Actin polymerization induced by GTPγS in permeabilized neutrophils is induced and maintained by free barbed ends

Abstract

To address the mechanisms through which agonists stimulate actin polymerization, we examined the roles of monomer sequestering proteins and free barbed ends on actin polymerization induced by guanosine 5'-3-O- (thio)triphosphate (GTPγS) in neutrophils permeabilized with streptolysin O. Addition of profilin (without GTPγS) caused a net decrease in F-actin. Thus, merely making profilin available in the cell was not sufficient to induce actin polymerization. On the other hand, addition of profilin hardly affected the polymerization induced by GTPγS, while thymosin β4 or DNase I decreased this polymerization. These data suggested that GTPγS induced polymerization by increasing the availability of barbed ends. In the presence of cytochalasin B, profilin did inhibit polymerization induced by GTPγS, demonstrating that GTPγS did not inhibit profilin's monomer sequestering ability. The F-actin induced by GTPγS was not limited by a time-dependent loss of G-actin or G-proteins from permeabilized cells since, following stimulation with suboptimal concentrations of GTPγS, addition of more GTPγS induced further polymerization. Barbed ends remained free after F-actin reached plateau since (a) cytochalasin B caused depolymerization of induced F-actin and (b) profilin did not depolymerize induced F-actin unless the cells were first treated with cytochalasin to cap barbed ends. The data indicate that GTPγS maintains an increased level of F-actin by keeping at least a few barbed ends available for polymerization.