Outer Leaflet Of Plasma Membrane Research Articles

Regulation of the transbilayer movement of diacylglycerol in the plasma membrane

This mini-review presents recent advances in the regulation of the membrane transbilayer movement (or flip-flop) of diacylglycerol (DAG), a key intermediate in lipid metabolism and a second messenger in lipid-mediated signaling. Despite progresses in lipid biophysics and imaging, little is known about the DAG dynamics across the two leaflets of the plasma membrane in living cells. Previous model membrane studies with DAG analogs demonstrated their fast flip-flop suggesting that DAG is evenly distributed between the two leaflets of the plasma membrane. However, recent molecular dynamics simulations indicate that DAG transbilayer movement depends on the lipid environment surrounding the lipid, i.e. DAG flips more slowly across a more ordered "lipid raft-like" bilayer (enriched in sphingomyelin/cholesterol) than across a more fluid bilayer (composed of unsaturated glycerophospholipids). Furthermore using the yellow fluorescent protein-tagged C1AB domain from protein kinase C-γ (EYFP-C1AB) that selectively binds DAG, we recently proved that the sphingomyelin (SM) content in the plasma membrane outer leaflet regulates DAG transbilayer movement in Madin-Darby canine kidney cells treated with bacterial phosphatidylcholine-specific phospholipase C. The dose-dependent inhibition of DAG flip-flop by SM could be reproduced in model membranes using fluorescent short chain DAG analog. Regulation of DAG transbilayer movement by the outer leaflet SM content is expected to modify the downstream recruitment of C1-domain containing effectors, thus bringing new insights on the role of DAG dynamics in cell pathophysiology.

Structural elucidation of cell membrane-derived nanoparticles using molecular probes.

Cell membrane-derived nanoparticles (CNPs) are a novel class of materials and are superior to synthetic nanomaterials in certain aspects due to their biological origin. Although their medical applications have been actively explored, the fundamental structure of CNPs is rarely studied. For example, the membrane orientation of CNPs is critical for their pharmacokinetics, but the previous characterizations were mostly qualitative. Herein, we report a method to quantitatively study membrane orientation of CNPs by using a 6-FAM ssDNA probe and a BHQ1 ssDNA quencher with a complementary sequence. This method utilizes specific DNA hybridization and fluorescence resonance energy transfer between 6-FAM and BHQ1. When ssDNA probes are conjugated on cell membranes, the probe marks the outer leaflet of cell membranes. The fluorescence intensities of particle solutions before and after the addition of the ssDNA quencher can be measured to quantitatively determine the fraction of CNPs with a correct outside-out (also called right-side-out) membrane orientation. Red blood cell membrane-derived nanoparticles (RBC-NPs) were fabricated and determined to have an 84% correct orientation. The quenching of membrane-bound nitrobenzoxadiazole (NBD) was used to study the permeability of RBC-NPs. It was found that RBC-NPs have a significantly higher permeability to the NBD quencher, dithionite ions, compared to live cells and egg PC/cholesterol liposomes. The ubiquitous methods using molecular probes can elucidate some structural properties of CNPs in general, enabling direct comparisons among CNPs that are derived from different cells and convenient optimization of particle fabrication.

On multivalent receptor activity of GM1 in cholesterol containing membranes

Gangliosides located at the outer leaflet of plasma membrane are molecules that either participate in recognizing of exogenous ligand molecules or exhibit their own receptor activity, which are both essential phenomena for cell communication and signaling as well as for virus and toxin entry. Regulatory mechanisms of lipid-mediated recognition are primarily subjected to the physical status of the membrane in close vicinity of the receptor. Concerning the multivalent receptor activity of the ganglioside GM1, several regulatory strategies dealing with GM1 clustering and cholesterol involvement have been proposed. So far however, merely the isolated issues were addressed and no interplay between them investigated. In this work, several advanced fluorescence techniques such as Z-scan fluorescence correlation spectroscopy, Förster resonance energy transfer combined with Monte Carlo simulations, and a newly developed fluorescence antibunching assay were employed to give a more complex portrait of clustering and cholesterol involvement in multivalent ligand recognition of GM1. Our results indicate that membrane properties have an impact on a fraction of GM1 molecules that is not available for the ligand binding. While at low GM1 densities (~1 %) it is the cholesterol that turns GM1 headgroups invisible, at higher GM1 level (~4 %) it is purely the local density of GM1 molecules that inhibits the recognition. At medium GM1 content, cooperation of the two phenomena occurs. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

Electroporation of asymmetric phospholipid membranes.

