The continuous assembly and disassembly of actin filament networks is vital for cellular processes including division, growth, and motility. Network remodeling is facilitated by cofilins, a family of essential regulatory proteins that fragment actin filaments. Cofilin induces net structural changes in filaments that render them more compliant in bending and twisting. A model in which local stress accumulation at mechanical discontinuities, such as boundaries of bare and cofilin-decorated filament segments, accounts for the cofilin concentration dependence of severing, including maximal activity at sub-stoichiometric binding densities. Real-time imaging of cofilin-mediated filament severing supports the boundary-fracture model. The severing model predicts that fragmentation is promoted by factors modulating filament mechanics (e.g. tethering, cross-linking, or deformation), possibly explaining enhanced in vivo severing activities.

Cofilin is an essential actin filament severing protein that functions in the dynamic remodeling of the actin cytoskeleton. Filament severing activity is most efficient at sub-stoichiometric cofilin binding densities (i.e. <1 cofilin per actin filament subunit), and peaks when the number density of boundaries (i.e. junctions) between bare and cofilin-decorated segments is maximal. A model in which local topological and mechanical discontinuities lead to preferential fragmentation at boundaries accounts for available experimental data, including direct visualization of cofilin and actin during real-time severing events. The boundary-severing model predicts that ligands (e.g. other actin-binding proteins) that compete with cofilin for actin filament binding and modulate cofilin occupancy on filaments will alter the bare-decorated segment boundary density, and thus, the filament severing activity of cofilin. Here, we directly test this model prediction by evaluating the effects of phalloidin and myosin, two ligands that compete with cofilin for filament binding, on the actin filament binding and severing activities of cofilin. Our experiments demonstrate that competitive displacement of cofilin lowers cofilin occupancy and promotes severing when initial cofilin occupancy is high (i.e. >50%). Even in the presence of competitive ligands, maximum severing activity occurs when cofilin-decorated boundary density is highest, consistent with preferential fragmentation at boundaries. We propose a general “severodyne” framework for the modulation of cofilin-mediated actin filament severing by small molecule or actin-binding protein ligands that compete with cofilin for actin filament binding.

Cofilin/ADF proteins are actin-remodeling proteins, essential for actin disassembly in various cellular processes, including cell division, intracellular transport, and motility. Cofilins bind actin filaments cooperatively and sever them preferentially at boundaries between bare and cofilin-decorated (cofilactin) segments. The cooperative binding to actin has been proposed to originate from conformational changes that propagate allosterically from clusters of bound cofilin to bare actin segments. Estimates of the lengths over which these cooperative conformational changes propagate vary dramatically, ranging from 2 to >100 subunits. Here, we present a general, structure-based method for detecting from cryo-EM micrographs small variations in filament geometry (i.e. twist) with single subunit precision. How these variations correlate with regulatory protein occupancy reveals how far allosteric, conformational changes propagate along filaments. We used this method to determine the effects of cofilin on the actin filament twist. Our results indicate that cofilin-induced changes in filament twist propagate only 1-2 subunits from the boundary into the bare actin segment, independently of the boundary polarity (i.e. irrespective of whether or not the bare actin segment flanks the pointed or barbed end side of the boundary) and the pyrene fluorophore labeling of actin. These observations indicate that the filament twist changes abruptly at boundaries between bare and cofilin-decorated segments, thereby constraining mechanistic models of cooperative actin filament interactions and severing by cofilin. The methods presented here extend the capability of cryo-EM to analyze biologically relevant deviations from helical symmetry in actin as well as other classes of linear polymers.

Intrinsically disordered (ID) proteins function in the absence of a unique stable structure and appear to challenge the classic structure-function paradigm. The extent to which ID proteins take advantage of subtle conformational biases to perform functions, and whether signals for such mechanism can be identified in proteome-wide studies is not well understood. Of particular interest is the polyproline II (PII) conformation, suggested to be highly populated in unfolded proteins. We experimentally determine a complete calorimetric propensity scale for the PII conformation. Projection of the scale into representative eukaryotic proteomes reveals significant PII bias in regions coding for ID proteins. Importantly, enrichment of PII in ID proteins, or protein segments, is also captured by other PII scales, indicating that this enrichment is robustly encoded and universally detectable regardless of the method of PII propensity determination. Gene ontology (GO) terms obtained using our PII scale and other scales demonstrate a consensus for molecular functions performed by high PII proteins across the proteome. Perhaps the most striking result of the GO analysis is conserved enrichment (P < 10 28) of phosphorylation sites in high PII regions found by all PII scales. Subsequent conformational analysis reveals a phosphorylation-dependent modulation of PII, suggestive of a conserved ''tunability'' within these regions. In summary, the application of an experimentally determined polyproline II (PII) propensity scale to proteome-wide sequence analysis and gene ontology reveals an enrichment of PII bias near disordered phosphorylation sites that is conserved throughout eukaryotes.

