The two actin-binding regions on the myosin heads of cardiac muscle

In the presence of myosin S1 or myosin heads, actin filaments tend to form bundles. The biological meaning of the bundling of actin filaments has been unclear. In this study, we found that the cardiac myosin heads can form the bundles of actin filaments more rapidly than can skeletal S1, as monitored by light scattering and electron microscopy. Moreover, the actin bundles formed by cardiac S1 were found to be more stable against mechanical agitation. The distance between actin filaments in the bundles was approximately 20 nm, which is comparable to the length of a myosin head and two actin molecules. This suggests the direct binding of S1 tails to the adjacent actin filament. The "essential" light chain of cardiac myosin could be cross-linked to the actin molecule in the bundle. When monomeric actin molecules were added to the bundle, the bundles could be dispersed into individual filaments. The three-dimensional structure of the dispersed actin filaments was reconstructed from electron cryo-microscopic images of the single actin filaments dispersed by monomer actin. We were able to demonstrate that cardiac myosin heads bind to two actin molecules: one actin molecule at the conventional actin-binding region and the other at the essential light-chain-binding region. This capability of cardiac myosin heads to bind two actin molecules is discussed in view of lower ATPase activity and slower shortening velocity than those of skeletal ones.     

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.     

The Antiparallel Dimerization of Myosin X Imparts Bundle Selectivity for Processive Motility

Myosin X is an unconventional actin-based molecular motor involved in filopodial formation, microtubule-actin filament interaction, and cell migration. Myosin X is an important component of filopodia regulation, localizing to tips of growing filopodia by an unclear targeting mechanism. The native α-helical dimerization domain of myosin X is thought to associate with antiparallel polarity of the two amino acid chains, making myosin X the only myosin that is currently considered to form antiparallel dimers. This study aims to determine if antiparallel dimerization of myosin X imparts selectivity toward actin bundles by comparing the motility of parallel and antiparallel dimers of myosin X on single and fascin-bundled actin filaments. Antiparallel myosin X dimers exhibit selective processivity on fascin-bundled actin and are only weakly processive on single actin filaments below saturating [ATP]. Artificial forced parallel dimers of myosin X are robustly processive on both single and bundled actin, exhibiting no selectivity. To determine the relationship between gating of the reaction steps and observed differences in motility, a mathematical model was developed to correlate the parameters of motility with the biochemical and mechanical kinetics of the dimer. Results from the model, constrained by experimental data, suggest that the probability of binding forward, toward the barbed end of the actin filament, is lower in antiparallel myosin X on single actin filaments compared to fascin-actin bundles and compared to constructs of myosin X with parallel dimerization.     

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.     

Intrinsic dynamic behavior of fascin in filopodia

Recent studies showed that the actin cross-linking protein, fascin, undergoes rapid cycling between filopodial filaments. Here, we used an experimental and computational approach to dissect features of fascin exchange and incorporation in filopodia. Using expression of phosphomimetic fascin mutants, we determined that fascin in the phosphorylated state is primarily freely diffusing, whereas actin bundling in filopodia is accomplished by fascin dephosphorylated at serine 39. Fluorescence recovery after photobleaching analysis revealed that fascin rapidly dissociates from filopodial filaments with a kinetic off-rate of 0.12 s(-1) and that it undergoes diffusion at moderate rates with a coefficient of 6 microm(2)s(-1). This kinetic off-rate was recapitulated in vitro, indicating that dynamic behavior is intrinsic to the fascin cross-linker. A computational reaction-diffusion model showed that reversible cross-linking is required for the delivery of fascin to growing filopodial tips at sufficient rates. Analysis of fascin bundling indicated that filopodia are semiordered bundles with one bound fascin per 25-60 actin monomers.     

Myosin VI has a One Track Mind Versus Myosin Va When Moving on Actin Bundles or at an Intersection

Myosin VI (myoVI) and myosin Va (myoVa) serve roles both as intracellular cargo transporters and tethers/anchors. In both capacities, these motors bind to and processively travel along the actin cytoskeleton, a network of intersecting actin filaments and bundles that present directional challenges to these motors. Are myoVI and myoVa inherently different in their abilities to interact and maneuver through the complexities of the actin cytoskeleton? Thus, we created an in vitro model system of intersecting actin filaments and individual unipolar (fascin‐actin) or mixed polarity (α‐actinin‐actin) bundles. The stepping dynamics of individual Qdot‐labeled myoVI and myoVa motors were determined on these actin tracks. Interestingly, myoVI prefers to stay on the actin filament it is traveling on, while myoVa switches filaments with higher probability at an intersection or between filaments in a bundle. The structural basis for this maneuverability difference was assessed by expressing a myoVI chimera in which the single myoVI IQ was replaced with the longer, six IQ myoVa lever. The mutant behaved more like myoVI at actin intersections and on bundles, suggesting that a structural element other than the lever arm dictates myoVI's preference to stay on track, which may be critical to its role as an intracellular anchor .     

