Beata Bugyi | University of Pecs (original) (raw)

Papers by Beata Bugyi

Journal of Biological Chemistry, 2012

Journal of Cell Science

We provide novel insights into the mechanisms of actin-microtubule crosstalk in axonal growth con... more We provide novel insights into the mechanisms of actin-microtubule crosstalk in axonal growth cones by describing the role of a formin protein in Drosophila neurons.

Trends in cell biology, 2008

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 actin-binding 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.

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.

Thermochimica Acta, 2006

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 polymerisation. 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 characterise the effect of actin-binding proteins or peptides on the polymerisation kinetics.

Molecular Cell, 2007

The Spire protein, together with the formin Cappuccino and profilin, plays an important role in a... more The Spire protein, together with the formin Cappuccino and profilin, plays an important role in actin-based processes that establish oocyte polarity. Spire contains a cluster of four actinbinding WH2 domains. It has been shown to nucleate actin filaments and was proposed to remain bound to their pointed ends. Here we show that the multifunctional character of the WH2 domains allows Spire to sequester four G-actin subunits binding cooperatively in a tight SA 4 complex and to nucleate, sever, and cap filaments at their barbed ends. Binding of Spire to barbed ends does not affect the thermodynamics of actin assembly at barbed ends but blocks barbed end growth from profilin-actin. The resulting Spire-induced increase in profilin-actin concentration enhances processive filament assembly by formin. The synergy between Spire and formin is reconstituted in an in vitro motility assay, which provides a functional basis for the genetic interplay between Spire, formin, and profilin in oogenesis.

Journal of Thermal Analysis and Calorimetry, 2005

Summary The effect of pH was characterised on the thermal stability of magnesium saturated skele... more Summary The effect of pH was characterised on the thermal stability of magnesium saturated skeletal and cardiac α-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

Journal of Thermal Analysis and Calorimetry, 2005

Handbook of Thermal Analysis and Calorimetry, Volume 2, Applications to Inorganic and Miscella-ne... more Handbook of Thermal Analysis and Calorimetry, Volume 2, Applications to Inorganic and Miscella-neous Materials, by Michael E. Brown and Patrick K. Gallagher (Editors). ... Chapter 10 (by HJ Seifert) deals with a broad range of general inorganic and coordination compounds. ...

Journal of Thermal Analysis and Calorimetry, 2005

Summary In our present study we performed the detailed characterisation of jasplakinolide and ph... more Summary In our present study we performed the detailed characterisation of jasplakinolide and phalloidin on the thermal stability of actin filaments. The heat absorption curves were analysed by using the model established by Sanchez-Ruiz et al. [1]. The analysis provided the activation energies attributed to the heat denaturation of actin filaments in the absence and in the presence of toxins. The

Journal of Biological Chemistry, 2006

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 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 protomerprotomer 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.

FEBS Letters, 2008

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 treadmilling, 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.

Biophysical Reviews and Letters, 2009

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 ...

Biophysical Journal, 2011

Profilin regulates actin polymerization in cells playing important role in cell motility and divi... more Profilin regulates actin polymerization in cells playing important role in cell motility and division. Actin polymerization involves ATP hydrolysis which occurs both in the absence and the presence of profilin. There is a hypothesis suggested in literature that profilin promotes actin polymerization through direct transfer of the energy of this spontaneous hydrolysis to polymerization utilizing tight coupling of the hydrolysis and corresponding polymerization events. Recently we suggested an alternative hypothesis based on an indirect energy transfer and pointed out that recent experimental and theoretical findings require re-evaluation of the direct transfer hypothesis. Our thermodynamically rigorous model of actin steady state dynamics in the presence of profilin describes all events in terms of chemical reactions and allows both energy transfer mechanisms, each corresponding to certain ranges of the rate constants (parameters) for these reactions. In fact, the difference between the two mechanisms is defined by a single ratio r of the two rates: the rate of hydrolysis by the profilin-and-ATP-bound subunit at the filament end, and the rate of dissociation of this subunit (in complex with profilin) from the end. The direct transfer cannot exist unless r>1 while indirect transfer can occur at any r. Our model predicts specific shapes for the dependence of actin critical concentration on profilin concentration depending on the ratio r. For values r>1, curves have peaks, which persist for wide ranges of other model parameters. Our experimental dependence (obtained with our new technique) shows no peak but a steady decline, contrary to the direct transfer hypothesis. This result provides strong support for our indirect transfer hypothesis. Moreover, using our theory and routine experimental techniques we determined relative activities of the two specific molecular mechanisms of profilin action (both based on indirect transfer) predicted earlier with our model.

