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A troponin switch that regulates muscle contraction by stretch instead of calcium

2004; Springer Nature; Volume: 23; Issue: 4 Linguagem: Inglês

10.1038/sj.emboj.7600097

1460-2075

Bogos Agianian, Uroš Kržič, Feng Qiu, Wolfgang A. Linke, Kevin Leonard, Belinda Bullard,

Adhesion, Friction, and Surface Interactions

Article12 February 2004free access A troponin switch that regulates muscle contraction by stretch instead of calcium Bogos Agianian Bogos Agianian European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany Search for more papers by this author Uroš Kržič Uroš Kržič European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Feng Qiu Feng Qiu European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, GermanyPresent address: CABM, Rutgers University, Piscataway, NJ 08854, USA Search for more papers by this author Wolfgang A Linke Wolfgang A Linke Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, GermanyPresent address: University of Münster, Schlossplatz 5, D-48149 Münster, Germany Search for more papers by this author Kevin Leonard Kevin Leonard European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany Search for more papers by this author Belinda Bullard Corresponding Author Belinda Bullard European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany Search for more papers by this author Bogos Agianian Bogos Agianian European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany Search for more papers by this author Uroš Kržič Uroš Kržič European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Feng Qiu Feng Qiu European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, GermanyPresent address: CABM, Rutgers University, Piscataway, NJ 08854, USA Search for more papers by this author Wolfgang A Linke Wolfgang A Linke Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, GermanyPresent address: University of Münster, Schlossplatz 5, D-48149 Münster, Germany Search for more papers by this author Kevin Leonard Kevin Leonard European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany Search for more papers by this author Belinda Bullard Corresponding Author Belinda Bullard European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany Search for more papers by this author Author Information Bogos Agianian1,‡, Uroš Kržič1,2,‡, Feng Qiu1, Wolfgang A Linke2, Kevin Leonard1 and Belinda Bullard 1 1European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany 2Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany ‡These authors contributed equally to this work *Corresponding author. European Molecular Biology Laboratory. Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Tel.: +49-6221-387-268; Fax: +49-6221-387-306; E-mail: [email protected] The EMBO Journal (2004)23:772-779https://doi.org/10.1038/sj.emboj.7600097 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The flight muscles of many insects have a form of regulation enabling them to contract at high frequencies. The muscles are activated by periodic stretches at low Ca2+ levels. The same muscles also give isometric contractions in response to higher Ca2+. We show that the two activities are controlled by different isoforms of TnC (F1 and F2) within single myofibrils. F1 binds one Ca2+ with high affinity in the C-terminal domain and F2 binds one Ca2+ in the C-terminal domain and one exchangeable Ca2+ in the N-terminal domain. We have characterised the isoforms and determined their effect on the development of stretch-activated and Ca2+-activated tension by replacing endogenous TnC in Lethocerus flight muscle fibres with recombinant isoforms. Fibres with F1 gave stretch-activated tension and minimal isometric tension; those with F2 gave Ca2+-dependent isometric tension and minimal stretch-activated tension. Regulation by a TnC responding to stretch rather than Ca2+ is unprecedented and has resulted in the ability of insect flight muscle to perform oscillatory work at low Ca2+ concentrations, a property to which a large number of flying insects owe their evolutionary success. Introduction Striated muscles are regulated by changes in the concentration of Ca2+ in