Ay. Chan et al., Role of cofilin in epidermal growth factor-stimulated actin polymerizationand lamellipod protrusion, J CELL BIOL, 148(3), 2000, pp. 531-542
Stimulation of metastatic MTLn3 cells with epidermal growth factor (EGF) ca
uses a rapid and transient increase in actin nucleation activity resulting
from the appearance of free barbed ends at the extreme leading edge of exte
nding lamellipods. To investigate the role of cofilin in EGF-stimulated act
in polymerization and lamellipod extension in MTLn3 cells, we examined in d
etail the temporal and spatial distribution of cofilin relative to free bar
bed ends and characterized the actin dynamics by measuring the changes in t
he number of actin filaments. EGF stimulation triggers a transient increase
in cofilin in the leading edge near the membrane, which is precisely cotem
poral with the appearance of free barbed ends there. A deoxyribonuclease I
binding assay shows that the number of filaments per cell increases by 1.5-
fold after EGF stimulation. Detection of pointed ends in situ using deoxyri
bonuclease I binding demonstrates that this increase in the number of point
ed ends is confined to the leading edge compartment, and does not occur wit
hin stress fibers or in the general cytoplasm. Using a light microscope sev
ering assay, cofilin's severing activity was observed directly in cell extr
acts and shown to be activated after stimulation of the cells with EGF. Mic
roinjection of function-blocking antibodies against cofilin inhibits the ap
pearance of free barbed ends at the leading edge and lamellipod protrusion
after EGF stimulation. These results support a model in which EGF stimulati
on recruits cofilin to the leading edge where its severing activity is acti
vated, leading to the generation of short actin filaments with free barbed
ends that participate in the nucleation of actin polymerization.