Interaction of phalloidin with chemically modified actin

Modification of Tyr-69 with tetranitromethane impairs the polymerizability of actin in accordance with the previous report [Lehrer, S. S. and Elzinga, M. (1972) Fed. Proc. 31, 502]. Phalloidin induces this chemically modified actin to form the same characteristic helical thread-like structure as normal F-actin. The filaments bind myosin heads and activate the myosin ATPase activity as effectively as normal F-actin. When a dansyl group is introduced at the same point [Chantler, P. D. and Gratzer, W. B. (1975) Eur. J. Biochem. 60, 67-72], phalloidin still induces the polymerization. The filaments bind myosin heads and activate the myosin ATPase activity. These results indicate that Tyr-69 is not directly involved in either an actin-actin binding site or the myosin binding site on actin. Moreover, the results suggest that phalloidin binds to actin monomer in the presence of salt and its binding induces a conformational change in actin which is essential for polymerization, or that actin monomer fluctuates between in unpolymerizable and polymerizable form while phalloidin binds to actin only in the polymerizable form and its binding locks the conformation which causes the irreversible polymerization of actin. Modification of Tyr-53 with 5-diazonium-(1H)tetrazole blocks actin polymerization [Bender, N., Fasold, H., Kenmoku, A., Middelhoff, G. and Volk, K. E. (1976) Eur. J. Biochem. 64, 215-218]. Phalloidin is unable to induce the polymerization of this modified actin nor does it bind to it. Phalloidin does not induce the polymerization of the trypsin-digested actin core. These results indicate that the site at which phalloidin binds is involved in polymerization and the probable conformational change involved in polymerization may be modulated through this site.     

Influence of phalloidin on ATP hydrolysis during actin polymerization

The influence of phalloidin on the ATP hydrolysis associated with actin polymerization was investigated. Whereas in the absence of phalloidin actin-bound ATP was totally hydrolyzed during polymerization, ATP hydrolysis was not complete after actin polymerization in the presence of phalloidin: 5-10% of ATP remained unhydrolyzed and disappeared only after 2 days.     

The phalloidin binding site of F-actin.

Tritium-containing affinity-labelling derivatives of phalloidin, an alkylating iodoacetyl compound (EAL) and a photolabile, carbene generating diazirine (PAL), have been reacted with rabbit muscle actin, the former after protection of thiol groups with N-ethylmaleimide. Labelled peptides generated by tryptic and/or thermolysin digestion were isolated by paper peptide mapping and characterized by determination of their amino acid sequences. EAL binds to methionine-119 and methionine-355; PAL binds to glutamic acid-117. These residues are located in regions with extremely conserved amino acid sequences. The cleft between the two domains of the actin monomer is suggested as the possible binding site for phalloidin.     

Mechanism of action of phalloidin on the polymerization of muscle actin

Under conditions where muscle actin only partially polymerizes, or where it does not polymerize at all, a significant enhancement of polymerization was observed if equimolar phalloidin was also present. The increased extent of polymerization in the the presence of phalloidin can be explained by the reduced critical actin concentration of partially polymerized populations at equilibrium. Under such conditions, the rate of polymerization, as judged by the length of time to reach half the viscosity plateau, was found to be essentially independent of the phalloidin concentration. Moreover, the initial rate of polymerization of actin was also found to be independent of phalloidin concentration. However, phalloidin apparently causes a reduction in the magnitude of the reverse rates in the polymerization reaction, as was demonstrated by the lack of depolymerization of phalloidin-treated actin polymers. This effect of phalloidin is also supported by the identification of actin nuclei and short polymers in populations of G-actin incubated with phalloidin in the absence of added KCl. Our conclusion, then, is that phalloidin influences the polymerization of actin by stabilizing nuclei and polymers as they are formed.     

Rhodamine phalloidin F-actin: critical concentration versus tensile strength.

The mechanic and elastic properties of rhodamine phalloidin F-actin were investigated as a function of the ionic strength and in the absence of Mg2+. By increasing ionic strength from 3 to 19 mM, critical concentration decreased from 146 to 36 nM and the yield strength increased from 5.6 pN to 28.6 pN. At the ionic strength of 12-13 mM, the elastic modulus by stretching increased by 330-430 kP. nm-1 up to the break point, where it was 38-44.2 MP. The work required to break the filament, 403-439 kJ.M-1 provides an estimate of the free energy of annealing of rhodamine phalloidin F-actin, the annealing constant being 2.8 x 1074 M-1.     

