Three-dimensional image analysis of the complex of thin filaments and myosin molecules from skeletal muscle. IV. Reconstitution from minimal- and high-dose images of the actin-tropomyosin-myosin subfragment-1 complex

Three-dimensional images of the actin-tropomyosin-myosin subfragment-1 (S1) complex were reconstituted from both minimal- and high-dose electron micrographs by using a conventional reconstruction technique. Higher resolution (1/15 A-1) than those of the previous reconstructions was attained. A multi-domain structure similar to that of the actin-S1 complex described in the previous paper (1) was observed and a ne diagram of the multi-domain structure of the actin-tropomyosin-S1 complex is presented. The shape of S1 molecules in the rigor complex was clearly resolved. In a view perpendicular to the filament axis, S1 had an axially bent profile; only the tail portion, which was thin but was not small in diameter, was steeply inclined. These features were more prominent in the model from minimal-dose images than that from high-dose images.     

Heavy meromyosin and subfragment-1 from squid mantle myosin, and Ca-sensitivity of their Mg-ATPases

Heavy meromyosin (HMM) and subfragment-1 (S1) were obtained from squid mantle myosin by tryptic digestion and chymotryptic digestion, respectively. Squid HMM(T) and S1(CT) preparations contained stoichiometric amounts of the two types of light chain subunit; regulatory light chain, LC-2, and essential light chain, LC-1. No difference was detected in the chymotryptic digestibilities of squid mantle myosin in Ca-medium and in EDTA-medium. This is in contrast to the digestibility of scallop adductor myosin. The Mg-ATPase activity of HMM(T) alone and that of acto-HMM(T) were both sensitive to calcium ions. In contrast, the activity of S1(CT) alone and that of acto-S1(CT) were both insensitive to calcium ions. The affinity of HMM(T) for actin was not affected by calcium ions, but the amount of HMM(T) bound to actin was increased by calcium ions from 20% to 60% of the total amount of HMM(T). On the other hand, the actin affinity of S1(CT) and the amount of S1(CT) bound to actin were both unaffected by calcium ions. The role of calcium ions in the regulation of contraction in molluscan muscles is discussed.     

The effect of troponin-tropomyosin on the binding of heavy meromyosin to actin in the presence of ATP

In the presence of ATP and the absence of Ca2+, the binding of myosin subfragment-1 to actin is only slightly inhibited by troponin-tropomyosin, while the actin-activated subfragment-1 ATPase rate is 95% inhibited (Chalovich, J. M., Chock, P. B., and Eisenberg, E. (1981) J. Biol. Chem. 256, 575-578). On the other hand, it has been reported the troponin-tropomyosin markedly inhibits the binding of heavy meromyosin (HMM) to actin in the presence of ATP and the absence of Ca2+, providing that the HMM has intact light chain 2 (Wagner, P. D., and Stone, D. (1982) Biochemistry 22, 1334-1342). In the present study, we reinvestigated the binding of HMM with 85% intact light chain 2, to regulated actin. If we assume that only a single population of HMM is present, the binding constant of HMM to regulated actin at 19 mM ionic strength is only about 3 times larger in the presence of Ca2+ than in the absence of Ca2+ (2.4 X 10(4) M-1 compared to 8.8 X 10(3) M-1). On the other hand, if we correct for the population of HMM with degraded light chain 2, the difference in the binding constants in the presence and absence of Ca2+ may be as great as 5-fold. A double binding experiment also suggested that HMM with intact light chain 2 binds at most 5 times more strongly to regulated actin in the presence of Ca2+ than in its absence. We conclude that, just as with subfragment-1, the primary effect of troponin-tropomyosin in regulating the acto HMM ATPase activity is to inhibit a kinetic step in the ATPase cycle. However, our data with HMM also suggest that, in addition to this primary effect, troponin-tropomyosin may modulate the binding of the cross-bridge to actin in relaxed muscle to a small extent.     

Binding of myosin cross-bridges to thin filaments of rabbit skeletal muscle.

