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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

Side-binding proteins alter filament structure.(A) (left panels) Images of individual filaments attached to the surface using different side-binding proteins at the lowest or highest surface density of tethering protein. Scale bar: 5 μm. (right panels) Estimated apparent persistence length from the angular correlation along the filament contour length at the lowest (red) and the highest (blue) lattice-binding protein densities, and when the protein is present in solution (green). Error bars represent s.e.m. of more than 50 filaments measured per experimental condition. (B) Images from a movie of an individual growing actin filament under treadmilling conditions. The barbed-end is marked with a red arrowhead and pointed-end with a blue dot. The filament undergoes a fragmentation event (yellow star) at 488 s and afterwards depolymerizes from its new pointed-end while the newly created barbed-end does not elongate. The free-actin concentration was 400 nM. Time is given in seconds. Scale bar: 5 μm. (C) Characterization of the direction of barbed-end filament growth as a function of the tethering protein used (see ‘Materials and methods’ for details). Examples of each class are shown in the left panels. Scale bars: 3 μm. α-actinin was observed to grow almost exclusively in the counterclockwise direction. No preferred direction of growth is observed for the other side-binding proteins measured.DOI:http://dx.doi.org/10.7554/eLife.04599.017
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fig5: Side-binding proteins alter filament structure.(A) (left panels) Images of individual filaments attached to the surface using different side-binding proteins at the lowest or highest surface density of tethering protein. Scale bar: 5 μm. (right panels) Estimated apparent persistence length from the angular correlation along the filament contour length at the lowest (red) and the highest (blue) lattice-binding protein densities, and when the protein is present in solution (green). Error bars represent s.e.m. of more than 50 filaments measured per experimental condition. (B) Images from a movie of an individual growing actin filament under treadmilling conditions. The barbed-end is marked with a red arrowhead and pointed-end with a blue dot. The filament undergoes a fragmentation event (yellow star) at 488 s and afterwards depolymerizes from its new pointed-end while the newly created barbed-end does not elongate. The free-actin concentration was 400 nM. Time is given in seconds. Scale bar: 5 μm. (C) Characterization of the direction of barbed-end filament growth as a function of the tethering protein used (see ‘Materials and methods’ for details). Examples of each class are shown in the left panels. Scale bars: 3 μm. α-actinin was observed to grow almost exclusively in the counterclockwise direction. No preferred direction of growth is observed for the other side-binding proteins measured.DOI:http://dx.doi.org/10.7554/eLife.04599.017

Mentions: During the course of filament elongation analysis as a function of side-binding protein density on the surface (Figure 3), we noticed that filaments appeared more bent as the tether density increased. To quantify this curviness, we estimated an apparent persistence length ‘’ of individual filaments associated with different side-binding proteins (see ‘Materials and methods’ for details). The persistence length Lp (Boal, 2012) reflects the material properties of the filament, which are related to its structure (Chu and Voth, 2005, 2006; Pfaendtner et al., 2010), and has already been shown to be tunable by side-binding proteins (such as myosin or cofilin [McCullough et al., 2008; Murrell and Gardel, 2012; Bengtsson et al., 2013]). At the lowest side-binding protein density (∼10 molecules μm−2 or ∼0.1 molecules per filament micron), actin filaments had an of ∼18 μm and was independent of the associated protein (Figure 5A). At the highest densities (∼16,000 molecules·μm−2 or ∼100 molecules per filament micron), the presence of NEM-myosin decreased the to 4 ± 1 μm while it was reduced to 3 ± 1 μm, 5 ± 1 μm, and 2.2 ± 0.3 μm when using filamin, VASP and α-actinin, respectively (N > 50 for each condition, Figure 5A). Estimates for the persistence length of surface adsorbed filaments are consistent with what has been determined for freely fluctuating filaments (McCullough et al., 2008; Graham et al., 2014). We also tested the mechanical effect of the side binding proteins on the actin filament when the actin filaments were attached to the surface with a low density of NEM-myosin and the side-binding protein was present in solution. Again, we observed a decrease in the persistence length of about 30% for filamin, α-actinin, and NEM-myosin, whereas the effect was about 50% in the presence of VASP or VASP ΔGAB (Figure 5A).10.7554/eLife.04599.017Figure 5.Side-binding proteins alter filament structure.


