The involvement of the cytoskeleton in regulating IP3 receptor-mediated internal Ca2+ release in human blood platelets.

In this study we have used saponin to permeabilize platelet membranes in order to test directly the involvement of IP₃ in regulating internal Ca²⁺ release, and to measure IP₃ binding to its receptor. Our results indicate that platelet vesicles release Ca²⁺ as early as 3 seconds after IP₃ addition. Using [³H]IP₃, we have found that platelets contain a single class of high affinity IP₃ binding sites with a Kd of ～0.20 (± 0.01) nM. Immuno-blotting shows that platelets contain a 260 kDa polypeptide which shares immunological cross reactivity with brain IP₃ receptor. Immunofluorescence staining data indicate that the IP₃ receptor is preferentially located at the periphery of the platelet plasma membrane. Most importantly, both IP₃ binding and IP₃-induced Ca²⁺ release activities are significantly inhibited by cytochalasin D (a microfilament inhibitor) and colchicine (a microtubule inhibitor). These findings suggest that the cytoskeleton is involved in the regulation of IP₃ binding and IP₃ receptor-mediated Ca²⁺ release during platelet activation.     

Possible involvement of cytoskeleton in collagen-stimulated activation of phospholipases in human platelets

The action of phospholipases A2 and C in the course of collagen-stimulated platelet activation and the effect of cytochalasins on the responses were studied. Stimulation of human platelets with collagen was accompanied by aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release. However, in the presence of a cyclooxygenase inhibitor or a thromboxane A2 (TXA2) receptor antagonist, collagen induced only weak arachidonic acid release and weak inositol phosphate formation. The TXA2 mimetic agonist U46619 induced all the responses except for arachidonic acid release, which was induced by synergistic action of collagen and U46619. The result that U46619 did not induce arachidonic acid release despite the activation of phospholipase C suggested that arachidonic acid was not released via phospholipase C but by phospholipase A2. These findings suggested that collagen initially induced weak activation of phospholipases A2 and C and that further activation of phospholipase C as well as Ca2+ mobilization and aggregation were induced by TXA2, whereas further activation of phospholipase A2 required the synergistic action of collagen and TXA2. Platelets pretreated with cytochalasins did not respond to collagen. Further analysis revealed that the initial activation of phospholipases A2 and C was specifically inhibited by cytochalasins, but the responses induced by U46619 or a synergistic action of collagen and U46619 were not inhibited. Therefore, we proposed that interaction of collagen receptor with actin filaments might have some roles in the collagen-induced initial activation of phospholipases.     

Rapid transbilayer movement of phospholipids induced by an asymmetrical perturbation of the bilayer

Phospholipase D is used to convert egg phosphatidylcholine to phosphatidic acid in unilamellar vesicles. The transbilayer distribution of both lipids is determined by 31P NMR using paramagnetic ions. Phosphatidic acid formed in the outer monolayer is translocated to the inner monolayer with a halftime of 30-40 min or less. This is accompanied by an equally fast movement of part of the phosphatidylcholine from the inner to the outer monolayer. During these fast transbilayer movements the barrier properties of the vesicle bilayer are maintained.     

Interaction between phosphatidylserine and the isolated cytoskeleton of human blood platelets

Binding experiments were performed to demonstrate a direct interaction between cytoskeletons from human blood platelets and phosphatidylserine. A centrifugation technique using radiolabeled phosphatidylserine-vesicles and Triton X-100 insoluble residues from unstimulated human platelets was used to assess the binding. Interaction between cytoskeleton and phospholipid is demonstrated to be specific for phosphatidylserine. No binding was observed for phosphatidylcholine. The binding of phosphatidylserine was saturable and dependent on the concentration of cytoskeleton used. The interaction between phosphatidylserine and the cytoskeleton appeared to be completely reversible. The existence of a reversible and specific interaction between phosphatidylserine and the cytoskeleton of unstimulated platelets would suggest a role for the cytoskeleton in the maintenance of the asymmetric distribution of this lipid in the plasma membrane. We have previously shown (Comfurius et al. (1985) Biochim. Biophys. Acta 815, 143-148) that in activated platelets a strong correlation exists between degradation of platelet cytoskeletal proteins by the endogenous calcium-dependent proteinase (calpain) and exposure of phosphatidylserine at their outer surface. Nevertheless, hydrolysis of the isolated cytoskeleton by calpain did not result in a change in the parameters of the binding between phosphatidylserine and cytoskeleton. Also, sulfhydryl oxidation of the cytoskeleton by diamide did not affect its binding properties for phosphatidylserine, in spite of the fact that diamide treatment of platelets results in exposure of phosphatidylserine at the outer surface. Exposition of phosphatidylserine upon activation of platelets cannot be directly ascribed to a change in affinity or number of binding sites of the modified cytoskeleton as measured in model systems. However, it cannot be excluded that topological rearrangements of the cytoskeleton as occur within the cell during platelet activation lead to a decreased contact between cytoskeleton and lipid, irrespective of the binding parameters.     

