doi:10.1006/jmbi.2001.4897 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 311, 1027±1036

Crossbridge and Tropomyosin Positions Observed inNative, Interacting Thick and Thin Filaments

Roger Craig1* and William Lehman2

1Department of Cell BiologyUniversity of MassachusettsMedical School, WorcesterMA 01655, USA2Department of Physiology andBiophysics, Boston UniversitySchool of Medicine, BostonMA 02118, USA

E-mail address of the correspondRoger.Craig@umassmed.edu

0022-2836/01/051027±10 $35.00/0

Tropomyosin movements on thin ®laments are thought to sterically regu-late muscle contraction, but have not been visualized during active ®la-ment sliding. In addition, although 3-D visualization of myosincrossbridges has been possible in rigor, it has been dif®cult for thick ®la-ments actively interacting with thin ®laments. In the current study, usingthree-dimensional reconstruction of electron micrographs of interacting®laments, we have been able to resolve not only tropomyosin, but alsothe docking sites for weak and strongly bound crossbridges on thin ®la-ments. In relaxing conditions, tropomyosin was observed on the outerdomain of actin, and thin ®lament interactions with thick ®laments wererare. In contracting conditions, tropomyosin had moved to the innerdomain of actin, and extra density, re¯ecting weakly bound, cyclingmyosin heads, was also detected, on the extreme periphery of actin. Inrigor conditions, tropomyosin had moved further on to the inner domainof actin, and strongly bound myosin heads were now observed over thejunction of the inner and outer domains. We conclude (1) that tropomyo-sin movements consistent with the steric model of muscle contractionoccur in interacting thick and thin ®laments, (2) that myosin-inducedmovement of tropomyosin in activated ®laments requires strongly boundcrossbridges, and (3) that crossbridges are bound to the periphery ofactin, at a site distinct from the strong myosin binding site, at an earlystage of the crossbridge cycle.

# 2001 Academic Press

Keywords: muscle contraction; myosin; tropomyosin; actin; electronmicroscopy

*Corresponding author

Introduction

Contraction of muscle is brought about by cyclicinteraction of myosin crossbridges with actin ®la-ments in a process that converts the chemicalenergy in ATP into mechanical work. Crossbridge-actin interaction must necessarily be regulated ormuscles would be permanently contracted and ofno use, yet the regulatory mechanisms involvedare complex and our understanding of their struc-tural basis and of the crossbridge cycle itselfremains incomplete.

A widely held view of the crossbridge cycleinvolves an initial contact of the myosin head withactin (a collision complex), followed by the``power-stroke'', which drives the thick ®lamentspast the thin ®laments to generate tension andshortening.1 It has been proposed that the power-stroke involves a conformational transition

ing author:

between two crossbridge states, one weaklyattached to actin (the A state) and the otherstrongly attached (the R (rigor-like) state).1 ± 3 Struc-tural and mutational analyses have provided con-siderable insight into likely interactions betweenmyosin heads and actin in the different states ofthe crossbridge cycle. At the end of the power-stroke (the R state) the crossbridge is thought tobind strongly and stereospeci®cally to actin in astructure resembling the well established rigor con-formation, having an angled, tangential attachmentto actin, involving the upper and lower halves ofthe ``50 kDa'' domain on the myosin head and sub-domains 1 and 3 of actin.1,4 ± 7 The structure of theputative weakly attached state(s) immediately pre-ceding the power-stroke (the A state) is controver-sial. It is argued that it should also involve astereospeci®c attachment to actin if crossbridgesare to generate tension ef®ciently.1 However, sev-eral experiments suggest that heads can also attachin a non-stereospeci®c (disordered) way prior tothe power-stroke.8 ± 16 The putative non-speci®c col-

# 2001 Academic Press

1028 Crossbridges and Tropomyosin on Thin Filaments

lision complex which precedes the weakly attachedA state appears to involve electrostatic interactionsbetween clusters of negatively charged residues onsubdomain-1 of actin and the positively chargedloop between the 50 K and 20 K fragments of themyosin head.1,4,17,18 This initial, non-speci®c inter-action may serve to bring the myosin heads intothe general vicinity of the force-generating attach-ment site on actin. Details of these interactionshave been dif®cult to study and remain speculativebecause they involve transient, non-synchronouslyand weakly attached heads.

