Capacitation induces cyclic adenosine 3',5'-monophosphate-dependent,but apoptosis-unrelated,exposure of aminophospholipids at the apical head plasma membrane of boar sperm cells

The capacitating agent bicarbonate/CO(2) has been shown to induce profound changes in the architecture and dynamics within the sperm's plasma membrane lipid bilayer via a cAMP-dependent protein phosphorylation signaling pathway. Here we have investigated the effect of bicarbonate on surface exposure of endogenous aminophospholipids in boar spermatozoa, detecting phosphatidylserine (PS) with fluorescein-conjugated annexin V and phosphatidylethanolamine (PE) with fluorescein-conjugated streptavidin/biotinylated Ro-09-0198. Flow cytometric analyses revealed that incubation with 15 mM bicarbonate induced 30%-70% of live acrosome-intact cells to expose PE very rapidly; this exposure was closely related to a decrease in lipid packing order as detected by enhanced binding of merocyanine 540. PS exposure was detectable in the same proportion of cells, though its expression was slower. Confocal microscopy revealed that exposure of aminophospholipids in intact cells was restricted to the anterior acrosomal region of the head plasma membrane. Aminophospholipid exposure, merocyanine stainability, and a subsequent migration of cholesterol to the apical region of the head plasma membrane, were all under the control of the cAMP-dependent protein phosphorylation pathway. The close coupling of decreased lipid packing order with exposure of PE led us to conclude that bicarbonate was inducing phospholipid scrambling (i.e., collapse of asymmetric transverse distribution), and that the scrambling was a prerequisite for cholesterol relocation. There was no evidence whatever that the bicarbonate-induced scrambling was an apoptotic process. It was not accompanied by major loss of viability or by DNA degeneration or by loss of mitochondrial function, and it could not be blocked by the broad-specificity caspase inhibitors zVAD-fmk and BocD-fmk. In the absence of bicarbonate, scrambling could not be induced by the apoptotic agents UV, staurosporine, or cycloheximide. Bicarbonate-induced phospholipid scrambling thus appears to be an important and early physiological event in the capacitation process.     

Engagement of phospholipid scramblase 1 in activated cells: implication for phosphatidylserine externalization and exocytosis

Phosphatidylserine (PS) in quiescent cells is predominantly confined to the inner leaflet of the plasma membrane. Externalization of PS is a marker of apoptosis, exocytosis, and some nonapoptotic activation events. It has been proposed that PS externalization is regulated by the activity of PLSCR1 (phospholipid scramblase 1), a Ca(2+)-dependent endofacial plasma membrane protein, which is tyrosine-phosphorylated in activated cells. It is, however, unclear how the phosphorylation of PLSCR1 is related to its membrane topography, PS externalization, and exocytosis. Using rat basophilic leukemia cells as a model, we show that nonapoptotic PS externalization induced through the high affinity IgE receptor (FcepsilonRI) or the glycosylphosphatidylinositol-anchored protein Thy-1 does not correlate with enhanced tyrosine phosphorylation of PLSCR1. In addition, PS externalization in FcepsilonRI- or Thy-1-activated cells is not associated with alterations of PLSCR1 fine topography as detected by electron microscopy on isolated plasma membrane sheets. In contrast, activation by calcium ionophore A23187 induces changes in the cellular distribution of PLSCR1. We also show for the first time that in pervanadate-activated cells, exocytosis occurs even in the absence of PS externalization. Finally, we document here that tyrosine-phosphorylated PLSCR1 is preferentially located in detergent-insoluble membranes, suggesting its involvement in the formation of membrane-bound signaling assemblies. The combined data indicate that changes in the topography of PLSCR1 and its tyrosine phosphorylation, PS externalization, and exocytosis are independent phenomena that could be distinguished by employing specific conditions of activation.     