As plasma membranes of animal cells are known to be asymmetric, the transmembrane lipid asymmetry, being essential for many membranes' properties and functions, should be properly accounted for in model membrane systems. In this paper, we employ atomic-scale molecular dynamics simulations to explore electroporation phenomena in asymmetric model membranes comprised of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipid monolayers that mimic the outer and inner leaflets of plasma membranes, respectively. Our findings clearly demonstrate that the molecular mechanism of electroporation in asymmetric phospholipid membranes differs considerably from the picture observed for their single-component symmetric counterparts: The initial stages of electric-field-induced formation of a water-filled pore turn out to be asymmetric and occur mainly on the PC side of the PC/PE membrane. In particular, water molecules penetrate in the membrane interior mostly from the PC side, and the reorientation of lipid head groups, being crucial for stabilizing the hydrophilic pore, also takes place in the PC leaflet. In contrast, the PE lipid head groups do not enter the central region of the membrane until the water pore becomes rather large and partly stabilized by PC head groups. Such behavior implies that the PE leaflet is considerably more robust against an electric field most likely due to interlipid hydrogen bonding. We also show that an electric field induces asymmetric changes in the lateral pressure profile of PC/PE membranes, decreasing the cohesion between lipid molecules predominantly in the PC membrane leaflet. Overall, our simulations provide compelling evidence that the transmembrane lipid asymmetry can be essential for understanding electroporation phenomena in living cells.

The Drosophila melanogaster Phospholipid Flippase dATP8B Is Required for Odorant Receptor Function

The olfactory systems of insects are fundamental to all aspects of their behaviour, and insect olfactory receptor neurons (ORNs) exhibit exquisite specificity and sensitivity to a wide range of environmental cues. In Drosophila melanogaster, ORN responses are determined by three different receptor families, the odorant (Or), ionotropic-like (IR) and gustatory (Gr) receptors. However, the precise mechanisms of signalling by these different receptor families are not fully understood. Here we report the unexpected finding that the type 4 P-type ATPase phospholipid transporter dATP8B, the homologue of a protein associated with intrahepatic cholestasis and hearing loss in humans, is crucial for Drosophila olfactory responses. Mutations in dATP8B severely attenuate sensitivity of odorant detection specifically in Or-expressing ORNs, but do not affect responses mediated by IR or Gr receptors. Accordingly, we find dATP8B to be expressed in ORNs and localised to the dendritic membrane of the olfactory neurons where signal transduction occurs. Localisation of Or proteins to the dendrites is unaffected in dATP8B mutants, as is dendrite morphology, suggesting instead that dATP8B is critical for Or signalling. As dATP8B is a member of the phospholipid flippase family of ATPases, which function to determine asymmetry in phospholipid composition between the outer and inner leaflets of plasma membranes, our findings suggest a requirement for phospholipid asymmetry in the signalling of a specific family of chemoreceptor proteins.

Tuning voltage-gated channel activity and cellular excitability with a sphingomyelinase

