Mechanism of actin filament bundling by fascin

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four β-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in β-trefoil domains 1 and 3. The site in β-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in β-trefoil-3 is related by pseudo-2-fold symmetry to that in β-trefoil-1. The two sites are ～5 nm apart, resulting in a distance between actin filaments in the bundle of ～8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.     

Energetics and dynamics of constrained actin filament bundling

The formation of filopodia-like bundles from a dendritic actin network has been observed to occur in vitro as a result of branching induced by Arp2/3 complex. We study both the energetics and dynamics of actin filament bundling in such a network to evaluate their relative importance in bundle formation processes. Our model considers two semiflexible actin filaments fixed at one end and free at the other, described using a normal-mode approximation. This model is studied by both Brownian dynamics and free-energy minimization methods. Remarkably, even short filaments can bundle at separations comparable to their lengths. In the dynamic simulations, we evaluate the time required for the filaments to interact and bind, and examine the dependence of this bundling time on the filament length, the distance between the filament bases, and the cross-linking energy. In most cases, bundling occurs in a second or less. Beyond a certain critical distance, we find that the bundling time increases very rapidly with increasing interfilament separation and/or decreasing filament length. For most of the cases we have studied, the energetics results for this critical distance are similar to those obtained from dynamics simulations run for 10 s, suggesting that beyond this timescale, energetics, rather than kinetic constraints, determine whether or not bundling occurs. Over a broad range of conditions, we find that the times required for bundling from a network are compatible with experimental observations.     

Cysteine-rich protein 1 (CRP1) regulates actin filament bundling

Background  

Cysteine-rich protein 1 (CRP1) is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. CRP1 has been demonstrated to bind the actin-bundling protein α-actinin and proposed to modulate the actin cytoskeleton; however, specific regulatory mechanisms have not been identified.     

Isolation of an abundant 50,000-dalton actin filament bundling protein from Dictyostelium amoebae

A monomeric actin bundling protein with a native molecular weight of approximately 50,000 (ABP-50) has been isolated from amoebae of Dictyostelium discoideum. ABP-50 cross-links F-actin to form tightly packed bundles, some of which are highly ordered. It exhibits a Kd of 2.1 microM and a molar ratio to actin of 1:1 in bundles. Calcium and ATP at physiological concentrations have no effect on these activities. ABP-50 is immunologically unrelated to 30-kDa protein, a previously described bundling protein from Dictyostelium. Immunofluorescence with affinity-purified polyclonal antibodies indicates that ABP-50 is localized in regions of the amoeboid cell cortex containing actin bundles. The molar ratio of ABP-50 to actin is approximately 1:5 in vivo. Therefore, the abundance of ABP-50 suggests that it may be responsible for the majority of the bundling activity in these cells.     

Nebulin regulation of actin filament lengths: new angles

The highly organized arrays of thick and thin filaments found in striated muscles continue to be the subject of studies that yield groundbreaking concepts regarding cell motility. One example is the idea that massive, linearly extended polypeptides function as molecular rulers that set the length of polymeric filaments. Actin filaments that are polymerized in vitro exhibit wide variations in length, but many cells can assemble structures that contain actin filaments that are remarkably uniform. In striated muscles, the giant nebulin polypeptide extends the length of the actin filaments, and nebulin size has been correlated with actin filament lengths in muscles from different species. Here, I discuss a recent study by Gregorio and colleagues that demonstrates that nebulin knockdown leads to loss of actin filament-length regulation in cardiomyocytes, providing functional evidence that is consistent with the molecular ruler concept.     

Actin filament attachments for sustained motility in vitro are maintained by filament bundling

We reconstructed cellular motility in vitro from individual proteins to investigate how actin filaments are organized at the leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel thin glass nanofibers or beads coated with N-WASP WCA domains. Thin nanofibers produced wide comet tails that showed more structural variation in actin filament organization than did bead substrates. During sustained motility, physiological concentrations of Mg(2+) generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg(2+) abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, or filament capping. Analysis of similar motility of microspheres showed that loss of filament bundling did not affect actin shell formation or symmetry breaking but eliminated sustained attachments between the comet tail and the particle surface. Addition of Mg(2+), Lys-Lys(2+), or fascin restored both comet tail attachment and sustained particle motility in low Mg(2+) buffers. TIRF microscopic analysis of filaments captured by WCA-coated beads in the absence of Arp2/3, profilin, and CP showed that filament bundling by polycation or fascin addition increased barbed end capture by WCA domains. We propose a model in which CP directs barbed ends toward the leading edge and polycation-induced filament bundling sustains processive barbed end attachment to the leading edge.     

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F- actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.     

F actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling

Transverse sections though Drosophila bristles reveal 7-11 nearly round, plasma membrane-associated bundles of actin filaments. These filaments are hexagonally packed and in a longitudinal section they show a 12-nm periodicity in both the 1.1 and 1.0 views. From earlier studies this periodicity is attributable to cross-links and indicates that the filaments are maximally cross-linked, singed mutants also have 7-11 bundles, but the bundles are smaller, flattened, and the filaments within the bundles are randomly packed (not hexagonal); no periodicity can be detected in longitudinal sections. Another mutant, forked (f36a), also has 7-11 bundles but even though the bundles are very small, the filaments within them are hexagonally packed and display a 12-nm periodicity in longitudinal section. The singed-forked double mutant lacks filament bundles. Thus there are at least two species of cross-links between adjacent actin filaments. Hints of why two species of cross-links are necessary can be gleaned by studying bristle formation. Bristles sprout with only microtubules within them. A little later in development actin filaments appear. At early stages the filaments in the bundles are randomly packed. Later the filaments in the bundles become hexagonally packed and maximally cross-linked. We consider that the forked proteins may be necessary early in development to tie the filaments together in a bundle so that they can be subsequently zippered together by fascin (the singed gene product).     

Kinetics of filament bundling with attractive interactions

We study the kinetics of filament bundling by variable time-step Brownian-dynamics simulations employing a simplified attractive potential based on earlier atomic-level calculations for actin filaments. Our results show that collisions often cluster in time, due to memory in the random walk. The clustering increases the bundling opportunities. Small-angle collisions and collisions with short center-to-center distance are more likely to lead to bundling. Increasing the monomer-monomer attraction decreases the bundling time to a diffusional limit, which is determined by the capture cross-section and diffusion coefficients. The simulations clearly show that the bundling process consists of two sequential phases: rotation, by which two filaments align parallel to each other; and sliding, by which they maximize their contact length. Whether two filaments bundle or not is determined by the competition between rotation to a parallel state and escape. Increasing the rotational diffusion coefficient and attraction enhances rotation; decreasing attraction and increasing the translational diffusion coefficients enhance escape. Because of several competing effects, the filament length only affects the bundling time weakly.     

Role of actin C-terminus in regulation of striated muscle thin filament

In striated muscle, regulation of actin-myosin interactions depends on a series of conformational changes within the thin filament that result in a shifting of the tropomyosin-troponin complex between distinct locations on actin. The major factors activating the filament are Ca²⁺ and strongly bound myosin heads. Many lines of evidence also point to an active role of actin in the regulation. Involvement of the actin C-terminus in binding of tropomyosin-troponin in different activation states and the regulation of actin-myosin interactions were examined using actin modified by proteolytic removal of three C-terminal amino acids. Actin C-terminal modification has no effect on the binding of tropomyosin or tropomyosin-troponin + Ca²⁺, but it reduces tropomyosin-troponin affinity in the absence of Ca²⁺. In contrast, myosin S1 induces binding of tropomyosin to truncated actin more readily than to native actin. The rate of actin-activated myosin S1 ATPase activity is reduced by actin truncation both in the absence and presence of tropomyosin. The Ca²⁺-dependent regulation of the ATPase activity is preserved. Without Ca²⁺ the ATPase activity is fully inhibited, but in the presence of Ca²⁺ the activation does not reach the level observed for native actin. The results suggest that through long-range allosteric interactions the actin C-terminus participates in the thin filament regulation.     

Stimulation of actin polymerization by filament severing

The extent and dynamics of actin polymerization in solution are calculated as functions of the filament severing rate, using a simple model of in vitro polymerization. The model is solved by both analytic theory and stochastic-growth simulation. The results show that severing essentially always enhances actin polymerization by freeing up barbed ends, if barbed-end cappers are present. Severing has much weaker effects if only pointed-end cappers are present. In the early stages of polymerization, the polymerized-actin concentration grows exponentially as a function of time. The exponential growth rate is given in terms of the severing rate, and the latter is given in terms of the maximum slope in a polymerization time course. Severing and branching are found to act synergistically.     

An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin

Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca(2+) to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca(2+) showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.     

Calcium-dependent regulation of exocytosis

A rapid increase in intracellular calcium directly triggers regulated exocytosis. In addition, changes in intracellular calcium concentration can adjust the extent of exocytosis (quantal content) or the magnitude of individual release events (quantal size) in both the short- and long-term. It is generally agreed that calcium achieves this regulation via an interaction with a number of different molecular targets located at or near to the site of membrane fusion. We review here the synaptic proteins with defined calcium-binding domains and protein kinases activated by calcium, summarize what is known about their function in membrane fusion and the experimental evidence in support of their involvement in synaptic plasticity.     

Mutations in actin subdomain 3 that impair thin filament regulation by troponin and tropomyosin.

Thin filament-mediated regulation of striated muscle contraction involves conformational switching among a few quaternary structures, with transitions induced by binding of Ca(2+) and myosin. We establish and exploit Saccharomyces cerevisiae actin as a model system to investigate this process. Ca(2+)-sensitive troponin-tropomyosin binding affinities for wild type yeast actin are seen to closely resemble those for muscle actin, and these hybrid thin filaments produce Ca(2+)-sensitive regulation of the myosin S-1 MgATPase rate. Yeast actin filament inner domain mutant K315A/E316A depresses Ca(2+) activation of the MgATPase rate, producing a 4-fold weakening of the apparent Ca(2+) affinity and a 50% decrease in the MgATPase rate at saturating Ca(2+) concentration. Observed destabilization of troponin-tropomyosin binding to actin in the presence of Ca(2+), a 1.4-fold effect, provides a partial explanation. Despite the decrease in apparent MgATPase Ca(2+) affinity, there was no detectable change in the true Ca(2+) affinity of the thin filament, measured using fluorophore-labeled troponin. Another inner domain mutant, E311A/R312A, decreased the MgATPase rate but did not change the apparent Ca(2+) affinity. These results suggest that charged residues on the surface of the actin inner domain are important in Ca(2+)- and myosin-induced thin filament activation.     

