Drosophila melanogaster sarcomere length short (SALS) is a recently identified Wiskott-Aldrich sy... more Drosophila melanogaster sarcomere length short (SALS) is a recently identified Wiskott-Aldrich syndrome protein homo-logy 2 (WH2) domain protein involved in skeletal muscle thin filament regulation. SALS was shown to be important for the establishment of the proper length and organization of sarcom-eric actin filaments. Here, we present the first detailed characterization of the biochemical activities of the tandem WH2 domains of SALS (SALS-WH2). Our results revealed that SALS-WH2 binds both monomeric and filamentous actin and shifts the monomer-filament equilibrium toward the monomeric actin. In addition, SALS-WH2 can bind to but fails to depo-lymerize phalloidin-or jasplakinolide-bound actin filaments. These interactions endow SALS-WH2 with the following two major activities in the regulation of actin dynamics: SALS-WH2 sequesters actin monomers into non-polymerizable complexes and enhances actin filament disassembly by severing, which is modulated by tropomyosin. We also show that profilin does not influence the activities of the WH2 domains of SALS in actin dynamics. In conclusion, the tandem WH2 domains of SALS are multifunctional regulators of actin dynamics. Our findings suggest that the activities of the WH2 domains do not reconstitute the presumed biological function of the full-length protein. Consequently, the interactions of the WH2 domains of SALS with actin must be tuned in the cellular context by other modules of the protein and/or sarcomeric components for its proper functioning.
During muscle development, myosin and actin containing filaments assemble into the highly organiz... more 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.
Several cellular processes rely on the fine tuning of actin cytoskeleton. A central component in ... more 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.
Tropomyosins were first identified in neuronal systems
in 1973. Although numerous isoforms were f... more 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.
Background: The regulation of the conformational dynamics of cellular actin structures is poorly ... more 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.
During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chem... more During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the c-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P i release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P i release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P i from F-actin. The products of ATP hydrolysis were released by dena-turation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P i. The effect of both toxins can be sufficiently explained by local and mechanical blockade of P i dissociation.
Actin is a protein abundant in many cell types. Decades of investigations have provided evidence ... more 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 conforma-tional states of actin and its biological function.
At the leading edge of migrating cells, protrusive forces are
developed by the assembly of actin ... more At the leading edge of migrating cells, protrusive forces are developed by the assembly of actin filaments organised in a lamellipodial dendritic array at the front and a more distal lamellar linear array. Whether these two arrays are distinct or functionally linked and how they contribute to cell migration is an open issue. Tropomyosin severely inhibits lamellipodium formation and facilitates the lamellar array while enhancing migration, by a mechanism that is not understood. Here we show that the complex in vivo effects of tropomyosin are recapitulated in the reconstituted propulsion of neural Wiskott–Aldrich syndrome protein (N-WASP)-functionalised beads, which is based on the sole formation of a dendritic array of actin-related protein (Arp)2/3-branched filaments. Actin-depolymerising factor (ADF) and tropomyosin control the length of the actin tail. By competing with Arp2/3 during filament branching, tropomyosin displays opposite effects on propulsion depending on the surface density of N-WASP. Tropomyosin binding to the dendritic array is facilitated following filament debranching, causing its enrichment at the rear of the actin tail, like in vivo. These results unveil the mechanism by which tropomyosin generates two morphologically and dynamically segregated actin networks from a single one.
Recent advances in structural, biochemical, biophysical, and live cell imaging approaches have fu... more 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 protru-sion, powered by the turnover of a branched filament array. ATP hy-drolysis 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.
We characterized the properties of Drosophila melanogaster DAAM-FH2 and DAAM-FH1-FH2 fragments an... more 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. melano-gaster DAAM formin represents an extreme class of barbed end regulators gated by profilin.
Cell motility, one of the modular activities of living cells, elicits the response of the cell to... more Cell motility, one of the modular activities of living cells, elicits the response of the cell to extra-cellular signals, to move directionally, feed, divide or transport materials. The combined actions of molecular motors and re-modeling of the cytoskeleton generate forces and movement. Here we describe mechanistic approaches of force and movement produced by site-directed assembly of actin filaments. The insight derived from a biochemical analysis of the protein machineries involved in " actin-based motile processes " like cell protrusions, invaginations, organelle propulsion, is used to build reconstituted assays that mimic cellular processes, using several protein machineries known to initiate filament assembly by different mechanisms. Reconstitution of complex self-organized systems presents a broad variety of interests. Reconstituting actin-based movement of a functionalized particle from a minimum number of pure proteins, first used to prove the general thermodynamic principles at work in motility, then was the basis for fully controlled physical measurements of forces produced by polymerization of actin against an obstacle and of the mechanical properties of the resulting polymer arrays. In addition, measurements at the mesoscopic scale (trajectories, velocity, polymer mechanics, fluorescence of specifically labeled components of the actin array, use of mutated proteins) can provide further insight into the molecular mechanisms underlying motility.
