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

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 αß 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 αß Sp around a protofilament may form a loop that sustains and equilibrates tension. Sequential association of ß 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 ß 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 ß 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.

Mouse erythrocyte tropomodulin in the brain reported by lacZ knocked-in downstream from the E1 promoter.

Erythrocyte tropomodulin (E-Tmod, Tmod1) is a tropomyosin-binding protein that caps the slow-growing end of actin filaments. In erythrocytes, it may favor the formation of short actin protofilaments needed for elastic cell deformation. Previously we created a knockout mouse model in which lacZ was knocked-in downstream of the E1 promoter to report the expression of full length E-Tmod. Here we utilize E-Tmod(+/lacZ) mice to study E-Tmod expression patterns in the CNS. X-gal staining and in situ hybridization of adults revealed its restricted expression in the olfactory bulb, hippocampus, cerebral cortex, basal ganglia, nuclei of brain stem and cerebellum. In neonates, signals in the cortex and caudate putamen increased from days 15 to 40. Immunohistochemistry also revealed that signals for beta-galactosidase coincided with that of NeuN, a post-mitotic nuclear marker for neurons, but not that for GFAP+ astrocytes or APC+ oligodendrocytes, suggesting E-Tmod/lacZ-positive cells in the CNS were neurons. Large neurons, e.g., mitral cells in olfactory bulb and mossy cells in hilus of the dentate gyrus are among those that expressed very high levels of E-Tmod in the CNS.

Interactions of recombinant mouse erythrocyte transglutaminase with membrane skeletal proteins.

Transglutaminases (TGs) are a family of enzymes that catalyze the formation of covalent gamma-glutamyl-epsilon-lysine crosslinks between glutamine (Q) acyl-donors and lysine (K) acyl-acceptors. Here, we report the cDNA cloning of a TG from mouse reticulocytes, its 4.6-kb message size and high-yield synthesis of recombinant TG in yeast cultures. Its activity was assayed by crosslinking the amine of monodansylcadaverine (DC) onto casein and inside-out vesicles of erythrocytes. The latter contain TG substrates including the anion ion exchanger (AE1) or band 3, and the crosslinking activity was the highest at physiological [GTP] and [ATP] of erythrocytes. To study individually how TG interacts with band 3 and what role P4.2, a pseudo-TG that is normally associated with band 3, may play in their interaction, recombinant cytoplasmic domain of band 3 (cdb3) and P4.2 were also cloned by polymerase chain reaction from mouse reticulocytes, expressed and affinity-purified from Escherichia coli. Enzyme-linked immunosorbent assay and Western blot analysis revealed that increasing [CaCl(2)] enhanced TG-mediated crosslinking of DC to cdb3 but decreased TG binding to cdb3. P4.2 inhibited the TG-mediated crosslinking of cdb3 but stabilized the binding of TG to cdb3 in the presence of calcium. This in vitro study suggests a relationship among TG, cdb3 and P4.2 in erythrocyte membrane during calcium influx.

A hybrid model for erythrocyte membrane: a single unit of protein network coupled with lipid bilayer.

To investigate the nanomechanics of the erythrocyte membrane we developed a hybrid model that couples the actin-spectrin network to the lipid bilayer. This model features a Fourier space Brownian dynamics model of the bilayer, a Brownian dynamics model of the actin protofilament, and a modified wormlike-chain model of the spectrin (including a cable-dynamics model to predict the oscillation in tension). This model enables us to predict the nanomechanics of single or multiple units of the protein network, the lipid bilayer, and the effect of their interactions. The present work is focused on the attitude of the actin protofilament at the equilibrium states coupled with the elevations of the lipid bilayer through their primary linkage at the suspension complex in deformations. Two different actin-spectrin junctions are considered at the junctional complex. With a point-attachment junction, large pitch angles and bifurcation of yaw angles are predicted. Thermal fluctuations at bifurcation may lead to mode-switching, which may affect the network and the physiological performance of the membrane. In contrast, with a wrap-around junction, pitch angles remain small, and the occurrence of bifurcation is greatly reduced. These simulations suggest the importance of three-dimensional molecular junctions and the lipid bilayer/protein network coupling on cell membrane mechanics.