The binding of calcium to glycerinated muscle fibers in rigor. The effect of filament overlap.

The binding of Ca2+ to glycerinated rabbit psoas fibers of varying sarcomere length was measured with a double isotope technique and ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid buffers. Experiments were carried out under rigor conditions with fiber bundles pre-set at different lengths prior to extraction with detergent and glycerol. These experiments were designed to test whether rigor complex formation, determined by the degree of filament overlap, enhances Ca2+-receptor affinity in the intact filament lattice, as it does in reconstituted actomyosin systems. The Ca2+-receptor affinity, as indicated by the free Ca2+ concentration at half-saturation and by the slopes of Scatchard plots, was found to be relatively unaffected by variations in filament overlap. However, the maximum bound Ca2+ was significantly reduced in stretched fibers. With maximum filament overlap the bound Ca2+ was equivalent to 4 mol per mol troponin. When stretched to zero overlap the fibers bound a maximum of 3 mol Ca2+ per mol troponin. When fibers with maximum overlap were incubated in the presence of 5 mM MgATP there was a reduction in the number of Ca2+-binding sites equivalent to that caused by stretching the fibers. These findings, taken together with other data in the literature, suggest that in the intact filament lattice at least one of the Ca2+-binding sites is present only when cross-bridge attachments are formed.     

Cooperative interactions between calcium-binding sites on glycerinated muscle fibers. The influence of cross-bridge attachment

A double isotope technique and EGTA buffers were used to measure the binding of Ca2+ to rabbit psoas muscle fibers extracted with detergent and glycerol. These experiments were designed to test the effect of rigor complex formation, determined by the degree of filament overlap, on the properties of the Ca2+-binding sites in the intact filament lattice. In the presence of 5 mM MgCl2 (no ATP), reduction of filament overlap was associated with a reduced binding of Ca2+ over the entire range of free Ca2+ concentrations (5.10(-8)-2.10(-5) M). With maximum filament overlap (sarcomere length 2.1-2.2 micrometer) the maximum bound Ca2+ was equivalent to 4 mol Ca2+/mol troponin and there was significant positive interaction between binding sites, as shown by Scatchard and Hill plots. With no filament overlap (sarcomere length 3.8-4.4 micrometer) the maximum bound Ca2+ was equivalent to 3 mumole Ca2+/mol troponin and graphical analysis indicated a single class of non-interacting sites. The data provide evidence that when cross-bridge attachments between actin and myosin filaments are formed not only does an additional Ca2+ binding site appear, but cooperative properties are imposed upon the binding sites.     

Distributions of calcium in A and I bands of skinned vertebrate muscle fibers stretched to beyond filament overlap.

Measurements were made of the distributions of total calcium along the length of A and I bands in skinned frog semitendinosus muscles using electron probe x-ray microanalysis. Since calcium in the water space was kept below the detection limit of the technique, the signal was assumed to reflect the distribution of calcium bound to myofilament proteins. Data from sarcomeres with overlap between thick and thin filaments showed enhancement of calcium in this region, as previously demonstrated in rabbit psoas muscle fibers in rigor (Cantino, M. E., T. S. Allen, and A. M. Gordon. 1993. Subsarcomeric distribution of calcium in demembranated fibers of rabbit psoas muscle. Biophys. J. 64:211-222). Such enhancement could arise from intrinsic non-uniformities in calcium binding to either thick or thin filaments or from enhancement of calcium binding to either filament by rigor cross-bridge attachment. To test for intrinsic variations in calcium binding, calcium distributions were determined in fibers stretched to beyond filament overlap. Calcium binding was found to be relatively uniform along both thick and thin filaments, and therefore cannot account for the increased calcium observed in the overlap region. From these results it can be concluded that the observed enhancement of calcium is due to an increase in calcium binding to myofilaments as a result of rigor attachment of cross-bridges to actin. The source of the enhancement is most likely an increase in calcium binding to troponin, although enhancement of calcium binding to myosin light chains cannot be ruled out.     

