Cracked actin filaments as mechanosensitive receptors.

Actin filament networks are exposed to mechanical stimuli, but the effect of strain on actin filament structure has not been well established in molecular detail. This is a critical gap in understanding because the activity of a variety of actin-binding proteins has recently been determined to be altered by actin filament strain. We therefore used all-atom molecular dynamics simulations to apply tensile strains to actin filaments and find that changes in actin subunit organization are minimal in mechanically strained, but intact, actin filaments. However, a conformational change disrupts the critical D-loop to W-loop connection between longitudinal neighboring subunits, which leads to a metastable cracked conformation of the actin filament whereby one protofilament is broken prior to filament severing. We propose that the metastable crack presents a force-activated binding site for actin regulatory factors that specifically associate with strained actin filaments. Through protein-protein docking simulations, we find that 43 evolutionarily diverse members of the dual zinc-finger-containing LIM-domain family, which localize to mechanically strained actin filaments, recognize two binding sites exposed at the cracked interface. Furthermore, through its interactions with the crack, LIM domains increase the length of time damaged filaments remain stable. Our findings propose a new molecular model for mechanosensitive binding to actin filaments.

A step-by-step guide to fragmenting bundled actin filaments.

There has long been conflicting evidence as to how bundled actin filaments, found in cellular structures such as filopodia, are disassembled. In this issue, Chikireddy et al. (https://doi.org/10.1083/jcb.202312106) provide a detailed in vitro analysis of the steps involved in fragmentation of fascin-bundled actin filaments and propose a novel mechanism for severing two-filament bundles.

Prediction of the essential intermolecular contacts for side-binding of VASP on F-actin.

Vasodilator-stimulated phosphoprotein (VASP) family proteins play a crucial role in mediating the actin network architecture in the cytoskeleton. The Ena/VASP homology 2 (EVH2) domain in each of the four identical arms of the tetrameric VASP consists of a loading poly-Pro region, a G-actin-binding domain (GAB), and an F-actin-binding domain (FAB). Together, the poly-Pro, GAB, and FAB domains allow VASP to bind to sides of actin filaments in a bundle, and recruit profilin-G-actin to processively elongate the filaments. The atomic resolution structure of the ternary complex, consisting of the loading poly-Pro region and GAB domain of VASP with profilin-actin, has been solved over a decade ago; however, a detailed structure of the FAB-F-actin complex has not been resolved to date. Experimental insights, based on homology of the FAB domain with the C region of WASP, have been used to hypothesize that the FAB domain binds to the cleft between subdomains 1 and 3 of F-actin. Here, in order to develop our understanding of the VASP-actin complex, we first augment known structural information about the GAB domain binding to actin with the missing FAB domain-actin structure, which we predict using homology modeling and docking simulations. In earlier work, we used mutagenesis and kinetic modeling to study the role of domain-level binding-unbinding kinetics of Ena/VASP on actin filaments in a bundle, specifically on the side of actin filaments. We further look at the nature of the side-binding of the FAB domain of VASP at the atomistic level using our predicted structure, and tabulate effective mutation sites on the FAB domain that would disrupt the VASP-actin complex. We test the binding affinity of Ena with mutated FAB domain using total internal reflection fluorescence microscopy experiments. The binding affinity of VASP is affected significantly for the mutant, providing additional support for our predicted structure.

Arp2/3 complex- and formin-mediated actin cytoskeleton networks facilitate actin binding protein sorting in fission yeast.

While it is well-established that F-actin networks with specific organizations and dynamics are tightly regulated by distinct sets of associated actin-binding proteins (ABPs), how ABPs self-sort to particular F-actin networks remains largely unclear. We report that actin assembly factors Arp2/3 complex and formin Cdc12 tune the association of ABPs fimbrin Fim1 and tropomyosin Cdc8 to different F-actin networks in fission yeast. Genetic and pharmacological disruption of F-actin networks revealed that Fim1 is preferentially directed to Arp2/3-complex mediated actin patches, whereas Cdc8 is preferentially targeted to formin Cdc12-mediated filaments in the contractile ring. To investigate the role of Arp2/3 complex- and formin Cdc12-mediated actin assembly, we used four-color TIRF microscopy to observe the in vitro reconstitution of ABP sorting with purified proteins. Fim1 or Cdc8 alone bind similarly well to filaments assembled by either assembly factor. However, in 'competition' reactions containing both actin assembly factors and both ABPs, ∼2.0-fold more Fim1 and ∼3.5-fold more Cdc8 accumulates on Arp2/3 complex branch points and formin Cdc12-assembled actin filaments, respectively. These findings indicate that F-actin assembly factors Arp2/3 complex and formin Cdc12 help facilitate the recruitment of specific ABPs, thereby tuning ABP sorting and subsequently establishing the identity of F-actin networks in fission yeast.

