X-ray evidence for conformational changes in the myosin filaments of vertebrate striated muscle

J. Mol. Biol. (1975) 92, 113-143

X-ray Evidence for Conformational Changes in the Myosin Filaments of Vertebrate Striated Muscle J. C. HASELQROVE Medical Research Council Laboratory of iklolecular Biology Hills Road, Cambridge CB2 2&H, England (Received 26 July 1974) Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band. When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length : the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 A and 72 A are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 A, and an activated state which gives no layer lines but a meridional spacing of 144.8 A.

1. Introduction To understand fully the processes occurring during muscle contraction it is necessary to complement biochemical studies with knowledge of the structural changes that take place. X-ray studies of live, contracting vertebrate striated muscle give information about the structural changes occurring during contraction. In vertebrate striated muscle, axially oriented myosin filaments are laterally ordered into hexagonal lattices and interdigitate with arrays of actin filaments. The a&in-containing (thin) filaments consist of actin monomers arranged into a two-stranded helical structure (Hanson & Lowy, 1963) with tropomyosin molecules situated in the grooves of the filaments: troponin molecules are bound to the tropomyosin at 386 A intervals along the filament. The interaction of myosin with actin is controlled by calcium binding to the troponin molecules (see Ebashi & Endo, 1968) which in turn affect the position of 113 s

114

J. C. HASELGROVE

the tropomyosin molecules. Recent X-ray studies of contracting smooth and striated muscles have shown that the control mechanism is operated by tropomyosin moving to different positions on the filament and blocking the active site of actin (Huxley, 1971,1972; Haselgrove, 1972; Vibert et al., 1973). In contrast to the studies on thin filaments, much less is known about the structure of the thick (myosin) filaments. The myosin molecules are arranged longitudinally so that the heads which contain the actin-combining and ATPase sites project from the side of the filaments. (The heads are referred to as cross-bridges in structural studies.) In a relaxed live muscle the cross-bridges are arranged around the thick filaments in a helical structure (Huxley & Brown, 1967), probably in a 3-start 9, helix (Squire, 1973) and with a helix repeat of 429 8. It is thought that tension development and shortening of the muscle are generated by cross-bridges attaching to adjacent actin filaments and then moving to produce a relative longitudinal movement of the thick and thin filaments. It is of obvious importance to study the movement of the cross-bridges during contraction of living muscles; and this can be done by X-ray diffraction. Huxley & Brown (1967) studied in some detail the low angle X-ray patterns from live and rigor muscles and found that when a muscle contracts or passes into rigor the cross-bridges do in fact move. Their studies were made predominantly on muscles at rest length where the myosin and actin filaments were completely overlapped so they could not distinguish whether the movement of the cross-bridges was caused by interaction with actin or occurred independent,ly of such interaction. By stretching muscles, the extent of overlap of actin and myosin filaments is decreased, so the study of stretched muscle during contraction gives information about which changes are dependent on the interaction of actin and myosin. Haselgrove (1970) studied sartorius muscles stretched as far as possible (to about half-overlap) and showed that the movement of any individual cross-bridge is not dependent on its interacting directly with actin. Huxley (1972) studied the patterns from semitendinosus muscles which had been stretched until no actin-myosin overlap apparently existed, and he found that even in such stretched muscles the cross-bridges still move when the muscle is activated. Therefore cross-bridges are able to detect in some way (though possibly a rather indirect one) that the muscle has been stimulated. This paper presents my results (Haselgrove, 1970) which continue from the studies of Huxley & Brown (1967). The low angle diffraction patterns of live and rigor muscle have been recorded using muscles at different sarcomere lengths and compared with patterns from rest-length muscles. Interpretation of the diffraction patterns from muscles contracting at different lengths requires knowledge of the way the resting diffraction pattern depends on sarcomere length, so a detailed study is reported of the changes in diffraction pattern when live resting muscles are stretched. Many of the changes occurring when a muscle contracts or passes into rigor are independent of the length of the muscle, i.e. the changes are independent of the number of actin-myosin interactions. The basic similarity of patterns from muscles in rigor at lengths where actin and myosin filaments no longer overlap, and contracting muscles at any length shows that there is considerable similarity in the structure of the myosin filament backbone in each case. The meridional reflections at 143 a and 72 A change their spacing slightly when the muscle contracts or passes into rigor, and it is deduced from this that the myosin filament structure changes slightly. I suggested (Haselgrove, 1970) that this change might be the result of a control system operating in parallel with the actin-linked system, and this proposal is discussed further here.

