Skeletal muscle myosin subfragment-1 induces bundle formation by actin filaments

As is well known, the light scattering intensity of F-actin solutions increases immediately upon formation of the rigor complex with subfragment-1 (S-1). We have found that after the initial rise in scattering, there is a further gradual increase in scattering (we call it "super-opalescence"). Fluorescence and electron microscopic observations of acto-S-1 solutions showed that super-opalescence results from formation of actin filament bundles once S-1 binds to F-actin. The actin bundles possessed transverse stripes with a periodicity of about 350 A, which suggested that in the bundles actin filaments are arranged in parallel register. The rate of the initial process of bundle formation (i.e. side-by-side dimerization) could be approximately estimated by measuring the initial rate of super-opalescence (V0). V0 had a maximum (V0m) at a molar ratio of S-1 to actin of 1;6-1;7, regardless of the actin concentration, pH (6-8.5), Mg2+ concentration (up to 5 mM), or ionic strength (up to 0.3 M KC1). Lower pH, higher Mg2+ concentration, and higher ionic strength increased V0m; V0 was proportional to the square of the actin concentration, regardless of the solution conditions.     

Propagation of Acto-S-1 ATPase reaction-coupled conformational change in actin along the filament

F-Actin was partially cross-linked to myosin subfragment-1 (S-1) at various molar ratios (r = S-1/actin) with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The cross-linked acto-S-1 ATPase showed so called "super-activation," Vx. S-1 was added further to the cross-linked acto-S-1 and the ATPase activity, Vy, was measured. Since the added S-1 can interact only with the bare actin protomers within the cross-linked actin filament, the difference, delta V = Vy - Vx - Vs (where Vs is the ATPase activity of the additional S-1 alone), can indicate the state of the bare actin protomers while the cross-linked acto-S-1 is hydrolyzing ATP. With increasing r, delta V decreased much more rapidly than delta Vo(1 - r) (where delta Vo is delta V at r = 0) and reached a minimum around r = 0.15. As r increased further, delta V approached the level of delta Vo(1 - r). When SH1/SH2-blocked S-1 was cross-linked to F-actin, delta V decreased according to delta Vo(1 - r). Therefore, the large reduction of delta V, observed when intact S-1 was cross-linked, was coupled to the high ATPase activity of the cross-linked acto-S-1. Combining these data with other kinetic data, we could deduce that structural distortion in a cross-linked actin induced by the ATPase reaction of the S-1 partner propagated over several bare actin protomers along the filament and reduced their affinity for the S-1-ADP-Pi complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bundling of actin filaments by aorta caldesmon is not related to its regulatory function

Ca2+-sensitive thin filaments from vascular smooth muscle were disassembled into their constituent proteins, actin, tropomyosin and caldesmon. Caldesmon bound to both actin and to actin-tropomyosin and inhibited actin-tropomyosin activation of skeletal muscle myosin MgATPase. It also promoted the aggregation of actin or actin-tropomyosin into parallel aligned bundles. Quantitative electron microscopy measurements showed that with 1.1 microM actin-tropomyosin, 1.6 +/- 0.5% (n = 3) of the filaments were in bundles. At 0.073 microM, caldesmon inhibited MgATPase activity by 50%, whereas bundling was 3.0 +/- 1.3% (n = 4). At 0.37 microM caldesmon, MgATPase inhibition was 83% while 28.1 +/- 6.9% (n = 4) of filaments were in bundles. Experiments at 4.4 microM in which MgATPase and bundling were measured in the same samples gave similar results. Small bundles of 2-3 filaments showed the most frequent occurrence at 1.1 microM actin. At 4.4 microM actin the most common bundle size was 3-5 filaments, with the occasional occurrence of large bundles consisting of up to 120 filaments. The incidence of bundling was the same in the presence and absence of tropomyosin. Thus caldesmon can induce the formation of actin bundles but this property bears no relationship to its inhibition of MgATPase activity.     

Affinity of alpha-actinin for actin determines the structure and mechanical properties of actin filament gels.

Proteins that cross-link actin filaments can either form bundles of parallel filaments or isotropic networks of individual filaments. We have found that mixtures of actin filaments with alpha-actinin purified from either Acanthamoeba castellanii or chicken smooth muscle can form bundles or isotropic networks depending on their concentration. Low concentrations of alpha-actinin and actin filaments form networks indistinguishable in electron micrographs from gels of actin alone. Higher concentrations of alpha-actinin and actin filaments form bundles. The threshold for bundling depends on the affinity of the alpha-actinin for actin. The complex of Acanthamoeba alpha-actinin with actin filaments has a Kd of 4.7 microM and a bundling threshold of 0.1 microM; chicken smooth muscle has a Kd of 0.6 microM and a bundling threshold of 1 microM. The physical properties of isotropic networks of cross-linked actin filaments are very different from a gel of bundles: the network behaves like a solid because each actin filament is part of a single structure that encompasses all the filaments. Bundles of filaments behave more like a very viscous fluid because each bundle, while very long and stiff, can slip past other bundles. We have developed a computer model that predicts the bundling threshold based on four variables: the length of the actin filaments, the affinity of the alpha-actinin for actin, and the concentrations of actin and alpha-actinin.     