The crossbridge cycle, and hence contraction, isregulated by the binding of Ca2� to the troponin-tropomyosin complex on the thin ®lament.19 EarlyX-ray diffraction studies of muscle suggested thattropomyosin might inhibit contraction in therelaxed state (low Ca2�) by physically blocking theattachment of myosin heads to actin.20 ± 22 Three-dimensional reconstructions of electron micro-graphs of isolated thin ®laments have provideddirect support for this steric model of regulation.The reconstructions show that at low Ca2�, tropo-myosin physically blocks the strong myosin bind-ing site (the R site) on the outer and the junctionof the inner and outer domains of actin, whichwould prevent crossbridge cycling and inducerelaxation.7,24 ± 26 However, this position of tropo-myosin does not block putative electrostatic myo-sin binding sites on the periphery of actin.7 Theproposed collision complex would therefore still bepossible, consistent with the ®nding that weakbinding of myosin heads to actin can occur in theabsence of Ca2�.23 Activation of contraction byCa2� appears to occur in a two-step process. Three-dimensional reconstructions show that the bindingof Ca2� by troponin causes tropomyosin to moveazimuthally by about 25 � to a position on theinner domain of actin, uncovering most of thestrong myosin binding site, but still obscuring onecluster of amino acid residues involved in strongmyosin binding.7,24 This would enable partial bind-ing of myosin heads (the A state would be poss-ible), but would not fully switch on contraction(the transition to the R state would still be partiallyinhibited). Reconstruction of ®laments decoratedwith strongly bound myosin heads (S1) in theabsence of ATP reveals a further, myosin-inducedmovement of tropomyosin, which fully exposes themyosin binding site,7 which could fully activatecontraction. This model involving three regulatorystates of the thin ®lament is supported by recon-structions of thin ®laments containing a mutanttropomyosin,27 and is consistent with indepen-dently proposed models based on solution experi-ments,28,29 on X-ray diffraction data from intactmuscle,30,31 and on X-ray crystallographic datafrom tropomyosin.32 Hence the structural position-ing of tropomyosin presumably controls the tran-sitions made by myosin to the A and R states.

The above observations, however, raise threequestions, which we have studied here: (1) Arestrongly bound myosin heads required to move

tropomyosin to the fully activated position, or doweakly attached heads suf®ce? (2) Can the pro-posed sites of myosin binding on actin in theweakly bound states be con®rmed by direct obser-vation? (3) Are the preceding observations on tro-pomyosin position, made with puri®ed proteins,also true with native actin and myosin ®lamentsinteracting with each other as they do in intactmuscle? We have approached these questions bymaking ``native'' ®lament homogenates frommuscle under relaxing conditions and observingthe structure of thin ®laments interacting withmyosin ®laments during Ca2�-induced sliding.Filament homogenates from tarantula muscle havebeen used as tarantula thin ®laments are regulatedby troponin-tropomyosin, and the homogenateshave been well characterized structurally andenzymatically, providing clear ®lament images inrelaxed and contracting conditions, thus makingstructural analysis possible.33,34 Controls have beencarried out in the absence of Ca2� (relaxation), andafter exhaustion of ATP (rigor).

Results

Relaxed state

Filament homogenates in relaxing conditions(ATP, low Ca2�) had a slightly turbid appearanceby eye. When observed by negative staining in theelectron microscope, they showed many isolatedmyosin and actin ®laments. The myosin ®lamentsfrequently displayed the helical ordering of cross-bridges known to characterize the relaxed state.33,34

Actin ®laments were sometimes observed lyingalongside and parallel to myosin ®laments(Figure 1(a) and (d)). These presumably resultedfrom occasional weak interactions that have beenproposed to occur between actin ®laments andmyosin heads even in the relaxed state.35 ± 37 Alter-natively, they may re¯ect occasional damagedmyosin heads that do not dissociate from actin inresponse to ATP. The latter seems less likelybecause of the rapid and mild methods used toprepare the ®lament suspensions. Further, if suchheads existed, they might be expected to causelooping out of thin ®laments from myosin ®la-ments in activated preparations, and this was notobserved (Figure 1(b)).