Phospholipid flip-flop and phospholipid scramblase 1 (PLSCR1) co-localize to uropod rafts in formylated Met-Leu-Phe-stimulated neutrophils

Movement of phosphatidylserine (PS) to the plasma membrane outer leaflet is a nearly universal marker of apoptosis and occurs during activation of many cells. Neutrophils stimulated with the chemotactic peptide formylated Met-Leu-Phe (fMLP) demonstrated transient PS exposure. Stimulated outward movement of PS was accompanied by enhanced inward movement of several phosphorylcholine lipid probes and was associated with enhanced FM 1-43 staining indicative of phospholipid packing changes. Unlike apoptosis, inward movement of exogenously added fluorescent PS did not decline, and DNA was not cleaved during fMLP stimulation. Movement of phospholipids occurred within minutes following stimulation, was independent of endocytosis/pinocytosis, and was consistent with bidirectional, transbilayer phospholipid flip-flop. While the role of phospholipid scramblase 1 (PLSCR1) is controversial in flip-flop, we sought evidence for its role in enhanced phospholipid movements during fMLP stimulation. Using antibodies to the carboxyl-terminal domain of PLSCR1, its presence in the plasma membranes of non-permeabilized neutrophils was confirmed by flow cytometry. Additionally subcellular fractionation demonstrated that PLSCR1 was also located in secretory vesicles and tertiary and secondary granules. Activation of neutrophils with fMLP, however, did not significantly alter surface labeling suggesting that stimulated phospholipid flip-flop does not require additional mobilization of PLSCR1 to the plasma membrane. As expected for palmitoylated proteins, PLSCR1 was enriched in detergent-insoluble membranes and co-localized with raft markers at the neutrophil uropod after stimulation. Of note, PS exposure, phospholipid uptake, and FM 1-43 staining also localized to the uropod following stimulation demonstrating that both PLSCR1 and phospholipid flip-flop characterize this specialized domain of polarized neutrophils.     

Protein kinase C activation induces phosphatidylserine exposure on red blood cells

We have shown previously that red blood cells (RBCs) can be induced to influx Ca(2+) when treated with lipid mediators, such as lysophosphatidic acid and prostaglandin E(2), that are released during clot formation. Since calcium loading of RBCs can lead to both protein kinase C (PKC) activation and phosphatidylserine (PS) exposure, we decided to investigate the possible linkage between PKC activation and membrane PS scrambling using phorbol 12-myristate-13-acetate (PMA), a commonly used activator of PKC. Treatment of RBCs with PMA in a calcium-containing buffer caused immediate PS exposure in an RBC subpopulation. The size of the subpopulation did not change upon further incubation, indicating that not all RBCs are equally susceptible to this treatment. Using a fluorescent indicator, we found a subpopulation of RBCs with elevated intracellular calcium levels. In the absence of extracellular calcium, no PS exposure was found. However, we did find cells with high levels of calcium that did not expose PS, and a variable percentage of PS-exposing cells that did not show elevated calcium concentrations. Inhibition of PKC with either calphostin C, a blocker of the PMA binding site, or chelerythrine chloride, an inhibitor of the active site, diminished the level of formation of PS-exposing cells. However, the inhibitors had different effects on calcium internalization, indicating that a high calcium concentration alone was not responsible for inducing PS exposure in the absence of PKC activity. Moreover, PKC inhibition could prevent PS exposure induced by calcium and ionophore treatment of RBCs. We conclude that PKC is implicated in the mechanism of membrane phospholipid scrambling.     