Actomyosin interactions in the presence of ATP and the N-terminal segment of actin.

The binding of myosin subfragment 1 (S-1) to actin in the presence of ATP and the acto-S-1 ATPase activities of acto-S-1 complexes were determined at 5 degrees C under conditions of partial saturation of actin, up to 90%, by antibodies against the first seven N-terminal residues on actin. The antibodies [Fab(1-7)] inhibited strongly the acto-S-1 ATPase and the binding of S-1 to actin in the presence of ATP at low concentrations of S-1, up to 25 microM. Further increases in S-1 concentration resulted in a partial and cooperative recovery of both the binding of S-1 to actin and the acto-S-1 ATPase while causing only limited displacement of Fab(1-7) from actin. The extent to which the binding and the ATPase activity were recovered depended on the saturation of actin by Fab(1-7). The combined amounts of S-1 and Fab binding to actin suggested that the activation of the myosin ATPase activity was due to actin free of Fab. Examination of the acto-S-1 ATPase activities as a function of S-1 bound to actin at different levels of actin saturation by Fab(1-7) revealed that the antibodies inhibited the activation of the bound myosin. Thus, the binding of antibodies to the N-terminal segment of actin can act to inhibit both the binding of S-1 to actin in the presence of ATP and a catalytic step in ATP hydrolysis by actomyosin. The implications of these results to the regulation of actomyosin interaction are discussed.     

Potentiated state of the tropomyosin actin filament and nucleotide-containing myosin subfragment 1

The main purpose of this study was to determine whether potentiation of acto-S-1 ATPase activity (activity higher than that obtained with tropomyosin-free actin) could be caused by nucleotide-containing acto-S-1 complexes. In addition, we wanted to know whether these complexes also have a positive cooperative effect on their own apparent binding constant under conditions where nucleotide-free acto-S-1 complexes cause potentiation of ATPase activity. Using calcium-saturated troponin-tropomyosin actin filaments, we observed potentiation of ATPase activity in the presence of 5.0 mM magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP) and calculated that the ability of acto-S-1-AMPPNP complexes to cause potentiation must have been very similar to that of nucleotide-free acto-S-1 complexes. In extension of earlier studies, potentiated acto-S-1 ATPase activity was characterized by an increase in Vmax and, as observed before, a lowering of the apparent Km for subfragment 1 (S-1). Under conditions similar to those that produce the potentiation of acto-S-1 ATPase activity, the apparent actin binding constant of nucleotide-free S-1 was increased about 3-5 fold while the apparent binding constant of AMPPNP to actin-bound S-1 was reduced to (2.5-10) x 10(2) M-1 compared to that of about (1-5) x 10(3) M-1 for S-1 bound to tropomyosin-free actin. Under the same conditions, the apparent binding constant of S-1-AMPPNP to actin was not increased. We suggest that a potentiated state of the tropomyosin actin filament is produced by the cooperative action of acto-S-1 or acto-S-1-AMPPNP complexes. The potentiated state is characterized by an increase in the Vmax of the acto-S-1 ATPase activity, increased binding constants for S-1 and S-1-ADP, and increased binding of tropomyosin to actin.     

Actin filament bundling by fimbrin is important for endocytosis, cytokinesis, and polarization in fission yeast

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.     

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.     

Pre-steady-state kinetic evidence for a cyclic interaction of myosin subfragment one with actin during the hydrolysis of adenosine 5'-triphosphate.

A single cycle of adenosine 5'-triphosphate (ATP) hydrolysis by a complex of actin and myosin subfragment one (acto-S-1) was studied in a stopped-flow apparatus at low temperature and low ionic strength, using light scattering to monitor the interaction of S-1 with actin and fluorescence to detect the formation of fluorescent intermediates. Our results show that the addition of a stoichiometric concentration of ATP to the acto-S-1 causes a cycle consisting of first, a rapid dissociation of the S-1 from actin by ATP; second, a slower fluorescence change in the S-1 that may be related to the initial phosphate burst; and third, a much slower rate limiting recombination of the S-1 with actin. This latter step equals the acto-S-1 steady-state adenosine 5'-triphosphatase (ATPase) rate at both low and high actin concentrations, and like the steady-state ATPase levels off at a V max of 0.9s-1 at high actin concentration. Therefore, the release of adenosine 5'-diphosphate and inorganic phosphate is not the rate-limiting step in the acto-S-1 ATPase. Rather, a slow first-order step corresponding to the previously postulated transition from the refractory to the nonrefractory state precedes the rebinding of the S-1 to the actin during each cycle of ATP hydrolysis.     