Voltage-gated ion channels generate action potentials in excitable cells and help set the resting membrane potential in nonexcitable cells like lymphocytes. It has been difficult to investigate what kinds of phospholipids interact with these membrane proteins in their native environments and what functional impacts such interactions create. This problem might be circumvented if we could modify specific lipid types in situ. Using certain voltage-gated K+ (KV) channels heterologously expressed in Xenopus laevis oocytes as a model, our group has shown previously that sphingomyelinase (SMase) D may serve this purpose. SMase D is known to remove the choline group from sphingomyelin, a phospholipid primarily present in the outer leaflet of plasma membranes. This SMase D action lowers the energy required for voltage sensors of a KV channel to enter the activated state, causing a hyperpolarizing shift of the Q-V and G-V curves and thus activating them at more hyperpolarized potentials. Here, we find that this SMase D effect vanishes after removing most of the voltage-sensor paddle sequence, a finding supporting the notion that SMase D modification of sphingomyelin molecules alters these lipids’ interactions with voltage sensors. Then, using SMase D to probe lipid–channel interactions, we find that SMase D not only similarly stimulates voltage-gated Na+ (NaV) and Ca2+ channels but also markedly slows NaV channel inactivation. However, the latter effect is not observed in tested mammalian cells, an observation highlighting the profound impact of the membrane environment on channel function. Finally, we directly demonstrate that SMase D stimulates both native KV1.3 in nonexcitable human T lymphocytes at their typical resting membrane potential and native NaV channels in excitable cells, such that it shifts the action potential threshold in the hyperpolarized direction. These proof-of-concept studies illustrate that the voltage-gated channel activity in both excitable and nonexcitable cells can be tuned by enzymatically modifying lipid head groups.

Oxidative stress induced in Leishmania infatum by batzelladine L and norbatzelladine L alkaloids isolated from the marine sponge Monanchora arbuscula[manuell

Leishmaniasis and American Trypanosomiais are protozoan neglected diseases affecting about 12 and 28 million people yearly in developing countries, respectively, with few and toxic alternatives for treatment. The study of new cell death pathways in protozoan parasites is an important strategy to control the progress of infectious diseases. In the search towards new drug leads to treat parasitic infectious diseases, marine natural products are considered promising tools for the discovery of drug prototypes. In the present investigation, the alkaloids batzelladine L and norbatzelladine L, isolated from the marine sponge Monanchora arbuscula, were tested against Leishmania infantum and T. cruzi parasites. Both alkaloids displayed IC50 in the range between 1 to 4 µg/mL, with an IC50 against a mammalian cell in the range between of 13 to 44 µg/mL. Batzelladine L and norbatzelladine L altered the permeability of Leishmania plasma membrane, but norbatzelladine L induced a time-dependent and the highest penetration of the fluorescent dye SYTOX Green. Both batzelladine L and norbatzelladine L induced rapid depolarization of Leishmania mitochondrial membrane potential, resulting in an up-regulation of reactive oxygen species (ROS), leading to oxidative stress. Moreover, batzelladine L demonstrated the highest capacity to induce ROS production, in a 4,300-fold higher levels when compared to untreated parasites. Flow cytometry analysis demonstrated an intense exposure of phospatidylserine in the outer leaflet of plasma membrane of batzelladine L-treated parasites. The size of the side alkyl chain of the two guanidine alkaloids batzelladine L and norbatzelladine L seems to play a major role in the antiparasitic effect. Both alkaloids represent promising tools to study novel cellular death pathways in Leishmania parasites.

Cryopreservation increases coating of bull sperm by seminal plasma binder of sperm proteins BSP1, BSP3, and BSP5

Artificial insemination with frozen semen allows affordable, worldwide dissemination of gametes with superior genetics. Nevertheless, sperm are damaged by the cryopreservation process. Elucidating the molecular effects of cryopreservation on sperm could suggest methods for improving fertility of frozen/thawed semen. This study was undertaken to examine the effect of cryopreservation on the coating of sperm by binder of sperm (BSP) proteins in seminal plasma. BSP proteins are secreted by the seminal vesicles and coat the surface of sperm by partially intercalating into the outer leaflet of the sperm plasma membrane. The BSP proteins are known to play roles in the formation of the oviductal sperm storage reservoir and in sperm capacitation. We investigated the effects of cryopreservation on the sperm BSP protein coat using Bovipure to separate live sperm from extended semen and then assaying the amounts of BSP proteins on sperm using quantitative western blotting with custom-made antibodies against unique sequences of each BSP protein. Greater amounts of all three BSP proteins (BSP1, BSP3, and BSP5) were detected on frozen/thawed sperm than on fresh sperm. Furthermore, the reduction of BSP3 from 15 to 13 kDa in mass, which occurs during incubation of sperm under mild capacitating conditions, was enhanced by cryopreservation. We concluded that freezing alters the BSP protein coating on sperm, which could account in part for reduced fertility of cryopreserved semen samples.

