The performance of CA-125 measurement in the detection of endometriosis: a meta-analysis

The effects resulting from the removal of the N-terminus of heavy meromyosin (HMM) A1 light chain by papain digestion are investigated. The fluorometry of TRITC-phalloidin labelled actin in ghost fibers is used as a tool for sensing conformational changes of rigor complex of phosphorylated and dephosphorylated HMM with actin filament. The experiments were performed both under conditions assuring saturation of RLC with magnesium cation (4 mM EGTA) or calcium cation (0.1 mM CaCl2), and in constant presence of 1 mM magnesium chloride. HMM native and with A1 shortened from the N-terminus is used. As it was observed previously rigor complex of actin filament and native HMM shows sensitivity to the kind of cation saturating RLC and to the phosphorylation status of RLC. In particular, the sin2 theta parameter of actin bound rhodamine-phalloidin fluorescence polarization representing roughly the flexibility of actin filament HMM complex changes significantly with the changes of RLC phosphorylation and cation saturation. Removal of the N-terminus of A1 reduces this sensitivity to cation and phosphorylation both in the case of dephosphorylated and phosphorylated HMM. Our results suggest that the N-terminus of A1 plays significant role in the rigor interaction of myosin heads with actin and is involved in modulatory function of RLC in this interaction.     

High microfilament concentration results in barbed-end ADP caps

Current theory and experiments describing actin polymerization suggest that site-specific cleavage of bound nucleotide following F-actin filament formation causes the barbed ends of microfilaments to be capped first with ATP subunits, then with ADP bound to inorganic phosphate (ADP.Pi) at steady-state. The barbed ends of depolymerizing filaments consist of ADP subunits. The decrease in stability of the barbed-end cap accompanying the transition from ADP.Pi to ADP allows nucleotide hydrolysis and subsequent loss of Pi to regulate F-actin filament dynamics. We describe a novel computational model of nucleotide capping that simulates both the spatial and temporal properties of actin polymerization. This model has been used to test the effects of high filament concentration on the behavior of the ATP hydrolysis cycle observed during polymerization. The model predicts that under conditions of high microfilament concentration an ADP cap can appear during steady-state at the barbed ends of filaments. We show that the presence of the cap can be accounted for by a kinetic model and predict the relationship between the nucleotide concentration ratio [ATP]/[ADP], the F-actin filament concentration, and the steady-state distribution of barbed-end ADP cap lengths. The possible consequences of this previously unreported phenomenon as a regulator of cytoskeletal behavior are discussed.     

Influence of tightly bound Mg2+ and Ca2+, nucleotides, and phalloidin on the microsecond torsional flexibility of F-actin

To better understand the relationship between structure and molecular dynamics in F-actin, we have monitored the torsional flexibility of actin filaments as a function of the type of tightly bound divalent cation (Ca2+ or Mg2+) or nucleotide (ATP or ADP), the level of inorganic phosphate and analogues, KCl concentration, and the level of phalloidin. Torsional flexibility on the microsecond time scale was monitored by measuring the steady-state phosphorescence emission anisotropy (rFA) of the triplet probe erythrosin-5-iodoacetamide covalently bound to Cys-374 of skeletal muscle actin; extrapolations to an infinite actin concentration corrected the measured anisotropy values for the influence of variable amounts of rotationally mobile G-actin in solution. The type of tightly bound divalent cation modulated the torsional flexibility of F-actin polymerized in the presence of ATP; filaments with Mg2+ bound (rFA = 0.066) at the active site cleft were more flexible than those with Ca2+ bound (rFA = 0.083). Filaments prepared from G-actin in the presence of MgADP were more flexible (rFA = 0.051) than those polymerized with MgATP; the addition of exogenous inorganic phosphate or beryllium trifluoride to ADP filaments, however, decreased the filament flexibility (increased the anisotropy) to that seen in the presence of MgATP. While variations in KCl concentration from 0 to 150 mM did not modulate the torsional flexibility of the filament, the binding of phalloidin decreased the torsional flexibility of all filaments regardless of the type of cation or nucleotide bound at the active site. These results emphasize the dynamic malleability of the actin filament, the role of the cation-nucleotide complex in modulating the torsional flexibility, and suggest that the structural differences that have previously been seen in electron micrographs of actin filaments manifest themselves as differences in torsional flexibility of the filament.     

