Flexibility in actin-myosin motility system revealed by in vitro motility assay

Flexibility of myosin molecule was studied by in vitro motility assay in terms of the direction of actin movement. Electron microscopy showed that HMM scattered on a nitrocellulose surface can bind actin filaments and form arrowhead-like patterns. Actin filaments can move in both directions on tracks of HMM made on a nitrocellulose surface. Further, actin filaments can move bidirectionally along native thick filaments over their central bare zone. These observations indicate that there is considerable flexibility in a myosin molecule and that the direction of the movement is determined by the polarity of actin filaments.     

Effects of urea and guanidine hydrochloride on the sliding movement of actin filaments with ATP hydrolysis by myosin molecules

To evaluate the role of the hydration layer on the protein surface of actomyosin, we compared the effects of urea and guanidine-HCl on the sliding velocities and ATPase activities of the actin-heavy meromyosin (HMM) system. Both chemicals denature proteins, but only urea perturbs the hydration layer. Both the sliding velocity of actin filaments and actin-activated ATPase activity decreased with increasing urea concentrations. The sliding movement was completely inhibited at 1.0 M urea, while actin filaments were bound to HMM molecules fixed on the glass surface. Guanidine-HCl (0-0.05 M) drastically decreased both the sliding velocity and ATPase activation of acto-HMM complexes. Under this condition, actin filaments almost detached from HMM molecules. In contrast, the ATPase activity of HMM without actin filaments was almost independent of urea concentrations <1.0 M and guanidine-HCl concentrations <0.05 M. An increase in urea concentrations up to 2.0 M partly induced changes in the ternary structure of HMM molecules, while the actin filaments were stable in this concentration range. Hydration changes around such actomyosin complexes may alter both the stability of part of the myosin molecules, and the affinity for force transmission between actin filaments and myosin heads.     

Spatiotemporal Dynamics of Actomyosin Networks

Rhodamine–phalloidin-labeled actin filaments were visualized gliding over a skeletal heavy meromyosin (HMM)-coated surface. Experiments at low filament densities showed that when two filaments collided, their paths were affected in a manner that depended on collision angle. Some collisions resulted in complete alignment of the filament paths; in others, the filaments crossed over one another. Filament crossover or alignment was equally probable at ∼40° contact angle. Filaments often underwent significant bending during collision and analysis of filament shape indicated an energy requirement of ∼13 k_BT. Experiments were performed over a wide range of HMM surface density and actin filament bulk concentration. Actin filament gliding speed and path persistence plateaued above a critical HMM surface density, and at high (micromolar) actin filament concentrations, filament motion became dramatically aligned in a common direction. Spatiotemporal features of alignment behavior were determined by correlation analysis, supported by simulations. The thermal drift of individual filament tracks was suppressed as the population became more oriented. Spatial correlation analysis revealed that long-range alignment was due to incremental recruitment rather than fusion of locally ordered seed domains. The global alignment of filament movement, described by an “order parameter,” peaked at optimal actin concentrations and myosin surface densities, in contrast to previous predictions of a critical phase transition. Either hydrodynamic coupling or exchange of filaments between the surface bound and adjacent bulk phase layers might degrade order at high actin filament concentration, and high HMM surface densities might decrease alignment probability during collisions. Our results are compatible with generation of long-range order from mechanical interaction between individual actin filaments. Furthermore, we show that randomly oriented myosin motors align relatively short, submicrometer actin filaments into motile surface domains that extend over many tens of micrometers and these patterns persist for several minutes.     

