ATP-dependent mechanics of red blood cells

Abstract

Red blood cells are amazingly deformable structures able to recover their initial shape even after large deformations as when passing through tight blood capillaries. The reason for this exceptional property is found in the composition of the membrane and the membrane-cytoskeleton interaction. We investigate the mechanics and the dynamics of RBCs by a unique noninvasive technique, using weak optical tweezers to measure membrane fluctuation amplitudes with s temporal and sub nm spatial resolution. This enhanced edge detection method allows to span over >4 orders of magnitude in frequency. Hence, we can simultaneously measure red blood cell membrane mechanical properties such as bending modulus ‫؍‬ 2.8 ؎ 0.3 ؋ 10 ؊19 J ‫؍‬ 67.6 ؎ 7.2 kBT, tension ‫؍‬ 6.5 ؎ 2.1 ؋ 10 ؊7 N/m, and an effective viscosity eff ‫؍‬ 81 ؎ 3.7 ؋ 10 ؊3 Pa s that suggests unknown dissipative processes. We furthermore show that cell mechanics highly depends on the membrane-spectrin interaction mediated by the phosphorylation of the interconnection protein 4.1R. Inhibition and activation of this phosphorylation significantly affects tension and effective viscosity. Our results show that on short time scales (slower than 100 ms) the membrane fluctuates as in thermodynamic equilibrium. At time scales longer than 100 ms, the equilibrium description breaks down and fluctuation amplitudes are higher by 40% than predicted by the membrane equilibrium theory. Possible explanations for this discrepancy are influences of the spectrin that is not included in the membrane theory or nonequilibrium fluctuations that can be accounted for by defining a nonthermal effective energy of up to Eeff ‫؍‬ 1.4 ؎ 0.1 kBT, that corresponds to an actively increased effective temperature.erythrocyte ͉ membrane fluctuations ͉ nonequilibrium ͉ optical tweezer ͉ spectrin T he extraordinary deformability of RBCs is vital for their proper function, as it enables the cells to be elongated by more than twice their size when passing through m-sized capillaries. The elastic properties of RBCs are dominated by the interaction between the cell membrane and the underlying cytoskeleton, mainly consisting of spectrin, a long heterodimer that aligns tail to tail forming a 200-nm-long tetramer. Each spectrin filament interconnects to up to 5 other spectrins and is bound to the cell membrane via a protein complex consisting of protein 4.1R, actin, and glycophorin C (1). The plasma membrane fluctuations are related to RBCs' mechanical properties that have been extensively studied over the past three decades (2-6). It is well known that fluctuations of the membrane depend on its bending rigidity and its membrane tension (7). Furthermore, RBCs gain their elasticity from the elastic shear modulus of the cytoskeletal spectrin network (8, 9). Recent theoretical analysis (10) suggest that the spectrin cytoskeleton acts as a steric barrier restricting membrane undulations toward the network.Experiments by Tuvia and coworkers showed an ATP dependent effect by monitoring the static fluctuation amplit...

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).…”

“…Indeed, the ability of RBCs to undergo reversible large deformations cannot be rationalized on the basis of a fixed connectivity of the cytoskeleton, but instead requires a model that attributes metabolically driven forces to active remodeling of the RBC cytoskeleton (6,14). Therefore, RBC dynamics has been postulated to be metabolically regulated by continuous remodeling of the junctional nodes of the spectrin skeleton (6)(7)(8)14). Under the optical microscope, normal RBCs experience large membrane undulations at the equatorial emplacement, a phenomenon originally referred to as the RBC flicker (15,16).…”

Model for Probing Membrane-Cortex Adhesion by Micropipette Aspiration and Fluctuation Spectroscopy

“…3 a sets the largewavelength cutoff, q min~1 /R, and the short-wavelength cutoff is set by the spacing of the linkers: q max ¼ 2p/r 0 À1/2 . In fluctuation spectroscopy experiments, the laser focal diameter sets the limitation for the latter (43,44).…”

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

“…By comparing membrane fluctuations directly between healthy RBCs and cells depleted of ATP (adenosine triphosphate), recent experiments find a decrease of fluctuations upon ATP-depletion, hence suggesting that an active process may contribute to membrane undulations 6,[14][15][16] . 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.…”

“…Today, flickering analysis is envisaged as a promising tool for high-rate malaria diagnosis 4 . However, although equilibrium fluctuation models [5][6][7] have been used to infer mechanical properties of RBCs since the seminal work of Brochard & Lennon 8 , the fundamental question whether membrane fluctuations are driven by an active process or simply by thermal agitation remains controversial [9][10][11][12] .…”