[Abstract unavailable

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.

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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...

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