Effect of hypothyroidism on actin subdomain-1 movement induced by myosin subfragment-1 binding in fast and slow rat skeletal muscles

The orientation and mobility of an N-(iodoacetyl)-(1-naphtyl-5-sulpho-ethylenediamine) fluorescent probe (1.5-IAEDANS) specifically bound to Cys-374 of actin in ghost muscle fibers isolated from fast and slow rat muscles were studied by polarized fluorimetry in the absence and presence of a myosin subfragment-1 (S1) in intact rats and in animals with a gradual (2–5 weeks) reduction in the level of thyroid hormones (development of hypothyroidism). The binding of S1 to F-actin of ghost muscle fibers was shown to induce changes in the orientation of dipoles of the 1.5-IAEDANS fluorescent probe and in the relative amount of the randomly oriented fluorophores that indicates changes in actin subdomain-1 orientation and mobility resulting from formation of its strong binding with S1. This effect is markedly inhibited by the development of hypothyroidism. The maximal effect of hypothyroidism is observed after 34 days of the development of the disease. It is suggested that the change in the thyroid status in muscle inhibits the ability of F-actin to form strong binding with myosin, which is essential for the generation of force.     

Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy

To study the orientation and dynamics of myosin, we measured fluorescence polarization of single molecules and ensembles of myosin decorating actin filaments. Engineered chicken gizzard regulatory light chain (RLC), labeled with bisiodoacetamidorhodamine at cysteine residues 100 and 108 or 104 and 115, was exchanged for endogenous RLC in rabbit skeletal muscle HMM or S1. AEDANS-labeled actin, fully decorated with labeled myosin fragment or a ratio of approximately 1:1000 labeled:unlabeled myosin fragment, was adhered to a quartz slide. Eight polarized fluorescence intensities were combined with the actin orientation from the AEDANS fluorescence to determine the axial angle (relative to actin), the azimuthal angle (around actin), and RLC mobility on the <10 ms timescale. Order parameters of the orientation distributions from heavily labeled filaments agree well with comparable measurements in muscle fibers, verifying the technique. Experiments with HMM provide sufficient angular resolution to detect two orientations corresponding to the two heads in rigor. Experiments with S1 show a single orientation intermediate to the two seen for HMM. The angles measured for HMM are consistent with heads bound on adjacent actin monomers of a filament, under strain, similar to predictions based on ensemble measurements made on muscle fibers with electron microscopy and spectroscopic experiments.     

Caldesmon restricts the movement of both C- and N-termini of tropomyosin on F-actin in ghost fibers during the actomyosin ATPase cycle

New data on the movements of tropomyosin singly labeled at alpha- or beta-chain during the ATP hydrolysis cycle in reconstituted ghost fibers have been obtained by using the polarized fluorescence technique which allowed us following the azimuthal movements of tropomyosin on actin filaments. Pronounced structural changes in tropomyosin evoked by myosin heads suggested the "rolling" of the tropomyosin molecule on F-actin surface during the ATP hydrolysis cycle. The movements of actin-bound tropomyosin correlated to the strength of S1 to actin binding. Weak binding of myosin to actin led to an increase in the affinity of the tropomyosin N-terminus to actin with simultaneous decrease in the affinity of the C-terminus. On the contrary, strong binding of myosin to actin resulted in the opposite changes of the affinity to actin of both ends of the tropomyosin molecule. Caldesmon inhibited the "rolling" of tropomyosin on the surface of the thin filament during the ATP hydrolysis cycle, drastically decreased the affinity of the whole tropomyosin molecule to actin, and "freezed" tropomyosin in the position characteristic of the weak binding of myosin to actin.     