The thermodynamic properties of the cardiac and skeletal alpha-actin isoforms were studied to cha... more The thermodynamic properties of the cardiac and skeletal alpha-actin isoforms were studied to characterise the molecular bases of the functional differences between them with the method of differential scanning calorimetry (DSC). The thermal properties of the actin filaments were described in the presence of calcium and magnesium ions as well. Based on the calculated free energy changes the alpha-cardiac actin filaments appeared to be more stable in its physiologically more relevant, magnesium saturated form. The magnesium saturated form of the alpha-cardiac actin filaments seemed to be more stable compared to the calcium saturated form of it. The enthalpy and entropy changes could differentiate between the alpha-cardiac and alpha-skeletal actin isoforms and between the calcium and magnesium saturated cardiac actin isoforms as well. Our results can demonstrate that the few differences between the amino acid sequences of the alpha-actin isoforms have an influence on the thermal properties and maybe on the function of these proteins as well.
Recent live cell image analysis of actin dynamics in lamellipodia of motile cells has shown that ... more Recent live cell image analysis of actin dynamics in lamellipodia of motile cells has shown that regulated treadmil-ling, which supports actin-based propulsion of functionalized particles in biomimetic reconstituted motility assays, is also responsible for lamellipodia extension. In both cases, filaments are created by branching with Arp2/3 complex only at the membrane or particle surface, grow transiently and are capped; ADF/ cofilin enhances the treadmilling but does not sever filaments in the body of the meshwork. Differences between the cellular and biomimetic systems suggest that additional regulatory mechanisms take place in lamellipodia.
WASP-homology 2 (WH2) domains, which were first
identified in the WASP/Scar (suppressor of cAMP
r... more WASP-homology 2 (WH2) domains, which were first identified in the WASP/Scar (suppressor of cAMP receptor)/WAVE (WASP-family verprolin homologous protein) family of proteins, are multifunctional regulators of actin assembly. Two recently discovered actinbinding proteins, Spire and Cordon-bleu (Cobl), which have roles in axis patterning in developmental processes, use repeats of WH2 domains to generate a large repertoire of novel regulatory activities, including G-actin sequestration, actin-filament nucleation, filament severing and barbed-end dynamics regulation. We describe how these multiple functions selectively operate in a cellular context to control the dynamics of the actin cytoskeleton. In vivo, Spire and Cobl can synergize with other actin regulators. As an example, we outline potential methods to gain insight into the functional basis for reported genetic interactions among Spire, profilin and formin.
The members of the formin family nucleate actin polymerization and play essential roles in the re... more 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 confor-mation of actin filaments by using a temperature-dependent fluo-rescence 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.
Formins bind actin filaments and play an essential role in the regulation of the actin cytoskelet... more Formins bind actin filaments and play an essential role in the regulation of the actin cytoskeleton. In this work we describe details of the formin-induced conformational changes in actin filaments by fluorescence-lifetime and anisotropy-decay experiments. The results show that the binding of the formin homology 2 domain of a mammalian formin (mouse mDia1) to actin filaments resulted in a less rigid protein structure in the microenvironment of the Cys 374 of actin, weakening of the interactions between neighboring actin protomers, and greater overall flexibility of the actin filaments. The formin effect is smaller at greater ionic strength. The results show that formin binding to the barbed end of actin filaments is responsible for the increase of flexibility of actin filaments. One formin dimer can affect the dynamic properties of an entire filament. Analyses of the results obtained at various formin/actin concentration ratios indicate that at least 160 actin protomers are affected by the binding of a single formin dimer to the barbed end of a filament.
Diaphanous-related formins (Drf) are activated by Rho GTP binding proteins and induce polymerizat... more 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.