Cooperative interactions between calcium-binding sites on glycerinated muscle fibers the influence of cross-bridge attachment

A double isotope technique and EGTA buffers were used to measure the binding of Ca²⁺ to rabbit psoas muscle fibers extracted with detergent and glycerol. These experiments were designed to test the effect of rigor complex formation, determined by the degree of filament overlap, on the properties of the Ca²⁺-binding sites in the intact filament lattice. In the presence of 5 mM MgCl₂ (no ATP), reduction of filament overlap was associated with a reduced binding of Ca²⁺ over the entire range of free Ca²⁺ concentrations (5 · 10^?8 – 2 · 10^?5 M). With maximum filament overlap (sarcomere length 2.1–2.2 μm) the maximum bound Ca²⁺ was equivalent to 4 mol Ca²⁺/mol troponin and there was significant positive interaction between binding sites, as shown by Scatchard and Hill plots. With no filament overlap (sarcomere length 3.8–4.4 μm) the maximum bound Ca²⁺ was equivalent to 3 μmol Ca²⁺/mol troponin and graphical analysis indicated a single class of non-interacting sites. The data provide evidence that when cross-bridge attachments between actin and myosin filaments are formed not only does an additional Ca²⁺ binding site appear, but cooperative properties are imposed upon the binding sites.     

Calcium alone does not fully activate the thin filament for S1 binding to rigor myofibrils.

Skeletal muscle contraction is regulated by calcium via troponin and tropomyosin and appears to involve cooperative activation of cross-bridge binding to actin. We studied the regulation of fluorescent myosin subfragment 1 (fS1) binding to rigor myofibrils over a wide range of fS1 and calcium levels using highly sensitive imaging techniques. At low calcium and low fS1, the fluorescence was restricted to the actin-myosin overlap region. At high calcium and very low fS1, the fluorescence was still predominantly in the overlap region. The ratio of nonoverlap to overlap fluorescence intensity showed that increases in the fS1 level resulted in a shift in maximum fluorescence from the overlap to the nonoverlap region at both low and high calcium; this transition occurred at lower fS1 levels in myofibrils with high calcium. At a fixed fS1 level, increases in calcium also resulted in a shift in maximum fluorescence from the overlap region to the nonoverlap region. These results suggest that calcium alone does not fully activate the thin filament for rigor S1 binding and that, even at high calcium, the thin filament is not activated along its entire length.     

Cooperative effects of rigor and cycling cross-bridges on calcium binding to troponin C

The effects of rigor and cycling cross-bridges on distributions of calcium (Ca) bound within sarcomeres of rabbit psoas muscle fibers were compared using electron probe x-ray microanalysis. Calcium in the overlap region of rigor fibers, after correction for that bound to thick filaments, was significantly higher than in the I-band at all pCa levels tested between 6.9 and 4.8, but the difference was greatest at pCa 6.9. With addition of MgATP, differences were significant at high levels of activation (pCa 5.6 and 4.9); near and below the threshold for activation, Ca was the same in I-band and overlap regions. Comparison of Ca and mass profiles at the A-I junction showed elevation of Ca extending 55-110 nm (up to three regulatory units) into the I-band. Extraction of TnC-reduced I-band and overlap Ca in rigor fibers at pCa 5.6 to the same levels found in unextracted fibers at pCa 8.9, suggesting that variations reported here reflect changes in Ca bound to troponin C (TnC). Taken together, these observations provide evidence for near-neighbor cooperative effects of both rigor and cycling cross-bridges on Ca(2+) binding to TnC.     

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.     

Extensibility of the myofilaments in vertebrate skeletal muscle as revealed by stretching rigor muscle fibers

The extensibility of the myofilaments in vertebrate skeletal muscle was studied by stretching glycerinated rabbit psoas muscle fibers in rigor state and examining the resulting extension of sarcomere structures under an electron microscope. Although stretches applied to rigor fibers produced a successive yielding of the weakest sarcomeres, the length of the remaining intact sarcomeres in many myofibrils was fairly uniform, being definitely longer than the sarcomeres in the control, nonstretched part of rigor fibers. The stretch-induced increase in sarcomere length was found to be taken up by the extension of the H zone and the I band, whereas the amount of overlap between the thick and thin filaments did not change appreciably with stretches of 10-20%. The thick filament extension in the H zone was localized in the bare regions, whereas the thin filament extension in the I band appeared to take place uniformly along the filament length. No marked increase in the Z-line width was observed even with stretches of 20-30%. These results clearly demonstrate the extensibility of the thick and thin filaments. The possible contribution of the myofilament compliance to the series elastic component (SEC) in vertebrate skeletal muscle fibers is discussed on the basis of the electron microscopic data and the force-extension curve of the SEC in rigor fibers.     

Subsarcomeric distribution of calcium in demembranated fibers of rabbit psoas muscle.