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2. Methods (a) X-ray

techniques

All the present X-my patterns were taken using point-focusing mirror-monochromator cameras and fine-focus rotating-anode X-ray tubes operated at 40 kV and 20 mA. Details of these cameras are described by Huxley & Brown (1967) and Haselgrove (1970). Three cameras with different focal lengths were used depending on the requirements, since B long camera gives better resolution than a short camera but requires a much longer exposure. (i) 12 cm camera This was the shortest and fastest camera and was used to record patterns from contracting muscles. It would record uss,ble patterns in 15 min and would easily resolve the myosin layer lines in the diffraction pattern from a live muscle. The 143 A meridional reflection was easily visible just clear of the central blackening on the film, and reflections at higher orders of 429 A could also be seen on the meridian (Plate III). The camera consisted of EL3” cut quartz-crystal set 15 cm from the source to give a focus 26 cm from the crystal and a specimen-to-film distance of 12 cm. A 12-cm camera. using a “double mirror bender” with a monochromating crystal (Huxley & Brown, 1967) was also used, but due to the considerable extra effort involved in focusing and collimating s, doublemirror arrangement (focusing is described by Haselgrove, 1970) it wss only used when a short exposure time was of paramount importance such as in studies of actively contracting muscle. (ii) 35 cm-camera This camera gave a clear pattern of a live muscle in 6 h (Plate II). The specimen holder was an integral part of the evacuated film holder SO that the specimen-to-film distance wss fixed, and the camera was used for meesuring the small changes of spacing of meridional reflections when muscles pass into rigor. The camera consisted of a 5” cut quartz crystal placed 15 cm from the source to give a focus 45 cm from the crystal and a specimen film distance of 35 cm. (iii) 2 m camera Patterns recorded on this camera required at least resolution along the meridian (Plate I). A 7’ cut crystal a focus 200 cm from the crystal and a specimen-to-film (b) Study of X-ray

48 h exposure but had very high placed 63 cm from the source gave distance of 190 cm.

patterns

Patterns were recorded on Ilford “Industrial G” X-ray film, using 2 films in the cassette in the coventiond manner. When accurate comparisons of intensities or spacings of reflections from different patterns were required both patterns were recorded on different parts of the same film, thus avoiding errors due to differences in processing of separate films. Spacing measurements of X-ray reflections were made using a Nikon model 6C microcomparator. Much work was done to measure small changes in spacings of the strong meridional reflections at 143 A and 72 A when muscles contract or paes into rigor. For this investigation, patterns from a live and contracting (or rigor) muscle were recorded on one film, keeping the specimen-to-film distance constant, and the difference in the measured spacings of corresponding reflections recorded as a percentage change (although the patterns may have been from different muscles). The results were then analysed in terms of these individual changes rather than the mean values of absolute measurements thus avoiding errors due to film processing or errors in measuring specimen-to-film distances. A vacuum camera was used for recording the change of spacing when a muscle goes into rigor and the film was scored by a small metal length gauge before the first exposure and after the last exposure to check that the film dimensions had not changed during the experiments. All such exposures in evacuated cameras were made on 6hn stored in vacua beforehand and no change in film dimensions occurred during exposure. For measurement of absolute spacings, the spacing of the strong 143 A reflection of a rest-length live,