The ability of actin to form filaments is fundamental to its biological function and
often charac... more The ability of actin to form filaments is fundamental to its biological function and often characterised by various methods in vitro. One of the most frequently used methods capitalises on the observation that the fluorescence emission of a pyrene label on the Cys-374 residue of actin is enhanced by a factor of ~20 during polymerization. This method inherently involves the chemical modification of actin monomers with pyrene. It was reported earlier that the pyrene labelling of actin monomers has only small effect on the polymerisation and depolymerisation rates of actin, indicating that the method is suitable to characterize the effect of actin-binding proteins or peptides on the polymerisation kinetics. In our present work we tested the effect of the pyrene labelling on the thermal denaturation of actin filaments by using the method of differential scanning calorimetry (DSC). By recording the heat denaturation profiles of unlabelled and pyrene labelled actin filaments we observed that pyrene labelling shifted the melting point (Tm) of actin filaments from 66 oC to 68 oC. A similar effect was detected in the presence of equimolar concentration of phalloidin where the Tm shifted from 79 oC to 82 oC. We concluded that the observed pyrene labelling induced differences of the thermal denaturation of actin filaments were small. The DSC results, therefore, confirmed that the methods based on the measurements of pyrene intensity during actin polymerisation are suitable to characterise the polymerisation kinetics of actin under in vitro conditions.
The effect of pH was characterised on the thermal stability of magnesium saturated skeletal and c... more The effect of pH was characterised on the thermal stability of magnesium saturated skeletal and cardiac alpha-actin isoforms with differential scanning calorimetry (DSC) at pH 7.0 and 8.0. The calorimetric curves were further analysed to calculate the enthalpy and transition entropy changes. The activation energy was also determined to describe the energy consumption of the initiation of the thermal denaturation process. Although the difference in Tm values is too small to interpret the difference between the alpha-actin isoforms, the values of the activation energy indicated that the alpha-skeletal actin is probably more stable compared to the alpha-cardiac actin. The difference in the activation energies indicated that lowering the pH can produce a more stable protein matrix in both cases of the isoforms. The larger range of the difference in the values of the activation energies suggested that the alpha-cardiac actin is probably more sensitive to the change of the pH compared to the alpha-skeletal actin.
Drosophila melanogaster sarcomere length short (SALS) is a recently identified Wiskott-Aldrich sy... more Drosophila melanogaster sarcomere length short (SALS) is a recently identified Wiskott-Aldrich syndrome protein homo-logy 2 (WH2) domain protein involved in skeletal muscle thin filament regulation. SALS was shown to be important for the establishment of the proper length and organization of sarcom-eric actin filaments. Here, we present the first detailed characterization of the biochemical activities of the tandem WH2 domains of SALS (SALS-WH2). Our results revealed that SALS-WH2 binds both monomeric and filamentous actin and shifts the monomer-filament equilibrium toward the monomeric actin. In addition, SALS-WH2 can bind to but fails to depo-lymerize phalloidin-or jasplakinolide-bound actin filaments. These interactions endow SALS-WH2 with the following two major activities in the regulation of actin dynamics: SALS-WH2 sequesters actin monomers into non-polymerizable complexes and enhances actin filament disassembly by severing, which is modulated by tropomyosin. We also show that profilin does not influence the activities of the WH2 domains of SALS in actin dynamics. In conclusion, the tandem WH2 domains of SALS are multifunctional regulators of actin dynamics. Our findings suggest that the activities of the WH2 domains do not reconstitute the presumed biological function of the full-length protein. Consequently, the interactions of the WH2 domains of SALS with actin must be tuned in the cellular context by other modules of the protein and/or sarcomeric components for its proper functioning.
During muscle development, myosin and actin containing filaments assemble into the highly organiz... more 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.
Several cellular processes rely on the fine tuning of actin cytoskeleton. A central component in ... more 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.
Tropomyosins were first identified in neuronal systems
in 1973. Although numerous isoforms were f... more 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.
Background: The regulation of the conformational dynamics of cellular actin structures is poorly ... more 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.
During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chem... more During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the c-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P i release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P i release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P i from F-actin. The products of ATP hydrolysis were released by dena-turation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P i. The effect of both toxins can be sufficiently explained by local and mechanical blockade of P i dissociation.
Actin is a protein abundant in many cell types. Decades of investigations have provided evidence ... more 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 conforma-tional states of actin and its biological function.
