induced, transient membrane permeability to have evolutionary implications via the facilitation of horizontal gene transfer.

Preparation, functional characterization, and NMR studies of human KCNE1, a voltage-gated potassium channel accessory subunit associated with deafness and long QT syndrome.

KCNE1, also known as minK, is a member of the KCNE family of membrane proteins that modulate the function of KCNQ1 and certain other voltage-gated potassium channels (KV). Mutations in human KCNE1 cause congenital deafness and congenital long QT syndrome, an inherited predisposition to potentially life-threatening cardiac arrhythmias. Although its modulation of KCNQ1 function has been extensively characterized, many questions remain regarding KCNE1's structure and location within the channel complex. In this study, KCNE1 was overexpressed in Escherichia coli and purified. Micellar solutions of the protein were then microinjected into Xenopus oocytes expressing KCNQ1 channels, followed by electrophysiological recordings aimed at testing whether recombinant KCNE1 can co-assemble with the channel. Nativelike modulation of channel properties was observed following injection of KCNE1 in lyso-myristoylphosphatidylglycerol (LMPG) micelles, indicating that KCNE1 is not irreversibly misfolded and that LMPG is able to act as a vehicle for delivering membrane proteins into the membranes of viable cells. 1H-15N TROSY NMR experiments indicated that LMPG micelles are well-suited for structural studies of KCNE1, leading to assignment of its backbone resonances and to relaxation studies. The chemical shift data confirmed that KCNE1's secondary structure includes several alpha-helices and demonstrated that its distal C-terminus is disordered. Surprisingly, for KCNE1 in LMPG micelles, there appears to be a break in alpha-helicity at sites 59-61, near the middle of the transmembrane segment, a feature that is accompanied by increased local backbone mobility. Given that this segment overlaps with sites 57-59, which are known to play a critical role in modulating KCNQ1 channel activation kinetics, this unusual structural feature likely has considerable functional relevance.

Novel fluorescence assay using calcein‐AM for the determination of human erythrocyte viability and aging

A highly sensitive, fast, and simple flow cytometric assay to assess human red blood cell (RBCs) viability and aging is reported. The assay described in this report is based on the use of acetoxymethyl ester of calcein (calcein-AM), a fluorescein derivative and nonfluorescent vital dye that passively crosses the cell membrane of viable cells and is converted by cytosolic esterases into green fluorescent calcein, which is retained by cells with intact membranes and inactive multidrug resistance protein. The loss of calcein can be easily determined by flow cytometry, and the cytosolic localization of esterases was demonstrated by spectrofluorometric analyses. We found that RBCs incubated with Ca(2+), which induces a rapid and modulated self-death that shares several features with apoptosis (Bratosin et al., Cell Death Differ 2001;8:1143-1156), externalized phosphatidylserine and lost calcein staining and cytosolic adenosine triphosphate content. Double labeling using phycoerythrin-labeled annexin-V and calcein-AM showed that the decrease of esterase activity is an early event that precedes the externalization of phosphatidylserine residues. In addition, this assay allowed us to distinguish young and aged RBCs isolated by ultracentrifugation in a self-forming Percoll gradient and can be considered as a reliable marker of RBC aging. Calcein-AM assay may represent a wide application for assessing RBC viability, particularly in blood banks.

Ultrasound-mediated disruption of cell membranes. II. Heterogeneous effects on cells.

Ultrasound has been shown to reversibly and irreversibly disrupt membranes of viable cells through a mechanism believed to involve cavitation. Because cavitation is both temporally and spatially heterogeneous, flow cytometry was used to identify and quantify heterogeneity in the effects of ultrasound on molecular uptake and cell viability on a cell-by-cell basis for suspensions of DU145 prostate cancer and aortic smooth muscle cells exposed to varying peak negative acoustic pressures (0.6-3.0 MPa). exposure times (120-2,000 ms), and pulse lengths (0.02-60 ms) in the presence of Optison (1.7% v/v) contrast agent. Cell-to-cell heterogeneity was observed at all conditions studied and was classified into three subpopulations: nominal uptake (NUP), low uptake (LUP), and high uptake (HUP) populations. The average number of molecules within each subpopulation was generally constant: 10(4)-10(5) molecules/cell in NUP, approximately 10(6) molecules/cell in LUP, and approximately 10(7) molecules/cell in HUP. However, the fraction of cells within each subpopulation showed a strong dependence on both acoustic pressure and exposure time. Varying pulse length produced no significant effect. The distribution of cells among the three subpopulations correlated with acoustic energy exposure, which suggests that energy exposure may govern the ability of ultrasound to induce bioeffects by a nonthermal mechanism.

