Chemotactic Peptide-induced Conformation Changes in Neutrophil Actin

The effect of the chemotatic peptide, N-formylmethionylleucylphenylalanine (FMLP), on actin conformation in human neutrophils (PMN) was studied by flow cytometry using fluorescent 7-nitrobenz-2-oxa-l,3-diazole (NBD)-phallacidin to quantitate cellular Factin content. Uptake of NBD-phallacidin by fixed PMN was saturable and inhibited by fluid phase F-actin but not G-actin. Stimulation of PMN by >1 nM FMLP resulted in a dosedependent and reversible increase in F-actin in 70-95% of PMN by 30 s. The induced increase in F-actin was blocked by 30 #M cytochalasin B or by a t-BOC peptide that competitively inhibits FMLP binding. Under fluorescence microscopy, NBD-phallacidin stained, unstimulated PMN had faint homogeneous cytoplasmic fluorescence while cells exposed to FMLP for 30 s prior to NBD-phallacidin staining had accentuated subcortical fluorescence. In the continued presence of an initial stimulatory dose of FMLP, PMN could respond with increased F-actin content to the addition of an increased concentration of FMLP. Thus, FMLP binding to PMN induces a rapid transient conversion of unpolymerized actin to subcortical F-actin and repetitive stimulation of F-actin formation can be induced by increasing chemoattractant concentration. The directed movement of PMN in response to chemoattractant gradients may require similar rapid reversible changes in actin conformation. The force for polymorphonuclear neutrophil (PMN) movement is generated by its cytoplasmic microfilament lattice which has as its most prevalent component polymerized actin (1). As neutrophils initiate movement, actin microfilaments increase in number and distribute themselves toward the leading portion of the moving cell (2). For sustained movement to occur, the microfilament lattice must be continuously remodeled. Consequently, actin polymerization and microfilament production are important determinants of cell motility. Actin comprises 10% of neutrophil cytoplasmic protein (3) and has two interchangeable physical states with widely different polymerization potentials (4, 5). G-actin exists primarily as a monomer due to the high critical concentration required for its polymerization. F-actin has a lower critical polymerization concentration and at the concentration of actin in cytoplasm, oligomers and polymers form. The presence of high concentrations of potassium and magnesium in the cell favors the F-actin conformation (5), whereas, cytoplasmic proteins such as profilin stabilize G-actin (1, 6). Thus, a pool of G-actin monomers exist in equilibrium with F-actin oligomers and polymers. Actin monomers are added to microfilaments only after a change from the G to the F conformation (7-9). Our studies examined the effect of the chemoattractant molecule N-formylmethionylleucylphenylalanine (FMLP) on neutrophil actin content and distribution. To accomplish this, PMN were exposed to a fluorescent derivative of an acidic phallotoxin, 7-nitrobenz-2-oxa-l,3-diazole (NBD)-phallacidin (10), which binds with high affinity to F-actin but not Gactin. Flow cytometry permitted us to quantitate F-actin in large numbers of cells and to observe separate subsets of the cell response to FMLP. ~Abbreviations in this paper: FMLP, N-formylmethionylleucylphenylalanine; NBD, 7-nitrobenz-2-oxa-diazole; PMN, polymorphonuclear neutrophil. THE JOURNAL OF CELL BIOLOGY • VOLUME 99 SEPTEMBER 1984 1060-1065 1060 © The Rockefeller University Press 0021-9525184/0911060106 $1.00 on Jne 8, 2017 D ow nladed fom Published September 1, 1984