Spatial resolution of phospholipid scramblase 1 (PLSCR1), caspase-3 activation and DNA-fragmentation in the human hippocampus after cerebral ischemia

Reports on non-neural cells have shown that enhanced activity of the Ca(2+)-dependent/ATP-independent phospholipid scramblase (PLSCR1) is, at least in part, responsible for surface exposure of phosphatidylserine and the collapse of plasma membrane asymmetry in injured or apoptotic cells. To shed some light on mechanisms with a potential to lead to apoptotic death of human neurones following ischemic/hypoxic injury, we examined the immunoreactivity of hippocampal neurones for PLSCR1, caspase-3, cytochrome c and DNA-fragmentation in 22 individuals with clinically symptomatic cerebral ischemia after cardiac arrest or severe hypotension.WE FOUND: (1) significant differences in the percentage of PLSCR1-immunoreactive neurones between controls and short survivors; statistically strong differences between the frequency of immunoreactive neurones among the subfields studied with lowest levels in the CA3; preferential distribution of immunoreactive neurones in controls within the regio entorhinalis, subfield CA1, and hilum. Additionally, these areas exhibited staining of fibre bundles which probably correspond to perforant path, alvear path and collateral's of Schaffer, (2) caspase-3 was upregulated in a region-specific manner with marked activation in the selectively vulnerable hippocampal areas, (3) cytochrome c was redistributed, (4) DNA-fragmentation represented by scattered TUNEL-positive cells increased predominantly during the first 3 days after ischemia, and particularly in the regions of greatest susceptibility to hypoxic injury.This study presents the first evidence that PLSCR1, and probably remodelling of plasma membrane phospholipids (PL), plays a role in ischemic injury in the human hippocampus.     

The phospholipid scramblase PLSCR1 increases UV induced apoptosis primarily through the augmentation of the intrinsic apoptotic pathway and independent of direct phosphorylation by protein kinase C delta

Cell death by apoptosis can be caused by the DNA mutagen UV light whose exposure causes the direct activation of both the caspase 9 regulated cell damage intrinsic pathway and the caspase 8 regulated plasma membrane extrinsic pathway. We determined that increased activity of the plasma membrane phospholipid scramblase, PLSCR1, amplified UV mediated apoptosis primarily through the activation of the intrinsic apoptotic pathway. The caspase 8 inhibitor z-IETD-fmk was not as effective an inhibitor of PLSCR1 augmented UV induced apoptosis compared to treatment with caspase 3, caspase 9, or pan-caspase inhibitors. The inability of the caspase 8 inhibitor to decrease UV induced apoptosis was dependent on PLSCR1, as UV induced apoptosis was decreased by a similar amount in the control cells in the presence of inhibitors of caspase 8, caspase 9, caspase 3, or the pan-caspase inhibitor. PKC-delta directly phosphorylates human PLSCR1 resulting in increased PLSCR1 scramblase activity. PKC-delta can also be activated by caspase mediated cleavage resulting in the release of a constitutively active kinase domain. We observed that replacing the PKC-delta phosphorylation site of PLSCR1 with an alanine did not affect the ability of PLSCR1 to enhance UV induced apoptosis implying that PKC-delta does not directly phosphorylate PLSCR1 to increase plasma membrane scramblase activity during apoptosis. Cells transfected with a PLSCR1 mutant that contained an alanine substitution at its known PKC-delta phosphorylation site underwent UV induced apoptosis at a level similar to those transfected with wild type PLSCR1. The combined results indicate that UV exposure in cells possessing PLSCR1 increases apoptosis primarily by enhancement of the intrinsic apoptotic pathway, and also imply that the increased apoptosis observed upon exposure to UV light is not through direct phosphorylation of PLSCR1 by PKC-delta.     

Generation of singlet oxygen induces phospholipid scrambling in human erythrocytes

