Modeling rigor cross-bridge patterns in muscle I. Initial studies of the rigor lattice of insect flight muscle.

We have undertaken some computer modeling studies of the cross-bridge observed by Reedy in insect flight muscle so that we investigate the geometric parameters that influence the attachment patterns of cross-bridges to actin filaments. We find that the appearance of double chevrons along an actin filament indicates that the cross-bridges are able to reach 10--14 nm axially, and about 90 degrees around the actin filament. Between three and five actin monomers are therefore available along each turn of one strand of actin helix for labeling by cross-bridges from an adjacent myosin filament. Reedy's flared X of four bridges, which appears rotated 60 degrees at successive levels on the thick filament, depends on the orientation of the actin filaments in the whole lattice as well as on the range of movement in each cross-bridge. Fairly accurate chevrons and flared X groupings can be modeled with a six-stranded myosin surface lattice. The 116-nm long repeat appears in our models as "beating" of the 14.5-nm myosin repeat and the 38.5-nm actin period. Fourier transforms of the labeled actin filaments indicate that the cross-bridges attach to each actin filament on average of 14.5 nm apart. The transform is sensitive to changes in the ease with which the cross-bridge can be distorted in different directions.     

Confocal Microscopy Observations on Actin Cytoskeleton in the Pollen and Pollen Protoplast of Narcissus

Actin cytoskeleton was localized in the pollen and pollen protoplast of Narcissus cyclamineus using fluorescence labelled phalloidin andconfocal microscopy. In the hydrated pollen (before germination) actin filamem bundles were arranged in a parallel array and at right angles to the long axis of the pollen grain in the cortex. But at the germination pore region(or fur row) the actin filament bundles formed a reticulate network. In the centre of the grain there was also an actin filament network which was more open and had less bundles associated with it than the network underneath the furrow. When the pollen grain started to produce pollen tube, most(if not all) of the actin filament bundles in the pollen grain rearranged into a parallel array pointing towards the tube. The bundles in the array later elongated and extended into the pollen tube. In the pollen protoplast a very tightly-packed actin bundle network was present. Numerous branches and jonts of actin filament bundles could be seen in the network. If the protoplasts were fixed before staining, the bundles aggregated and the branches and joints became less obvious indicating that fixation had affected the nature and arrangement of the actin filament bundles. If the pollen protoplasts were bursted (using the osmotic shock technique) or extracted (using Triton X-100), fragments of actin filament bundles could still be found associated with the membrane ghost indicating that some of the actin filament bundles in the cortex were tightly attached to the membrane. Using a double staining technique, actin filaments and microtubules were co-localized in the pollen protoplast. The co-alignment of some of the actin filament bundles with the microtubule bundles suggested that the actin cytoskeleton and the microtubule cytoskeleton were not distributed at random but in a well organized and orchestrated manner [possibly under the control of a yet undiscovered structure(s). The actin filament cytoskeleton in the generative cells failed to stain either in pollen or pollen tube, but they became stained in the pollen protoplast. The actin cytoskeleton in the generative cell appeared as a loosely organized network made up of short and long actin filament bundles.     

Myosin V is a left-handed spiral motor on the right-handed actin helix

Myosin V is a two-headed, actin-based molecular motor implicated in organelle transport. Previously, a single myosin V molecule has been shown to move processively along an actin filament in discrete approximately 36 nm steps. However, 36 nm is the helical repeat length of actin, and the geometry of the previous experiments may have forced the heads to bind to, or halt at, sites on one side of actin that are separated by 36 nm. To observe unconstrained motion, we suspended an actin filament in solution and attached a single myosin V molecule carrying a bead duplex. The duplex moved as a left-handed spiral around the filament, disregarding the right-handed actin helix. Our results indicate a stepwise walking mechanism in which myosin V positions and orients the unbound head such that the head will land at the 11th or 13th actin subunit on the opposing strand of the actin double helix.     

