Chemotactic peptide-induced changes in neutrophil actin conformation.

Abstract

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.

Role of gelsolin interaction with actin in regulation and creation of actin nuclei in chemotactic peptide activated polymorphonuclear neutrophils.

“…This notion is not supported by the observation of GA interactions in FH-PMN (Howard et al, 1990a). However, interpretation of FH-PMN studies is confused by the high basal level of GA complex in basal FH-PMN (Wallace et al, 1985).…”

“…The results do not exclude the possibility that in FMLP activated PMNs net turnover of GA complexes between severing/capping and uncapping states in generation of nuclei plays a role in the regeneration of actin nuclei before subsequent ligand activations. However, they are not consistent with an initial net increase in gelsolin severing/capping to FMLP activated PMNs (Wallace et al, 1985). DISCUSSION One critical step in regulating the state of actin polymerization in nonmuscle cells is the creation and regulation of actin nuclei Pollard and Cooper, 1986).…”

“…Further studies should consider a developmental role for gelsolin, the de novo creation of nuclei from monomers and the role of oligomer sidebinding ponticulin-like proteins. Finally, despite the fact that gelsolin does not create the barbed nuclei in the FMLP-activated ETF-PMN, the rapid turnover of gelsolin between severing/nucleating and dissociated states could be involved in regenerating cryptic barbedend nuclei for actin polymerization in response to subsequent ligand activations as described in C5a activated cells previously exposed to FMLP (Wallace et al, 1985).…”

“…The affinity constant of actin monomer (G-actin) for the barbed-end is 10-fold greater than that for the pointed end (Korn et al, 1987) and under identical buffer conditions the critical concentration for polymerization at the two ends differ dramatically. Because the barbed end is the preferred end for filament growth and the majority (-8 80%) of the ligand activated actin polymerization in nonmuscle cells like platelets and PMNs occurs on barbed-end nuclei (Rao and Varani, 1982;Fechheimer and Zigmond, 1983;Fox and Phillips, 1983;Howard and Oresajo, 1985b;Wallace et al, 1985;Lind et al, 1987;Howard et al, 1990a,b), the creation and regulation of barbed-end nuclei is a critical step in regulating actin polymerization in PMNs.…”

“…Furthermore, gelsolin is present in blood cells, including platelets (Lind et al, 1982) and polymorphonuclear neutrophils (PMNs) (Yin et al, 1981). In these cells increase in [Ca++]i and phosphoinositide turnover (Pozzan et al, 1983;Cockcroft et al, 1985;Lapetina, 1990;Stephens et al, 1991) accompanies ligand activated actin polymerization at the barbed end of actin filaments (Rao and Varani, 1982;Fechheimer and Zigmond, 1983;Fox and Phillips, 1983;How-ard and Meyer, 1984;Howard and Oresajo, 1985a,b;Wallace et al, 1985), and GA complex dissociation temporally correlates with F-actin growth in these cells (Lind et al, 1987;Howard et al, 1990a). The in vitro effects of gelsolin on creation and regulation of actin nuclei paired with gelsolin's behavior in blood cells suggest that gelsolin is an excellent candidate to regulate actin polymerization in platelets and PMNs.…”