Structural changes in contractile proteins of muscle fibers studied by polarization ultraviolet fluorescence microscopy. X. The effect of ATP, Ca2+, pH change and ionic strength of the washing solution on the structural state of thick filaments

By the method of polarized ultraviolet fluorescence microscopy, effects of ATP, Ca2+, changes in pH and ionic strength of washing solution of the structural state of thick filaments in both actin-free muscle fibers of rabbit and anisotropic discs (A-discs) of glycerinated fibers of crab were studied. The dependence of tryptophan fluorescence anisotropy of thin filaments upon physico-chemical parameters (compounds) of washing solution has been found. The structural state of thick filaments was suggested to be influenced by ATP, Ca2+, changes in pH and ionic strength of washing solution.     

The effect of myosin light chain phosphorylation and Mg2+ on the conformation of myosin in thick filaments of glycerinated fibers of rabbit skeletal muscle

It has been shown by polarization microfluorimetry that phosphorylation of myosin light chain 2, in stretched single glycerinated fibers of rabbit skeletal muscle, results in changes in polarized fluorescence anisotropy of both the tryptophan residues of myosin molecules and the fluorescent label, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine, associated with the fast-reacting thiol group in myosin heads. These changes are also dependent on the presence or absence of Mg2+ in the medium: they are most pronounced in the presence of 5 mM MgCl2. It is assumed that both Mg2+ binding to myosin and phosphorylation of light chain 2 associated with myosin heads induce structural changes in myosin filaments of muscle fibres which are expressed as changes in the orientation of myosin heads and in the conformation of myosin rods.     

Structural changes in muscle fiber contractile proteins studied by polarization ultraviolet fluorescence microscopy. VIII. The effect of glutaraldehyde and phalloidine on F-actin conformation

Conformational changes in F-actin, induced by glutaraldehyde or phalloidin, were found in glycerinated m. psoas rabbit fibres (ghost and reconstructed fibres). It is shown that the conformational changes of F-actin decreased the thin filament flexibility as well as weakened fibre's contractility. It is assumed that the stabilization in F-actin structure may be an important factor involved in the mechanism of muscle contraction regulation.     

Overrelaxation of muscle models in media with high concentrations of calcium ions and exposed to MgATP in "contraction-inducing" concentrations]

Cross-striation pattern and sarcomere length in isolated myofibrils (both glycerinated and fresh) as well as isometric tension of glycerinated fibers of rabbit m. psoas are unaffected by an evaluation in ionic strength of CaCl2 up to 0.2 in the absence of ATP. An addition of MgATP (1 to 3mM) to the Ca2+ media induces the changes which have been shown to be characteristic of overrelaxation [1, 2]: A band shortening occurs followed by a complete plastification of the fibres. A tentative mechanism of the process is discussed in terms of spontaneous rearrangement of calcium myosinate packing in thick filaments that follows disrupting of rigor crossbridges with thin filaments under the action of ATP. Released calcium myosinate heads fail to form "active" bridges with actin; thick filaments undergo a conformational change resulted in their shattening due to increase in the equilibrium region of LMM tail overlap. The effects do not depend on ionic strength only: on replacing CaCl2 by KCl at equal ionic strength 0.2, an addition of ATP induces normal contraction instead of overrelaxation. A possibility is discussed that in a living muscle overrelaxation could provide a siding to prevent damage in case of emergency.     

Localized deposition of lead phosphate precipitate in glycerinated rabbit psoas muscle during ATP-induced contraction

The location of ATP-ase sites within sarcomeres was studied by allowing glycerinated rabbit psoas muscle to shorten under load in a solution containing ATP and lead nitrate. The lead phosphate precipitate formed during the contraction is located mainly within the A-band of shortened sarcomeres. In fibers glycerinated at rest length, the heaviest precipitate deposits are located at the center of the A-band, roughly corresponding to the double overlap zone, with lesser precipitate concentrations at the A-l boundary contraction bands. In fibers glycerinated at stretched length, shortened sarcomeres with heavy contraction bands at the A-l boundary and patent H-bands have heavy precipitate deposits over the contraction bands and no localization to the center of the A-band. The precipitate is thus seen to be most concentrated at those regions where one would expect to find the best contact between thick and thin filaments. Physiological experiments were performed. Evidence is presented that lead ions can substitute for calcium in the contraction process, altering the time course of contraction.     

