Lateral mobility of a lipid analog in the membrane of irreversible sickle erythrocytes

Abstract

The major feature of sickle cell anemia is the tendency of erythrocytes to sickle when exposed to decreased oxygen tension and to unsickle when reoxygenated. Irreversible sickle cells (ISCs) are sickle erythrocytes which retain bipolar elongated shapes despite reoxygenation. ISCs are believed to owe their biophysical abnormalities to acquired membrane alterations which decrease membrane deformability. While increased membrane surface viscosity has been measured in ISCs, the lateral dynamics of membrane lipids in these cells have not heretofore been examined. We have measured the lateral diffusion of the lipid analog 3,3′-dioctadecylindocyanine iodide (DiI) in the plasma membrane of intact normal erythrocytes, reversible sickle cells (RSCs), and irreversible sickle cells by fluorescence photobleaching recovery (FPR). The diffusion coefficients ± standard errors of the mean of DiI in intact normal red blood cells (RBCs), RSCs,and ISCs at 37°C are (8.06 ± 0.29) · 10 −9cells (RBCs), RSCs,and ISCs at 37°C are (8.06 ± 0.29) · 10 − cm 2 · s −1, (7.74 ± 0.22) · 10 −9 cm 2 · s −1 cm 2 · s −1, (7.74 ± 0.22) · 10 −9 cm 2 · s and (7.29 ± 0.24) · 10 −9 cm 2 · s −1, respectively. A similar decrease in the diffusion coefficient of DiI in the plasma membranes of the three cell types was observed at 4, 10, 17, 23, and 30°C. ANOVA analysis of the changes in DiI diffusion showed significant differences between the RBC and ISC membranes at all temperatures examined. The characteristic breaks in Arrhenius plots of the diffusion coefficients for the RBCs, RSCs, and ISCs occurred at 20, 19, and 18.6°C, respectively. Photobleaching recovery data were used to estimate (Boullier, J.A., Melnykovich, G. and Barisas, B.G. (1982) Biochim. Biophys. Acta 692, 278–286) the microviscosities of the plasma membranes of the three cell types at 25°C. We find significant differences between our microviscosity values and those obtained in previous fluorescence depolarization studies. However, both methods indicate qualitatively similar differences in membrane microviscosity amoung the various cell types.