Three-dimensional structure of nucleotide-bearing crossbridges in situ: oblique section reconstruction of insect flight muscle in AMPPNP at 23 degrees C

We have explored the three-dimensional structure of myosin crossbridges in situ in order to define the structural changes that occur when nucleotide binds to the myosin motor. When AMPPNP binds to rigor insect flight muscle, each half sarcomere lengthens by approximately 2.0 nm and tension is reduced by approximately 70% without a reduction in stiffness, suggesting partial reversal of the power stroke. We have obtained averaged oblique section three-dimensional reconstructions of mechanically monitored insect flight muscle in AMPPNP that permit simultaneous examination of all myosin crossbridges within the unit cell and direct comparison of calculated transforms with X-ray diagrams of the native fibers. Transforms calculated from the oblique section reconstruction of AMPPNP insect flight muscle at 23 degrees C show good agreement with native X-ray diagrams, suggesting that the average crossbridge forms in the reconstruction reflect the native structure. In contrast to the rigor lead and rear crossbridges in the double chevrons, the averaged reconstruction of AMPPNP fibers show only one crossbridge class, in the position of the rigor lead bridge. The portion of the crossbridge close to the thick filament appears broader than in rigor, and shows a small 0.5 to 1.0 nm M-ward shift of the regulatory domain region of myosin. In transverse view, AMPPNP "lead" crossbridges are less azimuthally bent than rigor. Fitting the atomic model of actomyosin subfragment 1 to the averaged crossbridges shows that the detectable differences between rigor bridges and between rigor and AMPPNP bridges occur in the alignment and angles of the regulatory domains and suggests that rear bridges are more strained than lead crossbridges. The apparent absence of rear bridges in AMPPNP in averaged reconstructions indicates detachment of a number of force-bearing bridges, which conflicts with the maintained stiffness of the fibers used for the reconstruction. This conflict may be explained if rigor rear bridges become distributed irregularly over more actin sites in AMPPNP, so that their average density is too low to appear in the averaged reconstructions. The reconstructions indicate that in insect flight muscle the response of in situ rigor crossbridges to AMPPNP binding is not uniform. Lead bridges persist but have altered structure in the light chain domain, whereas rear bridges detach and possibly redistribute. Shape changes in attached myosin heads within the myofibrillar lattice are in the appropriate direction and of the appropriate magnitude needed to explain the sarcomere lengthening. This could be a direct response to nucleotide binding, a passive response to rear bridge detachment, or a combination of both.     

Computer system modelling muscle work

In the work, an original computer system, called Muscle, is described. The Muscle allowed modelling of the whole thick filament with different arrangements of the myosin tails and myosin heads. Computer simulation and animation have revealed that only the model of the thick filament with asymmetrical configuration of the crossbridges has 3-fold rotational symmetry and assures matching between many thousands specific binding-sites on myosin heads and actin monomers. The Muscle delivered a number of arguments that the hypothesis of near parallel packing of the myosin tails, as well as oar-like or lever-arm-like work of the myosin crossbridges, is unfounded. We have presented advantages of the computer simulation in studies of complicated 3D molecular objects especially when direct observation is technically impossible.     

Elastic bending and active tilting of myosin heads during muscle contraction

Muscle contraction is driven by a change in shape of the myosin head region that links the actin and myosin filaments. Tilting of the light-chain domain of the head with respect to its actin-bound catalytic domain is thought to be coupled to the ATPase cycle. Here, using X-ray diffraction and mechanical data from isolated muscle fibres, we characterize an elastic bending of the heads that is independent of the presence of ATP. Together, the tilting and bending motions can explain force generation in isometric muscle, when filament sliding is prevented. The elastic strain in the head is 2.0-2.7 nm under these conditions, contributing 40-50% of the compliance of the muscle sarcomere. We present an atomic model for changes in head conformation that accurately reproduces the changes in the X-ray diffraction pattern seen when rapid length changes are applied to muscle fibres both in active contraction and in the absence of ATP. The model predictions are relatively independent of which parts of the head are assumed to bend or tilt, but depend critically on the measured values of filament sliding and elastic strain.     

Structural coupling of troponin C and actomyosin in muscle fibers

EPR of spin labeled TnC at Cys98 was used to explore the possible structural coupling between TnC in the thin filament and myosin trapped in the intermediate states of ATPase cycle. Weakly attached myosin heads (trapped by low ionic strength, low temperature and ATP) did not induce structural changes in TnC as compared to relaxed muscle, as spin labeled TnC displayed the same narrow orientational distribution [Li, H.-C., and Fajer, P. G. (1994) Biochemistry 33, 14324]. Ca2+-binding alone resulted in disordering of the labeled domain of TnC. Additional conformational changes of TnC occurred upon the attachment of strongly bound, prepower stroke myosin heads (trapped by AlF4-). These changes were not present in ghost fibers which myosin had been removed, excluding direct effects of AlF4- on the orientation of TnC in muscle fibers. The postpower stroke heads (rigor.ADP/Ca2+ and rigor/Ca2+) induced further changes in the orientational distribution of labeled domain of TnC irrespective of the degree of cooperativity in thin filaments. We thus conclude that troponin C in thin filaments detects structural changes in myosin during force generation, implying that there is a structural coupling between actomyosin and TnC.     