Cross-linking of myosin subfragment 1 (S1) with a molar excess of actin in vitro reveals the presence of an actin-S1-actin complex. It is absolutely essential that actin be present in molar excess over S1 so that the decoration of F-actin with S1 be incomplete. However, the excess of actin may not be available in the overlap zone of sarcomeres of skeletal muscle. We therefore found it necessary to test for the presence of the actin-S1-actin complex in vivo. Myofibrils from rabbit skeletal muscle were reacted with zero-length cross-linker, the products were resolved by polyacrylamide gel electrophoresis and analyzed by Western blots using antibodies against actin and against heavy and light chains of myosin. The cross-linking produced the evidence of formation of actin-S1-actin complex.     

Three-dimensional image analysis of the complex of thin filaments and myosin molecules from skeletal muscle. V. Assignment of actin in the actin-tropomyosin-myosin subfragment-1 complex

To assign the actin molecule in the three-dimensional image of the actin-tropomyosin-myosin subfragment-1 (actin-TM-S1) complex, the three-dimensional image of the actin-tropomyosin complex was correlated to that of actin-TM-S1. To assess the similarity of two structures in a quantitative manner, we used a normalized cross-correlation function ("similarity function"). The calculation of similarity indicated that domain A and domain B defined in (1, 2) correspond to actin-tropomyosin. This assignment indicates that one S1 molecule strongly interacts with only one actin molecule, but at least two regions of S1 contribute to the binding. Comparison of the reconstituted models of thin filaments with those of decorated thin filaments suggested a change in the shape of the actin molecule.     

LC20 and kinetics of gizzard myosin subfragment-1: digestion with papain vs. S. aureus protease.

Previous reports have shown that papain-digested gizzard subfragment-1 (PAP-S1) has a cleaved regulatory light chain (LC20), and Vmax similar to phosphorylated heavy meromyosin (HMM) (Greene et al., Biochemistry 22:530-535, 1983; Sellers et al., J. Biol. Chem. 257:13880-13883, 1982; Umemoto et al., J. Biol. Chem. 264:1431-1436, 1989], while S. aureus protease-digested S-1 (SAP-S1) has intact LC20, but Vmax closer to that of unphosphorylated HMM [Ikebe and Hartshorne, 1985]. To determine whether intact LC20 inhibits ATPase activity for subfragment-1 (S1), we compared the kinetic properties and structures of unphosphorylated PAP-S1 and SAP-S1. SDS-PAGE showed that SAP-S1 had 68 and 24 KDa heavy chain and 20 and 17 KDa light chain components. PAP-S1 (15 minutes digestion at 20 degrees C) also had 68 and 17 KDa bands, but the single 24 KDa band (24HC) was replaced by a group of 22-24 KDa fragments and LC20 was cleaved to a 16 KDa fragment. At 13 mM ionic strength, both PAP-S1 and SAP-S1 had Vmax similar to phosphorylated HMM (1.1-1.5 s-1). SAP-S1 had the same KATPase as phosphorylated HMM (38 microM actin), but KATPase for PAP-S1 was 3-fold stronger (11 microM actin). Subsequent digestion of SAP-S1 with papain did not significantly change Vmax, but as LC20 and 24HC were cleaved, both KATPase and Kbinding strengthened 3- to 5-fold. Thus, intact LC20 did not inhibit, and cleavage of LC20 did not increase Vmax for S1. Rather, papain cleavage of LC20 and 24HC was associated with strengthened actin binding.     

Three-dimensional image analysis of the complex of thin filaments and myosin molecules from skeletal muscle. I. Tilt angle of myosin subfragment-1 in the rigor complex

A three-dimensional image of the "rigor" complex of actin and chymotryptic myosin subfragment-1 was reconstituted from electron micrographs of negatively stained specimens. Data went out to 20 A radially and 26 A axially. The reconstituted images allowed us to deduce the angle between the major axis of the main part of myosin subfragment-1 and the axis of the actin helix. The subfragment-1 molecules were attached to the actin filament in a configuration in which they were tilted by only about 15 degrees from the plane perpendicular to the axis of the actin helix. The implication of the smaller tilt angle than the commonly accepted value is discussed.     

Rotational dynamics of spin-labeled F-actin in the sub-millisecond time range.