Rational design of fluorescent membrane probes for apoptosis based on 3-hydroxyflavone

Environment-sensitive probes constitute powerful tools for monitoring changes in the physico-chemical properties of cell plasma membranes. Among these probes, 3-hydroxyflavone probes are of great interest due to their dual emission and ratiometric response. Here, three probes derived from the parent F2N12S were designed, characterized and applied to monitor the membrane changes occurring during apoptosis. These three probes were designed to orient the dye vertically in the membrane. They differ by the length of their alkyl chains (from 4 to 8 carbons), which were included to optimize their affinity to the lipid membranes. Among these three probes, the one with medium chain length (hexyl) showed the best affinity to model and cell membranes, while the one with the longest alkyl chains (octyl) did not efficiently stain the membranes, probably due to aggregation. The new probes were found to be more sensitive than F2N12S to both the lipid phase and surface charge in lipid vesicles and to loss of lipid order in cell plasma membranes after cholesterol extraction. The one with the shortest (butyl) chains was found to be the most sensitive to apoptosis, while the one with medium-length (hexyl) chains was the brightest. Interestingly, apoptosis induced by different agents led to similar spectroscopic effects to those produced by the loss of lipid order and change in the surface charge, confirming that apoptosis decreases the lipid order and increases the negative surface charge in the outer leaflet of cell membranes. In conclusion, these studies report the relationship between the probe structures and their sensitivity to lipid order, surface charge and apoptosis and propose new probes for membrane research.

P2X receptor‐dependent erythrocyte damage by α‐hemolysin from Escherichia coli triggers phagocytosis by THP‐1 cells

The pore‐forming exotoxin α‐hemolysin from E. coli causes a significant volume reduction of human erythrocytes that precedes the ultimate swelling and lysis. This shrinkage results from activation of Ca2+‐sensitive K+ (KCa3.1) and Cl− channels (TMEM16A) and reduced functions of both of these channels potentiate the HlyA‐induced haemolysis. This means that Ca2+‐ dependent activation of KCa3.1 and TMEM16A protects the cells against early haemolysis. Simultaneous to the HlyA‐induced shrinkage the erythrocytes show increased exposure of phosphatidyl‐serine (PS) in the outer plasma membrane leaflet, which is known to be a keen trigger for phagocytosis.We hypothesize that exposure to HlyA elicits removal of the damaged erythrocytes by phagocytic cells. Cultured THP‐1 cells as a model for erythrocytal phagocytosis was verified by a variety of methods including live cell imaging. We consistently found the HlyA to very potently trigger phagocytosis of erythrocytes by THP‐1 cells. The HlyA‐induced phagocytosis was prevented by inhibition of KCa3.1, which is known to reduce PS‐exposure in human erythrocytes exposed to both ionomycin and HlyA. Moreover, we show that the P2X receptor inhibition, which prevents the cell damages caused by HlyA, also reduced that HlyA‐induced PS‐exposure and phagocytosis. Based on these results, we propose that erythrocytes, damaged by HlyA‐insertion, are effectively cleared from the blood stream. This mechanism will potentially reduce the risk of intravascular hemolysis

P2X Receptor-Dependent Erythrocyte Damage by α-Hemolysin from Escherichia coli Triggers Phagocytosis by THP-1 Cells

