UC San Diego

The erythrocyte membrane skeleton is a multi-protein complex providing mechanical properties and stability to erythrocytes. Defects in the skeleton can manifest in dysfunction and disease such as hemolytic anemia. Erythrocyte tropomodulin (E-Tmod) is a slow-growing end actin-capping protein and has been proposed that together with tropomyosin 5 or 5b they form a "molecular ruler" which dictates protofilament length of 37 nm in the network. In this study, the role for E-Tmod in the network organization and erythrocyte mechanics was investigated. The embryonic lethality of the E-Tmod⁻/⁻ mice was rescued by breeding with E-Tmod overexpressing transgenic (TOT) mice. The morphological changes in E-Tmod⁻/⁻ erythrocytes and the topology of the network were evaluated using light, scanning electron, and transmission electron microscopy. The mechanical properties of E-Tmod⁻/⁻ erythrocytes were quantified using micropipette aspiration and ektacytometry. Blood count analyses of E-Tmod⁻/⁻ erythrocytes showed a lower mean corpuscular volume (p < 0.015) and a higher percentage of [Mu]RBCs (p < 0.01) indicating mild spherocytosis. TEM analysis revealed a more compacted network with smaller openings. The denser network could be caused by membrane loss due to the irregular actin filament lengths without the regulation of E-Tmod. During circulation, such irregularity may have resulted in altered stress-strain distribution across spectrin tetramers in the network. Without the proper support of the network some lipid bilayer may be lost, resulting mild spherocytosis and increased oxidative stress. Approximately 10 times more dimerization of the 29 -kDa isoform of E-Tmod was detected in the cytosol as compared to E-Tmod⁺/⁺ and E-Tmod⁺/⁻ counterparts. In micropipette aspiration, E-Tmod⁻/⁻ erythrocytes had a higher elastic shear modulus of 7 pN/[Mu]m versus 5 pN/ [Mu]m for E-Tmod⁺/⁺ and E-Tmod⁺/⁻ erythrocytes. Ektacytometry also showed they were more rigid, having a lower integrated elongation index p < 0.0001). Both findings could be linked to spherocytosis or oxidation of the network. These findings demonstrate the importance of E-Tmod in the organization of the network and the cellular deformability. Without it, erythrocytes can suffer from loss of membrane, denser membrane skeleton, becoming spherocytic, as well as greater oxidative stress, and consequently, significantly less deformable than normal erythrocytes