Arabidopsis VILLIN1 generates actin filament cables that are resistant to depolymerization

Dynamic cytoplasmic streaming, organelle positioning, and nuclear migration use molecular tracks generated from actin filaments arrayed into higher-order structures like actin cables and bundles. How these arrays are formed and stabilized against cellular depolymerizing forces remains an open question. Villin and fimbrin are the best characterized actin-filament bundling or cross-linking proteins in plants and each is encoded by a multigene family of five members in Arabidopsis thaliana. The related villins and gelsolins are conserved proteins that are constructed from a core of six homologous gelsolin domains. Gelsolin is a calcium-regulated actin filament severing, nucleating and barbed end capping factor. Villin has a seventh domain at its C terminus, the villin headpiece, which can bind to an actin filament, conferring the ability to crosslink or bundle actin filaments. Many, but not all, villins retain the ability to sever, nucleate, and cap filaments. Here we have identified a putative calcium-insensitive villin isoform through comparison of sequence alignments between human gelsolin and plant villins with x-ray crystallography data for vertebrate gelsolin. VILLIN1 (VLN1) has the least well-conserved type 1 and type 2 calcium binding sites among the Arabidopsis VILLIN isoforms. Recombinant VLN1 binds to actin filaments with high affinity (K(d) approximately 1 microM) and generates bundled filament networks; both properties are independent of the free Ca(2+) concentration. Unlike human plasma gelsolin, VLN1 does not nucleate the assembly of filaments from monomer, does not block the polymerization of profilin-actin onto barbed ends, and does not stimulate depolymerization or sever preexisting filaments. In kinetic assays with ADF/cofilin, villin appears to bind first to growing filaments and protects filaments against ADF-mediated depolymerization. We propose that VLN1 is a major regulator of the formation and stability of actin filament bundles in plant cells and that it functions to maintain the cable network even in the presence of stimuli that result in depolymerization of other actin arrays.     

Real-Time Dynamics of Emerging Actin Networks in Cell-Mimicking Compartments

Understanding the cytoskeletal functionality and its relation to other cellular components and properties is a prominent question in biophysics. The dynamics of actin cytoskeleton and its polymorphic nature are indispensable for the proper functioning of living cells. Actin bundles are involved in cell motility, environmental exploration, intracellular transport and mechanical stability. Though the viscoelastic properties of actin-based structures have been extensively probed, the underlying microstructure dynamics, especially their disassembly, is not fully understood. In this article, we explore the rich dynamics and emergent properties exhibited by actin bundles within flow-free confinements using a microfluidic set-up and epifluorescence microscopy. After forming entangled actin filaments within cell-sized quasi two-dimensional confinements, we induce their bundling using three different fundamental mechanisms: counterion condensation, depletion interactions and specific protein-protein interactions. Intriguingly, long actin filaments form emerging networks of actin bundles via percolation leading to remarkable properties such as stress generation and spindle-like intermediate structures. Simultaneous sharing of filaments in different links of the network is an important parameter, as short filaments do not form networks but segregated clusters of bundles instead. We encounter a hierarchical process of bundling and its subsequent disassembly. Additionally, our study suggests that such percolated networks are likely to exist within living cells in a dynamic fashion. These observations render a perspective about differential cytoskeletal responses towards numerous stimuli.     

Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca(++)-dependent, actin-bundling/depolymerizing protein

The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (M(r)) polypeptide, villin. This report describes the purification and characterization of villin as a Ca(++)-dependent, actin bundling/depolymerizing protein. Then 100,000 g supernatant from a Ca(++) extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M(r) (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. This viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca(++) concentration in solution. At low free Ca(++) (less than 10(-6) M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca(++) (more than 10(-6) M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca(++) effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosly associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca(++)-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.     

F-actin bundling protein from Physarum polycephalum: purification and its capacity for co-bundling of actin filaments and microtubules

An F-actin bundling protein was isolated and purified from plasmodium of Physarum polycephalum. The F-actin bundling protein in Physarum extract was passed through a DEAE-cellulose column. After the protein in the fraction was treated with 6 M urea, it was purified by gel filtration on Sephacryl S-300 HR followed by chromatography on CM-Toyopearl (cation exchange) in the presence of 6 M urea. The purified protein gave a single band on SDS-PAGE, and the molecular weight was estimated to be 52,000. This F-actin bundling protein is referred to as the 52 kDa protein. Interestingly, the 52 kDa protein also induced bundling of microtubules. The formation of F-actin and microtubule bundles was Ca(2+)-insensitive, but depended on the salt concentration. Each bundle formed at NaCl concentrations less than 0.1 M. The 52 kDa protein cross-reacted with monoclonal antibody raised against a HeLa 55 kDa protein (an F-actin bundling protein from HeLa cells) (Yamashiro-Matsumura and Matsumura: J. Biol. Chem. 260:5087-5097, 1985). When the 52 kDa protein was added to a mixture of actin filaments and microtubules, co-bundles composed of both filaments formed. This is the first reported example in which an F-actin bundling protein induced co-bundling of actin filaments and microtubules.     

