Ca2+ Causes Release of Myosin Heads from the Thick Filament Surface on the Milliseconds Time Scale

doi:10.1016/S0022-2836(03)00098-6

J. Mol. Biol. (2003) 327, 145–158

Ca21 Causes Release of Myosin Heads from the Thick Filament Surface on the Milliseconds Time Scale Fa-Qing Zhao and Roger Craig* Department of Cell Biology University of Massachusetts Medical School 55 Lake Avenue N Worcester MA 01655-0106, USA

We have used electron microscopy to study the structural changes induced when myosin filaments are activated by Ca2þ. Negative staining reveals that when Ca2þ binds to the heads of relaxed Ca2þ-regulated myosin filaments, the helically ordered myosin heads become disordered and project further from the filament surface. Cryo-electron microscopy of unstained, frozen-hydrated specimens supports this finding, and shows that disordering is reversible on removal of Ca2þ. The structural change is thus a result of Ca2þ binding alone and not an artifact of staining. Comparison of the two techniques suggests that negative staining preserves the structure induced by Ca2þ-binding. We therefore used a timeresolved negative staining technique to determine the time scale of the structural change. Full disordering was observed within 30 ms of Ca2þ addition, and had started to occur within 10 ms, showing that the change occurs on the physiological time scale. Comparison with studies of single heavy meromyosin molecules suggests that an increased mobility of myosin heads induced by Ca2þ binding underlies the changes in filament structure that we observe. We conclude that the loosening of the array of myosin heads that occurs on activation is real and physiological; it may function to make activated myosin heads freer to contact actin filaments during muscle contraction. q 2003 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: muscle; myosin filament; activation; cryo-EM; time-resolved negative staining

Introduction Muscle contraction and many forms of cell motility are caused by cyclic interaction of myosin heads with actin, causing thick and thin filaments to slide past one another. Contraction is regulated by Ca2þ-sensitive molecular switches on the myosin or actin filaments, depending on the muscle and species.1 At low Ca2þ levels, actin – myosin interaction is inhibited and actin-activated myosin ATPase is low. At high Ca2þ concentrations, the inhibition of actin –myosin interaction is removed and myosin ATPase is activated by actin. There are two types of myosin-linked regulation. In one, occurring in vertebrate smooth and nonmuscle myosins, and in some vertebrate and Abbreviations used: RLC, regulatory light chain; HMM, heavy meromyosin; EM, electron microscopy; EGTA, [ethylenebis-(oxyethylenenitrilo)]tetraacetic acid; AP5A, diadenosine pentaphosphate. E-mail address of the corresponding author: [email protected]

invertebrate striated muscles, phosphorylation of the regulatory light chain (RLC) switches on or modulates contraction.2 – 8 In the other, occurring in molluscan and probably many other invertebrate myosins, contraction is initiated by the binding of Ca2þ to the myosin heads.1,9,10 In scallop striated muscle, this activates the ATPase of the myosin alone up to 100-fold,11,12 and also permits interaction with actin, further enhancing myosin ATPase.11,12 The structural basis of myosin-linked regulation is unclear. Observations of the two-headed fragment of the myosin molecule, heavy meromyosin (HMM) suggest that in the OFF state (low Ca2þ, low ATPase), myosin heads interact with each other and/or the myosin tail in a rigid structure that may switch off ATPase activity and prevent actin – myosin interaction.13 – 17 On activation by phosphorylation or by Ca2þ binding, these interactions appear to be weakened, allowing the heads to move more freely around their junction with the tail, possibly facilitating their interaction with actin during contraction.13 – 17 Previous

0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved

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Ca2þ-induced Release of Heads from Myosin Filaments

Figure 1. Purification of myosin filaments from P. magellanicus. (a) Negative staining of initial filament homogenate, showing thick (myosin) filaments (thick arrow), thin (actin) filaments (thin arrow) and sarcoplasmic reticulum fragments (arrowhead). Bar represents 500 nm. (b) Negative staining of purified myosin filaments (P2, below). (c) SDSPAGE of filament homogenate before and after purification (relative molecular mass shown in kDa, based on molecular weight standards). Lane 1, initial filament homogenate (cf. (a)). Lane 2, pellet from first high speed centrifugation (after resuspension and slow spin to remove material that did not resuspend, see Materials and Methods). Lane 3, supernatant from first centrifugation. Lane 4, pellet from second centrifugation (after resuspension and slow spin to remove material that did not resuspend). Lane 5, supernatant from second centrifugation. MHC, myosin heavy chain; PM, paramyosin; A, actin; Tm, tropomyosin; RLC, regulatory light chain; ELC, essential light chain. (d) Densitometry of lanes 1 – 5 in (c), showing levels of actin and myosin as a percentage of total actin þ myosin. FS, initial filament suspension; P1 (or 2), pellet from first (or second) high speed spin; SN1 (or 2), supernatant from first (or second) high speed spin.

