RESUMEN
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.
Asunto(s)
Factores Despolimerizantes de la Actina , Actinas , Citoesqueleto de Actina , Citoesqueleto , Orientación del AxónRESUMEN
Mical family enzymes are unusual actin regulators that prime filaments (F-actin) for disassembly via the site-specific oxidation of M44/M47. Filamentous actin acts as a substrate of Mical enzymes, as well as an activator of their NADPH oxidase activity, which leads to hydrogen peroxide generation. Mical enzymes are required for cytokinesis, muscle and heart development, dendritic pruning, and axonal guidance, among other processes. Thus, it is critical to understand how this family of actin regulators functions in different cell types. Vertebrates express six actin isoforms in a cell-specific manner, but MICALs' impact on their intrinsic properties has never been systematically investigated. Our data reveal the differences in the intrinsic dynamics of Mical-oxidized actin isoforms. Furthermore, our results connect the intrinsic dynamics of actin isoforms and their redox state with the patterns of hydrogen peroxide (H2O2) generation by MICALs. We documented that the differential properties of actin isoforms translate into the distinct patterns of hydrogen peroxide generation in Mical/NADPH-containing systems. Moreover, our results establish a conceptual link between actin stabilization by interacting factors and its ability to activate MICALs' NADPH oxidase activity. Altogether, our results suggest that the regulatory impact of MICALs may differ depending on the isoform-related identities of local actin networks.
Asunto(s)
Actinas , Peróxido de Hidrógeno , Animales , Actinas/metabolismo , Peróxido de Hidrógeno/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo , NADPH Oxidasas/metabolismoRESUMEN
Drebrin is a key regulator of actin cytoskeleton in neuronal cells which is critical for synaptic plasticity, neuritogenesis, and neuronal migration. It is also known to orchestrate a cross-talk between actin and microtubules. Decreased level of drebrin is a hallmark of multiple neurodegenerative disorders such as Alzheimer's disease. Despite its established importance in health and disease, we still have a lot to learn about drebrin's interactome and its effects on cytoskeletal dynamics. This review aims to summarize the recently reported novel effects of drebrin on actin and its regulators. Here I will also reflect on the most recent progress made in understanding of the role of drebrin isoforms and posttranslational modifications on its functionality.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Microtúbulos/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Neuropéptidos/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Animales , Movimiento Celular/fisiología , Humanos , Neuronas/citologíaRESUMEN
Dendritic spines are small protrusions of dendrites that are critical for synaptic transmission. The plasticity and stability of dendritic spines is tightly linked to actin cytoskeleton. However, our understanding of specific properties and the fine-tuning of neuronal actin structures is incomplete. Drebrin A is highly enriched in dendritic spines, but its effects on actin morphology, dynamics, and interplay with other actin regulators are yet to be clarified. Here we review recent advances in understanding drebrin effects on actin morphology and dynamics.
Asunto(s)
Actinas/química , Dendritas/metabolismo , Espinas Dendríticas/metabolismo , Neuropéptidos/química , Transmisión Sináptica/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Dendritas/genética , Ratones , Plasticidad Neuronal/genética , Neuronas/química , Neuronas/metabolismo , Neuronas/fisiología , Neuropéptidos/metabolismo , Sinapsis/química , Sinapsis/metabolismoRESUMEN
Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this "stiffness cation" unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics.
Asunto(s)
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Cationes/metabolismo , Movimiento Celular/fisiología , Cofilina 1/metabolismo , Modelos Moleculares , Citoesqueleto de Actina/ultraestructura , Cromatografía de Afinidad , Microscopía por Crioelectrón , Humanos , Saccharomyces cerevisiaeRESUMEN
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.
Asunto(s)
Actinas/genética , Actinas/metabolismo , Cationes/metabolismo , Modelos Moleculares , Polimerizacion , Aminoácidos/metabolismo , Animales , Fenómenos Biomecánicos , Biología Computacional , Fluorescencia , Conejos , TermodinámicaRESUMEN
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.
Asunto(s)
Proteínas del Tejido Nervioso/química , Neuropéptidos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Fibras de Estrés/química , Sustitución de Aminoácidos , Animales , Mutación Missense , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Estructura Secundaria de Proteína , Conejos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fibras de Estrés/genética , Fibras de Estrés/metabolismoRESUMEN
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.
Asunto(s)
Actinas/química , Actinas/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Microscopía de Fuerza Atómica/métodos , Unión ProteicaRESUMEN
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.