The rotational motions of F-actin filaments and myosin heads attached to them have been measured by saturation transfer electron paramagnetic resonance spectroscopy using spin-labels rigidly bound to actin, or to the myosin head region in intact myosin molecules, heavy meromyosin, and subfragment-1. The spin-label attached to F-actin undergoes rotational motion having an effective correlation time of the order of 10^?4 seconds. This cannot be interpreted as rotation of the entire F-actin filament or local rotation of the spin-label, but must represent an internal rotational mode of F-actin, possibly a bending or flexing motion, or a rotation of an actin monomer or a segment of it. The rate of this rotational motion is reduced approximately fourfold by myosin, HMM or S-1; HMM and S-1 are equally effective, on a molar basis, in slowing this rotation and both produce their maximal effect at a ratio of about one molecule of HMM or S-1 per ten actin monomers. With chymotryptic S-1, the effect is partially reversed at higher concentrations. With S-1 prepared with papain in the presence of Mg²⁺, the reversal is smaller, while with HMM or myosin there is no reversal at higher concentrations. Tropomyosin slightly decreases the actin rotational mobility, and the addition of HMM to the actin-tropomyosin complex produces a further slowing. The rotational correlation time for acto-HMM is the same whether the spin-label is on actin or HMM, indicating that the rotation of the head region of HMM when bound to F-actin is controlled by a mode of rotation within the F-actin filaments.     

Regulation of the actin-myosin interaction by titin.

Titin is known to interact with actin thin filaments within the I-band region of striated muscle sarcomeres. In this study, we have used a titin fragment of 800 kDa (T800) purified from striated skeletal muscle to measure the effect of this interaction on the functional properties of the actin-myosin complex. MALDI-TOF MS revealed that T800 contains the entire titin PEVK (Pro, Glu, Val, Lys-rich) domain. In the presence of tropomyosin-troponin, T800 increased the sliding velocity (both average and maximum values) of actin filaments on heavy-meromyosin (HMM)-coated surfaces and dramatically decreased the number of stationary filaments. These results were correlated with a 30% reduction in actin-activated HMM ATPase activity and with an inhibition of HMM binding to actin N-terminal residues as shown by chemical cross-linking. At the same time, T800 did not affect the efficiency of the Ca(2+)-controlled on/off switch, nor did it alter the overall binding energetics of HMM to actin, as revealed by cosedimentation experiments. These data are consistent with a competitive effect of PEVK domain-containing T800 on the electrostatic contacts at the actin-HMM interface. They also suggest that titin may participate in the regulation of the active tension generated by the actin-myosin complex.     

Electron paramagnetic resonance and nanosecond fluorescence depolarization studies on creatine-phosphokinase interaction with myosin and its fragments

Recent reports in the literature have indicated a physical association of creatinephosphokinase (CPK) with the tail portion of the myosin molecule. The present paper describes further studies on the interaction of CPK with myosin and myosin fragments, using the techniques of electron paramagnetic resonance (EPR) and nanosecond fluorescence depolarization. From EPR work, spin-labeled CPK appears to interact with myosin, tail-less myosin (heavy meromyosin [HMM]), and myosin heads (subfragment-1 [S1]), the extent of interaction being proportional to the S1 content of myosin or its fragments. Spin-labeled CPK did not evidence interaction with the headless myosin “rods”, with myosin tails (light meromyosin [LMM]), with S2 necks (which connect S1 to the rest of the myosin molecule), or with actin. When a fluorescent dye is directed to the essential ϵ-amino group of CPK, nanosecond fluorescence depolarization studies indicate a substantial interaction with myosin, HMM, and S1, but very little with F-actin. When the “fast-reacting” thiol of the S1 moiety or the “essential thiol” of CPK was labeled with either a fluorescent dye or a spin label, no interaction between CPK and myosin (or S1) was detected.     

Characterization of single actomyosin rigor bonds: load dependence of lifetime and mechanical properties

Load dependence of the lifetime of the rigor bonds formed between a single myosin molecule (either heavy meromyosin, HMM, or myosin subfragment-1, S1) and actin filament was examined in the absence of nucleotide by pulling the barbed end of the actin filament with optical tweezers. For S1, the relationship between the lifetime (tau) and the externally imposed load (F) at absolute temperature T could be expressed as tau(F) = tau(0).exp(-F.d/k(B)T) with tau(0) of 67 s and an apparent interaction distance d of 2.4 nm (k(B) is the Boltzmann constant). The relationship for HMM was expressed by the sum of two exponentials, with two sets of tau(0) and d being, respectively, 62 s and 2.7 nm, and 950 s and 1.4 nm. The fast component of HMM coincides with tau(F) for S1, suggesting that the fast component corresponds to single-headed binding and the slow component to double-headed binding. These large interaction distances, which may be a common characteristic of motor proteins, are attributed to the geometry for applying an external load. The pulling experiment has also allowed direct estimation of the number of myosin molecules interacting with an actin filament. Actin filaments tethered to a single HMM molecule underwent extensive rotational Brownian motion, indicating a low torsional stiffness for HMM. From these results, we discuss the characteristics of interaction between actin and myosin, with the focus on the manner of binding of myosin.     

