Kinetic Analysis ofF-actin Depolymerization in Pblymorphonuclear Leukocyte Lysates Indicates That Chemoattractant Stimulation Increases Actin Filament Number without Altering the Filament Length Distribution

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolymerizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined . F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin . Lysis in 1.2 M KCl and 10 14M DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization . To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monoHEMOATTRACTANTS induce migration of polymorphonuclear leukocytes (PMNs)' by stimulating formation of lamellipodia . Lamellipodia formation on cells exposed to chemoattractant occurs concomitantly with actin polymerization (Zigmond and Sullivan, 1979; Fechheimer and Zigmond, 1983 ; Wallace et al ., 1984 ; Howard and Oresajo, 1984a,b ; Rao and Varani, 1982) . The actin polymerization is required for lamellipodia extension since cytochalasin, a drug which inhibits actin polymerization, also prevents lamellipodia extension (White et al ., 1983 ; Cooper, 1987 ; Yahara et al ., 1982) . The mechanism through which chemoattractant causes an increase in cellular filamentous actin (F-actin) is unclear. The increase could be accomplished by increasing the monomer globular actin (G-actin) pool available for polymerization or by increasing the effective affinity of the filament for 1. Abbreviations used in this paper: CD, cytochalasin D; F-actin, filamentous actin; fNLLP, N-formylnorleucylleucylphenylalanine ; G-actin, globular actin; PMN, polymorphonuclear leukocyte . © The Rockefeller University Press, 0021-9525/91/11/677/11 $2 .00 TheJournal of Cell Biology, Volume 115, Number3, November 1991677-687 mer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting . Control cells had 1 .7 f 0.4 x 105 filaments with an average length of 0.29 f 0.09 um. Chemoattractant stimulation for 90 s at room temperature with 0.02 juM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a twofold increase in the total number of actin filaments to 4.0 f 0.5 x 10 5 filaments with an average length of 0.27 t 0.07 p.m . In both cases, most (rv80%) of the filaments were quite short (<0.18 Am). The length distributions of actin filaments in stimulated and control