Rapid fluorometric assay for cell viability and cell growth using nucleic acid staining and cell lysis agents

The purpose of this study was to establish a new method for rapidly and simply assessing cell viability and growth with objective validation if the assay system proceeded under suitable conditions of cell culture. In this method, a cell lysis agent was combined with a fluorescent probe for nucleic acid which exclusively passes through the disrupted membranes of dead cells but not intact membranes of viable cells. The distinctive feature of this probe is to possess a large fluorescence enhancement (460-fold) on binding to nucleic acid despite very low intrinsic fluorescence. In this fluorometric assay based on cell lysis and staining (FACLS), the fluorescence intensity was linearly related to total tumour cell number. This FACLS was also used to evaluate the chemosensitivity of MOLT-4 human leukaemia cells and to measure cell viability. The results were similar to those obtained by MTT colorimetric and trypan blue exclusion assays. The main advantage of this assay is its ability to measure simultaneously both cell viability and cell growth rapidly (within about 5 min) and simply (two steps) with validation of cell culture conditions in each microplate. This method could be widely applicable to cytotoxic evaluation of anticancer drugs and other chemicals.

Plasma membrane expression of heat shock protein 60 in vivo in response to infection.

Heat shock protein 60 (hsp60) is constitutively expressed in the mitochondria of eukaryotic cells. However, it has been identified in other subcellular compartments in several disease states and in transformed cells, and it is an immunogenic molecule in various infectious and autoimmune diseases. To better understand the factors that influence expression of hsp60 in normal cells in vivo, we analyzed its cellular and subcellular distribution in mice infected with the intracellular bacterium Listeria monocytogenes. Western blotting of subcellular fractionated spleen cells showed that although endogenous hsp60 was restricted to the mitochondria in noninfected animals, it was associated with the plasma membrane as a result of infection. The low levels of plasma membrane-associated hsp60 seen in the livers in noninfected animals subsequently increased during infection. Plasma membrane hsp60 expression did not correlate with bacterial growth, being most evident during or after bacterial clearance and persisting at 3 weeks postinfection. Using flow cytometry, we determined that Mac-1(+), T-cell receptor gammadelta(+), and B220(+) cells represented the major Hsp60(+) populations in spleens of infected mice. By contrast, B220(+) cells were the predominant hsp60(+) population in livers of infected mice. Of the immune cells analyzed, the kinetic profile of the gammadelta T-cell response most closely matched that of hsp60 expression in both the spleen and liver. Collectively, these findings show that during infection hsp60 can be localized to the plasma membrane of viable cells, particularly antigen-presenting cells, providing a means by which hsp60-reactive lymphocytes seen in various infectious disease and autoimmune disorders may be generated and maintained.

Time-resolved fluorometric assay for natural killer activity using target cells labelled with a fluorescence enhancing ligand

A time-resolved fluorometric assay for the measurement of natural killer cell activity against target cells labelled with the acetoxymethyl ester of the fluorescence enhancing ligand 2,2′:6′,2″-terpyridine-6,6″-dicarboxylic acid (TDA) is described. The hydrophobic esterified form of TDA (bis(acetoxymethyl)2,2′:6′,2″-terpyridine-6,6″-dicarboxylate, BATDA) diffuses readily through the cell membrane of viable cells. BATDA is hydrolysed by intracellular esterases resulting in accumulation of membrane impermeable TDA inside the target cells. After incubation of labelled K-562 cells with effector cells the TDA released from lysed cells into the supernatant is chelated with Eu 3+. The natural killer cell activity is then quantified by measuring the intense fluorescence of the EuTDA chelates formed. Target cells are rapidly labelled when incubated with BATDA, TDA is released from target cells faster than 51Cr, the spontaneous release permits a short-term release assay to be set up and the detection of EuTDA is fast (5 min/96 well plate). Furthermore, this non-radioactive method permits the use of complex culture media since, in contrast to methods based on prompt fluorometry, the problem with autofluorescence can be avoided by the use of time-resolved fluorometry.