Three-dimensional reconstructions of thin ®la-ments ¯anked by a myosin ®lament on either siderevealed typical actin subunits, with continuousstrands of density (tropomyosin molecules) lyingon the inner part of the outer domain of actin(Figure 2(a)). In transverse sections (Z-sections) ofthe reconstructions (Figure 3(a)(i)), additional den-sity (arrows) was visible on the surface that wasnot seen with actin alone (Figure 3(b)(i)). Thisadditional density was not as well resolved fromactin as in puri®ed ®laments, probably due to lessuniform staining in this more complex system(Figure 1, cf. refs 24,25). Delineating these differ-ences required a precise ®tting of actin and thin

Figure 1. Negatively stained, interacting thick and thin ®laments from tarantula muscle (protein is white). (a)Relaxing conditions: thick ®laments show helical order (evident by viewing at a glancing angle along the ®lamentaxis) and no obvious crossbridges attached to thin ®laments. (b) Activating conditions: frequent crossbridges extendfrom thick ®laments to thin ®laments, at varying angles with no apparent relationship to ®lament polarity. These areparticularly evident when viewed at a glancing angle along the ®lament axis. (c) Rigor conditions: thick ®lamentcrossbridges attach to thin ®laments at a more constant and acute angle, forming chevrons. Polarities of the ®lamentsshown (", pointed end up; #, pointed end down) are #,#,",#,#,",", respectively from left to right as determined bythe ®tting algorithms. (d) High magni®cation examples of (a)-(c). Scale bars represent 100 nm (a)-(c), 50 nm (d).

Crossbridges and Tropomyosin on Thin Filaments 1029

®lament reconstructions to each other. This wasaccomplished by using a real space ®ttingalgorithm38 designed to align only respective actindensities within each, i.e. those densities that werestrictly comparable. The additional densities foundonly on thin ®laments could then be isolated fromactin density by difference density analysis,26,39 inessence superimposing Z-sections of pure actin onthose of thin ®laments (at the same Z-level;Figure 3(b)(i)) and subtracting one from the other,generating a difference map (Figure 3(c)(i)). Theexact location of this non-actin density on the actinstructure was then revealed by superimposing thedifference density on the pure actin map(Figure 3(d)(i)). This con®rmed that the additionaldensity seen in relaxed ®laments was on the innerpart of the outer domain of actin (Figures 2(c) and3(d)(i)), in a similar position to that seen with iso-lated thin ®laments (i.e. with no interacting thick®laments) at low Ca2�.7,24,25 On rare occasions

additional, discontinuous density was also seen onthe extreme periphery of the outer domain of actin,but this was lost after ®laments were averagedtogether. It may represent rare, weakly boundcrossbridges captured in the relaxed state. Suchcrossbridges are presumably responsible for hold-ing thick and thin ®laments in parallel even in therelaxed state.

Activated state

When relaxed ®lament homogenates were acti-vated by addition of Ca2�, there was a slight butrapid increase in turbidity of the suspension, andvisible lumps were seen after a few seconds. Thiseffect was rapidly reversed by the addition ofexcess EGTA. By phase contrast light microscopy,a uniform ¯occulent appearance seen under relax-ing conditions shrank into a ®ne, ®brous mesh-work in contracting solution, consistent with active

Figure 2. Three-dimensionalreconstructions (surface views) ofthin ®laments interacting with thick®laments in relaxed, activating andrigor conditions. To clearly delin-eate actin, tropomyosin and myo-sin, these reconstructions have beenprepared by superimposing tropo-myosin and S1 difference densities(Figure 3) on 3D reconstructions ofpure actin, and using data onlyfrom layer-lines 1, 2, and 3 todepict the path of tropomyosin.26

Surface maps generated from theraw data are not informative, asthe boundaries between respectiveprotein densities are not wellde®ned (Figure 3(a)). (a) Relaxed,showing tropomyosin strands (red)over the inner part of the outerdomain of actin; (b) Ca2�-activated,with tropomyosin (yellow) on theinner domain of actin; (c) rigor,showing tropomyosin (green)

located further over on the inner domain of actin; (d) comparison of the three positions of tropomyosin; (e) activated®laments (cf. (b)), in this case contoured lower to show weaker additional density (blue; not seen with relaxed orrigor ®laments) observed on the extreme periphery of the outer domain of actin, which we interpret as the actin-bound end of actively cycling crossbridges. The lower contouring also causes tropomyosin to appear wider.

1030 Crossbridges and Tropomyosin on Thin Filaments

®lament sliding. In the motility assay, short frag-ments of rhodamine-phalloidin-labeled F-actin ®la-ments when added to the ®lament suspensioncould be seen moving in straight lines, consistentwith sliding along native myosin ®laments.

