Quantitative fluorescent speckle microscopy analysis of lamellipodial and lamellar F-actin flow (left) and F-actin turnover (center) reveals widening of lamellipodium and increased spatial overlap of lamellipodium and lamella (right) at the leading edge of epithelial cells with high cofilin activity. As a result of this reorganization, the overall protrusion efficiency decreases.

Image courtesy of Dr. G. Danuser, The Scripps Research Institute, La Jolla, CA, USA.

Two actin networks - the lamellipodium (Lp) and the lamella (Lm) - occupy the front position of migrating cells. The Lp is characterized by fast retrograde flow powered by F-actin polymerisation, and localises to a 1-3 m band near the leading edge. The Lm is a more stable network with slow retrograde flow driven by actomyosin contraction and occupies a wider band (15 m). How the dynamics of these two networks are regulated and how they control cell protrusion is unclear. In Developmental Cell, Delorme et al. now report that cofilin regulates the interaction of the Lp and Lm and that high cofilin activity reduces protrusion efficiency.

The authors first confirmed in PtK1 cells (marsupial kidney epithelial cells) that cofilin activity at the leading edge is regulated by a signalling cascade downstream of the Rac1 GTPase that involves the sequential activation of the p21-activated (Pak1) and LIM (LIMK1) kinases. Cofilin is inactivated by phosphorylation, and by interfering with different stages of this pathway the authors studied the effect that gradually increasing cofilin activity has on actin dynamics.

Inactivation of cofilin by expressing constitutively active Rac1 resulted in the stabilization of actin filaments and widening of the Lp. Conversely, inhibition of Pak1 enhanced cofilin activity, increased the rate of F-actin retrograde flow in the Lp and reduced its width. Higher levels of cofilin activity - using dominant-negative LIMK1, active cofiin phosphatase (CIN), or constitutively active cofilin - resulted in higher retrograde flow velocity throughout the entire protrusion. The authors found that this rapid flow corresponded to the Lp actin network but not the Lm network, showing that cofilin activation induces Lp expansion. Moreover, Pak1 inhibition reduced the flow of the Lm independently of cofilin through an as yet undefined pathway.

Analysis of actin turnover revealed that whereas actin polymerization was confined to a narrow band along the leading edge in cells with low cofilin activity, high cofilin activity resulted in the spreading of actin polymerization sites inside the cell protrusion. An increase in the width of the actin treadmilling array and a higher density of polymerization-competent filament barbed ends were found to be independent of the Arp2/3 complex - a known nucleator of actin polymerization. This finding is consistent with in vitro observations suggesting that cofilin can increase the number of polymerizing actin filament ends by its severing activity and/or bona fide nucleation of actin filaments.

But how does cofilin affect cell protrusion? Migrating cells coordinate the events of cell edge protrusion and retraction, and net velocity results when protrusion exceeds retraction. Increased cofilin activity did not significantly affect instantaneous edge velocities, but coordination was lost and protrusion efficiency decreased.

Cofilin is known for its actin-severing activity and has been previously reported to positively regulate cell protrusion by increasing actin turnover. This work shows that if cofilin is activated globally, F-actin nucleation prevails, retrograde flow accelerates and protrusion is inhibited. Additionally, the Lp extends further away from the cell edge and the spatial separation between the Lp and the Lm becomes less distinct. The authors propose that cofilin activity, regulated by Pak1, affects the interaction between the two actin networks at the leading edge and thus controls the efficiency of cell migration.

Author: Kim Baumann