Visualization of caldesmon binding to synthetic filaments of smooth muscle myosin

We have used synthetic filaments of unphosphorylated chicken gizzard myosin with a compact, highly ordered structure under relaxing conditions (in the absence of Ca²⁺ and in the presence of ATP) to visualize the mode of caldesmon binding to myosin filaments by negative staining and immunogold electron microscopy. We demonstrate that the addition of caldesmon to preformed myosin filaments leads to the appearance of numerous smooth projections curving out from the filament surface. The addition of caldesmon or its N-terminal fragment resulted in the partial masking of myosin filament periodicity. However, it did not change the inner structure of the filaments. It is demonstrated that most caldesmon molecules bind to myosin filaments through the N-terminal part, while the C-terminal parts protrude from the filament surface, as confirmed by immunoelectron microscopy visualization. Together with the available biochemical data on caldesmon binding to both actin and myosin and electron microscopic observations on the mode of caldesmon attachment to actin filaments with the C-termini of the molecules curving out from the filaments, the visualization of caldesmon attachment to myosin filaments completes the scenario of actin to myosin tethering by caldesmon. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of unphosphorylated smooth muscle myosin on caldesmon-mediated regulation of actin filament velocity

Summary The effect of smooth muscle myosin at different levels of light chain phosphorylation on caldesmon-mediated movement of actin filaments was investigated using an in vitro motility assay. Myosin at different levels of phosphorylation was obtained by mixing different proportions of fully phosphorylated and unphosphorylated myosin in monomeric form, while keeping the total myosin concentration constant. The average velocity of actin filaments containing tropomyosin was 1.20±0.046 m s^–1 at 30°C with fully phosphorylated myosin. This velocity was not altered when the percentage of unphosphorylated myosin coated on the nitrocellulose surface was increased to 80%; further increases lowered the velocity. When the actin filaments with caldesmon bound at stoichiometric levels were used, filament velocity was unaffected until 50% of the myosin was unphosphorylated, but further increases in the percentage of unphosphorylated myosin induced a decrease in the velocity, and at 95% unphosphorylated myosin, filament movement had ceased. The decreased filament velocity in the presence of caldesmon was also observed when phosphorylated myosin was mixed with myosin rod instead of unphosphorylated myosin, but was not observed when the 38 kDa caldesmon C-terminal fragment, which lacks the myosin-binding domain, was used instead of intact caldesmon. These data indicate that the decreased filament velocity in the presence of caldesmon reflects the mechanical load produced by the tethering of actin to myosin through the interaction of the caldesmon N-terminal domain and the myosin S-2 region. The tethering effect mediated by caldesmon may play a role in smooth muscle contraction when a large number of myosin heads are dephosphorylated, as in force maintenance.     

Ultrastructure,function and composition of smooth muscle

Filamentous myosin is present in both relaxed (myosin light chains unphosphorylated) and contracted (light chains phosphorylated) vascular smooth muscle. The organization of myosin and actin filaments and the insertion of the latter on cytoplasmic and plasma membrane bound dense bodies is consistent with a mini sarcomere-like organization and a sliding filament mechanism of contraction in smooth muscle. Mitochondria are high capacity, low affinity Ca stores in smooth muscle. They do not play a role in the regulation of cytoplasmic Ca²⁺ at physiological levels. The localization and Ca content of the junctional sarcoplasmatic reticulum (SR) is consistent with this organelle being the major intracellular source of activator Ca released by excitatory transmitters. Repeated contractions in the absence of extracellular Ca²⁺ (thought to represent recycling of intracellular activator Ca²⁺) can be demonstrated if the excitatory agent is not allowed to remain in contact with the smooth muscle throughout relaxation; the demonstration of “recycling” is facilitated if the efflux of cellular Ca²⁺ is blocked. The rise in total cytoplasmic calcium measured with electron probe analysis during a maintained (30 min) contracture in rabbit portal-anterior mesenteric vein smooth muscle (∼0.9 mmol/kg dry cytoplasm) is greater than the amount of Ca that could be bound to calmodulin.     

