Intense, reversible aggregation of intramembrane particles in non-haemolyzed human erythrocytes. A freeze-fracture study

Acridine orange, a strongly cationic, membrane-penetrating dye, caused intense aggregation of intramembrane particles in non-haemolyzed human erythrocytes at 5 mM concentration. Simultaneously with the particle aggregation, large, empty, intramembrane particle-free or particle-depleted vesicles were detached from the red cells. Washing the erythrocytes after Acridine orange treatment resulted in complete disaggregation of the intramembrane particles. Less cationic acridine dyes (9-aminoacridine, 5-aminoacridine and Quinacrine) caused much less conspicuous alterations. Rivanol (ethacrine lactate), on the one hand, caused intramembrane particle aggregation in human red cells as well as ribosome aggregation in rabbit reticulocytes when it was dissolved in lactate-buffered sucrose. On the other, Rivanol dissolved in phosphate-buffered saline failed to induce these alterations. Neuraminidase treatment had no effect on the intensity of Acridine orange-induced particle aggregation, but impeded disaggregation. Our results indicate that, in contrast to previous observations, intense and reversible clustering of intramembrane particles is certainly possible in non-haemolyzed erythrocytes. The intramembrane particle aggregation may be due primarily to perturbation of the inner red cell surface by strongly cationic dyes and the presence of sialic acid residues on the outer red cell surface seems to be essential for the reversibility of the process.