the fibres, which are activated when Ca2+ binds to the tropomyosin–troponin complex (Tm–Tn) on actin. Kinetic studies suggest that there are three states of the thin filament, referred to as blocked, closed and open (McKillop and Geeves, 1993; Maytum et al, 1999); these correspond to the B-state, C-state and M-state observed by electron microscopy (Lehman et al, 2000). According to the steric blocking model of regulation, in the absence of Ca2+, myosin-binding sites on the thin filament are blocked by Tm, which extends over seven actin monomers (the blocked state). Tn has three subunits, TnT, TnI and TnC (Greaser and Gergely, 1971); on activation, Ca2+ binds to TnC and changes in the structure of the whole complex result in movement of Tm, exposing myosin-binding sites. In the presence of Ca2+, there is an equilibrium between closed and open states of the thin filament. The closed state allows weak binding of crossbridges to actin; when a few crossbridges bind to the open state, there is a cooperative transition towards strong crossbridge binding, followed by the power stroke (Vibert et al, 1997; Maytum et al, 1999, 2003; Gordon et al, 2000; Lehman et al, 2000). In an alternative model, regulation is explained by an effect of Ca2+ binding to Tn on the kinetics of the transition from weak to strong crossbridge binding (Brenner, 1988; Chalovich, 1992; Gordon et al, 2000). To understand muscle regulation, details of the structural changes in Tn that cause movement of Tm are needed. Recently, a change in the orientation of the N-terminal domain of cardiac TnC on the thin filament following activation has been reported (Ferguson et al, 2003), and a crystal structure of the tertiary troponin complex has been determined (Takeda et al, 2003), which may lead to a better understanding of how TnC acts in the complex to trigger contraction. The flight muscles of many insects are not simply regulated by the Ca2+ switch proposed in the steric blocking model. The wing beat frequency of small insects is too high for individual contractions of the flight muscles to be activated by Ca2+, and in these muscles, oscillatory contractions are not synchronous with fluctuations in Ca2+ produced by nerve stimulation (Pringle, 1949). The wings of insects with asynchronous regulation are moved by resonant changes in the shape of the thorax produced by indirect flight muscle (IFM). The flight muscles contract in response to periodic stretches at Ca2+ concentrations too low to activate the fibres fully, and alternating stretch activation of opposing muscles produces oscillatory contractions (Pringle, 1978; Thorson and White, 1983; Tregear, 1983). This form of regulation is highly efficient and also occurs in larger insects with lower wing beat frequencies, such as the giant water bug, Lethocerus, which is commonly used for mechanical studies of IFM. IFM can also be activated to give twitches synchronous with nerve impulses, and at high stimulation frequencies these produce a tetanus (Esch et al, 1991; Heinrich, 1996). This type of activation is used during the 'warm-up' contractions that precede flight in large insects. Synchronous nerve impulses produce a tetanus simultaneously in opposing muscles, so that there is no resonant oscillation of the thorax such as occurs during flight (Esch et al, 1991; Heinrich, 1996). Isolated IFM fibres develop isometric tension at Ca2+ concentrations higher than that needed for stretch-activated contractions (Loxdale and Tregear, 1985; Kühn et al, 1985; Taylor et al, 1999); this type of activation (without stretch) is likely to be used in the Ca2+-activated twitches and tetani of IFM in vivo. Thus, IFM is activated in two different ways to produce different types of contraction. The troponin components of IFM differ from those in other muscles. In vertebrate fast skeletal muscle, TnC has two sites in the C-terminal domain that bind Ca2+ with high affinity and also bind Mg2+ (sites III and IV), and two sites of lower affinity in the N-terminal domain that bind Ca2+ reversibly during activation (sites I and II) (Potter and Gergely, 1975). Cardiac TnC has sites III and IV, and a single regulatory Ca2+-binding site in