Loss of membrane phospholipid asymmetry during activation of blood platelets and sickled red cells; mechanisms and physiological significance

Membrane phospholipid asymmetry is considered to be a general property of biological membranes. Detailed information is presently available on the non-random orientation of phospholipids in red cell- and platelet membranes. The outer leaflet of the lipid bilayer membrane is rich in choline-phospholipids, whereas amino-phospholipids are abundant in the inner leaflet. Studies with blood platelets have shown that these asymmetries are not maintained when the cells are activated in various ways. Undoing the normal asymmetry of membrane phospholipids in activated blood cells is presumably mediated by increased transbilayer movement of phospholipids. This process, which leads to increased exposure of negatively charged phosphatidylserine at the outer surface, plays an important physiological role in local blood clotting reactions. A similar phenomenon occurs in sickled red cells. Phospholipid vesicles breaking off from reversibly sickled cells contribute similarly to intravascular clotting in the crisis phase of sickle cell disease.The loss of membrane phospholipid asymmetry in activated platelets seems to be strictly correlated with degradation of cytoskeletal proteins by endogenous calpain. It is remarkable that membrane phospholipid asymmetry can be (partly) restored when activated platelets are treated with reducing agents. This leads to disappearance of phosphatidylserine from the outer leaflet where it was previously exposed during cell activation. These observations will be discussed in relation to two mechanisms which have been recognized to play a role in the regulation of membrane phospholipid asymmetry; i.e. the interaction of aminophospholipids to cytoskeletal proteins, and the involvement of a phospholipid-translocase catalyzing outward-inward transbilayer movement of amino-phospholipids.     

Distinct activation mechanisms of protein kinase B by growth-factor stimulation and heat-shock treatment

Protein kinase B (PKB) alpha, having the pleckstrin homology (PH) and catalytic domains in its amino- and carboxyl-terminal regions, respectively, is activated in the signaling pathway of growth factors as a downstream target of phosphatidylinositol 3-kinase and becomes an active form in heat-shocked cells in a manner independent of the lipid kinase. Therefore, the activation mechanisms of PKBalpha were compared in platelet-derived growth factor (PDGF)-stimulated and heat-shocked cells by monitoring the protein kinase activity and phosphorylation of the mutant molecules expressed in COS-7 cells. In heat-shocked cells, PKBalpha was activated to a certain level without phosphorylation on Thr-308 in the activation loop and on Thr-450 and Ser-473 in the carboxyl-terminal end region, which is critical for growth-factor-induced activation of PKBalpha. Metabolic labeling with (32)P-orthophosphate in the transfected cells revealed that there is no major phosphorylation site other than the three residues in PKBalpha. PKBalpha activated by heat shock was more stable than the enzyme stimulated by PDGF in the cells, and PKBalpha recovered from heat-shocked cells was resistant to the protein phosphatase treatment, whereas the enzyme obtained from the growth-factor-stimulated cells was inactivated by dephosphorylation. Heat shock also enhanced the association of the PH-domain fragment to the full-length PKBalpha in the transfected cells. On the other hand, the PH-domain fragment of PKBalpha, which moves from the cytosol to the plasma membrane upon PDGF stimulation by the interaction with the phosphatidylinositol 3-kinase products, did not translocate but stayed in the cytosol in heat-shocked NIH 3T3 cells. Furthermore, PKBalpha was associated with the nuclear region in heat-shocked cells, which is not observed in growth-factor-stimulated cells. These results indicate that heat shock induces the conformational change of PKBalpha that accompanies the protein complex formation and perinuculear/nuclear localization of the enzyme, to generate an active form by a mechanism distinct from that in the growth-factor-signaling pathway.     

