Disassembly of bundled F-actin and cellular remodeling via an interplay of Mical, cofilin, and F-actin crosslinkers.

Cellular form and function are controlled by the assembly and stability of actin cytoskeletal structures-but disassembling/pruning these structures is equally essential for the plasticity and remodeling that underlie behavioral adaptations. Importantly, the mechanisms of actin assembly have been well-defined-including that it is driven by actin's polymerization into filaments (F-actin) and then often bundling by crosslinking proteins into stable higher-order structures. In contrast, it remains less clear how these stable bundled F-actin structures are rapidly disassembled. We now uncover mechanisms that rapidly and extensively disassemble bundled F-actin. Using biochemical, structural, and imaging assays with purified proteins, we show that F-actin bundled with one of the most prominent crosslinkers, fascin, is extensively disassembled by Mical, the F-actin disassembly enzyme. Furthermore, the product of this Mical effect, Mical-oxidized actin, is poorly bundled by fascin, thereby further amplifying Mical's disassembly effects on bundled F-actin. Moreover, another critical F-actin regulator, cofilin, also affects fascin-bundled filaments, but we find herein that it synergizes with Mical to dramatically amplify its disassembly of bundled F-actin compared to the sum of their individual effects. Genetic and high-resolution cellular assays reveal that Mical also counteracts crosslinking proteins/bundled F-actin in vivo to control cellular extension, axon guidance, and Semaphorin/Plexin cell-cell repulsion. Yet, our results also support the idea that fascin-bundling serves to dampen Mical's F-actin disassembly in vitro and in vivo-and that physiologically relevant cellular remodeling requires a fine-tuned interplay between the factors that build bundled F-actin networks and those that disassemble them.

Rounding Out the Understanding of ACD Toxicity with the Discovery of Cyclic Forms of Actin Oligomers.

Actin is an essential element of both innate and adaptive immune systems and can aid in motility and translocation of bacterial pathogens, making it an attractive target for bacterial toxins. Pathogenic Vibrio and Aeromonas genera deliver actin cross-linking domain (ACD) toxin into the cytoplasm of the host cell to poison actin regulation and promptly induce cell rounding. At early stages of toxicity, ACD covalently cross-links actin monomers into oligomers (AOs) that bind through multivalent interactions and potently inhibit several families of actin assembly proteins. At advanced toxicity stages, we found that the terminal protomers of linear AOs can get linked together by ACD to produce cyclic AOs. When tested against formins and Ena/VASP, linear and cyclic AOs exhibit similar inhibitory potential, which for the cyclic AOs is reduced in the presence of profilin. In coarse-grained molecular dynamics simulations, profilin and WH2-motif binding sites on actin subunits remain exposed in modeled AOs of both geometries. We speculate, therefore, that the reduced toxicity of cyclic AOs is due to their reduced configurational entropy. A characteristic feature of cyclic AOs is that, in contrast to the linear forms, they cannot be straightened to form filaments (e.g., through stabilization by cofilin), which makes them less susceptible to neutralization by the host cell.

Tropomyosin isoforms regulate cofilin 1 activity by modulating actin filament conformation.

Tropomyosin and cofilin are involved in the regulation of actin filament dynamic polymerization and depolymerization. Binding of cofilin changes actin filaments structure, leading to their severing and depolymerization. Non-muscle tropomyosin isoforms were shown before to differentially regulate the activity of cofilin 1; products of TPM1 gene stabilized actin filaments, but products of TPM3 gene promoted cofilin-dependent severing and depolymerization. Here, conformational changes at the longitudinal and lateral interface between actin subunits resulting from tropomyosin and cofilin 1 binding were studied using skeletal actin and yeast wild type and mutant Q41C and S265C actins. Cross-linking of F-actin and fluorescence changes in F-actin labeled with acrylodan at Cys41 (in D-loop) or Cys265 (in H-loop) showed that tropomyosin isoforms differentially regulated cofilin-induced conformational rearrangements at longitudinal and lateral filament interfaces. Tryptic digestion of F-Mg-actin confirmed the differences between tropomyosin isoforms in their regulation of cofilin-dependent changes at actin-actin interfaces. Changes in the fluorescence of AEDANS attached to C-terminal Cys of actin, as well as FRET between Trp residues in actin subdomain 1 and AEDANS, did not show differences in the conformation of the C-terminal segment of F-actin in the presence of different tropomyosins ± cofilin 1. Therefore, actin's D- and H-loop are the sites involved in regulation of cofilin activity by tropomyosin isoforms.

