Polarized actin bundles formed by human fascin-1: their sliding and disassembly on myosin II and myosin V in vitro

Fascin-1 is a putative bundling factor of actin filaments in the filopodia of neuronal growth cones. Here, we examined the structure of the actin bundle formed by human fascin-1 (actin/fascin bundle), and its mode of interaction with myosin in vitro. The distance between cross-linked filaments in the actin/bundle was 8-9 nm, and the bundle showed the transverse periodicity of 36 nm perpendicular to the bundle axis, which was confirmed by electron microscopy. Decoration of the actin/fascin bundle with heavy meromyosin revealed that the arrowheads of filaments in the bundle pointed in the same direction, indicating that the bundle has polarity. This result suggested that fascin-1 plays an essential role in polarity of actin bundles in filopodia. In the in vitro motility assay, actin/fascin bundles slid as fast as single actin filaments on myosin II and myosin V. When myosin was attached to the surface at high density, the actin/fascin bundle disassembled to single filaments at the pointed end of the bundle during sliding. These results suggest that myosins may drive filopodial actin bundles backward by interacting with actin filaments on the surface, and may induce disassembly of the bundle at the basal region of filopodia.     

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.     

Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia

Growth cones at the distal tips of growing nerve axons contain bundles of actin filaments distributed throughout the lamellipodium and that project into filopodia. The regulation of actin bundling by specific actin binding proteins is likely to play an important role in many growth cone behaviors. Although the actin binding protein, fascin, has been localized in growth cones, little information is available on its functional significance. We used the large growth cones of the snail Helisoma to determine whether fascin was involved in temporal changes in actin filaments during growth cone morphogenesis. Fascin localized to radially oriented actin bundles in lamellipodia (ribs) and filopodia. Using a fascin antibody and a GFP fascin construct, we found that fascin incorporated into actin bundles from the beginning of growth cone formation at the cut end of axons. Fascin associated with most of the actin bundle except the proximal 6--12% adjacent to the central domain, which is the region associated with actin disassembly. Later, during growth cone morphogenesis when actin ribs shortened, the proximal fascin-free zone of bundles increased, but fascin was retained in the distal, filopodial portion of bundles. Treatment with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which phosphorylates fascin and decreases its affinity for actin, resulted in loss of all actin bundles from growth cones. Our findings suggest that fascin may be particularly important for the linear structure and dynamics of filopodia and for lamellipodial rib dynamics by regulating filament organization in bundles.     

Actin bundle architecture and mechanics regulate myosin II force generation

The actin cytoskeleton is a soft, structural material that underlies biological processes such as cell division, motility, and cargo transport. The cross-linked actin filaments self-organize into a myriad of architectures, from disordered meshworks to ordered bundles, which are hypothesized to control the actomyosin force generation that regulates cell migration, shape, and adhesion. Here, we use fluorescence microscopy and simulations to investigate how actin bundle architectures with varying polarity, spacing, and rigidity impact myosin II dynamics and force generation. Microscopy reveals that mixed-polarity bundles formed by rigid cross-linkers support slow, bidirectional myosin II filament motion, punctuated by periods of stalled motion. Simulations reveal that these locations of stalled myosin motion correspond to sustained, high forces in regions of balanced actin filament polarity. By contrast, mixed-polarity bundles formed by compliant, large cross-linkers support fast, bidirectional motion with no traps. Simulations indicate that trap duration is directly related to force magnitude and that the observed increased velocity corresponds to lower forces resulting from both the increased bundle compliance and filament spacing. Our results indicate that the microstructures of actin assemblies regulate the dynamics and magnitude of myosin II forces, highlighting the importance of architecture and mechanics in regulating forces in biological materials.     

Effects of jasplakinolide on the kinetics of actin polymerization. An explanation for certain in vivo observations

Jasplakinolide paradoxically stabilizes actin filaments in vitro, but in vivo it can disrupt actin filaments and induce polymerization of monomeric actin into amorphous masses. A detailed analysis of the effects of jasplakinolide on the kinetics of actin polymerization suggests a resolution to this paradox. Jasplakinolide markedly enhances the rate of actin filament nucleation. This increase corresponds to a change in the size of actin oligomer capable of nucleating filament growth from four to approximately three subunits, which is mechanistically consistent with the localization of the jasplakinolide-binding site at an interface of three actin subunits. Because jasplakinolide both decreases the amount of sequestered actin (by lowering the critical concentration of actin) and augments nucleation, the enhancement of polymerization by jasplakinolide is amplified in the presence of actin-monomer sequestering proteins such as thymosin beta(4). Overall, the kinetic parameters in vitro define the mechanism by which jasplakinolide induces polymerization of monomeric actin in vivo. Expected consequences of jasplakinolide function are consistent with the experimental observations and include de novo nucleation resulting in disordered polymeric actin and in insufficient monomeric actin to allow for remodeling of stress fibers.     

