Erythrocyte morphology reflects the transbilayer distribution of incorporated phospholipids.

Abstract

Abstract. 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 (t~/2 ~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, DLPCinduced 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. Cells 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.

Phosphatidylethanolamine Binding Is a Conserved Feature of Cyclotide-Membrane Interactions

“…The relative hemolytic activity correlates with the relative leakage efficiency trend in POPC/POPE/Chol/SM model membranes (Fig. 10), consistent with the ability of cyclotides to insert in the hydrophobic core and disrupt the RBC membrane, which includes phosphatidylcholine phospholipids, Chol, SM, and a small amount of PE-phospholipids in the outer leaflet (41,55).…”

“…Phospholipids with a PG headgroup are negatively charged and were included to evaluate the effects of charge. The effects of PE-phospholipids, which are also common in eukaryotic membranes (41) and previously shown to be required for membrane targeting by kB1 (44), we assumed that the addition of 10% POPE to the POPC/Chol/SM mixture would not change the overall fluidity of the lipid bilayer, i.e. this lipid system will be in a homogeneous liquid-ordered phase.…”

“…PC-containing phospholipids are the most common form of phospholipid in eukaryotic membranes (41). Thus, membranes composed of POPC, which forms zwitterionic bilayers with liquid disordered phase properties at 25°C, along with model membranes of increased complexity, including POPC/POPG (80:20 molar ratio) and POPC/Chol/SM (27:33:40), were included.…”

Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement

“…If the analogues are partially water-soluble, their spontaneous insertion in the membrane corresponds to a partition between the aqueous buffer and the hydrophobic bilayer. Their distribution between the two monolayers with time is determined either by selective destruction of the probe in the outer leaflet or by selective extraction of the analogue from the external monolayer using Kornberg and McConnell, 1971Yuann and Morse, 1991Cabral et al, 1987Homan and Pownall, 1988Rothman and Dawidowicz, 1975de Kruijff et al, 1978Eastman et al, 1991Rothman et al, 1976Daleke and Huestis, 1985Middelkoop et al, 1986Bergmann et al, 1984Bitbol and Devaux, 1988Morrot et al, 1989Crain and Zilversmit, 1980Sandra and Pagano, 1978Lipka et al, 1991Barsukov et al, 1986 back-exchange into acceptor structures such as liposomes or bovine serum albumin (Bergmann et al, 1984;Biitikofer et al, 1990;Calvez et al, 1988;Connor et al, 1990;Daleke and Huestis, 1989). In the case of erythrocytes, whose shape is sensitive to the relative amount of lipid in each of the two membrane leaflets according to the bilayer-couple hypothesis (Deuticke, 1968;Sheetz and Singer, 1974), these changes ( Figure 3) have also been used to follow the transmembrane motion of lipids (Daleke and Huestis, 1985;.…”

Effect of benzyl alcohol on phospholipid transverse mobility in human erythrocyte membrane

“…Fixed cells were washed twice in NaC1/Pi and analysed by light microscopy. Erythrocytes were assigned previously determined scores [17]. The average score for a field of 200 cells was used for the morphological index.…”