Electron tomography of insect flight muscle in rigor and AMPPNP at 23 degrees C

Treatment of rigor fibers of insect flight muscle (IFM) with AMPPNP at 23 degrees C causes a 70% drop in tension with little change in stiffness. In order to visualize the changes in crossbridge conformation and distribution that give rise to the mechanical response, we have produced three-dimensional reconstructions by tomography of both rigor and AMPPNP-treated muscle that do not average the repeating motifs of crossbridges, and thereby retain information on variability of crossbridge structure and distribution. Tomograms can be averaged when display of only the regular features is wanted. Tomograms of rigor IFM show double-headed lead and single-headed rear crossbridges. Tomograms of IFM treated with AMPPNP at 23 degrees C reveal many double-headed and some single-headed "lead" bridges but few crossbridges corresponding to the rear bridges of rigor. Instead, new non-rigor forms of variably angled crossbridges are found bound to actin sites not labeled with myosin heads in rigor. This indicates that the rear bridges of rigor have redistributed during the transition from rigor to the AMPPNP state, which could explain the maintenance of rigor stiffness despite the loss of tension. Comparison of in situ crossbridges in tomograms of rigor with atomic model of acto-S1, the complex formed by myosin subfragment 1 and actin, reveals that the regulatory domain of S1 would require significant bending and realignment to fit into both types of rigor crossbridges. The modifications are particularly significant for the rear bridges and suggest that differential strain in the regulatory domain of rear bridges may be the basis for their detachment and redistribution upon binding AMPPNP. Similar comparison using lead-type crossbridges in AMPPNP reveals departures from the rigor acto-S1 atomic model that include azimuthal straightening and a slight M-ward bending in the regulatory domain. Both the motor and regulatory domains of the new non-rigor crossbridges differ from those in the atomic model of acto-S1. A new crossbridge motif identified in AMPPNP-treated muscle consists of paired rigor-like and non-rigor crossbridges and suggests possible transitions in the myosin working stroke.     

Innocuous labeling of the subfragment-2 region of skeletal muscle heavy meromyosin with a fluorescent polyacrylamide nanobead and visualization of individual heavy meromyosin molecules

We have studied transglutaminase-catalyzed incorporation of monodansylcadaverine and monobiotincadaverine into rabbit skeletal muscle heavy meromyosin (HMM). The incorporation of dansylcadaverine reached saturation at 4 mol per 1 mol of HMM. An electrophoretogram of the chymotryptic digest of the dansyl-labeled HMM revealed that the labeling took place primarily in the S-2 region of HMM. Atomic force microscopic images and electron micrographs of the complexes of the biotinylated HMM and UltraAvidin-coated fluorescent polyacrylamide nanoparticles revealed that the biotinylated site on S-2 was very close to the C-terminus (near the S-2/light meromyosin junction). In keeping with this result, together with HMM's key sites being localized on the S-1 region, the enzymatic conjugation of biotincadaverine had no influence upon the actin-activated ATPase activity of HMM or upon the ability of HMM to actuate sliding of actin filaments in in vitro motility assay. Attachment of an UltraAvidin-coated fluorescent nanobead to the biotinylated HMM also did not alter the motile activity of HMM. Thus, we can optically pinpoint individual HMM molecules in a sample, which will facilitate handling and manipulation of single HMM molecules and observation of their functional behavior.     

Calorimetric studies of the thermal unfolding of smooth muscle myosin fragments and their complexes with ADP and phosphate analogs

The thermal unfolding of turkey gizzard smooth muscle myosin subfragment 1 (S1) and heavy meromyosin (HMM) in the absence of added nucleotides, in the presence of ADP, and in S1 or HMM ternary complexes with ADP and Pi analogs, orthovanadate (Vi), beryllium fluoride (BeFx), or aluminum fluoride (AlF4-), have been studied by differential scanning calorimetry (DSC). It has been shown that the formation of these ternary complexes causes significant structural changes in S1 or in the heads of HMM which are reflected in a pronounced increase of the protein thermal stability. The effect of BeFx was less distinct than that of AlF4- or Vi. Phosphorylation of regulatory light chains (RLC) in S1 or in HMM had practically no influence on these effects. In general, the changes caused by various Pi analogs in smooth muscle S1 or HMM were similar to those observed earlier with skeletal muscle S1 devoid of RLC. It is concluded that RLC and their phosphorylation do not significantly affect the character of structural changes induced in motor domains of the HMM heads by the formation of ternary complexes HMM--ADP--Vi, HMM--ADP--AlF4-, and HMM--ADP--BeFx--stable analogs of the intermediate states of the HMM ATPase reaction, HMM.ADP.Pi and HMM. ATP.     

