Antigen-stimulated trafficking from the recycling compartment to the plasma membrane in RBL mast cells.

Abstract

Binding of fluorescein isothiocyanate (FITC)-conjugated cholera toxin B subunit to ganglioside GM1 on RBL-2H3 cells at 37 degrees C results in labeling of the plasma membrane as well as a pool of perinuclear intracellular membranes identified as the endosomal recycling compartment. Antigen-mediated activation of IgE receptor signaling causes rapid, sustained outward trafficking of these labeled endosomes, that is monitored as an increase in FITC fluorescence due to relief of quenching in the acidic endosomes upon delivery to the plasma membrane. Stimulation of this process depends on the integrity of cholesterol-dependent lipid rafts and occurs in response to Ca2+-mobilizing thapsigargin as well as antigen. Inhibitors of some early signaling enzymes stimulated by FcepsilonRI, including Syk tyrosine kinase and phosphoinositide 3-kinase, have little or no effect on this trafficking response. Other signaling pathways, including activation of phospholipase C and Ca2+ influx, do not appear to be necessary for the initiation of the outward trafficking response, but they contribute to maintaining the sustained phase of this process. Consistent with this, antigen-stimulated ruffles are labeled with FITC-cholera toxin B in a Ca2+-dependent manner. Thus, this trafficking response provides a mechanism by which an internal membrane pool can contribute to plasma membrane remodeling during stimulated membrane ruffling, cell motility, and phagocytosis.