Effects of Cholesterol and Sitosterol on the dynamical and functional properties of plasma membranes in live cells

M.A. BAQUÉ,; N.M.C. CASADO; J. ANDINI; V. GIORIA; J. CLAUS; A. M. GENNARO

Montevideo, Uruguay

Congreso; XXXVI Reunión Anual de la Sociedad Argentina de Biofísica, 6th International Conference on Biological Physics y 5th Southern Cone Biophysics Congress; 2007

SAB- IUPAB- IUPAP

As insect cells lack the capability to synthesize sterols, the lipid profile of plasma membrane in cultured insect cells is strongly dependent on the culture medium composition. Sterols affect short and long range membrane order with a specific impact on its fluidity. It is also known that the function of membrane-bound enzymes, receptors and transport carriers is correlated with membrane fluidity. In previous studies we observed that cultures of the lepidopteran insect cell line UFL-AG-286 adapted to grow in a cholesterol-free culture medium are highly refractary to baculovirus infection. Moreover, the kinetics of baculovirus infection appeared to be influenced by the type of sterol, either cholesterol or sitosterol, used to feed UFL-AG-286 cells. The aim of this work is to correlate the dynamical properties of the plasma membrane of UFL-AG-286 cells, with the incorporation of different sterols, and their response to baculovirus infection. First, in order to gain insight into the influence of the type of sterol on the lipid packing of a model plasma membrane, egg PC/Cholesterol and egg PC/sitosterol unilamellar vesicles (LUV) with different sterol contents were studied by Electronic Paramagnetic Resonance (EPR). Both sterols decreased membrane fluidity with no differences for sterol/phospholipids ratio lower than 10:90, but at higher sterol proportions, LUVs containing cholesterol showed reduced fluidity. Thereafter, to know the influence of both the sterol content and the type of sterol on the dynamical and functional properties of plasma membranes in live cells, UFL-AG-286 cells were treated with methyl-beta-cyclodextrin, that reduced more than 80% of the cellular sterol content measured by gas chromatography. Then, two aliquots of treated cells were fed with either cholesterol or sitosterol emulsion, while a third aliquot of treated cells was not fed with sterols. Two aliquots of treated cells were fed with either cholesterol or sitosterol emulsion, while a third aliquot of treated cells was not fed with sterols. The membrane fluidity in each group of cells was estimated through EPR measurements with 5, 12 and 16-SASL as spin labels, while the susceptibility to viral infection was determined following the kinetics of AgMNPV virus adsorption. Both results will be correlated.