In the electron microscope, Ca2�-activated prep-arations now showed many thin ®laments lyingparallel to and alongside thick ®laments, appar-ently joined to them by bridges projecting at vary-ing angles from the thick ®lament surface(Figure 1(b) and (d)). Occasionally thin ®lamentswere bordered by thick ®laments on both sides.Three-dimensional reconstructions of such thin ®la-ments (Figures 2(b) and 3(ii)) aligned and analyzedas above again showed typical actin subunits, butin this case tropomyosin strands (yellow inFigure 2(b); arrows in Figure 3(ii)) were on theouter edge of the inner domain of actin. This is thesame position induced by Ca2� in isolated ®la-ments with no interacting crossbridges.7,24 Therewas no evidence that crossbridge attachment inthese activated preparations had displaced tropo-myosin further on to the inner domain as occurswith strong binding of myosin heads in isolated®laments decorated with myosin subfragment-1(S1) in the absence of ATP.7

In addition to tropomyosin, extra density wasalso observed in these activated ®laments on theextreme periphery of the outer domain of actin(Figures 2(e) and 3(ii)). This density (blue inFigure 2(e); arrowheads in Figure 3(ii)) was notobserved in averaged reconstructions of ®lamentsunder relaxing or rigor conditions. It therefore pre-sumably represents the average position of theactin-bound end of actively cycling myosin heads.

Rigor state

The rigor state (no ATP, �Ca2�) was induced intwo ways. Activated ®lament preparations wereincubated at room temperature for a prolongedperiod to allow ATP to be hydrolyzed (evidentfrom the ®laments forming a gel), or skinnedmuscle specimens were put into rigor prior to vig-orous homogenization to generate fragmentedrigor myo®brils. By either method, many thick-thin®lament interactions were again observed, but inthis case the heads had obvious polarity and mademore regular and more acutely angled attachmentsto actin than was seen with activated ®laments(Figure 1(c) and (d)). In three-dimensional recon-structions of thin ®laments interacting with a myo-sin ®lament on either side, tropomyosin was nowobserved to have moved further on to the innerdomain of actin than with activated ®laments(Figure 2(c) (green strand); Figure 3(iii) arrows), toa position similar to that induced in puri®ed thin®laments decorated with myosin subfragment-1 inthe absence of ATP.7 Additional density due to themyosin heads was also observed over the face ofthe outer domain of actin (subdomain-1) and onthe junction of subdomains-1 and -3 (Figure 3(iii)arrowheads and asterisks). This is the same pos-ition at which heads bind to actin in ®lamentsdecorated with S1 in the absence of ATP.7

Discussion

In this study we have observed the positions oftropomyosin and of myosin crossbridges in inter-acting thick and thin ®laments from striated

Figure 3. Transverse sections (Z-sections) of three-dimensional reconstructions of thin ®laments in (i) relaxed, (ii)activated, and (iii) rigor states (see Figure 2). (a) Original data from thin ®laments interacting with thick ®laments.Arrows and arrowheads point to additional density on surface not seen in reconstructions of pure actin (see (b)).(b) Z-sections of pure actin (closely spaced contours) superimposed on thin ®lament Z-sections from (a) at sameZ-level; (c) Z-section difference maps (obtained by subtracting pure actin Z-sections from thin ®lament Z-sections in(b)) reveal more clearly26,39 the non-actin components of the thin ®laments (arrows and arrowheads; cf. (a) and (b)).The tropomyosin and myosin densities shown are signi®cant at the 99.5 % con®dence level and above. (d) Superposi-tion of difference densities in (c) on pure actin map reveals more precisely than (a) and (b) the binding positions ofnon-actin components on the actin subdomain structure. Numbers on (d)(i) indicate actin subdomain locations.Arrows indicate tropomyosin and arrowheads mark myosin heads. Myosin heads are weak in rigor reconstruction;asterisks in (d)(iii) indicate their center of mass after enlargement to size commensurate with other thin ®lament com-ponents. Note: omission of data very close to the meridian (R < 0.07 nmÿ1) on the l � 1, 2, and 3 layer-lines had noeffect at all on the density ascribed to myosin heads. Thus any potential periodic, non-helical attachment of myosinheads to actin ``target areas''40 that might occur in our analysis, leading to near-meridional diffraction, has no effecton our interpretation of the location of myosin head density (cf. also ref. 24, Figure 2).

Crossbridges and Tropomyosin on Thin Filaments 1031

muscle. Previous work revealed the positions oftropomyosin and myosin heads in isolated orreconstituted thin ®laments examined in staticstates, using proteolytic fragments of myosin todetermine the effects of head binding.7,24 ± 26 Herewe have simulated more closely the conditions inintact muscle by studying minimally puri®ed(therefore close-to ``native'') thick and thin ®la-

ments in freshly prepared muscle homogenates.Our reconstructions were made from thin ®lamentslying alongside myosin ®laments and thereforepresumably undergoing similar structural inter-actions to those that would occur in the lattice pre-sent in intact muscle. The ionic conditions usedwere comparable to those occurring in vivo(m � 0.14 M, pH 7.0). Using this approach we stu-

1032 Crossbridges and Tropomyosin on Thin Filaments

died the structure of thin ®laments undergoinginteractions with thick ®laments in relaxed, con-tracting and rigor states.

