Analysis of the kinetics of band 3 diffusion in human erythroblasts during assembly of the erythrocyte membrane skeleton.

During definitive erythropoiesis, erythroid precursors undergo differentiation through multiple nucleated states to an enucleated reticulocyte, which loses its residual RNA/organelles to become a mature erythrocyte. Over the course of these transformations, continuous changes in membrane proteins occur, including shifts in protein abundance, rates of expression, isoform prominence, states of phosphorylation, and stability. In an effort to understand when assembly of membrane proteins into an architecture characteristic of the mature erythrocyte occurs, we quantitated the lateral diffusion of the most abundant membrane protein, band 3 (AE1), during each stage of erythropoiesis using single particle tracking. Analysis of the lateral trajectories of individual band 3 molecules revealed a gradual reduction in mobility of the anion transporter as erythroblasts differentiated. Evidence for this progressive immobilization included a gradual decline in diffusion coefficients as determined at a video acquisition rate of 120 frames/s and a decrease in the percentage of compartment sizes >100 nm. Because complete acquisition of the properties of band 3 seen in mature erythrocytes is not observed until circulating erythrocytes are formed, we suggest that membrane maturation involves a gradual and cooperative assembly process that is not triggered by the synthesis of any single protein.