MOLECULAR MODEL OF COFILIN REG IN ACTIN FILAMENT DYNAMICS & CELL FUNCTIONS

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The actin-based cytoskeleton plays an important role in cell locomotion. As cells move in response to external signals, the cytoskeleton is continuously remodeled: actin filament networks are formed at the frontal lamella, which attach to the underlying substratum through focal adhesions, thereby providing adhesive contacts for the traction necessary for retraction of the cell body. For this process to continue, actin subunits must be continuously recycled to the leading edge. Although the assembly rates of actin in vitro are extremely rapid, the pointed end disassembly rate constant measured in vitro (about 1 s-1) is far too slow to recycle the monomers for this process to continue. If net depolymerization occurs only by a treadmilling mechanism, it has been estimated that this rate would need to be nearer ~180 s-1 to maintain motion at 30 mm/min. The most obvious way to increase the disassembly rate is to sever filaments to expose new barbed ends, where the dissociation rate constant for ADP-actin subunits is much higher (> 7 s-1). The cofilin/ADF family of proteins were thought to be the most obvious candidates to do this because of their apparent ability to sever F-actin without capping. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Our structure shows cofilin binds F-actin cooperatively by bridging two longitudinally-associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter actin crossovers' (about 75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin competes with phalloidin for F-actin binding. This is the first demonstration of an actin-binding protein which competes for binding by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be remodeled as cells move in response to external signals.