Beáta Bugyi

Control of Actin Filament Treadmilling in Cell Motility

Recent advances in structural, biochemical, biophysical, and live cell imaging approaches have furthered our understanding of the molecular mechanisms by which regulated assembly dynamics of actin filaments drive motile processes. Attention is focused on lamellipodium protrusion, powered by the turnover of a branched filament array. ATP hydrolysis on actin is the key reaction that allows filament treadmilling. It regulates barbed-end dynamics and length fluctuations at steady state and specifies the functional interaction of actin with essential regulatory proteins such as profilin and ADF/cofilin. ATP hydrolysis on actin and Arp2/3 acts as a timer, regulating the assembly and disassembly of the branched array to generate tropomyosin-mediated heterogeneity in the structure and dynamics of the lamellipodial network. The detailed molecular mechanisms of ATP hydrolysis/Pi release on F-actin remain elusive, as well as the mechanism of filament branching with Arp2/3 complex or that of the formin-driven processive actin assembly. Novel biophysical methods involving single-molecule measurements should foster progress in these crucial issues.

The core FH2 domain of diaphanous-related formins is an elongated actin binding protein that inhibits polymerization

Diaphanous-related formins (Drf) are activated by Rho GTP binding proteins and induce polymerization of unbranched actin filaments. They contain three formin homology domains. Evidence as to the effect of formins on actin polymerization were obtained using FH2/FH1 constructs of various length from different Drfs. Here we define the core FH2 domain as a proteolytically stable domain of approximately 338 residues. The monomeric FH2 domains from mDia1 and mDia3 inhibit polymerization of actin and can bind in a 1:1 complex with F-actin at micromolar concentrations. The X-ray structure analysis of the domain shows an elongated, crescent-shaped molecule consisting of three helical subdomains. The most highly conserved regions of the domain span a distance of 75 A and are both required for barbed-end inhibition. A construct containing an additional 72 residue linker has dramatically different properties: It oligomerizes and induces actin polymerization at subnanomolar concentration.

Formins regulate actin filament flexibility through long range allosteric interactions

The members of the formin family nucleate actin polymerization and play essential roles in the regulation of the actin cytoskeleton during a wide range of cellular and developmental processes. In the present work, we describe the effects of mDia1-FH2 on the conformation of actin filaments by using a temperature-dependent fluorescence resonance energy transfer method. Our results revealed that actin filaments were more flexible in the presence than in the absence of formin. The effect strongly depends on the mDia1-FH2 concentration in a way that indicates that more than one mechanism is responsible for the formin effect. In accordance with the more flexible filament structure, the thermal stability of actin decreased and the rate of phosphate dissociation from actin filaments increased in the presence of formin. The interpretation of the results supports a model in which formin binding to barbed ends makes filaments more flexible through long range allosteric interactions, whereas binding of formin to the sides of the filaments stabilizes the protomer-protomer interactions. These results suggest that formins can regulate the conformation of actin filaments and may thus also modulate the affinity of actin-binding proteins to filaments nucleated/capped by formins.

Conformational dynamics of actin: Effectors and implications for biological function

Actin is a protein abundant in many cell types. Decades of investigations have provided evidence that it has many functions in living cells. The diverse morphology and dynamics of actin structures adapted to versatile cellular functions is established by a large repertoire of actin‐binding proteins. The proper interactions with these proteins assume effective molecular adaptations from actin, in which its conformational transitions play essential role. This review attempts to summarise our current knowledge regarding the coupling between the conformational states of actin and its biological function.

Characterization of the Biochemical Properties and Biological Function of the Formin Homology Domains of Drosophila DAAM

We characterized the properties of Drosophila melanogaster DAAM-FH2 and DAAM-FH1-FH2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. The results show that although the DAAM-FH2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the DAAM-FH1-FH2 fragment, a profilin-dependent increase in the formation of actin structures is observed. The trachea-specific expression of DAAM-FH1-FH2 also induces phenotypic effects, leading to the collapse of the tracheal tube and lethality in the larval stages. In vitro, both DAAM fragments catalyze actin nucleation but severely decrease both the elongation and depolymerization rate of the filaments. Profilin acts as a molecular switch in DAAM function. DAAM-FH1-FH2, remaining bound to barbed ends, drives processive assembly of profilin-actin, whereas DAAM-FH2 forms an abortive complex with barbed ends that does not support profilin-actin assembly. Both DAAM fragments also bind to the sides of the actin filaments and induce actin bundling. These observations show that the D. melanogaster DAAM formin represents an extreme class of barbed end regulators gated by profilin.

The other side of the coin: Functional and structural versatility of ADF/cofilins

Several cellular processes rely on the fine tuning of actin cytoskeleton. A central component in the regulation of this cellular machinery is the ADF-H domain proteins. Despite sharing the same domain, ADF-H domain proteins produce a diverse functional landscape in the regulation of the actin cytoskeleton. Recent findings emphasize that the functional and structural features of these proteins can differ not only between ADF-H families but even within the same family. The structural and evolutional background of this functional diversity is poorly understood. This review focuses on the specific functional characteristics of ADF-H domain proteins and how these features can be linked to structural differences in the ADF-H domain and also to different conformational transitions in actin. In the light of recent discoveries we pay special attention to the ADF/cofilin proteins to find tendencies along which the functional and structural diversification is governed through the evolution.

