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.

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.

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.

Profilin and Mical combine to impair F-actin assembly and promote disassembly and remodeling.

Cellular events require the spatiotemporal interplay between actin assembly and actin disassembly. Yet, how different factors promote the integration of these two opposing processes is unclear. In particular, cellular monomeric (G)-actin is complexed with profilin, which inhibits spontaneous actin nucleation but fuels actin filament (F-actin) assembly by elongation-promoting factors (formins, Ena/VASP). In contrast, site-specific F-actin oxidation by Mical promotes F-actin disassembly and release of polymerization-impaired Mical-oxidized (Mox)-G-actin. Here we find that these two opposing processes connect with one another to orchestrate actin/cellular remodeling. Specifically, we find that profilin binds Mox-G-actin, yet these complexes do not fuel elongation factors'-mediated F-actin assembly, but instead inhibit polymerization and promote further Mox-F-actin disassembly. Using Drosophila as a model system, we show that similar profilin-Mical connections occur in vivo - where they underlie F-actin/cellular remodeling that accompanies Semaphorin-Plexin cellular/axon repulsion. Thus, profilin and Mical combine to impair F-actin assembly and promote F-actin disassembly, while concomitantly facilitating cellular remodeling and plasticity.

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.

D-loop Dynamics and Near-Atomic-Resolution Cryo-EM Structure of Phalloidin-Bound F-Actin.

Detailed molecular information on G-actin assembly into filaments (F-actin), and their structure, dynamics, and interactions, is essential for understanding their cellular functions. Previous studies indicate that a flexible DNase I binding loop (D-loop, residues 40-50) plays a major role in actin's conformational dynamics. Phalloidin, a "gold standard" for actin filament staining, stabilizes them and affects the D-loop. Using disulfide crosslinking in yeast actin D-loop mutant Q41C/V45C, light-scattering measurements, and cryoelectron microscopy reconstructions, we probed the constraints of D-loop dynamics and its contribution to F-actin formation/stability. Our data support a model of residues 41-45 distances that facilitate G- to F-actin transition. We report also a 3.3-Å resolution structure of phalloidin-bound F-actin in the ADP-Pi-like (ADP-BeFx) state. This shows the phalloidin-binding site on F-actin and how the relative movement between its two protofilaments is restricted by it. Together, our results provide molecular details of F-actin structure and D-loop dynamics.

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.

Neuronal drebrin A directly interacts with mDia2 formin to inhibit actin assembly.

Dendritic spines (DS) are actin-rich postsynaptic terminals of neurons that are critical for higher-order brain functions. Maturation of DS is accompanied by a change in actin architecture from linear to branched filamentous structures. Presumably, the underlying cause of this is a switch in a mode of actin assembly from formin-driven to Arp2/3-mediated via an undefined mechanism. Here we present data suggesting that neuron-specific actin-binding drebrin A may be a part of such a switch. It is well documented that DS are highly enriched in drebrin A, which is critical for their plasticity and function. At the same time, mDia2 is known to mediate the formation of filopodia-type (immature) spines. We found that neuronal drebrin A directly interacts with mDia2 formin. Drebrin inhibits formin-mediated nucleation of actin and abolishes mDia2-induced actin bundling. Using truncated protein constructs we identified the domain requirements for drebrin-mDia2 interaction. We hypothesize that accumulation of drebrin A in DS (that coincides with spine maturation) leads to inhibition of mDia2-driven actin polymerization and, therefore, may contribute to a change in actin architecture from linear to branched filaments.

Catastrophic disassembly of actin filaments via Mical-mediated oxidation.