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness.

The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as "polymerization" and "stiffness" sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments.

Drebrin-induced stabilization of actin filaments.

Drebrin is a mammalian neuronal protein that binds to and organizes filamentous actin (F-actin) in dendritic spines, the receptive regions of most excitatory synapses that play a crucial role in higher brain functions. Here, the structural effects of drebrin on F-actin were examined in solution. Depolymerization and differential scanning calorimetry assays show that F-actin is stabilized by the binding of drebrin. Drebrin inhibits depolymerization mainly at the barbed end of F-actin. Full-length drebrin and its C-terminal truncated constructs were used to clarify the domain requirements for these effects. The actin binding domain of drebrin decreases the intrastrand disulfide cross-linking of Cys-41 (in the DNase I binding loop) to Cys-374 (C-terminal) but increases the interstrand disulfide cross-linking of Cys-265 (hydrophobic loop) to Cys-374 in the yeast mutants Q41C and S265C, respectively. We also demonstrate, using solution biochemistry methods and EM, the rescue of filament formation by drebrin in different cases of longitudinal interprotomer contact perturbation: the T203C/C374S yeast actin mutant and grimelysin-cleaved skeletal actin (between Gly-42 and Val-43). Additionally, we show that drebrin rescues the polymerization of V266G/L267G, a hydrophobic loop yeast actin mutant with an impaired lateral interface formation between the two filament strands. Overall, our data suggest that drebrin stabilizes actin filaments through its effect on their interstrand and intrastrand contacts.

Nanostructured self-assembly of inverted formin 2 (INF2) and F-actin-INF2 complexes revealed by atomic force microscopy.

Self-organization of cytoskeletal proteins such as actin and tubulin into filaments and microtubules is frequently assisted by the proteins binding to them. Formins are regulatory proteins that nucleate the formation of new filaments and are essential for a wide range of cellular functions. The vertebrate inverted formin 2 (INF2) has both actin filament nucleating and severing/depolymerizing activities connected to its ability to encircle actin filaments. Using atomic force microscopy, we report that a formin homology 2 (FH2) domain-containing construct of INF2 (INF2-FH1-FH2-C or INF2-FFC) self-assembles into nanoscale ringlike oligomeric structures in the absence of actin filaments, demonstrating an inherent ability to reorganize from a dimeric to an oligomeric state. A construct lacking the C-terminal region (INF2-FH1-FH2 or INF2-FF) also oligomerizes, confirming the dominant role of FH2-mediated interactions. Moreover, INF2-FFC domains were observed to organize into ringlike structures around single actin filaments. This is the first demonstration that formin FH2 domains can self-assemble into oligomers in the absence of filaments and has important implications for observing unaveraged decoration and/or remodeling of filaments by actin binding proteins.

Remodeling of actin filaments by ADF/cofilin proteins.

Cofilin/ADF proteins play key roles in the dynamics of actin, one of the most abundant and highly conserved eukaryotic proteins. We used cryoelectron microscopy to generate a 9-Å resolution three-dimensional reconstruction of cofilin-decorated actin filaments, the highest resolution achieved for a complex of F-actin with an actin-binding protein. We show that the cofilin-induced change in the filament twist is due to a unique conformation of the actin molecule unrelated to any previously observed state. The changes between the actin protomer in naked F-actin and in the actin-cofilin filament are greater than the conformational changes between G- and F-actin. Our results show the structural plasticity of actin, suggest that other actin-binding proteins may also induce large but different conformational changes, and show that F-actin cannot be described by a single molecular model.

Drebrin Protects Assembled Actin from INF2-FFC-mediated Severing and Stabilizes Cell Protrusions.

Highly specialized cells, such as neurons and podocytes, have arborized morphologies that serve their specific functions. Actin cytoskeleton and its associated proteins are responsible for the distinctive shapes of cells. The mechanism of their cytoskeleton regulation - contributing to cell shape maintenance - is yet to be fully clarified. Inverted formin 2 (INF2), one of the modulators of the cytoskeleton, is an atypical formin that can both polymerize and depolymerize actin filaments depending on its molar ratio to actin. Prior work has established that INF2 binds to the sides of actin filaments and severs them. Drebrin is another actin-binding protein that also binds filaments laterally and stabilizes them, but the interplay between drebrin and INF2 on actin filament stabilization is not well understood. Here, we have used biochemical assays, electron microscopy, and total internal reflection fluorescence microscopy imaging to show that drebrin protects actin filaments from severing by INF2 without inhibiting its polymerization activity. Notably, truncated drebrin - DrbA1-300 - is sufficient for this protection, though not as effective as the full-length protein. INF2 and drebrin are abundantly expressed in highly specialized cells and are crucial for the temporal regulation of their actin cytoskeleton, consistent with their involvement in peripheral neuropathy.

