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Side-binding proteins modulate actin filament dynamics.

Crevenna AH, Arciniega M, Dupont A, Mizuno N, Kowalska K, Lange OF, Wedlich-Söldner R, Lamb DC - Elife (2015)

Bottom Line: In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface.We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry.Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics.

View Article: PubMed Central - PubMed

Affiliation: Physical Chemistry, Department of Chemistry and Center for Nanoscience, Ludwig-Maximilians-Universität München, Munich, Germany.

ABSTRACT
Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics.

No MeSH data available.


Related in: MedlinePlus

Actin filament elongation as a function of the surface density of side-binding proteins.(A–B) The change in length, ΔL, as a function of time for a filament tether to the surface using α-actinin at (A) low (16.7 molecules/μm2 or 0.1 molecules per micron of filament) or (B) high (16,700 molecules/μm2 or 100 molecules per micron of filament) surface densities. (C–D) The distribution of elongation velocities measured at (C) low or (D) high density. The distribution is calculated by binning the instantaneous elongation velocity of more than 20 filaments into bins of 0.75 subunits/s. Solid lines are fits to Gaussian distributions. (E–F) The change in length, ΔL, as a function of time for a filament tether to the surface using NEM-myosin at (E) low (6.8 molecules/μm2 or 0.04 molecules per micron of filament) or (F) high (6800 molecules/μm2 or 40 molecules per micron of filament) surface densities. (G–H) Elongation velocity distribution of filaments using a NEM-myosin-coated surface at low (G) or high (H) density. The distribution is calculated by binning the instantaneous elongation velocity of more than 20 filaments into bins of 0.75 subunits/s. Solid lines are fits to Gaussian distributions. (I–J) The change in length, ΔL, as a function of time for a filament tether to the surface using VASP at (I) low (18.2 molecules/μm2 or 0.1 molecules per micron of filament) or (J) high (18,200 molecules/μm2 or 100 molecules per micron of filament) surface density. (K–L) Elongation velocity distribution of filaments using a VASP-coated surface at low (K) or high (L) density. The distribution is calculated by binning the instantaneous elongation velocity of more than 20 filaments into bins of 0.75 subunits/s and 10 subunits/s for the low- and high-density experiments, respectively. Solid lines are fits to Gaussian distributions. (M–N) The change in length, ΔL, as a function of time for a filament tether to the surface using VASP ΔGAB at (M) low (18.5 molecules/μm2 or 0.1 molecules per micron of filament) or (N) high (18,500 molecules/μm2 or 100 molecules per micron of filament) surface density. (O–P) Elongation velocity distribution of filaments using a VASP ΔGAB-coated surface at low (O) or high (P) density. The distribution is calculated by binning (0.75 sub/s and 10 sub/s bin size for low and high density, respectively) the instantaneous elongation velocity of more than 20 filaments. Solid lines are fits to Gaussian distributions.DOI:http://dx.doi.org/10.7554/eLife.04599.008
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fig3s1: Actin filament elongation as a function of the surface density of side-binding proteins.(A–B) The change in length, ΔL, as a function of time for a filament tether to the surface using α-actinin at (A) low (16.7 molecules/μm2 or 0.1 molecules per micron of filament) or (B) high (16,700 molecules/μm2 or 100 molecules per micron of filament) surface densities. (C–D) The distribution of elongation velocities measured at (C) low or (D) high density. The distribution is calculated by binning the instantaneous elongation velocity of more than 20 filaments into bins of 0.75 subunits/s. Solid lines are fits to Gaussian distributions. (E–F) The change in length, ΔL, as a function of time for a filament tether to the surface using NEM-myosin at (E) low (6.8 molecules/μm2 or 0.04 molecules per micron of filament) or (F) high (6800 molecules/μm2 or 40 molecules per micron of filament) surface densities. (G–H) Elongation velocity distribution of filaments using a NEM-myosin-coated surface at low (G) or high (H) density. The distribution is calculated by binning the instantaneous elongation velocity of more than 20 filaments into bins of 0.75 subunits/s. Solid lines are fits to Gaussian distributions. (I–J) The change in length, ΔL, as a function of time for a filament tether to the surface using VASP at (I) low (18.2 molecules/μm2 or 0.1 molecules per micron of filament) or (J) high (18,200 molecules/μm2 or 100 molecules per micron of filament) surface density. (K–L) Elongation velocity distribution of filaments using a VASP-coated surface at low (K) or high (L) density. The distribution is calculated by binning the instantaneous elongation velocity of more than 20 filaments into bins of 0.75 subunits/s and 10 subunits/s for the low- and high-density experiments, respectively. Solid lines are fits to Gaussian distributions. (M–N) The change in length, ΔL, as a function of time for a filament tether to the surface using VASP ΔGAB at (M) low (18.5 molecules/μm2 or 0.1 molecules per micron of filament) or (N) high (18,500 molecules/μm2 or 100 molecules per micron of filament) surface density. (O–P) Elongation velocity distribution of filaments using a VASP ΔGAB-coated surface at low (O) or high (P) density. The distribution is calculated by binning (0.75 sub/s and 10 sub/s bin size for low and high density, respectively) the instantaneous elongation velocity of more than 20 filaments. Solid lines are fits to Gaussian distributions.DOI:http://dx.doi.org/10.7554/eLife.04599.008