The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids

Lactococcus lactis possesses an ATP-binding cassette transporter, LmrA, which is a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein, and is able to transport a broad range of structurally unrelated amphiphilic drugs. A histidine tag was introduced at the N-terminus of LmrA to facilitate purification by nickel affinity chromatography. The histidine-tagged protein was overexpressed in L. lactis using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter. This system allowed us to get functional overexpression of LmrA up to a level of 30% of total membrane protein. For reconstitution, LmrA was solubilized with dodecylmaltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecylmaltoside-destabilized, preformed liposomes prepared from L. lactis phospholipids. The detergent was removed by adsorption onto polystyrene beads. The LmrA protein was reconstituted in a functional form, and mediated the ATP-dependent transport of the fluorescent substrate Hoechst-33342 into the proteoliposomes. Interestingly, reconstituted LmrA also catalyzed the ATP-dependent transport of fluorescent phosphatidylethanolamine, but not of fluorescent phosphatidylcholine. These data demonstrate that LmrA activity is independent of accessory proteins and support the notion that LmrA may be involved in the transport of specific lipids or lipid-linked precursors in L. lactis.     

Mathematical modelling of lipid transbilayer movement in the human erythrocyte plasma membrane

A model is presented to simulate transverse lipid movement in the human erythrocyte membrane. The model is based on a system of differential equations describing the time-dependence of phospholipid redistribution and the steady state distribution between the inner and outer membrane monolayer. It takes into account several mechanisms of translocation: (i) ATP-dependent transport via the aminophospholipid translocase; (ii) protein-mediated facilitated and (iii) carrier independent transbilayer diffusion. A reasonable modelling of the known lipid asymmetry could only be achieved by introducing mechanism (iii). We have called this pathway the compensatory flux, which is proportional to the gradient of phospholipids between both membrane leaflets. Using realistic model parameters, the model allows the calculation of the transbilayer motion and distribution of endogenous phospholipids of the human erythrocyte membrane for several biologically relevant conditions. Moreover, the model can also be applied to experiments usually performed to assess phospholipid redistribution in biological membranes. Thus, it is possible to simulate transbilayer motion of exogenously added phospholipid analogues in erythrocyte membranes. Those experiments have been carried out here in parallel using spin labeled lipid analogues. The general application of this model to other membrane systems is outlined.Abbreviations PBS phosphate buffered saline - DFP diisopropyl fluorophosphate - ESR electron spin resonance - RBC red blood cells - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - SM sphingomyelin - (0,2)PC 1-palmitoyl-2(4doxylpentanoyl)-PC - (0,2)PE 1-palmitoyl-2(4-doxylpentanoyl)-PE - (0,2) PS 1-palmitoyl-2(4-doxylpentanoyl)-PS     

The transbilayer movement of phosphatidylcholine in vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane

Vesicles have been prepared from 18 : 1_c/18 : 1_c-phosphatidylcholine with or without purified glycophorin or partially purified band 3 (obtained by organomercurial gel chromatography). The vesicles have been characterized by freeze-fracture electron microscopy, binding studies to DEAE-cellulose, ³¹P-NMR and K⁺ trap measurements. Pools of phosphatidylcholine available for exchange have been investigated using phosphatidylcholine exchange protein from bovine liver. The protein-containing vesicles both exhibit exchangeable pools larger than the fraction of phosphatidylcholine in the outer monolayer, whereas in the protein-free vesicles the exchangeable pool is consistent with the outer monolayer. The results indicate that both glycophorin and the partially purified band 3 preparation enhance the transbilayer movement of phosphatidylcholine.     