electron microscopic studies of myosin filaments, isolated directly from intact muscle, have shown that the compact, helically ordered arrangement of myosin heads that characterizes the relaxed state becomes disordered and the heads project further from the filament backbone when filaments are activated by phosphorylation or by Ca2þ binding.6,18 – 20 Similar results have been obtained with synthetic filaments made from purified myosin.21,22 It has been suggested that these effects represent a looser association of myosin heads with the filament backbone, which may in part underlie their increased interaction with actin in active muscle. Although these studies are sugges-

tive, their relevance to the structural changes occurring in intact muscle is not unequivocal. The experiments on native filaments were carried out on muscle homogenates containing actin as well as myosin filaments, leaving the possibility that interaction with actin may have contributed to the observed changes. All of the studies employed negative staining to observe the filaments: the changes observed could therefore be an artifact of staining. Reversibility of the structural change was not tested or was incomplete,6,18,19 leaving open the possibility that the changes were artifactual. Most importantly, for technical reasons the changes observed had many seconds or even minutes to

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Ca2þ-induced Release of Heads from Myosin Filaments

develop before the filaments were finally stained. This contrasts with a rate of activation of intact muscle on the millisecond time scale, thus again leaving open the question of the physiological significance of the structural changes observed. Here, we present observations carried out using a newly developed millisecond time-resolved negative staining technique,23,24 revealing that the changes occur on the millisecond time scale. Our observations have been carried out on purified native filaments, minimizing any influence of actin; cryo-electron microscopic observations of unstained and unfixed filaments confirm that the change is real (not a staining artifact) and that it is reversible. The Ca2þ-induced disorder is thus physiologically relevant and is likely to be a key feature of the activation of intact muscle. Correlation of our filament data with Ca2þ-induced structural changes in single HMM molecules,17 suggests a possible molecular basis for the change in filament structure.

Results Purification of scallop myosin filaments The filament suspension obtained by simple homogenization of permeabilized muscle under relaxing conditions contained not only the thick filaments of interest, but also large numbers of thin (actin-containing) filaments (Figure 1(a) and (c)), as found previously.18,25 To obtain thick filament images free of thin filaments and especially to remove any influence of thin filaments on thick filament structure, we therefore attempted to purify the thick filaments, even though this had met with only limited success in the past.26 Following two cycles of centrifugation we obtained a thick filament suspension showing very few thin filaments by electron microscopy (Figure 1(b)) and a level of actin approximately 2% of total actin þ myosin, based on gel densitometry (Figure 1(c) and (d)). This compares with 45% without purification (Figure 1(d)). Thus scallop myosin and actin filaments can be separated by simple centrifugation and resuspension. For reasons that are not clear, fragmentation of thin filaments by gelsolin prior to centrifugation, which greatly aids filament separation in other species,27 is not necessary for the removal of thin filaments in scallop muscle prepared as we have described. Ca21-activation induces disordering of heads in scallop myosin filaments Purified scallop myosin filaments in relaxing solution showed a well-ordered helical array of heads when observed by negative staining (Figure 2(a) and (c)), with a helical pitch of , 48 nm and axial periodicities of 14.5 nm and 29 nm.25 Thus the ordering present in the original suspension

survives the pipetting and vortexing methods used to purify the filaments. Similarly good ordering was also seen in unstained filaments suspended in vitreous ice across holes in the carbon support film (Figure 3(a) and (c); cf. Ref. 28). Helical ordering is therefore characteristic of the native relaxed filament as implied by X-ray diffraction patterns of intact scallop muscle;29 moreover negative staining effectively preserves this ordering. When relaxed filaments were rinsed on the grid with activating rinse (pCa , 4.0) for 10– 20 seconds before negative staining, the myosin heads lost their helical order (Figure 2(b) and (d); cf. Ref. 18). The edges of the filaments became rough, and the myosin heads appeared to move out from the filament backbone, resulting in an overall increase in filament diameter from 37 nm to 65 nm (Figure 4; Tables 1 and 2). This observation, obtained with purified myosin filaments, removes the possibility remaining from our earlier study18 that Ca2þ-induced movement of myosin heads was a result of their interaction with actin. Similar results were obtained by cryo-EM of unstained, vitrified specimens (Figure 3(b) and (d); Table 1). Thus the Ca2þ-induced movement of the myosin heads appears to be real, and not an artifact of staining. Ca21-induced disordering is reversible To help ascertain the physiological relevance of the Ca2þ-induced disordering, we also tested whether it was reversible on removal of Ca2þ. A partial reversal had been observed previously, but this was not consistent, possibly because the experiments were carried out on unpurified filaments attached to the carbon substrate of the EM grid.18 We therefore carried out activation and reversal on purified filaments in solution before preparing the grid. Filaments activated at pCa , 5.0 before staining showed disordered filaments as before (Figure 5(a) and (c)). When Ca2þ was lowered by addition of excess EGTA to activated suspensions (pCa 7.0), almost all the filaments reverted to a well ordered structure, similar in appearance to relaxed controls (Figure 5(b) and (d) cf. Figure 3(a) and (c)). We conclude that Ca2þ-induced disordering of the myosin heads is fully reversible on removal of Ca2þ. Ca21-induced structural changes occur on the millisecond time scale The activation experiments described above were carried out on a time scale of seconds, which is long compared with the physiological rate of muscle activation. We therefore developed a timeresolved negative staining technique allowing us to determine whether the Ca2þ-induced disordering also occurred on the physiological time scale (see Materials and Methods for details). The similarity of cryo-EM and negative staining

