Paulette Hervé

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.

Investigation on lipid asymmetry using lipid probes Comparison between spin-labeled lipids and fluorescent lipids

Synthetic lipids with a nitroxide or a fluorescent probe have been extensively used during the last 30 years to determine the transmembrane diffusion of phospholipids in artificial or biological membranes. However, the relevance of data obtained with these modified lipids has sometimes been questioned. Beside possible artefacts introduced by the reporter probe, synthetic lipids used in cells often contain a short fatty acid chain in the sn-2 position, which gives them higher water solubility than naturally occurring lipids. In the present review, we have attempted to give a critical appraisal. Main strategies are recalled and important discoveries obtained with lipid probes on transmembrane lipid traffic in eukaryotic cells are briefly summarized. Examples of artefacts caused by lipid probes are given. Comparisons between data obtained by different techniques such as ESR and fluorescence allow us to emphasize the complementary character of the two approaches and more generally show the necessity to use several probes before drawing conclusions concerning endogenous lipids. In spite of these pitfalls, overall, lipid probes have provided a wealth of useful information that, to date, cannot be obtained with unlabeled lipids.

Transmembrane redistribution of phospholipids of the human red cell membrane during hypotonic hemolysis.

The transmembrane distribution of spin-labeled phospholipids was measured in human erythrocytes before and after hypotonic hemolysis by electron paramagnetic resonance. With a first series of partially water soluble probes a complete randomization of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin analogues was achieved when cells were resealed in the absence of Mg-ATP or when the aminophospholipid translocase was inhibited by vanadate or calcium. If the ghosts were resealed with Mg-ATP inside, the transmembrane asymmetry of the aminophospholipids was reestablished. With long chain insoluble spin-labeled lipids complete randomization was obtained with the phosphatidylcholine analogue but even in the presence of vanadate only a small percentage (approx. 15%) of the spin-labeled phosphatidylserine flopped to the outer monolayer and comparable percentage of the spin-labeled sphingomyelin flipped to the inner monolayer, indicating a hierarchy in the phospholipid redistribution for these water insoluble lipids during hemolysis. The mechanism by which a selective randomization takes place is not known. It may involve phosphatidylserine-protein interactions in the inner leaflet and sphingomyelin-cholesterol or sphingomyelin-sphingomyelin interaction in the outer leaflet.

Transmembrane distribution and translocation of spin-labeled plasmalogens in human red blood cells

We have synthesized two new spin-labeled alkenylacyl phospholipids (plasmalogens) in order to investigate the transmembrane distribution and transport of this subclass of glycerophospholipids in human red blood cells. The plasmenylethanolamine analogue diffuses rapidly from the outer to the inner leaflet with a half time at 37 degrees C of 30 min comparable to that of the corresponding diacyl-phosphatidylethanolamine spin-label in an ATP-requiring and N-ethyl maleimide sensitive manner. The plateau corresponds to 79% of the aminophospholipids on the inner leaflet. By contrast, after 4 h incubation less than 20% of the plasmenylcholine spin-labels reach the interior. Thus plasmalogens behave as the corresponding diacyl-lipids. We infer that plasmenylethanolamine is transported from the outer to the inner leaflet of the red cell membrane by the aminophospholipid translocase.

Labeling of human erythrocyte membrane proteins by photoactivatable radioiodinated phosphatidylcholine and phosphatidylserine A search for the aminophospholipid translocase

We have synthesized radioiodinated photoactivatable phosphatidylcholine (125‐N3‐PC) and phosphatidylserine (125I‐N3‐PS). After incubation with red blood cells in the dark, the labeled PC could be extracted but not the corresponding PS molecule, indicating that the latter was transported by the aminophospholipid translocase, but not the former. When irradiated immediately after incorporation, N3‐PS, but not N3‐PC, partially blocked subsequent translocation of spin‐labeled aminophospholipids. Analysis of probe distribution by SDSpolyacrylamide gel electrophoresis revealed that 125I‐N3‐PS labeled seven membrane bound components with molecular masses between 140 and 27 kDa: one (or several) of these components should correspond to the aminophospholipid translocase.