Spatiotemporal Dynamics of Actomyosin Networks

Rhodamine–phalloidin-labeled actin filaments were visualized gliding over a skeletal heavy meromyosin (HMM)-coated surface. Experiments at low filament densities showed that when two filaments collided, their paths were affected in a manner that depended on collision angle. Some collisions resulted in complete alignment of the filament paths; in others, the filaments crossed over one another. Filament crossover or alignment was equally probable at ∼40° contact angle. Filaments often underwent significant bending during collision and analysis of filament shape indicated an energy requirement of ∼13 k_BT. Experiments were performed over a wide range of HMM surface density and actin filament bulk concentration. Actin filament gliding speed and path persistence plateaued above a critical HMM surface density, and at high (micromolar) actin filament concentrations, filament motion became dramatically aligned in a common direction. Spatiotemporal features of alignment behavior were determined by correlation analysis, supported by simulations. The thermal drift of individual filament tracks was suppressed as the population became more oriented. Spatial correlation analysis revealed that long-range alignment was due to incremental recruitment rather than fusion of locally ordered seed domains. The global alignment of filament movement, described by an “order parameter,” peaked at optimal actin concentrations and myosin surface densities, in contrast to previous predictions of a critical phase transition. Either hydrodynamic coupling or exchange of filaments between the surface bound and adjacent bulk phase layers might degrade order at high actin filament concentration, and high HMM surface densities might decrease alignment probability during collisions. Our results are compatible with generation of long-range order from mechanical interaction between individual actin filaments. Furthermore, we show that randomly oriented myosin motors align relatively short, submicrometer actin filaments into motile surface domains that extend over many tens of micrometers and these patterns persist for several minutes.     

Translational motion of actin filaments in the presence of heavy meromyosin and MgATP as measured by Doppler broadening of laser light scattering

Intensity fluctuations of laser light scattering were utilized in order to follow enhancement of translational motion of the actin-heavy meromyosin (HMM) complex in extremely dilute solutions accompanied by the hydrolysis of MgATP. Such enhancement was anticipated on the basis of the idea that active streaming along actin filaments should be associated with their mechanochemical reactivity. Native tropomyosin was added in order to stabilize actin in its filamentous form, thus allowing the reduction of actin concentration below 50 micrograms/ml to enable free movement of neighboring filaments and yet give a reliable signal. Analysis of the data in terms of Doppler broadening led to an approximate evaluation of the average velocity of translation of the mobile filaments. This velocity was found to increase with increasing HMM concentration up to a maximum attained at a molar ratio HMM/actin of 1:2, and then decreased. Total intensity measurements indicate that the mobile scatterer is actually a complex of HMM with an isolated actin filament. HMM subfragment-1 was found to be ineffective. These results suggest that cooperation between the two myosin heads is necessary for efficient induction of active streaming along isolated actin filaments.     

Subtilisin cleavage of actin inhibits in vitro sliding movement of actin filaments over myosin

Subtilisin cleaved actin was shown to retain several properties of intact actin including the binding of heavy meromyosin (HMM), the dissociation from HMM by ATP, and the activation of HMM ATPase activity. Similar Vmax but different Km values were obtained for acto-HMM ATPase with the cleaved and intact actins. The ATPase activity of HMM stimulated by copolymers of intact and cleaved actin showed a linear dependence on the fraction of intact actin in the copolymer. The most important difference between the intact and cleaved actin was observed in an in vitro motility assay for actin sliding movement over an HMM coated surface. Only 30% of the cleaved actin filaments appeared mobile in this assay and moreover, the velocity of the mobile filaments was approximately 30% that of intact actin filaments. These results suggest that the motility of actin filaments can be uncoupled from the activation of myosin ATPase activity and is dependent on the structural integrity of actin and perhaps, dynamic changes in the actin molecule.     

Motion of actin filaments in the presence of myosin heads and ATP.

We measured, by fluorescence correlation spectroscopy, the motion of actin filaments in solution during hydrolysis of ATP by acto-heavy meromyosin (acto-HMM). The method relies on the fact that the intensity of fluorescence fluctuates as fluorescently labeled actin filaments enter and leave a small sample volume. The rapidity of these number fluctuations is characterized by the autocorrelation function, which decays to 0 in time that is related to the average velocity of translation of filaments. The time of decay of the autocorrelation function of bare actin filaments in solution was 10.59 +/- 0.85 s. Strongly bound (rigor) heads slowed down the diffusion. Direct observation of filaments under an optical microscope showed that addition of HMM did not change the average length or flexibility of actin filaments, suggesting that the decrease in diffusion was not due to a HMM-induced change in the shape of filaments. Rather, slowing down of translational motion was caused by an increase in the volume of the diffusing complex. Surprisingly, the addition of ATP to acto-HMM accelerated the motion of actin filaments. The acceleration was the greatest at the low molar ratios of HMM:actin. Direct observation of filaments under an optical microscope showed that in the presence of ATP the average length of filaments did not change and that the filaments became stiffer, suggesting that acceleration of diffusion was not due to an ATP-induced increase in flexibility of filaments. These results show that some of the energy of splitting of ATP is impaired to actin filaments and suggest that 0.06 +/- 0.02 of HMM interferes with the diffusion of actin filaments during hydrolysis of ATP.     