Behavior of caldesmon upon interaction of thin filaments with myosin subfragment 1 in ghost fibers

Caldesmon is a component of smooth muscle thin filaments which inhibits their interaction with myosin. We have used polarized fluorescence technique to study the behavior of caldesmon during the interaction of myosin subfragment 1 (S1) with thin filaments reconstituted in rabbit skeletal muscle ghost fibers by incorporation of smooth muscle tropomyosin and caldesmon labeled with acrylodan at cysteine residue located in the C-terminal region. Significant changes in acrylodan fluorescence intensity upon addition of skeletal muscle S1 reflected substantial displacement of caldesmon from thin filaments, while alterations in the calculated fluorescence parameters indicated the simultaneous rearrangement of the remaining caldesmon fraction. The orientation of caldesmon in the S1-thin filament complex relative to the fiber axis changes by approximately 7 degrees and the mobility of the fluorescent probe by about 9%. The alterations in caldesmon orientation were proportional to the strength of S1 binding and diminished respectively upon addition of ADP and ADP-V(i). The changes in orientation of acrylodan-caldesmon evoked by the interaction of S1 with thin filaments were more pronounced than that in AEDANS-F-actin which suggests that the spatial arrangement of caldesmon in the complex is governed not only by F-actin but also by S1. The results may indicate that the changes in spatial arrangement of caldesmon are adjusted to the conformation of F-actin and S1 characteristic for particular steps of the ATP hydrolysis cycle.     

Effect of the C-terminal actin-binding sites of caldesmon on the interaction of actin with myosin

TRITC-phalloidin or FITC-labeled F-actin of ghost muscle fibers was bound to tropomyosin and C-terminal recombinant fragments of caldesmon CaDH1 (residues 506-793) or CaDH2 (residues 683-767). After that the fibers were decorated with myosin subfragment 1. In the absence of caldesmon fragments, subfragment 1 interaction with F-actin caused changes in parameters of polarized fluorescence, that were typical of "strong" binding of myosin heads to F-actin and of the "switched on" conformational state of actin. CaDH1 inhibited, whereas CaDH2 activated the effect of subfragment 1. It is suggested that C-terminal part of caldesmon may modulate the transition of F-actin subunits from the "switched on" to the "switched off" state.     

Cleavage of DNA-binding loops of actin by subtilisin prevent formation of a strong type of myosin binding with actin

In order to elucidate the role of DNA-binding loop of actin (amino acid residues 38-52) in mechanisms of muscle contraction, polarizational fluorimetry and ghost muscle fibers, containing thin filaments reconstructed by intact and subtilisin-cleaved G-actin were used. The thin filaments were modified by fluorescent probes rhodamin-phalloidin and 1,5-IAEDANS. Changes in orientation and mobility of the probes were considered as an indication of changes in actin conformation. The stage AM of ATP hydrolysis cycle was simulated. For this purpose, thin filaments were decorated by myosin subfragment-1 (S1) in the absence of nucleotide. It has been shown that S1 binding to actin is accompanied by changes in orientation and mobility of the fluorescent probes. For intact filaments, the changes of these parameters indicate the formation of a strong binding between S1 and actin. Cleavage of DNA-binding loop by subtilisin markedly inhibits this effect. The cleavage of actin by subtilisin has also been shown to diminish the changes in fiber birefringence, which takes place at the formation of F-actin-S1 complex in the muscle fiber. The spatial organization of the actin DNA-binding loop is suggested to play an important role in determining the character of myosin interaction with actin in the ATP hydrolysis cycle.     

The effect of caldesmon on actin-myosin interaction in skeletal muscle fibers

The effects of caldesmon on structural and dynamic properties of phalloidin-rhodamine-labeled F-actin in single skeletal muscle fibers were investigated by polarized microphotometry. The binding of caldesmon to F-actin in glycerinated fibers reduced the alterations of thin filaments structure and dynamics that occur upon the transition of the fibers from rigor to relaxing conditions. In fibers devoid of myosin and regulatory proteins (ghost fibers) the binding of caldesmon to F-actin precluded structural changes in actin filaments induced by skeletal muscle myosin subfragment 1 and smooth muscle tropomyosin. These results suggest that the restraint for the alteration of actin structure and dynamics upon binding of myosin heads and/or tropomyosin evoked by caldesmon can be related to its inhibitory effect on actin-myosin interaction.     

