Dynamical control of the shape and size of stereocilia and microvilli

We discuss theoretically the shape of actin-based protrusions such as stereocilia or microvilli that have important functions in many biological systems. These linear protrusions are dynamical structures continuously renewed by treadmilling: actin polymerizes at the tip of the cilium and depolymerizes in its bulk. They also often have a well-controlled length such as in the hair bundles of the inner ear cells where they appear in a graded staircase structure. Recent experimental results by another group of researchers show that the treadmilling velocity of the hair cell stereocilia is proportional to their length. We use generic arguments to describe the physics of stereocilia taking into account the effect of many individual proteins at a coarse-grained level by a few phenomenological parameters. At the tip of the cilium, we find that actin polymerization induces an effective pressure. Below the tip, the shape of the cilium is determined by depolymerization: Agreement with the observed shape requires that depolymerization occurs at least in two steps. Under these conditions, we calculate the cilium shape and provide physical grounds for the proportionality between treadmilling velocity and cilium length. We also calculate the penetration of the stereocilium in the actin cortical layer.     

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.     

Fascins, and their roles in cell structure and function

The fascins are a structurally unique and evolutionarily conserved group of actin cross-linking proteins. Fascins function in the organisation of two major forms of actin-based structures: dynamic, cortical cell protrusions and cytoplasmic microfilament bundles. The cortical structures, which include filopodia, spikes, lamellipodial ribs, oocyte microvilli and the dendrites of dendritic cells, have roles in cell-matrix adhesion, cell interactions and cell migration, whereas the cytoplasmic actin bundles appear to participate in cell architecture. We discuss the current understanding of the cellular mechanisms that regulate the binding of fascin to actin and how these processes contribute to the organisation or disassembly of cell protrusions. Although the in vivo roles of fascin have been studied principally in Drosophila, several human diseases are associated with inherited or acquired alterations in the expression of fascins. Strategies to modulate fascin-containing protrusions and thereby cell adhesive and migratory behaviour could have potential for therapeutic intervention in these conditions. The supplementary material referred to in this section can be found at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/2002/v24.350.html     

Actin and tubulin in teratocarcinoma cells. Amount and intracellular organization upon cytodifferentiation.

Embryonal carcinoma (EC) cells and differentiated derivatives grown in tissue culture have rather similar amounts of actin and tubulin. Indirect immunofluorescent microscopy with antibodies to actin shows striking differences in the actin organization in the different teratocarcinoma derivatives. In the EC cells, actin is found predominantly in ruffles and in surface protrusions, as well as in the cytoplasm, but microfilament bundles are not seen. Some of the differentiated clones contain strongly stained microfilament bundles; others contain actin arrangements which appear to be characteristic of the particular cell type. Indirect immunofluorescence microscopy with antibody to tubulin suggests that cytoplasmic microtubules are present both in the EC cells and in the various differentiated states studied. However, the ease with which microtubules can be documented is dependent on how cells are spread on the substratum. During in vitro differentiation of EC cells, changing patterns of actin distribution appear. Cells at the edge of the colony show the characteristic changes in microfilament and microtubular organization before those in the center.     

Anisotropic nucleation growth of actin bundle: a model for determining the well-defined thickness of bundles

Biopolymers such as DNA, F-actins, and microtubules, which are highly charged, rodlike polyelectrolytes, are assembled into architectures with defined morphology and size by electrostatic interaction with multivalent cations (or polycations) in vivo and in vitro. The physical origin to determine their morphology and size is not clearly understood yet. Our results show that the actin bundle formation consists of two stages: the thickness of actin bundles is determined nearly at the initial stage, while the length of actin bundles is determined later on. It is also found that the thickness of actin bundles decreases with the increase of polycation-mediated attraction between F-actins. From these results, we propose the anisotropic nucleation-growth mechanism, in which the thickness of actin bundles is determined by critical nucleus size, whereas the length of actin bundles is determined by the concentration of free actins relative to nucleus concentration. Observing that polycations are concentrated in some sites of actin bundles, which are thought to be nucleation sites to initiate the formation of actin bundles, supports this model. This anisotropic nucleation-growth mechanism of actin bundles can be broadly applied to the self-assembly of rodlike polyelectrolytes.     

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.     

