Calcium regulation of thin filament movement in an in vitro motility assay.

The ability of calcium to regulate thin filament sliding velocity was studied in an in vitro motility assay system using cardiac troponin and tropomyosin and rhodamine-phalloidin-labeled skeletal actin and skeletal heavy meromyosin to propel the filaments. Measurements showed that both the number of thin filaments sliding and their sliding speed (Sf) were dependent on the calcium concentration in the range of pCa 5 to 9. Thin filament motility was completely inhibited only if troponin and tropomyosin were added at a concentration of 100 nM to the motility assay solution and the pCa was more than 8. The filament sliding speed was dependent on the pCa in a noncooperative fashion (Hill coefficient = 1) and reached maximum at 5 microns/s at a pCa of 5. The number of filaments moving uniformly decreased from > 90% at pCa 5-6 to near zero in less than 1 pCa unit. This behavior may be explained by a hypothesis in which the regulatory proteins control the number of cross-bridge heads interacting with the thin filaments rather than the rate at which they individually hydrolyze ATP or translocate the thin filaments.     

Assessment of the mechanical activity of cardiac isomyosins V1 and V3 by the in vitro motility assay with regulated thin filament

The dependences of thin filament sliding velocity on the calcium concentration in solution (pCa 5 to 8) for rabbit cardiac myosin isoforms V1 and V3 were determined in a set of experiments using an in vitro motility assay with a reconstructed thin filament. The constructed pCa-versus-velocity curves had a sigmoid shape. It was demonstrated that the sliding velocity of regulated thin filament at the saturating calcium concentration (pCa 5) did not differ from the actin sliding velocity for each isoform. The determined values of Hill’s cooperativity coefficient for isomyosins V1 and V3 were 1.04 and 0.75, respectively. It was demonstrated that isomyosin V3 was more sensitive to calcium as compared with isomyosin V1. Using the same assay, the dependence of thin filament sliding velocity on the concentration of the actin-binding protein α-actinin (analog of a force-velocity dependence) was determined at the saturating calcium concentration for each myosin isoform (V1 and V3). The results suggest that the calcium regulation of V1 and V3 contractile activity follows different mechanisms.     

The C-terminus of troponin T is essential for maintaining the inactive state of regulated actin

Striated muscle contraction is regulated by the actin binding proteins tropomyosin and troponin. Defects in these proteins lead to myopathies and cardiomyopathies. Deletion of the 14 C-terminal residues of cardiac troponin T leads to hypertrophic cardiomyopathy. We showed earlier that regulated actin containing Δ14 TnT was more readily activated than wild-type regulated actin. We suggested that the equilibria among the inactive (blocked), intermediate (closed or calcium), and active (open or myosin) states was shifted to the active state. We now show that, in addition, such regulated actin filaments cannot enter the inactive or blocked state. Regulated actin containing Δ14 TnT had ATPase activities in the absence of Ca2+ that were higher than wild-type filaments but far below the fully active rate. The rapid dissociation of S1-ATP from regulated actin filaments containing Δ14 TnT and acrylodan-labeled tropomyosin did not show the fluorescence increase characteristic of moving to the inactive state. Replacing wild-type TnI with S45E TnI, that favors the inactive state, did not restore the fluorescence change. We conclude that TnT has a previously unrecognized role in forming the inactive state of regulated actin.     

On the relation between filament overlap and the number of calcium-binding sites on glycerinated muscle fibers.

The formation of rigor complexes between the thick and thin filaments of glycerinated rabbit psoas muscle fibers causes the fibers to bind more calcium at any given level of free calcium. I studied the maximum amount of calcium bound as a function of filament overlap under rigor conditions. Fibers stretched to zero filament overlap (sarcomere length greater than 3.8 micron) bound exactly 75% as much calcium as fibers with maximum overlap. Between these extremes a linear relationship was found between maximum bound calcium and the length of the overlap zone. The results support the hypothesis that in the intact filament lattice one of the four calcium-binding sites of troponin depends for its existence on attachment between myosin and actin. In addition, the linear relation between maximum bound calcium and filament overlap is consistent with the assumption that the cooperative effect of rigor complex formation on calcium binding is limited to the binding site in the immediate vicinity of the rigor complex.     

