Modulation of Erythrocyte Membrane Mechanical Function by β-Spectrin Phosphorylation and Dephosphorylation

Abstract

The mechanical properties of human erythrocyte membrane are largely regulated by submembranous protein skeleton whose principal components are alpha- and beta-spectrin, actin, protein 4.1, adducin, and dematin. All of these proteins, except for actin, are phosphorylated by various kinases present in the erythrocyte. In vitro studies with purified skeletal proteins and various kinases has shown that while phosphorylation of these proteins can modify some of the binary and ternary protein interactions, it has no effect on certain other interactions between these proteins. Most importantly, at present there is no direct evidence that phosphorylation of skeletal protein(s) alters the function of the intact membrane. To explore this critical issue, we have developed experimental strategies to determine the functional consequences of phosphorylation of beta-spectrin on mechanical properties of intact erythrocyte membrane. We have been able to document that membrane mechanical stability is exquisitely regulated by phosphorylation of beta-spectrin by membrane-bound casein kinase I. Increased phosphorylation of beta-spectrin decreases membrane mechanical stability while decreased phosphorylation increases membrane mechanical stability. Our data for the first time demonstrate that phosphorylation of a skeletal protein in situ can modulate physiological function of native erythrocyte membrane.

Equilibrium physics breakdown reveals the active nature of red blood cell flickering

“…The validity of previous studies, which used equilibrium theories to extract mechanical information on the RBC membrane, has therefore to be critically checked. The comparison of the dissipative response for the three conditions (zero, partial and full ATP depletion) in figure 2c confirms the strong metabolic dependence of passive membrane properties noted previously 6,12,18,22,23 .…”

“…However, the passive mechanical properties of the RBC membrane themselves depend strongly on ATP, as illustrated by the stiffening of the RBC membrane on starvation [17][18][19]6,15 , that correlates with a sudden transition from discocyte to spiculated echinocyte shapes 20,21 . Therefore a direct and compelling explanation for the decrease of fluctuations observed in ATP-depleted cells is the stiffening of the membrane 22,23 rather than the loss of putative active (that is, non-equilibrium) fluctuations. To make a different assessment of the involvement of metabolic activity in flickering, an attractive approach was used by Tuvia et al 14 : at equilibrium, the mean-square fluctuations amplitude is a thermodynamic variable, which, by definition, cannot depend on the dynamics, and in particular on medium viscosity.…”

Direct Cytoskeleton Forces Cause Membrane Softening in Red Blood Cells

“…This phosphorylation is catalyzed by protein kinase C (PKC), which disassembles the spectrin/actin/4.1 trimer, the essential cytoskeletal complex that determines the mechanical stability of the RBC membrane (12). Indeed, PKC activation is known to lead to a decreased overall stability of the membrane skeleton (12,53,54), a structural effect that is consistent with a measurable increase of the dynamic fluctuations of the RBC membrane, as revealed by Betz et al (7). Therefore, assuming the currently accepted mechanochemical model of the 4.1 nodes (51,53), every unpinning event bears a reaction kicking force that should be stressed on the lipid membrane upon dissociation of a spectrin filament from the junctional complex (14,26).…”

“…The elastic properties of RBCs are dominated by the interaction between the lipid bilayer and the underlying spectrin cytoskeleton (8,9), which is a dynamical meshwork mainly consisting of spectrin filaments linked by reconfigurable junctional complexes (5,6). The transient binding capacity of these complexes depends on their phosphorylation state (10)(11)(12). This structural network endows the spectrin skeleton with the basic role of globally imparting structural rigidity to the cell membrane (13) and locally regulating its flexibility through reversible phosphorylation at the anchoring nodes (6,14).…”

Identification by Differential Display of Transcripts Regulated during Hematopoietic Differentiation

“…Ten of these represented repetitive elements, eight were mitochondrial sequences, and several were homologous to already-described genes. These genes included some which are known to be found in hematopoietic cells-for example, the α chain of the IL-3 receptor [2][3][4]; topoisomerase IIβ, which catalyzes the interconversion of topological isomers of DNA and is expressed in hematopoietic cell lines and leukemias [17] and is indeed a target of antineoplastic reagents; the adducin γ chain [18], which has been described to be present in the erythrocyte cytoskeleton [19]; and annexin II, which is a molecule widely expressed and described to have a role in the secretory process of neutrophils [20]. In addition, 16 sequences corresponded to expressed sequence tags (EST), sequences determined from the 5′ or 3′ ends of randomly chosen cDNA library clones within the framework of the Human Genome Project [21].…”