Rapid transbilayer movement of ceramides in phospholipid vesicles and in human erythrocytes

The transbilayer diffusion of unlabeled ceramides with different acyl chains (C6-Cer, C10-Cer, and C16-Cer) was investigated in giant unilamellar vesicles (GUVs) and in human erythrocytes. Incorporation of a very small percentage of ceramides (approximately 0.1% of total lipids) to the external leaflet of egg phosphatidylcholine GUVs suffices to trigger a shape change from prolate to pear shape vesicle. By observing the reversibility of this shape change the transmembrane diffusion of lipids was inferred. We found a half-time for unlabeled ceramide flip-flop below 1 min at 37 degrees C. The rapid diffusion of ceramides in a phosphatidylcholine bilayer was confirmed by flip-flop experiments with a spin-labeled ceramide analogue incorporated into large unilamellar vesicles. Shape change experiments were also carried out with human erythrocytes to determine the trans-membrane diffusion of unlabeled ceramides into a biological membrane. Addition of exogenous ceramides to the external leaflet of human erythrocytes did not trigger echinocyte formation immediately as one would anticipate from an asymmetrical accumulation of new amphiphiles in the outer leaflet but only after approximately 15 min of incubation at 20 degrees C in the presence of an excess of ceramide. We interpret these data as being indicative of a rapid ceramide equilibration between both erythrocyte leaflets as indicated also by electron spin resonance spectroscopy with a spin-labeled ceramide. The late appearance of echinocytes could reveal a progressive trapping of a fraction of the ceramide molecules in the outer erythrocytes leaflet. Thus, we cannot exclude the trapping of ceramides into plasma membrane domains.     

Rapid transbilayer movement of spin-labeled steroids in human erythrocytes and in liposomes

The transbilayer movement and distribution of spin-labeled analogs of the steroids androstane (SLA) and cholestane (SLC) were investigated in the human erythrocyte and in liposomes. Membranes were labeled with SLA or SLC, and the analogs in the outer leaflet were selectively reduced at 4C using 6-O-phenylascorbic acid. As shown previously, 6-O-phenylascorbic acid reduces rapidly nitroxides exposed on the outer leaflet, but its permeation of membranes is comparatively slow and thus does not interfere with the assay. From the reduction kinetics, we infer that transbilayer movement of SLA in erythrocytes is rapid at 4C with a half-time of approximately 4.3 min and that the probe distributes almost symmetrically between both halves of the plasma membrane. We have no indication that a protein-mediated transport is involved in the rapid transbilayer movement of SLA because 1) pretreatment of erythrocytes with N-ethyl maleimide affected neither flip-flop nor transbilayer distribution of SLA and 2) flip-flop of SLA was also rapid in pure lipid membranes. The transbilayer dynamics of SLC in erythrocyte membranes could not be resolved by our assay. Thus, the rate of SLC flip-flop must be on the order of, or even faster than, that of probe reduction rate on the exoplasmic leaflet (half-time approximately 0.5 min). The results are discussed with regard to the transbilayer dynamics of cholesterol.     

Erythrocyte morphology reflects the transbilayer distribution of incorporated phospholipids