Maintenance of phospholipid asymmetry of the plasma membrane is essential for cells to prevent phagocytic removal or acceleration of coagulation. Photodynamic treatment (PDT), which relies on the generation of reactive oxygen species to achieve inactivation of pathogens, might be a promising approach in the future for decontamination of red blood cell concentrates. To investigate whether PDT affects phospholipid asymmetry, erythrocytes were illuminated in the presence of 1,9-dimethyl-methylene blue (DMMB) as photosensitizer and subsequently labeled with FITC-labeled annexin V. This treatment resulted in about 10% annexin V positive cells, indicating exposure of phosphatidylserine (PS). Treatment of erythrocytes with N-ethylmaleimide (NEM) prior to illumination, to inhibit inward translocation of PS via the aminophospholipid translocase, resulted in enhanced PS exposure, while treatment with H(2)O(2) (previously shown to inhibit phospholipid scrambling) greatly diminished PS exposure, indicating the induction of phospholipid scrambling by PDT. Only erythrocytes illuminated in the presence of DMMB showed translocation of NBD-phosphatidylcholine (NBD-PC), confirming scrambling induction. Double label experiments indicated that PS exposure does not occur without concurrent scrambling activity. Induction of scrambling was only moderately affected by Ca(2+) depletion of the cells. In contrast, scavengers of singlet oxygen were found to prevent phospholipid scrambling induced by PDT. The results of this study show that phospholipid scrambling is induced in human erythrocytes by exposure to singlet oxygen.     

Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: functional relationship with zona binding and the zona-induced acrosome reaction

For sperm to successfully fertilize an oocyte, it needs to pass through certain steps prior to, during and after initial recognition of the zona pellucida (ZP). During capacitation, the surface of the sperm head becomes remodelled, priming it to bind to the ZP and subsequently to undergo the ZP-induced acrosome reaction. During capacitation, sperm ZP-binding proteins are ordered in functional protein complexes that only emerge at the apical tip of the sperm head plasma membrane; this is also functionally the exclusive sperm surface area involved in primary ZP binding. After primary ZP binding, the same area is probably involved in the induction of the acrosome reaction. A combination of biochemical and proteomic membrane protein techniques have enabled us to dissect and highly purify the apical sperm plasma membrane area from control and capacitated sperm cells. The actual ZP-binding proteins identified predominantly belonged to the sperm membrane-associated family members of spermadhesins (AQN-3) and were present in the aggregating lipid ordered membrane microdomains (lipid rafts) that emerged during in vitro capacitation in the apical ridge area of the sperm head plasma membrane. This clustering of these rafts was dependent on the presence of bicarbonate (involved in protein kinase A activation) and on the presence of albumin (involved in cholesterol removal). Remarkably, cholesterol removal was restricted to the non-raft membrane fraction of the sperm plasma membrane, but did not cause any depletion of cholesterol in the raft membrane fraction. Interestingly, sperm SNARE proteins (both VAMP from the outer acrosomal membrane, as well syntaxin from the apical sperm head plasma membrane) shared lateral redistribution properties, along with the ZP-binding protein complex and raft marker proteins. All of these were recovered after capacitation in detergent-resistant membrane preparations from sperm thought to represent membrane lipid rafts. We inferred that the capacitation-dependent formation of an aggregated lipid ordered apical ridge surface area in the sperm head plasma membrane was not only relevant for ZP-binding, but also for the ZP-induced acrosome reaction.     

Stimulation of phosphatidylserine biosynthesis and facilitation of UV-induced apoptosis in Chinese hamster ovary cells overexpressing phospholipid scramblase 1

Members of the phospholipid scramblase (PLSCR) family play active roles in altering lipid asymmetry at the plasma membrane including phosphatidylserine (PtdSer) exposure on the cell surface. To determine whether PtdSer biosynthesis and externalization are altered by PLSCR activities during apoptosis, Chinese hamster ovary K1 cell lines stably overexpressing PLSCR1 and PLSCR2 were established. PLSCR1 was localized on the plasma membrane, whereas PLSCR2 was predominantly in the nucleus. Cells overexpressing PLSCR1 showed suppressed growth, altered cell morphology, and higher basal levels of cell death. Following UV irradiation, these cells showed earlier and enhanced PtdSer exposure, increased caspase-3 activation, apoptotic nuclear changes, and PARP cleavage indicative of apoptosis. UV irradiation in cells overexpressing PLSCR1 led to a 4-fold stimulation of PtdSer synthesis (accompanied by increased movement of newly made PtdSer into microvesicles) relative to untreated PLSCR1 cells, whereas PtdSer formation in UV-irradiated vector control cells increased only by 2-fold. No differences in these responses were observed between PLSCR2-expressing cells and vector controls. PtdSer synthesis and its transbilayer movement stimulated by PLSCR1 overexpression were blocked by a caspase inhibitor along with progression of apoptosis. Thus, our studies showed that overexpression of PLSCR1 in Chinese hamster ovary K1 cells stimulated caspase-dependent PtdSer externalization and synthesis, implying an up-regulation of PtdSer formation in response to enhanced outward movement of this phospholipid to the cell surface during apoptosis. PLSCR1 also appears to influence progression of UV-induced apoptosis and could be a point of regulation or intervention during programmed cell death.     