Highly flexible PEG-LifeAct constructs act as tunable biomimetic actin crosslinkers.

In vitro studies of actin filament networks crosslinked with dynamic actin binding proteins provide critical insights into cytoskeletal mechanics as well as inspiration for new adaptive materials design. However, discontinuous variance in the physiochemical properties of actin binding proteins impedes holistic relationships between crosslinker molecular parameters, network structure, and mechanics. Bio-synthetic constructs composed of synthetic polymer backbones and actin binding motifs would enable crosslinkers with engineered physiochemical properties to directly target the desired structure-property relationships. As a proof of concept, bio-synthetic crosslinkers composed of highly flexible polyethylene glycol (PEG) polymers functionalized with the actin binding peptide LifeAct, are explored as actin crosslinkers. Using bulk rheology and fluorescence microscopy, these constructs are shown to modulate actin filament network structure and mechanics in a contour length dependent manner, while maintaining the stress-stiffening behavior inherent to actin filament networks. These results encourage the design of more diverse and complex peptide-polymer crosslinkers to interrogate and control semi-flexible polymer networks.

Multi-monoubiquitylation controls VASP-mediated actin dynamics.

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics.

Reconstitution of the transition from a lamellipodia- to filopodia-like actin network with purified proteins.

How cells utilize complex mixtures of actin binding proteins to assemble and maintain functionally diverse actin filament networks with distinct architectures and dynamics within a common cytoplasm is a longstanding question in cell biology. A compelling example of complex and specialized actin structures in cells are filopodia which sense extracellular chemical and mechanical signals to help steer motile cells. Filopodia have distinct actin architecture, composed of long, parallel actin filaments bundled by fascin, which form finger-like membrane protrusions. Elongation of the parallel actin filaments in filopodia can be mediated by two processive actin filament elongation factors, formin and Ena/VASP, which localize to the tips of filopodia. There remains debate as to how the architecture of filopodia are generated, with one hypothesis proposing that filopodia are generated from the lamellipodia, which consists of densely packed, branched actin filaments nucleated by Arp2/3 complex and kept short by capping protein. It remains unclear if different actin filament elongation factors are necessary and sufficient to facilitate the emergence of filopodia with diverse characteristics from a highly dense network of short-branched capped filaments. To address this question, we combined bead motility and micropatterning biomimetic assays with multi-color Total Internal Reflection Fluorescence microscopy imaging, to successfully reconstitute the formation of filopodia-like networks (FLN) from densely-branched lamellipodia-like networks (LLN) with eight purified proteins (actin, profilin, Arp2/3 complex, Wasp pWA, fascin, capping protein, VASP and formin mDia2). Saturating capping protein concentrations inhibit FLN assembly, but the addition of either formin or Ena/VASP differentially rescues the formation of FLN from LLN. Specifically, we found that formin/mDia2-generated FLNs are relatively long and lack capping protein, whereas VASP-generated FLNs are comparatively short and contain capping protein, indicating that the actin elongation factor can affect the architecture and composition of FLN emerging from LLN. Our biomimetic reconstitution systems reveal that formin or VASP are necessary and sufficient to induce the transition from a LLN to a FLN, and establish robust in vitro platforms to investigate FLN assembly mechanisms.

Multi-monoubiquitination controls VASP-mediated actin dynamics.

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitination regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitination impacts VASP activity was unknown. Here we show that mimicking multi-monoubiquitination of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitinated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitinated VASP maintained the ability to bind and protect barbed ends from capping protein. Lastly, we demonstrate the introduction of recombinant multi-monoubiquitinated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitination controls VASP-mediated actin dynamics.