Actomyosin motility on nanostructured surfaces

We have here, for the first time, used nanofabrication techniques to reproduce aspects of the ordered actomyosin arrangement in a muscle cell. The adsorption of functional heavy meromyosin (HMM) to five different resist polymers was first assessed. One group of resists (MRL-6000.1XP and ZEP-520) consistently exhibited high quality motility of actin filaments after incubation with HMM. A second group (PMMA-200, PMMA-950, and MRI-9030) generally gave low quality of motility with only few smoothly moving filaments. Based on these findings electron beam lithography was applied to a bi-layer resist system with PMMA-950 on top of MRL-6000.1XP. Grooves (100-200nm wide) in the PMMA layer were created to expose the MRL-6000.1XP surface for adsorption of HMM and guidance of actin filament motility. This guidance was quite efficient allowing no U-turns of the filaments and approximately 20 times higher density of moving filaments in the grooves than on the surrounding PMMA.     

Mitochondria-associated myosin 19 processively transports mitochondria on actin tracks in living cells

Mitochondria are fundamentally important in cell function, and their malfunction can cause the development of cancer, cardiovascular disease, and neuronal disorders. Myosin 19 (Myo19) shows discrete localization with mitochondria and is thought to play an important role in mitochondrial dynamics and function; however, the function of Myo19 in mitochondrial dynamics at the cellular and molecular levels is poorly understood. Critical missing information is whether Myo19 is a processive motor that is suitable for transportation of mitochondria. Here, we show for the first time that single Myo19 molecules processively move on actin filaments and can transport mitochondria in cells. We demonstrate that Myo19 dimers having a leucine zipper processively moved on cellular actin tracks in demembraned cells with a velocity of 50 to 60 nm/s and a run length of ∼0.4 μm, similar to the movement of isolated mitochondria from Myo19 dimer-transfected cells on actin tracks, suggesting that the Myo19 dimer can transport mitochondria. Furthermore, we show single molecules of Myo19 dimers processively moved on single actin filaments with a large step size of ∼34 nm. Importantly, WT Myo19 single molecules without the leucine zipper processively move in filopodia in living cells similar to Myo19 dimers, whereas deletion of the tail domain abolished such active movement. These results suggest that Myo19 can processively move on actin filaments when two Myo19 monomers form a dimer, presumably as a result of tail–tail association. In conclusion, Myo19 molecules can directly transport mitochondria on actin tracks within living cells.     

Heavy meromyosin induces sliding movements between antiparallel actin filaments

Suzuki et al. [Biochemistry 28, 6513-6518 (1989)] have shown that, when F-actin is mixed with inert high polymer, a large number of actin filaments closely align in parallel with overlaps to form a long and thick bundle. The bundle may be designated non-polar, as the constituent filaments are random in polarity (Suzuki et al. 1989). I prepared non-polar bundles of F-actin using methylcellulose (MC) as the high polymer, exposed them to heavy meromyosin (HMM) in the presence of ATP under a light microscope, and followed their morphological changes in the continuous presence of MC. It was found that bundles several tens of micrometers long contracted to about one-third the initial length, while becoming thicker, in half a minute after exposure to HMM. Subsequently, each bundle was split longitudinally into several bundles in a stepwise manner, while the newly formed ones remained associated together at one of the two ends. The product, an aster-like assembly of actin bundles, was morphologically quiescent; that is, individual bundles never contracted upon second exposure to HMM and ATP, although they were still longer than the F-actin used. Bundles in this state consisted of filaments with parallel polarity as examined by electron microscopy. This implies that non-polar bundles were transformed into assemblies of polar bundles with ATP hydrolysis by HMM. Importantly, myosin subfragment-1 caused neither contraction nor transformation. These results are interpreted as follows. In the presence of ATP, the two-headed HMM molecule was able to cross-bridge antiparallel actin filaments, as well as parallel ones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Up-and-down movement of a sliding actin filament in the in vitro motility assay

We observed a three-dimensional up-and-down movement of an actin filament sliding on heavy mero-myosin (HMM) molecules in an in vitro motility assay. The up-and-down movement occurred along the direction perpendicular to the planar glass plane on which the filament demonstrated a sliding movement. The height length of the up-and-down movement was measured by monitoring the extent of diminishing fluorescent emission from the marker attached to the filament in the evanescent field of attenuation. The height lengths whose distribution exhibits a local maximum were found around the two values, 150 nm and 90 nm, separately. This undulating three-dimensional movement of an actin filament suggests that the interactions between myosin (HMM) molecules and the actin filament may temporally be modulated during its sliding movement.     

