The effect of two actin depolymerizing factors (ADF/cofilins) on actin filament turnover : pH sensitivity of F‐actin binding by human ADF, but not of Acanthamoeba actophorin

Abstract

Actin depolymerizing factor (ADF) from vertebrates and actophorin from Acanthamoeba castellanii are members of a protein family that bind monomeric and polymeric actin and have been shown by microscopy to sever filaments. Here, we compare the properties of recombinant human ADF and actophorin using rabbit muscle actin. ADF binds tenfold more strongly than actophorin to monomeric actin (G‐actin)‐ATP, and both bind co‐operatively to F‐actin. ADF decorates filaments below pH 7.3 and induces substantial depolymerization at higher pH values [Hawkins, M., Pope, B., Maciver, S. K. & Weeds, A. G. (1993) Human actin depolymerizing factor mediates a pH‐sensitive destruction of actin filaments, Biochemistry 32, 9985−9993], but, at all pH values tested, actophorin binds to filaments in a similar manner to ADF at pH 6.5. Both proteins increase the depolymerization rate at the pointed ends of gelsolin‐capped filaments, but the effect of ADF is more marked at pH 8.0. Both proteins accelerate the nucleating activity when mixed with filamentous actin (F‐actin), but not with gelsolin‐capped filaments, and they rapidly decrease the lengths of filaments as evidenced by electron microscopy. Both of these effects are best explained by a weak severing activity. Our results are discussed in relation to earlier models and to the structural changes observed when ADF binds F‐actin [McGough, A., Pope, B., Chiu, W. & Weeds, A. (1997) Cofilin changes the twist of F‐actin : implications for actin filament dynamics and cellular function, J. Cell Biol. 138, 771−781]. We also discuss the relevance of these observations to their possible roles in facilitating actin turnover in cells, thereby regulating filament dynamics in cell motility.

ADF Proteins Are Involved in the Control of Flowering and Regulate F-Actin Organization, Cell Expansion, and Organ Growth in Arabidopsis

“…In some trichomes and stomatal guard cells, we observed complete disruption of the actin cytoskeleton as a consequence of AtADF1 overexpression. These findings are consistent with the ability of ADF proteins to stimulate F-actin depolymeriza- tion, which was predicted based on in vitro studies (Carlier et al, 1997;Maciver et al, 1998;Moriyama and Yahara, 1999), and also with the results of experiments in which the levels of ADF proteins were increased in single cells by microinjection (Nagaoka et al, 1995;Hussey et al, 1998) or transient transformation (Aizawa et al, 1996;Dong et al, 2001).…”

The Identification of a Second Cofilin Binding Site on Actin Suggests a Novel, Intercalated Arrangement of F-actin Binding

“…9), produces the change in filament twist by increasing the angle of rotation between each longitudinally associated actin. The high degree of cooperation evident in the binding of ADF-cofilins to F-actin (20,21,25) has been explained by the changed twist they induce in the filament (25). We propose that in opening up a space between two subunits at one side of the filament, this also places a strain on the opposite Inset, the interaction is detected from fluorescence polarization measurements.…”

“…Cofilin can be cross-linked to residues 1-12 on actin (58), and ADF can be cross-linked to cysteine 374 on actin (58). Also many have found that various fluorescent labels on cysteine 374 are quenched by ADF-cofilin binding (6,9,15,21,24,66).…”

“…Soon after the discovery of the first member of the family (10), several authors suggested that depolymerization occurred through severing (9,18). Evidence for a severing mechanism later came from videomicroscopy (19,20), but this was later challenged (6) as it was shown (6,21) that cofilins increased the off-rate from the pointed end of the filament. However, the two opinions are not necessarily exclusive (22)(23)(24), and a similar mechanism has been proposed for both events (23).…”

Experimental Evidence for the Limiting Role of Enzymatic Reactions in Chemoattractant-induced Pseudopod Extension in Human Neutrophils

“…G-actin in the neutrophil is sequestered by either profilin (33) or thymosin-␤4 (34). Profilin, a nucleotide exchange factor (35,36), recovers the ADP-bound actin after its release during cofilin-assisted severing of F-actin filaments (37). Profilin mediates the formation of ATP-actin, which is then added to free F-actin barbed ends (38).…”