The effect of Mg-ADP on the structural state of actin in the F-actin-myosin subfragment-1 complex]

Using polarized microfluorometry techniques, a study was made on the orientation and mobility of fluorescent probes 1,5-IAEDANS and rhomadin-phalloidin, located in various parts of actin, muscle fibers free of myosin, tropomyosin and troponin (ghost fibres) being used. It was found that the binding of a myosin subfragment 1 (S1) to actin induced changes in polarized fluorescence of the fibers. The analysis of these data showed that the formation of actin-S1 and actin-S1-ADP complexes in a muscle fiber resulted in a decrease in the angle between the thin filaments and the emission dipole of phalloidin-rhodamine, as well as in an increase of the mobility of this dye. In the experiments with the 1,5-IAEDANS label the angle of emission dipole increased, while the mobility of the label decreased. These changes were smaller in the presence of Mg-ADP than in its absence. It is assumed that the changes in actin monomer structure occur when a myosin head interacts with actin. These changes are expressed as those in orientation and mobility of large and small domains of actin in thin filaments. The domain orientation in actomyosin complex changes, influenced by Mg-ADP. The data obtained allow to propose the involvement of interdomain motions of some parts of actin monomer in the mechanisms of muscle contraction.     

Adiabatic compressibility of myosin subfragment-1 and heavy meromyosin with or without nucleotide.

The partial specific adiabatic compressibilities of myosin subfragment-1 (S1) and heavy meromyosin (HMM) of skeletal muscle in solution were determined by measuring the density and the sound velocity of the solution. The partial specific volumes of S1 and HMM were 0.713 and 0.711 cm3/g, respectively. The partial specific adiabatic compressibilities of S1 and HMM were 4.2 x 10(-12) and 2.9 x 10(-12) cm2/dyn, respectively. These values are in the same range as the most of globular proteins so far studied. The result indicates that the flexibility of S1 region almost equals to that of HMM. After binding to ADP.orthovanadate, S1 and HMM became softer than their complexes with ADP. The bulk moduli of S1 and HMM were of the order of (4-6) x 10(10) dyn/cm2, which are very comparable with the bulk modulus of muscle fiber.     

Proximity of reactive lysyl residue to the antigenic site in rabbit skeletal myosin against the monoclonal antibody (MF-18) generated to chicken skeletal myosin

MF-18, one of the monoclonal antibodies generated to chicken myosin, cross-reacted with rabbit skeletal myosin subfragment-1 (S1). Utilizing an improved procedure of immuno-blotting, a decrease in reactivity of MF-18 to S1 by trinitrophenylation was observed. This indicates that the reactive lysyl residue is very close to the hapten site. This is consistent with the evidence that the hapten site resides in the 26,000 dalton tryptic fragment of S1. Use of such antibodies as labels may open the way to determining the location of specific hapten sites in the three-dimensional image of actin-S1 complex reconstructed from the electron micrographs.     

Fluorescent staining of microfilaments with heavy meromyosin labeled with N-(7-dimethylamino-4-methylcoumarinyl) maleimide

For fluorescent staining of microfilaments in cells, heavy meromyosin (HMM) or subfragment-1 (S-1) was labeled with a novel thiol-directed fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide (DACM), instead of the usual dyes, such as fluorescein-isothiocyanate (FITC). DACM-labeled HMM or S-1 gave characteristic fluorescence patterns to a variety of cell types similar to those reported with the use of FITC-labeled HMM or S-1 or with immunofluorescence techniques using anti-actin antibody. The fluorescence of DACM was fairly photoresistant as compared with FITC, so that HMM or S-1 required only 1 mol of the dye per myosin head. Consequently, F-actin need not be used to preserve the actin binding activity of the myosin fragments when labeling with the dye.     