A number of observations imply that the inter-actions we have induced in our ®lament suspen-sions are physiologically relevant. The ATPase rateof tarantula myosin is low in the absence of Ca2�

and is activated �50-fold by actin-tropomyosinwhen Ca2� is present,34 providing biochemical sup-port for the actin-myosin interaction we observestructurally under contraction conditions. Thechanges in the physical properties of the ®lamentsuspensions we observe by eye under the threeconditions are consistent with the different levelsof interaction observed ultrastructurally (seeResults). The phase contrast microscope obser-vation of shrinkage of a uniform ¯occulent suspen-sion under relaxing conditions into a visiblemeshwork on addition of Ca2�, is consistent withactive ®lament sliding under contraction con-ditions. As the ®laments are free in solution, wewould expect that this sliding is isotonic althoughwe cannot rule out some drag by attached cross-bridges. Free sliding of thin ®laments past thick®laments is further suggested by our observationsusing the in vitro motility assay. Rhodamine-phal-loidin-labeled F-actin added to ®lament homogen-ates under activating conditions moved in straightlines, suggesting free sliding along myosin ®la-ments. After prolonged periods at room tempera-ture, Ca2�-activated ®lament homogenates formedgels, which were observed in the electron micro-scope to consist of bundles of actin and myosin®laments. The regular, acutely angled heads con-necting actin to myosin ®laments in these bundlesare similar to those occurring in rigor myo®brils,and in actin ®laments decorated with myosin sub-fragment-1 in the absence of ATP,6 consistent withthe assumption that ATP has been hydrolyzed byactin-myosin sliding in our preparations. Finally,because we have used myosin ®laments ratherthan subfragments (S1), we have been able tostudy crossbridge binding and tropomyosin pos-ition at physiological ionic strength and pH. Thiseliminates any possible structural artifacts thatmight occur at low ionic strength,13,41 which isrequired for S1 binding to actin in the presence ofATP; it also maintains the binding of troponin-tropomyosin, which dissociates from actin at lowionic strength.42

We have shown that when myosin and actin®laments interact under relaxing conditions, tropo-myosin occupies the same position as in myosin-free preparations of puri®ed thin ®laments at lowCa2�, where there are no actin-head interactions(compare refs 7, 24-26 with Figures 2 and 3). Ouruse of native ®laments and near-physiological con-ditions suggests that this is likely to be the positionthat tropomyosin occupies in living muscle in therelaxed state. Modeling of X-ray diffraction pat-terns of relaxed muscle supports this view.43,44

Thus the occasional, transient, weak binding ofheads (creating collision complexes) that presum-

ably underlies the relaxed interaction that wesometimes observe between actin and myosin ®la-ments has no effect on tropomyosin position. Thisis consistent with the ®nding that the location ofcrossbridge binding that we did occasionally detectin reconstructions of relaxed ®laments was on theperiphery of the outer domain of actin, at a sitedistinct from the tropomyosin-binding site (seealso below) but approximately coincident with thecharged region on subdomain-1 of actin that hasbeen proposed as the site of myosin head contactin the electrostatic collision complex.1,3 However,we cannot rule out the possibility of another struc-ture being responsible for the ®lament alignment.In actin ®laments interacting with myosin ®la-ments under Ca2�-activating conditions, whereactive crossbridge cycling and ®lament slidingappear to be taking place, we observe tropomyosinin the Ca2�-induced position previously observedwith puri®ed ®laments with no interacting head-s.7,24 ± 26 This therefore is presumably the averageposition occupied by tropomyosin in intact,actively shortening muscle. However, local andtransient effects of crossbridges may go undetectedby the averaging process. In rigor interacting ®la-ments, we ®nd that tropomyosin is displacedfurther on to the inner domain of actin, to themyosin-induced position seen previously withS1-decorated ®laments7 (Figures 2 and 3). We seeno evidence for displacement of tropomyosin tothis position in ®laments interacting with non-rigor, actively cycling crossbridges. This suggeststhat strongly bound heads are most likely requiredfor displacement of tropomyosin to this myosin-induced position, consistent with the view that themyosin binding site for strong attachment is onlyfully opened when tropomyosin is displacedfurther on to the inner domain from its Ca2� pos-ition, where it still partially blocks myosinbinding.1,7,45 Under our conditions of isotonic con-traction, strongly attached, post-power strokecrossbridges are transient and rare (estimated as10 % or less of the entire population of cross-bridges46 ± 48), which would explain why we do notdetect any movement of tropomyosin to the myo-sin-induced position. Even if a strongly attachedcrossbridge were located on an occasional thin ®la-ment, the effect of any associated local tropomyo-sin movement could be dampened out over therest of the ®lament and thus lost in the reconstruc-tion, which usually averages about six actin cross-over repeats. During isometric contraction, thenumber of force-bearing (strongly bound) cross-bridges increases,9,48 and the chance of detectingtheir effects would be greater. This is the case in X-ray diffraction studies of isometrically contractingmuscle, where it appears that actively cyclingcrossbridges do indeed induce tropomyosin move-ment to the myosin-induced position.45 Mimickingisometric conditions, for example by crosslinking®laments to each other,49 would not be straightfor-ward in our kind of experiment.

