Electron microscopic study of the calcium phosphate-induced aggregation and membrane destabilization of cytoskeleton-free erythrocyte vesicles.

Cytoskeleton-free vesicles derived from human erythrocytes were treated with trypsin, chymotrypsin, or neuraminidase followed by calcium, phosphate, or combined calcium/phosphate treatments in order to study the roles of cell surface proteins and glycoproteins in calcium/phosphate-induced cell aggregation and fusion. Vesicle aggregation (a necessary pre-cursor to membrane fusion) and subsequent membrane destabilization (an essential component of fusion) were examined by freeze-fracture electron microscopy. Enzymatic treatment alone had no effect on the morphology of the cytoskeleton-free vesicles. Neither did separate calcium nor phosphate treatments, although the treatment of the cytoskeleton-free vesicles with calcium did reduce their size slightly. Enzymatic pretreatment had no effect on the calcium-induced size changes. In contrast, the combination of calcium and phosphate drastically disrupted the membrane integrity of aggregated cytoskeleton-free vesicles at pH 7.8, although the effect was reduced at lower pH values. The extent of this membrane destabilization was independent of enzyme treatment. Our results indicate: (1) that the cell surface proteins and glycoproteins have only secondary effects on calcium/phosphate-induced cell aggregation and membrane destabilization, (2) that these processes primarily depend on the reaction between calcium and phosphate ions at the membrane surface, and (3) that cytoskeletal elements probably play no active (positive) role in the Ca2+/PO4(3-) induced erythrocyte membrane fusion process, apart from maintaining cell shape.