Charge effects modulate actin assembly by classic myelin basic protein isoforms

Myelin basic protein (MBP), a highly cationic structural protein of the myelin sheath, is believed to be associated with the cytoskeleton in vivo and interacts with actin in vitro, but little is known about the regulation of this interaction. The rate and extent of actin polymerization induced by 18.5 kDa MBP charge isomers were correlated to charge reduction by post-translational modifications. Increased ionic strength attenuated the initial rate but not the final extent of polymerization achieved. Reduced pH enhanced the rate and extent of polymerization, presumably via partial protonation of intrinsic histidyl residues. The polymerizing activities of the 21.5, 17, and 14 kDa MBP splice variants were not proportionate to their net charges or charge densities. The presence of at least one region derived from exon II or VI of the "classic" MBP gene was required for effective bundling as assessed by light scattering and transmission electron microscopy.     

Metal ions modulate the plastic nature of Mycobacterium tuberculosis chaperonin-10

Chaperonin-10s possess a highly flexible segment of approximately 10 residues that covers their dome-like structure and closes the central cavity of the chaperonin assembly. The dome loop is believed to contribute to the plasticity of their oligomeric structure. We have exploited the presence of a single tryptophan residue occurring in the dome loop of Mycobacterium tuberculosis chaperonin-10 (cpn-10), and through intrinsic fluorescence measurements show that in the absence of metal ions, the tryptophan is almost fully solvent exposed at neutral pH. The dome loop, however, assumes a closed conformation in the presence of metal ions, or at low pH. These changes are fully reversed in the presence of chelating agents such as EDTA, confirming the role of cations in modulating the metastable states of cpn-10.     

Metal ions modulate gene expression and accumulation of the mycotoxins aflatoxin and zearalenone

AIMS: To determine the modulating action of some metal ions (Zn+2, Fe+2, Cu+2) on gene expression of enzymes related to fungal growth and accumulation of the mycotoxins aflatoxin and zearalenone. METHODS AND RESULTS: The effect of the metal ions, as single or mixed treatments, was observed in submerged cultures of toxigenic Aspergillus flavus or Fusarium graminearum, which produce the mycotoxins aflatoxin or zearalenone, respectively. The enzyme-linked immunosorbent assay results showed that the single metals Zn+2 or Cu+2 stimulated aflatoxin accumulation while Cu+2 or Fe+2 stimulated zearalenone in fungal cultures. Single Zn+2 treatment also affected conidial differentiation and pigmentation. A cDNA suppression subtractive library was also produced and followed by sequencing of potential metal treatment-specific clones, thus determining induced genes. The genes uncovered included enzymes and regulators of cell growth and division, including many genes with unknown functions were uncovered. A Northern blot analysis was used to verify the expression pattern of the corresponding genes under metal treatment. The metal ions enhanced the expression of alcohol dehydrogenase Adh1 homologue by up to 33-fold in A. flavus and ca fourfold in F. graminearum. Encoding homologues of a neutral amino acid permease, were also used in the Northern analysis. However, the expression of the permease was not significantly affected by metal ion treatments. CONCLUSIONS: The results showed a significant effect of metal ions on expression of gene related to fungal growth, development, conidiation and production of both aflatoxin and zearalenone. SIGNIFICANT AND IMPACT OF THE STUDY: At the molecular and cellular level, the significant effects of metal ions on fungal growth and development, conidiation, and production of both aflatoxin and zearalenone were demonstrated.     

The effect of caldesmon on assembly and dynamic properties of actin

Our earlier fluorescence measurements using N-(1-pyrenyl)iodoacetamide-labeled actin revealed that caldesmon interacts with G-actin accelerating its nucleation at low salt concentration and causing polymerization in the absence of sale [Ga?azkiewicz, B., Mossakowska, M., Osińska, H. & Dabrowska, R. (1985) FEBS Lett. 184, 144-149]. In this work the caldesmon-induced process of actin polymerization as well as the dynamic properties of the polymers formed have been investigated with the use of fluorescence, electron paramagnetic resonance (EPR) and electron microscopy techniques. Fluorescence titration of N-(1-pyrenyl)iodoacetamide-labeled actin with caldesmon showed saturation of the polymerization at a 1:3 molar ratio of caldesmon/actin monomer. Parallel pelleting experiments revealed, however, that the process of polymer formation is biphasic and only at higher concentrations of caldesmon does the copolymer contain around one caldesmon/three actin monomers. At low concentration of caldesmon a complex of one caldesmon/nine actin monomers is formed. EPR spectroscopy, using maleimide spin label bound at Cys374 of actin, also indicated that one caldesmon molecule polymerizes nine actin monomers. Taken together, these results might suggest the existence of weak and strong forms of actin binding to caldesmon and detection of only the latter by the fluorescence method. Copolymers of actin and caldesmon are indistinguishable from actin polymerized by salt with respect to their appearance in the electron microscope and their ability to interact with heavy meromyosin, although they are characterized by lower torsional flexibility as indicated by immobilization of spin labels attached to actin.     