Cofilin-induced changes in F-actin detected via cross-linking with benzophenone-4-maleimide.

Cofilin is a member of the actin depolymerizing factor (ADF)/cofilin family of proteins. It plays a key role in actin dynamics by promoting disassembly and assembly of actin filaments. Upon its binding, cofilin has been shown to bridge two adjacent protomers in filamentous actin (F-actin) and promote the displacement and disordering of subdomain 2 of actin. Here, we present evidence for cofilin promoting a new structural change in the actin filament, as detected via a switch in cross-linking sites. Benzophenone-4-maleimide, which normally forms intramolecular cross-linking in F-actin, cross-links F-actin intermolecularly upon cofilin binding. We mapped the cross-linking sites and found that in the absence of cofilin intramolecular cross-linking occurred between residues Cys374 and Asp11. In contrast, cofilin shifts the cross-linking by this reagent to intermolecular, between residue Cys374, located within subdomain 1 of the upper protomer, and Met44, located in subdomain 2 of the lower protomer. The intermolecular cross-linking of F-actin slows the rate of cofilin dissociation from the filaments and decreases the effect of ionic strength on cofilin-actin binding. These results are consistent with a significant role of filament flexibility in cofilin-actin interactions.

Multiple forms of Spire-actin complexes and their functional consequences.

Spire is a WH2 domain-containing actin nucleator essential for establishing an actin mesh during oogenesis. In vitro, in addition to nucleating filaments, Spire can sever them and sequester actin monomers. Understanding how Spire is capable of these disparate functions and which are physiologically relevant is an important goal. To study severing, we examined the effect of Drosophila Spire on preformed filaments in bulk and single filament assays. We observed rapid depolymerization of actin filaments by Spire, which we conclude is largely due to its sequestration activity and enhanced by its weak severing activity. We also studied the solution and crystal structures of Spire-actin complexes. We find structural and functional differences between constructs containing four WH2 domains (Spir-ABCD) and two WH2 domains (Spir-CD) that may provide insight into the mechanisms of nucleation and sequestration. Intriguingly, we observed lateral interactions between actin monomers associated with Spir-ABCD, suggesting that the structures built by these four tandem WH2 domains are more complex than originally imagined. Finally, we propose that Spire-actin mixtures contain both nuclei and sequestration structures.

ATP and ADP actin states.

This minireview is dedicated to the memory of Henryk Eisenberg and honors his major contributions to many areas of biophysics and to the analysis of macromolecular states and interactions in particular. This work reviews the ATP and ADP states of a ubiquitous protein, actins, and considers the present evidence for and against unique, nucleotide-dependent conformations of this protein. The effects of ATP and ADP on specific structural elements of actins, its loops and clefts, as revealed by mutational, crosslinking, spectroscopic, and EPR methods are discussed. It is concluded that the existing evidence points to dynamic equilibria of these structural elements among various conformational states in both ATP- and ADP-actins, with the nucleotides impacting the equilibria distributions.

MICAL-mediated oxidation of actin and its effects on cytoskeletal and cellular dynamics.

Actin and its dynamic structural remodelings are involved in multiple cellular functions, including maintaining cell shape and integrity, cytokinesis, motility, navigation, and muscle contraction. Many actin-binding proteins regulate the cytoskeleton to facilitate these functions. Recently, actin's post-translational modifications (PTMs) and their importance to actin functions have gained increasing recognition. The MICAL family of proteins has emerged as important actin regulatory oxidation-reduction (Redox) enzymes, influencing actin's properties both in vitro and in vivo. MICALs specifically bind to actin filaments and selectively oxidize actin's methionine residues 44 and 47, which perturbs filaments' structure and leads to their disassembly. This review provides an overview of the MICALs and the impact of MICAL-mediated oxidation on actin's properties, including its assembly and disassembly, effects on other actin-binding proteins, and on cells and tissue systems.

Actin Bundles Dynamics and Architecture.

