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Electron microscopy of the actin-myosin head complex in the presence of ATP
J. Mol. Biol. (1992) 223, 391-397
COMMUNICATION
Electron
Microscopy
of the Actin-Myosin the Presence of ATP
Ling-Ling
Head Complex
in
Frado and Roger Craig?
Department of Cell Biology University of Massachusetts Medical School 55 Lake Avenue North, Worcester, MA 01655, U.S.A. (Received 15 April
1991; accepted 12 September 1991)
The structure of the actin-myosin head complex during the ATPase cycle has been studied by electron microscopy of negatively stained acto-heavy-meromyosin. In the absence of ATP, heavy meromyosin molecules generally showed a regular, angled appearance, with both heads attached to the actin filament. In the presence of ATP, attached molecules showed a less ordered structure, often with only one head attached. We conclude that configurations other than the rigor structure occur during the actomyosin cross-bridge cycle. Keywords: electron microscopy; actomyosin; cross-bridge cycle; structure; ATPase
The molecular mechanism of muscle contraction it thought to involve ATP-dependent structural changes in the myosin heads as they interact transiently with actin during the cross-bridge cycle. With each cycle of interaction between a myosin head and actin, one molecule of ATP is hydrolyzed, providing the energy for filament sliding (Cooke, 1986). The structure of the actin-myosin head complex at different stages of the cross-bridge cycle is unknown. The structure at the end of the power stroke, when the products of ATP hydrolysis have been lost and the affinity of myosin for actin is high, is thought to resemble the rigor structure, in which heads are attached to actin at a defined angle of about 45”, pointing towards the center of the sarcomere (Huxley, 1963; Reedy et al., 1965; Moore et al., 1970; Milligan & Flicker, 1987). Evidence concerning the structures of the more weakly bound states, actin-myosin-ATP (A.M.ATP$), and actinmyosin-ADP-phosphate (A.M.ADP.Pi), occurring at earlier stages of the cycle, is less definitive. Some experiments suggest a structure similar to that in rigor, while others imply a disordered structure (Cooke, 1986; Huxley & Kress, 1985; Thomas, 1987). For example, optical (Yanagida, 1984, 1985) and spin (Cooke et al., 1982) probes bound to myosin t Author
to whom all correspondence
should be
addressed. $ Abbreviations used: A.M.ATP, actin-myosin-ATP; A.M.ADP.Pi, actinmyosin-ADP-phosphate; e.p.r., electron paramagnetic resonance; ATPase, adenosine triphosphatase; HMM, heavy meromyosin; Sl, subfragment 1 of myosin.
heads have been found to be highly oriented in contracting muscle fibers, at the same angle as those in rigor, suggesting that there is no change in the orientation of the cross-bridges during the attached phase of contraction. On the other hand fluorescence resonance energy transfer studies of acto-Sl complexes and proteolytic studies of cross-linked acto-Sl suggest considerable differences in the structure of the acto-Sl complex between the weakly and strongly bound states (Trayer & Trayer, 1988; Duong & Reisler, 1989; Yamamoto, 1989). Numerous studies of cross-bridges using X-ray diffraction or electron paramagnetic resonance (e.p.r.) techniques also suggest that there are conformational differences between rigor and weakbinding cross-bridge states occurring during contraction (Matsuda & Podolsky, 1984; Xu et al., 1987; Lowy & Poulsen, 1987; Yu & Brenner, 1989; Yu et aE., 1990; Barnett & Thomas, 1989; Fajer et al., 1990). While such approaches make possible the study of intact contracting muscle, they do not provide a direct visual image of crossbridge structure. Although high-resolution electron microscopy does provide such images, it also has not been entirely satisfactory for studying the structures of the weakly bound states. For example, negative staining of the actomyosin complex during the adenosine triphosphatase (ATPase) cycle requires high concentrations of actin or myosin heads to maintain head binding in the presence of ATP. This requirement is incompatible with the low ‘concentrations generally necessary for ‘electron microscopy. To overcome this limitation, chemically cross-linked acto-Sl
391 0022-2836/92/02039147
$03.00/O
has been used to maintain
head binding
0 1992 Academic
Press Limited
in
L.-L.
Prado
and R. Craig
rigor buffer (5 m~~-.~aCI, 1 msr-A&i 21,; Figure 1. Electron micrographs of actin filaments decorated in low-salt 1 mm-dithiothreitol (DTT), 5 mx-sodium phosphate, pH 7.2) with an equimolar ratio of HMM heads, applied to a gl rid, then rapidly rinsed with the same solution followed immediately by staining with 1 TO uranyl acetate. (a) and (b) Fie ,ldS of fully decorat,ed actin filaments showing arrowhead structure with some less-decorated filaments in background. Cc)
Communication
the presence of ATP. Results with cross-linked acto-Sl using both negative staining (Craig et al., 1985) and cryo-electron microscopy (Applegate & Flicker, 1987) suggest that the A.M.ATP and A.M.ADP.Pi states are disordered, with the heads adopting a variety of angles and often looking shorter than in rigor. Disordering is also supported by e.p.r. studies of cross-linked acto-Xl in solution, which show microsecond rotational motions of Sl in the presence of ATP (Svensson & Thomas, 1986). Control experiments (Craig et al., 1985) suggested that the disordered heads were specifically attached states of the ATPase cycle (see also Duong & Reisler, 1989). However, the possibility could not be excluded that the disordered structures were simply heads tethered to actin by the cross-linker rather than specifically attached by non-covalent bonds (Zot et al., 1990). Rapid freezing/freeze-etching has also been used to study the conformational changes of Sl molecules bound (but not cross-linked) to actin filaments during ATP hydrolysis. Again the evidence is not clear. Sl bound to actin during ATP hydrolysis either looked similar to Sl in the absence of ATP (Pollard et al., 1985), or shorter and rounder than rigor Sl (Katayama, 1989).