Asunto(s)
Factores Despolimerizantes de la Actina/química , Actinas/química , Benzofenonas/química , Reactivos de Enlaces Cruzados/química , Maleimidas/química , Conformación Proteica , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Animales , Benzofenonas/metabolismo , Sitios de Unión , Reactivos de Enlaces Cruzados/metabolismo , Maleimidas/metabolismo , Modelos Moleculares , ConejosRESUMEN
Actin cytoskeleton is critical for neuronal shape and function. Drebrin and formins are key regulators of neuronal actin networks. Neuron-specific drebrin A is highly enriched in dendritic spines (postsynaptic terminals) of mature excitatory neurons. Decreased levels of drebrin in dendritic spines is a hallmark of Alzheimer's disease, epilepsy, and other complex disorders, which calls for better understanding of its regulatory functions. Drebrin A was previously shown to inhibit actin nucleation and bundling by the diaphanous formin-2 (mDia2) - an actin nucleator that is involved in the initiation of dendritic spines. Characterization of the molecular binding interface between mDia2 and drebrin is necessary to better understand the functional consequences of this interaction and its biological relevance. Prior work suggested a multi-pronged interface between mDia2 and drebrin, which involves both N-terminal and C-terminal regions of the drebrin molecule. Here we used mass spectrometry analysis, deletion mutagenesis, and an array of synthetic peptides of neuronal drebrin A to map its formin-binding interface. The mDia2-interacting interface on drebrin was narrowed down to three highly conserved 9-16 residue sequences that were used to identify some of the key residues involved in this interaction. Deletion of the C-terminal region of drebrin greatly reduces its binding to mDia2 and the extent of its inhibition of formin-driven actin assembly. Moreover, our experiments with formins from different subfamilies showed that drebrin is a specific rather than general inhibitor of these proteins. This work contributes to a molecular level understanding of the formin-drebrin interaction and will help to unravel its biological significance.
Asunto(s)
Actinas , Forminas , Neuropéptidos , Actinas/metabolismo , Neuronas/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismoRESUMEN
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.
Asunto(s)
Actinas/química , Modelos Moleculares , Neuropéptidos/química , Neuropéptidos/metabolismo , Citoesqueleto de Actina , Actinas/metabolismo , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Proteínas Mutantes/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Actinas/química , Actinas/ultraestructura , Microscopía de Fuerza Atómica/métodos , Neuropéptidos/química , Sitios de Unión , Unión Proteica , Conformación ProteicaRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Animales , Elasticidad , Humanos , Modelos Moleculares , Conformación Molecular , Docilidad , Conejos , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismoRESUMEN
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.
Asunto(s)
Actinas/química , Actinas/metabolismo , Nucleótidos/metabolismo , Actinas/genética , Actinas/ultraestructura , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Microscopía Electrónica , Mutagénesis Sitio-Dirigida , Mutación , Nucleótidos/química , Estructura Secundaria de Proteína , Tiazolidinas/química , Tiazolidinas/metabolismo , Levaduras/genética , Levaduras/metabolismo , Levaduras/ultraestructuraRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/metabolismo , Profilinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Orientación del Axón , Moléculas de Adhesión Celular/metabolismo , Forminas/metabolismo , Conos de Crecimiento/metabolismo , Humanos , Modelos Biológicos , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Oxidación-Reducción , Polimerizacion , Unión Proteica , Conejos , Semaforinas/metabolismoRESUMEN
The antiparallel dimer (APD) is a unique actin species, which can be detected in the early stages of actin polymerization. In this work, we introduce novel tools for examination of the effects of the APD on actin polymerization. We document that bifunctional methanothiosulfonate (MTS) reagents are an attractive alternative to the routinely used p-phenylene maleimide (pPDM) for APD detection, allowing for fast and efficient cross-linking under conditions of actin polymerization at neutral pH. We report also that pyrene-labeled yeast actin mutant A167C/C374A (C167PM) forms significant amounts of stable APD in solution, without chemical cross-linking or polymerization-affecting compounds, and that the kinetics of APD transformation and decay upon actin polymerization can be easily monitored. The dimerization of C167PM has been characterized in sedimentation equilibrium experiments (K(d) approximately 0.3 microM). This new system offers the advantage of assessing the effects of the APD under physiological conditions (pH, ionic strength, and Mg(2+) concentration) and testing for conformational transitions in the APD during nucleation-polymerization reactions or/and in the presence of actin-interacting factors. The results obtained using two different systems (C167PM actin and polylysine-induced polymerization of alpha-actin) show that the APD decays at a rate slower than that at which the filaments elongate, revealing its transient incorporation into filaments, and confirm that it inhibits the nucleation and elongation of actin filaments.
Asunto(s)
Actinas/química , Levaduras/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Dimerización , Cinética , Unión Proteica , Levaduras/genética , Levaduras/metabolismoRESUMEN
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.
Asunto(s)
Actinas/química , Actinas/metabolismo , Faloidina/química , Faloidina/metabolismo , Actinas/genética , Reactivos de Enlaces Cruzados/química , Microscopía por Crioelectrón/métodos , Desoxirribonucleasa I/metabolismo , Disulfuros/química , Modelos Moleculares , Mutación , Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , NADPH Deshidrogenasa/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Animales , Ratones , Proteínas Asociadas a Microtúbulos/química , NADPH Deshidrogenasa/química , Neuropéptidos/química , Unión Proteica , Dominios Proteicos , ConejosRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina/química , Factores Despolimerizantes de la Actina/química , Actinas/química , Proteínas de Unión al ADN/química , Multimerización de Proteína , Citoesqueleto de Actina/ultraestructura , Actinas/genética , Actinas/aislamiento & purificación , Actinas/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/ultraestructura , Metionina/química , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Unión Proteica/genética , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/ultraestructuraRESUMEN
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.