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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Magic angle spinning NMR structure of human cofilin-2 assembled on actin filaments reveals isoform-specific conformation and binding mode
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.
more » « less- NSF-PAR ID:
- 10381736
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)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.more » « less
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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.
