Summary Natural IgM antibodies secreted in the absence of antigenic challenge are important contributors to antimicrobial immunity and tissue homeostasis. Early studies had identified bone marrow and, to a lesser extent the spleen, as main tissue sources of this spontaneously secreted IgM. However, the responsible B cell subset has never been identified. Using multicolor flow cytometry, cell sorting and chimeric mice in which B-1 and B-2 cells and their secreted antibodies are distinguished by their Ig-allotype, we unequivocally identify the natural IgM secreting cells in spleen, and for the first time in the bone marrow as IgM+ IgDlo/−CD19hi CD43+ CD5+/− B-1 cells. The newly identified population of bone marrow B-1 cells shows many of the phenotypic characteristics of splenic B-1 cells but is distinct from B-1 cells in the peritoneal cavity, which generate at best very small amounts of IgM. Antibody-secreting spleen and bone marrow B-1 cells are distinct also from terminally differentiated plasma cells generated from antigen-induced conventional B cells, as they express high levels of surface IgM and CD19 and lack expression of CD138. Together the study identifies populations of non-terminally differentiated B-1 cells in spleen and bone marrow as the most significant producers of natural IgM.

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Clathrin-coated vesicles are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. AP-2 and AP180 are the resident coat proteins of clathrin-coated vesicles in nerve terminals, and interactions between these proteins could be important in vesicle dynamics. AP180 and AP-2 each assemble clathrin efficiently under acidic conditions, but neither protein will assemble clathrin efficiently at physiological pH. We find that there is a direct, clathrin-independent interaction between AP180 and AP-2 and that the AP180-AP-2 complex is more efficient at assembling clathrin under physiological conditions than is either protein alone. AP180 is phosphorylated in vivo, and in crude vesicle extracts its phosphorylation is enhanced by stimulation of casein kinase II, which is known to be present in coated vesicles. We find that recombinant AP180 is a substrate for casein kinase II in vitro and that its phosphorylation weakens both the binding of AP-2 by AP180 and the cooperative clathrin assembly activity of these proteins. We have localized the binding site for AP-2 to amino acids 623-680 of AP180. The AP180/AP-2 interaction can be disrupted by a recombinant AP180 fragment containing the AP-2 binding site, and this fragment also disrupts the cooperative clathrin assembly activity of the AP180-AP-2 complex. These results indicate that AP180 and AP-2 interact directly to form a complex that assembles clathrin more efficiently than either protein alone. Phosphorylation of AP180, by modulating the affinity of AP180 for AP-2, may contribute to the regulation of clathrin assembly in vivo.Clathrin-coated vesicles mediate protein and lipid transport between intracellular compartments in the regulated secretory and endocytic pathways of all eukaryotic cells (reviewed in Ref. 1). The outermost layer of a clathrin-coated vesicle consists of clathrin that has been polymerized into an icosahedral cage (2). Between the outer clathrin shell and the vesicle membrane lie the clathrin assembly proteins (3). Clathrin assembly proteins belong to one of two gene families, the tetrameric AP 1 family and the monomeric AP family. Three tetrameric APs have been described and designated AP-1, AP-2, and AP-3. AP-1 and AP-2 were first characterized in 1987 as major clathrin-coated vesicle coat proteins (4, 5). AP-3 has only recently been identified (6 -9) and also appears to be associated with clathrin-coated vesicles (10). Two members of the monomeric AP family have been described: AP180 and CALM. AP180 was independently discovered in a number of laboratories and was originally referred to as AP180 (11, 12), pp155 (13), NP185 (14), or F1-20 (15, 16). AP180, pp155, NP185, and F1-20 were found to be identical (17, 18) and given the name AP-3. However, in view of the decision to designate the newest member of the tetrameric AP family AP-3 (7, 8), we refer to the monomeric AP-3 as AP180. AP180 localizes to synapses (19 -21), while CALM is expressed ubiquitously (22). AP-1, AP-2, and AP180 localize to clathrin-coate...