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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

Two-color seeded assay for visualizing pointed-end growth from an actin filament seed.A schematic of the assay is shown. Actin filament fragments labeled in red with atto565 were used as seeds for filament growth in a solution of atto488-labeled (green) actin monomers. After 15 min, the reaction was stopped by addition of phalloidin and dilution to a final concentration below 200 nM of free monomers. Filaments formed during this time are therefore diluted and individual filaments can easily be visualized. The filaments exhibiting growth at the barbed-end or at both ends were counted. The last figure shows the results of the analysis: the observed (gray) frequency of filaments that exhibited pointed-end growth, 20% was significantly smaller than the predicted value (red) of 100% (n = 1000).DOI:http://dx.doi.org/10.7554/eLife.04599.016
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fig4s3: Two-color seeded assay for visualizing pointed-end growth from an actin filament seed.A schematic of the assay is shown. Actin filament fragments labeled in red with atto565 were used as seeds for filament growth in a solution of atto488-labeled (green) actin monomers. After 15 min, the reaction was stopped by addition of phalloidin and dilution to a final concentration below 200 nM of free monomers. Filaments formed during this time are therefore diluted and individual filaments can easily be visualized. The filaments exhibiting growth at the barbed-end or at both ends were counted. The last figure shows the results of the analysis: the observed (gray) frequency of filaments that exhibited pointed-end growth, 20% was significantly smaller than the predicted value (red) of 100% (n = 1000).DOI:http://dx.doi.org/10.7554/eLife.04599.016

Mentions: As an additional test to rule out any tether, surface or light-induced effect of the pausing, we used a two-color solution assay to investigate pointed-end growth. Here, a small seed (formed with atto565-labeled actin) was allowed to grow in solution for 15 min in the presence of atto488-labeled monomers, followed by stabilization, dilution, and visualization of the filaments (Figure 4—figure supplement 3). At a free actin concentration of 1 μM, the concentration used in solution to allow filament elongation, all pointed-ends are expected to grow at an average rate of ∼0.5 sub/s (Pollard, 1986). In contrast to this expectation, we observed that only 20% of the seeds grew at the pointed-end (N = 1000, Figure 4—figure supplement 3). This percentage is higher than we observe in the surface-based experiments, which could be due to annealing of filaments in solution (Sept et al., 1999; Andrianantoandro et al., 2001) or due to lack of the tethering protein. What is clear is that the non-elongating or paused state is not due to either surface or light-induced effects. Taken together, these results show that a single rate constant describes filament elongation kinetics from ATP-monomers in the absence of side-binding proteins and that the pointed-end has an intrinsic kinetically inactive state.


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)

Two-color seeded assay for visualizing pointed-end growth from an actin filament seed.A schematic of the assay is shown. Actin filament fragments labeled in red with atto565 were used as seeds for filament growth in a solution of atto488-labeled (green) actin monomers. After 15 min, the reaction was stopped by addition of phalloidin and dilution to a final concentration below 200 nM of free monomers. Filaments formed during this time are therefore diluted and individual filaments can easily be visualized. The filaments exhibiting growth at the barbed-end or at both ends were counted. The last figure shows the results of the analysis: the observed (gray) frequency of filaments that exhibited pointed-end growth, 20% was significantly smaller than the predicted value (red) of 100% (n = 1000).DOI:http://dx.doi.org/10.7554/eLife.04599.016
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4375888&req=5

fig4s3: Two-color seeded assay for visualizing pointed-end growth from an actin filament seed.A schematic of the assay is shown. Actin filament fragments labeled in red with atto565 were used as seeds for filament growth in a solution of atto488-labeled (green) actin monomers. After 15 min, the reaction was stopped by addition of phalloidin and dilution to a final concentration below 200 nM of free monomers. Filaments formed during this time are therefore diluted and individual filaments can easily be visualized. The filaments exhibiting growth at the barbed-end or at both ends were counted. The last figure shows the results of the analysis: the observed (gray) frequency of filaments that exhibited pointed-end growth, 20% was significantly smaller than the predicted value (red) of 100% (n = 1000).DOI:http://dx.doi.org/10.7554/eLife.04599.016
Mentions: As an additional test to rule out any tether, surface or light-induced effect of the pausing, we used a two-color solution assay to investigate pointed-end growth. Here, a small seed (formed with atto565-labeled actin) was allowed to grow in solution for 15 min in the presence of atto488-labeled monomers, followed by stabilization, dilution, and visualization of the filaments (Figure 4—figure supplement 3). At a free actin concentration of 1 μM, the concentration used in solution to allow filament elongation, all pointed-ends are expected to grow at an average rate of ∼0.5 sub/s (Pollard, 1986). In contrast to this expectation, we observed that only 20% of the seeds grew at the pointed-end (N = 1000, Figure 4—figure supplement 3). This percentage is higher than we observe in the surface-based experiments, which could be due to annealing of filaments in solution (Sept et al., 1999; Andrianantoandro et al., 2001) or due to lack of the tethering protein. What is clear is that the non-elongating or paused state is not due to either surface or light-induced effects. Taken together, these results show that a single rate constant describes filament elongation kinetics from ATP-monomers in the absence of side-binding proteins and that the pointed-end has an intrinsic kinetically inactive state.

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