LATERAL MOBILITY OF LIPID ANALOGS AND GPI-ANCHORED PROTEINS IN SUPPORTED BILAYERS DETERMINED BY FLUORESCENT BEAD TRACKING

Lipid analogues and glycosylphosphatidylinositol (GPI)-anchored protei ns incorporated in glass-supported phospholipid bilayers (SBL) were co upled to small (30 nm diameter) fluorescent beads whose motion in the liquid phase was tracked by intensified fluorescence video microscopy. Streptavidin (St), covalently attached to the carboxyl modified surfa ce of the polystyrene bead, bound either the biotinylated membrane com ponent, or a biotinylated monoclonal antibody (mAb) directed against a specific membrane constituent. The positions of the beads tethered to randomly diffusing membrane molecules were recorded at 0.2 sec interv als for about 1 min. The mean square displacement (rho) of the beads w as found to be a linear function of diffusion time t, and the diffusio n coefficient, D, was derived from the relation, rho(t) = 4Dt. The val ues of D for biotinylated phosphatidylethanolamine (Bi-PE) dispersed i n an egg lecithin: cholesterol (80:20%) bilayer obtained by this metho dology range from 0.05 to 0.6 mum2/sec with an average of (D) = 0.26 m um2/sec, similar to the value of [D] = 0.24 /mum2/sec for fluorescein- conjugated phosphatidylethanolamine (Fl-PE) linked to St-coupled beads by the anti-fluorescein mAb 4-4-20 or its Fab fragment. These values of D are comparable to those reported for Fl-PE linked to 30 nm gold p articles but are several times lower than that of Fl-PE in the same pl anar bilayer as measured by fluorescence photobleaching recovery, D = 1.3 mum2/sec. The mobilities of two GPI-anchored proteins in similar S BL were also determined by use of the appropriate biotinylated mAb and were found to be [D] = 0.25 and 0.56 mum2/sec for the decay accelerat ing factor (DAF, CD55) and the human FcgammaRIIIB (CD16) receptors, re spectively. The methodology described here is suitable for tracking an y accessible membrane component.