Cofilin and Arp2/3 complex in the initiation of lamellipodia protrusion
Ichetovkin, Ilia E.
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One of the earliest events in the process of cell motility is the massive generation of free actin barbed ends, which elongate to form filaments adjacent to the plasma membrane at the tip of the leading edge. Most eukaryotic cells rely on this localized actin polymerization to generate and sustain cell movement. Both cofilin and Arp2/3 complex have been proposed to contribute to barbed end formation during cell motility. Cofilin has been reported to depolymerize F-actin by either severing filaments to increase the number of depolymerizing ends or increasing the off-rate of monomers from F-actin without increasing the number of filament ends. We have compared directly the ability of native and recombinant cofilins from Dictyostelium to sever F-actin. Our results demonstrate that native cofilin has high level of severing activity. Off-rate over the concentration range of cofilin has not increased more than twofold beyond that observed in the absence of cofilin. Tethering filaments to the solid substrate increased their susceptibility to severing down to 9nM cofilin concentration range, or more than 100 fold compared to experiments with cofilin in solution. Tethered filaments are normally observed in vivo. Therefore, these results suggest that one of the roles of cofilin is severing to generate barbed ends in the absence of depolymerization.;In order to dissect the relative contributions of nucleation and branching to protrusive force, we prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. Side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. Microinjection of these antibodies into cells specifically inhibits lamellipod extension. However, it does not affect the EGF-stimulated appearance of free barbed ends in situ. That indicates that while Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. In order to determine if and how cofilin and Arp2/3, complex interact, we employed a light-microscope based assay to visualize actin polymerization directly in the presence of both proteins. The results indicate that cofilin generates barbed ends to increase the mass of freshly polymerized F-actin but does not directly affect the activity of Arp2/3 complex. However, newly polymerized F-actin supports the Arp2/3-induced branch formation at least 20 times more efficiently than older pre-polymerized filaments. The results suggest that in vivo cofilin contributes to barbed end formation by inducing the initial increase in the number of barbed ends, leading to a burst of new F-actin polymerization. That zone of brand new F-actin in turn supports higher levels of dendritic nucleation by active Arp2/3 complex.
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