Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility

Abstract

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 ADP-bound 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.

Figure 1

Interaction of ADF1 with G-actin. The quenching of fluorescence of 0.8 μM NBD-labeled MgATP–G-actin (○, abscissa bottom scale) or MgADP–G-actin (•, abscissa top scale) was measured at different concentrations of ADF, under physiological ionic conditions (0.1 M KCl, 1 mM MgCl₂, pH 7.8). Symbols are data; lines are calculated binding curves using values of K _d of 0.1 μM (•) and 8 μM (○).

Figure 2

Interaction of ADF1 with F-actin. (a) Sedimentation assay for binding of ADF1 to F-actin. The binding of ³⁵S-labeled ADF to F-actin was measured at the following concentrations of F-actin (μM): □, 2; ⋄, 5; ▵ and ▿, 10. •, 5 μM F-actin, 7.5 μM phalloidin. ▪, 7 μM F-actin-ADP-BeF₃. The interval between the two arrows represents the amount of unassembled actin found at steady state in the supernatant of sedimented samples. Thin lines represent the high-affinity titration curves that would be obtained if ADF bound tightly to F-actin exclusively in a 1:1 molar ratio. (Inset) SDS-PAGE pattern of actin in the supernatant of sedimented samples containing 7 μM F-actin and ADF (in μM, left to right): 0, 1, 2, 3, 4, 5, 6, 8, 18, 12, 15, and 17. (b and c) pH dependence of ADF1 interaction with F-actin. SDS-PAGE of the pellets and supernatants of F-actin (5 μM) assembled at pH 6.5 (b) or pH 8.3 (c) in the presence of ADF. Left to right lanes: whole actin (5 μM); samples containing 0, 1.5, 3, 4.5, 6, 7.5, and 9 μM ADF. ▪, densitometered actin bands in the supernatants (in μM); ○, amount of ADF–F-actin, in μM (from ³⁵S radioactivity measurements).

Figure 3

ADF1 binds to labeled F-actin with concomitant quenching of fluorescence followed by partial depolymerization. (a) Simultaneous recordings of light scattering (1) and pyrenyl fluorescence (2) upon addition of 3 μM ADF (arrow) to a 4.5 μM 100% pyrenyl-labeled F-actin solution. The curves are normalized by adjusting the light scattering and fluorescence intensities of F-actin recorded before addition of ADF to the same maximum level and subtracting the intensities corresponding to G-actin. (b) Same as in a, but 7.5 μM ADF was added to 13.5 μM fully labeled pyrenyl–F-actin. (c) Same as in a, with a 0.5% labeled pyrenyl– F-actin solution. (d) The extent of quenching of fluorescence of 100% labeled pyrenyl–F-actin (3.6 μM) upon binding ADF is plotted versus ADF concentration.

Figure 4

Effects of ADF1 on the polymerization of ATP- and ADP-actin. (a) Turbidimetric recording of the spontaneous polymerization of 9.6 μM MgATP-actin in the presence of ADF1. (b) Spontaneous polymerization of 20 μM MgADP-actin in the presence of ADF1. The concentrations of ADF1 (in μM) are indicated on the curves. (c) Critical concentration plots for actin assembly derived from turbidimetric measurements. The extent of turbidity change (A _t= − A _t=0) over the time course of polymerization was plotted for actin alone (○) and actin polymerized in the presence of a saturating (1.5 molar equivalent) amount of ADF (•).

Figure 5

ADF1 increases the rate of filament growth at the barbed end, not at the pointed end of actin filaments. (a) Barbed end growth. The initial rate of elongation of G-actin (3.3 μM) was measured turbidimetrically at the indicated concentrations of spectrin-actin seeds, and the following concentrations of ADF (μM): ▪, 0; ▵, 0.5; •, 1; ○, 1.5. (Bottom inset) Typical raw data at 0, 1, and 1.5 μM ADF. (Top inset) The increase in slope of the data shown in the main panel was plotted versus the concentration of MgATP-G-actin-ADF complex calculated using Eq. 3, with K = 8 μM. (b) Pointed end growth. The assembly of 6.5 μM MgATP–G-actin was initiated by the addition of 20 nM (1 and 3) or 4 nM (2 and 4) gelsolin-actin seeds in the absence (1 and 2) and in the presence (3 and 4) of 3 μM ADF. The reaction was started by addition of salt. The turbidity was measured before the addition of salt and subtracted from the polymerization time course. The dead time was 5 s.

Figure 6

ADF1 increases the rate of depolymerization from the pointed ends, not from the barbed ends of actin filaments. (a) Depolymerization from the pointed ends. ADF1 was added at the indicated concentrations to a solution of 3.5 μM F-actin and 7 nM gelsolin. The initial rate of depolymerization was measured turbidimetrically. Typical curves are shown in the inset. 0, no ADF; 1, 0.3 μM; 2, 2.4 μM; 3, 6.5 μM; 4, control curve without ADF in which depolymerization was induced by adding 26 μM Tβ₄. (The time course shown was recorded over 200 min, i.e., with a 10-fold contracted scale.) (b) Depolymerization from the barbed ends. Depolymerization of F-actin (6 μM) was induced by addition of 6 μM DNaseI in the absence (thin line) and in the presence (thick line) of 10 μM ADF.

Figure 7

ADF1 increases the treadmilling and steady state ATPase rates of F-actin. The rate of treadmilling (□) was derived from the decrease in fluorescence of εADP bound to F-actin (13 μM) after a chase of ATP. The rate of ATP hydrolysis (•) was measured under the same conditions (13.5 μM F-actin) in polymerization buffer containing 0.17 mM γ[³²P]ATP. A rate of 0.014 μM/min was measured in the absence of ADF.

Figure 8

ADF1 increases the rate of propulsion of Listeria monocytogenes in platelet extracts and shortens the length of the actin tails. (a and b) Images of Listeria moving in diluted platelet extracts (no ADF added). (c–e) Images of Listeria in diluted platelet extracts supplemented with 0.75 μM ADF. Times are indicated in min. The trajectories of bacteria are visualized as white lines. Bar, 5 μm.

Figure 9

Electron micrographs of F-actin and ADF–F-actin filaments. A solution of 4 μM F-actin polymerized in the absence (A) and in the presence (B) of 5 μM ADF1 in physiological ionic strength buffer, pH 7.8, was deposited on the grid using a largely truncated pipet tip and processed for negative staining as described (Carlier et al., 1994). Bar, 0.1 μm.

Figure 9

Electron micrographs of F-actin and ADF–F-actin filaments. A solution of 4 μM F-actin polymerized in the absence (A) and in the presence (B) of 5 μM ADF1 in physiological ionic strength buffer, pH 7.8, was deposited on the grid using a largely truncated pipet tip and processed for negative staining as described (Carlier et al., 1994). Bar, 0.1 μm.

Figure 10

ADF increases the treadmilling of actin filaments. T, D-Pi, and D represent the ATP, ADP-Pi, and ADP, respectively, bound to actin. The different sizes of the different species drawn are meant to give an idea of their relative steady-state concentrations.