The Stepping Pattern of Myosin X Is Adapted for Processive Motility on Bundled Actin

Myosin X is a molecular motor that is adapted to select bundled actin filaments over single actin filaments for processive motility. Its unique form of motility suggests that myosin X's stepping mechanism takes advantage of the arrangement of actin filaments and the additional target binding sites found within a bundle. Here we use fluorescence imaging with one-nanometer accuracy to show that myosin X takes steps of ∼18 nm along a fascin-actin bundle. This step-size is well short of the 36-nm step-size observed in myosin V and myosin VI that corresponds to the actin pseudohelical repeat distance. Myosin X is able to walk along bundles with this step-size if it straddles two actin filaments, but would be quickly forced to spiral into the constrained interior of the bundle if it were to use only a single actin filament. We also demonstrate that myosin X takes many sideways steps as it walks along a bundle, suggesting that it can switch actin filament pairs within the bundle as it walks. Sideways steps to the left or the right occur on bundles with equal frequency, suggesting a degree of lateral flexibility such that the motor's working stroke does not bias it to the left or to the right. On single actin filaments, we find a broad mixture of 10-20-nm steps, which again falls short of the 36-nm actin repeat. Moreover, the motor leans to the right as it walks along single filaments, which may require myosin X to adopt strained configurations. As a control, we also tracked myosin V stepping along actin filaments and fascin-actin bundles. We find that myosin V follows a narrower path on both structures, walking primarily along one surface of an actin filament and following a single filament within a bundle while occasionally switching to neighboring filaments. Together, these results delineate some of the structural features of the motor and the track that allow myosin X to recognize actin filament bundles.     

Role of fascin in filopodial protrusion

In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of filopodia, whereas the inactive fascin mutant S39E reduced filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in filopodia, with t(1/2) < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of filopodial actin filaments.     

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.     

The key feature for early migratory processes: Dependence of adhesion,actin bundles,force generation and transmission on filopodia

Migration of cells is one of the most essential prerequisites to form higher organisms and depends on a strongly coordinated sequence of processes. Early migratory events include substrate sensing, adhesion formation, actin bundle assembly and force generation. While substrate sensing was ascribed to filopodia, all other processes were believed to depend mainly on lamellipodia of migrating cells. In this work we show for motile keratinocytes that all processes from substrate sensing to force generation strongly depend on filopodial focal complexes as well as on filopodial actin bundles. In a coordinated step by step process, filopodial focal complexes have to be tightly adhered to the substrate and to filopodial actin bundles to enlarge upon lamellipodial contact forming classical focal adhesions. Lamellipodial actin filaments attached to those focal adhesions originate from filopodia. Upon cell progression, the incorporation of filopodial actin bundles into the lamellipodium goes along with a complete change in actin cross-linker composition from filopodial fascin to lamellipodial α-actinin. α-Actinin in turn is replaced by myosin II and becomes incorporated directly behind the leading edge. Myosin II activity makes this class of actin bundles with their attached FAs the major source of force generation and transmission at the cell front. Furthermore, connection of FAs to force generating actin bundles leads to their stabilization and further enlargement. Consequently, adhesion sites formed independently of filopodia are not connected to detectable actin bundles, transmit weak forces to the substrate and disassemble within a few minutes without having been increased in size.Key words: filopodia, focal complexes, cell migration, focal adhesion, myosin II, force, actin flow, maturation     

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.     

Retrograde flow and myosin II activity within the leading cell edge deliver F-actin to the lamella to seed the formation of graded polarity actomyosin II filament bundles in migrating fibroblasts

In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles.     

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.     

Arp2/3 branched actin network mediates filopodia-like bundles formation in vitro

During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia.     

Unidirectional sliding of myosin filaments along the bundle of F-actin filaments spontaneously formed during superprecipitation

I reported previously (Higashi-Fujime, S., 1982, Cold Spring Harbor Symp. Quant. Biol., 46:69-75) that active movements of fibrils composed of F-actin and myosin filaments occurred after superprecipitation in the presence of ATP at low ionic strengths. When the concentration of MgCl2 in the medium used in the above experiment was raised to 20-26 mM, bundles of F-actin filaments, in addition to large precipitates, were formed spontaneously both during and after superprecipitation. Along these bundles, many myosin filaments were observed to slide unidirectionally and successively through the bundle, from one end to the other. The sliding of myosin filaments continued for approximately 1 h at room temperature at a mean rate of 6.0 micron/s, as long as ATP remained in the medium. By electron microscopy, it was found that most F-actin filaments decorated with heavy meromyosin pointed to the same direction in the bundle. Myosin filaments moved actively not only along the F-actin bundle but also in the medium. Such movement probably occurred along F-actin filaments that did not form the bundle but were dispersed in the medium, although dispersed F-actin filaments were not visible under the microscope. In this case, myosin filament could have moved in a reverse direction, changing from one F-actin filament to the other. These results suggested that the direction of movement of myosin filament, which has a bipolar structure and the potentiality to move in both directions, was determined by the polarity of F-actin filament in action.     