Direct measurements were made of the Ca distribution within sarcomeres of glycerinated rabbit psoas muscle fibers in rigor using electron probe x-ray microanalysis. Both analogue raster analysis and digital x-ray imaging were used to quantitate the Ca distribution along thick and thin filaments as a function of the concentration of free Ca2+. Even when corrected for the estimated contribution of Ca bound to thick filaments, the Ca measured in the region of overlap between thick and thin filaments significantly exceeded the Ca in the I-band at subsaturating concentrations of free Ca2+. At saturating levels of free Ca2+, the excess Ca in the overlap region was diminished but still statistically significant. The data thus suggest that the formation of rigor linkages exerts multiple effects on the binding of Ca2+ to thin filaments in the overlap region by increasing the affinity of troponin C for Ca2+ and possibly by unmasking additional Ca2+ binding sites. The data also show that the cooperativity invested in the thin filaments is insufficient to permit the effects of rigor cross-bridge formation on Ca2+ binding to propagate far along the thin filaments into the I-band.     

Is the SII portion of the cross-bridge in glycerinated rabbit psoas fibers compliant in the rigor state?

To see whether the SII portion of the cross-bridge in rigor fibers is longitudinally compliant, we chemically cross-linked with dimethyl suberimidate the entire rod portion (including the SII portion) of myosin onto the surface of thick filaments in glycerinated rabbit psoas fibers, and studied the effect of the SII fixation on the stiffness of the rigor fibers. The cross-linking of fiber segments with full filament overlap increased the rigor stiffness by approximately 25%. Almost the same absolute amount of the stiffness increase was also observed in rigor fibers with half- or no filament overlap after the cross-linking, and a similar but somewhat larger increment of stiffness was observed in fiber segments cross-linked in relaxing solution. These results indicate that the stiffness increase is not produced by the fixation of the SII portion onto the thick filament surface, but is caused instead by the cross-linking of some parallel elastic elements in muscle, and therefore indicate that the SII portion of the cross-bridge is hardly longitudinally compliant in rigor fibers.     

Compliance of thin filaments in skinned fibers of rabbit skeletal muscle.

The mechanical compliance (reciprocal of stiffness) of thin filaments was estimated from the relative compliance of single, skinned muscle fibers in rigor at sarcomere lengths between 1.8 and 2.4 micron. The compliance of the fibers was calculated as the ratio of sarcomere length change to tension change during imposition of repetitive cycles of small stretches and releases. Fiber compliance decreased as the sarcomere length was decreased below 2.4 micron. The compliance of the thin filaments could be estimated from this decrement because in this range of lengths overlap between the thick and thin filaments is complete and all of the myosin heads bind to the thin filament in rigor. Thus, the compliance of the overlap region of the sarcomere is constant as length is changed and the decrease in fiber compliance is due to decrease of the nonoverlap length of the thin filaments (the I band). The compliance value obtained for the thin filaments implies that at 2.4-microns sarcomere length, the thin filaments contribute approximately 55% of the total sarcomere compliance. Considering that the sarcomeres are approximately 1.25-fold more compliant in active isometric contractions than in rigor, the thin filaments contribute approximately 44% to sarcomere compliance during isometric contraction.     

Structural transients of contractile proteins upon sudden ATP liberation in skeletal muscle fibers

Structural changes of contractile proteins were examined by millisecond time-resolved two-dimensional x-ray diffraction recordings during relaxation of skinned skeletal muscle fibers from rigor after caged ATP photolysis. It is known that the initial dissociation of the rigor actomyosin complex is followed by a period of transient active contraction, which is markedly prolonged in the presence of ADP by a mechanism yet to be clarified. Both single-headed (overstretched muscle fibers with exogenous myosin subfragment-1) and two-headed (fibers with full filament overlap) preparations were used. Analyses of various actin-based layer line reflections from both specimens showed the following: 1), The dissociation of the rigor actomyosin complex was fast and only modestly decelerated by ADP and occurred in a single exponential manner without passing through any detectable transitory state. Its ADP sensitivity was greater in the two-headed preparation but fell short of explaining the large ADP effect on the transient active contraction. 2), The decay of the activated state of the thin filament followed the time course of tension more closely in an ADP-dependent manner. These results suggest that the interplay between the reattached active myosin heads and the thin filament is responsible for the prolonged active contraction in the presence of ADP.     