Regulation of actin cytoskeleton by Rap1 binding to RacGEF1

Rap1 is rapidly and transiently activated in response to chemoattractant stimulation and helps establish cell polarity by locally modulating cytoskeletons. Here, we investigated the mechanisms by which Rap1 controls actin cytoskeletal reorganization in Dictyostelium and found that Rap1 interacts with RacGEF1 in vitro and stimulates F-actin polymerization at the sites where Rap1 is activated upon chemoattractant stimulation. Live cell imaging using GFP-coronin, a reporter for F-actin, demonstrates that cells expressing constitutively active Rap1 (Rap1CA) exhibit a high level of F-actin uniformly distributed at the cortex including the posterior and lateral sides of the chemotaxing cell. Examination of the localization of a PH-domain containing PIP3 reporter, PhdA-GFP, and the activation of Akt/Pkb and other Ras proteins in Rap1CA cells reveals that activated Rap1 has no effect on the production of PIP3 or the activation of Akt/Pkb and Ras proteins in response to chemoattractant stimulation. Rac family proteins are crucial regulators in actin cytoskeletal reorganization. In vitro binding assay using truncated RacGEF1 proteins shows that Rap1 interacts with the DH domain of RacGEF1. Taken together, these results suggest that Rap1-mediated F-actin polymerization probably occurs through the Rac signaling pathway by directly binding to RacGEF1.     

Regulation of transbilayer plasma membrane phospholipid asymmetry

Lipids in biological membranes are asymmetrically distributed across the bilayer; the amine-containing phospholipids are enriched on the cytoplasmic surface of the plasma membrane, while the choline-containing and sphingolipids are enriched on the outer surface. The maintenance of transbilayer lipid asymmetry is essential for normal membrane function, and disruption of this asymmetry is associated with cell activation or pathologic conditions. Lipid asymmetry is generated primarily by selective synthesis of lipids on one side of the membrane. Because passive lipid transbilayer diffusion is slow, a number of proteins have evolved to either dissipate or maintain this lipid gradient. These proteins fall into three classes: 1) cytofacially-directed, ATP-dependent transporters ("flippases"); 2) exofacially-directed, ATP-dependent transporters ("floppases"); and 3) bidirectional, ATP-independent transporters ("scramblases"). The flippase is highly selective for phosphatidylserine and functions to keep this lipid sequestered from the cell surface. Floppase activity has been associated with the ABC class of transmembrane transporters. Although they are primarily nonspecific, at least two members of this class display selectivity for their substrate lipid. Scramblases are inherently nonspecific and function to randomize the distribution of newly synthesized lipids in the endoplasmic reticulum or plasma membrane lipids in activated cells. It is the combined action of these proteins and the physical properties of the membrane bilayer that generate and maintain transbilayer lipid asymmetry.     

Lipid translocation across the plasma membrane of mammalian cells.

The plasma membrane, which forms the physical barrier between the intra- and extracellular milieu, plays a pivotal role in the communication of cells with their environment. Exchanging metabolites, transferring signals and providing a platform for the assembly of multi-protein complexes are a few of the major functions of the plasma membrane, each of which requires participation of specific membrane proteins and/or lipids. It is therefore not surprising that the two leaflets of the membrane bilayer each have their specific lipid composition. Although membrane lipid asymmetry has been known for many years, the mechanisms for maintaining or regulating the transbilayer lipid distribution are still not completely understood. Three major players have been presented over the past years: (1) an inward-directed pump specific for phosphatidylserine and phosphatidylethanolamine, known as aminophospholipid translocase; (2) an outward-directed pump referred to as 'floppase' with little selectivity for the polar headgroup of the phospholipid, but whose actual participation in transport of endogenous lipids has not been well established; and (3) a lipid scramblase, which facilitates bi-directional migration across the bilayer of all phospholipid classes, independent of the polar headgroup. Whereas a concerted action of aminophospholipid translocase and floppase could, in principle, account for the maintenance of lipid asymmetry in quiescent cells, activation of the scramblase and concomitant inhibition of the aminophospholipid translocase causes a collapse of lipid asymmetry, manifested by exposure of phosphatidylserine on the cell surface. In this article, each of these transporters will be discussed, and their physiological importance will be illustrated by the Scott syndrome, a bleeding disorder caused by impaired lipid scrambling. Finally, phosphatidylserine exposure during apoptosis will be briefly discussed in relation to inhibition of translocase and simultaneous activation of scramblase.     