MECHANISM OF SUPERCONTRACTION IN A STRIATED MUSCLE

The phenomenon of contraction of a striated muscle down to below 50 per cent rest length has been examined for the scutal depressor of the barnacle Balanus nubilus by a combination of phase contrast and electron microscopy. It was found that neurally evoked contraction down to 60 per cent rest length results from the shortening of the I band. At the same time the Z disc changes in structure by an active process which results in spaces opening up within it. Thick filaments can now pass through these spaces from adjacent sarcomeres, interdigitating across the discs. Interdigitation permits repetitive contraction in the living muscle to below 30 per cent rest length. In non-neurally evoked contractions most thick filaments do not find spaces in the Z disc and bend back, giving rise to contraction band artifacts. Expansion of the Z disc can be produced in glycerinated material by the addition of solutions containing a high concentration of ATP.     

Na+ and K+ binding by glycerinated muscle fibers with reciprocal cation concentrations in the medium

The binding of Na+ and K+ by glycerinated muscle fibres was observed at reserve concentrations of NaCl in the medium. Under external concentrations of Na+ of K+ up to 0.4-0.5 mM, a constant fraction (0.15-0.25 mmoles/kg dry weight of the fibres) bound by glycerinated fibres was revealed. With the increase of NaCl or KCl concentration in the medium up to 10 mM the concentration of bound cations increased too. The parameters of Na+ and K+ sorption by glycerinated models were calculated. The values of Na+ and K+ binding limits were 4.4 and 1.8 mmole/kg dry weight of the fibres and those of affinity, 3.2 and 4.1 kcal/mol, respectively. The binding of one cation took place in conditions when its concentration was 10,000-20,000 fold less than that of the other cation. This points to the fact that Na+ and K+ binding is highly specific and is carried out by different centres. It is suggested that myosin ATPase is a substratum binding Na+ and K+ in glycerinated muscle fibres at reverse ratio concentrations of these cations in the medium.     

Donnan potentials in rabbit psoas muscle in rigor.

Collins and Edwards (1971, Am. J. Physiol., 221:1130-1133) have shown that a tissue potential can be measured with microelectrodes in glycerinated muscle and that this potential is consistent with a Donnan equilibrium of small ions due to the concentration of net fixed electric charge on the contractile proteins. This approach has been combined with x-ray and light diffraction measurements of the muscle lattice dimensions, and the data are used to determine the thick filament charge and thin filament charge under a variety of different conditions. The thick filament charge is a function of the bathing solution, in particular its pH and ionic composition. These parameters are important in determining the volume of the equilibrium lattice and possibly are involved in the contraction mechanism itself.     

Effect of phosphorylation of light chain myosin and Ca2+ on the conformation of F-actin during skeletal muscle contraction

The dependence of polarized fluorescence of rhodaminylphalloin specifically bound to F-actin and the tension developed by a fiber upon phosphorylation of myosin (18.5 kD) light chains as well as on the concentration of free Ca2+ was observed during the contraction of glycerinated rabbit skeletal muscle fibers. Still greater changes in the polarized fluorescence and higher values of tension were recorded for fibers with phosphorylated light chains at low (0.6 microM) Ca2+ concentrations as well as for those with dephosphorylated light chains at high (10 microM) Ca2+ concentrations. It is concluded that phosphorylation of myosin light chains modulates skeletal muscle contraction. The mechanisms of modulation involve conformational changes in F-actin.     