Ena/VASP proteins can regulate distinct modes of actin organization at cadherin-adhesive contacts

Functional interactions between classical cadherins and the actin cytoskeleton involve diverse actin activities, including filament nucleation, cross-linking, and bundling. In this report, we explored the capacity of Ena/VASP proteins to regulate the actin cytoskeleton at cadherin-adhesive contacts. We extended the observation that Ena/vasodilator-stimulated phosphoprotein (VASP) proteins localize at cell-cell contacts to demonstrate that E-cadherin homophilic ligation is sufficient to recruit Mena to adhesion sites. Ena/VASP activity was necessary both for F-actin accumulation and assembly at cell-cell contacts. Moreover, we identified two distinct pools of Mena within individual homophilic adhesions that cells made when they adhered to cadherin-coated substrata. These Mena pools localized with Arp2/3-driven cellular protrusions as well as at the tips of cadherin-based actin bundles. Importantly, Ena/VASP activity was necessary for both modes of actin activity to be expressed. Moreover, selective depletion of Ena/VASP proteins from the tips of cadherin-based bundles perturbed the bundles without affecting the protrusive F-actin pool. We propose that Ena/VASP proteins may serve as higher order regulators of the cytoskeleton at cadherin contacts through their ability to modulate distinct modes of actin organization at those contacts.     

Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression

Remodeling of actin filaments is necessary for epithelial-mesenchymal transition (EMT); however, understanding of how this is regulated in real time is limited. We used an actin filament reporter and high-resolution live-cell imaging to analyze the regulated dynamics of actin filaments during transforming growth factor-β-induced EMT of mammary epithelial cells. Progressive changes in cell morphology were accompanied by reorganization of actin filaments from thin cortical bundles in epithelial cells to thick, parallel, contractile bundles that disassembled more slowly but remained dynamic in transdifferentiated cells. We show that efficient actin filament remodeling during EMT depends on increased expression of the ezrin/radixin/moesin (ERM) protein moesin. Cells suppressed for moesin expression by short hairpin RNA had fewer, thinner, and less stable actin bundles, incomplete morphological transition, and decreased invasive capacity. These cells also had less α-smooth muscle actin and phosphorylated myosin light chain in cortical patches, decreased abundance of the adhesion receptor CD44 at membrane protrusions, and attenuated autophosphorylation of focal adhesion kinase. Our findings suggest that increased moesin expression promotes EMT by regulating adhesion and contractile elements for changes in actin filament organization. We propose that the transciptional program driving EMT controls progressive remodeling of actin filament architectures.     

The cytoskeletal mechanisms of cell-cell junction formation in endothelial cells

The actin cytoskeleton and associated proteins play a vital role in cell-cell adhesion. However, the procedure by which cells establish adherens junctions remains unclear. We investigated the dynamics of cell-cell junction formation and the corresponding architecture of the underlying cytoskeleton in cultured human umbilical vein endothelial cells. We show that the initial interaction between cells is mediated by protruding lamellipodia. On their retraction, cells maintain contact through thin bridges formed by filopodia-like protrusions connected by VE-cadherin-rich junctions. Bridges share multiple features with conventional filopodia, such as an internal actin bundle associated with fascin along the length and vasodilator-stimulated phosphoprotein at the tip. It is striking that, unlike conventional filopodia, transformation of actin organization from the lamellipodial network to filopodial bundle during bridge formation occurs in a proximal-to-distal direction and is accompanied by recruitment of fascin in the same direction. Subsequently, bridge bundles recruit nonmuscle myosin II and mature into stress fibers. Myosin II activity is important for bridge formation and accumulation of VE-cadherin in nascent adherens junctions. Our data reveal a mechanism of cell-cell junction formation in endothelial cells using lamellipodia as the initial protrusive contact, subsequently transforming into filopodia-like bridges connected through adherens junctions. Moreover, a novel lamellipodia-to-filopodia transition is used in this context.     

Measuring Actin Flow in 3D Cell Protrusions

Actin dynamics is important in determining cell shape, tension, and migration. Methods such as fluorescent speckle microscopy and spatial temporal image correlation spectroscopy have been used to capture high-resolution actin turnover dynamics within cells in two dimensions. However, these methods are not directly applicable in 3D due to lower resolution and poor contrast. Here, we propose to capture actin flow in 3D with high spatial-temporal resolution by combining nanoscale precise imaging by rapid beam oscillation and fluctuation spectroscopy techniques. To measure the actin flow along cell protrusions in cell expressing actin-eGFP cultured in a type I collagen matrix, the laser was orbited around the protrusion and its trajectory was modulated in a clover-shaped pattern perpendicularly to the protrusion. Orbits were also alternated at two positions closely spaced along the protrusion axis. The pair cross-correlation function was applied to the fluorescence fluctuation from these two positions to capture the flow of actin. Measurements done on nonmoving cellular protrusion tips showed no pair-correlation at two orbital positions indicating a lack of flow of F-actin bundles. However, in some protrusions, the pair-correlation approach revealed directional flow of F-actin bundles near the protrusion surface with flow rates in the range of ∼1 μm/min, comparable to results in two dimensions using fluorescent speckle microscopy. Furthermore, we found that the actin flow rate is related to the distance to the protrusion tip. We also observed collagen deformation by concomitantly detecting collagen fibers with reflectance detection during these actin motions. The implementation of the nanoscale precise imaging by rapid beam oscillation method with a cloverleaf-shaped trajectory in conjunction with the pair cross-correlation function method provides a quantitative way of capturing dynamic flows and organization of proteins during cell migration in 3D in conditions of poor contrast.     

