Structural basis for the destabilization of F-actin by phosphate release following ATP hydrolysis.

The role of ATP hydrolysis in actin polymerization has been a puzzle, since it is known that polymer formation is possible without the ATPase activity and that the ATPase lags behind polymerization. We have used beryllium fluoride and G-ADP actin monomers to form F-ADP-BeF3- filaments that are a stable analog for either the ATP or the ADP-P(i) state. Electron microscopy and computed three-dimensional reconstruction have been used to compare this state to control actin, F-ADP, polymerized from G-ATP. We find, at a high degree of statistical significance, that subdomain-2 of the actin protomer in the ADP-BeF3- state is in a conformation very similar to that found in the atomic model for F-actin of Holmes and co-workers, but becomes disordered after the release of the phosphate. This breaks one of the longitudinal bonds in the filament, consistent with biochemical observations that phosphate release destabilizes F-actin. We have also found that lithium, which reduces the dissociation rate constant of actin filaments, induces a structural state indistinguishable from that of ADP-BeF3-. Further, in all states about ten C-terminal residues are displaced from the above mentioned model, but that the fit of the rest of the monomer is in excellent agreement, supporting the uniqueness of the solution they found and precluding a significantly different arrangement of the actin monomer in the filament.     

Polymerization of additional actin is not required for capping of surface antigens in B-lymphocytes

CH12 is a murine B-cell lymphoma whose surface immunoglobulin (sIg) and concanavalin A (Con A) receptors patch and cap readily. Actin may be involved in CH12 patching and capping, since fodrin and F-actin collect under the cap, and cytochalasin D inhibits sIg capping. We have examined the state of the actin cytoskeleton during patching and capping. A wide range of concentrations of rabbit anti-mouse antibody (RAM) and Con A were used to patch or cap CH12 cells. G-actin was quantitated by DNase I inhibition, F-actin was quantitated by fluorescence-activated cell sorter analysis of fluorescent phalloidin staining, and actin nucleation sites were measured by pyrene actin polymerization. None of these methods detected any significant changes in actin when compared to control cells or untreated cells, leading us to conclude that increased actin polymerization is not necessary for capping to occur. The significance of these data to the membrane flow and cytoskeletal models of capping is discussed.     

Effect of profilin on actin critical concentration: a theoretical analysis

To explain the effect of profilin on actin critical concentration in a manner consistent with thermodynamic constraints and available experimental data, we built a thermodynamically rigorous model of actin steady-state dynamics in the presence of profilin. We analyzed previously published mechanisms theoretically and experimentally and, based on our analysis, suggest a new explanation for the effect of profilin. It is based on a general principle of indirect energy coupling. The fluctuation-based process of exchange diffusion indirectly couples the energy of ATP hydrolysis to actin polymerization. Profilin modulates this coupling, producing two basic effects. The first is based on the acceleration of exchange diffusion by profilin, which indicates, paradoxically, that a faster rate of actin depolymerization promotes net polymerization. The second is an affinity-based mechanism similar to the one suggested in 1993 by Pantaloni and Carlier although based on indirect rather than direct energy coupling. In the model by Pantaloni and Carlier, transformation of chemical energy of ATP hydrolysis into polymerization energy is regulated by direct association of each step in the hydrolysis reaction with a corresponding step in polymerization. Thus, hydrolysis becomes a time-limiting step in actin polymerization. In contrast, indirect coupling allows ATP hydrolysis to lag behind actin polymerization, consistent with experimental results.     

An integrative simulation model linking major biochemical reactions of actin-polymerization to structural properties of actin filaments

We report on an advanced universal Monte Carlo simulation model of actin polymerization processes offering a broad application panel. The model integrates major actin-related reactions, such as assembly of actin nuclei, association/dissociation of monomers to filament ends, ATP-hydrolysis via ADP-Pi formation and ADP-ATP exchange, filament branching, fragmentation and annealing or the effects of regulatory proteins. Importantly, these reactions are linked to information on the nucleotide state of actin subunits in filaments (ATP hydrolysis) and the distribution of actin filament lengths. The developed stochastic simulation modelling schemes were validated on: i) synthetic theoretical data generated by a deterministic model and ii) sets of our and published experimental data obtained from fluorescence pyrene-actin experiments. Build on an open-architecture principle, the designed model can be extended for predictive evaluation of the activities of other actin-interacting proteins and can be applied for the analysis of experimental pyrene actin-based or fluorescence microscopy data. We provide a user-friendly, free software package ActinSimChem that integrates the implemented simulation algorithms and that is made available to the scientific community for modelling in silico any specific actin-polymerization system.     