Myosin filaments isolated from skinned amphibian smooth muscle cells are side-polar

Summary The structure of myosin filaments isolated from skinned toad stomach smooth muscle cells has been examined by electron microscopy as a step toward identifying thein vivo structure. When negatively stained following exposure to relaxing conditions, the filaments exhibited a continuous 14-nm axial repeat of crossbridge projections with no central bare zone. The filaments thus differed from the bipolar filaments found in striated muscle and displayed instead features resembling side-polar and mixed-polarity filament models. By rotation of isolated filaments around their longitudinal axes it was found that cross bridges occurred only along two sides of the filament, an arrangement consistent with the side-polar but not the mixed-polarity model. The polarity is thus similar to that proposed for ribbons (Small & Squire,J. molec. Biol. 67, (1972) 17–149) and for synthetic smooth muscle myosin filaments (Craig and Megerman,J. Cell Biol. 75, (1977) 990–996); their appearance in cross-section, however, shows that these structures are filaments (i.e. with two axes of similar dimensions) and not broad ribbons. As the filaments were derived directly from skinned cells which contracted and relaxed in response to physiological levels of MgATP and Ca²⁺ at rates comparable to those of native, isolated cells, this unusual arrangement of cross bridges appears to be an effective, functional form of myosin in the contractile apparatus. Side-polar filaments therefore merit consideration as plausible candidates for the native organization of myosin in vertebrate smooth muscle cells.     

Myosin light chain kinase: functional domains and structural motifs

Conventional myosin light chain kinase found in differentiated smooth and non-muscle cells is a dedicated Ca²⁺/calmodulin-dependent protein kinase which phosphorylates the regulatory light chain of myosin II. This phosphorylation increases the actin-activated myosin ATPase activity and is thought to play major roles in a number of biological processes, including smooth muscle contraction. The catalytic domain contains residues on its surface that bind a regulatory segment resulting in autoinhibition through an intrasteric mechanism. When Ca²⁺/calmodulin binds, there is a marked displacement of the regulatory segment from the catalytic cleft allowing phosphorylation of myosin regulatory light chain. Kinase activity depends upon Ca²⁺/calmodulin binding not only to the canonical calmodulin-binding sequence but also to additional interactions between Ca²⁺/calmodulin and the catalytic core. Previous biochemical evidence shows myosin light chain kinase binds tightly to actomyosin containing filaments. The kinase has low-affinity myosin and actin binding sites in Ig-like motifs at the N- and C-terminus, respectively. Recent results show the N-terminus of myosin light chain kinase is responsible for filament binding in vivo. However, the apparent binding affinity is greater for smooth muscle myofilaments, purified thin filaments, or actin-containing filaments in permeable cells than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. These results suggest a protein on actin thin filaments that may facilitate kinase binding. Myosin light chain kinase does not dissociate from filaments in the presence of Ca²⁺/calmodulin raising the interesting question as to how the kinase phosphorylates myosin in thick filaments if it is bound to actin-containing thin filaments.     

Cross-bridge movement in rat slow skeletal muscle as a function of calcium concentration

A single fibre bundle from rat soleus muscle was chemically skinned with saponin and the transfer of myosin heads from the thick filaments to the thin filaments at a sarcomere length of 2.4 μm was measured as a function of Ca²⁺ concentration using an x-ray diffraction method at 4–7 °C. In the relaxed state, the 1,0 spacing was 42.08 nm. The spacing showed no significant decrease when the Ca²⁺ concentration was below the threshold (−log₁₀ [Ca²⁺] or pCa 5.8). No significant transfer of the myosin heads occurred when the Ca²⁺concentration was below the threshold (pCa 5.8). When the muscle was maximally activated at pCa 4.4, the spacing decreased to 40.35 nm. During the maximum isometric contraction at pCa 4.4, 54.9 ± 6.5% (±SE of the mean) of the myosin heads were transferred to the thin filaments. The transfer of the myosin heads was approximately proportional to relative tension. These results suggest that myosin heads of both fast-twitch and slow-twitch skeletal muscles transferred on the common movement as a function of Ca²⁺ concentration. Received: 1 December 1995/Received after revision and accepted: 20 May 1996     

