Intertwined αβ spectrin meeting helical actin protofilament in the erythrocyte membrane skeleton: wrap-around vs. point-attachment

Abstract

Our 3-D model for a junctional complex (JC) in the erythrocyte membrane skeleton proposed that the helical actin protofilament functions as a mechanical axis for three pairs of αβ spectrin (Sp), and each pair wraps around the protofilament in a back-to-back fashion. The distal end of each Sp is further associated with the lipid bilayer by a suspension complex (SC). Here, we detail how splitting and rejoining of αβ Sp around a protofilament may form a loop that sustains and equilibrates tension. Sequential association of β and α Sp solves the challenge of constructing multiple loops along the protofilament, and topological connection facilitates their re-association. The wrap-around model minimizes the strain of the actin binding site on β Sp due to tension, redirection, or sliding of intertwined Sp. Pairing Sp balances the opposing forces and provides a mechanism for elastic recovery. The wrap-around junction thus provides mechanical advantages over a point-attachment junction in maintaining the integrity and functionality of the network. Severing α or β Sp may convert a wrapping-around junction to a point-attachment junction. In that case, a "bow up" motion of JC during deformation may disturb or flip the overlaid lipid bilayer, and mark stressed erythrocytes for phagocytosis.

Figure 1

Sketches of the erythrocyte membrane skeleton and the wrap-around model from large to small scale. (a) The organization of the skeletal network, showing JC, SC, and Sp; (b) A 3-D view of a JC with a protofilament associated with three pairs of Sp and 6 SC, forming a basic repeating unit. (The angle between JC and the lipid bilayer is not specified); (c) A protofilament having 12 individual G actin and 6 occupied (filled) binding sites for β Sp; (d) An enlarged section of the protofilament bound with the middle pair: Sp3 (α3β3) and Sp4 (α4β4). Sp3 wraps around a pair of G actin (3a/3b); Sp4 wraps around the next pair of G actin (4a/4b) in the opposite direction (a–d modified from Ref. 27); (e) The detail sketch of α and β spectrin, which split and rejoin to wrap the protofilament. The tail end begins with the non-homologous domains of N-β, including ABD (CH1 and CH2), and that of C-α, including EFs. Cys (*) may form disulfide bonds. The dimer nucleation site spans α1-4 and β18-21. This sketch appeared in Ref. without detailed description. The size of linkers (# of residues): N-β (50), between CH1 and CH2 (10), between CH2 and β1 (19), between homologous β domains (8); C-α (75), between EF1 and EF2 (14), between EFII and α20 (8), and between α homologous domains (8). Molecules and domains are not to scale. The intertwined feature is not shown for clarity purposes

Figure 2

Point-attachment vs. wrap-around. (a) In a point-attachment junction, the ABD binds to a single point (solid circle) on the front side of the protofilament and force applied through Sp directly affects the binding. The unfolding (and open/close configurations) of domains would be the primary protective measure. (b) In a wrap-around junction, the ABD binds to the back side of the protofilament. α and β spectrin are arranged in an anti-parallel orientation and resists sliding through steric hindrance which is enhanced by intertwining. The greater the extension, the tighter the intercalating would be. (c) Modified force-extension curve of single stranded spectrin. The saw-tooth pattern represents successive unfolding of single spectrin repeats. The point-attachment junction features an irreversible drop (a vertical line) at the threshold at the point of detachment between Sp and protofilament. The lower panel shows 3 domains in each pitch

Figure 3

The conversion of a wrap-around junction to a point-attachment junction. (a) A two layer network (consisting of 1 CU and 6 PU) under equibiaxial extension. (b) A pair of SC at the center of a Sp tetramer. (c) A JC wrapped around by a pair of Sp (see Fig. 1d). (d) Side view of a JC, showing 6 occupied ABD (solid circles), 3 in each opposing helix, spiral from one end to the other. (e) One ABD in a wrap-around junction before a single strand cleavage (e.g., on α spectrin) (open arrow). (f) The cleavage may free the f segment from the protofilament and roll the protofilament during extension, converting a wrap-around junction to a point-attachment junction. AC: actin protofilament. T: Sp tetramerization site. f segment: a segment of single stranded spectrin between ABD and the cleavage site (may consist only β spectrin or a combination of α and β spectrin connected at the tail end)

Figure 4

Snap shots of a membrane skeletal network in 5DOF and 6DOF simulations during equibiaxial extension (λ = 1.3). Upper panels are 5DOF simulations (wrap-around junction) of a network with three layers at 3, 6, and 9 s; lower panels are their corresponding 6DOF simulations (point-attachment junction) based on Ref. . Note the “bow up” motion of the protofilament in CU (in red) in the 6DOF simulation. The point end of the protofilament is indicated by a black sphere (E-Tmod). See supplementary data for more information and complete animations