393 In the work reported here, we describe a modified technique that allows us to observe the actinmyosin head complex in the presence of ATP by the high-resolution technique of negative staining without the use of cross-linkers. As was the case with cross-linked acto-Sl, we find non-rigor angles of head attachment in the presence of ATP. This work has been reported in preliminary form (Frado & Craig, 1991). For negative staining of acto-heavy-meromyosin in the absence of ATP, actin filaments were decorated in an ATP-free solution with an approximately equimolar amount of heavy meromyosin (HMM) heads. After applying the filaments to an electron microscope grid coated with a holey carbon film, the grid was rapidly rinsed (for 20 to 40 ms) with a lowsalt rigor buffer, and then immediately stained with uranyl acetate (see the legend to Fig. 1 for details). Decorated filaments showed the typical rigor appearance of polar arrowheads (Fig. l(a) to (c); Huxley, 1963; Moore et al., 1970). These were observed with greatest clarity in films of stain over holes in the carbon film. A significant number of naked actin filaments was also observed. These may be a result of forces that occur over the holes during rapid rinsing, since the same filaments were often
High magnification of arrowhead appearance. (d) to (g) Filaments were prepared as in (a) except they were decorated with a 1 : 10 molar ratio HMM heads/actin subunits; individual HMMs show regular, angled, Z-headed decoration. Scale bars represent: (a) and (b) 100 nm; (c) to (g) 50 nm. Actin (1.0 mg/ml) was mixed with chymotryptic HMM at an approximately equimolar ratio of heads (42 or 3.6 mg HMM/ml) to actin in low-salt rigor buffer; the lower ratio gave a cleaner background and most experiments were therefore carried out at this level of HMM. The mixture was left at room temperature for 1 h and then diluted fivefold with the rigor buffer before applying to the grid (the 1 h incubation was necessary to obtain the best decoration and cleanest background). 10 ~1 of the diluted sample was applied to a 400 mesh grid coated with a holey carbon film (Craig et aZ., 1980). After 10 to 60 s, the sample was rapidly rinsed with buffer then immediately stained with uranyl acetate using a technique devised by Dr Peter Knight. This consisted of drawing sequentially into a 1 ml Eppendorf pipet tip attached to a Pipetman: 0.2 ml 1 y0 uranyl acetate, 001 ml air and 0.05 ml low-salt rigor buffer (the air gap prevents the 2 solutions from mixing). The contents of the pipet tip were squirted rapidly on to the grid such that the grid was exposed to the rinse buffer very briefly (20 to 40 ms, measured using an oscilloscope) before being stained. After blotting off excess stain, the grid was allowed to dry in an 80% relative humidity chamber. The sample was observed with a JEOL 1OOCX electron microscope at 80 kV with an anticontamination device in place. Filaments lying in film of stain over holes in the carbon support were photographed using conventional electron doses. MgATP (0.2 to 1 mm) was added to the low-salt rigor buffer in experiments where we wanted to observe the acto-HMM during the ATPase cycle. High salt buffer (0.15 iv-NaCl replaces 5 mM-Nacl), with or without ATP, was used in control experiments. Rabbit myosin was prepared as described by Margossian & Lowey (1985). HMM was prepared by chymotryptic digestion of freshly prepared myosin according to the method of Okamoto & Sekine (1985) with the digestion time shortened to 10 min. HMM was precipitated with 60% (NH&SO, and desalted with a PD-10 column (Pharmacia) that was pre-equilibrated with 1 mM-DTT and 601 M-potassium phosphate buffer (pH 7.0). The HMM heavy chain ran as a single band at M, = 140,000 on SDS/polyacrylamide gel electrophoresis (Laemmli, 1970; data not shown). Actin was extracted and purified from a rabbit muscle acetone powder (Pardee & Spudich, 1982) by the method of Spudich & Watt (1971), and recycled just before use (Pardee & Spudich, 1982). The use of freshly prepared HMM with high ATPase activity and of newly recycled actin was essential for achieving the clean background that allowed us to distinguish true decoration from background contamination that was merely superimposed on the actin filaments. Protein concentrations were determined using extinction coefficients Ezso (1%) of 5.3, 6.0 and 11.1 cm-’ for myosin, HMM and actin, respectively. EDTA ATPase activity of myosin and HMM, and a&in-activated ATPase activity of HMM were determined by the method of Margossian & Lowey (1978). Inorganic phosphate was assayed according to the method of Taussky 85 Schorr (1953). In 1 experiment designed to remove any inactive HMM (with heads that might bind to actin even in the presence of ATP), 10 mg HMM was mixed with 0.36 mg actin (a 10 : 3 molar ratio of heads/a&in) in 1 ml of low-salt rigor buffer. Mg-ATP (10 mM) was added just before centrifugation and the mixture spun at 4°C at 150,000 g for 90 min in a Ti50 Beckman rotor. Although all ATP would be consumed early in the centrifugation and thus the pellet would contain fully decorated actin, we reasoned that the 1st molecules to bind to actin would be any HMM that possessed an ATP-intensive head. Thus all such molecules should be removed from solution, assuming they amount to less than 30% of the total heads.
394
A.-k.