Polylysine binding to unphosphorylated smooth muscle myosin enhances formation and stabilizes myosin filaments in vitro

Previously, we demonstrated that positively charged polylysine, our model for biological polyamines, activates the Mg2+ ATPase activity of unphosphorylated smooth muscle myosin and shifts the myosin conformation from the folded 10S to linear 6S form. These effects of polylysine were reversed by the oppositely charged heparin (Szymanski et al. (1993) Am J Physiol 265, C379). In the present report, we provide further information on polylysine binding to smooth muscle myosin, and test the hypothesis that polylysine binding to unphosphorylated myosin involves filament formation. To relate the effects of polylysine on contractility in smooth muscle to physiologically relevant material, we investigated the ability of naturally occurring positively charged polyamines, histones, cadaverine, putrescine and spermidine to activate the Mg2+ ATPase activity of unphosphorylated smooth muscle myosin. Our data show that polylysine binding to individual unphosphorylated myosin molecules stimulates formation of myosin filaments. Polylysine also interacts with myosin filaments, causing enhancement of their size and the numbers, and this could be reversed by heparin. Polylysine binding to myosin filaments made them more resistant to disassembly by high salt concentrations (KCl) or ATP. Naturally occurring polyamines in millimolar concentrations activate the Mg2+ ATPase activity of unphosphorylated smooth muscle myosin. We suggest that the electrostatic interactions between naturally occurring positively charged polyamines and unphosphorylated smooth muscle myosin may play a role in stabilization of thick filament structurein situ.     

Smooth muscle myosin filament assembly under control of a kinase-related protein (KRP) and caldesmon

Kinase-related protein (KRP) and caldesmon are abundant myosin-binding proteins of smooth muscle. KRP induces the assembly of unphosphorylated smooth muscle myosin filaments in the presence of ATP by promoting the unfolded state of myosin. Based upon electron microscopy data, it was suggested that caldesmon also possessed a KRP-like activity (Katayama et al., 1995, J Biol Chem 270: 3919–3925). However, the nature of its activity remains obscure since caldesmon does not affect the equilibrium between the folded and unfolded state of myosin. Therefore, to gain some insight into this problem we compared the effects of KRP and caldesmon, separately, and together on myosin filaments using turbidity measurements, protein sedimentation and electron microscopy. Turbidity assays demonstrated that KRP reduced myosin filament aggregation, while caldesmon had no effect. Additionally, neither caldesmon nor its N-terminal myosin binding domain (N152) induced myosin polymerization at subthreshold Mg²⁺ concentrations in the presence of ATP, whereas the filament promoting action of KRP was enhanced by Mg²⁺. Moreover, the amino-terminal myosin binding fragment of caldesmon, like the whole protein, antagonizes Mg²⁺-induced myosin filament formation. In electron microscopy experiments, caldesmon shortened myosin filaments in the presence of Mg²⁺ and KRP, but N152 failed to change their appearance from control. Therefore, the primary distinction between caldesmon and KRP appears to be that caldesmon interacts with myosin to limit filament extension, while KRP induces filament propagation into defined polymers. Transfection of tagged-KRP into fibroblasts and overlay of fibroblast cytoskeletons with Cy3KRP demonstrated that KRP colocalizes with myosin structures in vivo. We propose a new model that through their independent binding to myosin and differential effects on myosin dynamics, caldesmon and KRP can, in concert, control the length and polymerization state of myosin filaments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Thin-filament linked regulation of smooth muscle myosin

