We propose to continue our analysis of the fine structures of the network that lines the cytoplasmic surface of the human red cell membrane and to analyze the structural changes occurring to this skeletal network during the maturation of reticulocytes. The goal of this study is a better understanding of both the molecular architecture of the membrane skeleton and the fundamental basis for the durability, deformability, and elasticity of the normal red cell and the precise nature of the structural derangements in the skeletons of pathological red cells. We have shown by electron microscopic examination of negatively stained specimens that the skeleton is organized into short actin protofilaments interconnected by multiple strands of spectrin tetramer. The spectrin, which is the major element of the membrane skeleton, is in a compact or condensed form when freshly released from the static membranes but can be extended by both electrostatic and mechanical forces to the elongated and filamentous form commony observed for isolated spectrin. We propose to extend our investigation of the red cell membrane skeleton by examinung the following factors: (1) The conformation of spectrin and its contribution to the elasticity of the skeleton, (2) The basic organization of membrane skeleton of the human erythrocytes, and (3) The role of the skeleton in maintaining the shape and mechanical properties of the red cells. A variety of experimental approaches will be used including immunofluorescence microscopy, immunoelectron microscopy, and low-dose cryoelectronmicroscopy of frozen-hydrated specimens, as well as other biochemical and biophysical techniques. The principal aims will be to elucidate the precise nature and cellular significance of skeletal elasticity. Information gained from the above studies will help elucidate the nature of the structural derangement in the skeletons of pathological red cells and may also assist in the analysis of cytoskeletons of nucleated cells.
| 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 |