Actin filament assembly and disassembly are vital for cell functions. MICAL Redox enzymes are important post-translational effectors of actin that stereo-specifically oxidize actin's M44 and M47 residues to induce cellular F-actin disassembly. Here we show that Mical-oxidized (Mox) actin can undergo extremely fast (84 subunits/s) disassembly, which depends on F-actin's nucleotide-bound state. Using near-atomic resolution cryoEM reconstruction and single filament TIRF microscopy we identify two dynamic and structural states of Mox-actin. Modeling actin's D-loop region based on our 3.9 Å cryoEM reconstruction suggests that oxidation by Mical reorients the side chain of M44 and induces a new intermolecular interaction of actin residue M47 (M47-O-T351). Site-directed mutagenesis reveals that this interaction promotes Mox-actin instability. Moreover, we find that Mical oxidation of actin allows for cofilin-mediated severing even in the presence of inorganic phosphate. Thus, in conjunction with cofilin, Mical oxidation of actin promotes F-actin disassembly independent of the nucleotide-bound state.

Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy.

Cellular actin dynamics is an essential element of numerous cellular processes, such as cell motility, cell division and endocytosis. Actin's involvement in these processes is mediated by many actin-binding proteins, among which the cofilin family plays unique and essential role in accelerating actin treadmilling in filamentous actin (F-actin) in a nucleotide-state dependent manner. Cofilin preferentially interacts with older filaments by recognizing time-dependent changes in F-actin structure associated with the hydrolysis of ATP and release of inorganic phosphate (Pi) from the nucleotide cleft of actin. The structure of cofilin on F-actin and the details of the intermolecular interface remain poorly understood at atomic resolution. Here we report atomic-level characterization by magic angle spinning (MAS) NMR of the muscle isoform of human cofilin 2 (CFL2) bound to F-actin. We demonstrate that resonance assignments for the majority of atoms are readily accomplished and we derive the intermolecular interface between CFL2 and F-actin. The MAS NMR approach reported here establishes the foundation for atomic-resolution characterization of a broad range of actin-associated proteins bound to F-actin.

F-actin dismantling through a redox-driven synergy between Mical and cofilin.

Numerous cellular functions depend on actin filament (F-actin) disassembly. The best-characterized disassembly proteins, the ADF (actin-depolymerizing factor)/cofilins (encoded by the twinstar gene in Drosophila), sever filaments and recycle monomers to promote actin assembly. Cofilin is also a relatively weak actin disassembler, posing questions about mechanisms of cellular F-actin destabilization. Here we uncover a key link to targeted F-actin disassembly by finding that F-actin is efficiently dismantled through a post-translational-mediated synergism between cofilin and the actin-oxidizing enzyme Mical. We find that Mical-mediated oxidation of actin improves cofilin binding to filaments, where their combined effect dramatically accelerates F-actin disassembly compared with either effector alone. This synergism is also necessary and sufficient for F-actin disassembly in vivo, magnifying the effects of both Mical and cofilin on cellular remodelling, axon guidance and Semaphorin-Plexin repulsion. Mical and cofilin, therefore, form a redox-dependent synergistic pair that promotes F-actin instability by rapidly dismantling F-actin and generating post-translationally modified actin that has altered assembly properties.

Coronin Enhances Actin Filament Severing by Recruiting Cofilin to Filament Sides and Altering F-Actin Conformation.

High rates of actin filament turnover are essential for many biological processes and require the activities of multiple actin-binding proteins working in concert. The mechanistic role of the actin filament severing protein cofilin is now firmly established; however, the contributions of other conserved disassembly-promoting factors including coronin have remained more obscure. Here, we have investigated the mechanism by which yeast coronin (Crn1) enhances F-actin turnover. Using multi-color total internal reflection fluorescence microscopy, we show that Crn1 enhances Cof1-mediated severing by accelerating Cof1 binding to actin filament sides. Further, using biochemical assays to interrogate F-actin conformation, we show that Crn1 alters longitudinal and lateral actin-actin contacts and restricts opening of the nucleotide-binding cleft in actin subunits. Moreover, Crn1 and Cof1 show opposite structural effects on F-actin yet synergize in promoting release of phalloidin from filaments, suggesting that Crn1/Cof1 co-decoration may increase local discontinuities in filament topology to enhance severing.

Metavinculin Tunes the Flexibility and the Architecture of Vinculin-Induced Bundles of Actin Filaments.