The pore-forming exotoxin α-hemolysin from E. coli causes a significant volume reduction of human erythrocytes that precedes the ultimate swelling and lysis. This shrinkage results from activation of Ca2+-sensitive K+ (KCa3.1) and Cl− channels (TMEM16A) and reduced functions of either of these channels potentiate the HlyA-induced hemolysis. This means that Ca2+-dependent activation of KCa3.1 and TMEM16A protects the cells against early hemolysis. Simultaneous to the HlyA-induced shrinkage, the erythrocytes show increased exposure of phosphatidylserine (PS) in the outer plasma membrane leaflet, which is known to be a keen trigger for phagocytosis. We hypothesize that exposure to HlyA elicits removal of the damaged erythrocytes by phagocytic cells. Cultured THP-1 cells as a model for erythrocytal phagocytosis was verified by a variety of methods, including live cell imaging. We consistently found the HlyA to very potently trigger phagocytosis of erythrocytes by THP-1 cells. The HlyA-induced phagocytosis was prevented by inhibition of KCa3.1, which is known to reduce PS-exposure in human erythrocytes subjected to both ionomycin and HlyA. Moreover, we show that P2X receptor inhibition, which prevents the cell damages caused by HlyA, also reduced that HlyA-induced PS-exposure and phagocytosis. Based on these results, we propose that erythrocytes, damaged by HlyA-insertion, are effectively cleared from the blood stream. This mechanism will potentially reduce the risk of intravascular hemolysis.

Comparative study of membranotropic action of single- and multi-walled carbon nanotubes

The aim of the present work is the investigation of the interactions of single-walled and multi-walled carbon nanotubes (further referred as SWCNTs and MWCNTs, respectively) with bimolecular lipid model membrane (BLM) and cellular plasma membrane (PM). The findings demonstrate that both SWCNTs and MWCNTs (in concentration range of 10⁻⁴ to 10⁻¹ mg ml⁻¹) are capable to penetrate through the region of hydrophobic fatty acid residues of phospholipids and to form molecular associates in the bilayer that have conductive properties of molecular pores type. The formed pores were shown to enable phosphatidylserine externalization from inner to outer PM leaflet. Both types of CNTs increase the specific conductivity and decrease the specific capacity of BLM.

IFITM Proteins Restrict Viral Membrane Hemifusion

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection.

Chemical composition of volatile oil from Citrus limettioides and their inhibition of colon cancer cell proliferation

Volatile oil from Palestine sweet lime (PSL) was analyzed for their chemical constituents. A total of 24 compounds were identified in hydro-distilled oil by GC–MS with electron impact mode using Zebron (ZB)-5 and ZB wax columns. The PSL volatile oil included 13 monoterpenes, 9 sesquiterpenes, one acyclic ester and one straight chain hydrocarbon. Four compounds namely d-limonene, myrcene, citronellal and β-citronellol were further confirmed by NMR spectral analysis. In vitro treatment of PSL volatile oil exhibited dose dependent inhibition of colon cancer (SW480) cell proliferation. Induction of apoptosis by the PSL volatile oil was confirmed through Annexin V staining indicating translocation of phosphatidylserine from inner to outer leaflet of plasma membrane, upon incubation of cells with volatile oil at 50μg/mL. Treatment with PSL volatile oil suppressed the expression of both Cox-2 and IL-6 suggested the potential inhibition of inflammation and activation of apoptosis in human SW480 colon cancer cells. Suppression of Cox-2 and IL-6 expression in the cells treated with PSL volatile oil were significant compared to treatment with d-limonene (50ppm), one of the major constituent of citrus volatile oils. This is the first report on chemoprofiling and anti-proliferative activities of volatile oil from PSL.

Influence of glycosaminoglycans on lipid dynamics in supported phospholipid bilayers

