The dynamics of stimulated actin polymerization in human neutrophils.

Abstract

In this work, the chemoattractant stimulated actin polymerization response in human neutrophils is studied by investigating the development of a dynamic assay to measure the stimulated actin polymerization response in single cells and by developing and analyzing a mathematical model for describing this response. The development of the dynamic assay is based on the use of fluorescently labeled actin to follow the kinetics of actin polymerization, macromolecular loading techniques to load the labeled actin into neutrophils, and fluorescence microscopy coupled with digital image acquisition and analysis to record and measure the stimulated, fluorescent response in loaded neutrophils. Only small amounts of labeled actin were loaded into neutrophils and stimulated fluorescent responses were measured in only a few individual neutrophils. In addition, the time courses of these responses were slow compared to the rapid polymerization responses reported in the literature. The small amount of loaded actin is probably due to rapid actin polymerization in the extracellular buffer during loading, while the slow fluorescent responses indicate the labeled actin did not react similarly to native actin in neutrophils. A mathematical model is proposed for describing the dynamics of the chemoattractant stimulated actin polymerization response in human neutrophils. The model is divided into two parts: the signal transduction mechanism consisting of fast and slow signaling pathways which generate an activated signaling molecule; and the actin polymerization mechanism which consists of actin monomer/binding protein complexes whose dissociation is dependent on the concentration of signaling molecule. Model simulations agree with the experimentally observed characteristics of the N-formyl peptide stimulated actin polymerization response, the response decay following interruption of ligand binding, and ligand/receptor binding and dissociation data. The model predicts: the fast signaling pathway involves precoupled receptors and controls the rapid rise to a maximum in the response; the slow pathway involves free receptors and controls the slow depolymerization observed after the maximum; the number of precoupled receptors is only 5% of the initial number of surface receptors; and each receptor participating in signal transduction generates only one signaling molecule.