Cholesterol depletion affects membrane lipid order and GM1 localization in amphibian oocyte rafts

BUSCHIAZZO, J.; BONINI, I. C.; ANTOLLINI, S. S.; ALONSO, T. S.

Puerto Madryn, Chubut, Argentina

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2010

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

The fluorescent probe Laurdan was used to study the effect of progesterone-induced maturation and cholesterol depletion mediated by methyl-beta-cyclodextrin (MbetaCD) on biophysical properties of membranes from Rhinella arenarum ovarian oocytes. MbetaCD treatment was performed to determine changes in the localization of GM1 which could be indicative of membrane raft disorganization. The effect of maturation, induced either by progesterone or by ceramide, on the localization of GM1 was also analyzed. Functional studies regarding tyrosine phosphorylation were performed in light membranes (L) evidencing that multiple substrates and active tyrosine kinases are already organized in immature oocytes. Plasma membrane fractions showed two different thermotropic profiles of generalized polarization (GP) analyzed by direct excitation and by FRET. MbetaCD treatment decreased the GP value of L, indicating a greater lipid disorder whereas heavy membranes (H) evidenced an increase in membrane lipid order. Levels of ganglioside GM1 showed that after drug treatment the signal decreases in L. As a result, both fractions become more similar and this is reflected in their biophysical states. Progesterone modified plasma membrane fluidity increasing the lipid order of both fractions. Ceramide affected the distribution of GM1 among membrane fractions while progesterone seems not to affect membrane microdomain integrity.