Lipid raft heterogeneity in human peripheral blood T lymphoblasts: a mechanism for regulating the initiation of TCR signal transduction

Lateral mobility and spatial organization of proteins within the plasma membrane are likely to mediate the initial events coordinating T cell activation. Lipid rafts, distinct cholesterol/sphingolipid-rich membrane microdomains, provide a mechanism for this regulation by concentrating or excluding signaling proteins. We demonstrate in peripheral blood T cell lymphoblasts that immediate early phosphotyrosine signal transduction through the TCR complex is functionally dependent on a distinct population of lipid rafts. Specifically, cholesterol extraction destabilizes the membrane microdomains containing Lck, while the rafts containing the adapter protein linker for activation of T cells remain intact. Heterogeneity in the partitioning of these proteins in resting cells was confirmed by immunoelectron microscopy. After T cell activation, both Lck and the linker for activation of T cells colocalize to 50-100 nm microdomains in the plasma membrane, indicating that sequestration of these proteins into distinct lipid rafts may function to regulate the initiation of T cell signal transduction.     

Pseudoapoptosis induced by brief activation of ATP-gated P2X7 receptors

P2X7 receptors are ATP-gated ion channels primarily expressed on antigen-presenting immune cells where they play a role in the acute inflammatory response. These ion channels couple not only to influx of cations, including calcium, but also to rapid alterations in cell morphology (membrane blebbing, phosphatidylserine exposure, microvesicle shedding). These features resemble the extranuclear events associated with end stages of apoptosis but cell death does not occur if receptor activation is brief. Here we delineate two signaling pathways underlying these apoptotic-like processes. Loss of membrane asymmetry occurs within seconds, which directly triggers cytoskeletal disruption and zeiotic membrane blebbing; this is readily reversible and requires both calcium influx through P2X7 channels and mitochondrial calcium increase but is not associated with cytochrome c release. A slower, calcium-independent, ROCK-1-dependent cascade that does not involve rapid loss of membrane asymmetry but is associated with cytochrome c release is secondarily activated. The ROCK-1 pathway appears largely responsible for cell death, which occurs after prolonged stimulation of P2X7 receptors. We suggest that the former mechanism underlies the reversible pseudoapoptotic events induced by brief activation of P2X7 receptors.     

Scott syndrome, a bleeding disorder caused by defective scrambling of membrane phospholipids

Normal quescent cells maintain membrane lipid asymmetry by ATP-dependent membrane lipid transporters, which shuttle different phospholipids from one leaflet to the other against their respective concentration gradients. When cells are challenged, membrane lipid asymmetry can be perturbed resulting in exposure of phosphatidylserine [PS] at the outer cell surface. Translocation of PS from the inner to outer membrane leaflet of activated blood platelets and platelet-derived microvesicles provides a catalytic surface for interacting coagulation factors. This process is dramatically impaired in Scott syndrome, a rare congenital bleeding disorder, underscoring the indispensible role of PS in hemostasis. This also testifies to a defect of a protein-catalyzed scrambling of membrane phospholipids. The Scott phenotype is not restricted to platelets, but can be demonstrated in other blood cells as well. The functional aberrations observed in Scott syndrome have increased our understanding of transmembrane lipid movements, and may help to identify the molecular elements that promote the collapse of phospholipid asymmetry during cell activation and apoptosis.     

