Reconstitution of spectrin-deficient, spherocytic mouse erythrocyte membranes.

Abstract

A B S T R A C T To study directly the role of spectrin in erythrocyte membrane function, we have designed a reconstituted membrane system using erythrocyte membranes from spectrin-deficient mice and purified spectrin from normal mice. The normal spectrin is inserted into the spectrin-deficient spherocytes by exchange hemolysis. Thereafter, raising the ionic strength and temperature reseals the cells and, with time, facilitates binding of the spectrin to the spectrindeficient membranes. The binding is apparently specific as shown by its dependence upon the concentration of undenatured spectrin and the concentration of salt used, as well as by the immunofluorescent appearance of the reconstituted cells after treatment with specific antispectrin antibody.In terms of in vitro cellular behavior, the reconstituted preparations show marked changes in comparison to the untreated spherocytes. In particular, membrane stability, as measured by the reduction of myelin figure formation and lipid loss, is considerably enhanced. In addition, membrane fusion, which occurs readily with the untreated spherocytes, is virtually eliminated. Finally, the osmotic behavior of the native spherocytes is appreciably altered, such that the early phase of osmotically induced swelling, as measured in a highspeed stop-flow apparatus, is delayed and modified.Taken together, these findings indicate specific roles for spectrin in the stabilization of the erythrocyte membrane, in the limitation of membrane fusion, and in the modulation of the membrane's response to osmotic stress.

Restoration of normal membrane stability to unstable protein 4.1-deficient erythrocyte membranes by incorporation of purified protein 4.1.

“…One previous study has also defined a functional role for a skeletal protein by incorporating the protein into deficient erythrocytes (31). This involved incorporation of spectrin into spectrin-deficient (sph/sph) mutant mouse erythrocytes.…”

Abnormalities in membrane phospholipid organization in sickled erythrocytes.

“…The f:actors that coontrol the rearranigemiienit of phospholipids within the lipid bilayer (lurinig sicklinig are uinknowni. Evidence is graduially aciimmulating to suggest that spectrin miiay play ani important role in stabilizing membranie phosplholipid organ izatiomi in human RBC (11)(12)(13)(14)(15)(16). Spectrin has leen (leImlonlstrated to interact with plhospholipids in artificial membrane systemiis (12)(13)(14).…”

“…Since evidence is accumulating to suggest that membrane proteins, particularly spectrin, play an important role in stabilizing the organization of membrane phospholipids (11)(12)(13)(14)(15)(16), it is not surprising that abnormalities in the organization of certain phospholipid classes have been reported in sickle erythrocytes. In particular, the organization of amino phospholipids within the membrane, as detected by chemical probes, is abnormal (17,18).…”

The red cell membrane and its cytoskeleton

“…The worst affected have red cells with an immeasurably short survival time, which, when subjected to the slightest mechanical perturbation, disintegrate into vesicles and myelin forms. When the deficit is made good, using a transient haemolysis procedure to introduce spectrin into the cell, the anomalous mechanical properties are to a large extent mitigated (Shohet;. The cytoskeleton appears also to be the agency through which the cell exercises a number of biologically important functions, involving for instance the passage of signals across the membrane, as will be recounted.…”