Chemotactic peptide-induced changes in neutrophil actin conformation.
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-1,3-diazole (NBD)-phallacidin to quantitate cellular F-actin 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 greater than 1 nM FMLP resulted in a dose-dependent and reversible increase in F-actin in 70-95% of PMN by 30 s. The induced increase in F-actin was blocked by 30 microM 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.
D000199:Actins / Q000097:blood*; D000546:Amanitins; D002634:Chemotaxis, Leukocyte / Q000187:drug effects*; D003571:Cytochalasin B / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D011487:Protein Conformation
D000199:Actins; D000546:Amanitins; D005456:Fluorescent Dyes; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; C028877:7-nitrobenz-2-oxa-1,3-diazole-phallacidin