The key feature for early migratory processes

Migration of cells is one of the most essential prerequisites to form higher organisms and depends on a strongly coordinated sequence of processes. Early migratory events include substrate sensing, adhesion formation, actin bundle assembly and force generation. While substrate sensing was ascribed to filopodia, all other processes were believed to depend mainly on lamellipodia of migrating cells. In this work we show for motile keratinocytes that all processes from substrate sensing to force generation strongly depend on filopodial focal complexes as well as on filopodial actin bundles. In a coordinated step by step process filopodial focal complexes have to be tightly adhered to the substrate and to filopodial actin bundles to enlarge upon lamellipodial contact forming classical focal adhesions. Lamellipodial actin filaments attached to those focal adhesions originate from filopodia. Upon cell progression, the incorporation of filopodial actin bundles into the lamellipodium goes along with a complete change in actin cross-linker composition from filopodial fascin to lamellipodial α-actinin. α-Actinin in turn is replaced by myosin II and becomes incorporated directly behind the leading edge. Myosin II activity makes this class of actin bundles with their attached FAs the major source of force generation and transmission at the cell front. Furthermore, connection of FAs to force generating actin bundles leads to their stabilization and further enlargement. Consequently, adhesion sites formed independently of filopodia are not connected to detectable actin bundles, transmit weak forces to the substrate and disassemble within a few minutes without having been increased in size.     

Regulation of actin filament cross-linking and bundle shape in Drosophila bristles

Previous studies demonstrate that in developing Drosophila bristles, two cross-linking proteins are required sequentially to bundle the actin filaments that support elongating bristle cells. The forked protein initiates the process and facilitates subsequent cross-linking by fascin. Using cross-linker-specific antibodies, mutants, and drugs we show that fascin and actin are present in excessive amounts throughout bundle elongation. In contrast, the forked cross-linker is limited throughout bundle formation, and accordingly, regulates bundle size and shape. We also show that regulation of cross-linking by phosphorylation can affect bundle size. Specifically, inhibition of phosphorylation by staurosporine results in a failure to form large bundles if added during bundle formation, and leads to a loss of cross-linking by fascin if added after the bundles form. Interestingly, inhibition of dephosphorylation by okadaic acid results in the separation of the actin bundles from the plasma membrane. We further show by thin section electron microscopy analysis of mutant and wild-type bristles that the amount of material that connects the actin bundles to the plasma membrane is also limited throughout bristle elongation. Therefore, overall bundle shape is determined by the number of actin filaments assembled onto the limited area provided by the connector material. We conclude that assembly of actin bundles in Drosophila bristles is controlled in part by the controlled availability of a single cross-linking protein, forked, and in part by controlled phosphorylation of cross-links and membrane actin connector proteins.     

Persistence length of fascin-cross-linked actin filament bundles in solution and the in vitro motility assay

Background

Bundles of unipolar actin filaments (F-actin), cross-linked via the actin-binding protein fascin, are important in filopodia of motile cells and stereocilia of inner ear sensory cells. However, such bundles are also useful as shuttles in myosin-driven nanotechnological applications. Therefore, and for elucidating aspects of biological function, we investigate if the bundle tendency to follow straight paths (quantified by path persistence length) when propelled by myosin motors is directly determined by material properties quantified by persistence length of thermally fluctuating bundles.

Methods

Fluorescent bundles, labeled with rhodamine-phalloidin, were studied at fascin:actin molar ratios: 0:1 (F-actin), 1:7, 1:4 and 1:2. Persistence lengths (Lp) were obtained by fitting the cosine correlation function (CCF) to a single exponential function: < cos(θ(0) − θ(s)) > = exp(−s / (2Lp)) where θ(s) is tangent angle; s: path or contour lengths. < > denotes averaging over filaments.

Results

Bundle-Lp (bundles < 15 μm long) increased from ~ 10 to 150 μm with increased fascin:actin ratio. The increase was similar for path-Lp (path < 15 μm), with highly linear correlation. For longer bundle paths, the CCF-decay deviated from a single exponential, consistent with superimposition of the random path with a circular path as suggested by theoretical analysis.

Conclusions

Fascin–actin bundles have similar path-Lp and bundle-Lp, both increasing with fascin:actin ratio. Path-Lp is determined by the flexural rigidity of the bundle.

General significance

The findings give general insight into mechanics of cytoskeletal polymers that interact with molecular motors, aid rational development of nanotechnological applications and have implications for structure and in vivo functions of fascin–actin bundles.     

Molecular Architecture of Synaptic Actin Cytoskeleton in Hippocampal Neurons Reveals a Mechanism of Dendritic Spine Morphogenesis

Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility.     

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.