シグナル伝達と細胞機能調節 細胞外ＡＴＰの存在とその薬理作用および代謝

Cells are equipped with complex mechanisms for synthesis of ATP and try to keep the intracellular level of this compound, which is indispensable for maintenance of normal function and integrity, as constant as possible. Thus, it is generally believed that ATP rarely cross the plasma membrane of viable cells. However, since the first report of Holton in 1959, the release of nucleotides from cells has become an established fact, and the physiological role and metabolism of the released ATP has become an important topic; potent actions of extracellular purine nucleotides and nucleosides have been recognized for many years. In 1972, Burnstock demonstrated that ATP has a transmitter role in certain types of nerves and proposed a concept of purinergic nerve. The receptor for purine nucleotides, designated as P2, as opposed to P1, by Burnstock in 1978 was further subclassified in 1985 into P2X and P2Y by himself, and we now have a train of P2 receptors, such as P2S, P2T, P2Z and so forth. In this review, I summarized the characteristics of these purinoceptors. Pharmacological effects and metabolism of extracellular nucleotides were discussed and a brief mention was made of ecto-nucleotidases.

Analysis of mechanisms by which NK cells acquire increased cytotoxicity against class I MHC-eliminated targets

Acid treatment, where cells are exposed to 0.2 M citric acid buffer at pH 3 for 2 min, was described in a previous paper to be a method which specifically eliminates class I MHC antigens from the membrane of viable cells. We applied this method to characterize functional roles of class I MHC antigens on the target cells in NK cell cytotoxicity. When NK target cells, U937, Molt-4, and Raji, were subjected to acid treatment, the treated cells lost their surface class I MHC antigens and became more sensitive to NK cell killing. On the other hand, the susceptibility of K562 cells which initially lacked class I MHC antigens did not significantly change after such treatment. We then examined the mechanism which enables NK cells to become more cytotoxic against class I MHC antigen-eliminated target cells. Single cell binding assay and cold target inhibition assay demonstrated that class I MHC antigen-eliminated target cells did not acquire high binding affinity to NK cells. However, the interaction between NK cells and class I MHC antigen-eliminated targets resulted in a greater increase in production of NKCF-like factor than did the interaction between NK cells and untreated targets. Class I MHC antigen-eliminated targets did not acquire high killer susceptibility to NKCF-like factor. The present study utilizing the acid treatment method confirmed that surface class I MHC antigens on the targets are important immunoregulatory molecules not only for cytotoxic T lymphocytes but also for NK cells and elucidated some of the underlying mechanisms.

A solid-phase immunoassay to screen anti-HLA monoclonal antibodies

A solid-phase assay to detect anti-HLA monoclonal antibodies was developed. In this assay microtiter plates are coated with antigens solubilized from cultured lymphoid cells by sonication and then incubated with anti-HLA monoclonal antibodies. The antigen-antibody interaction is indicated by the development of color following the addition of peroxidase-conjugated anti-mouse Ig xenoantibodies and its substrate. The assay is rapid since it does not require centrifugations during the washing steps. Furthermore the assay is simple, reproducible and suitable to screen large numbers of samples and to detect antibodies recognizing determinants not exposed on the membrane of viable cells. The sensitivity of the assay is influenced by the pH of the buffer used to coat plates with antigens, by the number of cells used to prepare soluble antigens, by the incubation time of antigen preparations with plates and by the incubation time of antibody preparations with antigen-coated plates. Titration of anti-HLA monoclonal antibodies with known specificity and screening of hybridomas generated with splenocytes from mice immunized with cultured human lymphoid cells indicate that the sensitivity of the solid-phase assay is similar to that of the ELISA with lymphoid cells.

A simple method to eliminate the antigenicity of surface class I MHC molecules from the membrane of viable cells by acid treatment at pH 3

We describe here a simple, reproducible method which specifically eliminates the antigenecity of surface class I major histocompatibility complex (MHC) molecules by acid treatment at pH 3 from the membrane of viable cells. When fresh mononuclear cells (MNC) or established cultured cell lines were treated at 4°C for 2 min with citric acid buffer at pH 3 containing 1% bovine serum albumin, the antigenicity of class I MHC molecules, but not those of class II MHC and the other non-MHC antigens, was eliminated from the surface membrane without significant cell death. This method was effective for both human and murine cells with various origins. Monoclonal and polyclonal antibodies were used to identify the expression of surface antigens in conjunction with immunofluorescence tests. The eliminated antigenicity of human class I MHC antigens (i.e., HLA-A,B,C) on MNC regenerated when cells were incubated in the medium at 37°C for 10 h. The pretreatment of cells with emetine (10 −4 M), a protein synthesis inhibitor, was found to be effective in inhibiting this regeneration. The acid treatment method might be useful for future studies on the functional characterization of surface class I MHC antigens.