Analysis of tetramethylrhodamine-labeled actin polymerization and interaction with actin regulatory proteins

The hydrolysis of ATP accompanying actin polymerization destabilizes the filament, controls actin assembly dynamics in motile processes, and allows the specific binding of regulatory proteins to ATP- or ADP-actin. However, the relationship between the structural changes linked to ATP hydrolysis and the functional properties of actin is not understood. Labeling of actin Cys374 by tetramethylrhodamine (TMR) has been reported to make actin non-polymerizable and enabled the crystal structures of ADP-actin and 5'-adenylyl beta,gamma-imidodiphosphate-actin to be solved. TMR-actin has also been used to solve the structure of actin in complex with the formin homology 2 domain of mammalian Dia1. To understand how the covalent modification of actin by TMR may affect the structural changes linked to ATP hydrolysis and to evaluate the functional relevance of crystal structures of TMR-actin in complex with actin-binding proteins, we have analyzed the assembly properties of TMR-actin and its interaction with regulatory proteins. We show that TMR-actin polymerized in very short filaments that were destabilized by ATP hydrolysis. The critical concentrations for assembly of TMR-actin in ATP and ADP were only an order of magnitude higher than those for unlabeled actin. The functional interactions of actin with capping proteins, formin, actin-depolymerizing factor/cofilin, and the VCA-Arp2/3 filament branching machinery were profoundly altered by TMR labeling. The data suggest that TMR labeling hinders the intramolecular movements of actin that allow its specific adaptative recognition by regulatory proteins and that determine its function in the ATP- or ADP-bound state.     

Role of nucleotide hydrolysis in the polymerization of actin and tubulin

Here is presented a short survey of the main aspects of the involvement of nucleotide hydrolysis in the polymerization of actin and microtubules: 1) XTP hydrolysis is not tightly coupled to the polymerization process; XTP hydrolysis and phosphate release generate an unstable XDP-polymer which is maintained at steady state, in the presence of XTP, by terminal XTP-subunits; this feature can generate patterns of phase transitions of the polymer between stable and unstable conformations; 2) Interactions between subunits are involved in the mechanism of XTP hydrolysis; 3) XTP cleavage on the polymer is followed by the slow release of P_i; the structural and thermodynamic characteristics of the transient XDP-P_i-polymer may play a crucial role in the regulation of the dynamics of microtubules and actin filaments.     

Kinetics of Regulated Actin Transitions Measured by Probes on Tropomyosin

Changes in the muscle regulatory protein complex, troponin, are important for modulation of activity and may occur as a result of disease-causing mutations. Both increases and decreases in the rate of ATP hydrolysis by myosin may occur as dictated by changes in the distribution of actin-tropomyosin-troponin among its different states. It is important to measure the rates of transition among these states to study physiological adaptation and disease processes. We show here that acrylodan or pyrene probes on tropomyosin can be used to monitor the transition from active to intermediate and inactive states of actin-tropomyosin-troponin. Transitions measured in the absence of calcium had two phases, as previously reported for some other probes on troponin and actin. The first step was a rapid equilibrium that favored the formation of the intermediate state and had an apparent rate constant less than that of S1-ATP dissociation. The second fluorescence transition was slower, with an apparent constant that increased from ∼5 to 80/s over a range of 1-37°C. Only the initial rapid transition was seen in the presence of saturating calcium. The acrylodan probe had the advantage of yielding a larger signal than the pyrene probe. Furthermore, the acrylodan signal decreased in going from the active state to the intermediate state, and then increased upon going to the inactive state.     

Polymerization of ADP-actin and ATP-actin under sonication and characteristics of the ATP-actin equilibrium polymer

Polymerization under sonication has been developed as a new method to study the rapid polymerization of actin with a large number of elongating sites. The theory proposed assumes that filaments under sonication are maintained at a constant length by the constant input of energy. The data obtained for the reversible polymerization of ADP-actin under sonication have been successfully analyzed according to the proposed model and, therefore, validate the model. The results obtained for the polymerization of ATP-actin under sonication demonstrate the involvement of ATP hydrolysis in the polymerization process. At high actin concentration, polymerization was fast enough, as compared to ATP hydrolysis on the F-actin, to obtain completion of the reversible polymerization of ATP-actin before significant hydrolysis of ATP occurred. A critical concentration of 3 microM was determined as the ratio of the dissociation and association rate constants for the interaction of ATP-actin with the ATP filament ends in 1 mM MgCl2, 0.2 mM ATP. The plot of the rate of elongation of filaments versus actin monomer concentration exhibited an upward deviation at high actin concentration that is consistent with this result. The fact that F-actin at steady state is more stable than the ATP-F-actin polymer at equilibrium suggests that the interaction between ADP-actin and ATP-actin subunits at the end of the ATP-capped filament is much stronger than the interaction between two ATP-actin subunits.     