Stress fibers in situ in proximal tubules of the rat kidney

Actin bundles in proximal tubules of the rat kidney were examined by immunofluorescence and confocal laser microscopy with special reference to their three-dimensional distribution and identification as stress fibers. Renal tubular segments were prepared from the fresh renal cortex by simple homogenization and centrifugation, and fixed in formaldehyde for staining with fluorescent dye-labeled phalloidin. Segments of the proximal tubules could be identified easily on the bases of their diameter, the height of epithelial cells and prominent brush borders. Confocal laser microscopy clearly demonstrated the overall distribution of actin bundles in the whole-mount proximal tubular segments. Actin bundles in the basal cytoplasm of epithelial cells were observed to run parallel to each other and at a right angle to the tubular axis. In the stereo views reconstructed from serial optical sections, the basal actin bundles appeared as straight rods with both ends tapered. They varied in length and width and extended rather short distances of not more than 10 microns. Often, two or more actin bundles were longitudinally aligned in tandem. Some bundles showed irregular bandings along their length. Each bundle was composed of tightly packed actin filaments which could be decorated with heavy meromyosin subfragment-1 to display a bi-directional arrangement within the bundle. Immunostaining of cryostat sections showed that actin bundles contained myosin and vinculin. Enzymatically isolated proximal tubules contracted upon addition of Mg-ATP. These observations collectively suggest that the actin bundles at the base of renal proximal tubule epithelial cells can be listed among the examples of stress fibers in situ.     

Measuring Actin Flow in 3D Cell Protrusions

Actin dynamics is important in determining cell shape, tension, and migration. Methods such as fluorescent speckle microscopy and spatial temporal image correlation spectroscopy have been used to capture high-resolution actin turnover dynamics within cells in two dimensions. However, these methods are not directly applicable in 3D due to lower resolution and poor contrast. Here, we propose to capture actin flow in 3D with high spatial-temporal resolution by combining nanoscale precise imaging by rapid beam oscillation and fluctuation spectroscopy techniques. To measure the actin flow along cell protrusions in cell expressing actin-eGFP cultured in a type I collagen matrix, the laser was orbited around the protrusion and its trajectory was modulated in a clover-shaped pattern perpendicularly to the protrusion. Orbits were also alternated at two positions closely spaced along the protrusion axis. The pair cross-correlation function was applied to the fluorescence fluctuation from these two positions to capture the flow of actin. Measurements done on nonmoving cellular protrusion tips showed no pair-correlation at two orbital positions indicating a lack of flow of F-actin bundles. However, in some protrusions, the pair-correlation approach revealed directional flow of F-actin bundles near the protrusion surface with flow rates in the range of ∼1 μm/min, comparable to results in two dimensions using fluorescent speckle microscopy. Furthermore, we found that the actin flow rate is related to the distance to the protrusion tip. We also observed collagen deformation by concomitantly detecting collagen fibers with reflectance detection during these actin motions. The implementation of the nanoscale precise imaging by rapid beam oscillation method with a cloverleaf-shaped trajectory in conjunction with the pair cross-correlation function method provides a quantitative way of capturing dynamic flows and organization of proteins during cell migration in 3D in conditions of poor contrast.     

A Highlights from MBoC Selection: Nonmuscle myosin-2 contractility-dependent actin turnover limits the length of epithelial microvilli

Brush border microvilli enable functions that are critical for epithelial homeostasis, including solute uptake and host defense. However, the mechanisms that regulate the assembly and morphology of these protrusions are poorly understood. The parallel actin bundles that support microvilli have their pointed-end rootlets anchored in a filamentous meshwork referred to as the “terminal web.” Although classic electron microscopy studies revealed complex ultrastructure, the composition and function of the terminal web remain unclear. Here we identify nonmuscle myosin-2C (NM2C) as a component of the terminal web. NM2C is found in a dense, isotropic layer of puncta across the subapical domain, which transects the rootlets of microvillar actin bundles. Puncta are separated by ∼210 nm, the expected size of filaments formed by NM2C. In intestinal organoid cultures, the terminal web NM2C network is highly dynamic and exhibits continuous remodeling. Using pharmacological and genetic perturbations in cultured intestinal epithelial cells, we found that NM2C controls the length of growing microvilli by regulating actin turnover in a manner that requires a fully active motor domain. Our findings answer a decades-old question on the function of terminal web myosin and hold broad implications for understanding apical morphogenesis in diverse epithelial systems.     