Frado
fully decorated where they ran on to the carbon substrate, and specimens that were rinsed slowly yielded mostly fully decorated filaments over the holes. When filaments were decorated with a 1: 10 ratio of HMM heads to actin monomers; individual HMM molecules could be clearly seen bound to a&in, generally by both heads, at a roughly constant angle of about 45” to the filament axis (Fig. l(d) to (g)), as found by Craig et al. (1980). For negative staining of a&o-heavy-meromyosin in the presence of ATP, 0.2 to 1.0 miw-MgATP was included in the rinse buffer. Under these conditions, the level of HMM binding was considerably reduced, although many filaments still appeared relatively well decorated (Fig. 2(a),(b)). We estimate that up to 30% of the actin monomers were occupied in some filaments. The appearance of the bound HMM was quite different from that in the absence of ATP. Many attached HMMs had a fuzzy appearance from which it was difficult to discern the detailed structure of the molecule. When the attached HMMs were distinct, some appeared shorter or closer to the axis of the filament than those in rigor, while others remained elongated. Most did not show the regular 45” attachment angle of the rigor state, but more varied angles (Fig. 2(c) to (f)), although a minority did show a more rigor-like angle (Fig. 2(g)). In addition, the two-headed appearance of the rigor structure was generally not obvious, attachment usually occurring through only a single head. The extent of decoration seemed to diminish as the length of the ATP rinse increased, although some decoration was still apparent even after a one second rinse. The decoration was also less if the ATP was present in high salt (0.15 M-NaCl). The finding that actin is decorated with HMM in the presence of ATP is surprising, considering the weak affinity of these proteins for each other under these conditions. In interpreting the results, we should therefore consider possible, non-physiological reasons for the observations. One possibility is that the heads that remain attached in the presence of ATP are damaged and do not respond to ATP. This appears to be unlikely, as the ATPase activity of the preparations from which we obtained our electron microscopic results was generally high. EDTA ATPase activities were 21.5 mol and 21 mol ATP hydrolyzed per mol per second for myosin and HMM respectively, and actinactivated ATPase activity (V,,,) for HMM was 37 mol ATP hydrolyzed per mol per second, comparable to published values obtained under the same conditions (Margossian & Lowey, 1978). These activities suggest that there were few ATP-insensitive heads, yet it was not difficult to find quite large amounts of decoration in the presence of ATP. In addition, in occasional preparations where ATPase activity was low (results not shown), heads tended not to bind to actin even in the absence of ATP. In one experiment, any ATP-insensitive heads were first removed by centrifuging with actin in the presence of ATP (see legend, Fig. 1). A level of decoration was obtained with this HMM in the presence of ATP
and R. Craig
that was similar to that obtained with EMM not purified in this way, further suggesting that beads attached in the presence of ATP are uot inactive. Another possible cause of head attachment in the presence of ATE” is “precipitation” of heads on to the actin filaments during staining, for example due to the low pH (4.3) of the uranyl a.cetate stain. Some degree of precipitation (of already attached beads or background heads) may help to explain the fuzzy appearance of some decoration. However, precipitation seems unlikely to be the major cause of head attachment in ATP, because we have sometimes observed a high background of HMM with little apparent binding to actin. Less decoration is seen when ATP is in high salt (when the affinity of heads for actin is reduced), further suggesting that precipitation is not the main reason for the decoration. Furthermore, we often observe heads attached by their tips, suggesting a specific interaction with actin rather than precipitation, where all forms of interaction would be expected. We conclude that weakly bound a&o-HM&l complexes (a mixture of A.M.ATP and A.M.ADP.Pi states; see Adelstein & Eisenberg, 1980; Cooke; 1986) are “fixed” and preserved by rapid staining of aeto-HMM with uranyl acetate after brief (29 to 40 ms) exposure to ATPt. We assume that the attached heads observed must first have detached from actin, which happens rapidly (within 1 to 2 ms), and then reattached and be passing through the next, or possibly the next but one, ATPase cycle (Uyeda et al.; 1991). Reattachment occurs, even with the low affinity of HMM for actin in ATP, presumably because the local concentrations of actin a’nd HMM are high. This may occur because some molecules over the holes in the carbon film become trapped (and thereby concentrated), for
t That uranyl acetate “fixes” macromolecular assemblies is suggested by the excellent preservation shown by many structures when negatively stained wit,h uranyl acetate; the quality of preservation can be judged by comparing such negatively stained structures with X-ray diffraction data obtained from intact tissue and with cryo-electron microscopic images. For example, negatively stained arrowheads have Fourier transforms similar to those of frozen hydrated acto-Sl (Milligan & Flicker, 1987), and more labile structures such as myosin fila.ments have structures similar to those predicted from X-ray diffraction (e.g. compare Vibert & Craig (1983) with Wray et al. (1975)). If the stain had no fixation effect, myosin filaments would be expected to disassemble during drying of the stain (due to the increase in salt concentration). In addition, when actin filaments are decorated with Sl, rinsed with uranyl acetate, then treated with ATE’. following which they are negatively stained and dried, they show a fully decorated arrowhead appearance, indicating that treatment with uranyl acetate prevents dissociation of Sl from actin by ATP; uranyl acetate similarly protects actin filaments from disassembly at low ionic strength and MgZf paracrystals of actin from dissociation at low Mg ‘+ levels (P. Vibert, personal communication).
Communication
Figure 2. Negatively stained filaments prepared as in Fig. 1 (equimolar heads : actin), but with the rinse solution containing an additional 1 mivr-MgATP. (a) and (b) Fields showing considerable decoration of actin, but without the ordered arrowhead appearance. (c) to (g) High magnification images showing non-rigor angle of attachment of HMM ((c) to (f)) and some signs of rigor angle (g); attachment often appears to be via only one head ((d), (e) and (g)). Scale bars represent: (a) and (b) 100 nm; (c) to (g) 50 nm.
396
L.-L.
