Metabolic remodeling of the human red blood cell membrane

Abstract

The remarkable deformability of the human red blood cell (RBC) results from the coupled dynamic response of the phospholipid bilayer and the spectrin molecular network. Here we present quantitative connections between spectrin morphology and membrane fluctuations of human RBCs by using dynamic full-field laser interferometry techniques. We present conclusive evidence that the presence of adenosine 5'-triphosphate (ATP) facilitates non-equilibrium dynamic fluctuations in the RBC membrane that are highly correlated with the biconcave shape of RBCs. Spatial analysis of the fluctuations reveals that these non-equilibrium membrane vibrations are enhanced at the scale of spectrin mesh size. Our results indicate that the dynamic remodeling of the coupled membranes powered by ATP results in non-equilibrium membrane fluctuations manifesting from both metabolic and thermal energies and also maintains the biconcave shape of RBCs.

Fig. 1.

Effects of ATP on morphology and dynamic fluctuation in RBC membrane. Topography of a healthy RBC, (A) of an ATP-depleted RBC (irreversible-ATP group), (B) of an ATP-depleted RBC (metabolic-ATP group), (C, and of a RBC with recovered ATP level (+ATP group), (D) resp. (E–H) Instantaneous displacement maps of membrane fluctuation in the Fig. 1A–D, resp. The scale bar is 2 μm. The colorbar scales are in μm and nm, resp.

Fig. 2.

RMS displacements of membrane fluctuations for different ATP conditions: healthy RBCs, irreversibly ATP-depleted RBCs, metabolically ATP-depleted RBCs, and RBCs in which ATP was reintroduced to metabolically ATP-depleted RBCs. Each symbol represents an individual RBC and the horizontal line is the mean value.

Fig. 3.

Non-equilibrium dynamic in RBC membranes. Averaged non-Gaussian parameter, κ, versus a lag time, Δt, and a spatial frequency, q, for membrane fluctuation in healthy RBC, (A) irreversible ATP-depletion group, (B) metabolic ATP-depletion group, (C) and after reintroducing ATP to metabolic-depletion group, (D) resp. N = 40 RBCs per each group.

Fig. 4.

Correlation between biconcave shape and enhanced membrane fluctuations. (A–D) Averaged height as a function of the distance from the center of cells for healthy RBCs, (A) for RBCs in the irreversibly ATP-depleted group, (B) for RBCs in the metabolically ATP-depleted group, (C) and for RBCs in which ATP was reintroduced to the metabolically ATP-depleted group, (D) resp. (E-H) Averaged squared height fluctuations as a function of the distance from the center of cells in Fig. 3A–D, resp. Thick lines show the average value and the areas represent standard deviation for 40 RBCs.

Fig. 5.

Cytoskeletal structure and enhanced fluctuations. (A) Non-Gaussian parameter at short time delay (Δt < 0.1 sec) as a function of spatial wavelength. The presence of ATP lead to non-thermal fluctuations, especially at Λ = 361, 512, 680, 860, and 1030 nm. (B) Illustration showing the major proteins in the anchor complexes and the spectrin network. (C) Illustration showing the hexagonal lattice of spectrin network and a distance between neighboring junctions, Δ. (D) Non-Gaussian parameter at spatial wavelength (q = 15 rad/μm) as a function of a lag time. (E–F) Models explaining the non-equilibrium dynamics in RBC membranes by phosphorylation of PI (E) and by phosphorylation of protein-4.1 (F).