Quantitative Analysis of Approaches to Measure Cooperative Phosphate Release in Polymerized Actin

We use stochastic simulations that treat several experimental probes of actin dynamics to explore the extent to which phosphate dissociation in filamentous actin may be cooperative. Phosphate time-courses from polymerization and copolymerization experiments of ATP- and ADP-actin are studied, including the effects of variations in filament-number concentration as well as single-filament depolymerization time-courses. We find that highly cooperative models are consistent with the treated experimental data. We also find that some types of experiments that are believed to provide strong constraints on the cooperativity of actin hydrolysis models do not provide such constraints.     

Evidence for a novel,strongly bound acto-S1 complex carrying ADP and phosphate stabilized in the G680V mutant of Dictyostelium myosin II

Gly 680 of Dictyostelium myosin II sits at a critical position within the reactive thiol helices. We have previously shown that G680V mutant subfragment 1 largely remains in strongly actin-bound states in the presence of ATP. We speculated that acto-G680V subfragment 1 complexes accumulate in the A.M.ADP.P(i) state on the basis of the biochemical phenotypes conferred by mutations which suppress the G680V mutation in vivo [Wu, Y., et al. (1999) Genetics 153, 107-116]. Here, we report further characterization of the interaction between actin and G680V subfragment 1. Light scattering data demonstrate that the majority of G680V subfragment 1 is bound to actin in the presence of ATP. These acto-G680V subfragment 1 complexes in the presence of ATP do not efficiently quench the fluorescence of pyrene-actin, unlike those in rigor complexes or in the presence of ADP alone. Kinetic analyses demonstrated that phosphate release, but not ATP hydrolysis or ADP release, is very slow and rate limiting in the acto-G680V subfragment 1 ATPase cycle. Single turnover kinetic analysis demonstrates that, during ATP hydrolysis by the acto-G680V subfragment 1 complex, quenching of pyrene fluorescence significantly lags the increase of light scattering. This is unlike the situation with wild-type subfragment 1, where the two signals have similar rate constants. These data support the hypothesis that the main intermediate during ATP hydrolysis by acto-G680V subfragment 1 is an acto-subfragment 1 complex carrying ADP and P(i), which scatters light but does not quench the pyrene fluorescence and so has a different conformation from the rigor complex.     

A Nucleotide State-sensing Region on Actin

The nucleotide state of actin (ATP, ADP-P_i, or ADP) is known to impact its interactions with other actin molecules upon polymerization as well as with multiple actin binding proteins both in the monomeric and filamentous states of actin. Recently, molecular dynamics simulations predicted that a sequence located at the interface of subdomains 1 and 3 (W-loop; residues 165–172) changes from an unstructured loop to a β-turn conformation upon ATP hydrolysis (Zheng, X., Diraviyam, K., and Sept, D. (2007) Biophys. J. 93, 1277–1283). This region participates directly in the binding to other subunits in F-actin as well as to cofilin, profilin, and WH2 domain proteins and, therefore, could contribute to the nucleotide sensitivity of these interactions. The present study demonstrates a reciprocal communication between the W-loop region and the nucleotide binding cleft on actin. Point mutagenesis of residues 167, 169, and 170 and their site-specific labeling significantly affect the nucleotide release from the cleft region, whereas the ATP/ADP switch alters the fluorescence of probes located in the W-loop. In the ADP-P_i state, the W-loop adopts a conformation similar to that in the ATP state but different from the ADP state. Binding of latrunculin A to the nucleotide cleft favors the ATP-like conformation of the W-loop, whereas ADP-ribosylation of Arg-177 forces the W-loop into a conformation distinct from those in the ADP and ATP-states. Overall, our experimental data suggest that the W-loop of actin is a nucleotide sensor, which may contribute to the nucleotide state-dependent changes in F-actin and nucleotide state-modulated interactions of both G- and F-actin with actin-binding proteins.     