Origin of Twist-Bend Coupling in Actin Filaments

Actin filaments are semiflexible polymers that display large-scale conformational twisting and bending motions. Modulation of filament bending and twisting dynamics has been linked to regulatory actin-binding protein function, filament assembly and fragmentation, and overall cell motility. The relationship between actin filament bending and twisting dynamics has not been evaluated. The numerical and analytical experiments presented here reveal that actin filaments have a strong intrinsic twist-bend coupling that obligates the reciprocal interconversion of bending energy and twisting stress. We developed a mesoscopic model of actin filaments that captures key documented features, including the subunit dimensions, interaction energies, helicity, and geometrical constraints coming from the double-stranded structure. The filament bending and torsional rigidities predicted by the model are comparable to experimental values, demonstrating the capacity of the model to assess the mechanical properties of actin filaments, including the coupling between twisting and bending motions. The predicted actin filament twist-bend coupling is strong, with a persistence length of 0.15-0.4 μm depending on the actin-bound nucleotide. Twist-bend coupling is an emergent property that introduces local asymmetry to actin filaments and contributes to their overall elasticity. Up to 60% of the filament subunit elastic free energy originates from twist-bend coupling, with the largest contributions resulting under relatively small deformations. A comparison of filaments with different architectures indicates that twist-bend coupling in actin filaments originates from their double protofilament and helical structure.     

Role of intermonomer ionic bridges in the stabilization of the actin filament

Filament formation is required for most of the functions of actin. However, the intermonomer interactions that stabilize F-actin have not been elucidated because of a lack of an F-actin crystal structure. The Holmes muscle actin model suggests that an ionic interaction between Arg-39 of one monomer and Glu-167 of an adjacent monomer in the same strand contributes to this stabilization. Yeast actin has an Ala-167 instead. F-actin molecular dynamics modeling predicts another interaction between Arg-39 of one monomer and Asp-275 of an opposing strand monomer. In Toxoplasma gondii actin, which forms short stubby filaments, the Asp-275 equivalent is replaced by Arg leading to a potential filament-destabilizing charge-charge repulsion. Using yeast actin, we tested the effect of A167E as a potential stabilizer and A167R and D275R as potential filament disruptors. All mutations caused abnormal growth and mitochondrial malfunction. A167E and D275R actins polymerize normally and form relatively normal appearing filaments. A167R nucleates filaments more slowly and forms filament bundles. The R39D/A167R double mutant, which re-establishes an ionic bond in the opposite orientation, reverses this polymerization and bundling defect. Stoichiometric amounts of yeast cofilin have little effect on wild-type and A167E filaments. However, D275R and A167R actin depolymerization is profound with cofilin. Although our results suggest that disruption of an interaction between Arg-39 and Asp-275 is not sufficient to cause fragmentation, it suggests that it changes filament stability thereby disposing it for enhanced cofilin depolymerizing effects. Ala-167 results demonstrate the in vivo and in vitro importance of another potential Arg-39 ionic interaction.     

Human spire interacts with the barbed end of the actin filament

Spire is an actin nucleator that initiates actin polymerization at a specific place in the cell. Similar to the Arp2/3 complex, spire was initially considered to bind to the pointed end of the actin filament when it generates a new actin filament. Subsequently, spire was reported to be associated with the barbed end (B-end); thus, there is still no consensus regarding the end with which spire interacts. Here, we report direct evidence that spire binds to the B-end of the actin filament, under conditions where spire accelerates actin polymerization. Using electron microscopy, we visualized the location of spire bound to the filament by gold nanoparticle labeling of the histidine-tagged spire, and the polarity of the actin filament was determined by image analysis. In addition, our results suggest that multiple spires, linked through one gold nanoparticle, enhance the acceleration of actin polymerization. The B-end binding of spire provides the basis for understanding its functional mechanism in the cell.     

Structural basis of actin filament capping at the barbed-end: a cryo-electron microscopy study

The intracellular distribution and migration of many protein complexes and organelles is regulated by the dynamics of the actin filament. Many actin filament end-binding proteins play crucial roles in actin dynamics, since polymerization and depolymerization of actin protomers occur only at the filament ends. We present here an EM structure of the complex of the actin filament and hetero-dimeric capping protein (CP) bound to the barbed-end at 23 A resolution, by applying a newly developed methods of image analysis to cryo-electron micrographs. This structure was fitted by the crystal structure of CP and the proposed actin filament structure, allowing us to construct a model that depicts two major binding regions between CP and the barbed-end. This binding scheme accounted for the results of newly performed and previously published mutation experiments, and led us to propose a two-step binding model. This is the first determination of an actin filament end structure.     