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Ca2þ-induced Release of Heads from Myosin Filaments

Figure 2. Ca2þ-induced disordering of myosin heads observed by negative staining (protein light). Purified myosin filaments were applied to a grid in relaxing solution then rinsed with activating rinse (or relaxing rinse for control) before negative staining with 1% uranyl acetate. Time in rinse solution before staining was ,ten seconds. (a) Field of relaxed filaments (control). (b) Field of activated filaments. (c) and (d) Selected filaments at higher magnification. (c) Relaxed; helical tracks of myosin heads are visible as oblique, stainexcluding lines running at a small angle to the filament axis (best seen by viewing along the filament at a glancing angle); transverse striations at 14.5 nm and 29 nm intervals are also visible. (d) Activated; myosin heads spread away from the filament surface (outer edges marked by dotted lines in inset) exposing the filament backbone. Bars in (a) and (b) represent, 300 nm; in (c) and (d) represent 200 nm.

observations on Ca2þ-induced disordering suggests that 1% uranyl acetate effectively fixes the filaments (see Discussion), giving a valid indication of the degree of filament ordering in high and low Ca2þ states. A drop of relaxed filament suspension was first placed on a grid and the grid then flushed with activating rinse (pCa , 4.0) before being rapidly stained with 1% uranyl acetate. Activation for

10 ms, 30 ms or 50 ms before staining brought about a radical change in filament structure. Three different filament appearances were observed, the relative numbers depending on the activation time (Tables 1 and 2). In one (Figure 6(b)), the myosin heads became disordered, giving the edges of the filament a rough appearance, and appeared to move outwards, increasing the overall filament diameter close to that seen when slowly activated

Ca2þ-induced Release of Heads from Myosin Filaments

149

Figure 3. Ca2þ-induced disordering of heads observed by cryo-EM (protein dark). Purified filaments embedded in vitreous ice across holes in carbon support films. After applying filaments, grids were rinsed first with relaxing rinse, then for ,10 – 20 seconds with activating rinse (or relaxing rinse for control) before freezing. (a) Field of relaxed filaments (control), showing ordered structure. (b) Field of Ca2þ-activated filaments showing disordered heads projecting far from the filament backbone. (c) and (d) Selected filaments at higher magnification. (c) Relaxed. (d) Activated. Bars in (a) and (b) represent 300 nm; in (c) and (d) represent 200 nm.

filaments were observed by negative staining. A second appearance was similar to that of relaxed filaments, in which the heads remained helically ordered and close to the filament backbone (filament diameter , 36 nm). A third, intermediate, structure was also seen. In this structure, the helical ordering was lost, but the heads remained close to the filament backbone (diameter similar to relaxed filaments; Figure 6(e)). In filaments activated for only 10 ms before staining (Figure 6(d) and (e)), the majority showed the intermediate structure (Table 2), with small numbers appearing either ordered or fully disordered. Activation for either 30 ms or 50 ms (Figure 6(a) and (b)) resulted in a majority of filaments that were fully disordered, with small numbers of intermediate appearance and none ordered. In all cases, controls (Figure 6(c) and (f)),

in which relaxing rinse replaced activating rinse, showed . 90% helically ordered myosin filaments, with only occasional disordering (Table 2), demonstrating that the rapid flushing was not the cause of the disordering. The change in the numbers of filaments with these different appearances suggests that they represent progressive steps in the activation process. Activation therefore appears to involve an initial (by 10 ms) disordering of the myosin heads while they remain close to the filament backbone. The full change in structure, involving release of the heads from the filament surface, occurs by 30 ms. We conclude that disordering of myosin filaments occurs on the same time scale as activation of scallop muscle, in which peak twitch tension is reached in 90 ms,30 and so is likely to be physiologically relevant.

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Figure 4. Filament diameter as a function of time of activation. Note that filament diameters are significantly affected by the staining process (binding to grid surface, drying of stain (inducing flattening), etc). This is probably especially so for the activated filaments, where the heads are not strongly held on the filament surface. Thus the diameters of filaments activated for 30 ms and 50 ms appear to be the same within this experimental error. Some filaments activated for ten seconds can apparently increase in diameter even further than at 50 ms, although this increase is likely to be artifactual (see the text). Measurements were made from: relaxed: 42 filaments, two preparations; 10 ms activation: 85 filaments, three preparations; 30 ms activation: 77 filaments, three preparations; 50 ms activation: 26 filaments, one preparation; ten seconds activation: 26 filaments, three preparations.