Comparison of Ca2+-dependent effects of caldesmon-tropomyosin-calmodulin and troponin-tropomyosin complexes on the structure of F-actin in ghost fibers and its interaction with myosin heads

Comparison of two types of Ca2+-regulated thin filament, reconstructed in ghost fibers by incorporating either caldesmon-gizzard tropomyosin-calmodulin or skeletal muscle troponin-tropomyosin complex, was performed by polarized microphotometry. The changes in actin structure under the influence of these regulatory complexes, as well as those upon the binding of the myosin heads, were followed by measurements of F-actin intrinsic tryptophan fluorescence and the fluorescence of phalloidin-rhodamine complex attached to F-actin. The results show that in the presence of smooth muscle tropomyosin and calmodulin, caldesmon causes Ca2+-dependent alterations of actin conformation and flexibility similar to those induced by skeletal muscle troponin-tropomyosin complex. In both cases, transferring of the fiber from '-Ca2+' to '+Ca2+' solution increases the number of turned-on actin monomers. However, whereas troponin in the absence of Ca2+ potentiates the effect of skeletal muscle tropomyosin, caldesmon-calmodulin complex inhibits the effect of smooth muscle tropomyosin. This difference seems to be due to the qualitatively different alterations in the structure and flexibility of F-actin in ghost fibers evoked by smooth and skeletal muscle tropomyosins. Troponin can bind to F-actin-smooth muscle tropomyosin-caldesmon complex and, in the presence of Ca2+, release the restraint by caldesmon for S-1-induced alterations of conformation, and reduce that for flexibility of actin in ghost fibers. This effect seems to be related to the abolishment by troponin of the potentiating effect of tropomyosin on caldesmon-induced inhibition of actomyosin ATPase activity.     

The effect of the functional electrostimulation of rat fast and slow muscles on the structural state of actin in the thin filaments of a ghost muscle fiber]

The effect of electrostimulation of fast (EDL) and slow (SOL) rat muscles on the orientation and mobility of fluorescent probes rhodamine-phalloidine and 1.5-IAEDANS (N-iodoacetyl-N'-(5-sulpho-1-naphtyl)-ethylenediamine), located in various parts of actin molecule, has been studied by polarized microfluorimetry techniques. Muscles were stimulated at 20 Hz with the pulse width of 0.3 msec, some muscles were treated for 6 h during the first day, the other muscles for 6 h a day during the next 4 days before glycerinization. Then muscle fibres freed by the extraction of myosin, tropomyosin and troponin (ghost fibres) were used. It was shown that the binding of myosin subfragment 1 (S1) to actin induced the changes in polarized fluorescence of the fibres. The analysis of the obtained data showed that the formation of actomyosin complex in stimulated muscles resulted in increasing the angle between the thin filaments and the emission dipole of rhodamine-phalloidine, as well as in decreasing the mobility of this dye. In the experiments with the 1.5-IAEDANS label, the angle of the emission dipole decreased, while the label mobility increased. It was suggested that the orientation of domains in actomyosin complex changes following the electrostimulation to affect both the conformational state of F-actin in thin filaments of ghost fibres and actin-myosin interaction.     

Caldesmon inhibits formation of strongly bound myosin cross-bridges and activates an ability of weakly bound cross-bridges to transform actin monomers to the off-conformation

The effect of caldesmon and its actin-binding C-terminal 35 kDa fragment on conformational alterations of actin in a muscle fiber at relaxation, rigor and at simulation of strong and weak binding of myosin heads to actin was studied by polarizational fluorimetry technique. The strong and weak binding forms were mimicked during binding of F-actin of ghost muscle fibers to myosin subfragment-1 modified with NEM (NEM-S1) or pPDM (pPDM-S1), respectively. As a test for alterations in actin conformation, changes in orientation and mobility of a fluorescent probe, TRITC-phalloidin, bound specifically to F-actin were used. The results obtained have shown that during transition of the muscle fiber from the relaxed state into the rigor and during binding of actin filaments to NEM-S1, changes of polarization parameters take place, which are characteristic of formation between actin and myosin of the strong binding and of transformation of actin subunits from the "turned-off" (inactive) to the "turned-on" (active) conformation. Binding of pPDM-S1 to actin and relaxation of the muscle fiber are accompanied, on the contrary, by the changes of orientation and of the fluorescent probe mobility, which are typical of formation of the weak ("non-force-producing") form of actin-myosin binding and of transformation of actin subunits from the active conformation into the inactive one. Caldesmon and its C-terminal fragment markedly inhibit formation of the strong binding at rigor and activate transition of actin monomers to the switched off conformation at relaxation of muscle fiber. In parallel experiments, these regulatory proteins have been shown to inhibit an active force developed at the transition of a muscle fiber from relaxation to rigor. Besides, caldesmon and its fragment decrease the rate of actin filament sliding over myosin in an in vitro motility assay. Caldesmon is suggested to regulate the smooth muscle contraction in an allosterical manner. The alterations in actin conformation inhibit formation of strong binding of myosin cross bridges to actin and activate the ability of weakly bound cross bridges to switch actin monomers from the "on" to the "off" conformation.     

Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle

Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7 degrees relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**.M**.ADP.Pi (weak binding) and A--.M--.ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the "closed" position caldesmon "freezes" the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.     

40-kDa protein from thin filaments of the mussel Crenomytilus grayanus changes the conformation of F-actin during the ATPase cycle

Polarized fluorimetry was used to study in ghost muscle fibers the influence of a 40-kDa protein from the thin filaments of the mussel Crenomytilus grayanus on conformational changes of F-actin modified by the fluorescent probes 1,5-IAEDANS and FITC-phalloidin during myosin subfragment (S1) binding in the absence of nucleotides and in the presence of MgADP or MgATP. The fluorescence probes were rigidly bound with actin, which made the absorption and emission dipoles of the probes sensitive to changes in the orientation and mobility of both actin monomer and its subdomain-1 in thin filaments of the muscle fiber. On modeling different intermediate states of actomyosin, the orientation and mobility of oscillators of the dyes were changed discretely, which suggests multistep changes in the actin conformation during the cycle of ATP hydrolysis. The 40-kDa protein influenced the orientation and mobility of the fluorescent probes markedly, suppressing changes in their orientation and mobility in the absence of nucleotides and in the presence of MgADP, but enhancing these changes in the presence of MgATP. The calponin-like 40-kDa protein is supposed to prevent formation of the strong binding state of actomyosin in the absence of nucleotides and in the presence of MgADP but to activate formation of this state in the presence of MgATP.     

Hyperthyreosis inhibits the ability of actin to form strong-binding with myosin

The effect of hyperthyreosis development induced by the increase in thyroid hormones in rats (during 2-4 weeks) on the orientation and mobility of fluorescent probe N-(iodoacetyl)-(1-naphtyl-5-sulpho-ethylenediamine) specifically bound to Cys 374 of actin in ghost muscle fibers isolated from fast (EDL) and slow (SOL) rat muscles was studied. It was found that the binding of myosin subfragment-1 (S1) to F-actin induced the typical for the formation of strong binding actomyosin decrease in mobility of actin subdomain 1 and its rotation towards thin filament periphery. Development of hyperthyreosis markedly inhibited these phenomena. The maximal effect was observed after 21 days of disease development. It is suggested that one of the reasons of the contractile deficit of muscle in hyperthyreosis is inhibition of the strong binding between actin and myosin during ATPase cycle.     

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.     

Effect of caldesmon on the type of conformation changes in F-actin induced by the binding of myosin 1 subfragment

The effect of caldesmon on the conformational changes of F-actin caused by myosin subfragment 1 (S-1) binding was studied, using the polarized microfluorimetry method. It was demonstrated that the polarized fluorescence of rhodaminil-phalloin specifically bound to F-actin of pure actin filaments as well as of tropomyosin-containing actin filaments changes as a result of binding to S-1. The nature of these changes depends on the presence of caldesmon in the filaments. Caldesmon was supposed to modify the conformational changes in F-actin induced by S-1.     

