Biophys J, May 2001, p. 2120-2132, Vol. 80, No. 5

Cross-Correlation Analysis of Inner-Leaflet-Anchored Green Fluorescent Protein Co-Redistributed with IgE Receptors and Outer Leaflet Lipid Raft Components

Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853 USA

To investigate the structural basis for membrane interactions that occur between Lyn tyrosine kinase and IgE-FcRI 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-FcRI 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-FcRI and outer leaflet raft components, ganglioside GD_1b 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, _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- FcRI is due to their mutual co-association with lipid rafts.

Biophys J, May 2001, p. 2120-2132, Vol. 80, No. 5
© 2001 by the Biophysical Society   0006-3495/01/05/2120/13  $2.00

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