Reversible Ca21-induced disordering occurs in MgADP solutions, and on the millisecond time scale The disordering in myosin filaments that we have observed may result from a Ca2þ-induced change in the flexibility of the attachment of the myosin heads to the tail that has recently been described.17 To test this possible correlation between the responses of myosin filaments and isolated molecules to Ca2þ, we studied the effects

of Ca2þ on filament order using solutions in which ADP, the nucleotide used in the single molecule study, replaced ATP. Observations made by negative staining and cryo-EM were in agreement, and only the cryo-EM data are shown here. Scallop myosin filaments in MgADP relaxing solution appeared very similar to those in normal (MgATPcontaining) relaxing solution, with clear helical ordering of the myosin heads (Figure 7(a)). In the presence of Ca2þ, the heads became disordered, as observed before with MgATP (Figure 7(b)). This disordering was largely reversed when Ca2þ was chelated with excess EGTA (Figure 7(c)). A control, in which filaments were kept in a low Ca2þ rigor solution instead of ADP relaxing solution, showed disordering of heads (Figure 7(d)), as found previously under rigor conditions.18 This control demonstrates removal of ATP from the active site of the heads, supporting the conclusion that ADP is the bound nucleotide in the above experiments (Figure 7(a) –(c)). The time scale of the disordering in MgADP was found to be similar to that with MgATP, with Ca2þ inducing full disordering within 30 ms, and some initial disordering within 10 ms (Figure 8).

Discussion We have shown that approximately physiological levels of Ca2þ induce a rapid, major and reversible structural change in scallop myosin filaments, converting the ordered, helical array of myosin heads characteristic of relaxed filaments into a disordered array, projecting further from the filament backbone, in activated filaments. Our observations, made with purified native myosin filaments, are consistent with studies on synthetic filaments of purified scallop myosin,21 and suggest that such changes are a function solely of the binding of Ca2þ to the myosin heads, and do not result from interaction of the heads with actin filaments, a possibility that was not eliminated previously.18 The Ca2þ-induced disordering is seen not only by negative staining but also by cryo-EM of unstained, vitrified specimens. It is therefore

Table 1. Diameters in nm of relaxed filaments and of filaments activated for 10 ms, 30 ms and 50 ms and for , ten seconds Activated Relaxed Neg. stain Cryoa Relaxing solution ADP relaxing solution

37 ^ 2 36 ^ 3

42 ^ 3 42 ^ 2

10 ms Neg. stain

30 ms Neg. stain

50 ms Neg. stain

36 ^ 3 36 ^ 2

54 ^ 9 52 ^ 8

56 ^ 4 NDb

Ten seconds Neg. stain Cryoa 65 ^ 13 NDb

64 ^ 7 67 ^ 6

Diameters were measured between outer tips of myosin heads, in central third of a half filament, between bare zone and filament tip (mean ^ SD). Negative staining data are based on Figure 4. a Cryo-EM measurements were made only on relaxed and slowly-activated filaments. Note that the smaller diameter of relaxed filaments observed by negative staining is probably due to shrinkage that occurs during specimen drying, which is absent in frozenhydrated specimens. While such shrinkage would also be expected in activated filaments, it may be offset in this case by the binding of the released heads on to the carbon substrate, preventing the shrinkage. b ND, not done.

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Table 2. Distribution of filament structures after different times of activation

No. counted % Helically ordered % Partially disordered % Fully disordered

10 ms activation

10 ms control

30 ms activation

30 ms control

50 ms activation

50 ms control

Ten seconds activation

Ten seconds control

307 8 76

147 96 2

268 1 7

167 91 4

258 0 6

127 93 2

210 0 0

102 99 0

16

2

92

5

94

5

100

1

Filaments could be classified into one of three structures, whose proportions depended on the level and time of exposure to Ca2þ. The numbers of filaments displaying each structure in negative stain were counted for specimens that had been activated for 10 ms, 30 ms and 50 ms, and for ten seconds, or rinsed for the same times with relaxing rinse. Helically ordered filaments were those found in relaxing conditions and occasionally in activated specimens. They showed a clear, regular banding pattern (Figure 2)25 and smooth edges. In partially disordered filaments, found primarily after 10 ms activation, the banding was absent or weakened, but the diameter was the same as the ordered filaments and the surface was still relatively smooth. Fully disordered filaments predominated after $ 30 ms activation and showed rough edges, significantly increased diameter, and no regular banding.