Mechanism of actin filament nucleation

We used computational methods to analyze the mechanism of actin filament nucleation. We assumed a pathway where monomers form dimers, trimers, and tetramers that then elongate to form filaments but also considered other pathways. We aimed to identify the rate constants for these reactions that best fit experimental measurements of polymerization time courses. The analysis showed that the formation of dimers and trimers is unfavorable because the association reactions are orders of magnitude slower than estimated in previous work rather than because of rapid dissociation of dimers and trimers. The 95% confidence intervals calculated for the four rate constants spanned no more than one order of magnitude. Slow nucleation reactions are consistent with published high-resolution structures of actin filaments and molecular dynamics simulations of filament ends. One explanation for slow dimer formation, which we support with computational analysis, is that actin monomers are in a conformational equilibrium with a dominant conformation that cannot participate in the nucleation steps.     

VASP governs actin dynamics by modulating filament anchoring

Actin filament dynamics at the cell membrane are important for cell-matrix and cell-cell adhesions and the protrusion of the leading edge. Since actin filaments must be connected to the cell membrane to exert forces but must also detach from the membrane to allow it to move and evolve, the balance between actin filament tethering and detachment at adhesion sites and the leading edge is key for cell shape changes and motility. How this fine tuning is performed in cells remains an open question, but possible candidates are the Drosophila enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins, which localize to dynamic actin structures in the cell. Here we study VASP-mediated actin-related proteins 2/3 (Arp2/3) complex-dependent actin dynamics using a substrate that mimics the fluid properties of the cell membrane: an oil-water interface. We show evidence that polymerization activators undergo diffusion and convection on the fluid surface, due to continual attachment and detachment to the actin network. These dynamics are enhanced in the presence of VASP, and we observe cycles of catastrophic detachment of the actin network from the surface, resulting in stop-and-go motion. These results point to a role for VASP in the modulation of filament anchoring, with implications for actin dynamics at cell adhesions and at the leading edge of the cell.     

Role of gelsolin in the actin filament regulation of cardiac L-type calcium channels

The actin cytoskeleton is an important contributor to themodulation of the cell function. However, little is known about theregulatory role of this supermolecular structure in the membrane eventsthat take place in the heart. In this report, the regulation of cardiacmyocyte function by actin filament organization was investigated inneonatal mouse cardiac myocytes (NMCM) from both wild-type mice andmice genetically devoid of the actin filament severing protein gelsolin(Gsn/). Cardiac L-type calcium channel currents(I_Ca) wereassessed using the whole cell voltage-clamp technique. Addition of theactin filament stabilizer phalloidin to wild-type NMCM increasedI_Ca by 227% overcontrol conditions. The basalI_Ca ofGsn/ NMCM was 300% higher than wild-type controls. Thisincrease was completely reversed by intracellular perfusion of theGsn/ NMCM with exogenous gelsolin. Further, cytoskeletal disruption of either Gsn/ or phalloidin-dialyzedwild-type NMCM with cytochalasin D (CD) decreased the enhancedI_Ca by 84% and 87%, respectively. The data indicate that actin filament stabilization by either a lack of gelsolin or intracellular dialysis with phalloidin increase I_Ca,whereas actin filament disruption with CD or dialysis ofGsn/ NMCM with gelsolin decreaseI_Ca. We concludethat cardiac L-type calcium channel regulation is tightly controlled byactin filament organization. Actin filament rearrangement mediated by gelsolin may contribute to calcium channel inactivation.

     

A nonapeptide to the putative F-actin binding site of annexin-II tetramer inhibits its calcium-dependent activation of actin filament bundling.

A synthetic nonapeptide, Val-Leu-Ile-Arg-Ile-Met-Val-Ser-Arg, corresponding to residues 286-294 of annexin-II tetramer (A-IIt), was shown to completely inhibit the Ca(2+)-dependent bundling of F-actin by this protein. The inhibitory effect of the nonapeptide required preincubation with F-actin and was reversed by the addition of excess A-IIt. Kinetic analysis suggested that the nonapeptide reduced the K(0.5) but not the Vmax of F-actin bundling. In contrast, addition of excess nonapeptide to A-IIt-bundled F-actin did not reverse F-actin bundle formation. Although the nonapeptide produced a dose-dependent inhibition of A-IIt-dependent F-actin bundling, the binding of A-IIt to F-actin was not affected. These results identify a domain of A-IIt that is involved in the bundling activity of the protein and suggest that this domain binds transiently with F-actin, resulting in activation of the bundling activity of A-IIt.