Epidermal growth factor (EGF) stimulates both chemotaxis and the metastatic activity of mammary carcinoma cells. It regulates actin polymerization and promotes cellular protrusion by activating cofilin - an effect known to require phospholipase C (PLC). In The Journal of Cell Biology, Jacco van Reehen et al. now report that in rat MTLn3 carcinoma cells, cofilin is activated by its release from the plasma membrane as a result of reduced levels of phosphatidylinositol 4,5-bisphosphate (PIP₂).

PIP₂ (green) and cofilin (red) are associated in vivo in a compartment at the plasma membrane that releases active cofilin in response to stimulation of EGF receptors.

Image courtesy of Dr. J. van Rheenen, Albert Einstein College of Medicine of Yeshiva University, NY, USA.

Using a FRET-based PIP₂ assay, the authors first showed that EGF stimulation led to an increase in cell protrusion, which was accompanied by a substantial drop in PIP₂ levels. Both cell protrusion and PIP₂ hydrolysis were blocked upon PLC inhibition, suggesting that EGF-induced protrusion may result from PLC-catalysed hydrolysis of PIP₂.

The authors found that non-phosphorylated cofilin colocalized with PIP₂ at the plasma membrane in fixed cells, but that following a reduction in PIP₂ levels cofilin became activated and enriched at the leading edge of newly formed lamellipodia. When PLC activity was blocked, EGF was unable to induce the drop in PIP₂ levels and cofilin remained at the plasma membrane. These data suggest that non-phosphorylated cofilin is sequestered and inactivated at the plasma membrane by PIP₂, and that EGF-induced PIP₂ hydrolysis causes the release and activation of cofilin, which then translocates to the F-actin compartment.

The authors observed, using fluorescence imaging approaches, the rapid translocation of cofilin from the plasma membrane that was dependent on PLC-mediated PIP₂ hydrolysis. Cofilin was also released from the membrane upon PLC-independent PIP₂ reduction.

They also showed that endogenous cofilin binds to actin filaments shortly after EGF stimulation in fixed MTLn3 cells. Binding was reduced upon PLC inhibition, indicating that filamentous (F)-actin-bound cofilin originated from the plasma membrane. Furthermore, local application of EGF resulted in a local translocation of cofilin from the plasma membrane compartment to the F-actin compartment. Finally, the authors found that the actin-severing activity of cofilin increased after EGF treatment or PIP₂ reduction.

Taken together, this work provides the first in vivo evidence that cofilin is locally activated upon release from the plasma membrane as a result of a reduction in PIP₂ levels. Free active non-phosphorylated cofilin can then rapidly translocate to the cell front where it binds and severs F-actin to promote cell protrusion.

Author: Kim Baumann