B cell antibody responses are triggered by the binding of antigen to the clonally distributed B cell antigen receptors (BCRs). Over the last several years a great deal has been learned about the biochemistry of the complex signal cascades triggered by BCR antigen engagement. However, what remains relatively poorly understood are the molecular events that trigger the initiation of signaling. We understand that the BCR, like other members of the multichain immune recognition receptor family, is composed of a ligand binding chain, for the BCR a membrane form of Ig, mIg, that has a short cytoplasmic domain with no capacity to interact directly with the components of the signaling cascades. To do so the mIg associates with two additional chains, Ig alpha and Ig beta, that contain within their cytoplasmic domains immunoregulatory tyrosine activation motifs (ITAMs). The BCR has no inherent kinase activity but following antigen binding one of the first events observed is the phosphorylation of the BCR ITAMs by the Src-family kinase, Lyn. This project represents an approach to understanding the changes that occur in the BCR following antigen binding that allow Lyn to discriminate the antigen-bound BCR from the unbound BCR. Based on the crystal structure of antigens bound to antibodies there is at present no evidence for an antigen-induced structural change in the BCR that could propagate the information that the BCR has bound antigen from the BCR ectodomains, across the membrane, to the cytoplasmic domains. In the absence of such an allosteric effect of antigen binding one is left with antigen-induced clustering of the BCR as the trigger for the initiation of signaling. Thus, the goal of this project is to gain an understanding of how antigen induces BCRs to cluster and how BCR clustering leads to the initiation of signaling. ? ? The critical events that trigger signaling are likely to occur within seconds of antigen binding to the BCR and to be highly dynamic, involving many weak protein-protein and protein-lipid interactions. In general, the biochemical approaches that have been used so effectively to describe the BCR signaling pathways are inadequate to capture events that occur as rapidly and as transiently as those predicted to initiate BCR signaling. In addition, antigen binding to the B cell involves a dramatic spatial change in the BCRs resulting in their patching and capping and the formation of an immune synapse. All this potentially important spatial information is lost with the addition of detergents to cells for biochemical analyses. The use of detergents is particularly problematic in the study of the early membrane changes that accompany antigen binding by BCRs. Consequently we have taken advantage of live cell imaging technologies that allow analyses of the BCR and components of the BCR signaling pathway with the temporal and spatial resolution necessary to view the earliest events in B cell activation without the complications that the addition of detergents introduce. ? ? Over the last year we have applied the new technology of fluorescence resonance energy transfer (FRET) imaging that allows us to view the interactions of proteins and lipids in living cells, to better characterize the earliest events in antigen-driven BCR signaling, namely, the oligomerization of the receptor and its association with sphingolipid- and cholesterol-rich membrane microdomains termed lipid rafts. Using quantitative FRET imaging we showed that the BCR is a monomer of the surface of resting cells and that multivalent antigen binding results in the simultaneous phosphorylation of the receptors cytoplasmic domains of the BCR, a change in the conformation of the cytoplasmic domains of the BCR from a clustered to an open form and the rapid yet transient association with lipids that compose lipid rafts. These events precede the activation of downstream signaling events and require the continuous activity of Src-family kinases but not the binding of Syk. Thus, the initiation of BCR signaling is a remarkably dynamic process accompanied by reversible conformation changes and raft lipid associations induced by Src-family kinase activity. Efforts are under way to introduce appropriate FRET donor and acceptor pairs into transgenic mice to allow us to image signaling events in normal B cell subsets. To better resolve the spatial and temporal order of the early events in B cell activation we used total internal reflection fluorescence microscopy (TIRFM) in conjunction with FRET. We showed that in B cells responding to antigen presented on a planar lipid bilayer, simulating an antigen presenting cell, BCRs form signaling active microclusters by a mechanism that does not require physical crosslinking of BCRs by multivalent antigens. We observed that in response to either monovalent or multivalent antigens the BCRs accumulate and form microclusters at the initial points of contact of the B cell membrane with the bilayer. Clustering did not depend on a signaling active BCR revealing an intrinsic tendency of the BCR to cluster. The microclusters grow by trapping mobile BCR and the larger clusters are actively organized into an immune synapse. The kinetics of these events and signaling are identical for monovalent and multivalent antigens. Our findings that monovalent interactions between the BCR and antigen lead to B cell activation provides a mechanism by which B cells are able to respond to the topologically diverse, unpredictable world of pathogen antigens. These results may also be highly relevant to the mechanism by which T cell receptors cluster in response to monovalent peptide-MHC complexes on antigen presenting cell surfaces. ? ? We also use FRET in conjunction with TIRFM to describe the interaction of the BCR with a membrane raft lipid probe and the membrane raft lipid-tethered Src-family kinases, Lyn. We provide evidence for an ordered process in which the antigen-induced BCR microclusters form and then transiently associate with the raft lipid probe in a signaling- and cytoskeleton-independent fashion. Association with raft lipids leads to a more stable interaction of the clustered BCR with Lyn that persists as the BCR microclusters move to form a central synapse. Taken together these results provide a new view of the earliest events in BCR signaling, underscoring a role of BCR-lipid interactions in the formation of signaling active BCR complexes.
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