Plasma membrane organization affects lipid removal mediated by human apolipoprotein A-I

JAUREGUIBERRY, M. S.; SANCHEZ, S. A.; FINARELLI, G. S.; TRICERRI, M. A.; RIMOLDI. O. J.

Universidad de Buenos Aires, Buenos Aires

Workshop; 1st Workshop on Advanced Fluorescence Microscopy.; 2007

Departamento de Física, UBA

Cellular cholesterol efflux is central to the anti-atherogenic role of high density lipoproteins (HDL) and its major apolipoprotein, apolipoprotein A-I (apoA-I). Cholesterol efflux mediated by apoA-I can involve several different pathways, including an active efflux via the ABCA1 transporter, and passive pathways mediated by scavenger receptor BI. In order to evaluate the importance of membrane composition and lateral domain phase separation on apoA-I interactions and cholesterol mobilization, we constructed a permanent Chinese Hamster Ovary Cell line (CHO K1) overexpressing the rat Stearoyl CoA Desaturase gen (SCD). Cells transfected with an expression vector in mammalian hosts (containing or not the gen for the SCD), induced a stable increase in fatty acid unsaturation at the membrane phospholipids. SCD mRNA overexpression was characterized by northern blotting. We proved by western blotting with specific antibodies that, as expected, an increase in the expression level of SCD. SCD activity was evaluated by analysis of fatty acid composition at the plasma membrane, showing a relative increase of about 2.1 times the ratio 16:1/16:0 and 18:1/18:0. Moreover, ABCA1 was not modified in SCD-overexpressing cells respect to the control.In addition, incubation of SCD overexpressing cells with apoA-I, resulted in a decreased cholesterol removal as well as in the formation of lipid-apolipoprotein particles, in a process probably mediated by cellular cholesterol esterifcation via acyl cholesterol, acyl transferase. Furthermore, membrane fluidity was assessed by Laurdan General Polarization (GP) and 2-photon fluorescence microscopy, in control cells and in cells treated with methyl beta cyclo dextrines. Results are discussed in terms of lateral phase separation