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resting sartorius muscle was measured using the 2 m camera to be 143.4 A k 0.1 A (Cu Kai, h = I.5405 A) and this reflection was used to calibrate the other cameras. Intensity measurements were made using a Joyce-Loebl microcomparator (model IIIc). Density traces were made running along the meridian, or parallel to it at a radial distsnce corresponding to the position of maximum density of the 429 A layer-line. When investigating the changes in intensity of the myosin layer-lines, the analysis was performed on the intensities of the strong 429 A layer-line which could be measured with greater accuracy than the other layer-lines: the other layer-lines always behaved similarly to that at 429 A. Because of the very small size of the X-ray reflections (about 100 pm x 300 pm) it was and necessary to use 8 densitometer sampling spot with an area as large as the reflection, then assess the intensity of the reflection from the height rather than the area of the peak on the densitometer traces. Even so errors in measurement were probably only of the order of 6% (Wooster, 1964; Haselgrove, 1970).

(c) Specimen technique (i) Live m~cles. Resting and contracting isometrically Frog sartorius muscles and semitendinosus muscles from Rana pipiens and R. temporaria were dissected free from the frog and suspended horizontally, bathed in Ringer’s solution aa used by Haselgrove & Huxley (1973) in a Perspex cell, on the X-ray cameras at 6°C. Specimen techniques for resting and contracting muscles have been described by Haselgrove & Huxley (1973). Experiments commonly took 10 to 15 h to build up a 15 to 20-min exposure of a contracting muscle on the 12 cm camera. (ii) Live mu-&es undergoing oscillatory length changes Unstimulated live frog sartorius muscles were made to undergo small, rapid oscillations of length in the following way: a loop at one end of a fine nylon thread fitted over a pin mounted eccentrically on the face of & small disc; the thread passed through a guiding loop of wire and was connected to the tension bar passing into the cell to the unclamped end of the muscle (Haselgrove, 1970). By adjusting the guiding loop to lie on the axis of the muscle, continuous rotation of the wheel by an electric motor produced small oscillatory chs,nges in the length of the muscle. The frequency and amplitude of the oscillations were controlled by the frequency of rotation of the disc and the distance of the pin from its axis. Sarcomere length changes were usually about kO.1 pm; and the oscillations were studied by observing stroboscopically the light diffraction pattern given by the muscle.

Rigor muscles Iodoacetate rigor. Live frog sartorius and semitendinosus muscles were tied firmly at the appropriate length onto strips of Perspex. They were left at 4°C in frog Ringer solution for 24 h and were then transferred to a solution of 1 mM-iodoecetate in frog Ringer solution (pH djusted to pH 7.0 with NaOH) where they were left at 4°C until in rigor. Nwmal rigor. Live muscles, tied at the appropriate length on strips of Perspex, were left to stand in oxygen-free Ringer solution until they were in rigor. Glycerol rigor. Live muscles, tied at the appropriate length onto strips of Perspex, were soaked at 4°C in 8 solution of 60% glyoerol/50°~ 0.013 m-phosphate buffer (pH 7.0) for 24 h: the solution was then changed and the muscle soaked for 24 h more at 4°C before being stored at -20°C for at least 3 weeks. When being used, they were soaked for 0.5 h in 15% glycerol in standard salt before being transferred to the standard salt solution (pH 7.0) in which they were studied. (This is the method of Hanson t Huxley, 1955.) (iii)

(d) Sarcomere

length mecreurement

The sarcomere lengths of muscles were measured in situ on the X-ray camera from the light diffraction pattern as described previously (Haselgrove & Huxley, 1973). Since the samomere lengths of muscles were measured directly, the exact value of rest length, Lo (the ss,rcomere length of the muscle in the living, relaxed animal) is not important for the present work, but for convenience in discussion the following lengths are used: rest length for sartorius muscles, 2.2 pm (this is the sarcomere length et which the actin filaments reach as far &8 the bare zone on the myosin tilaments and all cross-bridges are

