Structural information reported by different spin probes in the membrane of insect cells

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

San Javier, Tucumán

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofisica

The lipid profile of plasma membrane in cultured insect cells is dependent on the culture medium composition, because these cells do not have the ability to synthesize sterols. In previous works, we adapted cells to grow in a cholesterol-free culture medium, and also in media containing cholesterol and sitosterol. Viral infection kinetics in the adapted cells showed to be influenced by the type of sterol used. We use spin label EPR spectroscopy in order to detect effects of the different sterols on the structural properties of the cell membranes. The liposoluble n-doxyl-stearic acid spin labels (n-SASL: 5, 12, 16) were first used, but no measurable differences in membrane fluidity among the three cell types were observed, probably as a result that these labels measure an average fluidity. In order to test the hypothesis that a probe structurally similar to the sterols would provide more reliable information regarding sterol rich lipid domains in the membrane, we then used the spin label 3β-doxyl-5α-cholestane (cholestane). As a first step, in order to evaluate the sensitivity of this label to changes in lipid fluidity, we studied unilamellar vesicles cholesterol/eggPC and -sitosterol/eggPC with different contents of sterol. Then, cholestane was incorporated to the adapted cells and their viability was determined using Trypan blue dye exclusion assay. We observed a dissimilar behavior in spin label incorporation to the different cell variants, supporting the hypothesis that cholestane is capable of reflecting the performance of different sterols in the cells membrane. Acknowledgements: work supported by CONICET and Universidad Nacional del Litoral.