The shape of most animal cells is controlled by the actin cortex, a thin network of dynamic actin filaments (F-actin) situated just beneath the plasma membrane. The cortex is held far from equilibrium by both active stresses and polymer turnover: Molecular motors drive deformations required for cell morphogenesis, while actin-filament disassembly dynamics relax stress and facilitate cortical remodeling. While many aspects of actin-cortex mechanics are well characterized, a mechanistic understanding of how nonequilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system of entangled F-actin, wherein the steady-state length and turnover rate of F-actin are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and assemble filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. We demonstrate that cofilin-mediated severing is a single-timescale mode of stress relaxation that tunes the low-frequency viscosity over two orders of magnitude. These findings serve as the foundation for understanding the mechanics of more physiological F-actin networks with turnover and inform an updated microscopic model of single-filament turnover. They also demonstrate that polymer activity, in the form of ATP hydrolysis on F-actin coupled to nucleotide-dependent cofilin binding, is sufficient to generate a form of active matter wherein asymmetric filament disassembly preserves filament number despite sustained severing.
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Stress relaxation in F-actin solutions by severing
Networks of filamentous actin (F-actin) are important for the mechanics of most animal cells. These cytoskeletal networks are highly dynamic, with a variety of actin-associated proteins that control cross-linking, polymerization and force generation in the cytoskeleton. Inspired by recent rheological experiments on reconstituted solutions of dynamic actin filaments, we report a theoretical model that describes stress relaxation behavior of these solutions in the presence of severing proteins. We show that depending on the kinetic rates of assembly, disassembly, and severing, one can observe both length-dependent and length-independent relaxation behavior.
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- Award ID(s):
- 1826623
- NSF-PAR ID:
- 10229919
- Date Published:
- Journal Name:
- Soft Matter
- Volume:
- 15
- Issue:
- 31
- ISSN:
- 1744-683X
- Page Range / eLocation ID:
- 6300 to 6307
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary Actin filament assembly in plants is a dynamic process, requiring the activity of more than 75 actin‐binding proteins. Central to the regulation of filament assembly and stability is the activity of a conserved family of actin‐depolymerizing factors (
ADF s), whose primarily function is to regulate the severing and depolymerization of actin filaments. In recent years, the activity ofADF proteins has been linked to a variety of cellular processes, including those associated with response to stress. Herein, a wheatADF Ta was identified and characterized.ADF 4,Ta encodes a 139‐amino‐acid protein containing five F‐actin‐binding sites and two G‐actin‐binding sites, and interacts with wheat (ADF 4Triticum aestivum ) Actin1 (TaACT 1),in planta . Following treatment of wheat, separately, with jasmonic acid, abscisic acid or with the avirulent race,CYR 23, of the stripe rust pathogenPuccinia striiformis f. sp.tritici , we observed a rapid induction in accumulation ofTa ADF 4mRNA . Interestingly, accumulation ofTa ADF 4mRNA was diminished in response to inoculation with a virulent race,CYR 31. Silencing ofTa resulted in enhanced susceptibility toADF 4CYR 23, demonstrating a role forTa in defense signaling. Using a pharmacological‐based approach, coupled with an analysis of host response to pathogen infection, we observed that treatment of plants with the actin‐modifying agent latrunculin B enhanced resistance toADF 4CYR 23, including increased production of reactive oxygen species and enhancement of localized hypersensitive cell death. Taken together, these data support the hypothesis thatTa ADF 4 positively modulates plant immunity in wheat via the modulation of actin cytoskeletal organization. -
Gov, Nir (Ed.)Actin networks are essential for living cells to move, reproduce, and sense their environments. The dynamic and rheological behavior of actin networks is modulated by actin-binding proteins such as α-actinin, Arp2/3, and myosin. There is experimental evidence that actin-binding proteins modulate the cooperation of myosin motors by connecting the actin network. In this work, we present an analytical mean field model, using the Flory-Stockmayer theory of gelation, to understand how different actin-binding proteins change the connectivity of the actin filaments as the networks are formed. We follow the kinetics of the networks and estimate the concentrations of actin-binding proteins that are needed to reach connectivity percolation as well as to reach rigidity percolation. We find that Arp2/3 increases the actomyosin connectivity in the network in a non-monotonic way. We also describe how changing the connectivity of actomyosin networks modulates the ability of motors to exert forces, leading to three possible phases of the networks with distinctive dynamical characteristics: a sol phase, a gel phase, and an active phase. Thus, changes in the concentration and activity of actin-binding proteins in cells lead to a phase transition of the actin network, allowing the cells to perform active contraction and change their rheological properties.more » « less
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Abstract Actin polymerization dynamics regulated by actin-binding proteins are essential for various cellular functions. The cofilin family of proteins are potent regulators of actin severing and filament disassembly. The structural basis for cofilin-isoform-specific severing activity is poorly understood as their high-resolution structures in complex with filamentous actin (F-actin) are lacking. Here, we present the atomic-resolution structure of the muscle-tissue-specific isoform, cofilin-2 (CFL2), assembled on ADP-F-actin, determined by magic-angle-spinning (MAS) NMR spectroscopy and data-guided molecular dynamics (MD) simulations. We observe an isoform-specific conformation for CFL2. This conformation is the result of a unique network of hydrogen bonding interactions within the α2 helix containing the non-conserved residue, Q26. Our results indicate F-site interactions that are specific between CFL2 and ADP-F-actin, revealing mechanistic insights into isoform-dependent F-actin disassembly.
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Abstract Microtubule (MT)‐associated proteins regulate the dynamic behavior of MTs during cellular processes. MT severing enzymes are the associated proteins which destabilize MTs by removing subunits from the lattice. One model for how severing enzymes remove tubulin dimers from the MT lattice is by unfolding its subunits through pulling on the carboxy‐terminal tails of tubulin dimers. This model stems from the fact that severing enzymes are AAA+ unfoldases. To test this mechanism, we apply pulling forces on the carboxy‐terminal regions of MT subunits using coarse grained molecular simulations. In our simulations, we used different MT lattices and concentrations of severing enzymes. We compare our simulation results with data from in vitro severing assays and find that the experimental data is best fit by a model of cooperative removal of protofilament fragments by severing enzymes, which depends on the severing enzyme concentration and placement on the MT lattice.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/c9sm01263j