Phosphorylation of the regulatory light chain subunit of smooth muscle myosin is sufficient, but not necessary for muscle contraction. It has been suggested that thin-filament regulation may also contribute to the regulation of contraction. A hallmark feature of regulated thin filaments, previously described for vertebrate skeletal muscle, is the capacity of strong-binding or rigor-like cross bridges to turn-on the actin filament. Turned-on thin filaments stimulate cross-bridge attachment even in the absence of calcium. The present study utilized an in vitro sliding-filament motility assay to test for thin-filament regulation of both unphosphorylated and phosphorylated smooth muscle myosins. Regulated thin-filaments were reconstituted from skeletal muscle actin and chicken gizzard smooth muscle tropomyosin (Tm_CG), and then turned-on either (1) by rigor cross bridges at low concentrations of MgATP, or (2) by adding N-ethyl-maleimide-modified skeletal subfragment S1(NEM-S1), which forms rigor-like bonds in the presence of MgATP. For control actin·Tm_CG filaments, force production by unphosphorylated myosin was 0.5% of that produced by thiophosphorylated myosin. The force exerted on actin·Tm filaments by both unphosphorylated and phosphorylated myosins was increased by reducing the [MgATP] to 10–100 M MgATP (rigor-dependent activation). Force was also increased by actin·Tm_CG filaments that had been turned-on by NEM-S1 binding, with force production by unphosphorylated myosin increased 80-fold vs. 2.3-fold for thiophosphorylated myosin. Tm_CG was required for increased force production with both low MgATP and NEM-S1. Unloaded filament velocity for NEM-S1-activated thin filaments was 0.72 m/sec with unphosphorylated myosin compared to 1.24 m/sec with thiophosphorylated myosin. Taken together, these results suggest that thin-filament regulation may play a role in the activation of both unphosphorylated and phosphorylated smooth muscle myosins and suggest a possible mechanism for activation of slowly cycling unphosphorylated cross bridges (i.e. latch-state) during tonic contractions of smooth muscle.     

Myosin Mg-ATPase of molluscan muscles is slightly activated by F-actin under catch state in vitro

Molluscan muscle twitchin, a titin/connectin-related giant protein, regulates interactions between actin and myosin filaments at low Ca²⁺ concentrations. When it is dephosphorylated, actin filaments tightly bind to myosin filaments, resulting in the catch state known as the state of high passive tension with very low energy consumption. Yet when twitchin is phosphorylated actin filaments detach from the myosin filaments, resulting in relaxation of the catch. Here, steady-state Mg-ATPase activities of purified myosin were measured under various conditions: without twitchin, with dephosphorylated twitchin, or with phosphorylated twitchin; with or without phalloidin-stabilized F-actin; and at various Ca²⁺ concentrations. At low Ca²⁺ concentration, Mg-ATPase was activated by F-actin only in the presence of dephosphorylated twitchin (catch state). The activation was about two orders lower than that fully activated by Ca²⁺ and F-actin. In the absence of F-actin, twitchin and its phosphorylation state did not affect Mg-ATPase activities in any of the conditions we tested. Based on these results, we propose a molecular mechanism for the catch, where twitchin alone does not interact with the myosin catalytic motor domain but its complex with F-actin does, forming the bridge between actin and myosin filaments and the myosin slowly hydrolyzes Mg-ATP in the catch state.     

Caldesmon binds to smooth muscle myosin and myosin rod and crosslinks thick filaments to actin filaments