Fine filaments in lymphatic endothelial cells

Several and various types of cells contain fine cytoplasmic filaments closely resembling the myofilaments of muscle cells (2, 18, 23, 24). In many of these cells and especially when cultured, it has been demonstrated that some of these filaments react with heavy meromyosin (HMM) in the same way as do the actin filaments of muscle cells (3, 6 7). This suggests that these filaments may be actinoid and form part of a contractile system. As fine intracytoplasmic filaments do occur in lymphatic endothelial cells (2, 14), we undertook an electron microscope investigation of their fine structure and their reaction on incubation with HMM and EDTA. We postulated that lymphatic endothelial cells possess a contractile filamentous system to which these filaments belong.     

Troponin is a potential regulator for actomyosin interactions

Troponin extracted from rabbit skeletal muscle directly binds to an actin filament in a molar ratio of 1:1 even in the absence of tropomyosin. An actin filament decorated with troponin did not exhibit significant difference from pure actin filaments in the maximum rate of actomyosin ATP hydrolysis and the sliding velocity of the filament examined by means of an in vitro motility assay. However, the relative number of troponin-bound actin filaments moving in the absence of calcium ions decreased to half that in their presence. The amount of HMM bound to the filaments was less than 4% of actin monomers in the presence of TNs. In addition, actin filaments could not move when Tn molecules were bound in the molar ratio of about 1:1 although they sufficiently bind to myosin heads. These results indicate that troponin can transform an actin monomer within a filament into an Off-state without sterically blocking of the myosin-binding sites with tropomyosin molecules.     

Intermonomer cross-linking of F-actin alters the dynamics of its interaction with H-meromyosin in the weak-binding state

Previous cross-linking studies [Kim E, Bobkova E, Hegyi G, Muhlrad A & Reisler E (2002) Biochemistry 41, 86-93] have shown that site-specific cross-linking among F-actin monomers inhibits the motion and force generation of actomyosin. However, it does not change the steady-state ATPase parameters of actomyosin. These apparently contradictory findings have been attributed to the uncoupling of force generation from other processes of actomyosin interaction as a consequence of reduced flexibility at the interface between actin subdomains-1 and -2. In this study, we use EPR spectroscopy to investigate the effects of cross-linking constituent monomers upon the molecular dynamics of the F-actin complex. We show that cross-linking reduces the rotational mobility of an attached probe. It is consistent with the filaments becoming more rigid. Addition of heavy meromyosin (HMM) to the cross-linked filaments further restricts the rotational mobility of the probe. The effect of HMM on the actin filaments is highly cooperative: even a 1 : 10 molar ratio of HMM to actin strongly restricts the dynamics of the filaments. More interesting results are obtained when nucleotides are also added. In the presence of HMM and ADP, similar strongly reduced mobility of the probe was found than in a rigor state. In the presence of adenosine 5'[betagamma-imido] triphosphate (AMPPNP), a nonhydrolyzable analogue of ATP, weak binding of HMM to either cross-linked or native F-actin increases probe mobility. By contrast, weak binding by the HMM/ADP/AlF4 complex has different effects upon the two systems. This protein-nucleotide complex increases probe mobility in native actin filaments, as does HMM + AMPPNP. However, its addition to cross-linked filaments leaves probe mobility as constrained as in the rigor state. These findings suggest that the dynamic change upon weak binding by HMM/ADP/AlF4 which is inhibited by cross-linking is essential to the proper mechanical behaviour of the filaments during movement.     