ANOMALOUS CONTRACTION OF INVERTEBRATE STRIATED MUSCLE

The phenomenon of A band shortening or contraction has been investigated in glycerinated myofibrils of Pecten irradians, Homarus americanus, Cambarus virilis, and Limulus polyphemus through the techniques of ultraviolet microbeam inactivation and polarization microscopy. With the former method, it has been shown that these muscles, even though exhibiting the shortening effect, contract in a manner consistent with only the sliding filament model. Intrinsic birefringence studies have indicated no significant changes in mass distribution or orientation within the shortened A bands. Except in the case of Limulus muscle, the shortening effect was seen only in contraction under tension. The magnitude of this anomalous phenomenon was dependent upon glycerination time and has been duplicated in rabbit psoas muscle through brief trypsin treatment. A band shortening could not be observed in glutaraldehyde-fixed muscle or in myofibrils glycerinated for only short periods. It has been concluded that the phenomenon of A band contraction is an artifact induced by the glycerination procedure, possibly through weakening of the sarcomere structure. However, the fact that the A band shortens under tension rather than lengthens poses an interesting paradox.     

Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin

F-actin in a glycerinated muscle fiber was specifically labeled with fluorescent phalloidin-(fluorescein isothiocyanate) FITC complex at 1:1 molar ratio. Binding of phalloidin-FITC to F-actin affected neither contraction of the fiber nor its regulation by Ca2+. Comparison of polarized fluorescence from phalloidin-FITC bound to F-actin in the relaxed state, rigor, and during isometric contraction of the fiber revealed that the changes in polarization accompanying activation are quantitatively as well as qualitatively different from those accompanying transition of the fiber from the relaxed state to rigor. The extent of the changes of polarized fluorescence during isometric contraction increased with decreasing ionic strength, in parallel with increase in isometric tension. On the other hand, polarized fluorescence was not affected by addition of ADP or by stretching of the fiber in rigor solution. It is concluded from these observations that conformational changes in F-actin are involved in the process of active tension development.     

Evidence for cooperative interactions of myosin heads with thin filament in the force generation of vertebrate skeletal muscle fibers

To examine the possibility of cooperative interactions between the two myosin heads in muscle contraction, Ca2+-activated force development, K+-EDTA-and Mg2+-ATPase activities, muscle fiber stiffness, and the velocity of unloaded shortening were measured on partially p-phenylenedimaleimide (p-PDM)-treated glycerinated muscle fibers, which contained a mixture of myosin molecules with zero, one, and two of their heads inactivated, and the relationships among these values (expressed relative to the control values) were studied. It was found that the magnitude of the Ca2+-activated isometric force development was proportional to the square of both K+-EDTA- and Mg2+-ATPase activities and also to the square of muscle fiber stiffness. If the two myosin heads in the glycerinated fibers are assumed to react independently with p-PDM, the above results strongly suggest that each myosin molecule in the thick filaments can generate force only when its two heads do not react with p-PDM, muscle fiber stiffness is determined by the total number of native heads, and there is no cooperative interaction between the two myosin heads in catalyzing ATP hydrolysis.     

Acridine orange accumulation in acid organelles of normal and vacuolated frog skeletal muscle fibres

The spatial distribution of acid membrane organelles and their relationships with normal and vacuolated transverse tubules has been studied in living frog skeletal muscle fibres using confocal microscopy. Acridine orange (AO) was used to evaluate acid compartments, while a lipophilic styryl dye, RH 414, was employed to stain the membranes of the T-system. AO accumulated in numerous spherical granules located near the poles of nuclei and between myofibrils where they were arranged in short parallel rows, triplets or pairs. AO granules could be divided into three groups: green (monomeric AO), red (aggregated AO), and mixed green/red. As demonstrated by lambda-scanning, most granules were mixed. Double staining of muscle fibres with AO and RH 414 revealed almost all AO granules located near the transverse tubules. Vacuolation of the T-system was induced by glycerol loading and subsequent removal. The close juxtaposition of AO granules and the T-system was preserved in vacuolated fibres. The lumens of vacuoles did not accumulate AO. It is concluded that AO granules represent an accumulation of AO in lysosome-related organelles and fragmented Golgi apparatus and a possible functional role of the spatial distribution of such acidic compartments is discussed.     