Biophysical Journal, 2006

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.

Applied Microbiology and Biotechnology, 2005

Biochemical and pharmacological properties of biosurfactants produced at 45°C temperature by Pseu... more Biochemical and pharmacological properties of biosurfactants produced at 45°C temperature by Pseudomonas aeruginosa mucoid (M) and non-mucoid (NM) strains, isolated from hydrocarbon-contaminated soil samples, were characterized. Both the strains secreted appreciable amount of biosurfactants (5.0-6.5 g/l), responsible for the reduction of surface tension of the medium from 68 to 29±0.5 mN/m post 96 h of growth. Maximum yield of biosurfactants was observed following the supplementation of NH 4 Cl and glycerol as nitrogenous source and carbon source, respectively. These thermostable biosurfactants exhibited strong emulsifying property and could release appreciable amount of oil from saturated sand-pack column. Pharmacological characterization of these biosurfactants revealed that they induced dose-dependent hemolysis and coagulation of platelet-poor plasma but were non-detrimental to chicken lung, liver, heart and kidney tissues. Our study has documented that biosurfactants from P. aeruginosa M and NM strains could be exploited for use in petroleum sectors as well in pharmaceutical industries.

fragment, a profilin-dependent increase in the formation of actin structures is observed. The tra... more 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.

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 actinbinding 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. Chua NH, Pantaloni D. 1997. Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility.

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 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. 449 Annu. Rev. Biophys. 2010.39:449-470. Downloaded from arjournals.annualreviews.org by 89.133.73.22 on 05/25/10. For personal use only.

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.

Journal of Biological Chemistry, 2012

Journal of Cell Science

We provide novel insights into the mechanisms of actin-microtubule crosstalk in axonal growth con... more We provide novel insights into the mechanisms of actin-microtubule crosstalk in axonal growth cones by describing the role of a formin protein in Drosophila neurons.

Trends in cell biology, 2008

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 actin-binding 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.

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.

Thermochimica Acta, 2006

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 polymerisation. 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 characterise the effect of actin-binding proteins or peptides on the polymerisation kinetics.

Molecular Cell, 2007

The Spire protein, together with the formin Cappuccino and profilin, plays an important role in a... more The Spire protein, together with the formin Cappuccino and profilin, plays an important role in actin-based processes that establish oocyte polarity. Spire contains a cluster of four actinbinding WH2 domains. It has been shown to nucleate actin filaments and was proposed to remain bound to their pointed ends. Here we show that the multifunctional character of the WH2 domains allows Spire to sequester four G-actin subunits binding cooperatively in a tight SA 4 complex and to nucleate, sever, and cap filaments at their barbed ends. Binding of Spire to barbed ends does not affect the thermodynamics of actin assembly at barbed ends but blocks barbed end growth from profilin-actin. The resulting Spire-induced increase in profilin-actin concentration enhances processive filament assembly by formin. The synergy between Spire and formin is reconstituted in an in vitro motility assay, which provides a functional basis for the genetic interplay between Spire, formin, and profilin in oogenesis.

Journal of Thermal Analysis and Calorimetry, 2005

Summary The effect of pH was characterised on the thermal stability of magnesium saturated skele... more Summary The effect of pH was characterised on the thermal stability of magnesium saturated skeletal and cardiac α-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

Journal of Thermal Analysis and Calorimetry, 2005

Handbook of Thermal Analysis and Calorimetry, Volume 2, Applications to Inorganic and Miscella-ne... more Handbook of Thermal Analysis and Calorimetry, Volume 2, Applications to Inorganic and Miscella-neous Materials, by Michael E. Brown and Patrick K. Gallagher (Editors). ... Chapter 10 (by HJ Seifert) deals with a broad range of general inorganic and coordination compounds. ...

