Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling

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Abstract
B cell responses are initiated by the clustering of the B cell receptor (BCR) by the binding of multivalent antigens. Clustering leads to phosphorylation of tyrosines in the cytoplasmic domains of the BCR by the inner plasma membrane leaflet-associated Src-family kinase Lyn. At present, little is known about the earliest events after BCR clustering that precede the BCR's phosphorylation by Lyn. Here we use fluorescence resonance energy transfer (FRET) in living cells to detect the interaction of the BCR with a Lyn-based membrane-targeted reporter in the first several seconds after BCR clustering. The results showed that, within seconds of antigen binding, the BCR selectively and transiently associated with the Lyn construct and that this association preceded by several seconds the triggering of Ca2+ fluxes and could be prolonged by the engagement of the B cell coreceptor complex, CD19/CD21. Thus, FRET measurements in living B cells revealed highly dynamic and regulated antigen-induced changes in the plasma membrane, allowing association of the BCR with the earliest components of its signaling cascade.