Glycosaminoglycans (GAGs) are important constituents of extracellular matrices (ECMs). As charged polymers, they do most likely influence lipid and protein dynamics in the outer leaflet of plasma membranes. In this study, we investigated their specific effect, depending on concentration, on lipid diffusion in model membranes. In our assay, GAGs are simply attached electrostatically to supported phospholipid (DOPC) bilayers doped with small amounts of cationic lipid (DOTAP) at physiological pH. Lipid dynamics are characterized via the diffusion of fluorescent lipid analogs (DiD/DiO), determined by fluorescence correlation spectroscopy (FCS). We find that diffusion of DiD is significantly affected by the attachment of GAG. Quite surprisingly, short chains (≤10 disaccharide units) of hyaluronic acid (unsulfated GAG) on the membrane surface affect the DiD diffusion coefficients stronger than medium or long chains (≥100 disaccharide units). In particular, short chains of hyaluronic acids at micromolar concentrations display a 2-fold decrease of the diffusion coefficients compared to the situation without GAG. At nanomolar concentrations of hyaluronic acid of both short and long chains, DiD diffusion remains unaltered. In contrast, sulfated GAGs, such as heparan sulfate (HS) and heparin, affect the lipid diffusion already at sub-micromolar concentrations, albeit not as strongly, with a less than 1.5 fold reduction of the diffusion coefficient. Chondroitin sulfate, another class of sulfated GAGs, did not impose any effect on DiD diffusion in the supported phospholipid bilayer at the concentrations studied. We also investigated desulfated heparin, to explore the role of sulfation and to compare its effect with HA. It is observed that heparin derivatives with lower degrees of sulfation have little effect on the lipid diffusion. Altogether, our results suggest that the presence of certain carbohydrate polymers in the ECM does have a noticeable effect on lipid dynamics in biological membranes.

Mechanical Compressibility of the Glycosylphosphatidylinositol (GPI) Anchor Backbone Governed by Independent Glycosidic Linkages

About 1% of the human proteome is anchored to the outer leaflet of cell membranes via a class of glycolipids called GPI anchors. In spite of their ubiquity, experimental information about the conformational dynamics of these glycolipids is rather limited. Here, we use a variety of computer simulation techniques to elucidate the conformational flexibility of the Man-α(1→2)-Man-α(1→6)-Man-α(1→4)-GlcNAc-α-OMe tetrasaccharide backbone 2 that is an essential and invariant part of all GPI-anchors. In addition to the complete tetrasaccharide structure, all disaccharide and trisaccharide subunits of the GPI backbone have been studied as independent moieties. The extended free energy landscape as a function of the corresponding dihedral angles has been determined for each glycosidic linkage relevant for the conformational preferences of the tetrasaccharide backbone (Man-α(1→2)-Man, Man-α(1→6)Man and Man-α(1→4)-GlcNAc). We compared the free energy landscapes obtained for the same glycosidic linkage within different oligosaccharides. This comparison reveals that the conformational properties of a linkage are primarily determined by its two connecting carbohydrate moieties, just as in the corresponding disaccharide. Furthermore, we can show that the torsions of the different glycosidic linkages within the GPI tetrasaccharide can be considered as statistically independent degrees of freedom. Using this insight, we are able to map the atomistic description to an effective, reduced model and study the response of the tetrasaccharide 2 to external forces. Even though the backbone assumes essentially a single, extended conformation in the absence of mechanical stress, it can be easily bent by forces of physiological magnitude.

Detection of apoptosis through the lipid order of the outer plasma membrane leaflet

Cell plasma membranes of living cells maintain their asymmetry, so that the outer leaflet presents a large quantity of sphingomyelin, which is critical for formation of ordered lipid domains. Here, a recently developed probe based on Nile Red (NR12S) was applied to monitor changes in the lipid order specifically at the outer leaflet of cell membranes. Important key features of NR12S are its ratiometric response exclusively to lipid order (liquid ordered vs. liquid disordered phase) and not to surface charge, the possibility of using it at very low concentrations (10–20nM) and the very simple staining protocol. Cholesterol extraction, oxidation and sphingomyelin hydrolysis were found to red shift the emission spectrum of NR12S, indicating a decrease in the lipid order at the outer plasma membrane leaflet. Remarkably, apoptosis induced by three different agents (actinomycin D, camptothecin, staurosporine) produced very similar spectroscopic effects, suggesting that apoptosis also significantly decreases the lipid order at this leaflet. The applicability of NR12S to detect apoptosis was further validated by fluorescence microscopy and flow cytometry, using the ratio between the blue and red parts of its emission band. Thus, for the first time, an environment-sensitive probe, sensitive to lipid order, is shown to detect apoptosis, suggesting a new concept in apoptosis sensing.