At the leading edge of migrating cells, protrusive forces are
developed by the assembly of actin ... more At the leading edge of migrating cells, protrusive forces are developed by the assembly of actin filaments organised in a lamellipodial dendritic array at the front and a more distal lamellar linear array. Whether these two arrays are distinct or functionally linked and how they contribute to cell migration is an open issue. Tropomyosin severely inhibits lamellipodium formation and facilitates the lamellar array while enhancing migration, by a mechanism that is not understood. Here we show that the complex in vivo effects of tropomyosin are recapitulated in the reconstituted propulsion of neural Wiskott–Aldrich syndrome protein (N-WASP)-functionalised beads, which is based on the sole formation of a dendritic array of actin-related protein (Arp)2/3-branched filaments. Actin-depolymerising factor (ADF) and tropomyosin control the length of the actin tail. By competing with Arp2/3 during filament branching, tropomyosin displays opposite effects on propulsion depending on the surface density of N-WASP. Tropomyosin binding to the dendritic array is facilitated following filament debranching, causing its enrichment at the rear of the actin tail, like in vivo. These results unveil the mechanism by which tropomyosin generates two morphologically and dynamically segregated actin networks from a single one.
Recent advances in structural, biochemical, biophysical, and live cell imaging approaches have fu... more 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 protru-sion, powered by the turnover of a branched filament array. ATP hy-drolysis 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.
We characterized the properties of Drosophila melanogaster DAAM-FH2 and DAAM-FH1-FH2 fragments an... more 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. melano-gaster DAAM formin represents an extreme class of barbed end regulators gated by profilin.
Cell motility, one of the modular activities of living cells, elicits the response of the cell to... more Cell motility, one of the modular activities of living cells, elicits the response of the cell to extra-cellular signals, to move directionally, feed, divide or transport materials. The combined actions of molecular motors and re-modeling of the cytoskeleton generate forces and movement. Here we describe mechanistic approaches of force and movement produced by site-directed assembly of actin filaments. The insight derived from a biochemical analysis of the protein machineries involved in " actin-based motile processes " like cell protrusions, invaginations, organelle propulsion, is used to build reconstituted assays that mimic cellular processes, using several protein machineries known to initiate filament assembly by different mechanisms. Reconstitution of complex self-organized systems presents a broad variety of interests. Reconstituting actin-based movement of a functionalized particle from a minimum number of pure proteins, first used to prove the general thermodynamic principles at work in motility, then was the basis for fully controlled physical measurements of forces produced by polymerization of actin against an obstacle and of the mechanical properties of the resulting polymer arrays. In addition, measurements at the mesoscopic scale (trajectories, velocity, polymer mechanics, fluorescence of specifically labeled components of the actin array, use of mutated proteins) can provide further insight into the molecular mechanisms underlying motility.
The thermodynamic properties of the cardiac and skeletal alpha-actin isoforms were studied to cha... more The thermodynamic properties of the cardiac and skeletal alpha-actin isoforms were studied to characterise the molecular bases of the functional differences between them with the method of differential scanning calorimetry (DSC). The thermal properties of the actin filaments were described in the presence of calcium and magnesium ions as well. Based on the calculated free energy changes the alpha-cardiac actin filaments appeared to be more stable in its physiologically more relevant, magnesium saturated form. The magnesium saturated form of the alpha-cardiac actin filaments seemed to be more stable compared to the calcium saturated form of it. The enthalpy and entropy changes could differentiate between the alpha-cardiac and alpha-skeletal actin isoforms and between the calcium and magnesium saturated cardiac actin isoforms as well. Our results can demonstrate that the few differences between the amino acid sequences of the alpha-actin isoforms have an influence on the thermal properties and maybe on the function of these proteins as well.
Recent live cell image analysis of actin dynamics in lamellipodia of motile cells has shown that ... more Recent live cell image analysis of actin dynamics in lamellipodia of motile cells has shown that regulated treadmil-ling, which supports actin-based propulsion of functionalized particles in biomimetic reconstituted motility assays, is also responsible for lamellipodia extension. In both cases, filaments are created by branching with Arp2/3 complex only at the membrane or particle surface, grow transiently and are capped; ADF/ cofilin enhances the treadmilling but does not sever filaments in the body of the meshwork. Differences between the cellular and biomimetic systems suggest that additional regulatory mechanisms take place in lamellipodia.
