EGF STIMULATES AN INCREASE IN ACTIN NUCLEATION AND FILAMENT NUMBER ATTHE LEADING-EDGE OF THE LAMELLIPOD IN MAMMARY ADENOCARCINOMA CELLS

Stimulation of metastatic MTLn3 cells with EGF causes the rapid extens ion of lamellipods, which contain a zone of F-actin at the leading edg e. In order to establish the mechanism for accumulation of F-actin at the leading edge and its relationship to lamellipod extension in respo nse to EGF, we have studied the kinetics and location of EGF-induced a ctin nucleation activity in MTLn3 cells and characterized the actin dy namics at the leading edge by measuring the changes at the pointed and barbed ends of actin filaments upon EGF stimulation of MTLn3 cells, T he major result of this study is that stimulation of MTLn3 cells with EGF causes a transient increase in actin nucleation activity resulting from the appearance of free barbed ends very close to the leading edg e of extending lamellipods. In addition, cytochalasin D causes a signi ficant decrease in the total F-actin content in EGF-stimulated cells, indicating that both actin polymerization and depolymerization are sti mulated by EGF, Pointed end incorporation of rhodamine-labeled actin b y the EGF stimulated cells is 2.12+/-0.47 times higher than that of co ntrol cells. Since EGF stimulation causes an increase in both barbed a nd pointed end incorporation of rhodamine-labeled actin in the same lo cation, the EGF-stimulated nucleation sites are more likely due either to severing of pre-existing filaments or de novo nucleation of filame nts at the leading edge thereby creating new barbed and pointed ends. The timing and location of EGF-induced actin nucleation activity in MT Ln3 cells can account for the observed accumulation of F-actin at the leading edge and demonstrate that this F-actin rich zone is the primar y actin polymerization zone after stimulation.