Reversible bleb formation in mast cells stimulated with antigen is Ca2+-calmodulin –dependent and bleb size is regulated by ARF6
Résumé
Mast cells stimulated with antigen undergo extensive changes in their cytoskeleton. We assess here the impact of actin-modifying drugs and report that in the presence of cytochalasin D, mast cells stop membrane ruffling but instead bleb. Bleb formation is reversible following washout of the cytochalasin D and occurs in an actin polymerization-dependent manner. Bleb formation is inhibited by expression of dominant-negative ezrin-T567D. Blebbing is also inhibited by blebbistatin, a myosin II inhibitor, implying myosin II activation in the process. We used a selection of inhibitors and observed that myosin II activation is dependent mainly on calcium-calmodulin with only a small contribution from Rho kinase. The signalling pathways stimulated by antigen include phospholipase C and D. Bleb formation was dependent on activation of phospholipase C but not phospholipase D. Primary alcohols, previously used as a means to reduce phospholipase D-derived PA, were potent inhibitors of membrane blebbing, but a more selective inhibitor of phospholipase D, FIPI, was without effect. FIPI also did not inhibit membrane ruffling or degranulation of mast cells, indicating that inhibition by primary alcohols works through an unidentified mechanism rather than via diversion of phospholipase D activity as assumed. We also examined the requirement for ARF6 and observed its expression led to an increase in bleb size and a further increase observed with the dominant-active mutant, Q67L-ARF6. Since T27N-ARF6 had no effect on bleb size, we conclude that ARF6 needs to be active to regulate the size of the blebs.
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