Microinjection of a monoclonal antibody against SPN antigen, now identified by peptide sequences as the NuMA protein, induces micronuclei in PtK2 cells

Abstract

Several high molecular mass proteins which relocate from the interphase nucleus to the spindle poles during mitosis have been defined by antibodies. Microinjection experiments have shown that at least the antigen defined by SPN antibody plays a functional role during mitosis. Recently the cDNA sequence for human NuMA antigen was established and epitopes for antibodies to centrophilin, and to 1F1 and 1H1 antigens were found to be included in the NuMA protein. Here we show that immunoprecipitated SPN antigen reacts with an autoimmune human NuMA serum. In addition three peptides derived from immunoprecipitated human SPN by cyanogen bromide cleavage and covering more than fifty amino acids show a perfect fit with the sequence predicted for NuMA protein. Thus SPN antigen and NuMA are the same protein. Injection of SPN-3 antibody into interphase or mitotic PtK2 cells results in cells with micronuclei. For cells injected in prophase, prometaphase or metaphase 90%, 78% and 77% display defective cytokinesis or yield daughter cells with micronuclei. In contrast only 16% of cells injected in anaphase are abnormal. Thus SPN/NuMA antigen may be required during early, but not during later, stages of mitosis. Surprising parallels are seen between the effects of microinjecting SPN-3 antibody and treatment with colcemid and taxol of PtK2 and HeLa cells. Our results identify an important role during mitosis for the SPN/NuMA antigen.