Kinetic analysis of F-actin depolymerization in the presence of platelet gelsolin and gelsolin-actin complexes

Platelet gelsolin (G), a 90,000-mol-wt protein, binds tightly to actin (A) and calcium at low ionic strength to form a 1:2:2 complex, GA2Ca2 (Bryan, J., and M. Kurth, 1984, J. Biol. Chem. 259:7480-7487). Chromatography of actin and gelsolin mixtures in EGTA-containing solutions isolates a stable binary complex, GA1Ca1 (Kurth, M., and J. Bryan, 1984, J. Biol. Chem. 259:7473-7479). The effects of platelet gelsolin and the binary gelsolin-actin complex on the depolymerization kinetics of rabbit skeletal muscle actin were studied by diluting pyrenyl F-actin into gelsolin or complex-containing buffers; a decrease in fluorescence represents disassembly of filaments. Dilution of F- actin to below the critical concentration required for filament assembly gave a biphasic depolymerization curve with both fast and slow components. Dilution into buffers containing gelsolin, as GCa2, increased the rate of depolymerization and gave a first order decay. The rate of decrease in fluorescence was found to be gelsolin concentration dependent. Electron microscopy of samples taken shortly after dilution into GCa2 showed a marked reduction in filament length consistent with filament severing and an increase in the number of ends. Conversely, occupancy of the EGTA-stable actin-binding site by an actin monomer eliminated the severing activity. Dilution of F-actin into the gelsolin-actin complex, either as GA1Ca1 or GA1Ca2, resulted in a decrease in the rate of depolymerization that was consistent with filament end capping. This result indicates that the EGTA-stable binding site is required and must be unoccupied for filament severing to occur. The effectiveness of gelsolin, GCa2, in causing filament depolymerization was dependent upon the ionic conditions: in KCI, actin filaments appeared to be more stable and less susceptible to gelsolin, whereas in Mg2+, actin filaments were more easily fragmented. Finally, a comparison of the number of kinetically active ends generated when filaments were diluted into gelsolin versus the number formed when gelsolin can function as a nucleation site suggests that gelsolin may sever more than once. The data are consistent with a mechanism where gelsolin, with both actin-binding sites unoccupied, can sever but not cap F-actin. Occupancy of the EGTA-stable binding site yields a gelsolin-actin complex that can no longer sever filaments, but can cap filament ends.     

Calcium-regulated conformational change in the C-terminal end segment of troponin I and its binding to tropomyosin

The troponin complex plays an essential role in the thin filament regulation of striated muscle contraction. Of the three subunits of troponin, troponin I (TnI) is the actomyosin ATPase inhibitory subunit and its effect is released upon Ca(2+) binding to troponin C. The exon-8-encoded C-terminal end segment represented by the last 24 amino acids of cardiac TnI is highly conserved and is critical to the inhibitory function of troponin. Here, we investigated the function and calcium regulation of the C-terminal end segment of TnI. A TnI model molecule was labeled with Alexa Fluor 532 at a Cys engineered at the C-terminal end and used to reconstitute the tertiary troponin complex. A Ca(2+) -regulated conformational change in the C-terminus of TnI was shown by a sigmoid-shape fluorescence intensity titration curve similar to that of the CD calcium titration curve of troponin C. Such corresponding Ca(2+) responses are consistent with the function of troponin as a coordinated molecular switch. Reconstituted troponin complex containing a mini-troponin T lacking its two tropomyosin-binding sites showed a saturable binding to tropomyosin at pCa 9 but not at pCa 4. This Ca(2+) -regulated binding was diminished when the C-terminal 19 amino acids of cardiac TnI were removed. These results provided novel evidence for suggesting that the C-terminal end segment of TnI participates in the Ca(2+) regulation of muscle thin filament through interaction with tropomyosin.     

Cooperative [Ca²+]-dependent regulation of the rate of myosin binding to actin: solution data and the tropomyosin chain model

The regulation of muscle contraction by calcium involves interactions among actin filaments, myosin-S1, tropomyosin (Tm), and troponin (Tn). We have extended our previous model in which the TmTn regulatory units are treated as a continuous flexible chain, and applied it to transient kinetic data. We have measured the time course of myosin-S1 binding to actin-Tm-Tn filaments in solution at various calcium levels with [actin]/[myosin] ratios of 10 and 0.1, which exhibit modest slowing as [Ca(2+)] is reduced and a lag phase at low calcium. These observations can be explained if myosin binds to actin in two steps, where the first step is rate-limiting and blocked by TmTnI at low calcium, and the second step is fast, reversible, and controlled by the neighboring configuration of coupled tropomyosin-troponin units. The model can describe the calcium dependence of the observed myosin binding reactions and predicts cooperative calcium binding to TnC with competition between actin and Ca-TnC for the binding of TnI. Implications for theories of thin-filament regulation in muscle are discussed.     