not an artifact of staining, a possibility that had also remained previously.18,21 It is likely, on the contrary, to be a real effect, being captured by vitrification of filaments under approximately physiological ionic conditions, and demonstrably reversible by the same technique. We attempted to test this conclusion using an independent, solution technique, by observing whether Ca2þ induced a change in sedimentation coefficient of the filaments. However, filaments suspended in activating solution for extended periods, as required for analytical ultracentrifugation, were found to aggregate, and it was therefore not possible to obtain independent data in this way. The results described above together suggest that the mobilization of myosin heads induced by the binding of Ca2þ is real, but they do not shed light on the timescale of the process and so do not demonstrate whether it is physiological. To answer this question, we therefore further developed a technique23,24 that we had described previously31 allowing us to stain filaments adhering to grids at defined, millisecond time points following an activating rinse. We found that the disordering and extension of the myosin heads from the filament surface that we had observed on the seconds time scale was strongly developed within 30 ms (with little or no further change between 30 ms and 50 ms), and had started to occur within 10 ms (Figures 4 and 6; Tables 1 and 2). This is well within the 90 ms time period that scallop muscle takes to reach peak twitch tension following electrical stimulation,30 suggesting that the disordering is physiologically relevant and would occur in living muscle upon activation. The rapidity of the disordering is consistent with a rapid rate of Ca2þ binding to the myosin head12,32 and with an upper limit of 6– 7 ms for a Ca2þ-induced conformational change that has been measured in scallop HMM using a fluorescence signal.12 The time scale of the disordering suggests that it precedes, and may be a prerequisite for, initial tension development when scallop muscle is activated. The Ca2þ-induced extension of the myosin heads to a radius of 27 nm from the filament axis (Table 1)

would place them, within 30 ms, right at the surface of the thin filament (in the lattice of intact muscle), as required for the generation of force (assuming a spacing between thick filament centers of 60 nm in scallop muscle and a thin filament diameter of 8 nm33). When filaments are allowed to remain activated for seconds, the myosin heads appear to project even further (up to 32 nm radius; Table 1), which would put them beyond the surface of the thin filament in intact muscle.33 While this extension reflects the potential of the myosin heads to project far from the filament, it appears unlikely that this extension is reached in living muscle, being constrained in space by the filament lattice and in time by the short duration of a muscle twitch. Our use of negative staining to determine the time scale of disordering assumes that this technique arrests and preserves (i.e. fixes) structures this rapidly. We have previously discussed evidence that uranyl acetate acts as a good molecular fixative, in addition to being an excellent negative stain.31 Our finding that myosin filaments have a different structure after 30 ms than after 10 ms activation suggests that its fixation effect is also extremely rapid. If the Ca2þ-induced structural change observed at 10 ms continued to develop after staining, we might expect that filaments observed at both times would appear similar. That they do not suggests that fixation occurs within milliseconds. We have confirmed the rapidity of fixation by uranyl acetate in a simple way, by reversing the order of the stain and activating solutions in the pipette.23,24 Relaxed filaments flushed with stain for only 10 ms before being rinsed for several seconds with activating solution retain their helical order. This result, and that obtained with a number of other systems, shows that uranyl acetate fixes protein structure, including both ionic and hydrophobic interactions, within 10 ms. Previous studies of scallop HMM (the soluble, two-headed fragment of myosin) have shown that Ca2þ causes a major re-orientation of the heads of isolated molecules in the presence of nucleotide.

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Ca2þ-induced Release of Heads from Myosin Filaments

Figure 5. Ca2þ-induced disordering of myosin heads is reversible. Purified filaments in relaxing solution were activated by addition of 100 mM CaAc2 to a total 1 mM concentration (pCa , 5.0) for ,one minute (control), then relaxed again by addition of 100 mM EGTA to a total 5 mM (pCa , 7.0) for , three minutes. Cryo-EM of filaments embedded in vitreous ice across holes in carbon support films. (a) Field of Ca2þ-activated filaments (control), showing disordered myosin heads. (b) Field of activated filaments re-relaxed by treatment with excess EGTA, showing ordered myosin heads. (c) and (d) Selected filaments at higher magnification. (c) Activated. (d) Re-relaxed. Bars in (a) and (b) represent 300 nm; in (c) and (d) represent 200 nm.

EM, proteolytic susceptibility and sedimentation studies showed that in relaxing conditions (MgATP, low Ca2þ), the two heads bend back towards the tail in a rigid structure (the OFF state), while in the presence of Ca2þ the heads appeared to become more flexibly attached (the ON state14,17). It is also possible that in the OFF state the heads not only bend towards the tail, but also interact with each other, as found with smooth muscle HMM.15,16,34 The OFF state was generated by a variety of ATP analogs as long as Ca2þ was absent, suggesting that it was a state distinct from

any of the states of the cross-bridge cycle.17 Communication between the two heads, reflected by cooperative interactions, occurs in the OFF state35 and is probably a requirement for generating the OFF structure, with the heads bent back. In the presence of Ca2þ, the molecule is switched ON (high ATPase) and the heads act independently,35 consistent with the increased flexibility observed.17 An obvious question is whether the two states of the heads in isolated molecules underlie the order and disorder that we have observed in the filaments. The single molecule observations were