the N-terminal domain (site II) (Johnson et al, 1980). We have shown that IFM has an unusual isoform of TnC with one Ca2+-binding site in the C-terminal domain (site IV) and no regulatory Ca2+ site in the N-terminal domain; an additional minor isoform has Ca2+ binding sites II and IV (Qiu et al, 2003). IFM TnT has a negatively charged extension at the C-terminus not present in vertebrate TnTs, and in Lethocerus the inhibitory component (TnH) is a fusion protein of TnI and a long C-terminal PA sequence rich in proline and alanine (Bullard et al, 1988; Qiu et al, unpublished data). Although it was likely that troponin regulated both types of contraction in IFM, it was not known how the complex could do this. Here, we characterise the two isoforms of TnC and study their independent effects on regulation. The function of the isoforms is investigated by measuring the Ca2+ sensitivity of thin filaments reconstituted with one or the other isoform, and mutant proteins are used to show the importance of Ca2+ sites II and IV in regulation. Finally, the effect of the isoforms on the mechanics of IFM fibres is investigated. The results enable us to propose a model for dual regulation in IFM. Results Stoichiometry and affinity of Ca2+ binding to TnC isoforms The formal names of Lethocerus TnCs, LiTnC4 and LiTnC1 (Qiu et al, 2003), have been replaced here by F1 and F2, respectively, indicating the number of Ca2+ ions bound to the IFM isoforms. Sequence analysis shows that F1 lacks key acidic residues at Ca2+-chelating positions (X, Y, Z, −Y, −X, −Z) of sites I, II and III, while F2 lacks these residues at sites I and III; therefore, F1 should bind one Ca2+ ion and F2 two. We previously found, using atomic absorption spectroscopy, that the mol bound Ca2+/mol protein was 1.01±0.37 for F1 and 1.86±0.58 (mean±s.d., n=6) for F2 (Qiu et al, 2003). To study Ca2+ binding further, the stoichiometry and the sites of bound Ca2+ were determined by mass spectrometry of recombinant F1, F2 and the mutants F1mIV and F2mII, with single residue substitutions (E to A) at the −Z position of the EF hand in Ca2+ -binding site IV and site II (Figure 1A–D). Comparison of the spectra of F1 in the presence of Ca2+ and apo F1 shows that there is an additional peak corresponding to F1 with one bound Ca2+. The spectrum of F2 with Ca2+ had two extra peaks compared to apo F2, corresponding to F2 with one or two bound Ca2+. The spectrum of F1mIV had no additional peak in the presence of Ca2+, while that of F2mII had a single additional peak; at saturating Ca2+, another peak appeared corresponding to F2mII with two Ca2+ (not shown). This confirmed predictions from the sequences that F1 binds one Ca2+ at site IV and F2 binds two at sites II and IV (Qiu et al, 2003.) Figure 1.Stoichiometry and affinity of Ca2+ binding to TnC isoforms. (A–D) ESI-MS analysis of apo (grey) and Ca2+-loaded (black) TnC: (A) F1, (B) F2, (C) F1mIV, (D) F2mII. Minor apo-species within the apo-protein sample that are additively oxidised [+O16] (grey stars) and their corresponding oxidised-protein-Ca2+ adducts (grey dots) are marked; black stars are apoprotein adducts that do not bind Ca2+. Schematic bar diagrams of TnC sequences show Ca2+-binding sites I–IV. Filled sites are shown in grey, arrows indicate mutated sites; (E) Ca2+ titration of CD change in F1 (grey squares), F2 (black circles), F2mII (grey diamonds); values are mean±s.d. (n=5). (F) Effect of Mg2+ (1 mM) on Ca2+ titration of the CD change in F1 and F2 (symbols as in (E); values are the mean of two estimations. For comparison, titrations in the absence of Mg2+ are shown without symbols. See text for details. Download figure Download PowerPoint The affinity of Ca2+-binding sites was estimated from changes in helical ellipticity measured by circular dichroism (CD) (Figure 1E). The CD change in F1 was fitted by a curve with a single transition of Kd 1.6(±0.1) × 10−7 M and Hill coefficient (nH) 0.98±0.05. The CD change in F2 had two transitions with Kd 2.6(±0.5) × 10−6 and 1.8(±0.4) × 10−4 M; nH was 0.9±0.1 and 1.25±0.20, respectively. The CD change in F2mII had a single transition with Kd 6.3(±0.6) × 10−6 M and nH 1.05±0.10. Values