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

Martin Fein, Jay Unkeless, Frank Chuang, Massimo Sassaroli, Rui da Costa, Heikki Väänänen, Josef Eisinger

Research output: Contribution to journalArticle

33 Scopus citations

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{variant}) 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{variant}(t) = 4 Dt. 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 Fc γRIIIB (CD16) receptors, respectively. The methodology described here is suitable for tracking any accessible membrane component.

Original languageEnglish (US)
Pages (from-to)83-92
Number of pages10
JournalThe Journal of Membrane Biology
Volume135
Issue number1
DOIs
StatePublished - Jul 1993
Externally publishedYes

Keywords

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

ASJC Scopus subject areas

  • Physiology
  • Cell Biology
  • Biophysics

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