Kinetics of actin depolymerization: influence of ions, temperature, age of F-actin, cytochalasin B and phalloidin

Actin, labelled with the fluorescent dye N-(3-pyrenyl)maleimide, was diluted below its critical concentration and depolymerization was followed by measuring the declining fluorescence intensity. The time courses of depolymerization were fitted to a sum of three exponentials. In most cases there was a fast initial phase followed by one or three slower ones. Increasing MgCl2 concentration slowed down depolymerization velocity, as did substitution of Tris-maleate buffer by phosphate buffer. Older F-actin preparations depolymerized more slowly than younger ones. Phalloidin strongly decreased depolymerization velocity even after sonication. In the presence of cytochalasin B depolymerization was more uniformly exponential than in the absence of cytochalasin B; overall depolymerization velocity was decreased by cytochalasin B. The results are discussed on the assumption that depolymerization kinetics reflect the length distribution of actin filaments during depolymerization.     

Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadate

Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68+/-22 microM and 17+/-2 microM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2mM concentration of "metavanadate" solution that contains ortho and metavanadate species, as observed by combining kinetic with (51)V NMR spectroscopy studies. Although at 25 degrees C, decameric vanadate (10 microM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 microM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the "decavanadate" interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems.     

Influence of phalloidin on both the nucleation and the elongation phase of actin polymerization

Phalloidin, a heptapeptide from the mushroom Amanita phalloides, increased the velocity of actin polymerization, but slightly decreased the velocity of elongation (polymerization onto sonicated F-actin). A plot of log polymerization velocity vs. log actin concentration was less steep in the presence of phalloidin than in its absence, suggesting that the filament nucleus is smaller in the presence of phalloidin than in its absence.     

Interaction of myosin subfragments with F-actin.

The effect of ionic strength, temperature, and divalent cations on the association of myosin with actin was determined in the ultracentrifuge using scanning absorption optics. The association constant (Ka) for the binding of heavy meromyosin (HmM) to F-actin was 1 X 10(7) M-1 at 20 degrees C, in 0.10 M KCl, 0.01 M imidazole (pH 7.0), 5 MM potassium phosphate, 1 mM MgCl2, and 0.3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Ka was the same for HMM prepared by trypsin or chymotrypsin. The affinity of subfragment 1 (S1) for actin under the same ionic conditions was 3 X 10(6) M-1. Varying the preparative procedure for S1 had little effect on Ka. The small difference in binding energy between HMM and S1 suggests that either only one head can bind strongly to actin at a time or that free energy is lost during the sterically unfavorable attachment of the two heads to actin.     

Adhesive F-actin waves: a novel integrin-mediated adhesion complex coupled to ventral actin polymerization

At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in "ventral F-actin waves" that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These "adhesive F-actin waves" require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization.     

Stabilization of actin by phalloidin: a differential scanning calorimetric study.

We have used differential scanning calorimetry to study the effects of phalloidin on F and G actin stability. For F actin, saturating concentrations of phalloidin induced an important shift on the transition temperature, Tm, from 69.5 degrees C to 83.5 degrees C. However, the calorimetric enthalpy remained unchanged. Using lower phalloidin concentrations, monomers linked to phalloidin, as well as neighboring unlinked monomers, were both stabilized. Contrary to previous reports, phalloidin was also shown to affect G actin, shifting its Tm from 59.5 degrees C to 75 degrees C. Two mechanisms are proposed to explain this finding: first, it could indicate a real interaction of phalloidin with G actin, and second, heating of the specimen during the scan could have induced polymerization of some G actin to the F form. The resulting F polymer would then interact with phalloidin, thus shifting the equilibrium between G and F actin towards the polymeric form.     

Interaction of ADP-ribosylated actin with actin binding proteins.