Mentions: At low tether densities (5–200 μm−2 or 0.025 to 1.0 molecules per micron of filament), the dynamics were independent of the tethering protein used. As an example, filamin is shown in Figure 3A–D. Elongating actin filaments (at a free actin monomer concentration of 1 μM) showed mostly kinetically active phases (Figure 3A), and the elongation velocity distributions were centered around ∼9 subunits·s−1 (Figure 3 and Figure 3—figure supplement 1). At high tether densities (600–18,000 μm−2 or 3–110 molecules per micron of filament), each side-binding protein tested generated a particular elongation behavior (Figure 3E and Figure 3—figure supplement 1). Using filamin, increasing the surface tether density decreased the mean elongation velocity of kinetically active phases (Figure 3B,D) and increased the fraction of time the filament spent in a paused state, i.e., the pausing probability ‘Pp’ (Figure 3B,D,F). In contrast, increasing the VASP or the VASP-ΔGAB density increased the elongation velocity while VASP also increased the Pp (Figure 3 and Figure 3—figure supplement 1). Higher surface concentrations of α-actinin or NEM-myosin had also an effect on the elongation velocity (Figure 3—figure supplement 1) and, in addition, the density of NEM-myosin had a strong effect on the Pp (Figure 3F and Figure 3—figure supplement 1).


Side-binding proteins modulate actin filament dynamics.

Crevenna AH, Arciniega M, Dupont A, Mizuno N, Kowalska K, Lange OF, Wedlich-Söldner R, Lamb DC - Elife (2015)