are mean±s.d. (n=5). There was no CD change for F1mIV because this mutant did not bind Ca2+. The absence of a second transition in F2mII shows that site II is the low-affinity site. Although both the single Ca2+ site in F1 and the higher affinity site in F2 are in position IV, the affinity of this site is more than 10 times lower in F2 and F2mII than in F1. The coordinating residues at site IV of F1 and F2 are identical (Qiu et al, 2003). Therefore, the observed difference in affinity is likely to be due to the effect of different noncoordinating residues within the Ca2+-binding loops of the two proteins, and/or the effect of sequence variation outside the EF hand. A similar effect has been observed in vertebrate TnCs (Wang et al, 1998; Tikunova et al, 2002). Mg2+ binds with low affinity to both TnC isoforms and this had a small effect on the affinity of Ca2+ binding. The effect of Mg2+ on Ca2+ binding to site IV is shown in Figure 1F. Mg2+ increased the Kd for Ca2+ binding to the single binding site of F1 to 3.1 × 10−7 M, and increased the Kd for binding to the high-affinity site of F2 to 6.2 × 10−6 M. The Kd for the low-affinity site of F2 was not changed significantly by Mg2+, decreasing slightly to 1.3 × 10−4 M. Assuming competition between Ca2+ and Mg2+ for the high-affinity sites (Potter and Gergely, 1975), these numbers yield KdMg ∼1 × 10−3 M for F1 and ∼7 × 10−4 M for F2. Mg2+ binding by F1 and F2 has been confirmed by mass spectroscopy (not shown). Regulation of actomyosin ATPase activity by TnC The regulation of actomyosin ATPase by isoforms of TnC was investigated (Figure 2A). Thin filaments were reconstituted with a Lethocerus Tm–Tn complex lacking TnC (Figure 2B, first lane) to which recombinant TnC was added. F2 produced strong Ca2+-dependent activation of the ATPase, likely to be due to an exchangeable Ca2+ at site II. F2mII produced partial activation, probably due to an increase in the Ca2+ affinity of the mutated site II when TnC was in the troponin complex. In contrast, F1 produced little activation: F1mIV had an insignificant effect on the ATPase, showing that site IV is needed for the small Ca2+-dependent effect of F1. Binding of F1, F2 and mutant TnCs to the other Lethocerus troponin components was tested with immobilised recombinant TnC (Figure 2B). All the TnCs bound TnT and TnH; therefore, effects on the ATPase were not due to lack of association with other troponin components, and a functional site IV is not necessary for binding. Figure 2.Effect of TnC isoforms on actomyosin ATPase. (A) Ca2+ dependence of ATPase with Tm–Tn reconstituted with F2 (black circles), F2mII (light grey triangles), F1 (dark grey squares) and F1mIV (inverted open triangles). K50 for F2 was 7.0 (±0.3) × 10−7 M; for F2mII it was 3.3 (±1.2) × 10−7 M and for F1 it was 1.4(±0.6) × 10−7 M (mean±s.d., n=3). (B) SDS–PAGE of Tm–Tn (lane 1) and TnC-binding assay. Lanes 2–6, fraction of Tm–Tn retained by TnCs on Ni-NTA-agarose beads; TnC isoforms on the beads are shown above the gel lanes (control, empty beads). TnT and TnH bind to all TnCs and the affinity is greater than that for Tm, which did not bind to the TnC-beads. Download figure Download PowerPoint Effect of TnC on the mechanical response of flight muscle fibres The function of the IFM TnC isoforms was studied by measuring the mechanical response of fibres before and after replacing the endogenous mixed TnC with single isoforms. Tension produced under isometric conditions and the transient response to a rapid stretch were recorded at different Ca2+ concentrations (see Materials and methods). TnC was extracted from fibre bundles with Na-orthovanadate, leaving TnT and TnH still bound to the fibres (Figure 3A). Added F1, F2 and the mutants F1mIV and F2mII bound to extracted fibres, but a third mutant, F1ΔC (lacking 18 residues from the C-terminus), did not, showing that the C-terminus is needed for association with thin filaments. F1ΔC also acts as a control demonstrating that association of the other TnCs is not due to nonspecific binding. Native fibres gave isometric contractions that decreased with decreasing Ca2+ concentration (Figure 3B). After extracting TnC, fibres