Cells use the actin cytoskeleton for many of their functions, including their division, adhesion, mechanosensing, endo- and phagocytosis, migration, and invasion. Actin bundles are the main constituent of actin-rich structures involved in these processes. An ever-increasing number of proteins that crosslink actin into bundles or regulate their morphology is being identified in cells. With recent advances in high-resolution microscopy and imaging techniques, the complex process of bundles formation and the multiple forms of physiological bundles are beginning to be better understood. Here, we review the physiochemical and biological properties of four families of highly conserved and abundant actin-bundling proteins, namely, α-actinin, fimbrin/plastin, fascin, and espin. We describe the similarities and differences between these proteins, their role in the formation of physiological actin bundles, and their properties-both related and unrelated to their bundling abilities. We also review some aspects of the general mechanism of actin bundles formation, which are known from the available information on the activity of the key actin partners involved in this process.

Parallel actin monomers in the 8S complex of actin-INF2.

Polymerization and depolymerization of actin play an essential role in eukaryotic cells. Actin exists in cells in both monomeric (G-actin) and filamentous (polymer, F-actin) forms. Actin binding proteins (ABPs) facilitate the transition between these two states, and their interactions with these two states of actin are critical for actin-based cellular processes. Rapid depolymerization of actin is assisted in the brain and/or other cells by its oxidation by the enzyme Mical (yielding Mox-actin), and/or by the binding of Inverted Formin 2 (INF2) - which can also accelerate filaments formation. At their stoichiometric molar ratio INF2 and actin yield the 8S complex (consisting of 4 actin monomers: 2 INF2 dimer molecules). Using biochemical and biophysical methods, we investigate the structural arrangement of actin in the 8S particles and the interaction of INF2 with actin and Mox-actin. To that end, we show 2 D class averages of 8S particles obtained by negative staining electron microscopy. We also show that: (i) 8S particles can seed rapid actin assembly; (ii) Mox-actin and INF2 form 8S particles at proteins ratios similar to those of unoxidized actin; (iii) chemical crosslinkings suggest that actin monomers are in a parallel orientation in the 8S particles of both actin and Mox-actin; and (iv) INF2 accelerates the disassembly of Mox-F-actin. Our results provide better understanding of actin's arrangement in the 8S particles formed during actin depolymerization and in the early polymerization stages of both actin and Mox-actin.Communicated by Ramaswamy H. Sarma.

Molecular cooperativity of drebrin1-300 binding and structural remodeling of F-actin.

Drebrin A, an actin-binding protein, is a key regulatory element in synaptic plasticity of neuronal dendrites. Understanding how drebrin binds and remodels F-actin is important for a functional analysis of their interactions. Conventionally, molecular models for protein-protein interactions use binding parameters derived from bulk solution measurements with limited spatial resolution, and the inherent assumption of homogeneous binding sites. In the case of actin filaments, their structural and dynamic states-as well as local changes in those states-may influence their binding parameters and interaction cooperativity. Here, we probed the structural remodeling of single actin filaments and the binding cooperativity of DrebrinA(1-300) -F-actin using AFM imaging. We show direct evidence of DrebrinA(1-300)-induced cooperative changes in the helical structure of F-actin and observe the binding cooperativity of drebrin to F-actin with nanometer resolution. The data confirm at the in vitro molecular level that variations in the F-actin helical structure can be modulated by cooperative binding of actin-binding proteins.

Structural states and dynamics of the D-loop in actin.

Conformational changes induced by ATP hydrolysis on actin are involved in the regulation of complex actin networks. Previous structural and biochemical data implicate the DNase I binding loop (D-loop) of actin in such nucleotide-dependent changes. Here, we investigated the structural and conformational states of the D-loop (in solution) using cysteine scanning mutagenesis and site-directed labeling. The reactivity of D-loop cysteine mutants toward acrylodan and the mobility of spin labels on these mutants do not show patterns of an α-helical structure in monomeric and filamentous actin, irrespective of the bound nucleotide. Upon transition from monomeric to filamentous actin, acrylodan emission spectra and electron paramagnetic resonance line shapes of labeled mutants are blue-shifted and more immobilized, respectively, with the central residues (residues 43-47) showing the most drastic changes. Moreover, complex electron paramagnetic resonance line shapes of spin-labeled mutants suggest several conformational states of the D-loop. Together with a new (to our knowledge) actin crystal structure that reveals the D-loop in a unique hairpin conformation, our data suggest that the D-loop equilibrates in F-actin among different conformational states irrespective of the nucleotide state of actin.

Atomic force microscopy reveals drebrin induced remodeling of f-actin with subnanometer resolution.

