Spin-labeled analogs of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine have been used to study phospholipid transverse diffusion and asymmetry in the human erythrocyte membrane. Ascorbate reduction was used to assess the transbilayer distribution of the labels. All three spin-labeled phospholipids initially incorporated into the outer leaflet of the membrane. On fresh erythrocytes at 50C, the phosphatidylcholine label remained mainly in the outer leaflet. In contrast, the phosphatidylserine and phosphatidylethanolamine labels underwent rapid transverse diffusion that led to their asymmetric distribution in favor of the inner leaflet. The latter effect was reversibly inhibited after ATP depletion of the erythrocytes and could be reproduced on resealed erythrocyte ghosts only if hydrolyzable Mg-ATP was included in the internal medium. It is suggested that an ATPdriven transport of amino phospholipids toward the inner leaflet could be the major cause of the phospholipid asymmetry in the erythrocyte membrane. It is also proposed that the same mechanism could explain the ATP requirement of the maintenance of the erythrocyte membrane discoid shape. that this method can be used successfully in erythrocytes, and the transverse diffusion of phosphatidylcholine has been measured. In the present study, the following phospholipid spin labels have been used.These phospholipids possess an unmodified polar head group R that can be either choline, serine, or ethanolamine and areAn asymmetric distribution of phospholipids exists between the two halves of many biological membranes (for review, see ref. 1). The human erythrocyte membrane appears to be the best characterized in this regard. Numerous studies using chemical labeling (2, 3) and phospholipases (4-6) indicate that, in this membrane, phosphatidylcholine and sphingomyelin are distributed in favor of the outer leaflet, while phosphatidylethanolamine and phosphatidylserine are mainly located in the inner leaflet.These observations raise the problem of how this asymmetry is maintained during the 120-day life span of the erythrocyte. The asymmetric distribution could in principle be maintained if transbilayer diffusion of phospholipids was negligible. However, for phosphatidylcholine, transbilayer "flip-flop" has been shown to occur with a half-time of 8-15 hr in the native membrane (7,8), although no such measurements exist for the three other major phospholipid species. A possible role of cytoskeletal proteins in maintaining the asymmetry in spite of transbilayer diffusion has been proposed by Haest et al. (9,10), but no direct evidence for such a process has been observed in the intact native membrane.In the present study, the formation and maintenance of phospholipid asymmetry in the erythrocyte membrane has been addressed by measuring the transverse diffusion of spin-labeled phospholipids bearing different polar head groups. The method, introduced by Kornberg and McConnell (11), is based on the accessibility of membrane-associated spin labe...

Engineered inorganic nanoparticles are essential components in the development of nanotechnologies. For applications in nanomedicine, particles need to be functionalized to ensure a good dispersibility in biological fluids. In many cases however, functionalization is not sufficient: the particles become either coated by a corona of serum proteins or precipitate out of the solvent. In the present paper, we show that by changing the coating of iron oxide nanoparticles from a low-molecular weight ligand (citrate ions) to small carboxylated polymers (poly(acrylic acid)), the colloidal stability of the dispersion is improved and the adsorption/internalization of iron toward living mammalian cells is profoundly affected. Citrate-coated particles are shown to destabilize in all fetal-calf-serum based physiological conditions tested, whereas the polymer coated particles exhibit an outstanding dispersibility as well as a structure devoid of protein corona. The interactions between nanoparticles and human lymphoblastoid cells are investigated by transmission electron microscopy and flow cytometry. Two types of nanoparticle/cell interactions are underlined. Iron oxides are found either adsorbed on the cellular membranes, or internalized into membrane-bound endocytosis compartments. For the precipitating citrate-coated particles, the kinetics of interactions reveal a massive and rapid adsorption of iron oxide on the cell surfaces. The quantification of the partition between adsorbed and internalized iron was performed from the cytometry data. The results highlight the importance of resilient adsorbed nanomaterials at the cytoplasmic membrane.

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