An official website of the United States government
Cellular remodeling of actin networks underlies cell motility during key morphological events, from embryogenesis to metastasis. In these transformations, there is an inherent competition between actin branching and bundling, because steric clashes among branches create a mechanical barrier to bundling. Recently, liquid-like condensates consisting purely of proteins involved in either branching or bundling of the cytoskeleton have been found to catalyze their respective functions. Yet in the cell, proteins that drive branching and bundling are present simultaneously. In this complex environment, which factors determine whether a condensate drives filaments to branch or become bundled? To answer this question, we added the branched actin nucleator, Arp2/3, to condensates composed of VASP, an actin bundling protein. At low actin to VASP ratios, branching activity, mediated by Arp2/3, robustly inhibited VASP-mediated bundling of filaments, in agreement with agent-based simulations. In contrast, as the actin to VASP ratio increased, addition of Arp2/3 led to formation of aster-shaped structures, in which bundled filaments emerged from a branched actin core, analogous to filopodia emerging from a branched lamellipodial network. These results demonstrate that multi-component, liquid-like condensates can modulate the inherent competition between bundled and branched actin morphologies, leading to organized, higher-order structures, similar to those found in motile cells.
more »
« less
Crowding alters F-actin secondary structure and hydration
Abstract Actin, an important component of eukaryotic cell cytoskeleton, regulates cell shape and transport. The morphology and biochemical properties of actin filaments are determined by their structure and protein-protein contacts. Crowded environments can organize filaments into bundles, but less is known about how they affect F-actin structure. This study used 2D IR spectroscopy and spectral calculations to examine how crowding and bundling impact the secondary structure and local environments in filaments and weakly or strongly bundled networks. The results reveal that bundling induces changes in actin’s secondary structure, leading to a decrease inβ-sheet and an increase in loop conformations. Strongly bundled networks exhibit a decrease in backbone solvent exposure, with less perturbedα-helices and nearly “locked”β-sheets. Similarly, the loops become less hydrated but maintain a dynamic environment. These findings highlight the role of loop structure in actin network morphology and stability under morphology control by PEG.
more »
« less
- PAR ID:
- 10505675
- Publisher / Repository:
- Nature Communications
- Date Published:
- Journal Name:
- Communications Biology
- Volume:
- 6
- Issue:
- 1
- ISSN:
- 2399-3642
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Cellular form and function are controlled by the assembly and stability of actin cytoskeletal structures—but disassembling/pruning these structures is equally essential for the plasticity and remodeling that underlie behavioral adaptations. Importantly, the mechanisms of actin assembly have been well-defined—including that it is driven by actin’s polymerization into filaments (F-actin) and then often bundling by crosslinking proteins into stable higher-order structures. In contrast, it remains less clear how these stable bundled F-actin structures are rapidly disassembled. We now uncover mechanisms that rapidly and extensively disassemble bundled F-actin. Using biochemical, structural, and imaging assays with purified proteins, we show that F-actin bundled with one of the most prominent crosslinkers, fascin, is extensively disassembled by Mical, the F-actin disassembly enzyme. Furthermore, the product of this Mical effect, Mical-oxidized actin, is poorly bundled by fascin, thereby further amplifying Mical’s disassembly effects on bundled F-actin. Moreover, another critical F-actin regulator, cofilin, also affects fascin-bundled filaments, but we find herein that it synergizes with Mical to dramatically amplify its disassembly of bundled F-actin compared to the sum of their individual effects. Genetic and high-resolution cellular assays reveal that Mical also counteracts crosslinking proteins/bundled F-actin in vivo to control cellular extension, axon guidance, and Semaphorin/Plexin cell-cell repulsion. Yet, our results also support the idea that fascin-bundling serves to dampen Mical’s F-actin disassembly in vitro and in vivo—and that physiologically relevant cellular remodeling requires a fine-tuned interplay between the factors that build bundled F-actin networks and those that disassemble them.more » « less
-