Actin ADP-ribosylated at Arg177 was previously shown not to polymerise after increasing the ionic strength, but to cap the barbed ends of filaments. Here we confirm that the polymerisation of ADP-ribosylated actin is inhibited, however, under specific conditions the modified actin copolymerises with native actin, indicating that its ability to take part in normal subunit interactions within filaments is not fully eliminated. We also show that ADP-ribosylated actin forms antiparallel but not parallel dimers: the former are not able to form filaments. ADP-ribosylated actin interacts with deoxyribonuclease I, vitamin D binding protein, thymosin beta(4), cofilin and gelsolin segment 1 like native actin. Interaction with myosin subfragment 1 revealed that the potential of the modified actin to aggregate into oligomers or short filaments is not fully eliminated.     

Polyamine-induced actin polymerization.

Muscle actin has been found to polymerize reversibly upon addition of low concentrations of polyamines. This polymerization, studied by centrifugation, has shown a linear relationship between the actin polymerization yield and the chain length of the polyamine. Among the biological polyamines tested, spermidine and spermine are the most efficient. The polymerization of actin can also be induced by the corresponding mono or diguanidine derivatives of these polyamines but monoamines or amino acids are inactive at the same concentration. The transformation of actin from a globular to a fibrous from upon addition of spermidine is also demonstrated by the changes in the near-ultraviolet circular dichoroic spectrum of this protein. Moreover, the polyamine-induced F -actin exhibits the same properties as the salt-induced F -actin: it strongly activates the Mg2+ -ATPase of myosin, its specific viscosity is enhanced to the same extent and electron micrographs show homogeneous thin filaments.     

F-actin binding region of SPIN90 C-terminus is essential for actin polymerization and lamellipodia formation

We recently reported that SPIN90 is able to bind with several proteins involved in regulating actin cytoskeleton networks, including dynamin, WASP, β PIX, and Nck. Based on these findings, we investigated how SPIN90 regulates the actin cytoskeleton and promotes actin assembly. This study demonstrated that aluminium fluoride-induced localization of SPIN90 to lamellipodia requires amino acids 582-722 at the SPIN90 C-terminus, which is also essential for F-actin binding and Arp2/3 complex mediated polymerization of actin into branched actin filaments. Furthermore, after deletion of the F-actin binding region (582-722 SPIN90) failed to localize at the membrane edge and was unable to promote lamellipodia formation, suggesting that the F-actin binding region in the SPIN90 C-terminus is essential for the formation of branched actin networks and regulation of the actin cytoskeleton at the leading edge of cells.     

Interaction of troponin components with F-actin and F-actin-tropomyosin complex.

1. Both TN-T and TN-I components of troponin interact with F-actin, causing its precipitation at 0.1 M KC1 and neutral pH in a form of highly ordered paracrystals, although the ability of TN-I component to precipitate of F-actin is much weaker. 2. F-actin paracrystals obtained in the presence of both TN-T and TN-I components consist of parallel arrays of F-actin filaments, although the fine structure is in each case different. 3.In the presence of tropomyosin in the proportion equal to that in muscle, less TN-T or TN-I component is needed to obtain full precipitation of F-actin. 4. Paracrystals of F-actin-tropomyosin-TN-T component and F-actin-tropomyosin-TN-I component show regular transverse striation spaced at about 380 A intervals. 5. The TN-C component of troponin solubilizes all precipitates of F-actin with TN-T or TN-I components, regardless of the presence of tropomyosin. 6. The results show that both TN-T or TN-I components can bind independently to F-actin-tropomyosin complex with the same periodicity, similar to that of the whole troponin in the living muscle.     

Differential response of fast and slow myosin ATPase from skeletal muscle to F-actin and to phalloidin F-actin

Fast muscle myosin responds in similar way to F-actin and to phalloidin F-actin. It is activated 7.5 fold at infinite F-actin concentration and 6.8 fold at infinite phalloidin F-actin. The actomyosin dissociation constants are 0.89±0.34 μM with F-actin and 0.90±0.71 μM with phalloidin F-actin. Slow muscle myosin responds differently to F-actin and to phalloidin F-actin. It is activated 3.76 fold at infinite F-actin concentration and only 2.27 fold at infinite phalloidin F-actin concentration. The actomyosin dissociation constants are 1.95±1.27 μM with F-actin and 0.27±0.16 μM with phalloidin F-actin. At first glance this means that substitution of F-actin with phalloidin F-actin magnifies the difference between fast muscle and slow muscle myosins. Furthermore the change of the dissociation constants may affect the contractile force of the attached crossbridge.