Rapid formation and high diffusibility of actin–cofilin cofilaments at low pH

Abstract

Cofilin is a small actin-binding protein that is known to bind both F-actin and G-actin, severing the former. The interaction of cofilin with actin is pH-sensitive, F-actin being preferentially bound at low pH and G-actin at higher pH, within the physiological range. Diffusion coefficients of F-actin with cofilin were measured by the fluorescence recovery after photobleaching (FRAP) technique. This has the potential for simultaneous and direct measurement of average polymer length via the average diffusion coefficient of the polymers (D LM ) as well as the fraction of polymerized actin, f LM , present in solution. In the range of cofilin±actin ratios up to 1 : 1 and at both pH 6.5 and pH 8.0, the diffusion coefficients of the polymers increased with the amount of cofilin present in the complex, in a co-operative manner to a plateau. We interpret this as indicating co-operative binding/severing and that filaments less than a certain length cannot be severed further. Under the conditions used here, filaments were found to be more motile at pH 6.5 than at pH 8.0. At pH 8.0, some actin is expected to be sequestered as ADP±actin±cofilin complexes, with the remaining actin being present as long slowly diffusing filaments. At pH 6.5, however, cofilin binds to F-actin to form short rapidly diffusing cofilaments. These filaments form very rapidly from cofilin±actin monomeric complexes, possibly indicating that this complex is able to polymerize without dissociation. These findings may be relevant to the nuclear import of actin±cofilin complexes.

A structural basis for the pH‐dependence of cofilin

“…2A) the calculated affinities were similar ( K d = 1 µ m ). This value is higher than that typically measured for actin‐ G–actin interaction at 0.1 µ m , probably as a result of the label as it is known that modification of Cys374 by other agents inhibits the interaction with cofilin [19]. We measured the affinity of binding of cofilin to unmodified ADP‐G‐actin and ATP‐G‐actin as a function of pH by direct means in order to confirm that the lack of pH sensitivity was not an artifact of labelled actin.…”

“…1). A significant fluorescent enhancement was observed only at pH 6.5, immediately after salt addition and before a significant amount of actin has polymerized [33], even if the very rapid kinetics of cofilin–actin polymerization at pH 6.5 are considered [19]. In a control experiment, no change was observed in the fluorescence intensity of FITC‐labeled cofilin used alone after salt addition to the sample (data not shown).…”

The second ADF/cofilin actin‐binding site exists in F‐actin, the cofilin–G‐actin complex, but not in G‐actin

“…However, Bonet et al . [28] have shown previously that actin labeled at cysteine 374 by a fluorescein reagent does not interact with cofilin. Therefore, we tested by ELISA the interaction of FITC‐labeled cofilin to coated dansylated G‐actin.…”

“…In agreement with Bonet et al . [28], we have found that the cofilin:G‐actin complex is able to polymerize without dissociation at pH 6.5. We can now see that a possible explanation for this is that cofilin binding G‐actin at site 1 induces a movement in subdomain 2 of actin that simultaneously exposes site 2 and moves subdomain into a position that allows two cofilin–actin complexes to dock.…”

Severing of F-actin by yeast cofilin is pH-independent

“…The overall slower F-actin shortening by cofilin at pH 6.8 than 7.8 has been attributed to either a combination of nucleation and depolymerization rates [Bonet et al, 2000;Blondin et al, 2002] or the reduced severing and depolymerization of filaments [Chen et al, 2004]. However, indirect assays of filament severing by ADF showed little, if any, pH-dependence of this process [Yeoh et al, 2002].…”