Bicarbonate Induces Membrane Reorganization and CBR1 and TRPV1 Endocannabinoid Receptor Migration in Lipid Microdomains in Capacitating Boar Spermatozoa

Mammalian spermatozoa acquire full fertilizing ability only after a morphofunctional maturation called "capacitation." During this process the high level of bicarbonate present within the upper female genital tract or in culture medium induces a marked reorganization of sperm membranes characterized by a biphasic behavior: In a few minutes, it promotes membrane phospholipid scrambling preliminary to the apical translocation of sterol that, 2-4?h later, enables spermatozoa to recognize zona pellucida after albumin-mediated cholesterol extraction. In the present research it was demonstrated that spermatozoa incubated with bicarbonate in protein-free media underwent a marked reorganization of lipid microdomains present in a detergent-resistant membrane fraction (DRM) isolated by ultracentrifugation on sucrose density gradient. In fact, bicarbonate exposed sperm (ES) cells, compared with ejaculated spermatozoa (nonexposed sperm [nES] cells), displayed an increase in protein DRM content and, in particular, in Cav-1 and CD55, markers of caveolae and lipid rafts, as well in acrosin-2, a marker of the outer acrosomal membrane (OAM). Moreover, the amount of certain proteins involved in capacitation, such as the endocannabinoid system receptors cannabinoid receptor type 1 (CBR1) and transient receptor potential cation channel 1 (TRPV1), increased in DRM obtained from ES. These data allow us to hypothesize that sperm membrane reorganization takes place even in the absence of extracellular proteins; that not only the plasma membrane but also the OAM participate in this process; and that important molecules playing a key role in inside-out signaling, such as the endocannbinoid receptors TRPV1 and CBR1, are involved in this event, with potentially important consequences on sperm function.     

AIF and Scythe (Bat3) Regulate Phosphatidylserine Exposure and Macrophage Clearance of Cells Undergoing Fas (APO-1)-Mediated Apoptosis

Phosphatidylserine (PS) exposure on the cell surface has been considered a characteristic feature of apoptosis and serves as a molecular cue for engulfment of dying cells by phagocytes. However, the mechanism of PS exposure is still not fully elucidated. Here we show that the cytosolic release from mitochondria of apoptosis-inducing factor (AIF) is required for PS exposure during death receptor-induced apoptosis and for efficient clearance of cell corpses by primary human macrophages. Fas-triggered PS exposure was significantly reduced upon siRNA-mediated silencing of AIF expression and by inhibition of the cytosolic translocation of AIF. In addition, AIF localizes to the plasma membrane upon Fas ligation and promotes activation of phospholipid scrambling activity. Finally, cytosolic stabilization of AIF through interaction with Scythe is shown to be involved in apoptotic PS exposure. Taken together, our results suggest an essential role for AIF and its binding partner Scythe in the pathway leading to apoptotic corpse clearance.     