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able to reach an actin filament): rest length for semitendinosus muscles 2.7 pm: nonoverlap length, 3.7 pm (the length at which actin and myosin filaments do just not overlap (Page & Huxley, 1963)). When semitendinosus muscles are stretched there is a large

spread in the sarcomere lengths of different fibres: a range of lengths from 3.7 pm to 4.0 pm in the same muscle is common. Therefore, when a muscle was to be studied at non-overlap

lengths,

the centre

region

of the muscle

(the region

through which the X-ray apparatus, and the muscle was only used if no fibres could be found which gave a diffraction pattern from sarcomeres shorter than 3.7 pm. On many muscles the lack of overlap was checked by fixing the muscles in situ in the X-ray cell and examining them in the electron microscope after embedding and sectioning.

beam would pass) was studied in detail with the light

3.

diffraction

Results

(a) General Axial cribed directly helpful Brown do the

diffraction patterns from live and rigor muscles have been studied and desin much detail by Huxley & Brown (1967). Because the present study follows from their work, brief summaries of their findings will be included where it is to do so. During this study most of the measurements made by Huxley and were repeated and all the values given here are the new ones, but in no case two sets of measurements differ significantly. (b) Live resting muscle

(i) Muscles at rest length Axial patterns from a resting sartorius muscle at rest length are shown in Plates I, II(a) and III(a) and axial spacings of the layer lines are given in Table 1. Two series of layer lines are distinguishable in the pattern and arise from the helical structures of the myosin and actin filaments. One series of layer lines indexes with an axial repeat of 429 A (see Plate II(a)) and arises from the myosin cross-bridges ordered into a helical configuration about the myosin filament backbone. Huxley & Brown (1967) interpreted this layer-line series as arising from cross-bridges ordered into a 6, helix with two cross-bridges diametrically opposite each other across the filament every 143 A along its length, and successive pairs rotated by 120” in azimuth. Recently Squire (1972,1973) pointed out that the layer lines could also arise from a four-strand 6, configuration of cross-bridges or a three-strand 9, configuration. The three-stranded 9, configuration also is consistent with the biochemically determined amounts of actin and myosin in the muscle (Tregear t Squire, 1973), and will be assumed to be the actual structure for the rest of this paper. (None of the ideas discussed later is dependent on the myosin structure being a three-stranded g1 helix, they can all be applied equally well to any of the possible structures.) Two layer lines with axial spacings of about 51 A and 59 A have long been known to arise from the actin filaments in the musle (Selby & Bear, 1956). These reflections are diffuse so it is very difficult to measure accurately their separation and thus determine the pitch of the actin helix. Huxley and Brown discussed this problem at length and decided that the pitch is about 2 x 360 A to 2 x 370 A. The actin pitch obtained here from the spacings of 59.5 and 512 A in Table 1 is 367 A which agrees well with Huxley and Brown’s values. Further out from the meridian than the first myosin layer line is a diffuse reflection with an axial spacing of about 400 A (464 A

118

J. C. HASELGROVE TABLE

Axial

1

spacings of low-angle X-ray reJections from relaxed frog sartorius muscles at rest length (A) Recorded olt 35 cm carnew

Meridian (4

214-3 143.4t 129.og 110.7 107.2 91.0 86.9 71++0*17

Myosin layer lines (4

Actin layer lines (4

429.2,t4 216.1 143.6

420*6

Indexing of meridian on 2296 4 (-4 Spacing

Order

(1’3)

143.4 127.6 109.3 104.3 91.8 86.0 71.7

107.6 86.0 71.6

(18)

(21) (22) (25) (27) (32)

69.5 51.2

(B) Recorded on, 2 m camera Meridian (4 600$+20

Indexing

on 2296 A (4

Spacing

Order

629.4 494.0

441.8)) 418.4 394.9s 1 383.0s

382.4

(6)

229.7

229.6

(10)

208.6 191.2

(11)

176.5 163.0

(13)

143.4

(1’3)

366.3 362.2 271.3 237.7

222.6 214.3 i 209.8

187.4 181.9 176.9 163.1 160.8 148.0

Unless otherwise

stated the probable

error on all reflections

(12) (15)

is about

*0.6%.

t The spacing of this reflection was calculated absolutely from measurements using the 2 m samera and using Cu Ka, = 1.5405 A. This reflection was then used to calibrate all other cameras. $ Very broad diffuse reflection. § Thought to be actin reflections. 11Doublets are shown bracketed.

