Role of Cofilin in Epidermal Growth Factor–Stimulated Actin Polymerization and Lamellipod Protrusion

Abstract

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.

ADF/cofilin family proteins control formation of oriented actin-filament bundles in the cell body to trigger fibroblast polarization

“…5EЈ). The spotty/granular appearance of ADF is similar to previously published images of AC in cells (Aizawa et al, 1996;Chan et al, 2000;Dawe et al, 2003;Mouneimne et al, 2006;Nishita et al, 2005;Svitkina and Borisy, 1999), including actin-colocalization studies (Aizawa et al, 1996;Chan et al, 2000;Svitkina and Borisy, 1999), and is probably indicative of the cooperative F-actin binding of ADF (Hayden et al, 1993), which, at substoichiometric amounts, gives local regions of saturation (McGough et al, 1997). Interestingly, once oriented actin bundles had formed (Fig.…”

“…Despite the clear role of AC in controlling cell polarity, studies of AC in the context of actin organization during cell polarization are limited; this process is either not the focus of the work (Ghosh et al, 2004;Mouneimne et al, 2006;Nishita et al, 2005) or the studies are preliminary (see PhDcit_addRef.doc). Furthermore, most studies focusing on the function of AC during motility have been limited to actin in the leading cell edge or lamellipodium (Chan et al, 2000;Dawe et al, 2003;Ghosh et al, 2004;Nishita et al, 2005;Zebda et al, 2000). By contrast, any role for AC in regulating actin organization within the cell body of migrating cells during cell polarization has yet to be explored.…”

Signaling mechanisms that regulate actin‐based motility processes in the nervous system

“…ADF also severs actin filaments, increasing the number of uncapped barbed ends that may undergo polymerization. Epidermal growth factor induces ADF-dependent increases in free barbed ends in MTLn3 cells (Chan et al 2000). While there is currently debate about which function of ADF is key to its facilitation of actin polymerization and membrane extension, the evidence indicates that ADF activity is required for actin-based motility processes.…”

Spatial and temporal regulation of actin polymerization for cytoskeleton formation through Arp2/3 complex and WASP/WAVE proteins

“…ADF/cofilin and gelsolin are candidate proteins for severing in the presence of signals [reviewed by Condeelis, 2001]. Actin filament severing and other activities of ADF/cofilin, such as depolymerization, are regulated by LIM-kinases through phosphorylation of the serine 3 residue of ADF/cofilin [Chan et al, 2000]. In this case, the phosphorylated form is inactive.…”