Frado
example at the air/buffer interface (only 1 side of the grid is rinsed with buffer), and their diffusion away into tbe bulk solution is thus restrictedt. That such trapping does indeed occur is sugggested by the observation that even after considerable rinsing with buffer, background proteins are usually still observed over the holes, sometimes in large quantities” Our results support the conclusions from earlier studies using cross-linked proteins (Craig et al., 1985; Applegate & Flicker, 198’i) tha,t the structure and/or orientation of the actin-myosin head complexes in the presenee of ATP are variable and different from those of rigor complexes. Trinick & White (1991) have found similar results using cryoelectron microscopy of acto-Sl frozen during steady-state ATP hydrolysis. Our results are also consistent with saturation transfer-e.p.r. studies of uncross-linked a&o-S1 in solution, showing that myosin heads undergo rotational motion when bound to actin during the ATPase cycle (Berger et al., 1989; Ostap & Thomas, 1991). In our study, HMMs in the presence of ATP often appeared to attach to actin at angles centered on about 90”, but this was not possible to quantify precisely owing to the shrinking and stretching of the films of stain over the holes in the carbon film that occurs during electron irradiation. Both heads of HMM bind to actin, at similar angles, in the absence of ATP. This is accommodated by distortion of the distal ends of the heads (Craig et al., 1980) and is energetically possible presumably due to the strength of the rigor bond. In the presence of ATP, two-headed binding does not appear to be common, presumably because the actin-head bond is too weak to bring about the required distortion in the distal part of the heads. The single-headed binding that we observe does not represent Sl, as SDS/polyacrylamide gel electrophoresis of our HMM shows a single heavy chain band at ilfr 140,000. Our results argue against the conclusions of Pollard et al. (1985) that weakly and strongly attached heads have similar appearances, and of Zot et al. (1990) that the non-rigor angles seen in crosslinked a&o-S1 in the presence of ATP are due to simple tethering of heads to actin. Our results also suggest tha,t some heads remain elongated in ATP, contrary to the conclusion of Katayama (1989). These discrepancies may result from our use of HMM, which has intact heads, while the other authors used Sl, which is proteolyzed at the headrod junction. Despite these differences, we have T If we assume, for example, that the interface is similar in thickness to the holey carbon film (of the order of 20 nm, or twice the thickness of an act’in filament) and that the length of actin filament per pm2 of holes is about, 14 pm (as measured), this gives an aetin concentration of about 400 ~BI, and this actin is initially fully decorated with HMM. These concentrations a,re high enough to give considerable HMM binding even in the presence of ATP (Pollard et az., 1985).
and
K.
Craiy
provided
furthor
weakly at’tached cycle is different states.
evidence that the structure of states of the act’omyosin ATPase from that of the strongly atta.cbed
We thank Dr Peter Vibert for his help in stages of this project, and Dr J. Leppo for rabbits for the preparation of actin and myosin. grateful to Dr Peter Knight for suggesting the rapid rinsing and staining used in this paper. was supportedd by NIH grant, AR3471 1.
the early providing We are method of This work
References Adelstein, R. S. & Eisenberg, E. (1980). Reguiation and kinetics of the actin-myosin-ATP interact’ion. Awnu. Rev. Biochem. 49; 921-956. Applegate, D. $ Flicker, P. (1987). New states of actomgosin. J. Biol. Chem. 262, 6856-6863. Barnet,t, V. A. & Thomas, D. D. (1989). Microsecond rotational motion of spin-labeled myosin heads during isometric muscle contraction. Rio$ys. J. 56, 517-523. Berger. C. L.. Svensson: E. C. & Thomas, 1). 6). (1989). Photolysis of a photolabile precursor of ATP (caged BTP) induces microsecond rotational motions of myosin heads bound to actin. Pzoc. Xat. Acad. Sci., U.S.A. 86, 8753-8757. Cooke, R. (1986). The mechanism of muscle contraction. C.R.C. Crdt. Rev. Biochem. 21; 533118. Cooke, R.. Crowder, M. S. & Thomas, D. D. (1982). Orientation of spin labels attached to cross-bridges in contracting muscle fibers. Nature (Londonj, 300, 776-778. Craig, R., Szent-GySrgyi, A. G., Reese, L., Flicker, P., Vibert, P. $ Cohen, C. (1980). Electron microscopy of thin filaments decorated with a Ca’+-regulated myosin. .I. Mol. Biol. 140, 35-55. Craig, R., Greene, I,. E. & Eisenberg, E. (1985). Structure of the actin-myosin complex in the presence of ATP. Proe. Nat. Acad. Sci.. U.S.A. 82; 3247-3251. Duong, A. M. & Reisler, E. (1989). Nucleotide-induced states of myosin subfragment 1 cross-linked to act’in. Biochemistry, 28? 3502-3509. Fajer, P. G., Fajer, E. A. & Thomas, D. D. (1990). Myosin heads have a broad orientational distribution during
isometric muscle cont,raction: time-resolved studies using caged ATE’. Proc. Nat. Acad.
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Sci,, U.S.A. 87, 5538-5542. Prado, L.-L. Y. & Craig, R. (1991). Structure of the actin-myosin head complex in the presence of ATP. Biophys. J. 59. 429a. Huxley, H. E. (1963). Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. J. Mol. Biol. 7, 281-308. Huxley, H. E. & Kress, M. (1985). Crossbridge behaviour during muscle contraction. J. Muscle Res. Cell Motil. 6, 153-161. Katayama, E. (1989). The effects of various nucleotidea on the structure of a&in-attached myosin subfragmen&l studied by quick freeze deep-etch electron microscopy. J. Biochem. 106, 751-770. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Xature (London), 227. 680-685. Lvwy, J. & Poulsen, F. R. (1987). X-ray study of myosin heads in contracting frog skeletal muscle. .J. Mol.
Biol. 194, 595-600.
Communication
Margosian, myosin
S. S. & Lowey, subfragments with
S. (1978). Interaction F-a&in. Biochemistry,
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5431-5439. Margossian, S. S. & Lowey, S. (1985). Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol. 85, 55-71. Matsuda, T. & Podolsky, R. J. (1984). X-ray evidence for two structural states of the actomyosin cross-bridge in muscle fibers. Proc. Nat. Acad. Sci., U.S.A. 81, 2364-2368 Milligan, R. A. & Flicker, P. F. (1987). Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy. J. Cell Biol.