A novel mechanism for the formation of actin-filament bundles by a nonprocessive formin

Actin-filament bundles (or cables) have a structural role during cell division and morphogenesis, but also serve as important "tracks" for the transport of materials during cytokinesis and polarized cell growth. However, the dynamic formation of these longitudinal actin-filament higher-order structures is not understood. Recently, several lines of evidence suggest that formins provide one avenue for the initiation of actin cables in vivo. A popular model for the mechanism of polymerization of actin filaments by formin involves the processive movement of formin attached at the barbed end of an elongating filament. In the present study, we use an in vitro system to reconstitute the dynamic formation of actin-filament bundles generated by Arabidopsis FORMIN1 (AFH1). To be able to visualize individual events in such a complex system, we used real-time evanescent-wave microscopy. Surprisingly, we find that AFH1 is a nonprocessive formin that moves from the barbed end to the side of an actin filament after the nucleation event. We show why this new mechanism of nucleation by a member of the formin family is important for bundle formation. Finally, we analyze the different parameters controlling the dynamic formation of such longitudinal actin-filament bundles.     

A polymorphism peculiar to bipolar actin bundles.

Both muscle and nonmuscle actins produced magnesium paracrystals which we found indistinguishable from one another. Contrary to some previous reports, calcium ions caused no change in filament organization for either type of actin. The most ordered paracrystals consisted of hexagonally packed filaments with opposite polarities. We suggest that this mode of packing permits a form of disorder not previously described, which may account for some puzzling aspects of earlier observations and may prove useful in analyzing actin bundles formed, for example, with erythrocyte band 4.9 protein.     

Thickness distribution of actin bundles in vitro

Bundles of filamentous actin form the primary building blocks of a broad range of cytoskeletal structures, including filopodia, stereocilia and microvilli. In each case, the cell uses specific associated proteins to tailor the dynamics, dimensions and mechanical properties of the bundles to suit a specific cellular function. While the length distribution of actin bundles was extensively studied, almost nothing is known about the thickness distribution. Here, we use high-resolution cryo-TEM to measure the thickness distribution of actin/fascin bundles, in vitro. We find that the thickness distribution has a prominent peak, with an exponential tail, supporting a scenario of an initial fast formation of a disc-like nucleus of short actin filaments, which only later elongates. The bundle thicknesses at steady state are found to follow the distribution of the initial nuclei indicating that no lateral coalescence occurs. Our results show that the distribution of bundles thicknesses can be controlled by monitoring the initial nucleation process. In vivo, this is done by using specific regulatory proteins complexes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Micromechanics and ultrastructure of actin filament networks crosslinked by human fascin: a comparison with alpha-actinin.

Fascin is an actin crosslinking protein that organizes actin filaments into tightly packed bundles believed to mediate the formation of cellular protrusions and to provide mechanical support to stress fibers. Using quantitative rheological methods, we studied the evolution of the mechanical behavior of filamentous actin (F-actin) networks assembled in the presence of human fascin. The mechanical properties of F-actin/fascin networks were directly compared with those formed by alpha-actinin, a prototypical actin filament crosslinking/bundling protein. Gelation of F-actin networks in the presence of fascin (fascin to actin molar ratio >1:50) exhibits a non-monotonic behavior characterized by a burst of elasticity followed by a slow decline over time. Moreover, the rate of gelation shows a non-monotonic dependence on fascin concentration. In contrast, alpha-actinin increased the F-actin network elasticity and the rate of gelation monotonically. Time-resolved multiple-angle light scattering and confocal and electron microscopies suggest that this unique behavior is due to competition between fascin-mediated crosslinking and side-branching of actin filaments and bundles, on the one hand, and delayed actin assembly and enhanced network micro-heterogeneity, on the other hand. The behavior of F-actin/fascin solutions under oscillatory shear of different frequencies, which mimics the cell's response to forces applied at different rates, supports a key role for fascin-mediated F-actin side-branching. F-actin side-branching promotes the formation of interconnected networks, which completely inhibits the motion of actin filaments and bundles. Our results therefore show that despite sharing seemingly similar F-actin crosslinking/bundling activity, alpha-actinin and fascin display completely different mechanical behavior. When viewed in the context of recent microrheological measurements in living cells, these results provide the basis for understanding the synergy between multiple crosslinking proteins, and in particular the complementary mechanical roles of fascin and alpha-actinin in vivo.     

