Evaluation of the Spectrin Compartment Size in Normal and Diseased Red Blood Cells by Single Particle Tracking.

In a process termed “hop diffusion”, membrane proteins diffuse randomly within a cytoskeletally-enclosed compartment over short time scales, periodically hopping from one compartment to the next. Band 3 diffuses in the erythrocyte membrane bilayer in a manner that is thought to be restricted by the size and stability of the spectrin network. Although a fraction of band 3 may exhibit reduced mobility due to its association with spectrin (mediated by proteins such as ankyrin and adducin), another subpopulation of band 3 is thought to diffuse freely within the spectrin compartments, hopping from one corral to the next by transient opening of the barrier (presumably a spectrin tetramer) separating adjacent corrals. Based on this hypothesis, measurement of the compartment size and hopping frequency of a band 3 molecule in the erythrocyte membrane can provide information on the structure and stability of the spectrin network. We have combined single particle tracking (SPT) techniques with high-speed video microscopy to determine the average compartment size and diffusion coefficient of the mobile fraction of band 3 in both normal and diseased red blood cells (RBCs). Streptavidin-linked quantum dots (525nm emission) and 40 nm fluorescent beads were attached to band 3 via a DIDS-biotin-linker, and fluorescence microscopy coupled to a high speed camera was used to determine the position of band 3 with a time resolution of 8 ms (120 fps). Labeling specificity was demonstrated by blotting with streptavidin-horseradish peroxidase, and flow cytometry studies established the binding constant of DIDS-biotin for band 3 in the intact cell of ∼1-2*10⁻⁵ M. For diffusion studies, RBCs were incubated with 10⁻¹¹ M conjugate in order to attach only one or two streptavidin-quantum dots per cell. SPT data were collected on both healthy and pathologic human RBCs, including HbSS (sickle cells), HbSC (sickle cell hemoglobin C), HbSB^o (sickle cell zero-beta-thalasamia), HbSB⁺ (sickle cell beta-plus-thalasamia), HS (hereditary spherocytosis), and HPP (hereditary pyropoikilocytosis). Movement within the observed compartments was found to be random, with an average diagonal length for the mobile fraction in healthy cells of ∼100nm and a diffusion coefficient of ∼1–2*10⁻¹⁰cm²/s. HbSS cells, however, displayed a comparatively smaller compartment size (∼60–80nm) with a lower diffusion coefficient (0.1−1*10⁻¹⁰cm²/s). In contrast, HPP cells exhibited an average compartment size of ∼133nm with a diffusion coefficient of 6*10⁻¹⁰cm²/s. SPT data further suggest the presence of multiple populations of band 3, exhibiting both free diffusion and restricted mobilities with different characteristics. Details of band 3 diffusion in all of the aforementioned diseased and normal blood cells will be provided, and a model accounting for the multiple populations of band 3 will be presented.