Ca2+ regulation of rabbit skeletal muscle thin filament sliding: role of cross-bridge number

We investigated how strong cross-bridge number affects sliding speed of regulated Ca(2+)-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca(2+). Second, we varied the number of Ca(2+)-activatable troponin complexes at maximal Ca(2+) using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A,D63A), a mutant deficient in Ca(2+) binding at both N-terminal, low affinity Ca(2+)-binding sites (xxsTnC-sTn). Sliding speed decreased nonlinearly as the proportion of WT sTn decreased. Speed of regulated thin filaments varied with pCa when filaments contained WT sTn but filaments containing only xxsTnC-sTn did not move. pCa(50) decreased by 0.12-0.18 when either heavy meromyosin density was reduced to approximately 60% or the fraction of Ca(2+)-activatable regulatory units was reduced to approximately 33%. Third, we exchanged mixtures of sTnC and xxsTnC into single, permeabilized fibers from rabbit psoas. As the proportion of xxsTnC increased, unloaded shortening velocity decreased nonlinearly at maximal Ca(2+). These data are consistent with unloaded filament sliding speed being limited by the number of cycling cross-bridges so that maximal speed is attained with a critical, low level of actomyosin interactions.     

Troponin is a potential regulator for actomyosin interactions

Troponin extracted from rabbit skeletal muscle directly binds to an actin filament in a molar ratio of 1:1 even in the absence of tropomyosin. An actin filament decorated with troponin did not exhibit significant difference from pure actin filaments in the maximum rate of actomyosin ATP hydrolysis and the sliding velocity of the filament examined by means of an in vitro motility assay. However, the relative number of troponin-bound actin filaments moving in the absence of calcium ions decreased to half that in their presence. The amount of HMM bound to the filaments was less than 4% of actin monomers in the presence of TNs. In addition, actin filaments could not move when Tn molecules were bound in the molar ratio of about 1:1 although they sufficiently bind to myosin heads. These results indicate that troponin can transform an actin monomer within a filament into an Off-state without sterically blocking of the myosin-binding sites with tropomyosin molecules.     

Calcium regulation of skeletal muscle thin filament motility in vitro.

Using an in vitro motility assay, we have investigated Ca2+ regulation of individual, regulated thin filaments reconstituted from rabbit fast skeletal actin, troponin, and tropomyosin. Rhodamine-phalloidin labeling was used to visualize the filaments by epifluorescence, and assays were conducted at 30 degrees C and at ionic strengths near the physiological range. Regulated thin filaments exhibited well-regulated behavior when tropomyosin and troponin were added to the motility solutions because there was no directed motion in the absence of Ca2+. Unlike F-actin, the speed increased in a graded manner with increasing [Ca2+], whereas the number of regulated thin filaments moving was more steeply regulated. With increased ionic strength, Ca2+ sensitivity of both the number of filaments moving and their speed was shifted toward higher [Ca2+] and was steepest at the highest ionic strength studied (0.14 M gamma/2). Methylcellulose concentration (0.4% versus 0.7%) had no effect on the Ca2+ dependence of speed or number of filaments moving. These conclusions hold for five different methods used to analyze the data, indicating that the conclusions are robust. The force-pCa relationship (pCa = -log10[Ca2+]) for rabbit psoas skinned fibers taken under similar conditions of temperature and solution composition (0.14 M gamma/2) paralleled the speed-pCa relationship for the regulated filaments in the in vitro motility assay. Comparison of motility results with the force-pCa relationship in fibers suggests that relatively few cross-bridges are needed to make filaments move, but many have to be cycling to make the regulated filament move at maximum speed.     

Regulation of actomyosin ATPase by a single calcium-binding site on troponin C from crayfish

Equilibrium-binding studies at 4 degrees C show that, in the instance of crayfish, troponin C contains only one Ca-binding site with an affinity in the range of physiological free [CA2+] (K = 2 X 10(5) M-1). At physiological levels of Mg2+, this site does not bind Mg2+. In the complexes of troponin C-troponin I, troponin and troponin-tropomyosin, the regulatory Ca-specific site exhibits a 10- to 20-fold higher affinity (K = 2-4 X 10(6) M-1). The latter affinity is reduced to that of troponin C upon incorporation of the troponin-tropomyosin complex into the actin filament (regulated actin), as determined at 4 degrees C by the double isotope technique. The Ca-binding constant is again shifted to a higher value (7 X 10(6) M-1) when regulated actin is associated with nucleotide-free myosin. Both crayfish myofibrils and rabbit actomyosin regulated by crayfish troponin-tropomyosin display a steep rise in ATPase activity with [Ca2+]. Comparison of the pCa/ATPase relationship and the Ca-binding properties at 25 degrees C for the crayfish troponin-regulated actomyosin indicates that while the threshold [Ca2+] for activation corresponds to the range of [Ca2+] where the regulatory site in its low affinity state (K = 1 X 10(5) M-1) starts to bind Ca2+ significantly, full activation is reached at [Ca2+] for which the Ca-specific site in its high affinity state (K = 3 X 10(6) M-1) approaches saturation. These results suggest that, in the actomyosin ATPase cycle, there are at least two calcium-activated states of regulated actin (one low and one high), the high affinity state being induced by interactions of myosin with actin in the cycle.     