Summary It is well established that caldesmon binds to actin (K _b–10⁷-10⁸ m ^–1) and to tropomyosin (K _b10⁶ m ^–1) and that it is a potent inhibitor of actomyosin ATPase. Caldesmon can also bind tightly to myosin. We investigated the binding of smooth muscle and nonmuscle caldesmon isoforms (CDh and CDl respectively) to myosin using proteins from sheep aorta. Both caldesmon isoforms bind to myosin with indistinguishable affinity. The affinity is about 10⁶ m ^–1 in low salt buffer, but is weakened by increasing [KCl] reaching 10⁵ mM^–1 in 100mm KCl. The stoichiometry of binding is about three caldesmon per myosin molecule. Stoichiometry and affinity are not dependent on whether myosin is phosphorylated nor on the presence of Mg²⁺ and ATP, provided the ionic strength is maintained constant. The caldesmon binding site of smooth muscle myosin is located in the S-2 region, consequently both HMM and myosin rod bind to caldesmon. Over a range of conditions myosin and myosin rod binding to caldesmon were indistinguishable. Skeletal muscle myosin has no caldesmon binding site. Smooth muscle myosin rods form side-polar filaments in low salt buffer in which the backbone packing of LMM into the filament shaft is clearly visible in negatively-stained electron microscopic images. Sometimes the S-2 portions can be seen frayed from the filament shaft. When caldesmon is bound the filament shaft appears to be about 20% thicker and the frayed effect is dramatically increased; long filamentous whiskers are often seen curving out from the filament shaft. Similar structures are observed with smooth muscle and with non-muscle caldesmon. Myosin also binds to caldesmon when it is incorporated into the thin filament; however, this interaction is qualitatively different. Measurements of smooth muscle HMM binding to native thin filaments in the presence of 3mm MgATP shows there is a high affinity binding (K_b=10⁶ m ^–1) which is independent of [Ca²⁺] and of the level of myosin phosphorylation. The stoichiometry is one HMM molecule per actin monomer which is equivalent to up to 14 HMM bound at high affinity per caldesmon. Negatively stained electron microscopic images of the HMM.ADP.Pi-thin filament complex have failed to show any attachment of HMM to the thin filaments. When rod filaments are added to actin plus caldesmon or to native thin filaments the rod filaments are strongly associated with the actin filament bundles. The majority of rod filaments are lined up parallel and in close proximity to actin filaments. Similar crosslinking is observed with non-muscle caldesmon. In the smooth muscle cell, caldesmon-containing thin filaments are found together with myosin filaments in the contractile domain in parallel arrays not unlike those shown in our synthetic systems. Thus caldesmon ought to be able to crosslink thick and thin filamentsin vivo.     

A novel Ca2+ binding protein associated with caldesmon in Ca2+-regulated smooth muscle thin filaments: evidence for a structurally altered form of calmodulin

Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca²⁺-binding protein. Thin filament activation of myosin MgATPase is Ca²⁺-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca²⁺-regulated at 25°C/50 mM KCl. We isolated the Ca²⁺-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25°C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca²⁺]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state.     

Formation of new quasi-crystalline ordered aggregates by gizzard myosin

Summary Turkey gizzard myosin was found to self-assemble into new polymorphic forms as detected by thin-section electron microscopy. In high ionic strength buffers (0.3 ihm KCl, pH 6.0), aggregates of sidepolar filaments were produced. Electron microscopy of thin sections revealed individual filaments with a 13.5 nm axial repeat. Under a number of conditions, with varying ionic strength, pH, MgCl₂ and ATP, the filaments assembled through the head region with the tail portion projecting out radially from the aggregate. The regions corresponding to heads and tails within the aggregates were established by immunoelectron microscopy using anti-Si and anti-LMM antibodies coupled to gold. These filaments often interacted to produce bilayer sheets, which, when cut perpendicular to the plane of the sheet, appeared as ladders. A hitherto unreported structure was obtained at 0.2m KCl (pH 8.0): myosin aggregated to generate a three-dimensional quasi-crystalline lattice with a 270 nm period. In these aggregates, myosin was arranged in an antiparallel fashion, stacked on one another, producing ribbon-like strips stabilized through non-covalent interactions between heads, thereby producing a crystalline lattice. Neither Mg²⁺ nor ATP were required for this form. Phosphorylation of the regulatory light chains or the cleavage of the heavy chains at a single site in the head region prevented myosin from assembling in the 3-D lattice form. Generally, unphosphorylated myosin produced periodic paracrystals at low ionic strength in the presence of 10 him MgCl₂, but as the ionic strength was increased the regular 3-D lattice became the predominant form. Some paracrystalline forms could be obtained at high ionic strength without magnesium with phosphorylated myosin.     