Novel Mode of Cooperative Binding between Myosin and Mg-actin Filaments in the Presence of Low Concentrations of ATP

Cooperative interaction between myosin and actin filaments has been detected by a number of different methods, and has been suggested to have some role in force generation by the actomyosin motor. In this study, we observed the binding of myosin to actin filaments directly using fluorescence microscopy to analyze the mechanism of the cooperative interaction in more detail. For this purpose, we prepared fluorescently labeled heavy meromyosin (HMM) of rabbit skeletal muscle myosin and Dictyostelium myosin II. Both types of HMMs formed fluorescent clusters along actin filaments when added at substoichiometric amounts. Quantitative analysis of the fluorescence intensity of the HMM clusters revealed that there are two distinct types of cooperative binding. The stronger form was observed along Ca²⁺-actin filaments with substoichiometric amounts of bound phalloidin, in which the density of HMM molecules in the clusters was comparable to full decoration. The novel, weaker form was observed along Mg²⁺-actin filaments with and without stoichiometric amounts of phalloidin. HMM density in the clusters of the weaker form was several-fold lower than full decoration. The weak cooperative binding required sub-micromolar ATP, and did not occur in the absence of nucleotides or in the presence of ADP and ADP-Vi. The G680V mutant of Dictyostelium HMM, which over-occupies the ADP-Pi bound state in the presence of actin filaments and ATP, also formed clusters along Mg²⁺-actin filaments, suggesting that the weak cooperative binding of HMM to actin filaments occurs or initiates at an intermediate state of the actomyosin-ADP-Pi complex other than that attained by adding ADP-Vi.     

Inorganic polyphosphate hydrolysis catalyzed by skeletal muscular actomyosin complexes is uncoupled with motility

Hydrolysis of the triphosphate moiety of ATP, catalyzed by myosin, induces alterations in the affinity of the myosin heads for actin filaments via conformational changes, thereby causing motility of the actomyosin complexes. To elucidate the contribution of the triphosphate group attached to adenosine, we examined the enzymatic activity of heavy meromyosin (HMM) with actin filaments for inorganic tripolyphosphate (3PP) using a Malachite green method and evaluated using fluorescence microscopy the effects of 3PP on actin filament motility on HMM-coated glass slides. In the presence of MgCl₂, HMM hydrolyzed 3PP at a maximum rate of 0.016 s⁻¹ HMM⁻¹, which was four times lower than the hydrolysis rate of ATP. Tetrapolyphosphate (4PP) was hydrolyzed at a rate similar to that of 3PP hydrolysis. The hydrolysis rates of 3PP and 4PP were enhanced by roughly 10-fold in the presence of actin filaments. In motility assays, the presence of polyphosphates did not lead to the sliding movement of actin filaments. Moreover, in the presence of ATP at low concentrations, the sliding velocity of actin filaments decreased as the concentration of added polyphosphate increased, indicating a competitive binding of polyphosphate to myosin heads with ATP. These results suggested that the energy produced by standalone triphosphate hydrolysis did not induce the unidirectional motion of actomyosin and that the link between triphosphate and adenosine was crucial for motility.     

The binding of myosin heads on heavy meromyosin and assembled myosin to actin in the presence of nucleotides. Measurements by the proteolytic rates method

The initial rates of tryptic digestion at the 50/20-kDa junction in myosin and myosin subfragment 1 were determined for the free proteins and their complexes with actin in the presence and absence of MgATP. The proteolytic reactions were carried out at 24 degrees C and under ionic strength conditions (mu) adjusted to 35, 60, and 130 mM. The percentages of myosin heads and myosin subfragment 1 bound to actin in the presence of MgATP were calculated from the rates of proteolysis for each set of digestion experiments. In all cases, the myosin heads in the synthetic filaments showed greater binding to actin than myosin subfragment 1. This binding difference was most prominent (3-fold) at mu = 130 mM. The binding of heavy meromyosin (HMM) to actin in the presence of MgADP was measured at 4 degrees C by ultracentrifugation and the proteolytic rates methods. Ultracentrifugation experiments determined the fraction of HMM molecules bound to actin in the presence of MgADP, whereas the proteolytic measurements yielded the information on the fraction of HMM heads bound to actin. Taken together, these measurements show that a significant fraction of HMM is bound to actin with only one head in the presence of MgADP under ionic conditions of 180 and 280 mM.     