Conformational changes of actin induced by strong or weak myosin subfragment-1 binding

Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S1 binding were studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labeled with NBD-Cl; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimid. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes). The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore location and binding site of actomyosin. Strong binding of S with actin leads to 1) a decrease in the orientation of oscillators of derivatives of falloidin (TRITC-falloidin, FITC-falloidin) and actin-bound nucleotide (e-ADP); 2) an increase in the orientation of dye oscillators located in the "front' surface of the small domain (where actin is viewed in the standard orientation with subdomains 1/2 and 3/4 oriented to the right and to the left, respectively); 3) a decrease in the angles of dye oscillators located on the "back" surface of subdomain-1. In contrast, a weak binding of S1 to actin induces the opposite effects in orientation of these probes. These data suggest that during the ATP hydrolysis cycle myosin heads induce a change in actin monomer (a tilt and twisting of its small domain). Presumably, these alterations in F-actin conformation play an important role in muscle contraction.     

Orientational distribution of spin-labeled actin oriented by flow.

Previous studies on spin-labeled F-actin (MSL-actin), using saturation transfer electron paramagnetic resonance (ST-EPR), have demonstrated that actin has submillisecond rotational flexibility and that this flexibility is affected by the binding of myosin and its subfragments. This rotational flexibility does not change during the active interaction of myosin heads, actin, and adenosine triphosphate. However, these ST-EPR studies, performed on randomly oriented actin, would not be sensitive to orientational changes on the millisecond time scale or slower. In the present study, we have clarified these results by performing conventional EPR experiments on MSL-actin oriented by flow to detect changes in the orientational distribution. We have determined the orientational distribution of the spin labels relative to the magnetic field (flow direction) by comparing experimental EPR spectra to simulated EPR spectra corresponding to known orientational distributions. Spectra acquired during flow indicate two populations of probes: a highly ordered population and a disordered population. For the ordered population (28% of the total spin concentration), the angle between the actin filament axis and the nitroxide z axis (theta) fits a Gaussian distribution centered at 32.0 +/- 0.9 degrees, with a full width at half maximum of 20.7 +/- 3.9 degrees. The angle between the nitroxide x axis and the projection of the field in the xy plane (phi) is centered at 37.5 +/- 9.2 degrees with a full width of 24.9 +/- 10.7 degrees. This orientational distribution is not significantly changed upon the binding of phalloidin or myosin subfragment 1 (S1), indicating that these proteins do not affect the axial orientation of actin subunits. Spectra of spin-labeled S1 (MSL-S1) bound to actin oriented by flow have about the same orientational distribution as MSL-S1 bound to actin in oriented fibers. Thus, the oriented fraction of flow-oriented actin filaments has nearly the same high degree of alignment as the actin filaments in muscle fibers.     

Orientation of actin filaments during motion in in vitro motility assay.

Rhodamine-phalloidin was added to F-actin, and the orientation of transition dipoles of the dye was measured in single actin filaments by polarization of fluorescence. Rhodamine-phalloidin was well immobilized on the surface of actin, indicating that changes in orientation of the dye reported changes in orientation of actin monomers. In stationary filaments the dipoles were inclined at 49.3 degrees with respect to the filament axis. The disorganization of dipoles in stationary filaments was insignificant. When the filaments were made to translate, the average orientation of the dye did not change, but disorganization slightly increased. Disorganization increased significantly when filaments were free in solution. We concluded that, within the accuracy of our measurements (approximately 18%), actin monomers did not undergo major reorientations during motion, but that binding of myosin heads deformed the structure of filaments.     

Conformational changes of contractile proteins accompanying modulation of skeletal muscle contraction. Polarized microfluorometry investigations

Results of studies on the modulation of skeletal muscle contraction by phosphorylation of myosin regulatory light chains and by exchange of magnesium for calcium in myosin heads were reviewed. The polarized fluorescence method was used in these studies, and conformational changes of contractile proteins accompanying modulation of skeletal muscle contraction were investigated. It was found that both the exchange of bound magnesium for calcium on myosin heads and the phosphorylation of myosin regulatory light chains control the ability of myosin heads to induce, upon binding to actin, conformational changes of thin filament leading to decrease or increase of its flexibility. The changes in actin filament flexibility may be caused by alteration of both the inter- and the intramonomer structural organization.     

Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin

Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.