Conformational changes in actin filaments induced by formin binding to the barbed end

Abstract

Formins bind actin filaments and play an essential role in the regulation of the actin cytoskeleton. In this work we describe details of the formin-induced conformational changes in actin filaments by fluorescence-lifetime and anisotropy-decay experiments. The results show that the binding of the formin homology 2 domain of a mammalian formin (mouse mDia1) to actin filaments resulted in a less rigid protein structure in the microenvironment of the Cys374 of actin, weakening of the interactions between neighboring actin protomers, and greater overall flexibility of the actin filaments. The formin effect is smaller at greater ionic strength. The results show that formin binding to the barbed end of actin filaments is responsible for the increase of flexibility of actin filaments. One formin dimer can affect the dynamic properties of an entire filament. Analyses of the results obtained at various formin/actin concentration ratios indicate that at least 160 actin protomers are affected by the binding of a single formin dimer to the barbed end of a filament.

FIGURE 1

Schematic representation of an actin filament. The tube representation shows five actin monomers in a filament. Dark circles indicate the approximate position of the Cys³⁷⁴ residue, which was labeled in this study with a fluorescence probe.

FIGURE 2

mDia1-FH2 affects the fluorescence lifetime of IAEDANS-actin filaments. (A) The frequency-dependent phase (solid circles) and modulation (crosses) data from fluorescence lifetime experiments with 20 μM actin in the absence of formin. Solid line indicates the results from double-exponential fits. The upper panel shows the differences between the measured values and those calculated from the nonlinear least-square analyses. (B) The mDia1-FH2 concentration dependence of the fluorescence lifetime measured at 20 μM actin. The figure shows the average lifetimes (solid circles) from double-exponential fits and the mean lifetimes (open circles) from Gaussian analyses. The experiments were carried out at 0.5 mM MgCl₂ and 10 mM KCl.

FIGURE 3

mDia1-FH2 influenced the anisotropy decay of IAEDANS-actin filaments. (A) The mDia1-FH2 concentration dependence of the longer rotational correlation times measured at 20 μM actin. (Inset) Values of the shorter rotational correlation times as a function of [mDia1-FH2]. (B) The formin dependence of the limiting anisotropy values determined from anisotropy decay experiments. (Inset) Formin dependence of the amplitudes of the shorter (open circles) and longer (solid circles) rotational correlation times. (C) The mDia-FH2 concentration dependence of the half-angle of the cone within which the fluorophore rotated (Eq. 4). The errors presented are standard deviations from at least three independent experiments. Experimental conditions were as in Fig. 2.

FIGURE 4

The effect of mDia1-FH2 on actin filaments was independent of the actin concentration. (A) The mDia1-FH2 concentration dependence of the longer rotational correlation times determined at 30 μM (open circles) and 40 μM (solid squares) actin concentrations. The dashed line indicates the results with 20 μM actin. (B) The mDia1-FH2 concentration dependence of the limiting anisotropy determined at 30 μM (open circles) and 40 μM (solid circles) actin. Dashed line indicates the results with 20 μM actin from Fig. 3 C. (Inset) Formin dependence of the amplitudes of the shorter (squares) and longer (triangles) rotational correlation times at 30 μM (open symbols) and 40 μM (solid symbols) actin. Dashed line indicates the results with 20 μM actin. The errors presented are standard deviations from at least three independent experiments. Experimental conditions were as in Fig. 2.

FIGURE 5

The effect of formins on actin filaments was dependent on ionic strength. The figure shows the mDia1-FH2 dependence of the longer rotational correlation times from fluorescence anisotropy decay experiments with 30 μM actin at 0.5 mM MgCl₂ and 10 mM KCl (solid circles; LS) or at 1 mM MgCl₂ and 50 mM KCl (open circles; HS). The errors presented are standard deviations from at least three independent experiments.

FIGURE 6

A scheme for the interpretation of the various modes of motion of actin. Actin protomers in the filament are symbolized with circles. Arrows indicate the approximate directions in which the actin can move in the different modes.