Control of morphology of resealed erythrocyte membrane

The ability of the erythrocyte to maintain its discoid shape after a wide variety of insults and stresses has stimulated much speculation about the molecular basis of this shape homeostasis. Isolated erythrocyte membranes (ghosts) are capable of morphologies and morphological changes similar to those observed in the intact erythrocyte. A method was developed to isolate ghosts so that their ability to regain and retain their discocytic shape could be controlled depending on the conditions of lysis.;Erythrocyte ghosts were prepared by hypotonic hemolysis in buffer which contained or omitted millimolar divalent magnesium cations. These membranes were incubated in isotonic medium in the presence of millimolar magnesium adenosine triphosphate. The shape change was monitored by phase contrast microscopy of fixed samples.;Ghosts lysed in the presence of divalent magnesium were better able to maintain the discocyte shape than those lysed without magnesium, for which much more stomatocytosis and endocytosis was observed. Increasing the temperature of lysis and the time spent in lysis buffer at elevated temperatures resulted in increases in endocytosis. Ghosts prepared from cells whose normal phospholipid asymmetry had been compromised were less capable of maintaining the discocyte shape even when divalent magnesium was present in the lysing buffer.;The conditions required to cause or enhance endocytosis correlated well with conditions which cause a disturbance in the normal phospholipid distribution. The metabolic requirement and inhibitors of ghost shape change are common to the putative aminophospholipid translocator, which catalyzes the movement of phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet of the lipid bilayer. Flipping of aminophospholipids scrambled to the outside during lysis and translocated to the inner monolayer during incubation with magnesium adenosine triphosphate provides a driving force for endocytosis, consistent with the bilayer couple hypothesis. The ability of the erythrocyte ghost to maintain its discocytic shape appears to depend on the maintenance of the normal phospholipid asymmetry at lysis.