Myosin heads contact with thin filaments in compressed relaxed skinned fibres of frog skeletal muscle

Summary When skinned skeletal muscle fibres with rest sarcomere length (L=2.5 m) are compressed by the addition of various concentrations ([PVP]) of polyvinylpyrrolidone, the relation between the 1,0 spacing (d) of thick filament lattice and [PVP] has been known to break at d of around 35 nm, resulting in a steeper slope of the relationship at d > 35 nm. To clarify the cause of this, X-ray diffraction and crosslinking experiments were carried out. Thed versus [PVP] relationship of stretched fibres (L=3.5m) breaks at a d of around 29 nm. The difference between these characteristic d values, 35-29=6 nm, is close to the diameter of thin filaments (8 nm). The crosslinking efficiency of formaldehyde between myosin heads and thin filament surface, measured by radial stiffness increase, was found to begin to markedly increase when the relaxed fibre with rest L was compressed to a d of nearly 35 nm. In addition to these results, the dversus [PVP] relationship obtained in rigor and in high [Mg²⁺] (30mM) relaxing solutions, and the crosslinking efficiency seen in high [Mg²⁺] solutions supported our previous hypothesis that in normal relaxing solution (containing 1mM Mg²⁺) the probability of myosin heads coming into contact with the thin filament surface abruptly increases at d near 35 nm in fibres with rest L.     

Probing the coupling of Ca2+ and rigor activation of rabbit psoas myofibrillar ATPase with ethylene glycol

We have exploited solvent perturbation to probe the coupling of Ca²⁺ and rigor activation of the ATPase of myofibrils from rabbit psoas. Three techniques were used: overall myofibrillar ATPases by the rapid-flow quench method; kinetics of the interaction of ATP with myofibrils by fluorescence stopped-flow; and myofibrillar shortening by optical microscopy. Because of its extensive use with muscle systems, ranging from myosin subfragment-1 to muscle fibres, we chose 40% ethylene glycol as the relaxing agent. At 4°C, the glycol had little effect on the myofibrillar ATPase at low [Ca²⁺], but at high [Ca²⁺] the activity was reduced 50-fold, close to the level found under relaxing conditions, and there was no shortening. However, the ATPase of chemically cross-linked myofibrils (permanently activated even without Ca²⁺) was reduced only 3–4-fold. The lesser reduction of the ATPase of permanently activated myofibrils was also observed in single turnover experiments in which activation occurs by a few heads in the rigor state activating the remaining heads. The addition of ADP, which also promotes strong head-thin filament interactions, also activated the ATPase but only in the presence of Ca²⁺. Further experiments revealed that in 40% ethylene glycol, Ca²⁺ does initiate shortening but only with the aid of strong interactions and at temperatures above 15°C. This confirms that in the organized and intact myofibril, Ca²⁺ and rigor activation are coupled, as proposed previously for regulated actomyosin subfragment-1. This revised version was published online in September 2006 with corrections to the Cover Date.     

The effect of calcium on the aggregation of chicken gizzard thin filaments

Summary Electron microscopy demonstrates that thin filaments isolated from chicken gizzard smooth muscle in the absence of Ca²⁺ are aggregated into networks. In contrast, thin filaments isolated in the presence of Ca²⁺ are dissociated from each other. Electron microscopy also reveals that the respective state of aggregation in each type of preparation is reversible and dependent on Ca²⁺ concentration. Corresponding viscosity measurements indicate that network formation is associated with an increase in thin filament viscosity. We propose that thin filament aggregationin vivo may be responsible for the tension maintenance of smooth muscle during relaxation.     

The chicken muscle thick filament: temperature and the relaxed cross-bridge arrangement

Summary Although chicken myosin S1 has recently been crystallized and its structure analysed, the relaxed periodic arrangement of myosin heads on the chicken thick filament has not been determined. We report here that the cross-bridge array of chicken filaments is temperature sensitive, and the myosin heads become disordered at temperatures near 4° C. At 25° C, however, thick filaments from chicken pectoralis muscle can be isolated with a well ordered, near-helical, arrangement of cross-bridges as seen in negatively stained preparations. This periodicity is confirmed by optical diffraction and computed transforms of images of the filaments. These show a strong series of layer lines near the orders of a 43 nm near-helical periodicity as expected from X-ray diffraction. Both analysis of phases on the first layer line, and computer filtered images of the filaments, are consistent with a three-stranded arrangement of the myosin heads on the filament.     

