Philippe F. Devaux

ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine.

ATP-binding-cassette transporter 1 (ABC1) has been implicated in processes related to membrane-lipid turnover. Here, using in vivo loss-of-function and in vitro gain-of-function models, we show that ABC1 promotes Ca2+-induced exposure of phosphatidylserine at the membrane, as determined by a prothrombinase assay, membrane microvesiculation and measurement of transbilayer redistribution of spin-labelled phospholipids. That ABC1 promotes engulfment of dead cells is shown by the impaired ability of ABC1-deficient macrophages to engulf apoptotic preys and by the acquisition of phagocytic behaviour by ABC1 transfectants. Release of membrane phospholipids and cholesterol to apo-AI, the protein core of the cholesterol-shuttling high-density lipoprotein (HDL) particle, is also ABC1-dependent. We propose that both the efficiency of apoptotic-cell engulfment and the efflux of cellular lipids depend on ABC1-induced perturbation of membrane phosphatidylserine turnover. Transient local exposure of anionic phospholipids in the outer membrane leaflet may be sufficient to alter the general properties of the membrane and thus influence discrete physiological functions.

Maintenance and consequences of membrane phospholipid asymmetry

The outer monolayer of animal plasma membranes is principally composed of choline-phospholipids while the amino-phospholipids reside in the inner monolayer. This anisotropic distribution is a steady state. The choline-lipids are submitted to a slow and passive transmembrane diffusion and the amino-lipids are inwardly transported by an ATP-dependent carrier, the amino-phospholipid translocase or ‘flippase’. The transport system has been characterized functionally and recently associated in red cells with a 110-kDa Mg-ATPase. Experiments indicate that the translocase can maintain by itself the amino-lipid asymmetry without requiring the help of cytoskeletal proteins. In the endoplasmic reticulum, phospholipids experience a facilitated diffusion involving a non-ATP-dependent ‘flippase’. A similar system exists in the liver canalicular plasma membrane. It has been demonstrated that the phosphatidylcholine secretion into the bile is under the control of the P-glycoprotein encoded by the mdr2 gene. The question arises whether the related protein from the mdr1 gene, which confers the multi-drug resistance to cells in which it is expressed, could also function as a lipid flippase. In response to some cellular events, such as blood platelet stimulation, the phospholipid asymmetry may suddenly collapse. The exact mechanism by which this randomization occurs is still unknown, but both a ‘scramblase’ protein and a minor lipid (PIP2) have been proposed as mediators of the event. Maintaining a plasma membrane asymmetry, even at the expense of energy consumption, is important for some cells: the appearance of phosphatidylserine into the outer membrane leaflet of blood cells generates a procoagulant surface catalysing the clot formation. It is also responsible for the recognition and phagocytosis of erythrocytes by macrophages. Finally, the amino-phospholipid translocase could play an important role in the control of membrane curvature, particularly in the initiation of vesicle formation.

Ion regulation of phosphatidylserine and phosphatidylethanolamine outside-inside translocation in human erythrocytes

In previous publications, we have shown, by using spin-labeled derivatives, that the translocation of phosphatidylserine and phosphatidylethanolamine from the outer to the inner monolayer of human erythrocyte membrane is a protein-mediated phenomenon, which requires hydrolisable Mg2+-ATP. The inhibition by intracellular Ca2+ (0.2 microM) or by extracellularly added vanadate (50 microM) was reported (Seigneuret, M. and Devaux, P.F. (1984) Proc. Natl. Acad. Sci. USA 81, 3751-3755; Zachowski, A., Favre, E., Cribier, S., Hervé, P. and Devaux, P.F. (1986) Biochemistry 25, 2585-2590). The present article gives further insight into the effects of intracellular and extracellular ions on the aminophospholipid translocation in human erythrocytes. By measuring the cell ATP concentration, we now show that the inhibitory effect of intracellular calcium on spin-labeled aminophospholipid translocation is partly due to the ATP depletion, which follows the increased consumption by the calcium pump. However, a direct inhibitory effect of cytosolic Ca2+ on the aminophospholipid translocase can be demonstrated by measuring the initial rate of aminophospholipid translocation in the presence of variable amounts of intracellular calcium, at fixed ATP concentrations. Moreover, the transmembrane equilibrium distribution of phosphatidylserine and phosphatidylethanolamine are affected differently by Ca2+: when cytosolic Ca2+ concentration is increased, alteration of phosphatidylethanolamine distribution begins as soon as the inward translocation is affected by Ca2+ (approx. 50 nM), whereas phosphatidylserine distribution remains unchanged within a large inhibitory range of cytosolic Ca2+ concentrations and decreases above 0.2 microM of free Ca2+ within the cytosol. Decrease of the intracellular Mg2+ concentration below its physiological value (approx. 2 mM) results in the inhibition of aminophospholipid inward transport, whereas increase of Mg2+ concentration does not modify this transport. If Mn2+ is substituted for Mg2+, part of the aminophospholipid translocation is maintained, whereas if Co2+ is substituted for Mg2+, the rapid translocation is completely abolished. Concentrations as high as a millimolar of extracellular Ca2+, Mg2+ or Mn2+ have no effect on the aminophospholipid translocation. The less usual cations Cr3+, Fe2+, Cu2+, Sn2+ and Eu3+ are also uneffective. With extracellular Ni2+ or Co2+, some inhibition can be observed, half inhibition by Ni2+ corresponding to 500 microM. Vanadyl (VO2+), on the other hand, is a potent inhibitor of the aminophospholipid translocation when applied on the extracellular surface, half-inhibition being reached around 30 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Translational Diffusion of Globular Proteins in the Cytoplasm of Cultured Muscle Cells