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

Dramatic changes in the structure of cell membranes on apoptosis allow easy, sensitive and non-destructive analysis of this process with the application of fluorescence methods. The strong plasma membrane asymmetry is present in living cells, and its loss on apoptosis is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the application of annexin V labeled with fluorescent dye is presently the most popular tool for its detection. Several methods have been suggested recently that offer important advantages over annexin V assay with the ability to study apoptosis by spectroscopy of cell suspensions, flow cytometry and confocal or two-photon microscopy. The PS exposure marks the integrated changes in the outer leaflet of cell membrane that involve electrostatic potential and hydration, and the attempts are being made to provide direct probing of these changes. This review describes the basic mechanisms underlying the loss of membrane asymmetry during apoptosis and discusses, in comparison with the annexin V-binding assay, the novel fluorescence techniques of detecting apoptosis on cellular membrane level. In more detail we describe the detection method based on smart fluorescent dye F2N12S incorporated into outer leaflet of cell membrane and reporting on apoptotic cell transformation by easily detectable change of the spectral distribution of fluorescent emission. It can be adapted to any assay format.

C. elegans Secreted Lipid-Binding Protein NRF-5 Mediates PS Appearance on Phagocytes for Cell Corpse Engulfment

During programmed cell death, apoptotic cells are rapidly removed by phagocytes. How dying cells are recognized remains poorly understood. Here we identify a secreted lipid transfer/LPS-binding family protein, NRF-5, which is required for efficient clearance of cell corpses. We observed that phosphatidylserine (PS), which is externalized to the outer leaflet of plasma membranes in apoptotic cells, is also detected on the surface of engulfing cells. Loss of NRF-5 function completely blocks PS appearance on engulfing cells but causes accumulation of PS on apoptotic cells, a phenotype observed in both ced-7(lf) and ttr-52(lf) mutants. The NRF-5 protein is expressed in and secreted from body wall muscle cells and clusters around apoptotic cells in a CED-7-dependent manner. NRF-5 associates with TTR-52, binds PS, and displays lipid transfer activity in vitro. Our data suggest that NRF-5 may act with CED-7 and TTR-52 to mediate PS transfer from apoptotic cells to engulfing cells and thus promotes engulfment by phagocytes.

Anti-tumor and pro-apoptotic activity of ethanolic extract and its various fractions from Polytrichum commune L.ex Hedw in L1210 cells

Ethnopharmacological relevancePolytrichum commune L.ex Hedw is a traditional Chinese herb for treatment of fever, hemostatic, uterine prolapse and especially for lymphocytic leukemia, but the antitumor effect and its potential mechanism remains unclear. Aims of the studyThe present study was to investigate the possible anti-proliferative activity of ethanolic extract and the organic fractions from P. commune on murine leukemia L1210 cells. Materials and methodsThe content of ethanolic extract and its fractions was performed on HPLC analysis with gradient elution. L1210 cells were treated with different concentrations of ethanolic extract and its fractions at different time intervals. Cell viability was evaluated using MTT assay. Apoptotic cell death was monitored by nuclear condensation and confirmed by exposure of phosphatidylserine to outer leaflet of plasma membrane. Loss of mitochondrial membrane potential was analyzed by flow cytometry using rhodamine 123 staining. ResultsThe obtained results showed that the cell viability of L1210 cells was reduced by ethanolic extract of P. commune in a concentration-dependent manner, and the IC50 value was about 77.22μg/ml at 24h post treatment. The ethylacetate fraction displayed higher anti-tumor effect than that of chloroform and butanol fractions with 32.29μg/ml (IC50 value, 48h). Microscopy studies revealed that ethanolic extract and ethylacetate fraction treated cells showed morphological characteristics of apoptosis such as chromatin condensation and DNA aggregation. Further, Annexin V-PE/ 7-AAD double staining showing the out leaflet of phosphatidylserine and the decline of mitochondrial membrane potential by flow cytometry confirmed that the extracts do, in fact, induce apoptosis in L1210 cells. ConclusionThis is the first report on anti-tumor and pro-apoptotic effect of P. commune in cultured leukemia cells, which provides scientific basis for its usefulness as traditional medicine. Further studies are needed to confirm the precise mechanism not only the crude extract but as well the monomeric chemical substances of Polytrichum commune L.ex Hedw.