Plasma membrane phospholipid scramblase 1 promotes EGF-dependent activation of c-Src through the epidermal growth factor receptor

Phospholipid scramblase (PLSCR1) is a multiply palmitoylated, calcium-binding endofacial membrane protein proposed to mediate transbilayer movement of plasma membrane phospholipids. PLSCR1 is a component of membrane lipid rafts and has been shown to both physically and functionally interact with activated epidermal growth factor (EGF) receptors and other raft-associated cell surface receptors. Cell stimulation by EGF results in Tyr phosphorylation of PLSCR1, its association with both Shc and EGF receptors, and rapid cycling of PLSCR1 between plasma membrane and endosomal compartments. We now report evidence that upon EGF stimulation, PLSCR1 is phosphorylated by c-Src, within the tandem repeat sequence 68VYNQPVYNQP77. The in vivo interaction between PLSCR1 and Shc requires the Src-mediated phosphorylation on tyrosines 69 and 74. In in vitro pull down studies, phosphorylated PLSCR1 was found to bind directly to Shc through the phosphotyrosine binding domain. Consistent with the potential role of PLSCR1 in growth factor signaling pathways, granulocyte precursors derived from mice deficient in PLSCR1 show impaired proliferation and maturation under cytokine stimulation. Using PLSCR1-/- embryonic fibroblasts and kidney epithelial cells, we now demonstrate that deletion of PLSCR1 from the plasma membrane reduces the activation of c-Src by EGF, implying that PLSCR1 normally facilitates receptor-dependent activation of this kinase. We propose that PLSCR1, through its interaction with Shc, promotes Src kinase activation through the EGF receptor.     

The cholesterol content of the erythrocyte membrane is an important determinant of phosphatidylserine exposure

Maintenance of the asymmetric distribution of phospholipids across the plasma membrane is a prerequisite for the survival of erythrocytes. Various stimuli have been shown to induce scrambling of phospholipids and thereby exposure of phosphatidylserine (PS). In two types of patients, both with aberrant plasma cholesterol levels, we observed an aberrant PS exposure in erythrocytes upon stimulation. We investigated the effect of high and low levels of cholesterol on the ATP-dependent flippase, which maintains phospholipid asymmetry, and the ATP-independent scrambling activity, which breaks down phospholipid asymmetry. We analyzed erythrocytes of a patient with spur cell anemia, characterized by elevated plasma cholesterol, and the erythrocytes of Tangier disease patients with very low levels of plasma cholesterol. In normal erythrocytes, loaded with cholesterol or depleted of cholesterol in vitro, the same analyses were performed. Changes in the cholesterol/phospholipid ratio of erythrocytes had marked effects on PS exposure upon cell activation. Excess cholesterol profoundly inhibited PS exposure, whereas cholesterol depletion led to increased PS exposure. The activity of the ATP‐dependent flippase was not changed, suggesting a major influence of cholesterol on the outward translocation of PS. The effects of cholesterol were not accompanied by eminent changes in cytoskeletal and membrane proteins. These findings emphasize the importance of cholesterol exchange between circulating plasma and the erythrocyte membrane as determinant for phosphatidylserine exposure in erythrocytes.     

Phospholipid scramblases: an overview

Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic organisms. They are believed to be involved in destroying plasma membrane phospholipid asymmetry at critical cellular events like cell activation, injury and apoptosis. However, a detailed mechanism of phospholipid scrambling still awaits a proper understanding. The most studied member of this family, phospholipid scramblase 1 (PLSCR1) (a 37kDa protein), is involved in rapid Ca2+ dependent transbilayer redistribution of plasma membrane phospholipids. Recently the function of PLSCR1 as a phospholipids translocator has been challenged and evidences suggest that PLSCR1 acts as signaling molecule. It has been shown to be involved in protein phosphorylation and as a potential activator of genes in response to interferon and other cytokines. Interferon induced rapid biosynthesis of PLSCR1 targets some of the protein into the nucleus, where it binds to the promoter region of inositol 1,4,5-triphosphate (IP3) receptor type 1 (IP3R1) gene and induces its expression. Palmitoylation of PLSCR1 acts as a switch, controlling its localization either to the PM or inside the nucleus. In the present review, we discuss the current understanding of PLSCR1 in relation to its trafficking, localization and signaling functions.     