105, 29-39. P. B., Huxley, H. E. & DeRosier, D. J. (1970). Three-dimensional reconstruction of F-actin, thin filaments and decorated thin filaments. J. Mol. Biol. 50, 279-295. Okamoto, Y. & Sekine, T. (1985). A streamlined method of subfragment one preparation from myosin. J. Biochem. 98, 1143-1145. Ostap, E. M & Thomas, D. D. (1991). Rotational dynamics of spin-labeled F-actin during activation of myosin Sl ATPase using caged ATP. Biophys. J. 59, 1235-1241. Pardee, J. D. & Spudich, J. A. (1982). Preparation of muscle actin. Methods Enzymol. 85, 164-181. Pollard, T. D., Weeds, A. G. & Huxley, H. E. (1985). A rapid-freezing/stopped-flow method to prepare the intermediates in the actin-myosin ATPase cycle for electron microscopy. Biophys. J. 47, 129a. Reedy, M. K., Holmes, K. C. & Tregear, R. T. (1965). Induced changes in orientation of the cross-bridges of glycerinated insect flight muscle. Nature (London), 207, 1276-1280. Spudich, J. A. & Watt, S. (1971). The regulation of rabbit skeletal muscle contraction. J. Biol. Chem. 246, 4866-487 1. Svensson, E. C. & Thomas, D. D. (1986). ATP induces microsecond rotational motions of myosin heads crosslinked to actin. Biophys. J. 50, 99991002. Taussky, H. H. & Schorr, E. (1953). A microcolorimetric method for the determination of inorganic phosphorus. J. Biol. Chem. 202, 675-685. Thomas, D. D. (1987). Spectroscopic probes of muscle cross-bridge rotation. Annu. Rev. Physiol. 49, 691l 709. Moore,
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H. R. & Trayer, I. P. (1988). Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between ‘weakly and strongly attached states. Biochemistry, 27, 5718-5727. Trinick, J. & White, H. (1991). Electron cryo-microscopy of actomyosin-Sl during steady state ATP hydrolysis. Biophys. J. 59, 410a. Uyeda, T. Q. P., Warrick, H. M., Kron, S. J. & Spudich, J. A. (1991). Quantized velocities at low myosin densities in an in vitro motility assay. Nature (London), 352, 307-311. Vibert, P. & Craig, R. (1983). Electron microscopy and image analysis of myosin filaments from scallop striated muscle. J. Mol. Biol. 165, 303-320. Wray, J. S., Vibert, P. J. & Cohen, C. (1975). Diversity of crossbridge configurations in invertebrate muscles. Nature (London), 257, 561-564. Xu, S., Kress, M. & Huxley, H. E. (1987). X-ray diffraction studies of the structural state of crossbridges in skinned frog sartorius muscle at low ionic strength. J. Muscle Res. Cell Motil. 8, 39-54. Yamamoto, K. (1989). Binding manner of actin to the lysine-rich sequence of myosin subfragment 1 in the presence and absence of ATP. Biochemistry, 28, 5573-5577. Yanagida, T. (1984). Angles of fluorescently labelled myosin heads and actin monomers in contracting and rigor stained muscle fiber. In Contractile Mechanisms in Muscle (Pollack, G. H. & Sugi, H.; eds), Plenum Publishing Corp, New York. Yanagida, T. (1985). Angle of active site of myosin heads in contracting muscle during sudden length changes. J. Muscle Res. Cell Motil. 6, 43-52. Yu, L. C. & Brenner, B. (1989). Structures of actomyosin crossbridges in relaxed and rigor muscle fibers. Biophys. J. 55, 441-453. Yu, L. C., Maeda, Y. & Brenner, B. (1990). Evidence for at least three distinct configurations of attached crossbridges. Biophys. J. 57, 409a. Zot, H. G., Maupin, P. & Pollard, T. D. (1990). Evidence that crosslinked acto-subfragment-l is dissociated but tethered in the presence of ATP. Biophys. J. 57, 410a. Trayer,
by H. E. Huxley
COMMUNICATION
Electron
Microscopy
of the Actin-Myosin the Presence of ATP
Ling-Ling
Head Complex
in
Frado and Roger Craig?
Department of Cell Biology University of Massachusetts Medical School 55 Lake Avenue North, Worcester, MA 01655, U.S.A. (Received 15 April
1991; accepted 12 September 1991)
The structure of the actin-myosin head complex during the ATPase cycle has been studied by electron microscopy of negatively stained acto-heavy-meromyosin. In the absence of ATP, heavy meromyosin molecules generally showed a regular, angled appearance, with both heads attached to the actin filament. In the presence of ATP, attached molecules showed a less ordered structure, often with only one head attached. We conclude that configurations other than the rigor structure occur during the actomyosin cross-bridge cycle. Keywords: electron microscopy; actomyosin; cross-bridge cycle; structure; ATPase
The molecular mechanism of muscle contraction it thought to involve ATP-dependent structural changes in the myosin heads as they interact transiently with actin during the cross-bridge cycle. With each cycle of interaction between a myosin head and actin, one molecule of ATP is hydrolyzed, providing the energy for filament sliding (Cooke, 1986). The structure of the actin-myosin head complex at different stages of the cross-bridge cycle is unknown. The structure at the end of the power stroke, when the products of ATP hydrolysis have been lost and the affinity of myosin for actin is high, is thought to resemble the rigor structure, in which heads are attached to actin at a defined angle of about 45”, pointing towards the center of the sarcomere (Huxley, 1963; Reedy et al., 1965; Moore et al., 1970; Milligan & Flicker, 1987). Evidence concerning the structures of the more weakly bound states, actin-myosin-ATP (A.M.ATP$), and actinmyosin-ADP-phosphate (A.M.ADP.Pi), occurring at earlier stages of the cycle, is less definitive. Some experiments suggest a structure similar to that in rigor, while others imply a disordered structure (Cooke, 1986; Huxley & Kress, 1985; Thomas, 1987). For example, optical (Yanagida, 1984, 1985) and spin (Cooke et al., 1982) probes bound to myosin t Author
to whom all correspondence
should be
addressed. $ Abbreviations used: A.M.ATP, actin-myosin-ATP; A.M.ADP.Pi, actinmyosin-ADP-phosphate; e.p.r., electron paramagnetic resonance; ATPase, adenosine triphosphatase; HMM, heavy meromyosin; Sl, subfragment 1 of myosin.