We show by high-resolution atomic force microscopy analysis that drebrin A (a major neuronal actin binding protein) induced F-actin structural and mechanical remodeling involves significant changes in helical twist and filament stiffness (+55% persistence length). These results provide evidence of a unique mechanical role of drebrin in the dendrites, contribute to current molecular-level understanding of the properties of the neuronal cytoskeleton, and reflect the role of biomechanics at the nanoscale, to modulate nanofilament-structure assemblies such as F-actin.

Cofilin-linked changes in actin filament flexibility promote severing.

The actin regulatory protein, cofilin, increases the bending and twisting elasticity of actin filaments and severs them. It has been proposed that filaments partially decorated with cofilin accumulate stress from thermally driven shape fluctuations at bare (stiff) and decorated (compliant) boundaries, thereby promoting severing. This mechanics-based severing model predicts that changes in actin filament compliance due to cofilin binding affect severing activity. Here, we test this prediction by evaluating how the severing activities of vertebrate and yeast cofilactin scale with the flexural rigidities determined from analysis of shape fluctuations. Yeast actin filaments are more compliant in bending than vertebrate actin filaments. Severing activities of cofilactin isoforms correlate with changes in filament flexibility. Vertebrate cofilin binds but does not increase the yeast actin filament flexibility, and does not sever them. Imaging of filament thermal fluctuations reveals that severing events are associated with local bending and fragmentation when deformations attain a critical angle. The critical severing angle at boundaries between bare and cofilin-decorated segments is smaller than in bare or fully decorated filaments. These measurements support a cofilin-severing mechanism in which mechanical asymmetry promotes local stress accumulation and fragmentation at boundaries of bare and cofilin-decorated segments, analogous to failure of some nonprotein materials.

A nucleotide state-sensing region on actin.

The nucleotide state of actin (ATP, ADP-P(i), or ADP) is known to impact its interactions with other actin molecules upon polymerization as well as with multiple actin binding proteins both in the monomeric and filamentous states of actin. Recently, molecular dynamics simulations predicted that a sequence located at the interface of subdomains 1 and 3 (W-loop; residues 165-172) changes from an unstructured loop to a beta-turn conformation upon ATP hydrolysis (Zheng, X., Diraviyam, K., and Sept, D. (2007) Biophys. J. 93, 1277-1283). This region participates directly in the binding to other subunits in F-actin as well as to cofilin, profilin, and WH2 domain proteins and, therefore, could contribute to the nucleotide sensitivity of these interactions. The present study demonstrates a reciprocal communication between the W-loop region and the nucleotide binding cleft on actin. Point mutagenesis of residues 167, 169, and 170 and their site-specific labeling significantly affect the nucleotide release from the cleft region, whereas the ATP/ADP switch alters the fluorescence of probes located in the W-loop. In the ADP-P(i) state, the W-loop adopts a conformation similar to that in the ATP state but different from the ADP state. Binding of latrunculin A to the nucleotide cleft favors the ATP-like conformation of the W-loop, whereas ADP-ribosylation of Arg-177 forces the W-loop into a conformation distinct from those in the ADP and ATP-states. Overall, our experimental data suggest that the W-loop of actin is a nucleotide sensor, which may contribute to the nucleotide state-dependent changes in F-actin and nucleotide state-modulated interactions of both G- and F-actin with actin-binding proteins.

Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin.

The Gram-negative bacterium Vibrio cholerae is the causative agent of a severe diarrheal disease that afflicts three to five million persons annually, causing up to 200,000 deaths. Nearly all V. cholerae strains produce a large multifunctional-autoprocessing RTX toxin (MARTX(Vc)), which contributes significantly to the pathogenesis of cholera in model systems. The actin cross-linking domain (ACD) of MARTX(Vc) directly catalyzes a covalent cross-linking of monomeric G-actin into oligomeric chains and causes cell rounding, but the nature of the cross-linked bond and the mechanism of the actin cytoskeleton disruption remained elusive. To elucidate the mechanism of ACD action and effect on actin, we identified the covalent cross-link bond between actin protomers using limited proteolysis, X-ray crystallography, and mass spectrometry. We report here that ACD catalyzes the formation of an intermolecular iso-peptide bond between residues E270 and K50 located in the hydrophobic and the DNaseI-binding loops of actin, respectively. Mutagenesis studies confirm that no other residues on actin can be cross-linked by ACD both in vitro and in vivo. This cross-linking locks actin protomers into an orientation different from that of F-actin, resulting in strong inhibition of actin polymerization. This report describes a microbial toxin mechanism acting via iso-peptide bond cross-linking between host proteins and is, to the best of our knowledge, the only known example of a peptide linkage between nonterminal glutamate and lysine side chains.