Cracked actin filaments as mechanosensitive receptors.

Actin filament networks are exposed to mechanical stimuli, but the effect of strain on actin filament structure has not been well-established in molecular detail. This is a critical gap in understanding because the activity of a variety of actin-binding proteins have recently been determined to be altered by actin filament strain. We therefore used all-atom molecular dynamics simulations to apply tensile strains to actin filaments and find that changes in actin subunit organization are minimal in mechanically strained, but intact, actin filaments. However, a conformational change disrupts the critical D-loop to W-loop connection between longitudinal neighboring subunits, which leads to a metastable cracked conformation of the actin filament, whereby one protofilament is broken prior to filament severing. We propose that the metastable crack presents a force-activated binding site for actin regulatory factors that specifically associate with strained actin filaments. Through protein-protein docking simulations, we find that 43 evolutionarily-diverse members of the dual zinc finger containing LIM domain family, which localize to mechanically strained actin filaments, recognize two binding sites exposed at the cracked interface. Furthermore, through its interactions with the crack, LIM domains increase the length of time damaged filaments remain stable. Our findings propose a new molecular model for mechanosensitive binding to actin filaments.

Acetylation of fission yeast tropomyosin does not promote differential association with cognate formins.

It was proposed from cellular studies that S. pombe tropomyosin Cdc8 (Tpm) segregates into two populations due to the presence or absence of an amino-terminal acetylation that specifies which formin-mediated F-actin networks it binds, but with no supporting biochemistry. To address this mechanism in vitro, we developed methods for S. pombe actin expression in Sf9 cells. We then employed 3-color TIRF microscopy using all recombinant S. pombe proteins to probe in vitro multicomponent mechanisms involving actin, acetylated and unacetylated Tpm, formins, and myosins. Acetyl-Tpm exhibits tight binding to actin in contrast to weaker binding by unacetylated Tpm. In disagreement with the differential recruitment model, Tpm showed no preferential binding to filaments assembled by the FH1-FH2-domains of two S. pombe formins, nor did Tpm binding have any bias towards the growing formin-bound actin filament barbed end. Although our in vitro findings do not support a direct formin-tropomyosin interaction, it is possible that formins bias differential tropomyosin isoform recruitment through undiscovered mechanisms. Importantly, despite a 12% sequence divergence between skeletal and S. pombe actin, S. pombe myosins Myo2 and Myo51 exhibited similar motile behavior with these two actins, validating key prior findings with these myosins that used skeletal actin.

Actin assembly requirements of the formin Fus1 to build the fusion focus.

In formin-family proteins, actin filament nucleation and elongation activities reside in the formin homology 1 (FH1) and FH2 domains, with reaction rates that vary by at least 20-fold between formins. Each cell expresses distinct formins that assemble one or several actin structures, raising the question of what confers each formin its specificity. Here, using the formin Fus1 in Schizosaccharomyces pombe, we systematically probed the importance of formin nucleation and elongation rates in vivo. Fus1 assembles the actin fusion focus, necessary for gamete fusion to form the zygote during sexual reproduction. By constructing chimeric formins with combinations of FH1 and FH2 domains previously characterized in vitro, we establish that changes in formin nucleation and elongation rates have direct consequences on fusion focus architecture, and that Fus1 native high nucleation and low elongation rates are optimal for fusion focus assembly. We further describe a point mutant in Fus1 FH2 that preserves native nucleation and elongation rates in vitro but alters function in vivo, indicating an additional FH2 domain property. Thus, rates of actin assembly are tailored for assembly of specific actin structures.

Mechanism of actin filament nucleation.

We used computational methods to analyze the mechanism of actin filament nucleation. We assumed a pathway where monomers form dimers, trimers, and tetramers that then elongate to form filaments but also considered other pathways. We aimed to identify the rate constants for these reactions that best fit experimental measurements of polymerization time courses. The analysis showed that the formation of dimers and trimers is unfavorable because the association reactions are orders of magnitude slower than estimated in previous work rather than because of rapid dissociation of dimers and trimers. The 95% confidence intervals calculated for the four rate constants spanned no more than one order of magnitude. Slow nucleation reactions are consistent with published high-resolution structures of actin filaments and molecular dynamics simulations of filament ends. One explanation for slow dimer formation, which we support with computational analysis, is that actin monomers are in a conformational equilibrium with a dominant conformation that cannot participate in the nucleation steps.