Ca2þ-induced Release of Heads from Myosin Filaments

153

Figure 6. Ca2þ-induced disordering of myosin filaments occurs on the millisecond time scale. Relaxed filaments placed on an EM grid were rapidly flushed with activating rinse (pCa , 4.0), for 10 ms or 30 ms, then with 1% uranyl acetate stain. (a) Oscilloscope trace showing elapsed time of 30 ms (horizontal bar) from contact of grid with activating rinse (start of trace, single arrowhead) to contact with stain (start of low amplitude portion of trace, double arrowhead); end of low amplitude trace (triple arrowhead) indicates end of stream of stain. (b) Filament activated for 30 ms; heads are disordered and project away from backbone. (c) 30 ms control, in which flush with relaxing rinse replaces flush with activating rinse; heads remain ordered. (d) Oscilloscope trace showing elapse of 10 ms (bar) from application of activating rinse to arrival of stain. (e) Filaments activated for 10 ms show an intermediate state of disorder, with loss of the regular pattern of heads but no overall increase in diameter. (f) 10 ms control remains ordered. Bar in (b), (c), (e) and (f) represents 200 nm.

carried out primarily with ADP rather than ATP to avoid complications due to ATP hydrolysis. We therefore tested filament suspensions in ADP and found that filaments in the absence of Ca2þ with ADP bound at the active site were helically ordered, and responded to Ca2þ by disordering, just as they did in ATP. These conditions closely paralleled those used for the single molecules,

thus strengthening the correlation between the behavior of the molecule and the filament. The simplest interpretation is that the cooperative, switched OFF, heads-back state underlies the compact, helically ordered filament structure found in the relaxed (low Ca2þ) state. The increased flexibility of the heads brought about when Ca2þ binds seems likely to be responsible for the disordering

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Ca2þ-induced Release of Heads from Myosin Filaments

Figure 7. Reversible Ca2þinduced disordering is observed when MgADP replaces MgATP on the myosin heads. (a) Cryo-electron micrograph of filament in ADP relaxing solution, showing helically ordered structure. This ordering was surprising, based on X-ray diffraction observations of rabbit muscle in the presence of ADP.36 We checked that it was not due to ATP contamination of ADP, by using the same ATP removal conditions as those used by Xu et al. (see Materials and Methods) and by using 50 mM instead of 5 mM ADP, reducing any ATP effect by 100-fold: we obtained the same helically ordered filaments. (b) Cryo-EM of filament activated in ADP relaxing solution by addition of Ca2þ to pCa , 5.0 (cf. Figure 5(c)), showing disordered heads. (c) Same as (b), only rerelaxed by addition of EGTA after activation (pCa , 7.0; cf. Figure 5(d)), showing that the heads become ordered again when Ca2þ is removed. (d) Negative staining of filaments in low Ca2þ rigor solution. In this control the filaments appeared disordered even in the absence of Ca2þ, as has been found previously in the absence of nucleotide.18 This confirms that our protocol fully removes ATP from the myosin heads, implying that in the presence of ADP only ADP would be bound to the heads. Bar represents 200 nm.

seen in activated filaments. In the studies of HMM the two conformations were found to equilibrate with a half-time of , 70 seconds,17 but the techniques did not reveal whether the structural change occurred on the millisecond (physiological) time scale. The correlation between filament and molecule described above would suggest that the molecular changes occur in milliseconds, leading to the changes observed at the filament level. This is also suggested by the rapidity of the Ca2þinduced structural change measured in HMM using a fluorescence signal.12 Our finding that filaments with ADP bound at the active site are helically ordered in the absence of Ca2þ (Figure 7(a)) was at first surprising, based on comparison with rabbit muscle, in which ADP produces disorder.40 Scallop muscle differs from rabbit muscle, however, in being Ca2þ-regulated.

In contrast with rabbit muscle, its relaxed (low Ca2þ) structure is not a part of the myosin ATPase cycle (see Discussion above). The relaxed (low Ca2þ) structures of (regulated) scallop filaments and (unregulated) rabbit filaments are therefore not directly comparable. We conclude that Ca2þ-activated myosin filaments respond to Ca2þ by a rapid mobilization of their heads, which is first visible within 10 ms and fully developed by 30 ms. In relaxed muscle the heads lie close to the filament backbone and are in an inactive state, with low ATPase activity and minimal interaction with actin. On Ca2þ binding, their interactions with each other and/or the filament backbone are abolished. This allows the heads to move freely, facilitating their interaction with actin and thus muscle contraction.

155

Ca2þ-induced Release of Heads from Myosin Filaments

Figure 8. Ca2þ-induced disordering of myosin filaments in MgADP solution occurs on the millisecond time scale. (a) Filaments activated for 30 ms; heads are disordered and project away from backbone. (b) Control filament, rapidly rinsed with ADP relaxing rinse for 30 ms; heads remain ordered. (c) Filaments activated for 10 ms, showing the intermediate state of disorder, with loss of the regular pattern of heads but no overall increase in diameter. (d) 10 ms control filaments remain ordered. Bar represents 200 nm.