produced no isometric or stretch-activated tension even at high Ca2+ concentration (Figure 3C). This was expected because TnT and the inhibitory component, TnH, remained bound to thin filaments. On adding F2, the fibres contracted isometrically in the presence of Ca2+ (Figure 3D); however, fibres with F2mII only produced significant tension at the highest Ca2+ concentration, probably due to some Ca2+ binding at site II (Figure 3E). In contrast to the effect of F2, fibres with added F1 developed little isometric tension and fibres with F1mIV did not respond at all (Figure 3F and G). Therefore, the exchangeable Ca2+ site II of F2 is essential for isometric contraction. This is in agreement with the effect of TnC isoforms on actomyosin ATPase. Figure 3.Effect of TnC isoforms on isometric tension in fibres. (A) Western blot of fibre bundles from which TnC was removed with vanadate (Vi) and replaced by different isoforms. First two lanes, fibres before and after removal of TnC; following lanes, replacement with TnC isoforms indicated; last lane, F1ΔC alone. Fibre bundles did not contain equal numbers of fibres. The blot was incubated in mixed anti-TnT, -TnH and -TnC. (B) Isometric tension in native fibres in solutions with decreasing free Ca2+ (1–7); fibres were put into relaxing solution between each contraction (see Methods). (C) Fibres after vanadate treatment, showing loss of tension response. (D–G) Isometric tension in vanadate-treated fibres with added TnC: (D) F2, (E) F2mII, (F) F1, (G) F1mIV; force bar in (B) also applies to traces (D–G) and time bars are 20 min. Spikes represent fibre stretching or buffer exchange. pCa from 1 to 7: 4.7, 5.5, 5.9, 6.1, 6.3, 6.6, 6.9. Download figure Download PowerPoint The response of fibres to a step change in length was measured during the plateau of isometric tension. The time course of the response was analysed as the sum of exponentials, and the amplitude (A3) of the delayed tension phase was taken as a measure of stretch activation with rate constant r3 (Kawai and Brandt, 1980; Thorson and White, 1983) (see Figure 4F, inset). For native fibres, stretch-activated tension superimposed on isometric tension was Ca2+ dependent (Figure 4A). Fibres substituted with F1 produced greater stretch-activated tension than native fibres, but fibres with F1mIV were not stretch activated, showing that Ca2+ binding to site IV is necessary (Figure 4B and C). In contrast, fibres substituted with F2 responded very little to stretch (Figure 4D). Stretch-activated tension was only partially recovered in fibres with F2mII (Figure 4E); therefore, lack of bound Ca2+ at site II does not reproduce the effect of F1. The rate of rise of delayed tension (r3) was Ca2+ dependent for native fibres (Abbott, 1973) (Figure 4F). F1-substituted fibres had a much lower r3 at all Ca2+ concentrations than native fibres, while for F2-substituted fibres, r3 of the small response peaked at a higher level. Fibres with F2mII had a low r3 similar to that of fibres with F1, except at high Ca2+, when isometric tension was high and stretch-activated tension was low. Thus, the amplitude of stretch-activated tension is inversely related to the rate of tension development, as found previously for the effect of phosphate on Lethocerus fibres (Peckham et al, 1990). Figure 4.Effect of TnC isoforms on stretch-activated tension. Transient tension responses to a rapid 1% step change in length (arrow) were recorded at the plateau of isometric tension, in solutions of decreasing Ca2+. (A) Native fibres, (B) fibres in which TnC was substituted with F1, (C) F1mIV, (D) F2, (E) F2mII. Phases 3 and 4 of the tension response were fitted by the sum of two exponential processes (Thorson and White, 1983), using a least-squares algorithm. Stretch activation is characterised by the amplitude (A3) and rate constant (r3) of phase 3 (see (F) inset). Tension was normalised to fibre cross-sectional area (Dickinson et al, 1997). Ca2+ concentrations (pCa) were: 4.7 (black), 5.5 (red), 5.9 (green), 6.1 (dark blue), 6.3 (sky blue), 6.6 (yellow), 6.9 (wine), relaxing (<pCa 7.5) (yellow ochre). The orange curve is the response at pCa 4.7 after vanadate