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

Extracellular fluid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body fluids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mecha- nism of this phenomenon, we examined the effect of EFM on mechanical fluctuations 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 car- boxymethyl-cellulose (100 kDa)), which differ in size, chem- ical nature, and in their capacity to increase fluid viscosity. The parameters of cell membrane fluctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, re- gardless of the cosolvent used to increase EFM. Because thermallydrivenmembranefluctuationscannotbedampedby elevation of EFM, the existence of am etabolic driving force is suggested. This is supported by the finding that in ATP- depleted red blood cells elevation of EMF did not affect cell membrane fluctuations. 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 fluctuations are driven by a metabolic driving force in addition to the thermal one. 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 thepossibilitythattheeffectofmacroviscosityoncellfunction istransducedthroughadirecteffectofEFMoncellmembrane dynamics. To examine this hypothesis, we investigated the effect of extracellular fluid viscosity on the cell membrane fluctuations (CMF). CMF, first explored in red blood cells (erythrocytes; RBCs) (19), consist of submicron, out-of-plane displacements of the cellmembraneinthefrequencyrangeof0.3-30Hz.CMFwere recentlyobservedindifferenttypesofnucleatedcells(20-23), suggestingthatCMFareacommoncharacteristicoftheliving cell. RBC plasma membrane and its underlying skeleton are relatively well characterized, making it an optimal cellular model system to study CMF. In the present study we examined the modulation of RBC membrane fluctuations by the macroviscosity of the external medium. The CMF were monitored by point dark field mi- croscopy (20-24), where a very small area (0.25 mm 2 )a t the cell edge was illuminated and the time-dependent intensity changes of the reflected and scattered light due to cell membranemovementinandoutofthefocusedlightspotwere recorded.