heads have been found to be highly oriented in contracting muscle fibers, at the same angle as those in rigor, suggesting that there is no change in the orientation of the cross-bridges during the attached phase of contraction. On the other hand fluorescence resonance energy transfer studies of acto-Sl complexes and proteolytic studies of cross-linked acto-Sl suggest considerable differences in the structure of the acto-Sl complex between the weakly and strongly bound states (Trayer & Trayer, 1988; Duong & Reisler, 1989; Yamamoto, 1989). Numerous studies of cross-bridges using X-ray diffraction or electron paramagnetic resonance (e.p.r.) techniques also suggest that there are conformational differences between rigor and weakbinding cross-bridge states occurring during contraction (Matsuda & Podolsky, 1984; Xu et al., 1987; Lowy & Poulsen, 1987; Yu & Brenner, 1989; Yu et aE., 1990; Barnett & Thomas, 1989; Fajer et al., 1990). While such approaches make possible the study of intact contracting muscle, they do not provide a direct visual image of crossbridge structure. Although high-resolution electron microscopy does provide such images, it also has not been entirely satisfactory for studying the structures of the weakly bound states. For example, negative staining of the actomyosin complex during the adenosine triphosphatase (ATPase) cycle requires high concentrations of actin or myosin heads to maintain head binding in the presence of ATP. This requirement is incompatible with the low ‘concentrations generally necessary for ‘electron microscopy. To overcome this limitation, chemically cross-linked acto-Sl
391 0022-2836/92/02039147
$03.00/O
has been used to maintain
head binding
0 1992 Academic
Press Limited
in
L.-L.
Prado
and R. Craig
rigor buffer (5 m~~-.~aCI, 1 msr-A&i 21,; Figure 1. Electron micrographs of actin filaments decorated in low-salt 1 mm-dithiothreitol (DTT), 5 mx-sodium phosphate, pH 7.2) with an equimolar ratio of HMM heads, applied to a gl rid, then rapidly rinsed with the same solution followed immediately by staining with 1 TO uranyl acetate. (a) and (b) Fie ,ldS of fully decorat,ed actin filaments showing arrowhead structure with some less-decorated filaments in background. Cc)
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the presence of ATP. Results with cross-linked acto-Sl using both negative staining (Craig et al., 1985) and cryo-electron microscopy (Applegate & Flicker, 1987) suggest that the A.M.ATP and A.M.ADP.Pi states are disordered, with the heads adopting a variety of angles and often looking shorter than in rigor. Disordering is also supported by e.p.r. studies of cross-linked acto-Xl in solution, which show microsecond rotational motions of Sl in the presence of ATP (Svensson & Thomas, 1986). Control experiments (Craig et al., 1985) suggested that the disordered heads were specifically attached states of the ATPase cycle (see also Duong & Reisler, 1989). However, the possibility could not be excluded that the disordered structures were simply heads tethered to actin by the cross-linker rather than specifically attached by non-covalent bonds (Zot et al., 1990). Rapid freezing/freeze-etching has also been used to study the conformational changes of Sl molecules bound (but not cross-linked) to actin filaments during ATP hydrolysis. Again the evidence is not clear. Sl bound to actin during ATP hydrolysis either looked similar to Sl in the absence of ATP (Pollard et al., 1985), or shorter and rounder than rigor Sl (Katayama, 1989).
393 In the work reported here, we describe a modified technique that allows us to observe the actinmyosin head complex in the presence of ATP by the high-resolution technique of negative staining without the use of cross-linkers. As was the case with cross-linked acto-Sl, we find non-rigor angles of head attachment in the presence of ATP. This work has been reported in preliminary form (Frado & Craig, 1991). For negative staining of acto-heavy-meromyosin in the absence of ATP, actin filaments were decorated in an ATP-free solution with an approximately equimolar amount of heavy meromyosin (HMM) heads. After applying the filaments to an electron microscope grid coated with a holey carbon film, the grid was rapidly rinsed (for 20 to 40 ms) with a lowsalt rigor buffer, and then immediately stained with uranyl acetate (see the legend to Fig. 1 for details). Decorated filaments showed the typical rigor appearance of polar arrowheads (Fig. l(a) to (c); Huxley, 1963; Moore et al., 1970). These were observed with greatest clarity in films of stain over holes in the carbon film. A significant number of naked actin filaments was also observed. These may be a result of forces that occur over the holes during rapid rinsing, since the same filaments were often
High magnification of arrowhead appearance. (d) to (g) Filaments were prepared as in (a) except they were decorated with a 1 : 10 molar ratio HMM heads/actin subunits; individual HMMs show regular, angled, Z-headed decoration. Scale bars represent: (a) and (b) 100 nm; (c) to (g) 50 nm. Actin (1.0 mg/ml) was mixed with chymotryptic HMM at an approximately equimolar ratio of heads (42 or 3.6 mg HMM/ml) to actin in low-salt rigor buffer; the lower ratio gave a cleaner background and most experiments were therefore carried out at this level of HMM. The mixture was left at room temperature for 1 h and then diluted fivefold with the rigor buffer before applying to the grid (the 1 h incubation was necessary to obtain the best decoration and cleanest background). 