Crossbridges and Tropomyosin on Thin Filaments 1033

In reconstructions of ®laments in rigor and acti-vating conditions, we have also looked for the pre-sence of density that might reveal the predominant(average) position of myosin head binding on actinin these states. Although actin subunits are notsaturated with myosin heads in these experiments,we have previously shown that binding of only afew myosin heads (in actin ®laments decoratedwith substoichiometric S1) appears in reconstruc-tions as a low density ``ghost'' form of the headsseen in a fully decorated ®lament.7 The reconstruc-tions we present here of actin ®laments interactingwith myosin ®laments in rigor show weakadditional density on the outer domain of actinand covering the junction of outer and innerdomains (Figure 3(iii)). The shape and location ofthis density corresponds to that seen in puri®ed®laments decorated with S1 in the absence of ATP,suggesting that it represents myosin heads. Thissimilarity in the binding position of stronglyattached myosin heads, whether they are free insolution or tethered to a myosin ®lament, is con-sistent with previous demonstrations of the ¯exi-bility of head attachment to the ®lament surface.50

In reconstructions of ®laments in which headswere cycling (ATP, Ca2�) and exhibited varying,predominantly more perpendicular angles ofattachment to actin (Figure 1(b) and (d)), we alsofound it possible to detect additional density. Thisdensity was greatest close to actin, where variabil-ity due to differing angles of attachment should beleast: we interpret it as the proximal ends ofactively cycling myosin heads. This additional den-sity was no longer in the rigor position but wasobserved on the extreme periphery of the outerdomain of actin, in the vicinity of negative chargeclusters on subdomain-1 (Figures 2(e) and 3(ii)), aconsensus binding site for a number of actin-bind-ing proteins.51 The predominance of this non-rigorposition of myosin head binding during the cross-bridge cycle is consistent with the view that signi®-cant numbers of pre-force heads may be weaklyattached to actin in contracting muscle while onlya small proportion at any time is strongly attachedand producing force.9,52 The position of bindingnear negative charge clusters on the extreme per-iphery of actin subdomain-1 suggests that we maybe directly observing the putative initial docking ofmyosin heads with actin (the weakly attached,non-stereospeci®cally bound, electrostatic collisioncomplex, which precedes the structure at the startof the power-stroke). This interaction has pre-viously been proposed to occur in this region ofactin, based on structural and mutationalstudies;1,3,4,17,18 if this concept is correct, our obser-vations provide the ®rst direct structural evidencein support of it. The positioning of tropomyosinover the inner part of the outer domain of actinwould explain why it has no effect on such initialmyosin head binding on the periphery of thisdomain,23 while it would affect transition to theproposed weak, stereospeci®c (A-state) or strongbinding state (R-state), which require the covered

sites on the inner part of the outer actindomain.1,3,7 The ®nding that most of the head den-sity is on the periphery of actin in actively interact-ing ®laments also accounts for the fact thattropomyosin is not displaced to the myosin-induced position on the inner domain of actin bythese weakly attached heads. Only when bridgesstrongly attach, competing for the same interfaceon actin partially occupied by tropomyosin in thehigh Ca2� state, would tropomyosin be displaced.An alternative interpretation of the head densitythat we cannot rule out is that it in fact representsthe start of the power-stroke itself, as a number ofstudies have suggested that this state is disordered(see Introduction).