WASP-homology 2 (WH2) domains, which were first
identified in the WASP/Scar (suppressor of cAMP
r... more WASP-homology 2 (WH2) domains, which were first identified in the WASP/Scar (suppressor of cAMP receptor)/WAVE (WASP-family verprolin homologous protein) family of proteins, are multifunctional regulators of actin assembly. Two recently discovered actinbinding proteins, Spire and Cordon-bleu (Cobl), which have roles in axis patterning in developmental processes, use repeats of WH2 domains to generate a large repertoire of novel regulatory activities, including G-actin sequestration, actin-filament nucleation, filament severing and barbed-end dynamics regulation. We describe how these multiple functions selectively operate in a cellular context to control the dynamics of the actin cytoskeleton. In vivo, Spire and Cobl can synergize with other actin regulators. As an example, we outline potential methods to gain insight into the functional basis for reported genetic interactions among Spire, profilin and formin.
The members of the formin family nucleate actin polymerization and play essential roles in the re... more 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 confor-mation of actin filaments by using a temperature-dependent fluo-rescence 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.
Formins bind actin filaments and play an essential role in the regulation of the actin cytoskelet... more Formins bind actin filaments and play an essential role in the regulation of the actin cytoskeleton. In this work we describe details of the formin-induced conformational changes in actin filaments by fluorescence-lifetime and anisotropy-decay experiments. The results show that the binding of the formin homology 2 domain of a mammalian formin (mouse mDia1) to actin filaments resulted in a less rigid protein structure in the microenvironment of the Cys 374 of actin, weakening of the interactions between neighboring actin protomers, and greater overall flexibility of the actin filaments. The formin effect is smaller at greater ionic strength. The results show that formin binding to the barbed end of actin filaments is responsible for the increase of flexibility of actin filaments. One formin dimer can affect the dynamic properties of an entire filament. Analyses of the results obtained at various formin/actin concentration ratios indicate that at least 160 actin protomers are affected by the binding of a single formin dimer to the barbed end of a filament.
Diaphanous-related formins (Drf) are activated by Rho GTP binding proteins and induce polymerizat... more 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.
The ability of actin to form filaments is fundamental to its biological function and
often charac... more The ability of actin to form filaments is fundamental to its biological function and often characterised by various methods in vitro. One of the most frequently used methods capitalises on the observation that the fluorescence emission of a pyrene label on the Cys-374 residue of actin is enhanced by a factor of ~20 during polymerization. This method inherently involves the chemical modification of actin monomers with pyrene. It was reported earlier that the pyrene labelling of actin monomers has only small effect on the polymerisation and depolymerisation rates of actin, indicating that the method is suitable to characterize the effect of actin-binding proteins or peptides on the polymerisation kinetics. In our present work we tested the effect of the pyrene labelling on the thermal denaturation of actin filaments by using the method of differential scanning calorimetry (DSC). By recording the heat denaturation profiles of unlabelled and pyrene labelled actin filaments we observed that pyrene labelling shifted the melting point (Tm) of actin filaments from 66 oC to 68 oC. A similar effect was detected in the presence of equimolar concentration of phalloidin where the Tm shifted from 79 oC to 82 oC. We concluded that the observed pyrene labelling induced differences of the thermal denaturation of actin filaments were small. The DSC results, therefore, confirmed that the methods based on the measurements of pyrene intensity during actin polymerisation are suitable to characterise the polymerisation kinetics of actin under in vitro conditions.
The effect of pH was characterised on the thermal stability of magnesium saturated skeletal and c... more The effect of pH was characterised on the thermal stability of magnesium saturated skeletal and cardiac alpha-actin isoforms with differential scanning calorimetry (DSC) at pH 7.0 and 8.0. The calorimetric curves were further analysed to calculate the enthalpy and transition entropy changes. The activation energy was also determined to describe the energy consumption of the initiation of the thermal denaturation process. Although the difference in Tm values is too small to interpret the difference between the alpha-actin isoforms, the values of the activation energy indicated that the alpha-skeletal actin is probably more stable compared to the alpha-cardiac actin. The difference in the activation energies indicated that lowering the pH can produce a more stable protein matrix in both cases of the isoforms. The larger range of the difference in the values of the activation energies suggested that the alpha-cardiac actin is probably more sensitive to the change of the pH compared to the alpha-skeletal actin.