Materials and Methods Solutions Rigor solution was 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 5 mM Pipes, 5 mM NaH2PO4, 1 mM NaN3 (pH 7.0). Relaxing solution was rigor solution with MgATP added to 5 mM. ADP relaxing solution was rigor solution containing 5 mM MgADP together with 100 mM AP5A (diadenosine pentaphosphate), 0.1 mg/ml hexokinase, and 1 mM glucose to remove any ATP contamination (we also carried out some experiments using slightly different amounts of AP5A, hexokinase and glucose40). Skinning solution was relaxing solution containing 0.1% (w/v) saponin. Rigor rinse contained 100 mM NaAc, 3 mM MgCl2, 0.2 mM EGTA, 2 mM imidazole, 1 mM NaN3 (pH 7.0). Relaxing rinse was rigor rinse containing 1 mM MgATP, and activating rinse was relaxing rinse with CaAc2 added to 0.4 mM total concentration (pCa , 4.0, calculated as described by Perrin & Sayce36). The use of chloride-based solutions in the filament preparation aids in dissociation of the actin filaments from the myosin filaments, but has been found to interfere with the helical ordering of the myosin heads.37 This problem is solved by rinsing grids with acetate-based solution before staining.6,37 Imidazole replaces phosphate and Pipes in the rinses as this has been found to result in improved spreading of stain.

Purification of scallop myosin filaments Sea scallops (Placopecten magellanicus) were obtained from the Marine Biological Laboratory, Woods Hole, MA and stored in a marine aquarium at 10 8C. All procedures were carried out at 0 – 4 8C except where otherwise noted. Strips of the striated adductor muscle 1 – 2 mm in diameter were dissected from active specimens at room temperature, tied to a plastic plate, and their membranes permeabilized by placing in a tube containing 6 ml skinning solution. After three to four hours rotation, the strips were placed in fresh relaxing solution and rotation was continued for three days, with a change to fresh relaxing solution each day (the three day incubation in relaxing solution was required to enable the subsequent purification of thick filaments, which had been only partially successful in a previous study that lacked this step).26 Filaments were prepared by first finely chopping an , 0.3 g strip of muscle with a scalpel in relaxing solution containing 3 mM EGTA, then homogenizing the fragments in 3 ml of the same relaxing solution, at setting 5 for two seconds using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). The homogenate was spun at 15,000g for two minutes in a microcentrifuge to remove large debris. A 1.0 ml aliquot of the supernatant (FS in Figure 1(d)), containing a suspension of thick and thin filaments, was purified by mixing with 2.6 ml relaxing

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solution, then centrifuging at 17,000g for 20 minutes at 4 8C (Beckman Optimae TL ultracentrifuge, Beckman Instruments, Palo Alto, CA). The pellet, containing mostly thick filaments, was resuspended in 1.0 ml relaxing solution by vigorous pipetting and vortexing, then spun as previously in the microcentrifuge to remove material that had not fully resuspended. A 0.6 ml aliquot of the supernatant (P1 in Figure 1(d)) was mixed with 2.6 ml relaxing solution and centrifuged at 17,000g for 20 minutes as before. The second pellet, containing more highly purified thick filaments, was resuspended as before in 0.6 ml relaxing solution, and again centrifuged to remove large particles. The supernatant (P2 in Figure 1(d)) containing thick filaments depleted of actin filaments was used for electron microscopy. The different steps of the purification were analyzed by negative staining EM (see below) and by SDSPAGE.41 Gel densitometry was carried out using a BioRad Fluor-S MultiImager (Hercules, CA). Experiments with ADP-containing solutions We wanted to determine whether the Ca2þ-induced structural changes that we observed with ATP also occurred with ADP. To achieve this we replaced MgATP with MgADP in the relevant solutions, and took additional precautions to ensure that any contaminating ATP was removed, so that competition with ADP for the active site on the myosin head would be eliminated. Contaminating ATP in ADP relaxing solution (Sigma ADP Cat. No. A-2754) amounted to ,0.7% (35 mM) by the luciferin-luciferase assay. To remove this contamination, hexokinase and glucose were added to the solutions, which were then kept for $ two hours at room temperature prior to use (see Solutions), to hydrolyze ATP to ADP (this reduced the ATP level to , 1 mM in 20 minutes by the same assay). AP5A was also included to inhibit any myokinase activity (producing ATP from ADP). Preparation of filaments for these experiments was carried out in the normal way, except that the pellets from the two high speed centrifugations were rinsed with ADP relaxing solution and then resuspended in this solution. After the second resuspension, the filaments were kept at room temperature for two hours. This was sufficient to allow the products of ATP hydrolysis to dissociate from the myosin heads and be replaced by ADP before preparing grids for microscopy (cf. Refs. 32,38). To further check that no MgATP was present on the myosin heads under the conditions of our experiments, we carried out a control experiment using rigor solution (no nucleotide) in place of MgADP relaxing solution, and found that the myosin heads became disordered, as expected when no nucleotide is bound to the heads.18 In this case the second pellet was rinsed with rigor solution and then resuspended in rigor solution containing 100 mM AP5A, 0.1 mg/ml hexokinase, and 1 mM glucose and left for two hours at room temperature. Electron microscopy

