CYTOSKELETAL PROTEIN CHANGES IN ACTIVATED NEUTROPHILS

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The assembly and disassembly cytoskeletal proteins is essential for cell activation and motility. Actin is a major cytoskeletel protein which exists in a monomeric (globular or G-actin) form as well as a polymeric (filamentous) form referred to 'as F-actin. In this project, we investigated actin polymerization changes in human neutrophils exposed to chemoattractants and chemically-modified chemoattractants. Two different techniques were used to evaluate actin polymerization. One technique utilized fluorochrome- conjugated phallacidin which specifically binds to F-actin. Neutrophils, that are activated, fixed permeabilized, and stained with the fluorescent phallacidin are analyzed flow cytometrically for changes in F-actin. The second technique required the precipitation of cytoskeletal-associated actin using an extraction procedure with triton X- 100 detergent. The extracted cytoskeletal Proteins were analyzed by -polyacrylamide gel electrophoresis and identified-by Western blotting. Neutrophils we-re 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 and we found that the rapid, initial phase of actin polymerization that occurred within 15 seconds after exposure to the stimuli, occurred with both forms of the N-formyl peptide. Significant differences in the kinetics of ac in depolymerization were found indicating that cells exposed to the non- chemotactic derivative exhibited a rapid Polymerization immediately followed b a depolymerization of actin. In contrast, cells exposed to the chemotactic form of the peptide exhibited a rapid Polymerization followed by a gradual depolymerization and repolymerization of actin. Further detailed studies are in progress to evaluate changes in other neutrophil cytoskeletal proteins exposed to these ligands. These studies should sights into the cytoskeletal protein changes involved in chemotaxis.