How actin binds and assembles onto plasma membranes from Dictyostelium discoideum

We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross-linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell.     

Structure and Dynamics of the Actin Filament

We used all-atom molecular dynamics simulations to investigate the structure and properties of the actin filament, starting with either the recent Oda model or the older Holmes model. Simulations of monomeric and polymerized actin show that polymerization changes the nucleotide-binding cleft, bringing together the Q137 side chain and bound ATP in a way that may enhance the ATP hydrolysis rate in the filament. Simulations with different bound nucleotides and conformations of the DNase I binding loop show that the persistence length of the filament depends only on loop conformation. Computational modeling reveals how bound phalloidin stiffens actin filaments and inhibits the release of γ-phosphate from ADP-P_i actin.     

Profilin promotes barbed-end actin filament assembly without lowering the critical concentration

The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin.4.1.Actin seeds were measured by right angle light scattering to avoid confounding effects of pyrenyl-actin, and KINSIM was used to analyze elongation progress curves. Without thymosin-beta4, both actin and Profilin.Actin (P.A) are competent in barbed-end polymerization, and kinetic simulations yielded the same bimolecular rate constant ( approximately 10 x 10(6) M(-1) s(-1)) for actin monomer or Profilin.Actin. When measured in the absence of profilin, actin assembly curves over a 0.7-4 microM thymosin-beta4 concentration range fit a simple monomer sequestering model (1 microM K(D) for Thymosin-beta4.Actin). The corresponding constant for thymosin-beta4.pyrenyl-Actin, however, was significantly higher ( approximately 9-10 microM), suggesting that the fluorophore markedly weakens binding to thymosin-beta4. With solutions of actin (2 microM) and thymosin-beta4 (2 or 4 microM), the barbed-end assembly rate rose with increasing profilin concentration (0.7-2 microM). Actin assembly in presence of thymosin-beta4 and profilin fit a simple thermodynamic energy cycle, thereby disproving an earlier claim (D. Pantaloni and M.-F. Carlier (1993) Cell 75, 1007-1014) that profilin promotes nonequilibrium filament assembly by accelerating hydrolysis of filament-bound ATP. Our findings indicate that profilin serves as a polymerization catalyst that captures actin monomers from Thymosin-beta4.Actin and ushers actin as a Profilin.Actin complex onto growing barbed filament ends.     

New Aspects of the Spontaneous Polymerization of Actin in the Presence of Salts

The mechanism of salt-induced actin polymerization involves the energetically unfavorable nucleation step, followed by filament elongation by the addition of monomers. The use of a bifunctional cross-linker, N,N′-(1,4-phenylene)dimaleimide, revealed rapid formation of the so-called lower dimers (LD) in which actin monomers are arranged in an antiparallel fashion. The filament elongation phase is characterized by a gradual LD decay and an increase in the yield of “upper dimers” (UD) characteristic of F-actin. Here we have used 90° light scattering, electron microscopy, and N,N′-(1,4-phenylene)dimaleimide cross-linking to reinvestigate relationships between changes in filament morphology, LD decay, and increase in the yield of UD during filament growth in a wide range of conditions influencing the rate of the nucleation reaction. The results show irregularity and instability of filaments at early stages of polymerization under all conditions used, and suggest that an earlier documented coassembling of LD with monomeric actin contributes to the initial disordering of the filaments rather than to the nucleation of polymerization. The effects of the type of G-actin-bound divalent cation (Ca²⁺/Mg²⁺), nucleotide (ATP/ADP), and polymerizing salt on the relation between changes in filament morphology and progress in G-actin-to-F-actin transformation show that ligand-dependent alterations in G-actin conformation determine not only the nucleation rate but also the kinetics of ordering of the filament structure in the elongation phase. The time courses of changes in the yield of UD suggest that filament maturation involves cooperative propagation of “proper” interprotomer contacts. Acceleration of this process by the initially bound MgATP supports the view that the filament-destabilizing conformational changes triggered by ATP hydrolysis and P_i liberation during polymerization are constrained by the intermolecular contacts established between MgATP monomers prior to ATP hydrolysis. An important role of contacts involving the DNase-I-binding loop and the C-terminus of actin is proposed.     