Neu1 sialidase and matrix metalloproteinase-9 cross-talk is essential for neurotrophin activation of Trk receptors and cellular signaling

Neurotrophin-induced Trk tyrosine kinase receptor activation and neuronal cell survival responses have been reported to be under the control of a membrane associated sialidase. Here, we identify an unprecedented membrane sialidase mechanism initiated by nerve growth factor (NGF) binding to TrkA to potentiate GPCR-signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP-9) activation to induce Neu1 sialidase activation in live primary neurons and TrkA- and TrkB-expressing cell lines. Central to this process is that Neu1/MMP-9 complex is bound to TrkA on the cell surface of naïve primary neurons and TrkA-expressing cells. Tamiflu completely blocks this sialidase activity in live TrkA-PC12 cells treated with NGF with an IC₅₀ of 3.876 μM with subsequent inhibition of Trk activation in primary neurons and neurite outgrowth in TrkA-PC12 cells. Our findings uncover a Neu1 and MMP-9 cross-talk on the cell surface that is critically essential for neurotrophin-induced Trk tyrosine kinase receptor activation and cellular signaling.     

Regulation of TRPV1 Ion Channel by Phosphoinositide (4,5)-Bisphosphate: THE ROLE OF MEMBRANE ASYMMETRY*

Membrane asymmetry is essential for generating second messengers that act in the cytosol and for trafficking of membrane proteins and membrane lipids, but the role of asymmetry in regulating membrane protein function remains unclear. Here we show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P₂) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in. We observed potentiation of capsaicin-activated TRPV1 currents by PI(4,5)P₂ in the intracellular leaflet of the plasma membrane but inhibition of capsaicin-activated currents when PI(4,5)P₂ was in both leaflets of the membrane, although much higher concentrations of PI(4,5)P₂ in the extracellular leaflet were required for inhibition compared with the concentrations of PI(4,5)P₂ in the intracellular leaflet that produced activation. Patch clamp fluorometry using a synthetic PI(4,5)P₂ whose fluorescence reports its concentration in the membrane indicates that PI(4,5)P₂ must incorporate into the extracellular leaflet for its inhibitory effects to be observed. The asymmetry-dependent effect of PI(4,5)P₂ may resolve the long standing controversy about whether PI(4,5)P₂ is an activator or inhibitor of TRPV1. Our results also underscore the importance of membrane asymmetry and the need to consider its influence when studying membrane proteins reconstituted into synthetic bilayers.     

Age-related and phenylhydrazine-induced activation of the membrane-associated cathepsin E in human erythrocytes

We examined the effects of cell aging and phenylhydrazine-induced oxidant damage on erythrocyte cathepsin E, which is present as a latent, membrane-associated enzyme in normal human erythrocytes. When young erythrocytes isolated from human mature erythrocytes by Percoll density gradient centrifugation were aged in vitro, the membrane-associated cathepsin E was progressively released from the membrane as an active enzyme. During the cell aging up to 100 h, about 40% of the membrane-associated enzyme was activated and solubilized. When phenylhydrazine was incubated with the erythrocytes, it also caused the activation and solubilization of cathepsin E in a dose-dependent and time-dependent manner. Exposure of erythrocytes to 2.5 mM phenylhydrazine for up to 2 h led to about 40% activation of the membrane-associated enzyme. Both aging and phenylhydrazine-treatment were accompanied with an increase in the association of the cytosolic proteins, primarily hemoglobin, with the membrane, which occurred prior to the release of cathepsin E from the membrane. A similar activation for the membrane-associated enzyme was observed with in vitro-aged hemoglobin-free membrane ghosts. Thus, the primary mechanism for activation of cathepsin E in the intact cells seems to be through lesion of the membrane framework that results from increased binding of hemoglobin to the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting employing polyclonal IgG antibodies for human spectrin and band 3 revealed that breakdown of the membrane proteins was enhanced in both aged and phenylhydrazine-treated cells. The relation between the cathepsin E activation and the membrane protein breakdown is discussed.     

