The mode of subunit addition regulates the processive elongation of actin filaments by formin.

Formins play crucial roles in actin polymerization by nucleating filaments and regulating their elongation. Formins bind the barbed ends of filaments via their dimeric FH2 domains, which step processively onto incoming actin subunits during elongation. Actin monomers can bind formin-bound barbed ends directly or undergo diffusion-mediated delivery through interactions with formin FH1 domains and profilin.

Actin filament dynamics at barbed ends: New structures, new insights.

The dynamic actin cytoskeleton contributes to many critical biological processes by providing the structural support underlying the morphology of most cells, facilitating intracellular transport, and generating forces required for cell motility and division. To execute many of these functions, actin monomers polymerize into polarized filaments that display different structural and biochemical properties at each end. Filament dynamics are regulated by diverse regulatory proteins which collaborate to dictate rates of elongation and disassembly, particularly at the fast-growing barbed (plus) end.

Computational tools for quantifying actin filament numbers, lengths, and bundling.

The actin cytoskeleton is a dynamic filamentous network that assembles into specialized structures to enable cells to perform essential processes. Direct visualization of fluorescently-labeled cytoskeletal proteins has provided numerous insights into the dynamic processes that govern the assembly of actin-based structures. However, accurate analysis of these experiments is often complicated by the interdependent and kinetic natures of the reactions involved.

Treatment with tumor-treating fields (TTFields) suppresses intercellular tunneling nanotube formation in vitro and upregulates immuno-oncologic biomarkers in vivo in malignant mesothelioma.

Disruption of intercellular communication within tumors is emerging as a novel potential strategy for cancer-directed therapy. Tumor-Treating Fields (TTFields) therapy is a treatment modality that has itself emerged over the past decade in active clinical use for patients with glioblastoma and malignant mesothelioma, based on the principle of using low-intensity alternating electric fields to disrupt microtubules in cancer cells undergoing mitosis. There is a need to identify other cellular and molecular effects of this treatment approach that could explain reported increased overall survival when TTFields are added to standard systemic agents.

Cooperative bundling by fascin generates actin structures with architectures that depend on filament length.

The assembly of actin-based structures with precisely defined architectures supports essential cellular functions, including motility, intracellular transport, and division. The geometric arrangements of the filaments within actin structures are stabilized via the association of crosslinking proteins, which bind two filaments simultaneously. Because actin polymerization and crosslinking occur concurrently within the dynamic environment of the cell, these processes likely play interdependent roles in shaping the architectures of actin-based structures.

Nucleation limits the lengths of actin filaments assembled by formin.

Formins stimulate actin polymerization by promoting both filament nucleation and elongation. Because nucleation and elongation draw upon a common pool of actin monomers, the rate at which each reaction proceeds influences the other. This interdependent mechanism determines the number of filaments assembled over the course of a polymerization reaction, as well as their equilibrium lengths.

A LINC between the nucleus and the cytoskeleton takes form(in).

Dynamic nuclear positioning requires the formation of robust connections between the cytoskeleton and components of the LINC complex, a protein assembly that spans the nuclear envelope. A new study by Lim et al. (2021) reveals the mechanism of association between the LINC complex proteins Nesprin-1/2 Giant and the cytoplasmic formin FHOD1.

Profilin's Affinity for Formin Regulates the Availability of Filament Ends for Actin Monomer Binding.

Nucleation-promoting proteins tightly regulate actin polymerization in cells. Whereas many of these proteins bind actin monomers directly, formins use the actin-binding protein profilin to dynamically load actin monomers onto their flexible Formin Homology 1 (FH1) domains. Following binding, FH1 domains deliver profilin-actin complexes to filament ends.

Competition for delivery of profilin-actin to barbed ends limits the rate of formin-mediated actin filament elongation.

Formins direct the elongation of unbranched actin filaments by binding their barbed ends and processively stepping onto incoming actin monomers to incorporate them into the filament. Binding of profilin to actin monomers creates profilin-actin complexes, which then bind polyproline tracts located in formin homology 1 (FH1) domains. Diffusion of these natively disordered domains enables direct delivery of profilin-actin to the barbed end, speeding the rate of filament elongation.

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends.

The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high-affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a ~ 6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations.

Mechanisms of formin-mediated actin assembly and dynamics.

Cellular viability requires tight regulation of actin cytoskeletal dynamics. Distinct families of nucleation-promoting factors enable the rapid assembly of filament nuclei that elongate and are incorporated into diverse and specialized actin-based structures. In addition to promoting filament nucleation, the formin family of proteins directs the elongation of unbranched actin filaments.

Latrunculin A Accelerates Actin Filament Depolymerization in Addition to Sequestering Actin Monomers.

Latrunculin A (LatA), a toxin from the red sea sponge Latrunculia magnifica, is the most widely used reagent to depolymerize actin filaments in experiments on live cells. LatA binds actin monomers and sequesters them from polymerization [1, 2]. Low concentrations of LatA result in rapid (tens of seconds) disassembly of actin filaments in animal [3] and yeast cells [2].

Dissection of two parallel pathways for formin-mediated actin filament elongation.

Formins direct the elongation of unbranched actin filaments that are incorporated into a diverse set of cytoskeletal structures. Elongation of formin-bound filaments occurs along two parallel pathways. The formin homology 2 (FH2) pathway allows actin monomers to bind directly to barbed ends bound by dimeric FH2 domains.

Gating mechanisms during actin filament elongation by formins.

