We propose a detailed analysis of the fine structure and deformability of the network of spectrin, actin, and band 4.1 which lines the cytoplasmic surface of the human red blood cell membrane. The goal of this work is a better understanding of normal red cell membrane durability, deformability and shape and its alterations in sickle cell disease. We have recently shown by electron microscopy of negatively-stained specimens that isolated red cell skeletons are constructed of actin filaments approximtely 50 nm in length linked by multiple spectrin tetramers at sites of association with oligomers of band 4.1. We propose to extend these initial studies as follows: (1) Examine skeletons prepared in isotonic saline plus 10 mM Mg++ so as to conserve their native structure and tropomyosin content against dissociations. (2) Generate and examine well-reserved unit fragments of the skeleton which have lost none of their spectrin, actin or band 4.1. (3) Test for the existence of cables of two or more spectrin molecules running between adjacent sites on neighboring actin filaments. (4) Generate, isolate and characterize native spectrin-oligomeric band 4.1 ensembles free of actin. (5) Measure the length of native (undegraded) actin protofilaments from red cells of varied age, phylogenetic origin and disease state. (6) Demonstrate definitively that the slow-growing ends of red cell actin protofilaments are free to elongate (i.e., bind G-actin). (7) Add G-actin onto actin filaments within red cells and determine whether spectrin migrates so as to bind to the new extensions. (8) Develop a quantitative assay for network deformability based on laser diffraction analysis of the shape of networks subjected to controlled shear stress. (9) Examine whether mechanical deformations or treatments which reduce cellular deformability alter the molecular ultrastructure of the skeletons. (10) Examine the network of reversibly and irreversibly sickle cells according to the approaches outlined above.
| Shen, B W; Josephs, R; Steck, T L (1986) Ultrastructure of the intact skeleton of the human erythrocyte membrane. J Cell Biol 102:997-1006 |