10 ~1 of the diluted sample was applied to a 400 mesh grid coated with a holey carbon film (Craig et aZ., 1980). After 10 to 60 s, the sample was rapidly rinsed with buffer then immediately stained with uranyl acetate using a technique devised by Dr Peter Knight. This consisted of drawing sequentially into a 1 ml Eppendorf pipet tip attached to a Pipetman: 0.2 ml 1 y0 uranyl acetate, 001 ml air and 0.05 ml low-salt rigor buffer (the air gap prevents the 2 solutions from mixing). The contents of the pipet tip were squirted rapidly on to the grid such that the grid was exposed to the rinse buffer very briefly (20 to 40 ms, measured using an oscilloscope) before being stained. After blotting off excess stain, the grid was allowed to dry in an 80% relative humidity chamber. The sample was observed with a JEOL 1OOCX electron microscope at 80 kV with an anticontamination device in place. Filaments lying in film of stain over holes in the carbon support were photographed using conventional electron doses. MgATP (0.2 to 1 mm) was added to the low-salt rigor buffer in experiments where we wanted to observe the acto-HMM during the ATPase cycle. High salt buffer (0.15 iv-NaCl replaces 5 mM-Nacl), with or without ATP, was used in control experiments. Rabbit myosin was prepared as described by Margossian & Lowey (1985). HMM was prepared by chymotryptic digestion of freshly prepared myosin according to the method of Okamoto & Sekine (1985) with the digestion time shortened to 10 min. HMM was precipitated with 60% (NH&SO, and desalted with a PD-10 column (Pharmacia) that was pre-equilibrated with 1 mM-DTT and 601 M-potassium phosphate buffer (pH 7.0). The HMM heavy chain ran as a single band at M, = 140,000 on SDS/polyacrylamide gel electrophoresis (Laemmli, 1970; data not shown). Actin was extracted and purified from a rabbit muscle acetone powder (Pardee & Spudich, 1982) by the method of Spudich & Watt (1971), and recycled just before use (Pardee & Spudich, 1982). The use of freshly prepared HMM with high ATPase activity and of newly recycled actin was essential for achieving the clean background that allowed us to distinguish true decoration from background contamination that was merely superimposed on the actin filaments. Protein concentrations were determined using extinction coefficients Ezso (1%) of 5.3, 6.0 and 11.1 cm-’ for myosin, HMM and actin, respectively. EDTA ATPase activity of myosin and HMM, and a&in-activated ATPase activity of HMM were determined by the method of Margossian & Lowey (1978). Inorganic phosphate was assayed according to the method of Taussky 85 Schorr (1953). In 1 experiment designed to remove any inactive HMM (with heads that might bind to actin even in the presence of ATP), 10 mg HMM was mixed with 0.36 mg actin (a 10 : 3 molar ratio of heads/a&in) in 1 ml of low-salt rigor buffer. Mg-ATP (10 mM) was added just before centrifugation and the mixture spun at 4°C at 150,000 g for 90 min in a Ti50 Beckman rotor. Although all ATP would be consumed early in the centrifugation and thus the pellet would contain fully decorated actin, we reasoned that the 1st molecules to bind to actin would be any HMM that possessed an ATP-intensive head. Thus all such molecules should be removed from solution, assuming they amount to less than 30% of the total heads.
394
A.-k.
Frado
fully decorated where they ran on to the carbon substrate, and specimens that were rinsed slowly yielded mostly fully decorated filaments over the holes. When filaments were decorated with a 1: 10 ratio of HMM heads to actin monomers; individual HMM molecules could be clearly seen bound to a&in, generally by both heads, at a roughly constant angle of about 45” to the filament axis (Fig. l(d) to (g)), as found by Craig et al. (1980). For negative staining of a&o-heavy-meromyosin in the presence of ATP, 0.2 to 1.0 miw-MgATP was included in the rinse buffer. Under these conditions, the level of HMM binding was considerably reduced, although many filaments still appeared relatively well decorated (Fig. 2(a),(b)). We estimate that up to 30% of the actin monomers were occupied in some filaments. The appearance of the bound HMM was quite different from that in the absence of ATP. Many attached HMMs had a fuzzy appearance from which it was difficult to discern the detailed structure of the molecule. When the attached HMMs were distinct, some appeared shorter or closer to the axis of the filament than those in rigor, while others remained elongated. Most did not show the regular 45” attachment angle of the rigor state, but more varied angles (Fig. 2(c) to (f)), although a minority did show a more rigor-like angle (Fig. 2(g)). In addition, the two-headed appearance of the rigor structure was generally not obvious, attachment usually occurring through only a single head. The extent of decoration seemed to diminish as the length of the ATP rinse increased, although some decoration was still apparent even after a one second rinse. The decoration was also less if the ATP was present in high salt (0.15 M-NaCl). The finding that actin is decorated with HMM in the presence of ATP is surprising, considering the weak affinity of these proteins for each other under these conditions. In interpreting the results, we should therefore consider possible, non-physiological reasons for the observations. One possibility is that the heads that remain attached in the presence of ATP are damaged and do not respond to ATP. This appears to be unlikely, as the ATPase activity of the preparations from which we obtained our electron microscopic results was generally high. EDTA ATPase activities were 21.5 mol and 21 mol ATP hydrolyzed per mol per second for myosin and HMM respectively, and actinactivated ATPase activity (V,,,) for HMM was 37 mol ATP hydrolyzed per mol per second, comparable to published values obtained under the same conditions (Margossian & Lowey, 1978). These activities suggest that there were few ATP-insensitive heads, yet it was not difficult to find quite large amounts of decoration in the presence of ATP. In addition, in occasional preparations where ATPase activity was low (results not shown), heads tended not to bind to actin even in the absence of ATP. In one experiment, any ATP-insensitive heads were first removed by centrifuging with actin in the presence of ATP (see legend, Fig. 1). A level of decoration was obtained with this HMM in the presence of ATP
and R. Craig