Changes in the structural state of myosin filaments in glycerol-treated fibers of rabbit skeletal muscles induced by phosphorylation of myosin light chains

Using the polarization microfluorimetry method, it was demonstrated that the increase in the degree of phosphorylation of myosin light chains (LC2) in extended single glycerinated fibers from rabbit psoas muscle changes the anisotropy of polarized fluorescence both tryptophan residue in the rod parts of the myosin molecule and the fluorescent label-N (iodoacetyl-aminoethyl)-5-naphthylamine-1-sulfonate (1,5-IAEDANS) bound to the SH1-group in myosin molecule heads. The changes in fluorescence anisotropy during LC2 phosphorylation were observed, when the measurements were performed only in the presence of 5 mM MgCl2. It was suggested that in the presence of MgCl2 the phosphorylation of LC2 associated with myosin heads changes their orientation and causes conformational shifts in the myosin filament core.     

Na+ and K+ binding by glycerinated muscle fibers at equal concentrations of the chlorides of these cations in the medium

The Na+ and K+ equilibrium distribution between the medium and glycerinated muscle fibres of the frog has been investigated under equal concentrations of NaCl and KCl in solutions. Concentrations of NaCl and KCl varied from 0.5-1.5 mkM till 50 mM. Ion strength (0.11) was constant owing to the imidazol--HCl buffer. The binding of Na+ and K+ by model fibres occurred in accordance with the Langmur equation. Two kinds of cation-binding sites were found. The one with a low limiting ion sorption (A infinity approximately 1.3 mmol/kg dry weight of fibres) and high affinities (-delta F0 approximately 4.3 kcal/mol) was saturated at 0.5 mM concentrations (Na+ = K+) in the medium, and the other--with A infinity exceeding the previous one by an order and low -delta F0 (2.5 kcal/mol) was discovered at Na+, K+-1-10 mM. At ion concentrations equal to 0.5-1 mM the Langmur-binding is disturbed. At Na+-K+ less than or equal to 1 mM Na+ bound:K+ bound approximately to 1:1. At higher concentrations of cations Na+ bound:K+ bound approximately equal to 3:2. It is concluded that at least part of the sites in model fibres is capable of interacting only with Na+, but not with K+. It is supposed that at equal concentrations of Na+ and K+ in the medium the cations are bound by Na+, K+-ATPase of glycerinated muscle fibres.     

Polarized ultraviolet fluorescence microscopic study of the structural changes in muscle fiber contractile proteins. V. The possible nature of the conformational rearrangements in heavy meromyosin in fiber relaxation]

The mode and degree of tryptophanyl orientation relative to muscle fiber axes within hydrophobic and hydrophylic sites of myosin macromolecule in the presence of a fluorescence quencher (acrylamide, NO-3) during rigor and relaxation of glycerinated muscle fibers were studied using the polarized ultraviolet fluorescent microscopy. It was shown that myosin tryptophanyls both in LMM and HMM are oriented with their short axes along the longer axis of muscle fiber. Tryptophanyls in LMM have a more pronounced anisotropy of orientation in comparison with the fluorophore orientation anisotropy in hydrophobic sites of HMM. During the muscle fiber relaxation, conformational changes in HMM take place owing to which a section of polypeptide chain with a hydrophilic fluorophore is probably submerged deep into the macromolecule and becomes unapprochable to the quencher.     