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.     

Putting a new twist on actin: ADF/cofilins modulate actin dynamics.

The actin-depolymerizing factor (ADF)/cofilins are a family of essential actin regulatory proteins, ubiquitous among eukaryotes, that enhance the turnover of actin by regulating the rate constants of polymerization and depolymerization at filament ends, changing the twist of the filament and severing actin filaments. Genetic and cell-biological studies have shown that an ADF/cofilin is required to drive the high turnover of the actin cytoskeleton observed in vivo. The activity of ADF/cofilin is regulated by a variety of mechanisms, including specific phosphorylation and dephosphorylation. This review addresses aspects of ADF/cofilin structure, dynamics, regulation and function.     

Molecular details of formin-mediated actin assembly

Formins are a large family of multi-domain polypeptides that form homodimers. The highly conserved formin homology 2 (FH2) domain and its neighboring formin homology 1 (FH1) domain, which are surrounded by regulatory domains, cooperate in rapidly assembling profilin-actin into long filaments while remaining continuously associated with the fast-growing barbed end. Recent biochemical, biophysical, theoretical and structural studies have concluded that diverse formins are mechanistically similar, but that the rates of various assembly states differ quantitatively, and have shed light on the mechanism of formin auto-regulation and activation by Rho GTPases.     

Mechanisms of cold-induced platelet actin assembly

Various agonists but also chilling cause blood platelets to increase cytosolic calcium, polymerize actin, and change shape. We report that cold increases barbed end nucleation sites in octyl glucoside-permeabilized platelets by 3-fold, enabling analysis of the intermediates of this response. Although chilling does not change polyphosphoinositide (ppI) levels, a ppI-binding peptide completely inhibits cold-induced nucleation. The C terminus of N-WASp, which inhibits the Arp2/3 complex, blocks nucleation by 40%; GDPbetaS, N17Rac and N17Cdc42 have no effects. Some gelsolin translocates to the detergent-insoluble cytoskeleton after cooling. Chilled platelets from gelsolin-deficient mice have approximately 50% fewer new actin nuclei compared with platelets from wild-type mice. EGTA completely inhibits gelsolin translocation into the cytoskeleton, and the small amount of gelsolin initially there becomes soluble. Chilling releases adducin from the detergent-resistant cytoskeleton. We conclude that platelet actin filament assembly induced by cooling involves ppI-mediated actin filament barbed end uncapping and de novo nucleation independently of surface receptors or downstream signaling intermediates besides calcium. The actin-related changes occur in platelets at temperatures below 37 degrees C, suggesting that the platelet may be more activable at temperatures at the body surface than at core temperature, thereby favoring superficial hemostasis over internal thrombosis.     

Mechanism of K+-induced actin assembly

The assembly of highly purified actin from Dictyostelium discoideum amoebae and rabbit skeletal muscle by physiological concentrations of KCI proceeds through successive stages of (a) rapid formation of a distinct monomeric species referred to as KCI-monomer, (b) incorporation of KCI-monomers into an ATP-containing filament, and (c) ATP hydrolysis that occurs significantly after the incorporation event. KCI-monomer has a conformation which is distinct from that of either conventional G- or F-actin, as judged by UV spectroscopy at 210-220 nm and by changes in ATP affinity. ATP is not hydrolyzed during conversion of G-actin to KCI-monomer. KCI-monomer formation precedes filament formation and may be necessary for the assembly event. Although incorporation of KCI-monomers into filaments demonstrates lagphase kinetics by viscometry, both continuous absorbance monitoring at 232 nm and rapid sedimentation of filaments demonstrate hyperbolic assembly curves. ATP hydrolysis significantly lags the formation of actin filaments. When half of the actin has assembled, only 0.1 to 0.2 mole of ATP are hydrolyzed per mole of actin present as filaments.     

Mechanisms of formin-mediated actin assembly and dynamics

Cellular viability requires tight regulation of actin cytoskeletal dynamics. Distinct families of nucleation-promoting factors enable the rapid assembly of filament nuclei that elongate and are incorporated into diverse and specialized actin-based structures. In addition to promoting filament nucleation, the formin family of proteins directs the elongation of unbranched actin filaments. Processive association of formins with growing filament ends is achieved through continuous barbed end binding of the highly conserved, dimeric formin homology (FH) 2 domain. In cooperation with the FH1 domain and C-terminal tail region, FH2 dimers mediate actin subunit addition at speeds that can dramatically exceed the rate of spontaneous assembly. Here, I review recent biophysical, structural, and computational studies that have provided insight into the mechanisms of formin-mediated actin assembly and dynamics.     

