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© The Rockefeller University Press,
0021-9525/2000//531 $5.00
The Journal of Cell Biology, Volume 148, Number 3,
, 2000 531-542
Original Article |
Role of Cofilin in Epidermal Growth Factor–Stimulated Actin Polymerization and Lamellipod Protrusion
condeeli{at}aecom.yu.edu
Stimulation of metastatic MTLn3 cells with epidermal growth factor (EGF) causes a rapid and transient increase in actin nucleation activity resulting from the appearance of free barbed ends at the extreme leading edge of extending lamellipods. To investigate the role of cofilin in EGF-stimulated actin polymerization and lamellipod extension in MTLn3 cells, we examined in detail the temporal and spatial distribution of cofilin relative to free barbed ends and characterized the actin dynamics by measuring the changes in the number of actin filaments. EGF stimulation triggers a transient increase in cofilin in the leading edge near the membrane, which is precisely cotemporal 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 deoxyribonuclease I binding demonstrates that this increase in the number of pointed ends is confined to the leading edge compartment, and does not occur within stress fibers or in the general cytoplasm. Using a light microscope severing assay, cofilin's severing activity was observed directly in cell extracts and shown to be activated after stimulation of the cells with EGF. Microinjection of function-blocking antibodies against cofilin inhibits the appearance of free barbed ends at the leading edge and lamellipod protrusion after EGF stimulation. These results support a model in which EGF stimulation recruits cofilin to the leading edge where its severing activity is activated, leading to the generation of short actin filaments with free barbed ends that participate in the nucleation of actin polymerization.
Key Words: metastasis • chemotaxis • F-actin severing • motility • actin-related protein (ARP) 2/3
© 2000 The Rockefeller University Press
The first two authors contributed equally to this work.The data in this paper was submitted by A.Y. Chan in partial fulfillment of the requirement for the Degree of Doctor of Philosophy in the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University.
Abbreviation used in this paper: Arp, actin-related protein.
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