The kinetics of chemotactic peptide-induced change in F-actin Content, F-actin distribution, and the shape of neutrophils

Abstract

Formyl-met-leu-phe (fMLP) induces actin assembly in neutrophils; the resultant increase in F-actin content correlates with an increase in the rate of cellular locomotion at fMLP concentrations ≤10-8 M (Howard, T. H., and W. H. Meyer, 1984, J. Cell Biol., 98:1265-1271). We studied the time course of change in F-actin content, F-actin distribution, and cell shape after fMLP stimulation. F-actin content was quantified by fluorescence activated cell sorter analysis of nitrobenzoxadiazole-phallacidin-stained cells (Howard, T. H., 1982, J. Cell Biol., 95(2, Pt. 2:327a). F-actin distribution and cell shape were determined by analysis of fluorescence photomicrographs of nitrobenzoxadiazole-phallacidin-stained cells. After fMLP stimulation at 25°C, there is a rapid actin polymerization that is maximal (up to 2.0 times the control level) at 45 s; subsequently, the F-actin depolymerizes to an intermediate F-actin content 5-10 rain after stimulation. The depolymerization of F-actin reflects a true decrease in F-actin content since the quantity of probe extractable from cells also decreases between 45 s and 10 min. The rate of actin polymerization (3.8 ± 0.3-4.4 ± 0.6% increase in F-actin/s) is the same for 10-10 - 10-6 M fMLP and the polymerization is inhibited by cytochalasin D. The initial rate of F-actin depolymerization (6.0 ± 1.0-30 ± 5% decrease in F-actin/min) is inversely proportional to fMLP dose. The F-actin content of stimulated cells at 45 s and 10 min is greater than control levels and varies directly with fMLP dose. F-actin distribution and cell shape also vary as a function of time after stimulation. 45 s after stimulation the cells are rounded and Factin is diffusely distributed; 10 min after stimulation the cell is polarized and F-actin is focally distributed. These results indicate that (a) actin polymerization and depolymerization follow fMLP stimulation in sequence, (b) the rate of depolymerization and the maximum and steady state F-actin content but not the rate of polymerization are fMLP dose dependent, and (c) concurrent with F-actin depolymerization, F-actin is redistributed and the cell changes shape. © 1985, Rockefeller University Press., All rights reserved.