Flicker spectroscopy of erythrocytes

Abstract

Frequency analysis of thermally excited surface undulations of erythrocytes leading to the flicker phenomenon is applied to determine biochemically and physically induced modulations of the membrane curvature elasticity. Flicker spectra of individual cells fixed to the window of a flow chamber by polylysine are taken by phase contrast microscopy, enabling investigations of the reversibility of the structural modifications. The spectra may be approximated by Lorentzian lines in most cases. By measuring the amplitude (at zero frequency) and the line width, effects of the structural changes on the curvature elastic constant, K _c , and the wavelength distribution of the undulations may be studied separately.

Effect of physically induced modifications: The temperature dependence of the flicker spectra are taken from 10°C to 37°C. Above 20°C, K _c decreases with increasing temperature whereas the reverse holds below this limit. The latter anomalous behaviour is explained in terms of a conformational change associated with protein and lipid lateral phase separation. The bending stiffness increases when the cells swell osmotically, owing to surface tension effects. The dependence of the flicker spectra on the viscosity of the suspension medium agrees with the theoretical prediction.

Biochemically and drug induced modifications: 5 vol‰ of ethanol leads to a pronounced and reversible suppression of the long wavelength undulations without altering the discoid cell shape and without affecting the bending stiffness appreciably. Adsorption of dextran to the glycocalix increases K _c by a factor of 1.6 at saturation. The bending stiffness is increased by a factor of 1.3 after cross-linking the proteins with the SH-oxidizing agent diamid. Injection of Ca⁺⁺ into the cell via ionophores evokes (within 10 min) the formation of fine — probably spectrin free — spicules. This leads to an increase in K _c by a factor of 1.3 which is explained in terms of a lateral condensation of the spectrin/actin network. The spicule formation and K _c change is completely reversible (within 2 min) after perfusion with Ca⁺⁺-free buffer. Cholesterol depletion leads first to a continuous increase in K _c without change of the cell shape whereas a sudden discocyte- to echinocyte transformation sets in below a critical steroid content. The latter transition is also observed in cell suspensions and is reminiscent of a phase transition. The anti-tumor drug actinomycin D evokes an increase in the bending stiffness K _c by a factor of two, suggesting that its effect is at least partially due to a modulation of the membrane structure. The α-receptor agonist leads to a remarkable increase in K _c (by about 25%) at 10^-4 M but the effect is not reversed by the α-antagonist prazosin, suggesting that the agonist exerts a non-specific effect.

A new technique, dynamic reflection interference contrast microscopy, is introduced by which absolute values of the amplitudes of the surface undulations and therefore K _c can be determined. The value obtained: K _c =5·10^-13 erg is about a factor of two larger then the bending stiffness of pure lipid bilayers. We suggest that the surface undulations may also be determined by lateral fluctuations of the quasi-two-dimensional spectrin/actin network.