treatment. (F) Effect of Ca2+ on r3 for native fibres (green) and fibres substituted with F1 (red), F2 (blue) and F2mII (orange). Values are mean±s.d. (n=7) for native fibres, and mean and range (n=2) for substituted fibres. Inset: typical fit of phases 3 and 4 of a stretch-activated tension curve (black) by two exponentials (red and green). Download figure Download PowerPoint The differences in the effect of F1, F2 and F2mII on isometric tension and stretch activation are shown in Figure 5. For native fibres, which gave both types of contraction, the stretch response reached a maximum at pCa 5.9; at higher Ca2+, stretch-activated tension fell as isometric tension increased (Figure 5A). For F1-substituted fibres in which isometric tension was low, stretch-activated tension was about 75% greater than in native fibres (Figure 5B). Conversely, for F2-substituted fibres, in which stretch activation was low, isometric tension increased to a greater maximum than in native fibres (Figure 5C). The Ca2+ dependence of isometric tension was also more cooperative in F2-substituted fibres than in native fibres (Figure 5A and C). Fibres with F2mII resembled fibres with F1 in the persistently low isometric tension at moderate Ca2+ levels, but stretch-activated tension was half that of F1-substituted fibres and isometric tension rose sharply at pCa 4.7 (Figure 5D). These results show that F1, which has no exchangeable Ca2+, is required for stretch activation, and F2 with one exchangeable Ca2+ is required for isometric tension. Lack of site II is not sufficient to produce the full stretch-activation response: a high-affinity site IV is also needed. Figure 5.Ca2+ dependence of isometric tension and stretch-activated tension. (A) Native fibres, (B) fibres substituted with F1, (C) F2, (D) F2mII. Isometric tension (grey squares) and stretch-activated tension (black circles) are normalised to the total tension in native fibres at pCa 4.7. Stretch-activated tension is A3 (see Figure 4); total tension is isometric tension+stretch-activated tension. Isometric tension in native fibres at pCa 4.7 is 107±24 kN/m2 (mean±s.d., n=7). Values in (B) (C) and (D) are the mean of two estimations using the same protocol. pCa50 for isometric tension is 5.65±0.35 (n=7) for native fibres and 5.8 for F2-substituted fibres; Hill coefficients are 1.95±0.07 and 2.8, respectively. Download figure Download PowerPoint Distribution of TnC isoforms in myofibrils The distribution of F1 and F2 in IFM myofibrils was determined by immunofluorescence microscopy with monoclonal antibodies specific to each isoform. The specificity of the antibodies is shown in Figure 6A. Myofibrils were double labelled with anti-F1 and anti-F2. Both isoforms are in the same sarcomere and labelling is in the position of the thin filaments (Figure 6B). Immunoelectron microscopy with Protein A gold also showed that F1 and F2 are distributed across the sarcomere; the density of gold particles was greater in fibres labelled with anti-F1 than with anti-F2, as expected from the relative amounts of the two isoforms (Figure 7). Figure 6.Position of F1 and F2 in myofibrils. (A) Western blot of F1 and F2 incubated with anti-F1 (left panel) and anti-F2 (right panel). (B) Lethocerus IFM myofibril double labelled with anti-F1 (rat antibody) and anti-F2 (mouse antibody), followed by FITC anti-rat and Texas red anti-mouse secondary antibodies. Overlay shows that both isoforms are in the same region of the sarcomere. White arrows mark the position of Z-discs. Scale bar 5 μm. Download figure Download PowerPoint Figure 7.Distribution of F1 and F2 in Lethocerus IFM. Cryosections of the dorsal longitudinal muscle were labelled with anti-F1 (A) or anti-F2 (B) and Protein A gold (10 nm). Scale bar 0.5 μm. Download figure Download PowerPoint Discussion The important conclusion from these results is that the regulatory protein, TnC, determines whether IFM will give stretch-activated or isometric contraction. Current models explain stretch activation in IFM either by an effect of strain on the thick filament, which could affect the kinetics of the crossbridge cycle (Thorson and White, 1983; Lund et al, 1987, 1988; Tawada and