Behavior of N-phenylmaleimide- and p-phenylenedimaleimide-reacted muscle crossbridge heads

The finding of Barnett et al. (Biophys. J. 61 (1992) 358) that NPM-reacted crossbridge heads do not bind strongly to actin in rigor solution is not easily interpreted in terms of the solution studies of Xie and Schoenberg (Biochemistry 37 (1998) 8048) who found strong binding of NPM-reacted myosin subfragment-1 to actin in solutions devoid of MgATP. For this reason, the current work uses stiffness measurement to re-investigate the binding of rabbit skeletal muscle crossbridges to actin in rigor solution. It is found that NPM-reacted crossbridge heads bind strongly to actin in rigor solution providing one is extremely careful to reduce MgATP contamination to levels well below those that would have a detectable effect on unmodified fibers. The reason for this is that NPM-reacted crossbridge heads, which hydrolyze MgATP extremely slowly, are especially susceptible to contaminant MgATP. The new fiber results show a strong correlation with the solution results. A further manifestation of this correlation is that pPDM-reacted crossbridge heads are different from NPM-reacted ones in that, like in solution, they remain weakly binding to actin even at extremely low MgATP levels. The findings suggest that the covalent crosslinking of SH1 and SH2 by pPDM is likely playing a significant role in locking pPDM-reacted crossbridge heads in a weakly binding conformation.     

Right-handed rotation of an actin filament in an in vitro motile system

Muscle contraction occurs by mutual sliding between thick (myosin) and thin (actin) filaments. But the physical and chemical properties of the sliding force are not clear; even the precise direction of sliding force generated at each cross-bridge is not known. We report here the use of a recently developed in vitro motile assay system to show supercoiling of an actin filament in which the front part of the filament was fixed to a glass surface through cross-linked heavy-meromyosin and the rear part was able to slide on a track of heavy-meromyosin. A left-handed single turn of superhelix formed just before supercoiling, suggesting that the sliding force has a right-handed torque component that induces the right-handed rotation of an actin filament around its long axis. The presence of the torque component in the sliding force will explain several properties of the contractile system of muscle.     

Electrostatic forces as a possible mechanism underlying skeletal muscle contraction

A possible mechanism is put forward to explain the sliding of thin filaments during muscle contraction. In our model, repulsion due to electrostatic forces is the mechanism which triggers crossbridges to cause the thin filaments to slide. The mechanism proposed could operate regardless of whether the myosin heads rotate or bend, although recent experimental evidence seems to confirm the latter action. In spite of its simplicity, the model prediction of the velocity of sliding of the thin filaments agrees well with experimental values from in vitro motility assays.     

Intrastrand cross-linked actin between Gln-41 and Cys-374. III. Inhibition of motion and force generation with myosin

Structural and functional properties of intrastrand, ANP (N-(4-azido-2-nitrophenyl)-putrescine) cross-linked actin filaments, between Gln-41 and Cys-374 on adjacent monomers, were examined for several preparations of such actin. Extensively cross-linked F-actin (with 12% un-cross-linked monomers) lost at 60 degrees C the ability to activate myosin ATPase at a 100-fold slower rate and unfolded in CD melting experiments at a temperature higher by 11 degrees C than the un-cross-linked actin. Electron microscopy and image reconstruction of these filaments did not reveal any gross changes in F-actin structure but showed a change in the orientation of subdomain 2 and a decrease in interstrand connectivity. Rigor and weak (in the presence of ATP) myosin subfragment (S1) binding and acto-S1 ATPase did not show major changes upon 50% and 90% ANP cross-linking of F-actin; the Kd and Km values were little affected by the cross-linking, and the Vmax decreased by 50% for the extensively cross-linked actin. The cross-linking of actin (50%) decreased the mean speed and the number of sliding filaments in the in vitro motility assays by approximately 35% while the relative force, as measured by using external load in these assays, was inhibited by approximately 25%. The mean speed of actin filaments decreased with the increase in their cross-linking and approached 0 for the 90% cross-linked actin. Also examined were actin filaments reassembled from cross-linked and purified ANP cross-linked dimers, trimers, and oligomers. All of these filaments had the same acto-S1 ATPase and rigor S1 binding properties but different behavior in the in vitro motility assays. Filaments made of cross-linked dimers moved at approximately 50% of the speed of the un-cross-linked actin. The movement of filaments made of cross-linked trimers was inhibited more severely, and the oligomer-made filaments did not move at all. These results show the uncoupling between force generation and other events in actomyosin interactions and emphasize the role of actin filament structure and dynamics in the contractile process.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.     