Cytoskeletal polymers play a fundamental role in the responses of cells to both external and internal stresses. Quantitative knowledge of the mechanical properties of those polymers is essential for developing predictive models of cell mechanics and mechano-sensing. Linear cytoskeletal polymers, such as actin filaments and microtubules, can grow to cellular length scales at which they behave as semiflexible polymers that undergo thermally-driven shape deformations. Bending deformations are often modeled using the wormlike chain model. A quantitative metric of a polymer's resistance to bending is the persistence length, the fundamental parameter of that model. A polymer's bending persistence length is extracted from its shape as visualized using various imaging techniques. However, the analysis methodologies required for determining the persistence length are often not readily within reach of most biological researchers or educators. Motivated by that limitation, we developed user-friendly, multi-platform compatible software to determine the bending persistence length from images of surface-adsorbed or freely fluctuating polymers. Three different types of analysis are available (cosine correlation, end-to-end and bending-mode analyses), allowing for rigorous cross-checking of analysis results. The software is freely available and we provide sample data of adsorbed and fluctuating filaments and expected analysis results for educational and tutorial purposes.

Actin assembly, filament mechanical properties, and interactions with regulatory proteins depend on the types and concentrations of salts in solution. Salts modulate actin through both nonspecific electrostatic effects and specific binding to discrete sites. Multiple cation-binding site classes spanning a broad range of affinities (nanomolar to millimolar) have been identified on actin monomers and filaments. This review focuses on discrete, low-affinity cation-binding interactions that drive polymerization, regulate filament-bending mechanics, and modulate interactions with regulatory proteins. Cation binding may be perturbed by actin post-translational modifications and linked equilibria. Partial cation occupancy under physiological and commonly used in vitro solution conditions likely contribute to filament mechanical heterogeneity and structural polymorphism. Site-specific cation-binding residues are conserved in Arp2 and Arp3, and may play a role in Arp2/3 complex activation and actin-filament branching activity. Actin-salt interactions demonstrate the relevance of ion-linked equilibria in the operation and regulation of complex biological systems.

Many biological processes, including cell division, growth, and motility, rely on rapid remodeling of the actin cytoskeleton and on actin filament severing by the regulatory protein cofilin. Phosphorylation of vertebrate cofilin at Ser-3 regulates both actin binding and severing. Substitution of serine with aspartate at position 3 (S3D) is widely used to mimic cofilin phosphorylation in cells and The S3D substitution weakens cofilin binding to filaments, and it is presumed that subsequent reduction in cofilin occupancy inhibits filament severing, but this hypothesis has remained untested. Here, using time-resolved phosphorescence anisotropy, electron cryomicroscopy, and all-atom molecular dynamics simulations, we show that S3D cofilin indeed binds filaments with lower affinity, but also with a higher cooperativity than wild-type cofilin, and severs actin weakly across a broad range of occupancies. We found that three factors contribute to the severing deficiency of S3D cofilin. First, the high cooperativity of S3D cofilin generates fewer boundaries between bare and decorated actin segments where severing occurs preferentially. Second, S3D cofilin only weakly alters filament bending and twisting dynamics and therefore does not introduce the mechanical discontinuities required for efficient filament severing at boundaries. Third, Ser-3 modification ( substitution with Asp or phosphorylation) "undocks" and repositions the cofilin N terminus away from the filament axis, which compromises S3D cofilin's ability to weaken longitudinal filament subunit interactions. Collectively, our results demonstrate that, in addition to inhibiting actin binding, Ser-3 modification favors formation of a cofilin-binding mode that is unable to sufficiently alter filament mechanical properties and promote severing.

Vinculin is an abundant protein found at cell-cell and cell-extracellular matrix junctions. In muscles, a longer splice-isoform of vinculin, metavinculin, is also expressed. The metavinculin-specific insert is part of the C-terminal tail domain, the actin-binding site of both isoforms. Mutations in the metavinculin-specific insert are linked to heart disease such as dilated cardiomyopathies. Vinculin tail domain (VT) both binds and bundles actin filaments. Metavinculin tail domain (MVT) binds actin filaments in a similar orientation but does not bundle filaments. Recently, MVT was reported to sever actin filaments. In this work, we asked how MVT influences F-actin alone or in combination with VT. Cosedimentation and limited proteolysis experiments indicated a similar actin binding affinity and mode for both VT and MVT. In real time TIRF microscopy experiments MVT’s severing activity was negligible. Instead, we found that MVT binding caused a two-fold reduction in F-actin’s bending persistence length and increased susceptibility to breakage. Perhaps MVT allows the load of muscle contraction to act as a signal to reorganize actin filaments. Using mutagenesis and site-directed labeling with fluorescence probes, we determined that MVT alters actin interprotomer contacts and dynamics, which presumably reflect the observed changes in bending persistence length. Finally, we found that MVT decreases the density and thickness of actin filament bundles generated by VT. Altogether, our data suggest that MVT alters actin filament flexibility and tunes filament organization in the presence of VT. Both of these activities are potentially important for muscle cell function.