Cross-correlation analysis of inner-leaflet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components

Biophys J. 2001 May;80(5):2120-32. doi: 10.1016/S0006-3495(01)76185-1.


To investigate the structural basis for membrane interactions that occur between Lyn tyrosine kinase and IgE-Fc(epsilon)RI or other components of lipid rafts, we prepared a green fluorescent protein analog of Lyn (PM-EGFP) and used cross-correlation analysis to quantify co-redistributions of aggregates that occur after IgE-Fc(epsilon)RI is cross-linked on the cell surface. PM-EGFP, which contains minimally the palmitoylation and myristoylation sites on Lyn, was compared with another inner leaflet probe, EGFP-GG, which contains a prenylation site and a polybasic sequence similar to K-ras. Confocal fluorescence microscopy was used to examine co-redistributions of these inner leaflet components with IgE-Fc(epsilon)RI and outer leaflet raft components, ganglioside GD1b and glycosylphosphotidylinositol-linked Thy-1, under conditions where the latter were cross-linked externally to form large patches at the cell surface. The cross-correlation analysis was developed and characterized with simulations representing cell surface distributions, and parameters from the cross-correlation curves, rho(o) (peak height) and A (peak area), were shown to be reliable measures of the extent of co-redistributed aggregates and their size. Cross-correlation analysis was then applied to quantify co-redistributions of the fluorescently labeled inner and outer leaflet components on RBL-2H3 cells. As visually observed and parameterized in this manner, PM-EGFP was found to co-redistribute with lipid rafts significantly more than EGFP-GG or an endogenous prenylated protein, Cdc42. These quantitative results are consistent with previous analyses of Lyn co-redistributions and support the hypothesis that the functionally important interaction of Lyn with cross-linked IgE- Fc(epsilon)RI is due to their mutual co-association with lipid rafts.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Biophysical Phenomena
  • Biophysics
  • Cell Line
  • Cell Membrane / metabolism
  • Cloning, Molecular
  • Green Fluorescent Proteins
  • Luminescent Proteins / chemistry*
  • Membrane Microdomains / chemistry*
  • Microscopy, Confocal
  • Models, Statistical
  • Molecular Sequence Data
  • Plant Physiological Phenomena
  • Plants / chemistry
  • Receptors, IgE / chemistry*
  • Recombinant Fusion Proteins / chemistry
  • Spectrometry, Fluorescence
  • Spectrophotometry
  • Tissue Distribution
  • cdc42 GTP-Binding Protein / chemistry
  • src-Family Kinases / chemistry


  • Luminescent Proteins
  • Receptors, IgE
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • lyn protein-tyrosine kinase
  • src-Family Kinases
  • cdc42 GTP-Binding Protein