Nonequilibrium fluctuations of mechanically stretched single red blood cells detected by optical tweezers

Abstract

We study the thermal and out-of-equilibrium mechanical dynamics of single, living human red blood cells (RBCs) by combining two-probe passive and active microrheology techniques. Both experiments were performed quasisimultaneously on the same cell using two identical polystyrene probes, biochemically attached to the cell membrane. We obtained compelling evidence of nonequilibrium fluctuations in the RBCs under physiological condition and without the influence of any external chemicals. The spectral distributions of metabolically driven forces and viscoelastic response were evaluated in the relaxed and stretched states, intended to simulate the varying natural environment of the cells during blood circulation. We found that the internally generated forces are more pronounced in the stretched state, suggesting a stress-dependent RBC activity.

Mechanical diagnosis of human erythrocytes by ultra-high speed manipulation unraveled critical time window for global cytoskeletal remodeling

“…Previously, the fast characteristic recovery time τ fast in the order of 0.1 s was evaluated from the viscoelastic shape recovery after a local deformation by micropipette aspiration 5 42 43 , the parachute-shaped deformation by multiple optical trapping 44 , and the entire shear deformation by Couette flow 45 . Recent shape fluctuation analyses suggested that the time window of dissociation-reassociation of a spectrin network is about 100 ms on a single node level 8 29 , whose ATP-dependent, non-equilibrium response could be evidenced by the violation of fluctuation-dissipation theorem 9 30 . However, although the timescale of fast recovery τ fast = 0.1–1 s apparently seems to agree, it is not valid to directly correlate the dissociation-reassociation of a single node and the global shape change.…”

“…The experimental strategy proposed in the present work enables to fill the gap between the fast ( τ fast = 0.1–1 s) and extremely slow ( τ ex-slow ~1000 s) shape recovery, unraveling a new time window reflecting the “global” cytoskeletal remodeling. An increasing number of studies suggested that the ATP-dependent deformation of a erythrocyte is originated from the cytoskeletal remodeling by using non-equilibrium contour fluctuation analysis 8 9 13 29 30 38 40 and shear deformation experiments 27 28 47 . Therefore, the significant delay of T c found for ATP-depleted erythrocytes can be attributed to the decrease in the connectivity between spectrin networks and the associated proteins, such as protein 4.1R 49 , band-3 50 51 , ankyrin 52 , actin 53 , and spectrin itself 54 .…”

“…There have been experimental and theoretical studies demonstrating that the remodeling of spectrin cytoskeleton depends on the concentration of adenosine triphosphate (ATP) 20 and shearing force 27 28 . Recent shape fluctuation analyses confirmed the discrepancy in the fluctuation spectrum between the intact and ATP-depleted states 8 29 and the violation of fluctuation-dissipation theorem 9 30 . The characteristic time window, about 100 ms, can be attributed to the dissociation-reassociation process of a single node of the triangular spectrin lattice.…”