Antibodies directed against N-terminal residues on actin do not block acto-myosin binding

Several studies using a variety of approaches have suggested a possible role for the amino-terminal residues of skeletal muscle actin in acto-myosin interaction. In order to assess the significance of acto-S-1 contacts involving the N-terminal segment of actin, we have prepared polyclonal antisera against a synthetic peptide corresponding to the seven amino-terminal residues of rabbit skeletal muscle actin (alpha-N-terminal peptide). Affinity-purified immunoglobulin (Ig) G (and Fab) prepared from these antisera reacts strongly and specifically with the amino-terminal segment of both G- and F-actin but not with myosin subfragment 1 (S-1). This specificity was determined by Western blot analysis of actin and its proteolytic fragments and the inhibition of the above reactivity by the alpha-N-terminal peptide. The alpha-N-terminal peptide did not interact with S-1 in solution, affect S-1 and actin-activated S-1 MgATPase, or cause dissociation of the acto-S-1 complex. In separate experiments F-actin could be cosedimented with S-1 and affinity-purified IgG or Fab by using an air-driven ultracentrifuge. Densitometric analysis of sodium dodecyl sulfate/polyacrylamide gels of pellet and supernatant fractions from such experiments demonstrated the binding of both S-1 and IgG or Fab to the same F-actin protomer. Our results suggest that, while the acidic N-terminal amino acids of actin may contact the myosin head, these residues cannot be the main determinants of acto-S-1 interaction.     

Actin crosslinking protein EF-1a of Dictyostelium discoideum has a unique bonding rule that allows square-packed bundles.

The elongation factor 1a (EF-1a) of Dictyostelium discoideum is an actin crosslinking protein that gives rise to a unique kind of actin bundle. Purified actin and EF-1a were allowed to form bundles and then were characterized by electron microscopy, computed diffraction analysis, and modeling. In these bundles crosslinked actin filaments are rotated by 90 degrees relative to each other, whereas other known crosslinking proteins require filaments to be unrotated. Bundles of actin EF-1a would tend to exclude other actin bundling proteins. EF-1a can thus regulate the state of the actin cytoskeleton as well as regulate protein synthesis.     

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.     

Tobacco WLIM1 is a novel F-actin binding protein involved in actin cytoskeleton remodeling

We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)-tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.     

Interplay between Brownian motion and cross-linking controls bundling dynamics in actin networks

Morphology changes in cross-linked actin networks are important in cell motility, division, and cargo transport. Here, we study the transition from a weakly cross-linked network of actin filaments to a heavily cross-linked network of actin bundles through microscopic Brownian dynamics simulations. We show that this transition occurs in two stages: first, a composite bundle network of small and highly aligned bundles evolves from cross-linking of individual filaments and, second, small bundles coalesce into the clustered bundle state. We demonstrate that Brownian motion speeds up the first stage of this process at a faster rate than the second. We quantify the time to reach the composite bundle state and show that it strongly increases as the mesh size increases only when the concentration of cross-links is small and that it remains roughly constant if we decrease the relative ratio of cross-linkers as we increase the actin concentration. Finally, we examine the dependence of the bundling timescale on filament length, finding that shorter filaments bundle faster because they diffuse faster.     

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.     

The myosin head can bind two actin monomers

Force impulse is thought to be generated in muscle when myosin head (S-1), while weakly bound to actin filament, undergoes orientational change to form a strong (rigor) bond with actin. There is ample evidence that this bond involves interaction of 1 myosin head with 1 actin monomer. However, X-ray diffraction data of muscle decorated with S-1, as well as recently proposed model of the thin filaments, suggested that each S-1 molecule interacted with two actin monomers. We reinvestigated this controversy and found that the stoichiometry of acto-S-1 bond depended on the relative amounts of actin and myosin present during titrations: when increasing amounts of actin were added to a fixed amount of S-1 (i.e. when myosin heads were initially in excess over actin), the saturating stoichiometry was 1 mol of S-1 per 1 mol of actin. However, when increasing amounts of S-1 were added slowly to a fixed amount of F-actin (i.e. when actin was initially in excess over S-1), the stoichiometry at saturation was 1 mol of S-1 per 2 mols of actin. The ability of S-1 to bind either one or two actin monomers suggests a way that force could be generated during muscle contraction.