Disturbance of endomembrane trafficking by brefeldin A and calyculin A reorganizes the actin cytoskeleton of <Emphasis Type="Italic">Lilium longiflorum</Emphasis> pollen tubes

We investigated the effect of brefeldin A on membrane trafficking and the actin cytoskeleton of pollen tubes of Lilium longiflorum with fluorescent dyes, inhibitor experiments, and confocal laser scanning microscopy. The formation of a subapical brefeldin A-induced membrane aggregation (BIA) was associated with the formation of an actin basket from which filaments extended towards the tip. The orientation of these actin filaments correlated with the trajectories of membrane material stained by FM dyes, suggesting that the BIA-associated actin filaments are used as tracks for retrograde transport. Analysis of time series indicated that these tracks (actin filaments) were either stationary or glided along the plasma membrane towards the BIA together with the attached membranes or organelles. Disturbance of the actin cytoskeleton by cytochalasin D or latrunculin B caused immediate arrest of membrane trafficking, dissipation of the BIA and the BIA-associated actin basket, and reorganization into randomly oriented actin rods. Our observations suggest that brefeldin A causes ectopic activation of actin-nucleating proteins at the BIA, resulting in retrograde movement of membranes not only along but also together with actin filaments. We show further that subapical membrane aggregations and actin baskets supporting retrograde membrane flow can also be induced by calyculin A, indicating that dephosphorylation by type 2 protein phosphatases is required for proper formation of membrane coats and polar membrane trafficking.     

The tail of myosin reduces actin filament velocity in the in vitro motility assay

It has been observed that heavy meromyosin (HMM) propels actin filaments to higher velocities than native myosin in the in vitro motility assay, yet the reason for this difference has remained unexplained. Since the major difference between these two proteins is the presence of the tail in native myosin, we tested the hypothesis that unknown interactions between actin and the tail (LMM) slow motility in native myosin. Chymotryptic HMM and LMM were mixed in a range of molar ratios (0-5 LMM/HMM) and compared to native rat skeletal myosin in the in vitro motility assay at 30 degrees C. Increasing proportions of LMM to HMM slowed actin filament velocities, becoming equivalent to native myosin at a ratio of 3 LMM/HMM. NH4+ -ATPase assays demonstrated that HMM concentrations on the surface were constant and independent of LMM concentration, arguing against a simple displacement mechanism. Relationships between velocity and the number of available heads suggested that the duty cycle of HMM was not altered by the presence of LMM. HMM prepared with a lower chymotrypsin concentration and with very short digestion times moved actin at the same high velocity. The difference between velocities of actin filament propelled by HMM and HMM/LMM decreased with increasing ionic strength, suggesting that ionic bonds between myosin tail and actin filaments may play a role in slowing filament velocity. These data suggest the high velocities of actin filaments over HMM result from the absence of drag generated by the myosin tail, and not from proteolytic nicking of the motor domain.     

The rigor configuration of smooth muscle heavy meromyosin trapped by a zero-length cross-linker

When chicken gizzard heavy meromyosin (HMM) in its rigor complex with actin was reacted with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), HMM cross-linked with actin but also the two heads of the HMM molecule cross-linked to each other [Onishi, H., Maita, T., Matsuda, G., & Fujiwara, K. (1989) Biochemistry 28, 1898-1904, 1905-1912]. By ultracentrifugal fractionation of the EDC-treated acto-HMM in the presence of Mg-ATP, we obtained a preparation enriched for gizzard HMM with cross-linked heads. When HMM molecules in this preparation were rotary-shadowed and observed in an electron microscope, many head pairs were in contact with each other. The amount of HMM with cross-linked heads determined by electron microscopy was equal to that of the cross-linked NH2-terminal 24K tryptic fragments of HMM heavy chains determined by NaDodSO4 gel electrophoresis, indicating that this cross-linking is primarily responsible for the contact observed between two HMM heads. Most pairs of the contacted heads originated in the same HMM molecule, although a few pairs belonged to different HMM molecules. Cross-linking between the two heads of the same HMM molecule appeared to occur within the distal, more globular half of each head. However, the cross-linking sites were located at different positions within the globular portion. The actin-activated Mg-ATPase activity of the HMM sample treated with EDC in the presence of actin increased in a biphasic manner, depending on the concentration of F-actin, with two apparent association constants: 2.9 x 10(4) M-1 and one much less than 1 x 10(4) M-1. Since the apparent association constant obtained with the HMM control was similar to the latter value, the association constant for HMM molecules with cross-linked heads was identified to be the former value. The binding of HMM to actin was thus strengthened at least by a factor of 3 by the cross-linking between two HMM heads. These results suggest that HMM heads are trapped by treatment with EDC in the rigor complex configuration and that this configuration is retained even after the HMM has been released from actin. The EDC reactivity of rabbit skeletal muscle HMM, however, was different from that of chicken gizzard HMM. The treatment of acto-HMM complexes with EDC did not generate cross-linking between two skeletal muscle HMM heads.