Plasma membrane organization is essential for balancing competing pseudopod- and uropod-promoting signals during neutrophil polarization and migration

Exposure of neutrophils to chemoattractant induces cell polarization and migration. These behaviors require the asymmetric activation of distinct signaling pathways and cytoskeletal elements in the protruding pseudopod at the front of cells and the retracting uropod at the rear. An important outstanding question is, how does the organization of the plasma membrane participate in establishing asymmetry during polarization and migration? To answer this question, we investigated the function of cholesterol, a lipid known to influence membrane organization. Using controlled cholesterol depletion, we found that a cholesterol-dependent membrane organization enabled cell polarization and migration by promoting uropod function and suppressing ectopic pseudopod formation. At a mechanistic level, we showed that cholesterol was directly required for suppressing inappropriate activation of the pseudopod-promoting Gi/PI3-kinase signaling pathway. Furthermore, cholesterol was required for dampening Gi-dependent negative feedback on the RhoA signaling pathway, thus enabling RhoA activation and uropod function. Our findings suggest a model in which a cholesterol-dependent membrane organization plays an essential role in the establishment of cellular asymmetry by balancing the activation and segregating the localization of competing pseudopod- and uropod-inducing signaling pathways during neutrophil polarization and migration.     

The glycosyl phosphatidylinositol anchor is critical for Ly-6A/E-mediated T cell activation.

Ly-6E, a glycosyl phosphatidylinositol (GPI)-anchored murine alloantigen that can activate T cells upon antibody cross-linking, has been converted into an integral membrane protein by gene fusion. This fusion product, designated Ly-6EDb, was characterized in transiently transfected COS cells and demonstrated to be an integral cell surface membrane protein. Furthermore, the fusion antigen can be expressed on the surface of the BW5147 class "E" mutant cell line, which only expresses integral membrane proteins but not GPI-anchored proteins. The capability of this fusion antigen to activate T cells was examined by gene transfer studies in D10G4.1, a type 2 T cell helper clones. When transfected into D10 cells, the GPI-anchored Ly-6E antigen, as well as the endogenous GPI-anchored Ly-6A antigen, can initiate T cell activation upon antibody cross-linking. In contrast, the transmembrane anchored Ly-6EDb antigen was unable to mediate T cell activation. Our results demonstrate that the GPI-anchor is critical to Ly-6A/E-mediated T cell activation.     

Functional implications of plasma membrane condensation for T cell activation

The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.     

Generation of asymmetry during development. Segregation of type-specific proteins in Caulobacter.

An essential event in developmental processes is the introduction of asymmetry into an otherwise undifferentiated cell population. Cell division in Caulobacter is asymmetric; the progeny cells are structurally different and follow different sequences of development, thus providing a useful model system for the study of differentiation. Because the progeny cells are different from one another, there must be a segregation of morphogenetic and informational components at some time in the cell cycle. We have examined the pattern of specific protein segregation between Caulobacter stalked and swarmer daughter cells, with the rationale that such a progeny analysis would identify both structurally and developmentally important proteins. To complement the study, we have also examined the pattern of protein synthesis during synchronous growth and in various cellular fractions. We show here, for the first time, that the association of proteins with a specific cell type may result not only from their periodicity of synthesis, but also from their pattern of distribution at the time of cell division. Several membrane-associated and soluble proteins are segregated asymmetrically between progeny stalked and swarmer cells. The data further show that a subclass of soluble proteins becomes associated with the membrane of the progeny stalked cells. Therefore, although the principal differentiated cell types possess different synthetic capabilities and characteristic proteins, the asymmetry between progeny stalked and swarmer cells is generated primarily by the preferential association of specific soluble proteins with the membrane of only one daughter cell. The majority of the proteins which exhibit this segregation behavior are synthesized during the entire cell cycle and exhibit relatively long, functional messenger RNA half-lives.     

The heterocytotoxicity of human serum. II. Role of natural antibody and of the classical and alternative complement pathways.