Distribution of actin bundles in Bowman's capsule of rat kidney

In this study we define the distribution of actin bundle arrangement in Bowman's capsule of rat renal corpuscles. Parietal cells of Bowman's capsule were examined by conventional light microscopy, electron microscopy and confocal microscopy. Within each parietal cell individual actin bundles are arranged in a parallel fashion running the length of the cell. Computer reconstructions obtained using confocal microscopy clearly show the lengths of actin bundles to be arranged, on a capsule level, end-to-end, at angles and perpendicular to bundles in adjacent cells. The bundles stain positively for non-muscle myosin and vinculin. The presence and arrangement of actin bundles in parietal cells is consistent with a role in reinforcing capsule structure.     

Does self-organized criticality drive leading edge protrusion?

Arp2/3 complex nucleates dendritic actin networks and plays a pivotal role in the formation of lamellipodia at the leading edge of motile cells. Mouse fibroblasts lacking functional Arp2/3 complex have the characteristic smooth, veil-like lamellipodial leading edge of wild-type cells replaced by a massive, bifurcating filopodia-like protrusions (FLPs) with fractal geometry. The nanometer-scale actin-network organization of these FLPs can be linked to the fractal geometry of the cell boundary by a self-organized criticality through the bifurcation behavior of cross-linked actin bundles. Despite the pivotal role of the Arp2/3 complex in cell migration, the cells lacking functional Arp2/3 complex migrate at rates similar to wild-type cells. However, these cells display defects in the persistence of a directional movement. We suggest that Arp2/3 complex suppresses the formation of FLPs by locally fine-tuning actin networks and favoring dendritic geometry over bifurcating bundles, giving cells a distinct evolutionary edge by providing the means for a directed movement.     

Spine-scale reorientation inApedinella radians (Pedinellales,Chrysophyceae): the microarchitecture and immunocytochemistry of the associated cytoskeleton

Summary Motile unicells ofApedinella radians have the extraordinary ability to instantaneously reorient six elongate spine-scales located on the cell surface. Extracellular striated fibrous connectors (termed microligaments) attach spine-scales to discrete regions of the plasma membrane underlain by intricate cytoplasmic plaques. A complex cytoskeleton is associated with the plaques and appears responsible for spine-scale movement. Three cytoskeletal proteins have thus far been identified by immunofluorescence using anti-tubulin, anti-actin, and anti-centrin. The three-dimensional configuration of the cytoskeleton has been established and consists of filamentous bundles of actin and centrin which form stellate systems interconnecting the plaques. Additionally, there is a network of microtubular triads which originate on the surface of the nuclear envelope and subtend the plasma membrane and also support several tentacular protrusions. It is proposed that contraction of the actin and/or centrin filamentous bundles is responsible for the reorientation of the spine-scales.     

Actin is not required for nanotubular protrusions of primary astrocytes grown on metal nano-lawn

We used sub-micron metal rod decorated surfaces, 'nano-lawn' structures, as a substrate to study cell-to-cell and cell-to-surface interactions of primary murine astrocytes. These cells form thin membranous tubes with diameters of less than 100 nm and a length of several microns, which make contact to neighboring cells and the substrate during differentiation. While membrane protrusions grow on top of the nano-lawn pillars, nuclei sink to the bottom of the substrate. We observed gondola-like structures along those tubes, suggestive of their function as transport vehicles. Elements of the cytoskeleton such as actin fibers are commonly believed to be essential for triggering the onset and growth of tubular membrane protrusions. A rope-pulling mechanism along actin fibers has recently been proposed to account for the transport or exchange of cellular material between cells. We present evidence for a complementary mechanism that promotes growth and stabilization of the observed tubular protrusions of cell membranes. This mechanism does not require active involvement of actin fibers as the formation of membrane protrusions could not be prevented by suppressing polymerization of actin by latrunculin B. Also theoretically, actin fibers are not essential for the growing and stability of nanotubes since curvature-driven self-assembly of interacting anisotropic raft elements is sufficient for the spontaneous formation of thin nano-tubular membrane protrusions.     

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.     

Filopodia are required for cortical neurite initiation

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.     

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.