Placing our observations in the context of currentmodels for the crossbridge cycle1,3 suggests the fol-lowing scheme for contraction. In relaxed muscle,myosin heads remain on average helically orderedand close to the thick ®lament backbone but maketransient, non-stereospeci®c electrostatic inter-actions with the charged periphery of actin. Ourobservations showing thick and thin ®lamentsinteracting under relaxing conditions provide directsupport for the view that such interactions occur inrelaxed muscle under physiological ionic con-ditions.35-37 Although tropomyosin does not pre-vent the formation of these interactions, it couldprevent the power-stroke by physically blockingthe proposed stereospeci®c weak and the strongmyosin binding sites on the inner part of subdo-main-1 (i.e. the A and the R sites). On activation, thebinding of Ca2� by troponin causes tropomyosin tomove to the inner domain of actin. This opens upmost of the strong myosin binding site, allowingweakly attached bridges on the periphery of actinnow to form putative weak stereospeci®c inter-actions with clusters of amino acids on the innerpart of subdomain 1 of actin (the A-state). Strongbinding and the power-stroke (the R state) occurwhen weakly attached heads displace tropomyosinto the myosin-induced position, opening up the fullactin interface, or when rapid, transient movementsof tropomyosin about its site on the inner domainspontaneously open the R-site for strong head bind-ing. In slowly or isometrically contracting muscle,such strongly bound heads may hold tropomyosinin this myosin-induced position, thus facilitatingthe power-stroke of nearby weakly attached heads.In isotonically contracting muscle, where the num-ber of strongly attached myosin heads is small, suchcooperativity will be reduced.

Materials and Methods

Solutions

Rigor solution contained 100 mM NaCl, 3 mM MgCl2,0.2 mM EGTA, 5 mM Pipes, 5 mM NaH2PO4, 1 mMNaN3 (pH 7.0). Relaxing solution was the same as rigorsolution with MgATP added to 5 mM. Activating sol-ution was the same as relaxing solution with CaCl2

added to 0.4 mM (pCa � 4.2, calculated according to ref.

1034 Crossbridges and Tropomyosin on Thin Filaments

53). Skinning solution was relaxing solution containing0.1 % saponin. The ionic strength of relaxing, activatingand skinning solution was �0.14.53 To improve stainingand structural regularity of ®laments, solutions used torinse EM grids before staining had most of the Cl ÿ

replaced by Ac ÿ, and the and the Pipes/phosphate buf-fer was replaced by imidazole (pH 7.0).33 Thus rigorrinse contained 100 mM NaAc, 3 mM MgCl2, 0.2 mMEGTA, 2 mM imidazole, 1 mM NaN3 (pH 7.0); relaxingrinse was the same with MgATP added to 1 mM; andactivating rinse was the same as relaxing rinse withCaCl2 added to 0.4 mM.

Filament preparation

Relaxed ®lament homogenates were prepared from theleg muscles (femur) of tarantulas (various species; Caroli-na Biological Supply Co., Burlington, NC) by modi®-cations to methods previously described.33,34 Muscleswere chemically demembranated by agitation in skinningsolution for three to four hours at 4 �C, rinsed overnightin relaxing solution with continued agitation, then usedimmediately or stored up to three days on ice. Approxi-mately 0.2-0.3 g (wet wt) muscle was ®nely chopped witha scalpel blade then homogenized in 3 ml relaxing sol-ution for one second at setting 5 of a Polytron homogen-izer (Brinkmann Instruments, Inc., Westbury, NY), usingthe small probe. The homogenate was centrifuged for twominutes in a microfuge to remove large debris. Activationwas achieved by adding 100 mM CaCl2 to the diluted orundiluted relaxed ®lament preparation to a ®nal 0.4 mMtotal Ca2� (pCa � 4.2), leaving at room temperature(�22 �C) for ®ve to ten minutes, then preparing gridsimmediately or storing on ice until grid preparation.34

Filament interaction on addition of Ca2� was suggestedby an immediate slight clouding of the suspension andthe formation of visible particles. Rigor was achievedeither by leaving activated suspensions at room tempera-ture for two hours so that all ATP was hydrolyzed (a vis-ible gel was formed after this time), or by washing andincubating skinned muscles in rigor solution overnight,then vigorously homogenizing (15 seconds at Polytronsetting 5) to produce myo®brils fragmented into layersthin enough for electron microscopy.