Calcium-triggered movement of regulated actin in vitro. A fluorescence microscopy study

We previously reported setting up an in vitro system for the observation of actin filament sliding along myosin filaments. The system involved a minute amount of fluorescently labelled F-actin, and its movement was monitored by fluorescence microscopy. Here, we report observations of the Ca2+-dependent movement of F-actin complex with tropomyosin plus troponin (regulated actin) added to the movement system in place of pure F-actin. In a wide range of pCa (-log10[Ca2+]) between 3 and 5.5 at 30 degrees C, regulated actin filaments moved rapidly, and the average velocity depended little on the Ca2+ concentration (about 7.5 microns/s). However, when the Ca2+ concentration was decreased to pCa = 5.8 or lower, the filaments suddenly stopped moving. In striking contrast to these observations, unregulated actin moved rapidly within the whole pCa range examined, the average velocity (about 7.5 microns/s) being essentially Ca2+-independent. These observations indicate that (1) tropomyosin-troponin actually gave Ca2+-sensitivity to F-actin, and (2) the movement system was regulated by Ca2+ in an on-off fashion within a narrow range of Ca2+ concentration. In a pCa range between 5.8 and 6.0, regulated actin filaments did not exhibit thermal motion; instead, they had fixed positions in the specimen, possibly because they remained associated with myosin filaments in the background, without sliding past each other. Although regulated actin moved fast in the presence of 1 mM-CaCl2 (pCa = 3) at 30 degrees C, it became entirely non-motile as the temperature was decreased to 25 degrees C or lower. Such a sharp movement/temperature relation was never found for unregulated actin. We assayed regulated actin-activated myosin ATPase in the same conditions as used for microscopy, and found that the ATPase activity depended both on pCa and on the temperature considerably less than the movement of regulated actin. The results suggest that the sliding velocity in the in vitro system would not be proportional to the rate of actin-activated ATPase.     

Cooperative [Ca]-Dependent Regulation of the Rate of Myosin Binding to Actin: Solution Data and the Tropomyosin Chain Model

The regulation of muscle contraction by calcium involves interactions among actin filaments, myosin-S1, tropomyosin (Tm), and troponin (Tn). We have extended our previous model in which the TmTn regulatory units are treated as a continuous flexible chain, and applied it to transient kinetic data. We have measured the time course of myosin-S1 binding to actin-Tm-Tn filaments in solution at various calcium levels with [actin]/[myosin] ratios of 10 and 0.1, which exhibit modest slowing as [Ca²⁺] is reduced and a lag phase at low calcium. These observations can be explained if myosin binds to actin in two steps, where the first step is rate-limiting and blocked by TmTnI at low calcium, and the second step is fast, reversible, and controlled by the neighboring configuration of coupled tropomyosin-troponin units. The model can describe the calcium dependence of the observed myosin binding reactions and predicts cooperative calcium binding to TnC with competition between actin and Ca-TnC for the binding of TnI. Implications for theories of thin-filament regulation in muscle are discussed.     

Structural Basis for the Activation of Muscle Contraction by Troponin and Tropomyosin

The molecular regulation of striated muscle contraction couples the binding and dissociation of Ca²⁺ on troponin (Tn) to the movement of tropomyosin on actin filaments. In turn, this process exposes or blocks myosin binding sites on actin, thereby controlling myosin crossbridge dynamics and consequently muscle contraction. Using 3D electron microscopy, we recently provided structural evidence that a C-terminal extension of TnI is anchored on actin at low Ca²⁺ and competes with tropomyosin for a common site to drive tropomyosin to the B-state location, a constrained, relaxing position on actin that inhibits myosin-crossbridge association. Here, we show that release of this constraint at high Ca²⁺ allows a second segment of troponin, probably representing parts of TnT or the troponin core domain, to promote tropomyosin movement on actin to the Ca²⁺-induced C-state location. With tropomyosin stabilized in this position, myosin binding interactions can begin. Tropomyosin appears to oscillate to a higher degree between respective B- and C-state positions on troponin-free filaments than on fully regulated filaments, suggesting that tropomyosin positioning in both states is troponin-dependent. By biasing tropomyosin to either of these two positions, troponin appears to have two distinct structural functions; in relaxed muscles at low Ca²⁺, troponin operates as an inhibitor, while in activated muscles at high Ca²⁺, it acts as a promoter to initiate contraction.     