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Papers by Beáta Bugyi
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.
developed by the assembly of actin filaments organised in
a lamellipodial dendritic array at the front and a more
distal lamellar linear array. Whether these two arrays are
distinct or functionally linked and how they contribute to
cell migration is an open issue. Tropomyosin severely
inhibits lamellipodium formation and facilitates the lamellar
array while enhancing migration, by a mechanism that
is not understood. Here we show that the complex in vivo
effects of tropomyosin are recapitulated in the reconstituted
propulsion of neural Wiskott–Aldrich syndrome
protein (N-WASP)-functionalised beads, which is based
on the sole formation of a dendritic array of actin-related
protein (Arp)2/3-branched filaments. Actin-depolymerising
factor (ADF) and tropomyosin control the length of the
actin tail. By competing with Arp2/3 during filament
branching, tropomyosin displays opposite effects on propulsion
depending on the surface density of N-WASP.
Tropomyosin binding to the dendritic array is facilitated
following filament debranching, causing its enrichment at
the rear of the actin tail, like in vivo. These results unveil
the mechanism by which tropomyosin generates two
morphologically and dynamically segregated actin networks
from a single one.
functional differences between them with the method of differential scanning calorimetry (DSC). The thermal properties of the
actin filaments were described in the presence of calcium and magnesium ions as well. Based on the calculated free energy changes
the alpha-cardiac actin filaments appeared to be more stable in its physiologically more relevant, magnesium saturated form. The magnesium
saturated form of the alpha-cardiac actin filaments seemed to be more stable compared to the calcium saturated form of it. The
enthalpy and entropy changes could differentiate between the alpha-cardiac and alpha-skeletal actin isoforms and between the calcium and
magnesium saturated cardiac actin isoforms as well.
Our results can demonstrate that the few differences between the amino acid sequences of the alpha-actin isoforms have an influence
on the thermal properties and maybe on the function of these proteins as well.
identified in the WASP/Scar (suppressor of cAMP
receptor)/WAVE (WASP-family verprolin homologous
protein) family of proteins, are multifunctional regulators
of actin assembly. Two recently discovered actinbinding
proteins, Spire and Cordon-bleu (Cobl), which
have roles in axis patterning in developmental processes,
use repeats of WH2 domains to generate a large
repertoire of novel regulatory activities, including
G-actin sequestration, actin-filament nucleation, filament
severing and barbed-end dynamics regulation.
We describe how these multiple functions selectively
operate in a cellular context to control the dynamics of
the actin cytoskeleton. In vivo, Spire and Cobl can synergize
with other actin regulators. As an example,
we outline potential methods to gain insight into the
functional basis for reported genetic interactions among
Spire, profilin and formin.
often characterised by various methods in vitro. One of the most frequently used
methods capitalises on the observation that the fluorescence emission of a pyrene
label on the Cys-374 residue of actin is enhanced by a factor of ~20 during
polymerization. This method inherently involves the chemical modification of actin
monomers with pyrene. It was reported earlier that the pyrene labelling of actin
monomers has only small effect on the polymerisation and depolymerisation rates of actin, indicating that the method is suitable to characterize the effect of actin-binding proteins or peptides on the polymerisation kinetics.
In our present work we tested the effect of the pyrene labelling on the thermal
denaturation of actin filaments by using the method of differential scanning
calorimetry (DSC). By recording the heat denaturation profiles of unlabelled and
pyrene labelled actin filaments we observed that pyrene labelling shifted the melting
point (Tm) of actin filaments from 66 oC to 68 oC. A similar effect was detected in the presence of equimolar concentration of phalloidin where the Tm shifted from 79 oC to 82 oC. We concluded that the observed pyrene labelling induced differences of the thermal denaturation of actin filaments were small. The DSC results, therefore, confirmed that the methods based on the measurements of pyrene intensity during actin polymerisation are suitable to characterise the polymerisation kinetics of actin under in vitro conditions.
scanning calorimetry (DSC) at pH 7.0 and 8.0. The calorimetric curves were further analysed to calculate the enthalpy and
transition entropy changes. The activation energy was also determined to describe the energy consumption of the initiation of the
thermal denaturation process.
Although the difference in Tm values is too small to interpret the difference between the alpha-actin isoforms, the values of the activation
energy indicated that the alpha-skeletal actin is probably more stable compared to the alpha-cardiac actin. The difference in the activation
energies indicated that lowering the pH can produce a more stable protein matrix in both cases of the isoforms. The larger
range of the difference in the values of the activation energies suggested that the alpha-cardiac actin is probably more sensitive to the
change of the pH compared to the alpha-skeletal actin.