Formins play an important role in the polymerization of unbranched actin filaments, and particular formins slow elongation by 5-95%. We studied the interactions between actin and the FH2 domains of formins Cdc12, Bni1 and mDia1 to understand the factors underlying their different rates of polymerization. All-atom molecular dynamics simulations revealed two factors that influence actin filament elongation and correlate with the rates of elongation.

Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins.

When tagged with a fluorescent protein, actin is not fully functional, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly in vitro and in fission yeast cells. mEGFP-LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring.

Abl2/Abl-related gene stabilizes actin filaments, stimulates actin branching by actin-related protein 2/3 complex, and promotes actin filament severing by cofilin.

Both Arp2/3 complex and the Abl2/Arg nonreceptor tyrosine kinase are essential to form and maintain diverse actin-based structures in cells, including cell edge protrusions in fibroblasts and cancer cells and dendritic spines in neurons. The ability of Arg to promote cell edge protrusions in fibroblasts does not absolutely require kinase activity, raising the question of how Arg might modulate actin assembly and turnover in the absence of kinase function. Arg has two distinct actin-binding domains and interacts physically and functionally with cortactin, an activator of the Arp2/3 complex.

Electrostatic interactions between the Bni1p Formin FH2 domain and actin influence actin filament nucleation.

Formins catalyze nucleation and growth of actin filaments. Here, we study the structure and interactions of actin with the FH2 domain of budding yeast formin Bni1p. We built an all-atom model of the formin dimer on an Oda actin filament 7-mer and studied structural relaxation and interprotein interactions by molecular dynamics simulations.

Aip1 promotes actin filament severing by cofilin and regulates constriction of the cytokinetic contractile ring.

Aip1 (actin interacting protein 1) is ubiquitous in eukaryotic organisms, where it cooperates with cofilin to disassemble actin filaments, but neither its mechanism of action nor its biological functions have been clear. We purified both fission yeast and human Aip1 and investigated their biochemical activities with or without cofilin. Both types of Aip1 bind actin filaments with micromolar affinities and weakly nucleate actin polymerization.

Interaction of profilin with the barbed end of actin filaments.

Profilin binds not only to actin monomers but also to the barbed end of the actin filament, where it inhibits association of subunits. To address open questions about the interactions of profilin with barbed ends, we measured the effects of a wide range of concentrations of Homo sapiens profilin 1 on the rate of elongation of individual skeletal muscle actin filaments by total internal reflection fluorescence microscopy. Much higher concentrations of profilin were required to stop elongation by AMP-PNP-actin monomers than ADP-actin monomers.

Tension modulates actin filament polymerization mediated by formin and profilin.

Formins promote processive elongation of actin filaments for cytokinetic contractile rings and other cellular structures. In vivo, these structures are exposed to tension, but the effect of tension on these processes was unknown. Here we used single-molecule imaging to investigate the effects of tension on actin polymerization mediated by yeast formin Bni1p.

Determinants of Formin Homology 1 (FH1) domain function in actin filament elongation by formins.

Formin-mediated elongation of actin filaments proceeds via association of Formin Homology 2 (FH2) domain dimers with the barbed end of the filament, allowing subunit addition while remaining processively attached to the end. The flexible Formin Homology 1 (FH1) domain, located directly N-terminal to the FH2 domain, contains one or more stretches of polyproline that bind the actin-binding protein profilin. Diffusion of FH1 domains brings associated profilin-actin complexes into contact with the FH2-bound barbed end of the filament, thereby enabling direct transfer of actin.

The leucine-rich repeat domain of Internalin B folds along a polarized N-terminal pathway.

The leucine-rich repeat domain of Internalin B is composed of seven tandem leucine-rich repeats, which each contain a short beta strand connected to a 3(10) helix by a short turn, and an N-terminal alpha-helical capping motif. To determine whether folding proceeds along a single, discrete pathway or multiple, parallel pathways, and to map the structure of the transition state ensemble, we examined the effects of destabilizing substitutions of conserved residues in each repeat. We find that, despite the structural redundancy among the repeats, folding proceeds through an N-terminal transition state ensemble in which the extent of structure formation is biased toward repeats one and two and includes both local and interrepeat interactions.

Folding thermodynamics and kinetics of the leucine-rich repeat domain of the virulence factor Internalin B.

Although the folding of alpha-helical repeat proteins has been well characterized, much less is known about the folding of repeat proteins containing beta-sheets. Here we investigate the folding thermodynamics and kinetics of the leucine-rich repeat (LRR) domain of Internalin B (InlB), an extracellular virulence factor from the bacterium Lysteria monocytogenes. This domain contains seven tandem leucine-rich repeats, of which each contribute a single beta-strand that forms a continuous beta-sheet with neighboring repeats, and an N-terminal alpha-helical capping motif.

Protein folding and stability using denaturants.

Measurements of protein folding and thermodynamic stability provide insight into the forces and energetics that determine structure, and can inform on protein domain organization, interdomain interactions, and effects of mutations on structure. This chapter describes methods, theory, and data analysis for the most accessible means to determine the thermodynamics of protein folding: chemical denaturation. Topics include overall features of the folding reaction, advances in instrumentation, optimization of reagent purity, mechanistic models for analysis, and statistical and structural interpretation of fitted thermodynamic parameters.

Repeat-protein folding: new insights into origins of cooperativity, stability, and topology.

Although our understanding of globular protein folding continues to advance, the irregular tertiary structures and high cooperativity of globular proteins complicates energetic dissection. Recently, proteins with regular, repetitive tertiary structures have been identified that sidestep limitations imposed by globular protein architecture. Here we review recent studies of repeat-protein folding.