ATP-dependent mechanics of red blood cells

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 μs 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 κ = 2.8 ± 0.3 × 10−19J = 67.6 ± 7.2 kBT, tension σ = 6.5 ± 2.1 × 10−7N/m, and an effective viscosity ηeff = 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.

[1]  Thorsten Auth,et al.  Fluctuations of coupled fluid and solid membranes with application to red blood cells. , 2007, Physical review. E, Statistical, nonlinear, and soft matter physics.

[2]  V. Ohanian,et al.  Analysis of the ternary interaction of the red cell membrane skeletal proteins spectrin, actin, and 4.1. , 1984, Biochemistry.

[3]  N. Mohandas,et al.  Modulation of Erythrocyte Membrane Mechanical Function by Protein 4.1 Phosphorylation* , 2005, Journal of Biological Chemistry.

[4]  A. Cumano,et al.  Forced Unfolding of Proteins Within Cells , 2007 .

[5]  A. Zilman,et al.  Hydrodynamics of confined membranes. , 2004, Physical review. E, Statistical, nonlinear, and soft matter physics.

[6]  F. MacKintosh,et al.  Nonequilibrium Mechanics of Active Cytoskeletal Networks , 2007, Science.

[7]  J. Fournier,et al.  Fluctuation spectrum of fluid membranes coupled to an elastic meshwork: jump of the effective surface tension at the mesh size. , 2003, Physical review letters.

[8]  E. Evans,et al.  Mechanical properties of the red cell membrane in relation to molecular structure and genetic defects. , 1994, Annual review of biophysics and biomolecular structure.

[9]  S Levin,et al.  Cell membrane fluctuations are regulated by medium macroviscosity: evidence for a metabolic driving force. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[10]  F. MacKintosh,et al.  Viscoelastic properties of actin-coated membranes. , 2001, Physical review. E, Statistical, nonlinear, and soft matter physics.

[11]  Yeang H. Ch'ng,et al.  A widely expressed betaIII spectrin associated with Golgi and cytoplasmic vesicles. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[12]  E. Sackmann,et al.  Measurement of erythrocyte membrane elasticity by flicker eigenmode decomposition. , 1995, Biophysical journal.

[13]  Kim Parker,et al.  Fluctuations of the red blood cell membrane: relation to mechanical properties and lack of ATP dependence. , 2008, Biophysical journal.

[14]  Nir S Gov,et al.  Active elastic network: cytoskeleton of the red blood cell. , 2007, Physical review. E, Statistical, nonlinear, and soft matter physics.

[15]  F. Brochard,et al.  Frequency spectrum of the flicker phenomenon in erythrocytes , 1975 .

[16]  E. Evans Bending elastic modulus of red blood cell membrane derived from buckling instability in micropipet aspiration tests. , 1983, Biophysical journal.

[17]  G. Cokelet,et al.  Rheological Comparison of Hemoglobin Solutions and Erythrocyte Suspensions , 1968, Science.

[18]  Suliana Manley,et al.  Optical measurement of cell membrane tension. , 2006, Physical review letters.

[19]  N. Gov,et al.  Red blood cell membrane fluctuations and shape controlled by ATP-induced cytoskeletal defects. , 2005, Biophysical journal.

[20]  A J Hudspeth,et al.  Comparison of a hair bundle's spontaneous oscillations with its response to mechanical stimulation reveals the underlying active process , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Subra Suresh,et al.  Cytoskeletal dynamics of human erythrocyte , 2007, Proceedings of the National Academy of Sciences.

[22]  D. Discher,et al.  Deformation-enhanced fluctuations in the red cell skeleton with theoretical relations to elasticity, connectivity, and spectrin unfolding. , 2001, Biophysical journal.

[23]  Francesco S. Pavone,et al.  Calibration of optical tweezers with positional detection in the back focal plane , 2006, physics/0603037.

[24]  Milner,et al.  Dynamical fluctuations of droplet microemulsions and vesicles. , 1987, Physical review. A, General physics.

[25]  E. Evans,et al.  Molecular maps of red cell deformation: hidden elasticity and in situ connectivity. , 1994, Science.

[26]  W. Helfrich Elastic Properties of Lipid Bilayers: Theory and Possible Experiments , 1973, Zeitschrift fur Naturforschung. Teil C: Biochemie, Biophysik, Biologie, Virologie.

[27]  Seifert Dynamics of a bound membrane. , 1994, Physical review. E, Statistical physics, plasmas, fluids, and related interdisciplinary topics.

[28]  A. Baines,et al.  Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. , 2001, Physiological reviews.

[29]  K. Neuman,et al.  Optical trapping. , 2004, The Review of scientific instruments.

[30]  N. Gov,et al.  Pinning of fluid membranes by periodic harmonic potentials. , 2004, Physical review. E, Statistical, nonlinear, and soft matter physics.

[31]  Simultaneous manipulation and detection of living cell membrane dynamics. , 2007, Optics letters.

[32]  Jacques Prost,et al.  Fluctuation-magnification of non-equilibrium membranes near a wall , 1998 .

[33]  A. Zilman,et al.  Cytoskeleton confinement and tension of red blood cell membranes. , 2003, Physical review letters.