SHAPE, F-ACTIN, AND SURFACE-MORPHOLOGY CHANGES DURING CHEMOTACTIC PEPTIDE-INDUCED POLARITY IN HUMAN NEUTROPHILS

Background: The exposure of human neutrophils to uniform concentration s of chemoattractants, such as N-formyl peptides, induces morphologica l cell polarization. In this study we report the temporal sequence of changes in cell shape, F-actin, and cell surface morphology during cel lular polarization induced by N-formylmethionyl-leucyl-phenylalanine ( fMLP) in human neutrophils in suspension. Methods: Neutrophil shape ch anges induced by 10(-8) M fMLP were observed with DIC microscopy. Size and cellular granularity were analyzed by flow cytometry measuring th eir forward and side scattered Light, To visualize F-actin distributio n, neutrophils were labeled with the fluorescence probe FITC-phalloidi n, and were examined with fluorescence and confocal laser scanning mic roscopy, Cell surface morphology was assessed with scanning electron m icroscopy (SEM). Results: The stimulation of round-smooth neutrophils with nanomolar concentrations (10(-8) M) of fMLP in suspension induced a temporal sequence of morphological changes during cell polarization , characterized by 1) increase in size as determined by forward angle scattered light, 2) rapid redistribution of F-actin from a diffuse cyt oplasmic localization to the cell periphery, and 3) rapid reorganizati on of cell surface morphological features, with accumulation of plasma membrane in the front of polar cells. Four cell shapes were identifie d with SEM after stimulation of round-smooth neutrophils: round-ridged , round-ruffled, nonpolar ruffled, and polar cells. These cell shapes were correlated with a cortical localization, focal aggregates, and mu ltipolar distribution of F-actin, In polar neutrophils, F-actin became concentrated in the front of the cell. Conclusions: These findings sh ow the relation between reorganization of the microfilamentous cytoske leton and modifications in cell shape and surface features during cell polarization induced after fMLP activation in neutrophils. This appro ach offers a powerful tool for further analysis of receptor distributi on in polarized, motile neutrophils. (C) 1995 Wiley-Liss, Inc.