Study of structural changes of contractile muscle proteins with the aid of polarization ultraviolet fluorescence microscopy. 1. Conformational changes of F-actin in the muscle fiber caused by ATP and its analogs]

Increase of anisotropy of F-actin fluorescence of balanus and rabbit muscle fibers under the influence of ATP, AMP and pyrophosphate in EGTA presence was detected by means of the polarized ultraviolet (UV) fluorescent microscopy methods. The fluorescence anisotropy changes are assumed to be associated with the conformational changes in the actin. ATP cause more noticeable changes of actin structure, than pyrophosphate and AMP. The conformational changes in the actin of balanus and rabbit muscle fibres were similar. ATP and its analogs induced also decrease of UV fluorescence anisotropy of A-band which appears to be associated with conformational changes in myosin. It was siggested that the changes in fluorescence of anisotropy of A-bands are due to structural changes in both HMM and LMM parts of myosin molecule.     

The disappearance of the dependence of actin-myosin interaction on the phosphorylation of myosin light chains in the "freezing" of the structure of heavy meromyosin by a bifunctional reagent

Using glycerinated muscle fibers, free of myosin, tropomyosin and troponin, a study was made of the structural state of F-actin modified by N-(iodoacetyl)-N'-(1-naphthyl-5-sulfo)-ethylendiamine (1.5-IAEDANS) and by rhodaminyl--phalloin at decoration of thin filaments with a proteolytic fragment of myosin--heavy meromyosin containing phosphorylated and dephosphorylated myosin light chains. The heavy meromyosin used has three SH-groups of heavy chain SH1, SH2 and SH chi modified by bifunctional reagent N,N'-n-phenylmaleimide (SH1-SH2, SH2-SH chi). At decoration of thin filaments with heavy meromyosin, some changes in polarized fluorescence of rhodaminyl--phalloin and 1.5-IAEDANS independent of phosphorylation of myosin light chains were found. Fluorescence anisotropy of the fiber was found to depend primarily on the character of heavy chain of SH-group modification. The ability of heavy chains to change their conformations is supposed to play an important role in the mechanism of myosin system modulation of muscle contraction.     

Motion of myosin cross-bridges in skeletal muscle fibers studied by time-resolved fluorescence anisotropy decay

The time-resolved fluorescence polarization anisotropy signal has been measured from fluorescent-labeled myosin cross-bridges in single glycerinated muscle fibers in the relaxed and rigor states. In one experimental configuration, the polarization of the excitation light and the fiber axis are aligned, and the anisotropy is sensitive to rotational motions of the probes about axes other than the fiber axis. The rotational correlation times are approximately 1000 ns for relaxed fibers and greater than 7000 ns for rigor fibers. In another experimental configuration, the excitation light polarization is perpendicular to the fiber axis, and its propagation vector has a component parallel to the fiber axis so that the anisotropy is sensitive to probe rotational motion about different axes, including the fiber axis. In this configuration, the rotational correlation times are approximately 300 ns for both relaxed and rigor fibers. The theory of rotational diffusion in a potential described in a related paper [Burghardt, T.P. (1985) Biophys. J. (in press)] is applied to the relaxed fiber data.     

The mechanism of the movement of leucocytes

In a study of the movement of human leucocytes it was clarified that characteristic contraction waves were observed on the cell surface during movement and an initial morphological change directly related to the appearance of the wave originated in the surface of the granuloplasm and not in the cell membrane. From these findings, together with physicochemical properties of the contractile protein from equine leucocytes, it was proposed that the wave observed in moving leucocytes might be conducted, in some way, by contraction and relaxation of the contractile protein in the cells. Myosin A and actin as constituents of the contractile protein were extracted separately from leucocytes in polymerized form, which resemble myosin aggregate and F-actin from muscle, respectively. The thick and thin filaments of about 150 and 80 Å in diameter were observed in glycerinated leucocytes with electron microscopy. When glycerinated leucocytes were incubated with heavy meromyosin (HMM) from rabbit skeletal myosin A, the thin filaments developed a structure resembling the ‘arrowhead structure’ of the HMM F-actin complex in vitro. The thick filaments seemed to correspond to myosin aggregates and the thin ones to filaments containing F-actin.