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by Huxley & Brown: 420 A here). It has long been difficult to determine its origin because the spacing is significantly different from both the myosin and actin filament repeats. There now seems general agreement (Huxley & Brown, 1967; Vibert et al., 1972) that this layer line consists of the first layer lines from the myosin and the actin structures overlapping to give one broad peak with its centre between the position of the two constituent peaks. The studies of contracting muscle and rigor muscles (described later) show that the spacing of the 143 A meridional reflection is about 1% greater than in a live resting muscle. This effect might have been explained if the 143 A reflection were a doublet with reflections at about 143 and 145 A: an increase in the relative intensities of the latter when muscles contract would change the spacing of the reflection as seen on a camera which could not resolve them. A detailed study of the meridional pattern of resting sartorius muscles was therefore made using the 2 m camera, and showed no reflection with a spacing of 145 A, but showed that the 143 A reflection is a doublet with a strong reflection at 143.4 A and a much weaker one at 141.4 A (Plate I, Fig. 1, Table 1). Since the strongest reflection is at the longer spacing it is not posaible to explain the increase in spacing when a muscle contracts in terms of changes of intensity of these two reflections. The general appearance of the meridional pattern at very high resolution (Pig. 1) indicates that reflections from the molecular packing in the actin and myosin filaments are sampled by interference functions arising from the separation of the two halves of the actin or mysoin filaments. At present the detailed structure of the meridional pattern has defined complete analysis although the nature of some pairs of reflections

Fm. 1. Densitometer trace along the meridian of diffraction pattern taken with the 2 m camera of a frog sartorius muscle relaxed at rest-length. The reflections at 143.4, 214.3 and 442 A are indicated.

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C.

HASELGROVE

have been explained. Huxley and Brown concluded that the doublet at 383 and 395 w comes from the actin filaments, and Rome (1972aJ973) has shown that the doublet at 442 and 418 d is associated with the recently discovered C-protein in the thick filament (Starr & Offer, 1971; Offer, 1972). Rome suggested that the doublet arises because the principal reflection is sampled due to the separation of the two regions in the myosin filament where C-protein occurs. The pair of reflections at 143.4 and 141.4 A are similar in appearance and separation (about 10,000 A-l) to the pair at 214.3 and 209.8 A. It seems likely that both these pairs of reflections arise for the same reason; namely the main reflections from the cross-bridges ordered on one-half of the myosin filament interfere with the reflections from the cross-bridges on the other half, because the cross-bridge structures on opposite ends of the A filament are not separated axially by a distance that is an integral multiple of the basic cross-bridgr repeat. (For example, calculations with some trial models show that reflections at 143.4, 141.1 and 215.1, 210.2 A can be obtained from two sets of cross-bridges with a centre-centre distance of 9034 A, but sampling of the 400 L%reflection then occurs at 452, 430, 411 A which are not the observed position. It is not possible using a simple sampling system to account simultaneously for all the three sets of doublets at 430,214 and 143 A.) The meridional pattern in the region between 150 A and 50 A has been studied on a 35-cm camera where the resolution is not sufficient to see the fine sampling of the reflections due to the separation of the two halves of the filaments. It is possible to index most of these reflections on a long repeat of 2295 A (Table 1). This repeat is 16 times the cross-bridge repeat of 143.4 A and Squire has suggested recently (Squire, 1971,1972) that long repeats of 8 and 16 subunit-repeats are a common feature of the myosin packing in different muscles. It seems reasonable to assume therefore that these reflections arise from the myosin filament structure, although one of the actin reflections (at 383 A) will also index on this long repeat, and higher orders (192, 127 A, etc.) of this reflection will do so too, so great care must be taken when identifying the source of reflections solely by their indexing. Very faintly visible on patterns of rest-length sartorius muscles is a weak meridional reflection with a spacing of 129 & This reflection also indexes on the long myosin repeat of 2295 A, and its intensity depends very much on the state of the muscle, being clearly visible in stretched and in rigor muscles. It was earlier thought (Haselgrove, 1970) that this is a myosin reflection but it is now thought to be an actin reflection and will be discussed later. Finally, a very broad diffuse reflection with a spacing of about 600 A is clearly visible on the meridian of patterns taken with a 2 m camera (Plate I). The axial and radial widths of the reflection are about 200 A and 500 A, respectively and are much wider than the meridional reflections arising from the actin or myosin filaments. The origin of the reflection remains unknown. Frog semitendinosus muscles were studied at rest length (about 2.7 pm) to find out whether the pattern was the same as that from sartorius muscles at rest length. The patterns are identical to the resolution of the 35 cm cameras on which the patterns were taken. (ii) Live muscles stretched above their resting length Before it is possible to interpret the changes in the diffraction patterns when muscles contract or go into rigor at different lengths, it is necessary to know what

