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Cross-linking-induced association of membrane IgM (mIgM) with the cytoskeleton is well documented. However, its functional significance during B cell activation is not yet understood. One possible need for mIgM/cytoskeleton interactions may be to recruit the B cell receptor (BCR)-associated signaling molecules to the cytoskeletal matrix for the propagation of downstream signaling. We first verified whether BCR-associated Ig alpha translocates to the cytoskeleton together with mIgM in polyclonal anti-IgM-treated murine B lymphoma cell line, BAL17.7.1. Co-capping experiments and the purification of the membrane skeleton under conditions that preserve IgM-Ig alphabeta) interactions confirmed that Ig alpha translocates to the cytoskeleton as part of the BCR complex. Furthermore, two BCR-associated kinases that are known to play critical roles in anti-IgM-induced B cell signaling, the src family kinase Lyn and the non-src family kinase Syk, accumulate in the membrane skeleton shortly after BCR cross-linking, when most of IgM and Ig alpha accumulate in this fraction. The kinetics of recruitment of the bulk of Ig alpha, Lyn, and Syk into the membrane skeleton appeared to precede the accumulation of their hypertyrosine-phosphorylated forms, suggesting that activation of the BCR-associated signaling molecules occurs in this fraction. These data suggest that cross-linked mIgM translocating to the membrane skeleton serves as a vehicle for active signaling molecules to be recruited to this vicinity. This may promote B cell activation events by providing high affinity interactions between signaling molecules and their substrates supported by the cytoskeletal matrix.

The B cell Ag receptor complex consists of membrane-associated Ig (mIg), Ig alpha, and Ig beta, associated molecules that have been implicated in transducing the activation signal that occurs upon receptor cross-linking. The role of the Ig alpha beta heterodimer in mediating binding to the cytoskeleton is unknown. We studied the ligand-induced association of mIgM with the cytoskeleton following receptor cross-linking in mIgM-expressing B lymphoma lines by biochemical assays, FACS analysis, and electron microscopy. Cytoskeletal association is not detected in unstimulated cells, but occurs rapidly upon anti-IgM-mediated cross-linking. Ig alpha is absent from the cytoskeleton-mIgM complex. To further analyze the possible role of Ig alpha beta in cytoskeletal binding, a surface Ig alpha beta-negative plasmacytoma line was transfected with a mutant form of mIgM (IgM-MutA). IgM-MutA is expressed on the surface despite the lack of Ig alpha beta, and the cytoskeletal binding occurred to a similar extent as in Ig-alpha-positive cell lines. In another transfectant expressing a mutated form of human mIgM (YS:VV), which does not have the capacity to bind to Ig alpha beta, the association of the receptor with the cytoskeleton appeared to be more extensive (100%) and faster than with mouse mIgM. These data indicate that Ig-associated Ig alpha beta proteins are not required for mIgM association with the cytoskeleton.