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)

Side-binding proteins alter filament structure.(A) (left panels) Images of individual filaments attached to the surface using different side-binding proteins at the lowest or highest surface density of tethering protein. Scale bar: 5 μm. (right panels) Estimated apparent persistence length from the angular correlation along the filament contour length at the lowest (red) and the highest (blue) lattice-binding protein densities, and when the protein is present in solution (green). Error bars represent s.e.m. of more than 50 filaments measured per experimental condition. (B) Images from a movie of an individual growing actin filament under treadmilling conditions. The barbed-end is marked with a red arrowhead and pointed-end with a blue dot. The filament undergoes a fragmentation event (yellow star) at 488 s and afterwards depolymerizes from its new pointed-end while the newly created barbed-end does not elongate. The free-actin concentration was 400 nM. Time is given in seconds. Scale bar: 5 μm. (C) Characterization of the direction of barbed-end filament growth as a function of the tethering protein used (see ‘Materials and methods’ for details). Examples of each class are shown in the left panels. Scale bars: 3 μm. α-actinin was observed to grow almost exclusively in the counterclockwise direction. No preferred direction of growth is observed for the other side-binding proteins measured.DOI:http://dx.doi.org/10.7554/eLife.04599.017
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Related In: Results  -  Collection

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fig5: Side-binding proteins alter filament structure.(A) (left panels) Images of individual filaments attached to the surface using different side-binding proteins at the lowest or highest surface density of tethering protein. Scale bar: 5 μm. (right panels) Estimated apparent persistence length from the angular correlation along the filament contour length at the lowest (red) and the highest (blue) lattice-binding protein densities, and when the protein is present in solution (green). Error bars represent s.e.m. of more than 50 filaments measured per experimental condition. (B) Images from a movie of an individual growing actin filament under treadmilling conditions. The barbed-end is marked with a red arrowhead and pointed-end with a blue dot. The filament undergoes a fragmentation event (yellow star) at 488 s and afterwards depolymerizes from its new pointed-end while the newly created barbed-end does not elongate. The free-actin concentration was 400 nM. Time is given in seconds. Scale bar: 5 μm. (C) Characterization of the direction of barbed-end filament growth as a function of the tethering protein used (see ‘Materials and methods’ for details). Examples of each class are shown in the left panels. Scale bars: 3 μm. α-actinin was observed to grow almost exclusively in the counterclockwise direction. No preferred direction of growth is observed for the other side-binding proteins measured.DOI:http://dx.doi.org/10.7554/eLife.04599.017
Mentions: During the course of filament elongation analysis as a function of side-binding protein density on the surface (Figure 3), we noticed that filaments appeared more bent as the tether density increased. To quantify this curviness, we estimated an apparent persistence length ‘’ of individual filaments associated with different side-binding proteins (see ‘Materials and methods’ for details). The persistence length Lp (Boal, 2012) reflects the material properties of the filament, which are related to its structure (Chu and Voth, 2005, 2006; Pfaendtner et al., 2010), and has already been shown to be tunable by side-binding proteins (such as myosin or cofilin [McCullough et al., 2008; Murrell and Gardel, 2012; Bengtsson et al., 2013]). At the lowest side-binding protein density (∼10 molecules μm−2 or ∼0.1 molecules per filament micron), actin filaments had an of ∼18 μm and was independent of the associated protein (Figure 5A). At the highest densities (∼16,000 molecules·μm−2 or ∼100 molecules per filament micron), the presence of NEM-myosin decreased the to 4 ± 1 μm while it was reduced to 3 ± 1 μm, 5 ± 1 μm, and 2.2 ± 0.3 μm when using filamin, VASP and α-actinin, respectively (N > 50 for each condition, Figure 5A). Estimates for the persistence length of surface adsorbed filaments are consistent with what has been determined for freely fluctuating filaments (McCullough et al., 2008; Graham et al., 2014). We also tested the mechanical effect of the side binding proteins on the actin filament when the actin filaments were attached to the surface with a low density of NEM-myosin and the side-binding protein was present in solution. Again, we observed a decrease in the persistence length of about 30% for filamin, α-actinin, and NEM-myosin, whereas the effect was about 50% in the presence of VASP or VASP ΔGAB (Figure 5A).10.7554/eLife.04599.017Figure 5.Side-binding proteins alter filament structure.

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