PLATES

I--\‘1

I

.1II thn ?(-ray patterns have been reproduced by a masking technique similar to that drsc~~ibrrcl by Tibert et trl. (1973) and no “shading” was used in the printing stages, so the layer lines of one photograph hare visually the correct relative intensity. and patterns which are to be compared with each ot,her have been treat,4 identically during processing. In all pattnms the vertical axis i* the meridian which is the same tlirection as the axis of the mnscl~~.

1438 -

A M

la&-

PLATE

I

layer lines (b) Pettl graphed SC but the rn~

143

-

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changes (if any) occur when the resting live muscle changes length. If, for example, the cross-bridges in the H-zone of a stretched, resting muscle were always disordered, then only those cross-bridges in the region where actin and myosin overlap would give rise to the layer-line pattern; so a study of the change in this pattern at different sarcomere lengths would give no information about the cross-bridges uninfluenced by actin in the H-zone. Huxley and Brown found that the intensity distribution of the layer lines remained unchanged when a sartorius muscle is stretched by up to 2076 (i.e. up to a sarcomere length of about 2.7 pm) but they did not study the absolute intensity of the reflection and thus investigate the number of cross-bridges ordered enough to contribute to the pattern. This problem was therefore investigated in detail and the X-ray patterns of live resting muscles were studied at all lengths at which contracting or rigor muscles were studied: sartorius muscles were stretched up to 3.0 pm (the longest possible before they broke) and semitendinosus muscles were stretched until all fibres in the region studied had sarcomere lengths of 3.7 pm or longer, so that overlap of actin and myosin filaments was abolished. One problem in comparing the absolute intensity of a given reflection at different sarcomere lengths is that when a muscle is stretched, the amount of material in the X-ray beam decreases by an unknown amount, and this effect alone makes the whole diffraction pattern weaker. If the X-ray beam crosssection is smaller than the muscle and if the muscle becomes thinner uniformly as it is stretched, then the intensity of any given reflection due to this effect alone should bc proportional to l/d s, where s is the sarcomere length of the muscle. As a sartorius muscle is stretched up to 3-O pm the intensity of the 59 A actin layer line decreases in such a way that its intensity is proportional to l/v’s as predicted above (Fig. 2). It was therefore assumed that the structure of the actin filament is independent of the sarcomere length of the muscle, and the 59 L%layer line was used as a scale value when comparing the myosin reflections. In contrast to the behaviour of the actin layer lines it was surprising to find that if a muscle is stretched soon after dissection the intensity of the myosin reflections decreased significantly with respect to the actin reflections. The behaviour of the myosin reflections varied considerably from muscle to muscle but seemed to depend on the earlier treatment of the muscle. Three situations were studied: (1) stretching a sartorius muscle within a few hours of dissection; (2) leaving the dissected sartorius muscle at rest length in frog Ringer solution for 24 hours before stretching it, and (3) stretching semitendinosus muscles to sarcomere lengths beyond 3.7 pm under conditions which will be described later. In order that patterns of sartorius muscles could be recorded in times much shorter than the periods that the muscles were equilibrated and to enable several patterns to be recorded from one muscle, the sartorius muscles were studied with the 12 cm camera which would record a pattern in less than half an hour, but which had relatively poor resolution. Patterns from the semitendinosus muscles on the other hand were recorded on a 35 cm camera using exposure times of several hours. (A) Sartorius muscles stretched soon after dissection. Sartorius muscles could be stretched to about 2.6 pm (as was done by Huxley & Brown) with no changes in the pattern other than the expected general slight decrease in the absolute intensity. On further stretching to a sarcomere length of about 3.0 pm the intensity of the actin layer lines continued to decrease slowly by the expected amount (Fig. 2) while the intensity of the myosin reflections decreased considerably more. If the intensity of

