The role of cofilin in EGF -stimulated actin polymerization in mammary adenocarcinoma cells
Chan, Yan Fan (Amanda)
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Cell motility plays an important role in many basic biological processes including embryogenesis, neurite growth, wound healing, inflammation and cancer metastasis. To study the regulation of cancer cell motility, in particular mechanism of agonist stimulated actin polymerization, we characterized the motile response of MTLn3 cells, a metastatic cell line derived from the 13762 rat mammary adenocarcinoma, to EGF. EGF stimulates F-actin rich lamellipod extension and chemotaxis in MTLn3 cells. Actin polymerization is necessary for these EGF-stimulated responses because cytochalasin D inhibits the EGF-stimulated, extension, increases in F-actin in lamellipods, and chemotaxis. In order to establish the mechanism for accumulation of F-actin at the leading edge and its relationship to lamellipod extension in response to EGF, we have studied the kinetics and location of EGF-induced actin nucleation activity in MTLn3 cells. Stimulation of metastatic MTLn3 cells with EGF causes a transient increase in actin nucleation activity resulting from the appearance of free barbed and pointed ends very close to the leading edge (nucleation zone) of extending lamellipods. An approximate 1.5 fold increase in number of filaments detected by direct binding of DNase I to pointed ends was observed at the time of barbed and pointed end appearance in EGF stimulated MTLn3 cells. In situ measurement of pointed ends using DNase I immunofluorescence 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.;The increase in the number of filaments, after EGF stimulation lead us to examine the role of cofilin in EGF induced actin polymerization. EGF stimulation triggers a brief transient increase in cofilin at the nucleation zone that is precisely cotemporal with the kinetic appearance of the free barbed ends. The spatial and temporal correspondence of these events at the actin nucleation zone after EGF stimulation, suggests that an EGF stimulated cofilin severing activity causes the increase of filament numbers in MTLn3 cells. To test this hypothesis, a light microscope assay was used to visualize directly the severing of exogenous rhodamine-labeled F-actin. Results from this assay show that cofilin severing activity is activated after EGF stimulation. The presence of an EGF-inducible cofilin severing activity as well as the colocalization of cofilin with barbed ends at the leading edge strongly suggest that cofilin is involved in the generation of free barbed ends during EGF stimulation. Our results support a model in which EGF stimulation recruits cofilin to the leading edge where its severing activity is activated. This results in the generation of short actin filaments with free barbed ends that can be captured by Arp2/3 complex to nucleate actin polymerization at the tip of the lamellipod.