BIPHASIC FILAMENTOUS-ACTIN POLYMERIZATION IN ACTIVATED NEUTROPHILS

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Actin is a major cytoskeletal protein which exists in a monomeric (G- actin) form as well as a polymer or filamentous form (F-actin) and is essential for cell activation and motility. Actin polymerization in chemoattractant-stimulated human neutrophils (PMN) was evaluated with two different techniques. One technique used fluorochrome-conjugated phallacidin to specifically stain F-actin. The activated PMN were fixed, permeabilized, and stained with the fluorescent phallacidin and analyzed flow cytometrically for changes in F-actin content. In the second technique, cytoskeletal associated actin was precipitated using an extraction procedure with triton X-100 detergent. The extracted cytoskeletal proteins were analyzed by polyacrylamide gel electrophoresis and actin was identified by Western blotting. PMN were exposed to chemotactic N-formyl peptide and to an oxidized N-formyl peptide derivative that binds to membrane receptors but fails to elicit chemotactic responsiveness. The kinetics of actin polymerization were evaluated using both techniques. A rapid, initial phase of actin polymerization occurred within 15 seconds after exposure to both forms of the N-formyl peptide. The chemotactic form the N-formyl peptide stimulated a biphasic response in which a second polymerization phase occurred approximately 2 minutes after stimulation, whereas the nonchemotactic form of the peptide failed to elicit a biphasic response. These results indicate that chemoattractant-stimulated PMN have cyclic F-actin responses to chemoattractant stimulation. The initial phase of the F-actin response is related to receptor-ligand binding, whereas the second F-actin phase appears to be related to events accompanying PMN motility. Some of the results were published in paper this year (Cancer Invest. 8 (6): 651-654, 1990).