Cell membrane fluctuations are regulated by medium macroviscosity: Evidence for a metabolic driving force

Abstract

Extracellular f luid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body f luids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mechanism of this phenomenon, we examined the effect of EFM on mechanical f luctuations of the cell membrane of human erythrocytes. The macroviscosity of the external medium was varied by adding to it various macromolecules [dextrans (70, 500, and 2,000 kDa), polyethylene glycol (20 kDa), and carboxymethyl-cellulose (100 kDa)], which differ in size, chemical nature, and in their capacity to increase f luid viscosity. The parameters of cell membrane f luctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, regardless of the cosolvent used to increase EFM. Because thermally driven membrane f luctuations cannot be damped by elevation of EFM, the existence of a metabolic driving force is suggested. This is supported by the finding that in ATPdepleted red blood cells elevation of EMF did not affect cell membrane f luctuations. This study demonstrates that (i) EFM is a regulator of membrane dynamics, providing a possible mechanism by which EFM affects cell membrane activities; and (ii) cell membrane f luctuations are driven by a metabolic driving force in addition to the thermal one.The viscosity of body fluids is determined by the level of macromolecules consisting of proteins, lipoproteins, and polysacharides (1). Accordingly, elevated plasma viscosity has been observed in various diseases associated with increased levels of proteins and lipoproteins, such as diabetes, hyperlipidemia, macroglobulinemia, multiple myeloma, nephrosis, and others (1-5). Various studies have shown that solvent viscosity affects protein dynamics and reactions (6-10). However, in these studies the solvent viscosity was modified by the addition of high concentrations of small cosolvents such as glycerol and sucrose, producing relatively high viscosity levels. This is incompatible with physiological and pathological states, where fluid viscosity is altered by small concentrations of large macromolecules (1). Other studies, in which the viscosity was elevated by macromolecular cosolvents, have shown that extracellular fluid macroviscosity (EFM) is a regulator of cellular processes, such as secretion of renin (11) and lipoproteins (12), phospholipase A 2 activity at the cell membrane (13, 14), and ganglioside metabolism (15). In search of the mechanism of this phenomenon, the effect of macroviscosity, as modified by macromolecules, on isolated proteins in aqueous solutions was examined (16,17). It was found that the effect of solvent viscosity decreases with increasing molecular weight of the cosolvent and is practically diminished when the cosolvent molecular weight exceeds that of the protein. Because the activity of cell membrane enzymes is known to be sensitive to the physical properties of the membrane (18), we considered the possibility that the...

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. To make a different assessment of the involvement of metabolic activity in flickering, an attractive approach was used by Tuvia et al 14 : at equilibrium, the mean-square fluctuations amplitude is a thermodynamic variable, which, by definition, cannot depend on the dynamics, and in particular on medium viscosity. Tuvia et al 14 reported a viscosity dependence of the mean-square fluctuation amplitude in a frequency range of 0.3-30Hz on changing the suspending medium and therefore drew the conclusion of a metabolic origin of flickering.…”

“…To make a different assessment of the involvement of metabolic activity in flickering, an attractive approach was used by Tuvia et al 14 : at equilibrium, the mean-square fluctuations amplitude is a thermodynamic variable, which, by definition, cannot depend on the dynamics, and in particular on medium viscosity. Tuvia et al 14 reported a viscosity dependence of the mean-square fluctuation amplitude in a frequency range of 0.3-30Hz on changing the suspending medium and therefore drew the conclusion of a metabolic origin of flickering. However, recent analysis of fluctuations over a larger bandwidth (0.1-1000Hz) shows no detectable dependence of fluctuation amplitudes on medium viscosity 6 , as also confirmed here (Supplementary Figure S1 and SI Appendix C), hence making the conclusions of Tuvia et al equivocal.…”

Атомно-Силовая Микроскопия Набухания И Упрочнения Закрепленных На Подложке Интактных Эритроцитов

“…В АСМ исследовании [9] сообщалось, что интактные эритроциты демонстрировали на подложке необычную, распластанную плоскую форму, что связывалось с воз-росшим натяжением мембраны клетки, реагирующей на контакт с полилизином. Отмечалось [10], что на под-ложках, обработанных концентрированным раствором полилизина, провоцируется разрыв мембраны и лизис эритроцитов. Если же брать разбавленный раствор с концентрацией менее 1 mg/ml, то покрытие подложки полилизином становится не сплошным, и падает надеж-ность иммобилизации эритроцитов.…”

“…В ис-следованиях [10,15] было замечено, что на подложках, обработанных раствором полилизина в концентраци-ях 100 µg/ml, эритроциты меняли форму, а при кон-центрации 250 µg/ml происходил лизис клеток. В нашей работе брались на порядок меньшие концентрации по-лилизина, но отклонения формы эритроцитов от тра-диционной дисковидной двояковогнутой геометрии не исчезали.…”