Actin dynamics rapidly reset chemoattractant receptor sensitivity following adaptation in neutrophils

Neutrophils are cells of the innate immune system that hunt and kill pathogens using directed migration. This process, known as chemotaxis, requires the regulation of actin polymerization downstream of chemoattractant receptors. Reciprocal interactions between actin and intracellular signals are thought to underlie many of the sophisticated signal processing capabilities of the chemotactic cascade including adaptation, amplification and long-range inhibition. However, with existing tools, it has been difficult to discern actin''s role in these processes. Most studies investigating the role of the actin cytoskeleton have primarily relied on actin-depolymerizing agents, which not only block new actin polymerization but also destroy the existing cytoskeleton. We recently developed a combination of pharmacological inhibitors that stabilizes the existing actin cytoskeleton by inhibiting actin polymerization, depolymerization and myosin-based rearrangements; we refer to these processes collectively as actin dynamics. Here, we investigated how actin dynamics influence multiple signalling responses (PI3K lipid products, calcium and Pak phosphorylation) following acute agonist addition or during desensitization. We find that stabilized actin polymer extends the period of receptor desensitization following agonist binding and that actin dynamics rapidly reset receptors from this desensitized state. Spatial differences in actin dynamics may underlie front/back differences in agonist sensitivity in neutrophils.     

Direct measurement of the lamellipodial protrusive force in a migrating cell

There has been a great deal of interest in the mechanism of lamellipodial protrusion (Pollard, T., and G. Borisy. 2003. Cell. 112:453-465). However, one of this mechanism's endpoints, the force of protrusion, has never been directly measured. We place an atomic force microscopy cantilever in the path of a migrating keratocyte. The deflection of the cantilever, which occurs over a period of approximately 10 s, provides a direct measure of the force exerted by the lamellipodial leading edge. Stall forces are consistent with approximately 100 polymerizing actin filaments per micrometer of the leading edge, each working as an elastic Brownian ratchet and generating a force of several piconewtons. However, the force-velocity curves obtained from this measurement, in which velocity drops sharply under very small loads, is not sensitive to low loading forces, and finally stalls rapidly at large loads, are not consistent with current theoretical models for the actin polymerization force. Rather, the curves indicate that the protrusive force generation is a complex multiphase process involving actin and adhesion dynamics.     

ATP-dependent membrane assembly of F-actin facilitates membrane fusion

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.     

Probing actin and liposome interaction of talin and talin-vinculin complexes: a kinetic, thermodynamic and lipid labeling study.

Talin purified from human platelets and chicken gizzard smooth muscle is an actin and lipid binding protein. Here, we have investigated the effect of vinculin on (a) talin-nucleated actin polymerization and (b) insertion of talin into lipid bilayers. Calorimetric data show ternary complex formation between talin, vinculin, and actin. Actin-talin, actin-vinculin and actin-(talin-vinculin) binding and rate constants as well as actin polymerization rates for all three protein species have been determined by steady state titration, stopped-flow, and fluorescence assay. In contrast to an increase of the polymerization rate by a factor of less than 2 for actin-talin and actin-(talin-vinculin) when lowering the temperature, we measured a decrease in rates for actin alone and actin-vinculin. The overall equilibrium constants (Keq) in the van't Hoff plot proved linear and were of one-step reactions. Thermodynamic data exhibited signs of van der Waal's binding forces. Using the photoactivatable lipid analogue [3H]PTPC/11, which selectively labels membrane-embedded hydrophobic domains of proteins, we also show that talin partially inserts into the hydrophobic bilayer of liposomes. This insertion occurs in a similar manner irrespective of preincubation with vinculin.     

Real-time monitoring of actin polymerization in living cells using split luciferase

Real-time monitoring of actin polymerization in living cells is beneficial for characterizing cellular activities such as migration, proliferation, and death. We developed new bioluminescence-based probe proteins that enable the monitoring of actin polymerization in living cells. Unlike other ordinary split luciferase probes, our probes were incorporated in endogenous actin filament that enabled it to measure the actin polymerization quantitatively. The probe proteins exhibited a dose-responsive decrease in photon emission intensity in response to the filamentous (F)-actin-disrupting agent latrunculin A. This technique has a high sensitivity with a high signal-to-noise ratio and is nontoxic compared with other methods of monitoring actin polymerization in living cells. Using this technique, we succeeded in monitoring the F-actin level in living cells during apoptosis progression induced by UV irradiation continuously for 12 h. F-actin was transiently upregulated after UV irradiation. Since UV-induced cell death was enhanced by treatment with latrunculin A during the period which F-actin is increased, transient upregulation of F-actin after UV is likely a protective reaction against UV-induced cell death. Our novel technique is an effective tool for investigating actin polymerization in living cells.