Video Essay Compartmentalization of the Erythrocyte Membrane by the Membrane Skeleton: Intercompartmental Hop Diffusion of Band 3□V

Recent developments in microscopic instrumentation and probes have allowed the observation and manipulation of the movement of membrane proteins and lipids in the plasma membrane at the level of single molecules. These experiments are performed by tracking small colloidal gold particles attached to specific membrane proteins and lipids (single particle tracking) (Jacobson et al., 1995; Sheets et al., 1995; Kusumi and Sako, 1996; Saxton and Jacobson, 1997) or by dragging particle–protein complexes along the surface of the plasma membrane using laser tweezers (also termed an “optical trap” or OT) (Edidin et al., 1991; Kusumi and Sako, 1996; Kusumi et al., 1998). In optimal cases, a single protein molecule is attached to a 40-nm-diameter colloidal gold probe (Tomishige et al., 1998). These single molecule experiments have demonstrated that most membrane-spanning proteins do not undergo simple diffusion, but that a significant number are locally slowed or stopped, some are temporarily confined, and others are transported unidirectionally (Kusumi et al., 1993; Sheets et al., 1997; Sako et al., 1998; Simson et al., 1998). Previously, these processes had not been observed, because the methods used, e.g., fluorescence recovery after photobleaching, recorded the ensemble-averaged behavior of a large number of molecules. The underlying cellular mechanisms that induce nonrandom diffusion have remained largely unknown; however, we and others found that properties of the membrane skeleton network fundamentally affect the movement and distribution of certain membrane proteins, such as transferrin receptor, a2-macroglobulin receptor, MHC class I molecules, E-cadherin, and band 3, through corralling and binding effects imposed by the membrane skeleton network (Edidin et al., 1991; Luna and Hitt, 1992; Kusumi et al., 1993; Sako and Kusumi, 1994, 1995; Sako et al., 1998; Tomishige et al., 1998). The results are particularly clear with band 3 in human erythrocyte ghost membranes, where the specimen consists of a lipid bilayer and its underlying membrane skeleton, which has been biochemically and biophysically well characterized (Golan, 1989; Bennett, 1990; Bennett and Gilligan, 1993; Mohandas and Evans, 1994). The results obtained by these studies support a “membrane skeleton fence model” in which the cytoplasmic portion of a membrane protein collides with the membrane skeletal “meshwork” because of steric hindrance, thus resulting in the temporal confinement of the protein within the mesh (compartment, Figure 1A). Membrane proteins escape from these compartments and hop to adjacent ones because of the dynamic properties of the membrane skeleton. We predict that time-dependent fluctuations in the distance between the membrane and the skeleton are large or the membrane–skeleton network connections form and break continuously, as expected in a chemical equilibrium, providing membrane proteins with the opportunity to pass through the mesh barrier. In the case of band 3, macroscopic diffusion within the erythrocyte membrane occurs as the result of a series of such intercompartmental hops. The scientific content of this work has been published previously (Tomishige et al., 1998), and the purpose of the present essay is to give the reader a glimpse of the raw data from this study, as recorded by both normal and high-speed video microscopy. We hope the video sequences attached to this article will help the viewing audience develop a feel for how band 3 interacts with the membrane skeleton and undergoes intercompartmental hop diffusion within the erythrocyte ghost membrane.