A mechanism with severing near barbed ends and annealing explains structure and dynamics of dendritic actin networks
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Evaluation Summary:
In this work, Holz and colleagues develop a computational stochastic model of lamellipodial actin network growth and turnover to address an unresolved important question: how do these networks remain wide enough, maintain angular order, and actually increase the filament length behind the leading edge? They compare the filament organization and rate of incorporation/detachment of actin subunits with experimental data published in the literature. A main result from this study is that frequent filament fragmentation and annealing are key events in the reorganization of branched actin networks. The paper is well written, contains very thorough and fair literature review, is accurate, well documented. The result is novel and significant.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Single molecule imaging has shown that part of actin disassembles within a few seconds after incorporation into the dendritic filament network in lamellipodia, suggestive of frequent destabilization near barbed ends. To investigate the mechanisms behind network remodeling, we created a stochastic model with polymerization, depolymerization, branching, capping, uncapping, severing, oligomer diffusion, annealing, and debranching. We find that filament severing, enhanced near barbed ends, can explain the single molecule actin lifetime distribution, if oligomer fragments reanneal to free ends with rate constants comparable to in vitro measurements. The same mechanism leads to actin networks consistent with measured filament, end, and branch concentrations. These networks undergo structural remodeling, leading to longer filaments away from the leading edge, at the +/-35° orientation pattern. Imaging of actin speckle lifetimes at sub-second resolution verifies frequent disassembly of newly-assembled actin. We thus propose a unified mechanism that fits a diverse set of basic lamellipodia phenomenology.
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Evaluation Summary:
In this work, Holz and colleagues develop a computational stochastic model of lamellipodial actin network growth and turnover to address an unresolved important question: how do these networks remain wide enough, maintain angular order, and actually increase the filament length behind the leading edge? They compare the filament organization and rate of incorporation/detachment of actin subunits with experimental data published in the literature. A main result from this study is that frequent filament fragmentation and annealing are key events in the reorganization of branched actin networks. The paper is well written, contains very thorough and fair literature review, is accurate, well documented. The result is novel and significant.
(This preprint has been reviewed by eLife. We include the public reviews from the …
Evaluation Summary:
In this work, Holz and colleagues develop a computational stochastic model of lamellipodial actin network growth and turnover to address an unresolved important question: how do these networks remain wide enough, maintain angular order, and actually increase the filament length behind the leading edge? They compare the filament organization and rate of incorporation/detachment of actin subunits with experimental data published in the literature. A main result from this study is that frequent filament fragmentation and annealing are key events in the reorganization of branched actin networks. The paper is well written, contains very thorough and fair literature review, is accurate, well documented. The result is novel and significant.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this work, Holz and colleagues develop a computational stochastic model of lamellipodial growth and turnover. The aim of this work is to compare the filament organization and rate of incorporation/detachment of actin subunits with experimental data published in the literature. This model includes many reactions: actin polymerization, depolymerization, filament branching by the Arp2/3 complex, capping, uncapping, severing, oligomer diffusion, annealing, and debranching.
One of the difficulties of such model is to constrain as many parameters as possible. Thus, the first part of this study works on the dimensionality of the model, and determines that correct filament orientation pattern relative to the membrane requires a quasi-2D model, where new filaments are limited to branching within 10{degree sign} of …
Reviewer #1 (Public Review):
In this work, Holz and colleagues develop a computational stochastic model of lamellipodial growth and turnover. The aim of this work is to compare the filament organization and rate of incorporation/detachment of actin subunits with experimental data published in the literature. This model includes many reactions: actin polymerization, depolymerization, filament branching by the Arp2/3 complex, capping, uncapping, severing, oligomer diffusion, annealing, and debranching.
One of the difficulties of such model is to constrain as many parameters as possible. Thus, the first part of this study works on the dimensionality of the model, and determines that correct filament orientation pattern relative to the membrane requires a quasi-2D model, where new filaments are limited to branching within 10{degree sign} of the lamellipodial plane, a rather reasonable assumption for such flat structures.
The second part of this work treats network rearrangement and dynamics during treadmilling. Most of the parameters are set to estimated values or values published in the literature. Floating parameters are severing rates (random or biased toward barbed ends) and maximum fragment size in order to test the importance of fragmentation and reannealing in the reorganization of these actin networks. The authors demonstrate that frequent severing and annealing are necessary conditions to model correctly the dynamics of actin subunits along the lamellipodium, the presence of non-negligible amount of uncapped barbed ends along the lamellipodium, and the structural remodeling of actin networks.
The last part of the manuscript reports new speckle microscopy experiments performed at faster 0.1s time intervals. These experiments confirm that a surprisingly high fraction of actin speckles are disassembled shortly after actin filament assembly, which is supported by the model.
One the one hand, I am impressed by these simulations, which I find very informative and provide comprehensive understanding of the reactions at play. On the other hand, multi-parameter simulations raise necessarily questions about the choices of hypotheses and parameter values (and the sensitivity of this model to fluctuations of these parameters). I appreciate parameter scans which offer a visible way to follow the behavior of the system.
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Reviewer #2 (Public Review):
This is an excellent modeling study addressing the unresolved important question about lamellipodial actin network: how does the network remain wide enough, maintains angular order, and actually increases the filament length behind the leading edge?
The modeling approach is straightforward: use Monte Carlo simulations to grow actin networks in 2d and 3d by a combination of stochastic branching, capping and elongation. Such models were used before many times, but the key here is to add fragmentation and annealing of oligomers (short filaments). The authors show that this addition is the key to explain many observations and measurements, including speckle dynamics, long filaments behind the leading edge, etc. Zcomparison with the structure of the lamellipodia from 2 different cell types allows to test a couple …
Reviewer #2 (Public Review):
This is an excellent modeling study addressing the unresolved important question about lamellipodial actin network: how does the network remain wide enough, maintains angular order, and actually increases the filament length behind the leading edge?
The modeling approach is straightforward: use Monte Carlo simulations to grow actin networks in 2d and 3d by a combination of stochastic branching, capping and elongation. Such models were used before many times, but the key here is to add fragmentation and annealing of oligomers (short filaments). The authors show that this addition is the key to explain many observations and measurements, including speckle dynamics, long filaments behind the leading edge, etc. Zcomparison with the structure of the lamellipodia from 2 different cell types allows to test a couple of different parameter sets.
The paper is well written, contains very thorough and fair literature review, accurate, well documented. The result is novel and significant.
I don't have any critical comments.
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