Formin Cdc12's specific actin assembly properties are tailored for cytokinesis in fission yeast.

Formins generate unbranched actin filaments by a conserved, processive actin assembly mechanism. Most organisms express multiple formin isoforms that mediate distinct cellular processes and facilitate actin filament polymerization by significantly different rates, but how these actin assembly differences correlate to cellular activity is unclear. We used a computational model of fission yeast cytokinetic ring assembly to test the hypothesis that particular actin assembly properties help tailor formins for specific cellular roles. Simulations run in different actin filament nucleation and elongation conditions revealed that variations in formin's nucleation efficiency critically impact both the probability and timing of contractile ring formation. To probe the physiological importance of nucleation efficiency, we engineered fission yeast formin chimera strains in which the FH1-FH2 actin assembly domains of full-length cytokinesis formin Cdc12 were replaced with the FH1-FH2 domains from functionally and evolutionarily diverse formins with significantly different actin assembly properties. Although Cdc12 chimeras generally support life in fission yeast, quantitative live-cell imaging revealed a range of cytokinesis defects from mild to severe. In agreement with the computational model, chimeras whose nucleation efficiencies are least similar to Cdc12 exhibit more severe cytokinesis defects, specifically in the rate of contractile ring assembly. Together, our computational and experimental results suggest that fission yeast cytokinesis is ideally mediated by a formin with properly tailored actin assembly parameters.

LIM domain proteins in cell mechanobiology.

The actin cytoskeleton is important for maintaining mechanical homeostasis in adherent cells, largely through its regulation of adhesion and cortical tension. The LIM (Lin-11, Isl1, MEC-3) domain-containing proteins are involved in a myriad of cellular mechanosensitive pathways. Recent work has discovered that LIM domains bind to mechanically stressed actin filaments, suggesting a novel and widely conserved mechanism of mechanosensing. This review summarizes the current state of knowledge of LIM protein mechanosensitivity.

Stir it up: The role of actin in mitochondrial mixing during mitosis.

During symmetric cell division, it is important that daughter cells receive not only equal genetic information, but also equal allocations of organelles. Recently, in Nature,Moore et al. (2021) identify three complementary F-actin networks that help ensure proper mixing and distribution of functionally equivalent mitochondria to daughter cells.

F-Actin Cytoskeleton Network Self-Organization Through Competition and Cooperation.

Many fundamental cellular processes such as division, polarization, endocytosis, and motility require the assembly, maintenance, and disassembly of filamentous actin (F-actin) networks at specific locations and times within the cell. The particular function of each network is governed by F-actin organization, size, and density as well as by its dynamics. The distinct characteristics of different F-actin networks are determined through the coordinated actions of specific sets of actin-binding proteins (ABPs). Furthermore, a cell typically assembles and uses multiple F-actin networks simultaneously within a common cytoplasm, so these networks must self-organize from a common pool of shared globular actin (G-actin) monomers and overlapping sets of ABPs. Recent advances in multicolor imaging and analysis of ABPs and their associated F-actin networks in cells, as well as the development of sophisticated in vitro reconstitutions of networks with ensembles of ABPs, have allowed the field to start uncovering the underlying principles by which cells self-organize diverse F-actin networks to execute basic cellular functions.

Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism.

The actin cytoskeleton assembles into diverse load-bearing networks, including stress fibers (SFs), muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl- 1, and Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force sensitivity. Zyxin rapidly localizes via its LIM domains to failing SFs in cells, known as strain sites, to initiate SF repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites (SFSS) is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We identify that the LIM domain-containing region of 18 proteins from the Zyxin, Paxillin, Tes, and Enigma proteins accumulate to SFSS. Moreover, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to SFSS in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. We then used sequence and domain analysis to demonstrate that tandem LIM domains contribute additively, for SFSS localization. Employing in vitro reconstitution, we show that the LIM domain-containing region from mammalian zyxin and fission yeast Pxl1 binds to mechanically stressed F-actin networks but does not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress.