Transferrin receptors on tumor and bone marrow cells: lack of involvement as target structure for natural killer cells.

Two different experimental approaches based on the specificity of monoclonal antibodies (mAbs) have been taken to verify the hypothesis that the transferrin receptor (TfR) on proliferating cells serves as a common target structure for natural killer (NK) cells. Thus, by the lysostrip technique the TfR was removed from the surface of K562 and Molt4 tumor cells by incubation with two different anti-TfR mAbs. The effect of removal of the TfR was controlled by uptake of radiolabeled transferrin, and by binding of non-cross-reacting monoclonal anti-TfR receptor antibodies. Though the modulation of TfR on the membrane of viable cells was nearly complete, the cells remained fully susceptible to NK lysis. The second approach consisted in removal of TfR-bearing cells from bone marrow cell suspensions by an indirect rosetting technique. Using mAbs bound to ox erythrocytes the rosetted TfR-bearing cells could be removed from bone marrow cell suspension by density centrifugation with an efficiency of greater than 99%. It could be shown that both fractions, TfR+ and TfR- cells, could be lysed to the same degree by NK cells. Thus, the evidence obtained speaks against a role of TfR as a recognition structure for NK cells.

Freeze-Fracture Cytochemical Study of Membrane Systems in Human Epidermis Using Filipin As a Probe for Cholesterol

Filipin (a polyene antibiotic) interacts specifically with cholesterol in membranes, producing characteristic 25 nm-diameter deformation (pitlike lesions) within the membrane plane detectable by freeze-fracture electron microscopy. Utilizing this probe, the distribution of cholesterol molecules in membranes and in lamellar structures between horny cells was investigated in human skin. The plasma membranes of basal, spinous, and granular cells reacted extensively with filipin except for desmosomal membrane portions. However, the plasma membranes of horny cells were rarely labeled with filipin, while lamellar structures between horny cells were well labeled. These observations indicate the distinct difference in susceptibility to filipin among the plasma membranes of viable cells and horny cells, and the lipid lamellar structures. Whenever horny cell plasma membranes were affected with filipin, they revealed a low deformability showing shallow pits or low protrusions. This low deformability may be due to greater membrane rigidity rather than a lower content of cholesterol, although the possibility of a low amount of cholesterol cannot be excluded. Lamellar bodies in granular cells were well labeled in the limiting membranes but poorly labeled in the internal lamellar structures. The regions of gap junctions were absolutely unlabeled. Filipin-cholesterol complexes were produced very close to the junctional strands but did not appear to disrupt the junctional structure of tight junctions. Nuclear membranes were affected only in the outer membrane with filipin. These results suggest that keratinocytes undergo a distinctive reduction in membrane deformability or in free-cholesterol content at the transition from living to dead cells, and display a heterogeneity in cholesterol distribution in human epidermal cell membranes.

Uptake of Polynucleotides by Mammalian Cells XV. Properties and Function of a DNA-Protein Complex Situated in the Outer Membrane of Ehrlich Ascites Tumor Cells

1) DNA-protein complexes are supposed to be original constituents of the membrane of Ehrlich ascites tumor cells. These complexes can be attacked at the surface of viable cells by DNase or protease. The DNA is partially embedded in protein structures. 2) The net charge of this complex is of major importance for the RNA uptake capacity of the cells. Negatively charged DNA which is situated at the surface hinders RNA uptake. This is the explanation for the stimulation of RNA uptake by DNase or the decrease in RNA uptake after protease treatment. 3) Upon treatment of DNA-deficient complexes with homologous or heterologous DNA the original RNA uptake capacity of the cells is restored but the original conformation of the complex cannot be regained. 4) The DNase action on the complex is temperature dependent in a sigmoidal fashion. It is markedly slowed down at temperatures below 12 degrees C. This implies that structural changes in the complex occur at this transition temperature which make surface DNA susceptible to DNase. This effect can only be observed in original structures but not in reconstituted ones. 5) Polyanion treatment of the cells [poly(L-lysine)] which increases their RNA uptake capacity, most probably does not interact with the DNA-protein complex. Poly(L-lysine) appears to act at other membrane sites. 6) The DNA-protein complex has been investigated entirely in situ, i.e. situated in the membrane of viable cells.