Liquid-like protein condensates perform diverse physiological functions. Previous work showed that VASP, a processive actin polymerase, forms condensates that polymerize and bundle actin. To minimize their curvature, filaments accumulated at the inner condensate surface, ultimately deforming the condensate into a rod-like shape, filled with a bundle of parallel filaments. Here we show that this behavior does not require proteins with specific polymerase activity. Specifically, we found that condensates composed of Lamellipodin, a protein that binds actin but is not an actin polymerase, were also capable of polymerizing and bundling actin filaments. To probe the minimum requirements for condensate-mediated actin bundling, we developed an agent-based computational model. Guided by its predictions, we hypothesized that any condensate-forming protein that binds actin could bundle filaments through multivalent crosslinking. To test this idea, we added an actin-binding motif to Eps15, a condensate-forming protein that does not normally bind actin. The resulting chimera formed condensates that drove efficient actin polymerization and bundling. Collectively, these findings broaden the family of proteins that could organize cytoskeletal filaments to include any actin-binding protein that participates in protein condensation.more » « less
-
ABSTRACT Actin microfilaments (F-actin) serve as the track for directional movement of organelles in plant cells. In actively growing plant cells, F-actin often form robust bundles that trespass the cellular dimension. To test how the F-actin network was employed for peroxisome movement, we wished to disturb actin organization by genetically compromising the function of villin (VLN) proteins that serve as the primary bundling factor inArabidopsis thalianacells. To do so, we isolated T-DNA insertional mutants in threeVLNgenes that were most actively expressed in vegetative tissues. We found that thevln4mutation greatly enhanced the growth defects caused by thevln2 vln3double mutant as thevln2 vln3 vln4triple mutant had a great reduction of organ growth and formed heavily deformed tissues. Both VLN2 and VLN4 proteins were detected on bundled F-actin filaments. Compared to the wild-type cells, the double and triple mutants exhibited progressively reduction of stable F-actin bundles and had fine F-actin filaments undergo rapid remodeling. The defective F-actin network did not prevent peroxisomes from taking on both rapid and slow movements along the F-actin tracks. However, we found that compromised F-actin bundling caused significant reductions in the speed of peroxisome movement and the displacement distance of peroxisome positions. Using a correlation analysis method, we also demonstrated that the complex heterogeneous peroxisome movement may be classified into clusters reflecting the directionality of peroxisome movement. The triple mutant suffered from a significant reduction of peroxisomes exhibiting long-range and linear movement. Our results provided insights into how VLN-dependent F-actin organization was coupled with the complex patterns of peroxisome movement.more » « less
-
Merks, Roeland M.H. (Ed.)Cross-linked actin networks are the primary component of the cell cytoskeleton and have been the subject of numerous experimental and modeling studies. While these studies have demonstrated that the networks are viscoelastic materials, evolving from elastic solids on short timescales to viscous fluids on long ones, questions remain about the duration of each asymptotic regime, the role of the surrounding fluid, and the behavior of the networks on intermediate timescales. Here we perform detailed simulations of passively cross-linked non-Brownian actin networks to quantify the principal timescales involved in the elastoviscous behavior, study the role of nonlocal hydrodynamic interactions, and parameterize continuum models from discrete stochastic simulations. To do this, we extend our recent computational framework for semiflexible filament suspensions, which is based on nonlocal slender body theory, to actin networks with dynamic cross linkers and finite filament lifetime. We introduce a model where the cross linkers are elastic springs with sticky ends stochastically binding to and unbinding from the elastic filaments, which randomly turn over at a characteristic rate. We show that, depending on the parameters, the network evolves to a steady state morphology that is either an isotropic actin mesh or a mesh with embedded actin bundles. For different degrees of bundling, we numerically apply small-amplitude oscillatory shear deformation to extract three timescales from networks of hundreds of filaments and cross linkers. We analyze the dependence of these timescales, which range from the order of hundredths of a second to the actin turnover time of several seconds, on the dynamic nature of the links, solvent viscosity, and filament bending stiffness. We show that the network is mostly elastic on the short time scale, with the elasticity coming mainly from the cross links, and viscous on the long time scale, with the effective viscosity originating primarily from stretching and breaking of the cross links. We show that the influence of nonlocal hydrodynamic interactions depends on the network morphology: for homogeneous meshworks, nonlocal hydrodynamics gives only a small correction to the viscous behavior, but for bundled networks it both hinders the formation of bundles and significantly lowers the resistance to shear once bundles are formed. We use our results to construct three-timescale generalized Maxwell models of the networks.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s42003-023-05274-3