The state of the filament

Movement is a defining characteristic of life. Macroscopic motion is driven by the dynamic interactions of myosin with actin filaments in muscle. Directed polymerization of actin behind the advancing membrane of a eukaryotic cell generates microscopic movement. Despite the fundamental importance of actin in these processes, the structure of the actin filament remains unknown. The Holmes model of the actin filament was published 15 years ago, and although it has been widely accepted, no high-resolution structural data have yet confirmed its veracity. Here, we review the implications of recently determined structures of F-actin-binding proteins for the structure of the actin filament and suggest a series of in silico tests for actin-filament models. We also review the significance of these structures for the arp2/3-mediated branched filament.     

Thiol oxidation of actin produces dimers that enhance the elasticity of the F-actin network

Slow oxidation of sulfhydryls, forming covalently linked actin dimers and higher oligomers, accounts for increases in the shear elasticity of purified actin observed after aging. Disulfide-bonded actin dimers are incorporated into F-actin during polymerization and generate cross-links between actin filaments. The large gel strength of oxidized actin (>100 Pa for 1 mg/ml) in the absence of cross-linking proteins falls to within the theoretically predicted order of magnitude for uncross-linked actin filament networks (1 Pa) with the addition of sufficient concentrations of reducing agents such as 5 mM dithiothreitol or 10 mM beta-mercaptoethanol. As little as 1 gelsolin/1000 actin subunits also lowers the high storage modulus of oxidized actin. The effects of gelsolin may be both to increase filament number as it severs F-actin and to cover the barbed end of an actin filament, which otherwise might cross-link to the side of another filament via an actin dimer. These new findings may explain why previous studies of actin rheology report a wide range of values when purified actin is polymerized without added regulatory proteins.     

Talin anchors and nucleates actin filaments at lipid membranes. A direct demonstration.

Platelet talin nucleates actin assembly as we show here directly by using rhodamine-phalloidin labelling of actin filaments. Nucleation by talin still occurs after reconstitution into liposomal bilayers. This is also demonstrated directly after protein-lipid double labelling and light microscopic imaging. Talin, thus, is the first actin binding protein for which anchoring and nucleation of actin filament growth at lipid interfaces have been visualized.     

The effect of microtubule disruption on the distribution of the cytoskeletal proteins, vinculin, alpha-actinin, and actin in normal and RSV-transformed fibroblasts

By double indirect immunofluorescence and interference electron microscopy, we have observed the effect of microtubule disruption by antimitotic drugs and coldness treatment on the distribution of adhesion sites and of the three cytoskeletal proteins, vinculin, alpha-actinin, and actin in normal rat cells and in rat cells transformed by Rous sarcoma virus. This study shows that the state of organization of the microtubule--intermediate filament complex modulates the location and the arrangement of intracellular structures containing vinculin, alpha-actinin, and actin in normal as well as in transformed cells. The most important alterations are observed in transformed cells on the distribution of the rosette clusters that have been shown to characterize the transformation by Rous sarcoma virus [8]. These results suggest the microtubule-intermediate filament complex is directly or indirectly connected with the microfilament network.     

Cofilin tunes the nucleotide state of actin filaments and severs at bare and decorated segment boundaries

Actin-based motility demands the spatial and temporal coordination of numerous regulatory actin-binding proteins (ABPs), many of which bind with affinities that depend on the nucleotide state of actin filament. Cofilin, one of three ABPs that precisely choreograph actin assembly and organization into comet tails that drive motility in vitro, binds and stochastically severs aged ADP actin filament segments of de novo growing actin filaments. Deficiencies in methodologies to track in real time the nucleotide state of actin filaments, as well as cofilin severing, limit the molecular understanding of coupling between actin filament chemical and mechanical states and severing. We engineered a fluorescently labeled cofilin that retains actin filament binding and severing activities. Because cofilin binding depends strongly on the actin-bound nucleotide, direct visualization of fluorescent cofilin binding serves as a marker of the actin filament nucleotide state during assembly. Bound cofilin allosterically accelerates P(i) release from unoccupied filament subunits, which shortens the filament ATP/ADP-P(i) cap length by nearly an order of magnitude. Real-time visualization of filament severing indicates that fragmentation scales with and occurs preferentially at boundaries between bare and cofilin-decorated filament segments, thereby controlling the overall filament length, depending on cofilin binding density.     

