Previous studies showed that crosslinking of IgE-Fc(epsilon)RI complexes on RBL-2H3 mast cells causes their association with isolated detergent-resistant membranes, also known as lipid rafts, in a cholesterol-dependent process that precedes initiation of signaling by these receptors. To investigate these interactions on intact cells, we examined the co-redistribution of raft components with crosslinked IgE-Fc(epsilon)RI using confocal microscopy. After several hours of crosslinking at 4 degrees C, the glycosylphosphatidylinositol-linked protein Thy-1 and the Src-family tyrosine kinase Lyn co-redistribute with IgE-Fc(epsilon)RI in large patches at the plasma membrane. Under these conditions, F-actin also undergoes dramatic co-segregation with Fc(epsilon)RI and raft components but is dispersed following a brief warm-up to 37 degrees C. When crosslinking of IgE-Fc(epsilon)RI is initiated at higher temperatures, co-redistribution of raft components with patched Fc(epsilon)RI is not readily detected unless stimulated F-actin polymerization is inhibited by cytochalasin D. In parallel, cytochalasin D converts transient antigen-stimulated tyrosine phosphorylation to a more sustained response. Sucrose gradient analysis of lysed cells reveals that crosslinked IgE-Fc(epsilon)RI remains associated with lipid rafts throughout the time course of the transient phosphorylation response but undergoes a time-dependent shift to higher density that is prevented by cytochalasin D. Our results indicate that interactions between Lyn and crosslinked IgE-Fc(epsilon)RI are regulated by stimulated F-actin polymerization, and this is best explained by a segregation of anchored raft components from more mobile ones.

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