Two-step ligand binding and cooperativity. A model to describe the cooperative binding of myosin subfragment 1 to regulated actin.

The binding of actin to myosin subfragment 1 (S1) has been shown to occur as a two-step reaction. In the first step actin is weakly bound and then the complex isomerizes to the "rigor type" acto-S1 complex (Coates, J. H., A. H. Criddle, and M. A. Geeves, 1985 Biochem. J., 232:351-356). We propose here a model in which troponin/tropomyosin (Tn/Tm) controls the actin-S1 interaction by inhibiting the isomerization step. In this model the (actin)7 Tn/Tm unit is assumed to exist in two states: open and closed. S1 can bind to either of the two states but only the open form allows the isomerization reaction to take place. We demonstrate that this model can account for the cooperative binding of S1 and S1 nucleotide complexes to actin. The model provides a way of integrating both the effects of calcium and nucleotide on actin-S1 interactions.     

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.     

Superprecipitation induced by soluble myosin fragments.

Superprecipitation (s.p.) took place when $\text{both}$ an active myosin fragment [heavy meromyosin (HMM) or HMM subfragment-1 (S-1)] and an inactivated myosin were added to actin. The duration of the “clearing phase” decreased, while the rate and extent of s.p. increased up to a constant value when the myosin fragment concentration was raised. The extent and rate were higher while the delay time shorter for HMM, as compared to S-1 at the same concentration, No s.p. could be detected when: a) an inactivated myosin fragment or the ATPase apyrase was used; b) MgATP was replaced by Mg-pyrophosphate; c) the ability of myosin to form “rigor” complex with actin has been abolished. It is concluded that the soluble myosin fragment is probably involved in the mechanochemical process associated with s.p..     

Actin-caldesmon-myosin-subfragment-1 ternary complex viewed by electron microscopy. Competitive actin binding region for caldesmon and myosin subfragment-1

An earlier electron microscopic study using different caldesmon forms complexed with actin revealed that the aggregates produced display regular periodic striation after antibody labeling of the 35-kDa caldesmon fragment. This approach provides further evidence that a caldesmon fragment, even as small as 15 kDa, can induce actin filaments to assemble into bundles. The observed difference in the compactness of these structures, depending on the use of the 15-kDa fragment instead of the 35-kDa fragment, suggests the existence of more than one actin-binding site in the caldesmon molecule. In this study, the caldesmon-induced process of F-actin association was investigated in the presence of skeletal myosin subfragment-1, using light-scattering methods, cosedimentation experiment and electron microscopic techniques. We show that the actin-caldesmon association is partially destabilized in the presence of subfragment-1 and this leads to a ternary complex formation. Immunogold labelling of the actin filaments still reveals the presence of caldesmon within this structure. This latter result strengthens the hypothesis that actin has a site(s) able to bind both caldesmon and myosin subfragment-1, as detected by recent NMR observations. This evidence is discussed with respect to the regulatory function of caldesmon during smooth muscle contraction.     

Myosin subfragment binding for the localization of actin-like microfilaments in cultured cells. A light and electron microscope study

Fluorescein-labeled heavy meromyosin subfragment-1 (F-S-1) has been purified by ion exchange chromatography and characterized in terms of its ability to bind specifically to actin. F-S-1 activates the Mg++-adenosine triphosphatase activity of rabbit skeletal muscle actin and decorates actin as shown by negative stains and thin sections of rabbit actin and rat embryo cell microfilament bundles, respectively. Binding of F-S-1 to cellular structures is prevented by pyrophosphate and by competition with excess unlabeled S-1. The F-S-1 is used in light microscope studies to determine the distribution of actin-containing structures in wnterphase and mitotic rat embryo and rat kangaroo cells. Interphase cells display the familiar pattern of fluorescent stress fibers. Chromosome-to-pole fibers are fluorescent in mitotic cells. The glycerol extraction procedures employed provide an opportunity to examine cells prepared in an identical manner by light and electron microscopy. The latter technique reveals that actin-like microfilaments are identifiable in spindles of glycerinated cells before and after addition of S-1 or HMM. In some cases, microfilaments appear to be closely associated with spindle microtubles. Comparison of the light and electron microscope results aids in the evaluation of the fluorescent myosin fragment technique and provides further evidence for possible structural and functional roles of actin in the mitotic apparatus.