Polymerization and depolymerization of V159N actin were measured by light scattering at 400 nm. (A) Polymerization of 5 μM wild-type or V159N actin was initiated by the addition of 0.1 M KCl, 2 mM MgCl₂, and 0.5 mM ATP. (B) Depolymerization was measured by diluting 5 μM F-actin to 0.5 μM in the polymerization buffer. The half time of depolymerization for wild-type actin is 114 s (n = 4, SD = 18), and for V159N actin is 355 s (n = 5, SD = 155). (C) Cofilin-accelerated depolymerization was observed by diluting 5 μM F-actin to 0.5 μM in polymerization buffer containing 0.5 μM yeast cofilin. (D) The critical concentration was measured by pelleting filamentous actin from steady-state reactions containing different concentrations of actin. Supernatants and pellets were analyzed by SDS/PAGE, and actin was quantified by densitometry. The critical concentration of V159N was measured three times, and the values ranged from 0 to 20 nM. The critical concentration of wild-type yeast actin is 160 nM.

Depolymerization of gelsolin-capped actin filaments. (A) Latrunculin A (50 μM) was added to gelsolin-capped filaments, and depolymerization was monitored with pyrene fluorescence. The half time of depolymerization is 96 s for wild-type (wt) filaments and 230 s for V159N filaments. (B) To compare length distributions, actin filaments used for the depolymerization assay were stained with rhodamine phalloidin and observed by fluorescence microscopy. (Bar = 5 μm.)

P_i release during actin filament assembly. Polymerization of actin (25 μM) was monitored by light scattering at 400 nm, and P_i release measured with the EnzChek phosphate assay kit (Molecular Probes). The amount of total polymerized actin then was measured by pelleting the final polymerization mix and measuring the protein concentration of the pellet by using an SDS gel containing actin standards. (A) Wild-type yeast actin. (B) V159N actin. (C) V159N actin plus 25 μM cofilin. The solid lines represent actin assembly and the circles are free P_i.

Electron micrographs of yeast wild-type F-actin (a), ADP-BeF₄⁻ filaments (b), and V159N filaments (c). Tobacco mosaic virus particles with a helical pitch of 23 Å were used as a size standard (c). (Bar in a = 1,000 Å.)

Three-dimensional electron microscopy reconstructions and actin filament models. Reconstructions of yeast actin filaments (a–f) compared with models generated from β-actin (g–l). The wild-type yeast actin, shown as a surface view (d) and in a single cross section (a), has an open nucleotide-binding cleft (arrow, d). The open cleft results in a lack of density between subdomains 2 (S2) and 4 (S4), as seen in cross section (a). Subdomains 1 (S1′) and 3 (S3′) from a subunit on the opposite strand also are labeled in a, and four subdomains from a single actin subunit are labeled in e. When the wild-type F-ADP filaments are incubated with BeF₄⁻ (b and e), the nucleotide-binding cleft closes. The V159N mutant actin (c and f) also shows a closure of the nucleotide-binding cleft. The opening of the nucleotide-binding cleft also can be seen in a crystal structure of nonmuscle β-actin (27) (h and i). The G-actin subunit from this crystal structure (h) has been oriented to the Lorenz et al. model for F-actin (26), using a least-squares alignment of Cα atoms in subdomains 1, 3, and 4 (3.3-Å rms deviation). The filament axis after such an alignment is shown by the vertical line (h, front view; i, side view), and the open arrows in g indicate the corresponding views of the subunits in a low-resolution filament surface generated from the atomic model. The corresponding “closed” state of β-actin (26) after alignment to the Lorenz et al. model (3.1-Å rms deviation) is shown in k and l, with the low-resolution surface generated from this subunit in j. The main difference between these two forms is the rotation of subdomain 2 by about 15°, shown by the arrow in i. (The scale bar in a is 50 Å and applies to a–c.)