Myosin and Tropomyosin Stabilize the Conformation of Formin-nucleated Actin Filaments

Background: The regulation of the conformational dynamics of cellular actin structures is poorly understood. Results: Myosin and tropomyosin stabilize the conformation of formin-nucleated flexible actin filaments. Conclusion: Actin-binding proteins can play a central role in the establishment of the conformational properties of actin filaments. Significance: Our results add to our understanding of the mechanisms regulating the conformational and functional versatility of the actin cytoskeleton. The conformational elasticity of the actin cytoskeleton is essential for its versatile biological functions. Increasing evidence supports that the interplay between the structural and functional properties of actin filaments is finely regulated by actin-binding proteins; however, the underlying mechanisms and biological consequences are not completely understood. Previous studies showed that the binding of formins to the barbed end induces conformational transitions in actin filaments by making them more flexible through long range allosteric interactions. These conformational changes are accompanied by altered functional properties of the filaments. To get insight into the conformational regulation of formin-nucleated actin structures, in the present work we investigated in detail how binding partners of formin-generated actin structures, myosin and tropomyosin, affect the conformation of the formin-nucleated actin filaments using fluorescence spectroscopic approaches. Time-dependent fluorescence anisotropy and temperature-dependent Förster-type resonance energy transfer measurements revealed that heavy meromyosin, similarly to tropomyosin, restores the formin-induced effects and stabilizes the conformation of actin filaments. The stabilizing effect of heavy meromyosin is cooperative. The kinetic analysis revealed that despite the qualitatively similar effects of heavy meromyosin and tropomyosin on the conformational dynamics of actin filaments the mechanisms of the conformational transition are different for the two proteins. Heavy meromyosin stabilizes the formin-nucleated actin filaments in an apparently single step reaction upon binding, whereas the stabilization by tropomyosin occurs after complex formation. These observations support the idea that actin-binding proteins are key elements of the molecular mechanisms that regulate the conformational and functional diversity of actin filaments in living cells.

DAAM Is Required for Thin Filament Formation and Sarcomerogenesis during Muscle Development in Drosophila

During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin.

Purification of Tropomyosin Br-3 and 5NM1 and Characterization of Their Interactions with Actin

Tropomyosins were first identified in neuronal systems in 1973. Although numerous isoforms were found and described since then, many aspects of their function and interactions remained unknown. Tropomyosin isoforms show different sorting pattern in neurogenesis. As one example, TM5NM1/2 is present in developing axons, but it is replaced by TMBr‐3 in mature neurons, suggesting that these tropomyosin isoforms contribute differently to the establishment of the functional features of the neuronal actin networks. We developed a method for the efficient purification of TMBr‐3 and TM5NM1 as recombinant proteins using bacterial expression system and investigated their interactions with actin. We found that both isoforms bind actin filaments, however, the binding of TM5NM1 was much stronger than that of TMBr‐3. TMBr‐3 and TM5NM1 modestly affected actin assembly kinetics, in an opposite manner. Consistently with the higher affinity of TM5NM1 it inhibited actin filament disassembly more efficiently than TMBr‐3. Similarly to other previously studied tropomyosins TM5NM1 inhibited the Arp2/3 complex‐mediated actin assembly. Notably, TMBr‐3 did not influence the Arp2/3 complex‐mediated polymerization. This is a unique feature of TMBr‐3, since so far it is the only known tropomyosin supporting the activity of the Arp2/3 complex, indicating that TMBr‐3 may colocalize and work simultaneously with Arp2/3 complex in neuronal cells.

The formin DAAM is required for coordination of the actin and microtubule cytoskeleton in axonal growth cones

ABSTRACT Directed axonal growth depends on correct coordination of the actin and microtubule cytoskeleton in the growth cone. However, despite the relatively large number of proteins implicated in actin−microtubule crosstalk, the mechanisms whereby actin polymerization is coupled to microtubule stabilization and advancement in the peripheral growth cone remained largely unclear. Here, we identified the formin Dishevelled-associated activator of morphogenesis (DAAM) as a novel factor playing a role in concerted regulation of actin and microtubule remodeling in Drosophila melanogaster primary neurons. In vitro, DAAM binds to F-actin as well as to microtubules and has the ability to crosslink the two filament systems. Accordingly, DAAM associates with the neuronal cytoskeleton, and a significant fraction of DAAM accumulates at places where the actin filaments overlap with that of microtubules. Loss of DAAM affects growth cone and microtubule morphology, and several aspects of microtubule dynamics; and biochemical and cellular assays revealed a microtubule stabilization activity and binding to the microtubule tip protein EB1. Together, these data suggest that, besides operating as an actin assembly factor, DAAM is involved in linking actin remodeling in filopodia to microtubule stabilization during axonal growth. Highlighted Article: As well as operating as an actin assembly factor, the formin DAAM is involved in linking actin remodeling in filopodia to microtubule stabilization during axonal growth in Drosophila.