Kawai, 1990), or by recruitment of crossbridges when fibres are stretched (Wray, 1979; Thorson and White, 1983). Certain properties of IFM myosin are necessary for maximum oscillatory power output by Drosophila flight muscle: myosin kinetics are affected by phosphorylation of a light chain (MLC2) (Tohtong et al, 1995), and a converter domain with the particular sequence in IFM myosin is needed for optimum performance (Swank et al, 2002). It has also been suggested that a link between thick and thin filaments, through the PA-rich extensions in TnH (Bullard et al, 1988; Reedy et al, 1994) or MLC2 (Tohtong et al, 1995), could transmit the effect of strain from thick to thin filaments. However, our results show that in IFM fibres with native thick filaments, it is the thin filament that determines the response to stretch or Ca2+. A possible explanation for the inability of F1-substituted fibres to produce isometric tension and F2-substituted fibres to produce stretch-activated tension is that crossbridges producing one type of tension are not available to produce the other. However, simple competition is unlikely to be the reason for the specificity of TnCs, because even at low levels of isometric tension F2-substituted fibres are incapable of producing appreciable stretch-activated tension. Both TnC isoforms are found in the same myofibril and in the same sarcomere within the myofibril, and it is likely that each thin filament contains a mixture of isoforms. The ratio of F1 to F2 is approximately 5:1 (Qiu et al, 2003), but it is not known whether the distribution on the thin filament is random or periodic. The two isoforms probably regulate the same thin filament independently. F1 is expected to regulate at low Ca2+ levels, priming the filament for activation by stretch, and F2 at higher Ca2+, producing isometric tension. A possible model for the way in which F1 and F2 can act independently on the same filament is one where the two isoforms locally produce different states of the filament. The extent to which crossbridge target sites on actin were blocked by Tm would depend on whether the sites were under the local control of Tn containing F1 or F2. It is known that different states of the thin filament are produced by cardiac TnC, which has one exchangeable Ca2+, and skeletal TnC, which has two (Maytum et al, 2003). In the presence of Ca2+, both cardiac and skeletal Tn activate the thin filament to the same extent, but in the absence of Ca2+, the thin filament is less inhibited by cardiac Tn. Interpreted by the three-state model for regulation, in the absence of Ca2+, a greater proportion of the cardiac thin filament is in the closed or open state, compared with the skeletal thin filament (Maytum et al, 2003). A similar, but more pronounced, effect may be produced in IFM thin filaments by Tn complexes containing F1, which has a single high-affinity Ca2+ site and no exchangeable Ca2+. At low Ca2+ concentrations, most of the thin filament would be in the less inhibited state, primed for myosin binding and the transition to the open state. In IFM, crossbridges on the thick filament and target sites on actin have the same lattice positions and, on stretching, the two could be brought into register (Wray, 1979). If regions of the thin filament were already in the closed or open state, the proximity of crossbridges and target sites might be sufficient to activate the muscle, even at low Ca2+ concentrations. A further parallel with cardiac Tn may be drawn from the observation that the equilibrium between closed and open states in the cardiac thin filament is insensitive to Ca2+ (Maytum et al, 2003). The model proposed here could be tested by comparing the affinity of myosin S1 for thin filaments regulated by F1- or F2-troponin, in the presence and absence of Ca2+. The model predicts that, in the absence of Ca2+, S1 would have a higher affinity for thin filaments with F1-troponin. Another test would be to compare the position of Tm in electron micrographs of thin filaments regulated by F1 or F2. In the absence of Ca2+, Tm in F1-regulated filaments might be nearer the C- and M-positions, which correspond to closed and open states.