Maximal activation of skeletal muscle thin filaments requires both rigor myosin S1 and calcium

The regulation by calcium and rigor-bound myosin-S1 of the rate of acceleration of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) release from myosin-mdADP-P(i) by skeletal muscle thin filaments (reconstituted from actin-tropomyosin-troponin) was measured using double mixing stopped-flow fluorescence with the nucleotide substrate 2'-deoxy-3'-O-(N-methylanthraniloyl). The predominant mechanism of regulation is the acceleration of product dissociation by a factor of approximately 200 by thin filaments in the fully activated conformation (bound calcium and rigor S1) relative to the inhibited conformation (no bound calcium or rigor S1). In contrast, only 2-3-fold regulation is due to a change in actin affinity such as would be expected by "steric blocking" of the myosin binding site of the thin filament by tropomyosin. The binding of one ligand (either calcium or rigor-S1) produces partial activation of the rate of product dissociation, but the binding of both is required to maximally accelerate product dissociation to a rate similar to that obtained with F-actin in the absence of regulatory proteins. The data support an allosteric regulation model in which the binding of either calcium or rigor S1 alone to the thin filament shifts the equilibrium in favor of the active conformation, but full activation requires binding of both ligands.     

Filament interaction monitored by light scattering in skinned fibers

The intensity of light scattered by chemically skinned rabbit psoas fibers in relaxed, rigor, and activated states was monitored at 90 degrees to the incident beam. In the relaxed state, scattering varied in proportion to the volume of muscle in the beam. Scattering increased to 2.3 times the resting value when rigor was induced by withdrawal of MgATP or when the myofibrils were activated by the caffeine-induced release of Ca from the sarcoplasmic reticulum. The rigor-induced increase in scattering decreased monotonically when MgATP was reintroduced stepwise (0-100 microM). This decrease in scattering was accompanied by an increase in tension up to an optimum MgATP level of approximately 10 microM, and then tension decreased at higher concentrations (10-100 microM). The increase in scattering during both rigor and activation was dependent upon fiber length. At lengths when thick-thin filament overlap was near zero, the light signal due to rigor and activation fell to within 10% of the signal for the relaxed fiber at that length. The signal during rigor increased only minimally (approximately 10%) when stretch (approximately 1%) was applied. This increase in signal was small despite a measured 5- to 10-fold increase in tension and an estimated twofold increase in stiffness. Thus, the increased light scattering caused by rigor and activation depends on filament overlap and not tension, stiffness, or substrate binding.     

Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament.

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin responsible for initiating the cascade of events resulting in contraction of striated muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers. The properties of TnC extraction have not been characterized at the structural level, nor have the interactions of TnC with the native myofibrillar thin filament been studied. To address these issues, fluorescein-labeled TnC, in conjunction with high-resolution digital fluorescence microscopy, was used to characterize TnC binding to myofibrils and to determine the randomness of TnC extraction. Fluorescein-5-maleimide TnC (F5M TnC) retained biological activity, as evidenced by reconstitution of Ca(2+)-dependent ATPase activity in extracted myofibrils and binding to TnI in a Ca(2+)-sensitive manner. The binding of F5M TnC to highly extracted myofibrils at low Ca2+ was restricted to the overlap region under rigor conditions, and the location of binding was not influenced by F5M TnC concentration. The addition of myosin subfragment 1 to occupy all actin sites resulted in F5M TnC being bound in both the overlap and nonoverlap regions. However, very little F5M TnC was bound to myofibrils under relaxing conditions. These results suggest that strong binding of myosin heads enhances TnC binding. At high Ca2+, the pattern of F5M TnC binding was concentration dependent: binding was restricted to the overlap region at low F5M TnC concentration, whereas the binding propagated into the nonoverlap region at higher levels. Analysis of fluorescence intensity showed the greatest binding of F5M TnC at high Ca2+ with S1, and these conditions were used to characterize partially TnC-extracted myofibrils. Comparison of partially extracted myofibrils showed that low levels of extraction were associated with greater F5M TnC being bound in the nonoverlap region than in the overlap region relative to higher levels of extraction. These results show that TnC extraction is not random along the length of the thin filament, but occurs more readily in the nonoverlap region. This observation, in conjunction with the influence of rigor heads on the pattern of F5M TnC binding, suggests that strong myosin binding to actin stabilizes TnC binding at low Ca2+.     

Modulation of contractile activation in skeletal muscle by a calcium-insensitive troponin C mutant

Calcium controls the level of muscle activation via interactions with the troponin complex. Replacement of the native, skeletal calcium-binding subunit of troponin, troponin C, with mixtures of functional cardiac and mutant cardiac troponin C insensitive to calcium and permanently inactive provides a novel method to alter the number of myosin cross-bridges capable of binding to the actin filament. Extraction of skeletal troponin C and replacement with functional and mutant cardiac troponin C were used to evaluate the relationship between the extent of thin filament activation (fractional calcium binding), isometric force, and the rate of force generation in muscle fibers independent of the calcium concentration. The experiments showed a direct, linear relationship between force and the number of cross-bridges attaching to the thin filament. Further, above 35% maximal isometric activation, following partial replacement with mixtures of cardiac and mutant troponin C, the rate of force generation was independent of the number of actin sites available for cross-bridge interaction at saturating calcium concentrations. This contrasts with the marked decrease in the rate of force generation when force was reduced by decreasing the calcium concentration. The results are consistent with hypotheses proposing that calcium controls the transition between weakly and strongly bound cross-bridge states.     