122

J. C’. HASELGROVE

(b)

60 r,

40 20 0

22

4-g--.+%.-,. I I 2.4 2.6 Scrcomere

,--0-oI I 2-0 3.0

length (pm)

FIG. 2. Intensity measurements of the reflections in the diffraction patterns from one musale at different sarcomere lengths. The exposure time for all patterns w&s the same. (0) Musale Ttretched between successive points; (0) muscle shortened between successive points. (a) Meridional relkction at 143 A, measured on the seoond film. (b) (O), (a), Myosin 429 A layer line. (0) ( n ), Actin layer line at 59 A. The solid lines represent the relation Ial/z/s whioh would be expected if the only effect of stretching is that the muscle becomes thinner.

the myosin reflections at each sarcomere length are scaled by the intensity of the actin 59 A layer line to correct for changes in the muscle thickness, then at 2.7 pm the myosin layer-line intensity is 100% of that at 2.2 pm but an increase in saroomere length of only O-35 to 3.05 pm causes the intensity to drop to 60%. Stretching the muscle has far more effect on the meridional reflections than on layer lines, for at 3.05 pm the 143 A meridional reflection is only 25% as strong (relative to the actin layer line) as it is at 2.2 pm. The meridional reflections near 107 A and 86 A often disappeared completely when the muscle was stretched to 3-O pm although this disappearance may have been due to the relatively poor resolution ofthe 12 cm camera. Although the meridional reflections became broader across the meridian as the muscle was stretched, there was never any indication of broadening along the meridian. These changes in the pattern when a muscle was stretched were not permanent for they could be reversed either by allowing the muscle to shorten again or by holding the muscle stretched for a day or two. When a stretched muscle with weak myosin reflections was allowed to shorten to rest length again the layer-line reflections reverted to their original intensity (Fig. 2), indicating that the regular helical ordering of the myosin cross-bridges had not been destroyed permanently by stretching. The meridional reflections on the other hand, did not recover immediately when the muscle was