The transbilayer distribution of exogenous phospholipids incorporated into human erythrocytes is monitored through cell morphology changes and by the extraction of incorporated 14C-labeled lipids. Dilauroylphosphatidylserine (DLPS) and dilauroylphosphatidylcholine (DLPC) transfer spontaneously from sonicated unilamellar vesicles to erythrocytes, inducing a discocyte-to-echinocyte shape change within 5 min. DLPC-induced echinocytes revert slowly (t1/2 approximately 8 h) to discocytes, but DLPS-treated cells revert rapidly (10-20 min) to discocytes and then become invaginate stomatocytes. The second phase of the phosphatidylserine (PS)-induced shape change, conversion of echinocytes to stomatocytes, can be inhibited by blocking cell protein sulfhydryl groups or by depleting intracellular ATP or magnesium (Daleke, D. L., and W. H. Huestis. 1985. Biochemistry. 24:5406-5416). These cell shape changes are consistent with incorporation of phosphatidylcholine (PC) and PS into the membrane outer monolayer followed by selective and energy-dependent translocation of PS to the membrane inner monolayer. This hypothesis is explored by correlating cell shape with the fraction of the exogenous lipid accessible to extraction into phospholipid vesicles. Upon exposure to recipient vesicles, DLPC-induced echinocytes revert to discoid forms within 5 min, concomitant with the removal of most (88%) of the radiolabeled lipid. On further incubation, 97% of the foreign PC transfers to recipient vesicles. Treatment of DLPS-induced stomatocytes with acceptor vesicles extracts foreign PS only partially (22%) and does not affect cell shape significantly. Cell treated with inhibitors of aminophospholipid translocation (sulfhydryl blockers or intracellular magnesium depletion) and then incubated with either DLPS or DLPC become echinocytic and do not revert to discocytic or stomatocytic shape for many hours. On treatment with recipient vesicles, these echinocytes revert to discocytes in both cases, with concomitant extraction of 88-99% of radiolabeled PC and 86-97% of radiolabeled PS. The accessibility of exogenous lipids to extraction is uniformly consistent with the transbilayer lipid distribution inferred from cell shape changes, indicating that red cell morphology is an accurate and sensitive reporter of the transbilayer partitioning of incorporated exogenous phospholipids.     

The transbilayer distribution of phospholipids in disc membranes is a dynamic equilibrium evidence for rapid flip and flop movement.

We studied the transbilayer redistribution of phospholipids in bovine rod outer segment membranes on thoroughly washed, Ficoll-floated osmotically intact disc vesicles; freshly prepared membranes separated from the disc stack by osmotic shock; and intact disc stacks with a permeabilized plasma membrane (A-discs, B-discs C-discs, respectively). In all cases, spin-labelled phospholipid analogues (SL-PL) with choline, serine and ethanolamine head groups (PtdCho, PtdSer and PtdEtn, respectively) were taken up into the outer leaflet of the membranes by > 90% and within less than 30 s after SL-PL addition, as deduced from the disappearance of spin-label from the suspension medium and from the specific ESR spectrum of membrane-associated spin-label. Using BSA extraction, the amount of SL-PL in the outer leaflet of the bilayer was determined. It decreased with a mean half-time of < 5 min at 25 degrees C, indicating rapid redistribution of all spin-labelled phospholipids into the inner leaflet of the disc membranes. After 1 h, PtdCho and PtdEtn were distributed almost symmetrically, whereas PtdSer was 35 : 65% (in/out). Using subsequent incubation with BSA, the outward movement (flop) of the analogues was observed directly, demonstrating that inward and outward movements proceed in thermodynamic equilibrium. No effect of N-ethylmaleimide or ATP on the redistribution could be measured, which makes it unlikely that energy-consuming translocase or flippase processes are involved in the redistribution in the dark. We reason that the solubilization zone around the photoreceptor rhodopsin may be the locus of rapid redistribution of the highly unsaturated disc phospholipid.     

Ca2+ induces transbilayer redistribution of all major phospholipids in human erythrocytes.

Elevating cytoplasmic Ca2+ levels in erythrocytes activates a pathway for transbilayer diffusion of plasma membrane phospholipids. The use of spin-labeled and fluorescent phospholipid analogues revealed that the pathway permits diffusion of all the major classes of phospholipids and does not distinguish between the two types of probes. Diffusion was bidirectional, began immediately upon elevation of cytoplasmic [Ca2+] above 50-100 microM, persisted as long as the [Ca2+] remained elevated, and disappeared promptly when Ca2+ levels fell. Diffusion was unaffected by conditions which suppress shedding of vesicles, discounting this event as a requisite for phospholipid reorientation induced by Ca2+.     

Glycophorin facilitates the transbilayer movement of phosphatidylcholine in vesicles

The rate of transbilayer movement of dioleoylphosphatidylcholine in sonicated lipid vesicles is enhanced by at least two orders of magnitude upon incorporation of glycophorin in the bilayer.     

