Structural and Mechanical Heterogeneity of the Erythrocyte Membrane Reveals Hallmarks of Membrane Stability

Abstract

The erythrocyte membrane, a metabolically regulated active structure that comprises lipid molecules, junctional complexes, and the spectrin network, enables the cell to undergo large passive deformations when passing through the microvascular system. Here we use atomic force microscopy (AFM) imaging and quantitative mechanical mapping at nanometer resolution to correlate structure and mechanics of key components of the erythrocyte membrane, crucial for cell integrity and function. Our data reveal structural and mechanical heterogeneity modulated by the metabolic state at unprecedented nanometer resolution. ATP-depletion, reducing skeletal junction phosphorylation in RBC cells, leads to membrane stiffening. Analysis of ghosts and shear-force opened erythrocytes show that, in the absence of cytosolic kinases, spectrin phosporylation results in membrane stiffening at the extracellular face and a reduced junction remodeling in response to loading forces. Topography and mechanical mapping of single components at the cytoplasmic face reveal that, surprisingly, spectrin phosphorylation by ATP softens individual filaments. Our findings suggest that, besides the mechanical signature of each component, the RBC membrane mechanics is regulated by the metabolic state and the assembly of its structural elements.

Involvement of Calcium, Reactive Oxygen Species, and ATP in Hexavalent Chromium-Induced Damage in Red Blood Cells

“…Moreover, reduced ATP depletion can maintain RBCs morphology in response to Cr 6+ exposure, suggesting that ATP depletion may not be just a by-pass result of increased Ca 2+ influx during the process of Cr 6+ -induced eryptosis. This result was consistent with the report that ATP is important for maintaining membrane dynamic equilibrium and cytoskeleton in RBCs [43,45,49]. The ROS toxicity effect on RBCs after Cr 6+ exposure was not reversed by additional antioxidants.…”

“…Rapid ATP depletion is caused by excessive activation of the Ca 2+ pump and has been considered a result of Ca 2+ influx [38,44]. A recent study reported that ATP depletion affects phosphorylation levels of the 4.1R and spectrin proteins, which are key proteins in the RBCs cytoskeletal network [45]. Depletion of the ATP level to below 25% that of normal RBCs eventually leads to cell membrane skeleton fracture and irreversible eryptosis of spike-shaped RBCs [20].…”

Quantitative biomolecular imaging by dynamic nanomechanical mapping

“…Scheuring et al found that the ability of ATP to modulate RBC membrane stability is to govern the different components as a unit instead of to influence the mechanical properties of individual components themselves. 64 They also found that the mechanical properties of membrane proteins can be considered as the signature of molecular bio-function. 65 In addition to native proteins, the misfolding amyloid proteins have also been characterized by advanced FS.…”

Rapid recognition and functional analysis of membrane proteins on human cancer cells using atomic force microscopy

“…Since the advent, researchers have applied PFT imaging to investigate the physicochemical properties of different biological samples, e.g., membrane proteins (bacteriorhodopsin on the purple membrane of bacterial (Rico et al, 2011;Medalsy et al, 2011), erythrocyte membrane (Picas et al, 2013) and living cells (keratinocyte (Heu et al, 2012), yeast , and Escherichia coli bacteria . However, these researches were commonly performed using ordinary tips (Heu et al, 2012;Rico et al, 2011;Medalsy et al, 2011;Picas et al, 2013) that cannot identify the specific membrane proteins on the cell surface. In recent studies, researchers have applied PFT imaging to investigate the molecular interactions on bacterial cells using functionalized tips.…”