Three-dimensional structure of a single filament in the Limulus acrosomal bundle: scruin binds to homologous helix-loop-beta motifs in actin

Frozen, hydrated acrosomal bundles from Limulus sperm were imaged with a 400 kV electron cryomicroscope. Segments of this long bundle can be studied as a P1 crystal with a unit cell containing an acrosomal filament with 28 actin and 28 scruin molecules in 13 helical turns. A novel computational procedure was developed to extract single columns of superimposed acrosomal filaments from the distinctive crystallographic view. Helical reconstruction was used to generate a three-dimensional structure of this computationally isolated acrosomal filament. The scruin molecule is organized into two domains which contact two actin subunits in different strands of the same actin filament. A correlation of Holmes' actin filament model to the density in our acrosomal filament map shows that actin subdomains 1, 2, and 3 match the model density closely. However, actin subdomain 4 matches rather poorly, suggesting that interactions with scruin may have altered actin conformation. Scruin makes extensive interactions with helix-loop-beta motifs in subdomain 3 of one actin subunit and in subdomain 1 of a consecutive actin subunit along the genetic filament helix. These two actin subdomains are structurally homologous and are closely spaced along the actin filament. Our model suggests that scruin, which is derived from a tandemly duplicated gene, has evolved to bind structurally homologous but non-identical positions across two consecutive actin subunits.     

Effects of latrunculin B on the actin cytoskeleton and hyphal growth in Phytophthora infestans

The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 μM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 μM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin cytoskeleton plays a role in preventing the movement of nuclei towards the hyphal tip.     

Effect of tensile force on the mechanical behavior of actin filaments

Actin filaments are the most abundant components of the cellular cytoskeleton, and play critical roles in various cellular functions such as migration, division and shape control. In these activities, mechanical tension causes structural changes in the double-helical structure of the actin filament, which is a key modulator of cytoskeletal reorganization. This study performed large-scale molecular dynamics (MD) and steered MD simulations to quantitatively analyze the effects of tensile force on the mechanical behavior of actin filaments. The results revealed that when a tensile force of 200pN was applied to a filament consisting of 14 actin subunits, the twist angle of the filament decreased by approximately 20°, corresponding to a rotation of approximately -2° per subunit, representing a critical structural change in actin filaments. Based on these structural changes, the variance in filament length and twist angle was found to decrease, leading to increases in extensional and torsional stiffness. Torsional stiffness increased significantly under the tensile condition, and the ratio of filament stiffness under tensile force to that under no external force increased significantly on longer temporal scales. The results obtained from this study contribute to the understanding of mechano-chemical interactions concerning actin dynamics, showing that increased tensile force in the filament prevents actin regulatory proteins from binding to the filament.     

The interaction of capping protein with the barbed end of the actin filament

The interaction of capping protein (CP) with actin filaments is an essential element of actin assembly and actin-based motility in nearly all eukaryotes. The dendritic nucleation model for Arp2/3-based lamellipodial assembly features capping of barbed ends by CP, and the formation of filopodia is proposed to involve inhibition of capping by formins and other proteins. To understand the molecular basis for how CP binds the barbed end of the actin filament, we have used a combination of computational and experimental approaches, primarily involving molecular docking and site-directed mutagenesis. We arrive at a model that supports all of our biochemical data and agrees very well with a cryo-electron microscopy structure of the capped filament. CP interacts with both actin protomers at the barbed end of the filament, and the amphipathic helix at the C-terminus of the β-subunit binds to the hydrophobic cleft on actin, in a manner similar to that of WH2 domains. These studies provide us with new molecular insight into how CP binds to the actin filament.     