that was similar to that obtained with EMM not purified in this way, further suggesting that beads attached in the presence of ATP are uot inactive. Another possible cause of head attachment in the presence of ATE” is “precipitation” of heads on to the actin filaments during staining, for example due to the low pH (4.3) of the uranyl a.cetate stain. Some degree of precipitation (of already attached beads or background heads) may help to explain the fuzzy appearance of some decoration. However, precipitation seems unlikely to be the major cause of head attachment in ATP, because we have sometimes observed a high background of HMM with little apparent binding to actin. Less decoration is seen when ATP is in high salt (when the affinity of heads for actin is reduced), further suggesting that precipitation is not the main reason for the decoration. Furthermore, we often observe heads attached by their tips, suggesting a specific interaction with actin rather than precipitation, where all forms of interaction would be expected. We conclude that weakly bound a&o-HM&l complexes (a mixture of A.M.ATP and A.M.ADP.Pi states; see Adelstein & Eisenberg, 1980; Cooke; 1986) are “fixed” and preserved by rapid staining of aeto-HMM with uranyl acetate after brief (29 to 40 ms) exposure to ATPt. We assume that the attached heads observed must first have detached from actin, which happens rapidly (within 1 to 2 ms), and then reattached and be passing through the next, or possibly the next but one, ATPase cycle (Uyeda et al.; 1991). Reattachment occurs, even with the low affinity of HMM for actin in ATP, presumably because the local concentrations of actin a’nd HMM are high. This may occur because some molecules over the holes in the carbon film become trapped (and thereby concentrated), for
t That uranyl acetate “fixes” macromolecular assemblies is suggested by the excellent preservation shown by many structures when negatively stained wit,h uranyl acetate; the quality of preservation can be judged by comparing such negatively stained structures with X-ray diffraction data obtained from intact tissue and with cryo-electron microscopic images. For example, negatively stained arrowheads have Fourier transforms similar to those of frozen hydrated acto-Sl (Milligan & Flicker, 1987), and more labile structures such as myosin fila.ments have structures similar to those predicted from X-ray diffraction (e.g. compare Vibert & Craig (1983) with Wray et al. (1975)). If the stain had no fixation effect, myosin filaments would be expected to disassemble during drying of the stain (due to the increase in salt concentration). In addition, when actin filaments are decorated with Sl, rinsed with uranyl acetate, then treated with ATE’. following which they are negatively stained and dried, they show a fully decorated arrowhead appearance, indicating that treatment with uranyl acetate prevents dissociation of Sl from actin by ATP; uranyl acetate similarly protects actin filaments from disassembly at low ionic strength and MgZf paracrystals of actin from dissociation at low Mg ‘+ levels (P. Vibert, personal communication).
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Figure 2. Negatively stained filaments prepared as in Fig. 1 (equimolar heads : actin), but with the rinse solution containing an additional 1 mivr-MgATP. (a) and (b) Fields showing considerable decoration of actin, but without the ordered arrowhead appearance. (c) to (g) High magnification images showing non-rigor angle of attachment of HMM ((c) to (f)) and some signs of rigor angle (g); attachment often appears to be via only one head ((d), (e) and (g)). Scale bars represent: (a) and (b) 100 nm; (c) to (g) 50 nm.
396
L.-L.
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example at the air/buffer interface (only 1 side of the grid is rinsed with buffer), and their diffusion away into tbe bulk solution is thus restrictedt. That such trapping does indeed occur is sugggested by the observation that even after considerable rinsing with buffer, background proteins are usually still observed over the holes, sometimes in large quantities” Our results support the conclusions from earlier studies using cross-linked proteins (Craig et al., 1985; Applegate & Flicker, 198’i) tha,t the structure and/or orientation of the actin-myosin head complexes in the presenee of ATP are variable and different from those of rigor complexes. Trinick & White (1991) have found similar results using cryoelectron microscopy of acto-Sl frozen during steady-state ATP hydrolysis. Our results are also consistent with saturation transfer-e.p.r. studies of uncross-linked a&o-S1 in solution, showing that myosin heads undergo rotational motion when bound to actin during the ATPase cycle (Berger et al., 1989; Ostap & Thomas, 1991). In our study, HMMs in the presence of ATP often appeared to attach to actin at angles centered on about 90”, but this was not possible to quantify precisely owing to the shrinking and stretching of the films of stain over the holes in the carbon film that occurs during electron irradiation. Both heads of HMM bind to actin, at similar angles, in the absence of ATP. This is accommodated by distortion of the distal ends of the heads (Craig et al., 1980) and is energetically possible presumably due to the strength of the rigor bond. In the presence of ATP, two-headed binding does not appear to be common, presumably because the actin-head bond is too weak to bring about the required distortion in the distal part of the heads. The single-headed binding that we observe does not represent Sl, as SDS/polyacrylamide gel electrophoresis of our HMM shows a single heavy chain band at ilfr 140,000. Our results argue against the conclusions of Pollard et al. (1985) that weakly and strongly attached heads have similar appearances, and of Zot et al. (1990) that the non-rigor angles seen in crosslinked a&o-S1 in the presence of ATP are due to simple tethering of heads to actin. Our results also suggest tha,t some heads remain elongated in ATP, contrary to the conclusion of Katayama (1989). These discrepancies may result from our use of HMM, which has intact heads, while the other authors used Sl, which is proteolyzed at the headrod junction. Despite these differences, we have T If we assume, for example, that the interface is similar in thickness to the holey carbon film (of the order of 20 nm, or twice the thickness of an act’in filament) and that the length of actin filament per pm2 of holes is about, 14 pm (as measured), this gives an aetin concentration of about 400 ~BI, and this actin is initially fully decorated with HMM. These concentrations a,re high enough to give considerable HMM binding even in the presence of ATP (Pollard et az., 1985).
and
K.
Craiy
provided
furthor
weakly at’tached cycle is different states.
evidence that the structure of states of the act’omyosin ATPase from that of the strongly atta.cbed
We thank Dr Peter Vibert for his help in stages of this project, and Dr J. Leppo for rabbits for the preparation of actin and myosin. grateful to Dr Peter Knight for suggesting the rapid rinsing and staining used in this paper. was supportedd by NIH grant, AR3471 1.
the early providing We are method of This work
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by H. E. Huxley