Why Are Two Different Cross-linkers Necessary for Actin Bundle Formation In Vivo and What Does Each Cross-link Contribute?

In developing Drosophila bristles two species of cross-linker, the forked proteins and fascin, connect adjacent actin filaments into bundles. Bundles form in three phases: (a) tiny bundles appear; (b) these bundles aggregate into larger bundles; and (c) the filaments become maximally cross-linked by fascin. In mutants that completely lack forked, aggregation of the bundles does not occur so that the mature bundles consist of <50 filaments versus ∼700 for wild type. If the forked concentration is genetically reduced to half the wild type, aggregation of the tiny bundles occurs but the filaments are poorly ordered albeit with small patches of fascin cross-linked filaments. In mutants containing an excess of forked, all the bundles tend to aggregate and the filaments are maximally crossbridged by fascin. Alternatively, if fascin is absent, phases 1 and 2 occur normally but the resultant bundles are twisted and the filaments within them are poorly ordered. By extracting fully elongated bristles with potassium iodide which removes fascin but leaves forked, the bundles change from being straight to twisted and the filaments within them become poorly ordered. From these observations we conclude that (a) forked is used early in development to aggregate the tiny bundles into larger bundles; and (b) forked facilitates fascin entry into the bundles to maximally cross-link the actin filaments into straight, compact, rigid bundles. Thus, forked aligns the filaments and then directs fascin binding so that inappropriate cross-linking does not occur.     

Disruption of the cytoskeleton during Semaphorin 3A induced growth cone collapse correlates with differences in actin organization and associated binding proteins

Repulsive guidance cues induce growth cone collapse or collapse and retraction. Collapse results from disruption and loss of the actin cytoskeleton. Actin‐rich regions of growth cones contain binding proteins that influence filament organization, such as Arp2/3, cortactin, and fascin, but little is known about the role that these proteins play in collapse. Here, we show that Semaphorin 3A (Sema 3A), which is repulsive to mouse dorsal root ganglion neurons, has unequal effects on actin binding proteins and their associated filaments. The immunofluorescence staining intensity of Arp‐2 and cortactin decreases relative to total protein; whereas in unextracted growth cones fascin increases. Fascin and myosin IIB staining redistribute and show increased overlap. The degree of actin filament loss during collapse correlates with filament superstructures detected by rotary shadow electron microscopy. Collapse results in the loss of branched f‐actin meshworks, while actin bundles are partially retained to varying degrees. Taken together with the known affects of Sema 3A on actin, this suggests a model for collapse that follows a sequence; depolymerization of actin meshworks followed by partial depolymerization of fascin associated actin bundles and their movement to the neurite to complete collapse. The relocated fascin associated actin bundles may provide the substrate for actomyosin contractions that produce retraction. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Long continuous actin bundles in Drosophila bristles are constructed by overlapping short filaments

The actin bundles essential for Drosophila bristle elongation are hundreds of microns long and composed of cross-linked unipolar filaments. These long bundles are built from much shorter modules that graft together. Using both confocal and electron microscopy, we demonstrate that newly synthesized modules are short (1-2 microm in length); modules elongate to approximately 3 microm by growing over the surface of longitudinally adjacent modules to form a graft; the grafted regions are initially secured by the forked protein cross-bridge and later by the fascin cross-bridge; actin bundles are smoothed by filament addition and appear continuous and without swellings; and in the absence of grafting, dramatic alterations in cell shape occur that substitutes cell width expansion for elongation. Thus, bundle morphogenesis has several components: module formation, elongation, grafting, and bundle smoothing. These actin bundles are much like a rope or cable, made by overlapping elements that run a small fraction of the overall length, and stiffened by cross-linking.     

F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.     

Formation of filopodia-like bundles in vitro from a dendritic network

We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)-activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)-coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.     

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.     

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.     