Mechanically tuning actin filaments to modulate the action of actin-binding proteins

In cells, the actin cytoskeleton is regulated by an interplay between mechanics and biochemistry. A key mechanism, which has emerged based on converging indications from structural, cellular, and biophysical data, depicts the actin filament as a mechanically tunable substrate: mechanical stress applied to an actin filament induces conformational changes, which modify the binding and the regulatory action of actin-binding proteins. For a long time, however, direct evidence of this mechanotransductive mechanism was very scarce. This situation is changing rapidly, and recent in vitro single-filament studies using different techniques have revealed that several actin-binding proteins are able to sense tension, curvature, and/or torsion, applied to actin filaments. Here, we discuss these recent advances and their possible implications.     

Measurements of spatiotemporal changes in G-actin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension

To understand the intracellular role of G-actin concentration in stimulus-induced actin assembly and lamellipodium extension during cell migration, we developed a novel technique for quantifying spatiotemporal changes in G-actin concentration in live cells, consisting of sequential measurements of fluorescent decay after photoactivation (FDAP) of Dronpa-labeled actin. Cytoplasmic G-actin concentrations decreased by ～40% immediately after cell stimulation and thereafter the cell area extended. The extent of stimulus-induced G-actin loss and cell extension correlated linearly with G-actin concentration in unstimulated cells, even at concentrations much higher than the critical concentration of actin filaments, indicating that cytoplasmic G-actin concentration is a critical parameter for determining the extent of stimulus-induced G-actin assembly and cell extension. Multipoint FDAP analysis revealed that G-actin concentration in lamellipodia was comparable to that in the cell body. We also assessed the cellular concentrations of free G-actin, profilin- and thymosin-β4-bound G-actin, and free barbed and pointed ends of actin filaments by model fitting of jasplakinolide-induced temporal changes in G-actin concentration.     

Calcium control of Saccharomyces cerevisiae actin assembly.

Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo.     

Processive acceleration of actin barbed-end assembly by N-WASP

Neuronal Wiskott–Aldrich syndrome protein (N-WASP)–activated actin polymerization drives extension of invadopodia and podosomes into the basement layer. In addition to activating Arp2/3, N-WASP binds actin-filament barbed ends, and both N-WASP and barbed ends are tightly clustered in these invasive structures. We use nanofibers coated with N-WASP WWCA domains as model cell surfaces and single-actin-filament imaging to determine how clustered N-WASP affects Arp2/3-independent barbed-end assembly. Individual barbed ends captured by WWCA domains grow at or below their diffusion-limited assembly rate. At high filament densities, however, overlapping filaments form buckles between their nanofiber tethers and myosin attachment points. These buckles grew ∼3.4-fold faster than the diffusion-limited rate of unattached barbed ends. N-WASP constructs with and without the native polyproline (PP) region show similar rate enhancements in the absence of profilin, but profilin slows barbed-end acceleration from constructs containing the PP region. Increasing Mg²⁺ to enhance filament bundling increases the frequency of filament buckle formation, consistent with a requirement of accelerated assembly on barbed-end bundling. We propose that this novel N-WASP assembly activity provides an Arp2/3-independent force that drives nascent filament bundles into the basement layer during cell invasion.     

Extension of filopodia by motor-dependent actin assembly.

A variety of mechanisms have been proposed to explain the forward extension of cytoplasm in advancing cells and axonal growth cones, including actin polymerization and osmotic swelling. Based on our observations of the filopodia of cultured neuronal growth cones, we propose a mechanism involving motor-induced extension and retraction. We observed that filopodia (actin-based protrusions 0.2-0.5 mu in diameter) extend and retract from growth cone lamellae at the same rate. Further, force is generated at the tips of filopodia which is sufficient to produce compressive buckling of the proximal portion of the filopodium. From our analysis of these movements we suggest that a motor protein powers both the extension and retraction of filopodia.     

Detection of actin assembly by fluorescence energy transfer

Fluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide). Donor- labeled actin and acceptor-labeled actin were coassembled. The dependence of the transfer efficiency on the mole fraction of acceptor- labeled actin showed that the radial coordinate of the label at cysteine 373 is approximately 35 A, which means that this site is located near the outer surface of the filament. The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.     

Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts

Cadherin cell adhesion molecules are major determinants of tissue patterning which function in cooperation with the actin cytoskeleton. In the context of stable adhesion, cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis. Here actin polymerization has been proposed to drive cell surfaces together, although F-actin reorganization also occurs as cell contacts mature. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. We sought to analyze the relationship between cadherin homophilic binding and cytoskeletal activity during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere. We therefore activated homophilic ligation using a specific functional recombinant protein. We report the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator and demonstrate that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface.     

The effect of methylglyoxal on actin

Studying the gelation of Ehrlich ascites tumor cell extracts at various methyglyoxal concentrations, an increase of the gelled protein fraction, composed mainly of actin, was found at 10^?7 M to 10^?5 M. When methylglyoxal was added to intact tumor cells, the filamentous portion of cytoplasmic actin was increased at 10^?7 M to 10^?6 M concentrations. Furthermore, certain functional properties of purified skeletal muscle actin were also affected by μM concentrations of methylglyoxal; the speed of actin polymerization was facilitated and more filamentous actin formed in polymerizing conditions. The possible mode of methylglyoxal action is discussed.