Articulation and flexibility in the physical linkage between band 3 and the cytoskeleton in the human erythrocyte

摘要

The erythrocyte membrane with its underlying cytoskeleton undergoes gross deformation during passage of the cell through fine capillary networks in the circulation. The physical linkages between the cytoskeleton and the membrane must involve substantial internal flexibility or points of articulation within and between molecules in the system. We have used time-resolved phosphorescence spectroscopy to examine three aspects of this dynamic flexibility on the microsecond to millisecond time scale: (1) the rotational diffusion and internal flexibility of F-actin, (2) the intrinsic flexibility of spectrin in solution and when reconstituted to erythrocyte membranes, and (3) internal flexibility in the cytoplasmic domain of band three. The results show that the spectrin dimer displays substantial flexibility on the microsecond time scale: this flexibility is largely retained when spectrin is reassociated with the erythrocyte membrane. There is also evidence that the cytoplasmic domain of band three itself is flexible since the rotational correlation time for band three labeled through this domain (via a phosphorescently labeled monoclonal Fab) is significantly shorter than when the protein is labeled directly at the external face of its transmembrane domain. Short actin filaments possess limited torsional motion on the submicrosecond time scale and are unlikely to contribute to the flexibility of the cytoskeleton.