Cytoskeletal bundle mechanics

The mechanical properties of cytoskeletal actin bundles play an essential role in numerous physiological processes, including hearing, fertilization, cell migration, and growth. Cells employ a multitude of actin-binding proteins to actively regulate bundle dimensions and cross-linking properties to suit biological function. The mechanical properties of actin bundles vary by orders of magnitude depending on diameter and length, cross-linking protein type and concentration, and constituent filament properties. Despite their importance to cell function, the molecular design principles responsible for this mechanical behavior remain unknown. Here, we examine the mechanics of cytoskeletal bundles using a molecular-based model that accounts for the discrete nature of constituent actin filaments and their distinct cross-linking proteins. A generic competition between filament stretching and cross-link shearing determines three markedly different regimes of mechanical response that are delineated by the relative values of two simple design parameters, revealing the universal nature of bundle-bending mechanics. In each regime, bundle-bending stiffness displays distinct scaling behavior with respect to bundle dimensions and molecular composition, as observed in reconstituted actin bundles in vitro. This mechanical behavior has direct implications on the physiological bending, buckling, and entropic stretching behavior of cytoskeletal processes, as well as reconstituted actin systems. Results are used to predict the bending regimes of various in vivo cytoskeletal bundles that are not easily accessible to experiment and to generate hypotheses regarding implications of the isolated behavior on in vivo bundle function.     

Actin from Thyone sperm assembles on only one end of an actin filament: a behavior regulated by profilin

Thyone sperm were demembranated with Triton X-100 and, after washing, extracted with 30 mM Tris at pH 8.0 and 1 mM MgCl2. After the insoluble contaminants were removed by centrifugation, the sperm extract was warmed to 22 degrees C. Actin filaments rapidly assembled and aggregated into bundles when KCl was added to the extract. When we added preformed actin filaments, i.e., the acrosomal filament bundles of Limulus sperm, to the extract, the actin monomers rapidly assembled on these filaments. What was unexpected was that assembly took place on only one end of the bundle--the end corresponding to the preferred end for monomer addition. We showed that the absence of growth on the nonpreferred end was not due to the presence of a capper because exogenously added actin readily assembled on both ends. We also analyzed the sperm extract by SDS gel electrophoresis. Two major proteins were present in a 1:1 molar ratio: actin and a 12,500-dalton protein whose apparent isoelectric point was 8.4. The 12,500-dalton protein was purified by DEAE chromatography. We concluded that it is profilin because of its size, isoelectric point, molar ratio to actin, inability to bind to DEAE, and its effect on actin assembly. When profilin was added to actin in the presence of Limulus bundles, addition of monomers on the nonpreferred end of the bundle was inhibited, even though actin by itself assembled on both ends. Using the Limulus bundles as nuclei, we determined the critical concentration for assembly off each end of the filament and estimated the Kd for the profilin-actin complex (approximately 10 microM). We present a model to explain how profilin may regulate the extension of the Thyone acrosomal process in vivo: The profilin-actin complex can add to only the preferred end of the filament bundle. Once the actin monomer is bound to the filament, the profilin is released, and is available to bind to additional actin monomers. This mechanism accounts for the rapid rate of filament elongation in the acrosomal process in vivo.     

Movement of the actin filament bundle in Mytilus sperm: a new mechanism is proposed

An actin filament bundle approximately 2-5 microns in length is present in the sperm of the blue mussel, Mytilus. In unfired sperm this bundle extends from the midpiece through a canal in the center of the nucleus to terminate on the membrane limiting the inside of the cone-shaped acrosomal vacuole. The bundle is composed of 45-65 actin filaments which are hexagonally packed and regularly cross-bridged together to form an actin paracrystal so well ordered that it has six nearly equal faces. Upon induction of the acrosomal reaction, a needle-like process is formed in a few seconds. Within this process is the actin filament bundle which appears unchanged in filament number and packing as determined by optical diffraction methods. Using fluorescein-conjugated phalloidin we were able to establish that the bundle does not change length but instead is projected anteriorly out of the midpiece and nuclear canal like an arrow. Existing mechanisms to explain this extension cannot apply. Specifically, the bundle does not increase in length (no polymerization), does not change its organization (no change in actin twist), does not change filament number (no filament sliding), and cannot move by myosin (wrong polarity). Thus we are forced to look elsewhere for a mechanism and have postulated that at least a component of this movement, or cell elongation, is the interaction of the actin filament bundle with the plasma membrane.