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Lateral mobility of lipid analogues and GPI-anchored proteins in supported bilayers determined by fluorescent bead tracking

Abstract

Lipid analogues and glycosylphosphati-dylinositol (GPI)-anchored proteins incorporated in glass-supported phospholipid bilayers (SBL) were coupled 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 surface of the polystyrene bead, bound either the biotinylated membrane component, 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 intervals for about l min. The mean square displacement (ϱ) of the beads was found to be a linear function of diffusion time t, and the diffusion coefficient, D, was derived from the relation, ϱ(t) = 4Dt. The values of D for biotinylated phosphatidylethanolamine (Bi-PE) dispersed in an egg lecithin: cholesterol (80:20%) bilayer obtained by this methodology range from 0.05 to 0.6 μm2/sec with an average of 〈D〉 = 0.26 μm2/sec, similar to the value of 〈D〉 = 0.24 μm2/sec for fluorescein-conjugated phosphati-dylethanolamine (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 particles but are several times lower than that of Fl-PE in the same planar bilayer as measured by fluorescence photobleaching recovery, D = 1.3 μm2sec. The mobilities of two GPI-anchored proteins in similar SBL were also determined by use of the appropriate biotinylated mAb and were found to be 〈D〉 = 0.25 and 0.56 μm2/sec for the decay accelerating factor (DAF, CD55) and the human FcγRIIIB (CD16) receptors, respectively. The methodology described here is suitable for tracking any accessible membrane component.

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This work was supported by National Institutes of Health grants 1R24 RR05272 and AI-24322.

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Fein, M., Unkeless, J., Chuang, F.Y.S. et al. Lateral mobility of lipid analogues and GPI-anchored proteins in supported bilayers determined by fluorescent bead tracking. J. Membarin Biol. 135, 83–92 (1993). https://doi.org/10.1007/BF00234654

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Key words

  • Single particle tracking
  • Lateral diffusion
  • Fluorescent beads
  • Brownian motion
  • Fluorescence imaging
  • Membrane fluidity