Distribution of F-actin elongation sites in lysed polymorphonuclear leukocytes parallels the distribution of endogenous F-actin.

Abstract

We compared, on lysed polymorphonuclear leukocytes (PMNs), the spatial distributions of sites that nucleate actin polymerization with the spatial distribution of endogenous F-actin. Sites nucleating polymerization of exogenous actin were detected by incubating lysed cells with rhodamine-labeled G-actin under polymerizing conditions. Endogenous F-actin was stabilized and stained by lysis of cells into fluorescein-labeled (FITC) phalloidin. We found the distributions of rhodamine and fluorescein intensities in a given cell, resting or stimulated with chemoattractant, to be similar. Thus, after lysis the number of sites able to nucleate actin polymerization is proportional to the local F-actin concentration. Quantitative fluorescence microscopic analysis also demonstrated that (1) if cells were stimulated with chemoattractant shortly before lysis, the total fluorescence per cell of both fluorophores went up; (2) if peptide was diluted shortly before lysis, the endogenous F-actin in the lamellae was dramatically reduced, but nucleation sites persisted, giving a high rhodamine to fluorescein ratio; and (3) there was a small increase in the ratio of rhodamine (exogenously grown actin) to fluorescein (endogenous F-actin) in a region near the lamellar/endoplasm border, centripetal to regions of the highest concentration of endogenous F-actin. The rhodamine signal appeared to be due to in situ actin polymerization probably nucleated by existing free barbed ends, since (1) the rhodamine signal increased linearly with time with no detectable lag if the actin concentration was above that of the critical concentration of the barbed end; (2) the rhodamine signal was dramatically reduced if lysates were incubated with gelsolin-actin complex (which stably caps barbed ends), then washed before the rhodamine G-actin was added; and (3) the number of nucleation sites at the time of lysis is similar to the number of the barbed ends of actin filaments determined by the kinetics of depolymerization [Cano et al., 1991]. The fact that the distribution of exogenous actin polymerization paralleled the endogenous F-actin suggests that the number of free barbed ends per F-actin is roughly constant. If all filament ends were free, or if a constant fraction of the filaments ends were free, these data would suggest that the mean filament length is roughly constant throughout the cell.