Imaging Of The Diffusion Of Individual Band 3 Molecules On Whole Erythrocytes From Patients With Hereditary Hemolytic Disorders

The plasma membrane of the human erythrocyte is a composite structure that consists of a fluid lipid bilayer (including membrane-embedded and membrane-associated proteins) and a sub-surface scaffolding consisting of a six-fold network of the tetrameric flexible structural protein spectrin. The spectrin network is pinned to the fluid bilayer through a series of transmembrane and membrane associated proteins both at the six-fold junctions and near the mid-points along the spectrin tetramers between junctions. Common to both of these pinning points is the presence of the transmembrane anion exchange protein band 3 (AE1). Band 3 ties the six-fold junction to the bilayer through interaction with the protein adducin and ties the midpoint to the bilayer through interaction with ankyrin. Much of the remarkable mechanical characteristics of the red cell have been attributed to this membrane architecture. Further, red cells become fragile in pathologies known to disrupt the spectrin network or its pinning points to the membrane. To assess changes in the structure of the plasma membrane of pathologic red cells at the single molecule level, the mobility of individual band 3 molecules was observed on normal red cells as well as those from patients with several types of hereditary diseases including spherocytosis, elliptocytosis, and pyropokilocytosis. Specifically, individual band 3 molecules on whole red cells were labeled by quantum dots through the band 3 inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS). We will present data on the mobility of band 3 in each of these cell types recorded at video imaging rates of 120 fps. The observation that membrane pathologies can be distinguished by the mobility of individual membrane molecules suggests that single particle tracking might constitute a useful tool for characterizing the “health” of a membrane.