Regulation of actin and secretion by Rho and calmodulin

Activities of small, Rho-related proteins are essential for the secretory function of mast cells. In addition, these GTPases also regulate distinct and specific steps in the reorganisation of actin cytoskeleton: Rho controls de novo actin polymerisation, while Rac is required for relocalisation of the released cortical filaments. Here I have shown that in the presence of calcium, Rho has an additional role, that of promoting cortical F-actin disassembly. When actin cortex is stabilised by phalloidin, Rho-induced enhancement of secretion is still apparent. This suggests that secretion and cortical F-actin disassembly are regulated independently. Another mechanism that controls actin cortex is dependent on calmodulin. Calmodulin is mandatory for calcium induced cortical F-actin disassembly. Inhibition of calmodulin by specific calmodulin-binding peptides prevents the calcium-induced loss of F-actin from permeabilised mast cells. On the other hand, addition of exogenous calmodulin greatly promotes this effect. Inhibition of endogenous Rho does not prevent calmodulin-induced enhancement of F-actin disassembly. Results suggest that Rho and calmodulin control a common target via independent pathways. This target is likely to be myosin light chain since inhibitors of myosin light chain kinase prevented cortical disassembly. A model is proposed in which myosin-based contractility, activated by Rho and/or calcium-calmodulin, is required for the cortical F-actin disassembly. Calmodulin or its inhibitors have no effect on secretion from permeabilised mast cells. This confirms the independence of regulatory pathways leading to the final steps of exocytosis and to the cytoskeleton. However, secretory responses of intact cells to activation by antigen or polycationic compounds are inhibited by cell permeant calmodulin inhibitors (W7 and myristylated calmodulin-binding peptides). This is not due to inhibition of calcium influx or IP3 production. Thus, signal transduction from the activated receptor to exocytosis does require calmodulin