Lipid traffic: the ABC of transbilayer movement

Membrane lipids do not spontaneously exchange between the two leaflets of lipid bilayers because the polar headgroups cannot cross the hydrophobic membrane interior. Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other. In addition, cellular membranes contain proteins that facilitate a passive equilibration of lipids between the two membrane halves. In recent years, a growing number of proteins have been put forward as lipid translocators or facilitators. Unexpectedly, some of these appear to be required for efficient translocation of lipids lacking bulky headgroups, like cholesterol and fatty acids. The candidate lipid translocators identified so far belong to large protein families whose other members include pumps for amphiphilic molecules like bile salts and drugs.     

The rapid transbilayer movement of thiocholesterol in small unilamellar phospholipid vesicles

Cholesterol is a major component of biological membranes, yet there is very little information concerning its distribution across the membrane. Recent experiments in our laboratory, using cholesterol oxidase, have demonstrated that cholesterol can undergo a rapid transbilayer movement in lecithin-cholesterol vesicles in a half-time of 1 min or less at 37°C. In order to support this conclusion, we have sought other approaches to the measurement of this process. We now report our finding that the transbilayer movement of thiocholesterol in phospholipid vesicles occurs in a half-time of 1 min or less at 20°C.     

The interfacial conformation and transbilayer movement of diacylglycerols in phospholipid bilayers

The interaction of diacylglycerols, primarily 1,2-dilauroyl-sn-glycerol (1,2-DLG), with egg phosphatidylcholine (PC) bilayers was studied by NMR spectroscopy and other physical techniques. In the low proportions used (less than or equal to 20 mol % with respect to total lipid), 1,2-DLG formed bilayers with PC with no hexagonal phase separation, as assessed by light, polarizing and electron microscopy, and 31P and 13C NMR spectroscopy. The 13C-carbonyl chemical shift of 90% [13C]carbonyl 1,2-DLG was monitored in small unilamellar vesicles as a function of relative DLG content (1.5-20%) and temperature (10-55 degrees C). The chemically inequivalent sn-1 and sn-2 carbonyls gave a single, narrow resonance in vesicles, in contrast to neat 1,2-DLG and 1,2-DLG in organic solvents, whose spectra showed two well-separated carbonyl resonances. The chemical shift of 1,2-DLG in PC shows that the carbonyl groups are proximal to the aqueous interface, necessitating orientation of the DLG molecule along the normal to the bilayer. Both carbonyl groups are H-bonded to H2O, but the secondary ester (sn-2) carbonyl is relatively more hydrated than the primary ester (sn-1) carbonyl. The 13C-carbonyl chemical shift data further suggest that the interfacial conformation resembles that of crystalline and liquid crystalline lamellar 1,2-dilauroyl-sn-glycero-3-phosphatidylethanolamine and certain PCs, in which the glycerol backbone is perpendicular to the bilayer plane. This conformation is different from that of crystalline 1,2-dilauroyl-sn-glycerol, in which the glycerol backbone is parallel to the bilayer plane. Between 1.5 and 8% DLG in vesicles, the chemical shift of the 1,2-DLG carbonyl at a given temperature was constant. However, above 8% DLG the chemical shift at each temperature increased with increasing DLG concentration, suggesting increased hydration at higher DLG content. At low temperatures 13C NMR spectra of vesicles with the highest proportions of 1,2-DLG studied (15 and 20%) showed two DLG carbonyl resonances, which most likely represent 1,2-DLG on outer and inner leaflets of the vesicle bilayer. The two peaks collapsed into a single resonance by 38 degrees C, at which temperature the two environments equilibrate with a rate constant of approximately 60 s-1 (t1/2 approximately 10 ms). Thus, transbilayer movement of DLG is extremely fast compared with phospholipids. In vesicles the 1,3-isomer of DLG exhibited a narrow carbonyl peak slightly downfield from that of 1,2-DLG. Acyl chain migration from 1,2-DLG to 1,3-DLG was monitored directly in the vesicle by time-dependent NMR measurements.