Actin protofilament orientation at the erythrocyte membrane

The short actin filaments in the erythrocyte's membrane skeleton are shown to be largely oriented tangent to the lipid bilayer. Actin "proto"-filament s have previously been described as junctional centers intertriangulated by spectrin; however, the protofilaments may simultaneously serve as pinning centers between the network and the overlying bilayer. The latter function now seems of particular importance because near-normal network assembly has been reported with transgenic mouse sphero-erythrocytes that lack the prim ary linkage protein Band 3. To assess possible physical constraints on acti n protofilaments in intact membranes, fluorescence polarization microscopy (FPM) has been used to study rhodamine phalloidin-labeled red cell ghosts. A basis for interpreting FPM images of cells is provided by FPM applied to isolated actin filaments. These are labeled with the same rhodamine probes and imaged at various orientations with respect to the polarizers, includin g filament orientations perpendicular to the image plane. High aperture and fluorophore conjugation effects are found to be minimal, enabling developm ent of a simple, semi-empirical model which indicates that protofilaments a re generally within similar to 20 degrees of the membrane tangent plane.