Pseudomonas aeruginosa exoenzyme Y directly bundles actin filaments.

Pseudomonas aeruginosa uses a type III secretion system (T3SS) to inject cytotoxic effector proteins into host cells. The promiscuous nucleotidyl cyclase, exoenzyme Y (ExoY), is one of the most common effectors found in clinical P. aeruginosa isolates. Recent studies have revealed that the nucleotidyl cyclase activity of ExoY is stimulated by actin filaments (F-actin) and that ExoY alters actin cytoskeleton dynamics in vitro, via an unknown mechanism. The actin cytoskeleton plays an important role in numerous key biological processes and is targeted by many pathogens to gain competitive advantages. We utilized total internal reflection fluorescence microscopy, bulk actin assays, and EM to investigate how ExoY impacts actin dynamics. We found that ExoY can directly bundle actin filaments with high affinity, comparable with eukaryotic F-actin-bundling proteins, such as fimbrin. Of note, ExoY enzymatic activity was not required for F-actin bundling. Bundling is known to require multiple actin-binding sites, yet small-angle X-ray scattering experiments revealed that ExoY is a monomer in solution, and previous data suggested that ExoY possesses only one actin-binding site. We therefore hypothesized that ExoY oligomerizes in response to F-actin binding and have used the ExoY structure to construct a dimer-based structural model for the ExoY-F-actin complex. Subsequent mutational analyses suggested that the ExoY oligomerization interface plays a crucial role in mediating F-actin bundling. Our results indicate that ExoY represents a new class of actin-binding proteins that modulate the actin cytoskeleton both directly, via F-actin bundling, and indirectly, via actin-activated nucleotidyl cyclase activity.

Chlamydomonas reinhardtii formin FOR1 and profilin PRF1 are optimized for acute rapid actin filament assembly.

The regulated assembly of multiple filamentous actin (F-actin) networks from an actin monomer pool is important for a variety of cellular processes. Chlamydomonas reinhardtii is a unicellular green alga expressing a conventional and divergent actin that is an emerging system for investigating the complex regulation of actin polymerization. One actin network that contains exclusively conventional F-actin in Chlamydomonas is the fertilization tubule, a mating structure at the apical cell surface in gametes. In addition to two actin genes, Chlamydomonas expresses a profilin (PRF1) and four formin genes (FOR1-4), one of which (FOR1) we have characterized for the first time. We found that unlike typical profilins, PRF1 prevents unwanted actin assembly by strongly inhibiting both F-actin nucleation and barbed-end elongation at equimolar concentrations to actin. However, FOR1 stimulates the assembly of rapidly elongating actin filaments from PRF1-bound actin. Furthermore, for1 and prf1-1 mutants, as well as the small molecule formin inhibitor SMIFH2, prevent fertilization tubule formation in gametes, suggesting that polymerization of F-actin for fertilization tubule formation is a primary function of FOR1. Together, these findings indicate that FOR1 and PRF1 cooperate to selectively and rapidly assemble F-actin at the right time and place.

Mechanical and kinetic factors drive sorting of F-actin cross-linkers on bundles.

In cells, actin-binding proteins (ABPs) sort to different regions to establish F-actin networks with diverse functions, including filopodia used for cell migration and contractile rings required for cell division. Recent experimental work uncovered a competition-based mechanism that may facilitate spatial localization of ABPs: binding of a short cross-linker protein to 2 actin filaments promotes the binding of other short cross-linkers and inhibits the binding of longer cross-linkers (and vice versa). We hypothesize this sorting arises because F-actin is semiflexible and cannot bend over short distances. We develop a mathematical theory and lattice models encompassing the most important physical parameters for this process and use coarse-grained simulations with explicit cross-linkers to characterize and test our predictions. Our theory and data predict an explicit dependence of cross-linker separation on bundle polymerization rate. We perform experiments that confirm this dependence, but with an unexpected cross-over in dominance of one cross-linker at high growth rates to the other at slow growth rates, and we investigate the origin of this cross-over with further simulations. The nonequilibrium mechanism that we describe can allow cells to organize molecular material to drive biological processes, and our results can guide the choice and design of cross-linkers for engineered protein-based materials.