Kinetic Analysis of the Interaction of Actin-depolymerizing Factor (ADF)/Cofilin with G- and F-Actins

Abstract

The thermodynamics and kinetics of actin interaction with Arabidopsis thaliana actin-depolymerizing factor (ADF) 1 , human ADF, and S6D mutant ADF 1 protein mimicking phosphorylated (inactive) ADF are examined comparatively. ADFs interact with ADP⅐G-actin in rapid equilibrium (k ؉ ‫؍‬ 155 M ؊1 ⅐s ؊1 and k ؊ ‫؍‬ 16 s ؊1 at 4°C under physiological ionic conditions). The kinetics of interaction of plant and human ADFs with F-actin are slower and exhibit kinetic cooperativity, consistent with a scheme in which the initial binding of ADF to two adjacent subunits of the filament nucleates a structural change that propagates along the filament, allowing faster binding of ADF in a "zipper" mode. ADF binds in a non-cooperative faster process to gelsolin-capped filaments or to subtilisin-cleaved F-actin, which are structurally different from standard filaments (Orlova, A., Prochniewicz, E., and Egelman, E. H. (1995) J. Mol. Biol. 245, 598 -607). In contrast, the binding of phalloidin to F-actin cooperatively inhibits its interaction with ADF. The ADF-facilitated nucleation of ADP⅐actin self-assembly indicates that ADF stabilizes lateral interactions in the filament. Plant and human ADFs cause only partial depolymerization of F-actin at pH 8, consistent with identical functions in enhancing F-actin dynamics. Phosphorylation does not affect ADF activity per se, but decreases its affinity for actin by 20-fold.A large body of evidence supports the view that the rapid turnover of actin filaments drives actin-based motility processes such as the forward movement of the leading edge of the lamellipodium of locomoting cells, the propulsive movement of Listeria monocytogenes, the movement of cortical actin patches in yeast, or the extension of the growth cone (1). Filaments turn over via a treadmilling mechanism, whereby the steady growth of barbed ends is fed by the subunits depolymerizing from the pointed ends (2, 3). While barbed end growth, which provides the motile force (4), is restricted to specialized regions of the cell such as the leading edge (5), depolymerization may occur from all pointed ends in the cell medium. The observed rates of actin-based movement fall in the range 1-20 m/min, corresponding to treadmilling rates of 7-130 subunits/s/filament, i.e. 1-2 orders of magnitude higher than the treadmilling rate measured in vitro in solutions of pure actin (6).Actin-binding proteins of the ADF 1 /cofilin family have recently been demonstrated to enhance the treadmilling of Factin in vitro by 25-fold and consistently to increase the rate of Listeria propulsion in platelet extracts (7). We proposed (7) that these in vitro properties of ADF accounted for the enhancement of motility of Dictyostelium discoideum (8) due to ADF overexpression and for its high level of expression in early development (9). Consistently, ADF was shown to be responsible for the high rate of filament turnover in yeast (10).It was initially thought that ADF depolymerized F-actin rapidly due to a severing activity (11)(12)(13)(14). Severing of the f...

The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

“…Moreover, the high dissociation rate constant of the ADF/ cofilin-ADP-G-actin complex will facilitate rapid exchange of ADP-G-actin between ADF/cofilin and twinfilin. (The off-rate for Arabidopsis ADF1-ADP-G-actin complex was 12-16 s Ϫ1 at 4°C and would be significantly higher at higher temperatures [Ressad et al, 1998].) The relatively slow k off rates of twinfilinactin monomer complexes, together with fast k off rates of ADF/ cofilin-actin monomer complexes, provides kinetic evidence to support the argument that ADP-G-actin is recycled from ADF/ cofilin to twinfilin.…”

“…Our results further suggest that the affinity of twinfilin for ADP-G-actin is somewhat higher than that of ADF/cofilins for ADP-G-actin, because the mean value of measurements for a variety of isoforms of ADF and cofilin is ϳ0.15 M (Blanchoin and Pollard, 1998;Ressad et al, 1998;Vartiainen et al, 2002;Yeoh et al, 2002). This, together with a relatively high cellular concentration of twinfilin, suggests that twinfilin might displace ADF/cofilin from ADP-actin (Palmgren et al, 2001).…”

“…ADF/cofilin promotes the dissociation of ADP-G-actin at the minus end of the filament with a rate in excess of 9 s Ϫ1 (Carlier et al, 1997). Because ADF/cofilins dissociate rapidly from G-actin (Ressad et al, 1998) and compete in G-actin binding with twinfilin, the actin monomer may be delivered to twinfilin, which leaves ADF/cofilin free to associate with actin filaments and promote a new round of depolymerization. Twinfilin may first associate with an actin monomer through its N-terminal ADF-H domain, which promotes a conformational change and a consequent delivery of the monomer to the C-terminal ADF-H domain.…”

“…These reactions may be catalyzed by profilin. The references for the kinetic and equilibrium parameters for the ADF/cofilin-actin and profilin-actin complexes are from Carlier et al, 1997;Blanchoin and Pollard, 1998;Ressad et al, 1998;Perelroizen et al, 1996;and Gutsche-Perelroizen et al, 1999. ADP-G-actin by twinfilin will reduce the concentration of ADF/cofilin-ADP-G-actin complexes and thereby reduce both the addition of complexes to filament ends and the spontaneous nucleation of these complexes, which is a highly cooperative process in its concentration dependence (Yeoh et al, 2002). Moreover, the high dissociation rate constant of the ADF/ cofilin-ADP-G-actin complex will facilitate rapid exchange of ADP-G-actin between ADF/cofilin and twinfilin.…”

A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

“…However, we cannot be confident that this assay is in fact measuring barbed end capping rather than severing by Aip1p. Although the small amount of cofilin used in this experiment should not allow extensive Aip1p-induced severing, the cooperative nature of cofilin binding to filaments is likely to saturate short stretches of actin filaments (McGough et al, 1997;Ressad et al, 1998;Pope et al, 2000), creating ideal sites for Aip1p severing. We propose two scenarios of how minimal Aip1p-induced severing could lead to extensive disassembly of filaments in this assay.…”