Structural interactions between actin,tropomyosin, caldesmon and calcium binding protein and the regulation of smooth muscle thin filaments

The basic structure and functional properties of smooth muscle thin filaments were established about 10 years ago. Since then we and others have been working on the details of how tropomyosin, caldesmon and the Ca²⁺-binding protein regulate actin interaction with myosin. Our work has tended to emphasize the similarities between caldesmon and troponin function whilst others have been more concerned with the differences. The need to resolve the resulting differences has stimulated us to find new and more direct ways of investigating the mechanism of thin filament regulation. In recent years an apparent divergence has opened up between functional measurements, which indicate an allosteric-cooperative regulatory mechanism in which caldesmon and Ca²⁺-binding protein control actin—tropomyosin state in the same way as troponin, and structural measurements which show thin filament structures unlike striated muscle thin filaments. The challenge is to interpret function in terms of structure. We have combined functional studies with expression and mutagenesis of caldesmon and with structural methods including X-ray crystalography of tropomyosin—caldesmon crystals, electron microscopy and helical reconstruction of actin—tropomyosincaldesmon complexes and high resolution nuclear magnetic resonance spectroscopy of the C-terminus of caldesmon in interaction with actin and calmodulin. We have used this information to propose a structural mechanism for caldesmon regulation of the smooth muscle thin filament.     

Mechanism of phosphorylation of the regulatory light chain of myosin from tarantula striated muscle

Contraction is modulated in many striated muscles by Ca²⁺-calmodulin dependent phosphorylation of the myosin regulatory light chain (RLC) by myosin light chain kinase. We have investigated the biochemical mechanism of RLC phosphorylation in tarantula muscle to better understand the basis of myosin-linked regulation. In an earlier study it was concluded that the RLC occurred as two species, both of which could be phosphorylated, potentiating contraction. Here we present evidence that only a single species exists, and that this can be phosphorylated at one or two sites. In relaxed muscle we find evidence for a substantial level of basal phosphorylation at the first site. This is augmented on activation, followed by partial phosphorylation of the second site. We find in addition that Ca²⁺ has a dual effect on light chain phosphorylation, depending on its concentration. At low concentration (relaxing conditions) only basal phosphorylation is observed, while at higher concentrations (activating conditions) RLC phosphorylation is stimulated. At still higher Ca²⁺ concentrations we find partial inhibition of RLC phosphorylation, suggesting an additional mechanism by which the muscle cell can fine tune contractile activity by controlling the level of free Ca²⁺.     

Smooth,cardiac and skeletal muscle myosin force and motion generation assessed by cross-bridge mechanical interactions in vitro

Summary Differences in the mechanical properties of mammalian smooth, skeletal, and cardiac muscle have led to the proposal that the myosin isozymes expressed by these tissues may differ in their molecular mechanics. To test this hypothesis, mixtures of fast skeletal, V1 cardiac, V3 cardiac and smooth muscle (phosphorylated and unphosphorylated) myosin were studied in an in vitro motility assay in which fluorescently-labelled actin filaments are observed moving over a myosin coated surface.Pure populations of each myosin produced actin filament velocities proportional to their actin-activated ATPase rates. Mixtures of two myosin species produced actin filament velocities between those of the faster and slower myosin alone. However, the shapes of the myosin mixture curves depended upon the types of myosins present. Analysis of myosin mixtures data suggest that: (1) the two myosins in the mixture interact mechanically and (2) the same force-velocity relationship describes a myosin's ability to operate over both positive and negative forces. These data also allow us to rank order the myosins by their average force per cross-bridge and ability to resist motion (phosphorylated smooth > skeletal = V3 cardiac > V1 cardiac). The results of our study may reflect the mechanical consequence of multiple myosin isozyme expression in a single muscle cell.