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.
developed by the assembly of actin filaments organised in
a lamellipodial dendritic array at the front and a more
distal lamellar linear array. Whether these two arrays are
distinct or functionally linked and how they contribute to
cell migration is an open issue. Tropomyosin severely
inhibits lamellipodium formation and facilitates the lamellar
array while enhancing migration, by a mechanism that
is not understood. Here we show that the complex in vivo
effects of tropomyosin are recapitulated in the reconstituted
propulsion of neural Wiskott–Aldrich syndrome
protein (N-WASP)-functionalised beads, which is based
on the sole formation of a dendritic array of actin-related
protein (Arp)2/3-branched filaments. Actin-depolymerising
factor (ADF) and tropomyosin control the length of the
actin tail. By competing with Arp2/3 during filament
branching, tropomyosin displays opposite effects on propulsion
depending on the surface density of N-WASP.
Tropomyosin binding to the dendritic array is facilitated
following filament debranching, causing its enrichment at
the rear of the actin tail, like in vivo. These results unveil
the mechanism by which tropomyosin generates two
morphologically and dynamically segregated actin networks
from a single one.
functional differences between them with the method of differential scanning calorimetry (DSC). The thermal properties of the
actin filaments were described in the presence of calcium and magnesium ions as well. Based on the calculated free energy changes
the alpha-cardiac actin filaments appeared to be more stable in its physiologically more relevant, magnesium saturated form. The magnesium
saturated form of the alpha-cardiac actin filaments seemed to be more stable compared to the calcium saturated form of it. The
enthalpy and entropy changes could differentiate between the alpha-cardiac and alpha-skeletal actin isoforms and between the calcium and
magnesium saturated cardiac actin isoforms as well.
Our results can demonstrate that the few differences between the amino acid sequences of the alpha-actin isoforms have an influence
on the thermal properties and maybe on the function of these proteins as well.
identified in the WASP/Scar (suppressor of cAMP
receptor)/WAVE (WASP-family verprolin homologous
protein) family of proteins, are multifunctional regulators
of actin assembly. Two recently discovered actinbinding
proteins, Spire and Cordon-bleu (Cobl), which
have roles in axis patterning in developmental processes,
use repeats of WH2 domains to generate a large
repertoire of novel regulatory activities, including
G-actin sequestration, actin-filament nucleation, filament
severing and barbed-end dynamics regulation.
We describe how these multiple functions selectively
operate in a cellular context to control the dynamics of
the actin cytoskeleton. In vivo, Spire and Cobl can synergize
with other actin regulators. As an example,
we outline potential methods to gain insight into the
functional basis for reported genetic interactions among
Spire, profilin and formin.
often characterised by various methods in vitro. One of the most frequently used
methods capitalises on the observation that the fluorescence emission of a pyrene
label on the Cys-374 residue of actin is enhanced by a factor of ~20 during
polymerization. This method inherently involves the chemical modification of actin
monomers with pyrene. It was reported earlier that the pyrene labelling of actin
monomers has only small effect on the polymerisation and depolymerisation rates of actin, indicating that the method is suitable to characterize the effect of actin-binding proteins or peptides on the polymerisation kinetics.
In our present work we tested the effect of the pyrene labelling on the thermal
denaturation of actin filaments by using the method of differential scanning
calorimetry (DSC). By recording the heat denaturation profiles of unlabelled and
pyrene labelled actin filaments we observed that pyrene labelling shifted the melting
point (Tm) of actin filaments from 66 oC to 68 oC. A similar effect was detected in the presence of equimolar concentration of phalloidin where the Tm shifted from 79 oC to 82 oC. We concluded that the observed pyrene labelling induced differences of the thermal denaturation of actin filaments were small. The DSC results, therefore, confirmed that the methods based on the measurements of pyrene intensity during actin polymerisation are suitable to characterise the polymerisation kinetics of actin under in vitro conditions.
scanning calorimetry (DSC) at pH 7.0 and 8.0. The calorimetric curves were further analysed to calculate the enthalpy and
transition entropy changes. The activation energy was also determined to describe the energy consumption of the initiation of the
thermal denaturation process.
Although the difference in Tm values is too small to interpret the difference between the alpha-actin isoforms, the values of the activation
energy indicated that the alpha-skeletal actin is probably more stable compared to the alpha-cardiac actin. The difference in the activation
energies indicated that lowering the pH can produce a more stable protein matrix in both cases of the isoforms. The larger
range of the difference in the values of the activation energies suggested that the alpha-cardiac actin is probably more sensitive to the
change of the pH compared to the alpha-skeletal actin.