Capacitation-like changes in equine spermatozoa following cryopreservation

The primary objective of this study was to assess plasma membrane characteristics and activation of signal transduction pathways in equine spermatozoa during both in vitro capacitation and cryopreservation. Significant plasma membrane restructuring, as assessed by measurement of plasma membrane lipid disorder and phospholipid scrambling, was not observed until after cryopreservation and subsequent thawing (P < 0.05). Although in vitro capacitated cells also displayed increased plasma membrane lipid disorder and phospholipid scrambling (P < 0.05), it appeared that regulation of these events in in vitro capacitated versus cryopreserved equine spermatozoa was not identical. Addition of 5 microM staurosporine to the capacitation media reduced plasma membrane phospholipid scrambling (P < 0.05), but supplementation to the freezing extender prior to cryopreservation did not. Furthermore, progesterone was able to induce a greater degree of acrosomal exocytosis in in vitro capacitated versus frozen/thawed spermatozoa. Expression of phospholipid scramblase, a protein thought to be important in plasma membrane phospholipid scrambling, did not differ between treatments. Comparison of protein tyrosine phosphorylation patterns between in vitro capacitated and cryopreserved cells demonstrated a divergence in signal transduction. Cellular signaling in in vitro capacitated equine spermatozoa appeared to be in part dependent on activation of the cAMP/PKA pathway, whereas signaling in cryopreserved cells seemed to proceed predominantly through alternative pathways. Taken together, these data support the idea that capacitation and "cryocapacitation" are not equivalent processes.     

Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosis results from phospholipid scrambling and alters cell-surface morphology

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosylphosphorylcholine (C(6)-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca(2+) ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-beta-cyclodextrin, which is indicative of a loss of tight SM-cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C(6)- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.     

Role of cAMP-dependent protein kinase in the regulation of platelet procoagulant activity

The membrane microparticle (MP) formation and phosphatidylserine (PS) exposure evoked by platelet activation provide catalytic surfaces for thrombin generation. Several reports have indicated the effects of cAMP-elevating agents on agonist-induced MP formation and PS exposure; however, the mechanism still remains unclear. Here we show that inhibition of basal cyclic AMP-dependent protein kinase (PKA) activity incurred platelet MP formation and PS exposure. Pretreatment of platelets with cAMP-elevating agent, forskolin, abolished thrombin plus collagen-induced MP formation and PS exposure, and obviously decreased calcium ionophore-evoked MP shedding. Moreover, the inhibitory effects of forskolin on agonists-induced MP formation and PS exposure were reversed by the PKA inhibitor H89. PKA inhibitor-induced MP formation was dose-dependently inhibited by calpain inhibitor MDL28170, and forskolin abrogated thrombin plus collagen-induced calpain activation. In conclusion, PKA plays key roles in the regulation of platelet MP formation and PS exposure. PKA-mediated MP shedding is dependent on calpain activation.     

