An abrupt inhibition of cell surface and secretory functions occurs at the onset of mitosis. In particular, treatment of mitotic RBL-2H3 rat basophilic leukemia cells with antigen no longer stimulates membrane ruffling, fluid uptake and the release of immune mediators by degranulation. Nevertheless, cell surface receptors for antigen are present on mitotic RBL-2H3 cells and the pathways leading to membrane responses can be stimulated by TPA, a tumor promoter that directly activates protein kinase C. It thus appears that the transmembrane coupling of antigen-receptor interaction to the generation of signals for membrane and secretory responses may be specifically inhibited during mitosis. In Chinese Hamster Ovary (CHO) cells, mitotic membrane functions are reactivated by fusing mitotics with interphase cells, presumably because signalling molecules are contributed by the interphase partner. The experiments proposed here test the hypothesis that membrane transduction pathways are inhibited during mitosis and they explore the mechanism and significance of this inhibition. First, we will confirm and extend the evidence for mitotic membrane reactivation in CHO and RBL-2H3 cells by treatments that bypass or modulate ligand-activated membrane transduction pathways. Using RBL-2H3 as a model system, we will then determine directly if antigen-induced inositol phospholipid turnover and Ca2+ mobilization are inhibited during mitosis; we will explore changes in the properties of membrane-associated GTP-binding proteins that might contribute to the predicted failure of antigen-induced membrane and secretory responses; and we will determine if changes in the kinetics of antigen-IgE-receptor binding and in antigen-stimulated F-actin assembly are correlated with the inhibition of membrane transduction. These studies are expected to provide direct evidence for the specific and reversible inhibition of membrane transduction during mitosis. They may establish the mitotic cell as an important tool for continuing analyses of the membrane pathways that couple ligand binding events to cellular responses in animal cells.
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