Ca2þ-induced Release of Heads from Myosin Filaments

Time-resolved negative staining For this work we developed a simple procedure enabling us to capture structural intermediates by rapidly staining grids at pre-determined millisecond time intervals following an activating rinse. The procedure was based on a previously described technique31 first suggested to us by Dr Peter Knight and is described in full elsewhere.24 In outline, 6 ml of filament suspension in relaxing solution was first applied to a carbon-coated grid and the grid given an initial wash with relaxing rinse to lower the EGTA level to 0.2 mM. The grid was then flushed sequentially with activating rinse followed by 1% uranyl acetate stain. This was achieved using a Gilson Pipetman fitted with a 1 ml plastic tip. A volume of 520 ml of stain was first drawn up into the tip, followed by 60 ml of air then 40 ml rinsing solution, the air functioning to separate the stain from the rinsing solution. With the pipette tip almost contacting the EM grid, the piston of the pipette was rapidly pushed down, flushing the grid first with rinsing solution followed rapidly by stain. The grid was dried at 80% relative humidity as above. Control of the rapid expulsion of rinsing solution and stain was achieved by attaching the pipette to a pneumatic piston whose movement was brought about by pressurized nitrogen. When the control button was depressed, the piston rapidly expelled the contents of the pipette tip over the surface of the grid. The simple design of this procedure, using a single pipette tip to hold both rinse and stain, ensured that the stain followed the exact path of the rinse. The flushing time (the time between initial contact of the rinse solution with the grid and initial contact of the stain) was varied by altering either the volume of rinse solution in the pipette tip or the pressure of the nitrogen. The apparatus was calibrated to generate flushing times of 10 ms, 30 ms or 50 ms, which were displayed on an oscilloscope24 (Figure 6(a) and (d)). When examining grids rapidly flushed with rinse then stain (especially in the 10 ms experiments), we noticed that in many areas filaments were aligned with each other. Such alignment was not observed in conventionally stained grids, suggesting that it was due to the rapid flow of solutions across the grid. This therefore provided a useful guide to the regions of the grid that had been most directly exposed to the rinse solution, and data were collected only from these areas. Preparation of frozen-hydrated specimens A 6 ml aliquot of filament suspension was applied to a 400 mesh grid coated with a holey carbon film that had been rendered hydrophilic by glow discharge in n-amylamine vapor for three minutes before use. After allowing the filaments to adsorb to the grid for 30 seconds, the grid was rinsed with the appropriate solution, then placed in a humidity chamber (,70% relative humidity). The grid was blotted to a thin film using Whatman No. 42 filter paper, then immediately plunged under gravity into liquid ethane cooled by liquid nitrogen. Grids were stored under liquid nitrogen.

Routine negative staining An aliquot of 6 ml of filament suspension was placed on a holey carbon film supporting a thin carbon film (floated from mica) on a 400-mesh grid. The grid was then rinsed with eight drops of the appropriate rinsing solution and stained with 1% (w/v) uranyl acetate. Drying was carried out at ,80% relative humidity to improve stain spreading.

Microscopy Conventional dose images of negatively stained specimens were recorded on Kodak 4489 film at 80 kV on a Philips (FEI, Hillsboro, OR) CM10 electron microscope. ˚ 2) images of filaments suspended in Low dose (, 6 e2/A vitreous ice over holes in the carbon support film were

157

Ca2þ-induced Release of Heads from Myosin Filaments

recorded on Kodak SO-163 film in a Philips CM120 cryoelectron microscope at 80 kV with a magnification of 28,000 £ and a defocus of 2.3 mm using a Gatan (Pleasanton, CA) 626DH cryo-holder at , 2 184 8C. Low dose micrographs were developed for 12 minutes in full strength D19 developer. Magnifications were calibrated using tropomyosin paracrystals having a 39.5 nm repeat.39 Filament measurements Because the distribution and spreading of stain affects filament appearance, only filaments that showed “good” negative staining (thick and evenly spread) were measured. Filament diameters were measured from negatives enlarged 36 £ on the screen of a microfilm reader. On a transparency attached to the screen, lines were drawn along the outer edges of the myosin heads on each side of the filament, and the distance between the lines measured (see Figure 2(d) inset). Measurements were taken from three positions in the central third of a half filament between the bare zone and the filament tip.

7. 8.

9. 10. 11. 12.

13.

Acknowledgements We thank Dr Peter Knight for his original suggestion of the time-resolved negative staining technique, Drs Rau´l Padro´n, Josh Singer and Hui Zou for help with calibrating the timing, and Ms Maria-Elena Zoghbi for measuring ATP contamination of ADP and its rate of removal by hexokinase-glucose. We also thank Drs Michael Geeves and Leepo Yu for providing preprints of papers before publication, and Dr Andrew Szent-Gyo¨rgyi for encouragement, advice and critical reading of the manuscript. This work was carried out using the Core Electron Microscopy Facility of the University of Massachusetts Medical School and was supported by NIH grants AR34711, and Shared Instrumentation Grant RR08426 for the purchase of the cryo-electron microscope.

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Edited by W. Baumeister (Received 20 August 2002; received in revised form 7 January 2003; accepted 13 January 2003)