Actin filament elongation as a function of the surface density of side-binding proteins.(A–B) The change in length, ΔL, as a function of time for a filament tether to the surface using α-actinin at (A) low (16.7 molecules/μm2 or 0.1 molecules per micron of filament) or (B) high (16,700 molecules/μm2 or 100 molecules per micron of filament) surface densities. (C–D) The distribution of elongation velocities measured at (C) low or (D) high density. The distribution is calculated by binning the instantaneous elongation velocity of more than 20 filaments into bins of 0.75 subunits/s. Solid lines are fits to Gaussian distributions. (E–F) The change in length, ΔL, as a function of time for a filament tether to the surface using NEM-myosin at (E) low (6.8 molecules/μm2 or 0.04 molecules per micron of filament) or (F) high (6800 molecules/μm2 or 40 molecules per micron of filament) surface densities. (G–H) Elongation velocity distribution of filaments using a NEM-myosin-coated surface at low (G) or high (H) density. The distribution is calculated by binning the instantaneous elongation velocity of more than 20 filaments into bins of 0.75 subunits/s. Solid lines are fits to Gaussian distributions. (I–J) The change in length, ΔL, as a function of time for a filament tether to the surface using VASP at (I) low (18.2 molecules/μm2 or 0.1 molecules per micron of filament) or (J) high (18,200 molecules/μm2 or 100 molecules per micron of filament) surface density. (K–L) Elongation velocity distribution of filaments using a VASP-coated surface at low (K) or high (L) density. The distribution is calculated by binning the instantaneous elongation velocity of more than 20 filaments into bins of 0.75 subunits/s and 10 subunits/s for the low- and high-density experiments, respectively. Solid lines are fits to Gaussian distributions. (M–N) The change in length, ΔL, as a function of time for a filament tether to the surface using VASP ΔGAB at (M) low (18.5 molecules/μm2 or 0.1 molecules per micron of filament) or (N) high (18,500 molecules/μm2 or 100 molecules per micron of filament) surface density. (O–P) Elongation velocity distribution of filaments using a VASP ΔGAB-coated surface at low (O) or high (P) density. The distribution is calculated by binning (0.75 sub/s and 10 sub/s bin size for low and high density, respectively) the instantaneous elongation velocity of more than 20 filaments. Solid lines are fits to Gaussian distributions.DOI:http://dx.doi.org/10.7554/eLife.04599.008
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fig3s1: Actin filament elongation as a function of the surface density of side-binding proteins.(A–B) The change in length, ΔL, as a function of time for a filament tether to the surface using α-actinin at (A) low (16.7 molecules/μm2 or 0.1 molecules per micron of filament) or (B) high (16,700 molecules/μm2 or 100 molecules per micron of filament) surface densities. (C–D) The distribution of elongation velocities measured at (C) low or (D) high density. The distribution is calculated by binning the instantaneous elongation velocity of more than 20 filaments into bins of 0.75 subunits/s. Solid lines are fits to Gaussian distributions. (E–F) The change in length, ΔL, as a function of time for a filament tether to the surface using NEM-myosin at (E) low (6.8 molecules/μm2 or 0.04 molecules per micron of filament) or (F) high (6800 molecules/μm2 or 40 molecules per micron of filament) surface densities. (G–H) Elongation velocity distribution of filaments using a NEM-myosin-coated surface at low (G) or high (H) density. The distribution is calculated by binning the instantaneous elongation velocity of more than 20 filaments into bins of 0.75 subunits/s. Solid lines are fits to Gaussian distributions. (I–J) The change in length, ΔL, as a function of time for a filament tether to the surface using VASP at (I) low (18.2 molecules/μm2 or 0.1 molecules per micron of filament) or (J) high (18,200 molecules/μm2 or 100 molecules per micron of filament) surface density. (K–L) Elongation velocity distribution of filaments using a VASP-coated surface at low (K) or high (L) density. The distribution is calculated by binning the instantaneous elongation velocity of more than 20 filaments into bins of 0.75 subunits/s and 10 subunits/s for the low- and high-density experiments, respectively. Solid lines are fits to Gaussian distributions. (M–N) The change in length, ΔL, as a function of time for a filament tether to the surface using VASP ΔGAB at (M) low (18.5 molecules/μm2 or 0.1 molecules per micron of filament) or (N) high (18,500 molecules/μm2 or 100 molecules per micron of filament) surface density. (O–P) Elongation velocity distribution of filaments using a VASP ΔGAB-coated surface at low (O) or high (P) density. The distribution is calculated by binning (0.75 sub/s and 10 sub/s bin size for low and high density, respectively) the instantaneous elongation velocity of more than 20 filaments. Solid lines are fits to Gaussian distributions.DOI:http://dx.doi.org/10.7554/eLife.04599.008
Mentions: At low tether densities (5–200 μm−2 or 0.025 to 1.0 molecules per micron of filament), the dynamics were independent of the tethering protein used. As an example, filamin is shown in Figure 3A–D. Elongating actin filaments (at a free actin monomer concentration of 1 μM) showed mostly kinetically active phases (Figure 3A), and the elongation velocity distributions were centered around ∼9 subunits·s−1 (Figure 3 and Figure 3—figure supplement 1). At high tether densities (600–18,000 μm−2 or 3–110 molecules per micron of filament), each side-binding protein tested generated a particular elongation behavior (Figure 3E and Figure 3—figure supplement 1). Using filamin, increasing the surface tether density decreased the mean elongation velocity of kinetically active phases (Figure 3B,D) and increased the fraction of time the filament spent in a paused state, i.e., the pausing probability ‘Pp’ (Figure 3B,D,F). In contrast, increasing the VASP or the VASP-ΔGAB density increased the elongation velocity while VASP also increased the Pp (Figure 3 and Figure 3—figure supplement 1). Higher surface concentrations of α-actinin or NEM-myosin had also an effect on the elongation velocity (Figure 3—figure supplement 1) and, in addition, the density of NEM-myosin had a strong effect on the Pp (Figure 3F and Figure 3—figure supplement 1).

Bottom Line: In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface.We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry.Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics.

View Article: PubMed Central - PubMed

Affiliation: Physical Chemistry, Department of Chemistry and Center for Nanoscience, Ludwig-Maximilians-Universität München, Munich, Germany.

ABSTRACT
Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics.

No MeSH data available.


Related in: MedlinePlus