Cross-linking of IgE receptors (FCπRI) by multivalent antigens in basophils results in the activation of phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K). PLC λ activation leads to the production of inositol 1,4,5-triphosphate (IP₃) and release of calcium from intracellular stores triggering exocytosis of secretory granules. Previous studies have shown that activation of the FCπRI leads to changes in IP₃ receptor (IP₃R) distribution in rat basophilic leukaemia cells (RBL-2H3), a mast cell line. At rest the IP₃R distribution is homogenous throughout the cell; upon activation the IP₃Rs cluster and form discrete spots of about 1 mm diameter (Wilson et al. 1998). We have examined the potential role of actin-myosin in this process.

In our experiments, RBL-2H3 cells were primed with anti-DNP-IgE (1 mg ml^-1) for 12Ð24 h and activated with the multivalent antigen DNP-BSA (1 mg ml^-1) for 1 h. We then localised IP₃R type II, calreticulin, BiP, actin and myosin II using GFP constructs and immunocytochemistry. All experiments were carried out at least twice and all observations consistently seen in at least three images.

After cell activation IP₃R type II receptors aggregate, as shown by Wilson et al. (1998). However, we also saw that these receptor aggregates appear to cluster preferentially towards one region of the cell, usually the granular region. Staining of F-actin with phalloidin, or actin-GFP, showed actin throughout the cell with a concentration in the subplasmalemmal region. We visualised the distribution of myosin IIA using an antibody and GFP construct (Wei & Adelstein, 2000). Myosin II was mainly found in the cell periphery. The concentrations of either actin or myosin II were not associated with the IP₃R aggregates. These data suggest that actin and myosin II are not the sole mediators of IP₃R distribution and indeed may not play any role. We then tested a role for actin using Latrunculin, an agent that depolymerises the actin cytoskeleton. We show that Latrunculin-B (50 mM, 1 h) dramatically reduced actin but actually enhanced IP₃R clustering. Under conditions of cell surface receptor stimulation, remodelling of the actin cytoskelton is regulated by PI3K, which can be inhibited by wortmannin. Wortmannin (100 nM, 1 h) did not prevent the aggregation of the IP₃R but did prevent the IP₃Rs from concentrating in the granular region of the cell. We are conducting experiments to test for the selectivity of these effects on IP₃R distribution.

This work was funded by the MRC.