Changes in the lateral filament spacing of skinned muscle fibres when cross-bridges attach

When a skinned fibre prepared from frog skeletal muscle goes from the relaxed to the rigor state at a sarcomere length of about 2.2 μm, the 1, 0 transverse spacing of the filament lattice, measured by X-ray diffraction, decreases by about 11%. In measurements at various sarcomere lengths, the decrease in the spacing was approximately proportional to the degree of overlap between the thick and thin filaments. This suggests that the shrinkage of the lattice is caused by a lateral force produced by cross-bridges. In order to estimate the magnitude of the lateral force, the decrease of spacing between relaxed and rigor states was compared with the shrinkage caused osmotically by adding a high molecular weight polymer, polyvinylpyrrolidone, to the bathing solution. The results indicate that the lateral force produced per unit length of thick filament in the overlap zone is of the same order of magnitude as the axially directed force produced during maximum isometric contraction (10^?10 to 10^?9 N/μm).Experiments in the presence of a high concentration of polyvinylpyrrolidone (100 g/l) show that when the lattice spacing is decreased osmotically beyond a certain value, the lateral force produced when the fibre goes into rigor changes its direction, causing the lattice to swell. This result can be explained by assuming that there is an optimum interfilament spacing at which the cross-bridges produce no lateral force. At other spacings, the lateral force tends to displace the filament lattice toward that optimum value.     

Differences in the Charge Distribution of Glycerol-Extracted Muscle Fibers in Rigor, Relaxation, and Contraction

Glycerol-extracted rabbit psoas muscle fibers were impaled with KCl-filled glass microelectrodes. For fibers at rest-length, the potentials were significantly more negative in solutions producing relaxation than in solutions producing either rigor or contraction; further the potentials in the latter two cases were not significantly different. For stretched fibers, with no overlap between thick and thin filaments, the potentials did not differ in the rigor, the relaxation, or the contraction solutions. The potentials measured from fibers in rigor did not vary significantly with the sarcomere length. For relaxed fibers, however, the potential magnitude decreased with increasing sarcomere length. The difference between the potentials measured for rigor and relaxed fibers exhibited a nonlinear relationship with sarcomere length. The potentials from calcium-insensitive fibers were less negative in both the rigor and the relaxation solutions than those from normal fibers. When calcium-insensitive fibers had been incubated in Hasselbach and Schneider's solution plus MgCl₂ or Guba-Straub's solution plus MgATP the potentials recorded upon impalement were similar in the rigor and the relaxation solution to those obtained from normal fibers in the relaxed state. It is concluded that the increase in the negative potential as the glycerinated fiber goes from rigor to relaxation may be due to an alteration in the conformation of the contractile proteins in the relaxed state.     

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.     

Regulation of binding of subfragment 1 in isolated rigor myofibrils

A steric-hindrance model has been used to explain the regulation of muscle contraction by tropomyosin-troponin complex. The regulation of binding was studied by microscopic observation of mixtures of fluorescent subfragment 1 (S1) with rigor myofibrils at different actin- to-S1 ratios and in the presence and absence of calcium. Procedures were adapted to protect the critical thiols of S1 before conjugation to thiol-specific fluorochromes, this giving fluorescent S1 with unaltered enzyme activity. S1 binding was greatest in the I band (except at the Z- lines) in the presence of calcium regardless of the [S1]. The patterns in the absence of calcium depended on the actin-to-S1 ratios: low [S1], binding in the myosin-actin overlap region; intermediate [S1], highest binding at the A-I junction; high [S1], greatest binding in the I-band. The two distinct binding patterns observed at low [S1] were demonstrated by dual-channel fluorescence microscopy when myofibrils were sequentially incubated with fluorescent S1 without calcium followed by a different fluorescent S1 with calcium. These observations support the concept of rigor activation of actin sites. The change in the pattern upon increasing [S1] without calcium demonstrate cooperative interactions along the thin filament. However, these interactions (under the conditions used without calcium) do not appear to extend over greater than 2-3 tropomyosin-troponin-7 actin functional units.