Light microscopy

Phase contrast observations of ®lament homogenateswere made by placing a drop of suspension on a glassslide previously washed with 10 mM EGTA to removecontaminating Ca2�, and covering with an EGTA-washed coverslip. Observations were made by eye as thethin layer of homogenate was irrigated with activatingsolution. The motility assay54,55 was carried out by pla-cing a drop of ®lament homogenate in relaxing solutioninto a motility chamber (without nitrocellulose coating),followed by addition of rhodamine/phalloidin-treated F-actin (sheared to generate short ®laments) and myosinlight chain kinase and calmodulin.34 Motion of rhoda-mine-labeled actin ®laments was observed by ¯uor-escence microscopy. Attempts to observe movement ofendogenous native thin ®laments by labeling them withrhodamine/phalloidin were unsuccessful.

Electron microscopy

Relaxed or activated ®laments were diluted 10 to20-fold in relaxing or activating solution, respectively

(either at room temperature or 0 �C) immediately priorto grid preparation. 5 ml of ®lament suspension wasapplied to an electron microscope grid coated with a per-forated thick carbon ®lm supporting a thin carbon ®lm.After adherence of ®laments to the ®lm for 30 seconds,the grid was rinsed with the appropriate rinsing solutionand negatively stained with 1 % (w/v) uranyl acetate. Toimprove stain spreading, drying was carried out at�80 % relative humidity. Low dose (�1200 e ÿ/nm2)electron micrographs of ®laments lying on thin carbonover the holes were recorded at 60,000� on a PhilipsCM120 electron microscope operated at 80 kV. Thin ®la-ments ¯anked on both sides by a myosin ®lament werechosen for analysis because they should have the mostsymmetrical stain distribution, the greatest number ofcrossbridge attachments and the most symmetricalorganization of tropomyosin strands on the two long-pitch actin helices. Previous studies indicate that nega-tive staining with uranyl acetate preserves moleculardetails of muscle ®laments in both static .(7 cf. 26) andprobably also dynamic states(10 cf. 11; 13 cf. 41) Cryo-EMwas precluded since contrast in the images was insuf®-cient to assess alignment and attachments between®laments.

Image analysis

Thin ®laments were analyzed only when they met thefollowing criteria: (1) they lay between pairs of thick ®la-ments; (2) they were well centered and free of distortionsthat sometimes occurred when distances between neigh-boring thick and thin ®laments varied along their length;(3) they were evenly and not too heavily stained (thestain meniscus associated with the ¯anking thick ®la-ments was often too thick to enable details of the thin®lament in between to be discerned, and such ®lamentscould not be processed). Well-stained ®laments weredigitized with an Eikonix 1412 CCD camera with a pixelsize corresponding to 0.70 nm in the specimen. Slightlycurved ®laments were straightened by applying spline®tting algorithms.56 They were boxed so as to isolate theactin ®laments and attached crossbridges from the shaftof the myosin ®laments on either side. Helical recon-struction and averaging of reconstructions was carriedout by standard methods57 ± 60 as described previously.7,61

Nine examples of ®laments stained under contractingand rigor conditions and seven under relaxing con-ditions were chosen for averaging based on their respect-ive phase agreement from sets of 68, 21 and 42 ®lamentsmade from three muscle preparations. Averaged datasets were aligned to each other and to actin reconstruc-tions using a real space alignment algorithm38,60 whichallowed only those parts of maps attributable to actin(and therefore directly comparable) to be aligned usingobjective criteria. This was necessary as surface densities(tropomyosin and myosin) in the average reconstructionsvaried in shape, position and composition and wouldcompromise alignment if included. Once aligned to eachother and to actin, difference density analysis betweenreconstructions could be made as previously, helping todelineate more clearly low density information comingfrom tropomyosin and myosin heads.26,39,62,63 The stat-istical signi®cance of densities in reconstructions wascomputed from the standard deviations associated withcontributing points using a Student's t-test.6,64 Surfacerendering of reconstructions was performed using AVS53-D visualization software (Advanced Visual Systems,Inc., Waltham, MA).

Crossbridges and Tropomyosin on Thin Filaments 1035

Acknowledgments

We thank Drs David DeRosier and David Morgan forsharing ®lament alignment programs used in this study,Dr James Sellers for help with the motility assay, Norber-to Gherbesi for help with ®lament preparations and pho-tography, and Rachel Horowitz for surface rendering ofour reconstructions using AVS. We also thank Drs PeterVibert and Larry Tobacman for their comments on themanuscript. Electron microscopy was carried out withthe support of the Core Electron Microscopy Facility ofthe University of Massachusetts Medical School. Thiswork was supported by NIH grants AR34711 (R.C.),HL36153 (W.L.) and a Shared Instrumentation Grant(RR08426) for the purchase of the electron microscope.

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Edited by W. Baumeister

(Received 8 March 2001; received in revised form 20 June 2001; accepted 22 June 2001)