Zero-mode waveguides visualize the first steps during gelsolin-mediated actin filament formation

Actin filament dynamics underlie key cellular processes. Although the elongation of actin filaments has been extensively studied, the mechanism of nucleation remains unclear. The micromolar concentrations needed for filament formation have prevented direct observation of nucleation dynamics on the single molecule level. To overcome this limitation, we have used the attoliter excitation volume of zero-mode waveguides to directly monitor the early steps of filament assembly. Immobilizing single gelsolin molecules as a nucleator at the bottom of the zero-mode waveguide, we could visualize the actin filament nucleation process. The process is surprisingly dynamic, and two distinct populations during gelsolin-mediated nucleation are observed. The two populations are defined by the stability of the actin dimers and determine whether elongation occurs. Furthermore, by using an inhibitor to block flattening, a conformational change in actin associated with filament formation, elongation was prevented. These observations indicate that a conformational transition and pathway competition determine the nucleation of gelsolin-mediated actin filament formation.     

Coarse-grained Modeling and Simulation of Actin Filament Behavior Based on Brownian Dynamics Method

The actin filament, which is the most abundant component of the cytoskeleton, plays important roles in fundamental cellular activities such as shape determination, cell motility, and mechanosensing. In each activity, the actin filament dynamically changes its structure by polymerization, depolymerization, and severing. These phenomena occur on the scales ranging from the dynamics of actin molecules to filament structural changes with its deformation due to the various forces, for example, by the membrane and solvent. To better understand the actin filament dynamics, it is important to focus on these scales and develop its mathematical model. Thus, the objectives of this study were to model and simulate actin filament polymerization, depolymerization, and severing based on the Brownian dynamics method. In the model, the actin monomers and the solvent were considered as globular particles and a continuum, respectively. The motion of the actin molecules was assumed to follow the Langevin equation. The polymerization, which increases the filament length, was determined by the distance between the center of the actin particle at the barbed end and actin particles in the solvent. The depolymerization, which decreases the filament length, was modeled such that the number of dissociation particles from the filament end per unit time was constant. In addition, the filament severing, in which one filament divides into two, was modeled to occur at an equal rate along the filament. Then, we simulated the actin filament dynamics using the developed model, and analyzed the filament elongation rate, its turnover, and the effects of filament severing on the polymerization and depolymerization. Results indicated that the model reproduced the linear dependence of the filament elongation on time, filament turnover process by polymerization and depolymerization, and acceleration of the polymerization and depolymerization by severing, which qualitatively agreed with those observed in experiments.     

Importance of a Lys113–Glu195 Intermonomer Ionic Bond in F-actin Stabilization and Regulation by Yeast Formins Bni1p and Bnr1p

Proper actin cytoskeletal function requires actin''s ability to generate a stable filament and requires that this reaction be regulated by actin-binding proteins via allosteric effects on the actin. A proposed ionic interaction in the actin filament interior between Lys¹¹³ of one monomer and Glu¹⁹⁵ of a monomer in the apposing strand potentially fosters cross-strand stabilization and allosteric communication between the filament interior and exterior. We interrupted the potential interaction by creating either K113E or E195K actin. By combining the two, we also reversed the interaction with a K113E/E195K (E/K) mutant. In all cases, we isolated viable cells expressing only the mutant actin. Either single mutant cell displays significantly decreased growth in YPD medium. This deficit is rescued in the double mutant. All three mutants display abnormal phalloidin cytoskeletal staining. K113E actin exhibits a critical concentration of polymerization 4 times higher than WT actin, nucleates more poorly, and forms shorter filaments. Restoration of the ionic bond, E/K, eliminates most of these problems. E195K actin behaves much more like WT actin, indicating accommodation of the neighboring lysines. Both Bni1 and Bnr1 formin FH1-FH2 fragment accelerate polymerization of WT, E/K, and to a lesser extent E195K actin. Bni1p FH1-FH2 dramatically inhibits K113E actin polymerization, consistent with barbed end capping. However, Bnr1p FH1-FH2 restores K113E actin polymerization, forming single filaments. In summary, the proposed ionic interaction plays an important role in filament stabilization and in the propagation of allosteric changes affecting formin regulation in an isoform-specific fashion.