Muscle proteins--their actions and interactions

Muscle contracts by the myosin cross-bridges "rowing' the actin filaments past the myosin filaments. In the past year many structural details of this mechanism have become clear. Structural studies indicate distinct states for myosin S1 in the rigor, ATP or "down' conformation and in the products complex (ADP.Pi) or "up' to state. Crystallographic studies substantiate this classification and yield details of the transformation. The isomerization "up' to "down' is the power stroke of muscle. This consists in the main of large changes of angle of the "lever arm' (at the distal part of the myosin head) which can account for an 11 nm power stroke.     

Effect of lateral forces on the movement of myosin-coated beads on actin cables studied using a centrifuge microscope

We developed an in vitro motility assay system, in which myosin-coated polystyrene beads were made to slide on actin filament arrays (actin cables) in giant algal cells and subjected to centrifugal forces, which were parallel to the direction of bead movement to serve as external loads on actin-myosin sliding (Oiwa et al. (1990) Proc Natl Acad Sci USA 87: 7893-7897), and succeeded in determining the steady-state force-velocity relation of ATP-dependent actin-myosin sliding. To give further information about the properties of actin-myosin sliding, we have applied centrifugal forces, in parallel with the plane of actin-myosin sliding but at right angles with the direction of bead movement, and have found that such "lateral" centrifugal forces reduced the velocity of bead movement. In addition, we have also found that the velocity of bead movement is reduced more markedly with lateral forces applied from the left side of the bead ("left" lateral forces) than those applied from the right side of the bead ("right" lateral forces). These results are discussed in connection with the direction of sliding force generated by the myosin heads on the bead which interact with the right-handed double helix of actin monomers constituting actin filaments.     

Role of the Dictyostelium 30 kDa protein in actin bundle formation

We have studied the formation of bundles in mixtures of actin with the Dictyostelium 30 kDa actin-bundling protein as a function of 30 kDa protein concentration, actin concentration, and filament length. The presence of the 30 kDa protein promotes formation of filament bundles at actin concentrations and filament lengths that are not spontaneously aligned into liquid crystalline domains in the absence of the 30 kDa protein. Bundle formation in the presence of the 30 kDa protein was observed over a broad range of actin filament lengths and concentrations. Bundling was filament length dependent, and short filaments were more efficiently bundled. Bundles formed at actin concentrations as low as 2 microM. The volume fraction of the bundled portion and concentrations of actin and the 30 kDa protein in the bundled portion were measured using a sedimentation assay. Bundles have concentrations of actin and 30 kDa protein that are 10-20 and 5-20 times, respectively, greater than that of the bulk solution. Computer modeling reveals that bundling of actin by a bundling protein increases both the mean length and the polydispersity of the length distribution, factors which lower the actin concentration required for spontaneous alignment within the bundle. We propose that entropy-driven spontaneous ordering may contribute to bundle formation in two ways. Bundling of actin creates longer aggregates with a more polydisperse length distribution in which actin aligns spontaneously within the bundle at very low concentrations. In addition, bundling creates locally high concentrations of actin within these aggregates that will spontaneously align, providing an additional driving force for bundle ordering.     

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

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 degrees C. At 25 degrees 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.     

Viscoelasticity of actin-gelsolin networks in the presence of filamin

Cross-linking of actin filaments by filamin by means of frequency-dependent rheology yields an increase in the filament's elasticity and stiffness. Higher cross-linker (filamin) ratios are required for mean actin-filament lengths of 5-6 microm than for random-length distribution of actin filaments. The loss modulus (i.e. the viscous portion) in the region of the internal-chain dynamics [G"(omega) approximately omega(alpha)] is influenced by the cross-linking of filaments, and with an increasing molar ratio of filamin/actin a reduction of alpha is observed. Rheological measurements reveal that actin networks are already formed at the polymerizing stage at a molar ratio of filamin/actin of less than 1:100, and electron micrographs show phase separation of actin/filament networks of low density and of actin/filament bundles.