Kinases, phosphatases and proteases during sperm capacitation

Fertilization is the process by which male and female haploid gametes (sperm and egg) unite to produce a genetically distinct individual. In mammals, fertilization involves a number of sequential steps, including sperm migration through the female genital tract, sperm penetration through the cumulus mass, sperm adhesion and binding to the zona pellucida, acrosome exocytosis, sperm penetration through the zona and fusion of the sperm and egg plasma membranes. However, freshly ejaculated sperm are not capable of fertilizing an oocyte. They must first undergo a series of biochemical and physiological changes, collectively known as capacitation, before acquiring fertilizing capabilities. Several molecules are required for successful capacitation and in vitro fertilization; these include bicarbonate, serum albumin (normally bovine serum albumin, BSA) and Ca(2+). Bicarbonate activates the sperm protein soluble adenylyl cyclase (SACY), which results in increased levels of cAMP and cAMP-dependent protein kinase (PKA) activation. The response to bicarbonate is fast and cAMP levels increase within 60?s followed by an increase in PKA activity. Several studies with an anti-phospho-PKA substrate antibody have demonstrated a rapid increase in protein phosphorylation in human, mouse and boar sperm. The target proteins of PKA are not known and the precise role of BSA during capacitation is unclear. Most of the studies provide support for the idea that BSA acts by removing cholesterol from the sperm. The loss of cholesterol has been suggested to affect the bilayer of the sperm plasma membrane making it more fusogenic. The relationship between cholesterol loss and the activation of the cAMP/PKA pathway is also unclear. During early stages of capacitation, Ca(2+) might be involved in the stimulation of SACY, although definitive proof is lacking. Protein tyrosine phosphorylation is another landmark of capacitation but occurs during the late stages of capacitation on a different time-scale from cAMP/PKA activation. Additionally, the tyrosine kinases present in sperm are not well characterized. Although protein phosphorylation depends upon the balanced action of protein kinases and protein phosphatase, we have even less information regarding the role of protein phosphatases during sperm capacitation. Over the last few years, several reports have pointed out that the ubiquitin-proteasome system might play a role during sperm capacitation, acrosome reaction and/or sperm-egg fusion. In the present review, we summarize the information regarding the role of protein kinases, phosphatases and the proteasome during sperm capacitation. Where appropriate, we give examples of the way that these molecules interact and regulate each other's activities.     

Phosphatidylserine exposure during apoptosis is a cell-type-specific event and does not correlate with plasma membrane phospholipid scramblase expression

Phosphatidylserine (PS) exposure on the surface of cells has been considered a characteristic feature of apoptosis. However, we demonstrate herein that externalization of PS occurs in a cell-type-specific, albeit caspase-dependent, manner. Moreover, we could find no correlation in six different cell lines between the level of expression of the phospholipid (PL) scramblase and the capacity of these cells to externalize PS during apoptosis. Overexpression of PL scramblase in Raji cells, which exhibit low constitutive expression of this enzyme, by retroviral transduction of PL scramblase or treatment of the cells with interferon-alpha, failed to confer the capacity to expose PS in response to apoptotic stimuli. However, the lack of PS exposure in some cell types was not due to their inability to translocate PS molecules to the cell surface, since incubation with thiol reactive agents, such as N-ethylmaleimide, disulfiram and diamide, yielded rapid and pronounced PS exposure in all cell lines. These data suggest that plasma membrane PS exposure is not an obligatory component of the apoptotic phenotype, and that PL scramblase is not the sole determinant of PS externalization in apoptotic cells when this occurs.     

Phospholipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages

Phospholipid Scramblase 1 (PLSCR1) was initially characterized as a type II transmembrane protein involved in bilayer movements of phospholipids across the plasma membrane leading to the cell surface exposure of phosphatidylserine, but other cellular functions have been ascribed to this protein in signaling processes and in the nucleus. In the present study, expression and functions of PLSCR1 were explored in specialized phagocytic cells of the monocyte/macrophage lineage. The expression of PLSCR1 was found to be markedly increased in monocyte-derived macrophages compared to undifferentiated primary monocytes. Surprisingly, this 3-fold increase in PLSCR1 expression correlated with an apparent modification in the membrane topology of the protein at the cell surface of differentiated macrophages. While depletion of PLSCR1 in the monocytic THP-1 cell-line with specific shRNA did not inhibit the constitutive cell surface exposure of phosphatidylserine observed in differentiated macrophages, a net increase in the FcR-mediated phagocytic activity was measured in PLSCR1-depleted THP-1 cells and in bone marrow-derived macrophages from PLSCR1 knock-out mice. Reciprocally, phagocytosis was down-regulated in cells overexpressing PLSCR1. Since endogenous PLSCR1 was recruited both in phagocytic cups and in phagosomes, our results reveal a specific role for induced PLSCR1 expression in the modulation of the phagocytic process in differentiated macrophages.