B-cell hybrids expressing both a membrane-associated and secreted form of immunoglobulin have been generated by the fusion of antigen-primed splenic lymphocytes with either drug-resistant B-lymphomas or myelomas. These hybrids are specific for the charged hapten phthalate and after cloning, the hybrids have been shown to be phenotypically stable for two years. We are using these hybrids to study and record biochemical and ultrastructural events that occur following cross-linking of hapten-binding receptors with hapten-protein conjugates. In an attempt to understand transmembrane signaling of B-cells, one focus is to determine whether plasma membrane proteins as well as cytoplasmic cytoskeletal proteins become associated with B-cell receptors following antigen binding. Directly related to this focus is our desire to establish how the receptor-ligand complex that forms on the surface of the hybrid is internalized, and what its fate is after internalization. In the course of our studies with the B-cell hybrids three unexpected observations have been made and will be investigated further. Firstly, we have observed that spectrin, now recognized to be an important membrane-associated cytoskletal protein in lymphocytes, exists in a capped configuration in a particular subset of lymphocytes found within lymphoid organs. In neonatal mouse thymus, spectrin is observed as a coincidental cap with H-2 antigen. Immature B cells grown in culture can also be distinguished by the observation that their spectrin is in the form of a discrete cap rather than in the diffuse contiguration seen in mature B lymphocytes. Thus, spectrin distribution may serve as a useful marker for analysis of lymphocyte differentiation. Secondly, secreted immunoglobulin appears to be released from a restricted domain in the membrane of the B-cell hybrids. This implies that there may be a significant rearrangement of membrane proteins and associated cytoskeletal elements in this restricted region of secretion. And finally, the fusion of the B-cell hybrids to penultimately differentiated B-cell lines indicated that there is a segregation of the expression of the membrane-associated immunoglobulins while the secreted form of immunoglobulin is co-expressed in the """"""""tribrids"""""""". It is expected that the information derived from our proposed studies (involving both biochemical and ultrastructural analysis) will provide a better understanding of antigen-induced B-cell activation, B-cell differentiation and a molecular characterization of a B-cell function.
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