Mouse lymphoid cells and tumor cell lines were employed as target cells for the investigation of the mechanism of heterocytotoxicity in human serum samples. It was shown that the heterocytotoxic effects were due to two differing mechanisms. Cytotoxicity was mediated in part, by activation of the alternative complement pathway on target cell membrane, a process which was antibody-independent. A second mechanism of cytotoxicity was induced by natural antibodies to a target cell, which probably mediated activation of the classical complement pathway. These data may shed light on the frequently observed cytotoxicity in mammalian sera for various target cells.     

Eryptosis in hereditary spherocytosis and thalassemia: role of glycoconjugates

The present work is aimed to study the mechanism of faster erythrocyte clearance in hereditary spherocytosis (HS), a heterogeneous disorders characterized by alterations in the proteins of the red cell membrane skeleton along with different kinds of thalassemia. The maximum exposure of phosphatidylserine (PS) is found in HS compared to those in both α- and β-thalassemia. Interestingly, in HS more PS exposed cells were found in younger erythrocytes compared to normal and the thalassemics where aged cells showed higher loss of PS asymmetry. Loss of sialic acid and GlcNAc bearing glycoconjugates, presumably the glycophorins, was also found upon aging. The loss of PS asymmetry together with the cell surface glycoproteins mediated by membrane vesiculation, seemed to play key role in early clearance of erythrocytes from circulation following a mechanism similar to HbEβ-thalassemia.     

Polarity mediates asymmetric trafficking of the Gbeta heterotrimeric G-protein subunit GPB-1 in C. elegans embryos

Asymmetric cell division is an evolutionarily conserved process that gives rise to daughter cells with different fates. In one-cell stage C. elegans embryos, this process is accompanied by asymmetric spindle positioning, which is regulated by anterior-posterior (A-P) polarity cues and driven by force generators located at the cell membrane. These force generators comprise two Gα proteins, the coiled-coil protein LIN-5 and the GoLoco protein GPR-1/2. The distribution of GPR-1/2 at the cell membrane is asymmetric during mitosis, with more protein present on the posterior side, an asymmetry that is thought to be crucial for asymmetric spindle positioning. The mechanisms by which the distribution of components such as GPR-1/2 is regulated in time and space are incompletely understood. Here, we report that the distribution of the Gβ subunit GPB-1, a negative regulator of force generators, varies across the cell cycle, with levels at the cell membrane being lowest during mitosis. Furthermore, we uncover that GPB-1 trafficks through the endosomal network in a dynamin- and RAB-5-dependent manner, which is most apparent during mitosis. We find that GPB-1 trafficking is more pronounced on the anterior side and that this asymmetry is regulated by A-P polarity cues. In addition, we demonstrate that GPB-1 depletion results in the loss of GPR-1/2 asymmetry during mitosis. Overall, our results lead us to propose that modulation of Gβ trafficking plays a crucial role during the asymmetric division of one-cell stage C. elegans embryos.     

Membrane expression of soluble endotoxin-binding proteins permits lipopolysaccharide signaling in Chinese hamster ovary fibroblasts independently of CD14.

The activation of phagocytes by lipopolysaccharide (LPS) has been implicated in the pathogenesis of Gram-negative sepsis. Although the interaction between CD14 and LPS is a key event in the signaling cascade, the molecular mechanism by which cellular activation occurs remains obscure. We hypothesized that the main function of CD14 was to bind LPS and transfer it to a second receptor, which then initiates the subsequent signal for cellular activation. Thus, surface binding of LPS to the cell membrane would be the critical step that CD14 carries out. To test this hypothesis, we examined the activity of two other proteins known to bind LPS, lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein. We found that when these normally soluble proteins were expressed in Chinese hamster ovary-K1 fibroblasts as glycosylphosphatidylinositol-anchored proteins, both could substitute for CD14 in initiating LPS signaling. Pharmacological studies with synthetic lipid A analogues demonstrated that these surface expressed LPS-binding proteins had characteristics that were qualitatively identical to membrane CD14. These data support the hypothesis that a receptor distinct from CD14 functions as the actual signal transducer and suggest that surface binding of LPS to the cell membrane is the crucial first step for initiating downstream signaling events.