Arthrin: a new actin-like protein in insect flight muscle

There are one or more proteins of 50,000 to 60,000 Mr in the thin filaments of insect flight muscle. A protein of 55,000 Mr has been isolated from insect fibrillar flight muscle and called arthrin. Despite its higher molecular weight, arthrin is in many ways like actin. The amino acid composition of arthrin was similar to that of actin. There were similarities in the peptides produced by digesting the denatured proteins and mild digestion of polymerized proteins cleaved similar-sized fragments from arthrin and actin. Polymerized arthrin activated the Mg2+ ATPase of myosin to the same extent as actin and the ATPase was regulated by rabbit or Lethocerus troponin and tropomyosin. Arthrin did not itself act as troponin-T. Electron microscopy of negatively stained specimens showed that arthrin and actin filaments were similar in structure and that arthrin could be decorated by rabbit subfragment-1 to form normal-looking arrowheads. Arthrin formed paracrystals at an optimum concentration of MgCl2 (25 mM) that was somewhat lower than the optimum for actin paracrystals. Optical diffraction showed that the structure of the paracrystals was similar to those formed from actin. The mass of arthrin and actin filaments relative to phage fd was measured by scanning transmission electron microscopy; the relative mass of arthrin and actin was 1.33, in agreement with molecular weight estimations. Therefore arthrin has the properties of a heavy form of actin. The proportion of actin, arthrin and troponin-T in Lethocerus myofibrils was six moles of actin to one mole of arthrin and one mole of troponin-T. The function of arthrin is not known.     

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 Hill model for binding myosin S1 to regulated actin is not equivalent to the McKillop-Geeves model

The Hill two-state cooperativity model and the McKillop-Geeves (McK-G) three-state model predict very similar binding traces of myosin subfragment 1 (S1) binding to regulated actin filaments in the presence and absence of calcium, and both fit the experimental data reasonably well [Chen et al., Biophys. J., 80, 2338-2349]. Here, we compared the Hill model and the McK-G model for binding myosin S1 to regulated actin against three sets of experimental data: the titration of regulated actin with S1 and the kinetics of S1 binding of regulated actin with either excess S1 to actin or excess actin to S1. Each data set was collected for a wide range of specified calcium concentrations. Both models were able to generate reasonable fits to the time course data and to titration data. The McK-G model can fit all three data sets with the same calcium-concentration-sensitive parameters. Only K(B) and K(T) show significant calcium dependence, and the parameters have a classic pCa curve. A unique set of the Hill model parameters was extremely difficult to estimate from the best fits of multiple sets of data. In summary, the McK-G cooperativity model more uniquely resolves parameters estimated from kinetic and titration data than the Hill model, predicts a sigmoidal dependence of key parameters with calcium concentration, and is simpler and more suitable for practical use.     

Structural basis for the regulation of muscle contraction by troponin and tropomyosin

The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca²⁺ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosin-crossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca²⁺ binding to TnC (the Ca²⁺ sensor of troponin that relieves inhibition) presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI (the “inhibitory” subunit of troponin) with tropomyosin and actin that cause tropomyosin movement are unknown, and thus, the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI, and then, 3D electron microscopy was used to resolve where troponin is anchored on actin-tropomyosin. Electron microscopy reconstruction showed how TnI binding to both actin and tropomyosin at low Ca²⁺ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus, the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated steric interference of actin-myosin interaction that regulates muscle contraction.     

The thin filament of vertebrate skeletal muscle co-operatively activates as a unit

We find that extraction of as little as one troponin C molecule per troponin-tropomyosin strand on a thin filament reduces the slope of the pCa/tension relation. We interpret this to mean that the regulatory units along a thin filament of rabbit psoas fibers are linked co-operatively so that a thin filament activates as a unit. The presence of extended co-operativity explains why the pCa/tension relation in skinned fibers has a slope much higher than predicted by binding of Ca2+ to one regulatory unit. Replacement of the extracted troponin C with purified troponin C fully reverses the effect of extraction and shows it to be the essential Ca2+ binding protein responsible for the steep slope of the pCa/tension relation.