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 total internal reflection fluorescence microscopy experiments, MVT's severing activity was negligible. Instead, we found that MVT binding caused a 2-fold reduction in F-actin's bending persistence length and increased susceptibility to breakage. 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. Perhaps MVT allows the load of muscle contraction to act as a signal to reorganize actin filaments.

Cryo-EM reveals different coronin binding modes for ADP- and ADP-BeFx actin filaments.

Essential cellular processes involving the actin cytoskeleton are regulated by auxiliary proteins that can sense the nucleotide state of actin. Here we report cryo-EM structures for ADP-bound and ADP-beryllium fluoride (ADP-BeFx, an ADP-Pi mimic)-bound actin filaments in complex with the ß-propeller domain of yeast coronin 1 (crn1), at 8.6-Å resolution. Our structures reveal the main differences in the interaction of coronin with the two nucleotide states of F-actin. We derived pseudoatomic models by fitting the atomic structures of actin and coronin into the EM envelopes and confirmed the identified interfaces on actin by chemical cross-linking, fluorescence spectroscopy and actin mutagenesis. The models offer a structural explanation for the nucleotide-dependent effects of coronin on cofilin-assisted remodeling of F-actin.

Drebrin inhibits cofilin-induced severing of F-actin.

Molecular cross-talk between neuronal drebrin A and cofilin is believed to be a part of the activity-dependent cytoskeleton-modulating pathway in dendritic spines. Impairments in this pathway are implicated also in synaptic dysfunction in Alzheimer's disease, Down syndrome, epilepsy, and normal aging. However, up to now the molecular interplay between cofilin and drebrin has not been elucidated. TIRF microscopy and solution experiments revealed that full length drebrin A or its actin binding core (Drb1-300) inhibits, but do not abolish cofilin-induced severing of actin filaments. Cosedimentation experiments showed that F-actin can be fully occupied with combination of these two proteins. The dependence of cofilin binding on fractional saturation of actin filaments with drebrin suggests direct competition between these two proteins for F-actin binding. This implies that cofilin and drebrin can either overcome or reverse the allosteric changes in F-actin induced by the competitor's binding. The ability of cofilin to displace drebrin from actin filaments is pH dependent and is facilitated at acidic pH (6.8). Pre-steady state kinetic experiments reveal that both binding and dissociation of drebrin to/from actin filaments is faster than that reported for cooperative binding of cofilin. We found, that drebrin displacement by cofilin is greatly inhibited when actin severing is abolished, which might be linked to the cooperativity of drebrin binding to actin filaments. Our results contribute to molecular understanding of the competitive interactions of drebrin and cofilin with actin filaments.

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.

INF2-mediated severing through actin filament encirclement and disruption.

BACKGROUND: INF2 is a formin protein with the unique ability to accelerate both actin polymerization and depolymerization, the latter requiring filament severing. Mutations in INF2 lead to the kidney disease focal segmental glomerulosclerosis (FSGS) and the neurological disorder Charcot-Marie Tooth disease (CMTD). RESULTS: Here, we compare the severing mechanism of INF2 with that of the well-studied severing protein cofilin. INF2, like cofilin, binds stoichiometrically to filament sides and severs in a manner that requires phosphate release from the filament. In contrast to cofilin, however, INF2 binds ADP and ADP-Pi filaments equally well. Furthermore, two-color total internal reflection fluorescence (TIRF) microscopy reveals that a low number of INF2 molecules, as few as a single INF2 dimer, are capable of severing, while measurable cofilin-mediated severing requires more extensive binding. Hence, INF2 is a more potent severing protein than cofilin. While a construct containing the FH1 and FH2 domains alone has some severing activity, addition of the C-terminal region increases severing potency by 40-fold, and we show that the WH2-resembling DAD motif is responsible for this increase. Helical 3D reconstruction from electron micrographs at 20 Å resolution provides a structure of filament-bound INF2, showing that the FH2 domain encircles the filament. CONCLUSIONS: We propose a severing model in which FH2 binding and phosphate release causes local filament deformation, allowing the DAD to bind adjacent actin protomers, further disrupting filament structure.

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.