Journal of Thermal Analysis and Calorimetry, 2005

Summary In our present study we performed the detailed characterisation of jasplakinolide and ph... more Summary In our present study we performed the detailed characterisation of jasplakinolide and phalloidin on the thermal stability of actin filaments. The heat absorption curves were analysed by using the model established by Sanchez-Ruiz et al. [1]. The analysis provided the activation energies attributed to the heat denaturation of actin filaments in the absence and in the presence of toxins. The

Journal of Biological Chemistry, 2006

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 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 protomerprotomer 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.

FEBS Letters, 2008

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 treadmilling, 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.

Biophysical Reviews and Letters, 2009

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 ...

Biophysical Journal, 2011

Profilin regulates actin polymerization in cells playing important role in cell motility and divi... more Profilin regulates actin polymerization in cells playing important role in cell motility and division. Actin polymerization involves ATP hydrolysis which occurs both in the absence and the presence of profilin. There is a hypothesis suggested in literature that profilin promotes actin polymerization through direct transfer of the energy of this spontaneous hydrolysis to polymerization utilizing tight coupling of the hydrolysis and corresponding polymerization events. Recently we suggested an alternative hypothesis based on an indirect energy transfer and pointed out that recent experimental and theoretical findings require re-evaluation of the direct transfer hypothesis. Our thermodynamically rigorous model of actin steady state dynamics in the presence of profilin describes all events in terms of chemical reactions and allows both energy transfer mechanisms, each corresponding to certain ranges of the rate constants (parameters) for these reactions. In fact, the difference between the two mechanisms is defined by a single ratio r of the two rates: the rate of hydrolysis by the profilin-and-ATP-bound subunit at the filament end, and the rate of dissociation of this subunit (in complex with profilin) from the end. The direct transfer cannot exist unless r>1 while indirect transfer can occur at any r. Our model predicts specific shapes for the dependence of actin critical concentration on profilin concentration depending on the ratio r. For values r>1, curves have peaks, which persist for wide ranges of other model parameters. Our experimental dependence (obtained with our new technique) shows no peak but a steady decline, contrary to the direct transfer hypothesis. This result provides strong support for our indirect transfer hypothesis. Moreover, using our theory and routine experimental techniques we determined relative activities of the two specific molecular mechanisms of profilin action (both based on indirect transfer) predicted earlier with our model.

Biophysical Journal, 2006

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.

Applied Microbiology and Biotechnology, 2005

Biochemical and pharmacological properties of biosurfactants produced at 45°C temperature by Pseu... more Biochemical and pharmacological properties of biosurfactants produced at 45°C temperature by Pseudomonas aeruginosa mucoid (M) and non-mucoid (NM) strains, isolated from hydrocarbon-contaminated soil samples, were characterized. Both the strains secreted appreciable amount of biosurfactants (5.0-6.5 g/l), responsible for the reduction of surface tension of the medium from 68 to 29±0.5 mN/m post 96 h of growth. Maximum yield of biosurfactants was observed following the supplementation of NH 4 Cl and glycerol as nitrogenous source and carbon source, respectively. These thermostable biosurfactants exhibited strong emulsifying property and could release appreciable amount of oil from saturated sand-pack column. Pharmacological characterization of these biosurfactants revealed that they induced dose-dependent hemolysis and coagulation of platelet-poor plasma but were non-detrimental to chicken lung, liver, heart and kidney tissues. Our study has documented that biosurfactants from P. aeruginosa M and NM strains could be exploited for use in petroleum sectors as well in pharmaceutical industries.

fragment, a profilin-dependent increase in the formation of actin structures is observed. The tra... more 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.

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 actinbinding 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. Chua NH, Pantaloni D. 1997. Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility.

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 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. 449 Annu. Rev. Biophys. 2010.39:449-470. Downloaded from arjournals.annualreviews.org by 89.133.73.22 on 05/25/10. For personal use only.

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.