Modulated fringe pattern photobleaching (MFPP) was used to measure the translational diffusion of microinjected fluorescein isothiocyanate (FITC)-labeled proteins of different sizes in the cytoplasm of cultured muscle cells. This technique, which is an extension of the classical fluorescence recovery after photobleaching (FRAP) technique, allows the measurement of the translational diffusion of macromolecules over several microns. Proteins used had molecular masses between 21 and 540 kDa. The results clearly indicated that the diffusivity of the various proteins is a decreasing function of their hydrodynamic radius. This decrease is more rapid with globular proteins than with FITC-labeled dextrans (, Biophys. J. 70:2327-2332), most likely because, unlike globular proteins, dextrans are randomly coiled macromolecules with a flexible structure. These data do not exclude the possibility of a rapid diffusion over a short distance, unobservable with our experimental set-up, which would take place within the first milliseconds after bleaching and would correspond to the diffusion in restricted domains followed by impeded diffusion provoked by the network of microtubules, microfilaments, and intermediate filaments. Thus our results may complement rather than contradict those of Verkman and collaborators (, J. Cell Biol. 138:1-12). The biological consequence of the size-dependent restriction of the mobility of proteins in the cell cytoplasm is that the formation of intracellular complexes with other proteins considerably reduces their mobility.

Transmembrane distribution of sterol in the human erythrocyte

The transbilayer cholesterol distribution of human erythrocytes was examined by two independent techniques, quenching of dehydroergosterol fluorescence and fluorescence photobleaching of NBD-cholesterol. Dehydroergosterol in conjunction with leaflet selective quenching showed that, at equilibrium, 75% of the sterol was localized to the inner leaflet of resealed erythrocyte ghosts. NBD-cholesterol and fluorescence photobleaching displayed two diffusion values in both resealed ghosts and intact erythrocytes. The fractional contribution of the fast and slow diffusion constants of NBD-labelled cholesterol represent its inner and outer leaflet distribution. At room temperature the plasma membrane inner leaflet of erythrocyte ghosts as well as intact erythrocytes cells contained 78% of the plasma membrane sterol. The erythrocyte membrane transbilayer distribution of sterol was independent of temperature. In conclusion, dehydroergosterol and NBD-cholesterol data are consistent with an enrichment of cholesterol in the inner leaflet of the human erythrocyte.

How lipid flippases can modulate membrane structure

Phospholipid flippases, are proteins able to translocate phospholipids from one side of a membrane to the other even against a gradient of concentration and thereby able to establish, or annihilate, a transmembrane asymmetrical lipid distribution. This lipid shuttling forms new membrane structures, in particular vesicles, which are associated with diverse physiological functions in eukaryotic cells such as lipid and protein traffic via vesicles between organelles or towards the plasma membrane, and the stimulation of fluid phase endocytosis. The transfer of lipids is also responsible for the triggering of membrane associated events such as blood coagulation, the recognition and elimination of apoptotic or aged cells, and the regulation of phosphatidylserine dependent enzymes. Exposure of new lipid-head groups on a membrane leaflet by rapid flip-flop can serve as a specific signal and, upon recognition, can be the cause of physiological modifications. Membrane bending is one of the mechanisms by which such activities can be triggered. We show that the lateral membrane tension is an important physical factor for the regulation of the size of the membrane invaginations. Finally, we suggest in this review that this diversity of functions benefits from the diversity of the lipids existing in a cell and the ability of proteins to recognize specific messenger molecules.

Partial purification and characterization of the human erythrocyte Mg2+ -ATPase A candidate aminophospholipid translocase

A Mg2+‐ATPase‐enriched fraction was obtained from solubilized human erythrocyte membranes by ammonium sulphate precipitation and anion‐exchange chromatography. The solubilized enzyme, of apparent molecular weight 120 kDa, requires phosphatidylserine to be fully active. Phosphatidylethanolamine but not other anionic phospholipids can only partially restore the activity. The Mg‐ATPase has a low affinity for Mg2+‐ATP and is inhibited by fluoride, vanadate, vanadyl and calcium ions. From these characteristics, we infer that this Mg2+‐ATPase is the same protein as the aminophospholipid translocase which regulates the membrane phospholipid transverse distribution in human erythrocytes by actively transporting aminophospholipids from the outer to the inner monolayer.

Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and 31P-nuclear magnetic resonance

Abstract It has been reported that repetitive freeze-thaw cycles of aqueous suspensions of dioleoylphosphatidylcholine form vesicles with a diameter smaller than 200 nm. We have applied the same treatment to a series of phospholipid suspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoylphosphatidic acid (DOPC/DOPA) mixtures. Freeze-fracture electron microscopy revealed that these unsaturated lipids form unilamellar vesicles after 10 cycles of freeze-thawing. Both electron microscopy and broad-band 31P NMR spectra indicated a disparity of the vesicle sizes with a highest frequency for small unilamellar vesicles (diameters ≤30 nm) and a population of larger vesicles with a frequency decreasing exponentially as the diameter increases. From 31P NMR investigations we inferred that the average diameter of DOPC/DOPA vesicles calculated on the basis of an exponential size distribution was of the order of 100 nm after 10 freeze-thaw cycles and only 60 nm after 50 cycles. Fragmentation by repeated freeze-thawing does not have the same efficiency for all lipid mixtures. As found already by others, fragmentation into small vesicles requires the presence of salt and does not take place in pure water. Repetitive freeze-thawing is also efficient to fragment large unilamellar vesicles obtained by filtration. If applied to sonicated DOPC vesicles, freeze-thawing treatment causes fusion of sonicated unilamellar vesicles into larger vesicles only in pure water. These experiments show the usefulness of NMR as a complementary technique to electron microscopy for size determination of lipid vesicles. The applicability of the freeze-thaw technique to different lipid mixtures confirms that this procedure is a simple way to obtain unilamellar vesicles.

Transmembrane diffusion of fluorescent phospholipids in human erythrocytes

The outside-inside passage and transmembrane equilibrium distribution of several amphiphilic fluorescent phospholipids were examined in human erythrocytes. The results were compared with previous kinetic data obtained with spin-labeled phospholipids and with the equilibrium distribution of endogenous lipids in erythrocytes. When a nitro benzoxadiazole (NBD) was at the terminal position of a 6 carbon beta-chain, the outside-inside diffusion of the fluorescent phosphatidylserine (PS) analogue was slower, and the plateau lower than with long chain radioactive PS or spin-labeled PS. The corresponding phosphatidylethanolamine (PE) did not flip nor did the phosphatidylcholine (PC) analogue. With a NBD at the 12th carbon of a 18C alpha-chain, the amino-derivatives behaved more like endogenous PS and PE, i.e. they accumulated rapidly on the inner monolayer; however, the phosphatidylcholine analogue reached a plateau corresponding to 50% inside within 2 h at 37 degrees C, indicative of an abnormal rapid diffusion. In the latter case, changing the beta-chain from four to eight carbons had no influence on this rapid diffusion. We conclude that when the NBD is close to the glycerol moiety, it diminishes the affinity of the aminophospholipids for the aminophospholipid translocase. When it is close to the methyl terminal of an acyl chain, there is an acceleration of the spontaneous flip-flop. Presumably the polarity of the NBD is responsible for an unconventional orientation of the flexible acyl chain, thereby causing the transmembrane destabilization of the phospholipid. Overall these results illustrate the respective roles of spontaneous diffusion and translocase activity on transmembrane equilibrium distribution of phospholipids. They also show that NBD derivatives should be used cautiously as indicators of endogenous phospholipids.

Alteration of the aminophospholipid translocase activity during in vivo and artificial aging of human erythrocytes

Human erythrocytes were separated into three density groups representing different age groups. Phospholipid outside-inside translocation rates and equilibrium distribution were determined in each group with spin-labeled phosphatidylserine (PS*), phosphatidylethanolamine (PE*), and phosphatidylcholine (PC*), at 37 degrees C and 4 degrees C. At both temperatures, the initial velocity of aminolipid translocation was reduced in the more dense (older) cells. The equilibrium distribution was not significantly modified for PS*, but a larger fraction of PE* remained on the outer monolayer of the more dense cells. PC* transmembrane diffusion was identical in the three fractions. Cytosolic ATP, which is required for aminophospholipid translocation, was not responsible for the variability of the density separated cells since ATP enrichment did not cancel the differences between top and bottom fractions, although it equalized the ATP concentration of the various fractions. Variations in the level of intracellular Ca2+ could also be excluded. Thus, the enzyme aminophospholipid translocase seemed to be directly altered in aged cells, possibly due to oxidation caused by lipid peroxidation products. Experiments with malonyldialdehyde or H2O2 treated cells confirmed this interpretation and suggest that defects in endogenous lipid asymmetry observed in aged human erythrocytes may be due to altered activity of the translocase.