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123

shortened. If, instead of being allowed to shorten, the muscle was held stretched for 20 to 30 hours, the intensity of all the myosin layer lines and meridional reflections increased, while the absolute intensity of the actin reflections remained constant, so that the diffraction pattern reverted to its original appearance at rest length (Plate III). Consider now the structural changes in the muscle that could give rise to such changes in intensity of the meridional reflections. First, because the meridian is the transform of the projection of the muscle structure onto the muscle axis, a longitudinal disorder of the filament lattice will smear out the significant periodic changes in electron density and so greatly decrease the intensity of the meridional reflections: lateral disordering of the filament lattice is also known to occur in stretched muscles (Haselgrove & Huxley, 1973) and will also cause a decrease in the meridional reflections because the lattice sampling will be decreased. Second, a random axial movement of the cross-bridges from their resting positions on the myosin filaments would cause a decrease in the intensity of the meridional reflections and also of the layer lines-as indeed happens. But when the muscle is shortened, the layer lines return immediately to their original intensity (indicating that the cross-bridges are back in their original positions) but the meridional reflections remain weak (Fig. 2). This suggests that the movement of cross-bridges along the filaments is not the sole reason for the drop in intensity of the meridional reflections, and it is therefore thought that the large (persistent) drop in the intensity of the meridional reflections is due mainly to disordering of the filament lattice. A third effect could also change the intensity of the meridional reflections with very little change in the layer-line pattern : tilting the muscle by a few degrees so that the muscle axis is not perpendicular to the X-ray beam will cause the very sharp meridional reflections to move off the Ewald sphere and hence change the observed intensity, while the layer lines are not similarly affected. (However, the geometry of the X-ray cameras makes it most unlikely that this effect is occurring here.) Thus, all these different changes can affect the intensity of the meridional reflections. Since the present experiments do not allow an unambiguous interpretation of which effects are occurring, then throughout the rest of this paper the changes in intensity of the meridional reflections will be reported as they were observed but no interpretation attempted. Of the structural changes described above the only one which will cause a significant change in the observed intensity of the layer lines is a movement of the cross-bridges from their well ordered resting position around the myosin filaments. The question then to be answered is : which cross-bridges are moving and what sort of movement is occurring? The effect of stretch on the pattern cannot be explained by the assumption that all the cross-bridges undergo small random movements near their resting positions, for the effect of such movements would be that the layer lines further from the origin would decrease in intensity much more than the layer lines close to the origin (the crystallographic temperature effect). Measurement of the patterns shows that all the layer lines decrease by equivalent amounts, so uniform loss of intensity of the myosin layer lines can only be explained by large movements of some of the crossbridges away from their resting positions to positions where they no longer contribute to the layer-line pattern. The ability to stretch muscles regularly to about 2.6 pm before any change in the pattern occurs, indicates that there is no direct dependence of the intensity on the number of cross-bridges in the actin-myosin overlap region, since at 2.6 pm only about 70% of the cross-bridges are in this region yet the pattern is still

124

,J. C. HARELGROVE

relatively as strong as a rest length pattern. Thus, for short stretches at least, the cross-bridges in the H-zone of a muscle are as well ordered as the bridges in the overlap zone. The possibility cannot be completely excluded that for longer stretches, the cross-bridges lying in the centre portion of the H-zone become disordered while all the others remain well ordered, but such a long range influence of the actin filament on the myosin filaments seems most unlikely. Thus there is no direct evidence showing which cross-bridges move, but it seems that the ones that do move are not all in the II-zone. This problem will be considered further in the Discussion. (B) Sartorius muscle stretched 24 hours after dissection. The experiments with stretched muscle described above show that time can have a large effect on the structural organization of a stretched muscle, so the effect of leaving a dissected muscle unstretched overnight was investigated to see if the muscle could then be stretched without affecting the diffraction pattern. Most of the muscles that were left for 24 hours before stretching could indeed then be stretched to a sarcomere length of about 3-O pm with no change in the diffraction pattern other than the expected overall slight decrease in the intensity, and a slight broadening of the meridional reflections. When the muscle was then held fixed for a further day the meridional reflections became sharper until the diffraction pattern was indistinguishable from that of a muscle at rest length. Some muscles, however, when left for a day before stretching, still responded like a muscle that was stretched soon after dissection, i.e. the myosin pattern decreased in intensity far more than the slight decrease in the actin pattern. Huxley (1972) has also obtained somewhat variable results when stretching semitendinosus muscles which had equilibrated for some time at rest length, and he found that using a dilute Ringer solution helped him to obtain strong patterns from stretched muscles. The ability to stretch muscles without affecting the diffraction pattern indicates that neither withdrawal of the actin filaments from the myosin filament lattice nor a decrease in the separation of filaments directly cause the crossbridges to move in muscles that are affected by stretching. The axial spacings of the actin and myosin reflections were measured from the patterns of sartorius muscles stretched soon after dissection, and of those stretched after a waiting period. Within the accuracy of measurement of spacings from patterns recorded with the short 12 cm camera there is no change in the spacings of any of the reflections upon stretching the muscle. (C) Semitendinosus muscles stretched to non-overlap length. The experiments with stretched sartorius muscles indicated that although changes can occur in the pattern when a muscle is stretched the changes are not permanent, and that a stretched muscle can give a diffraction pattern identical to that of a rest length muscle. Sartorius muscles however cannot be stretched up to or beyond non-overlap length and it was of importance to see if the myosin cross-bridge arrangement was totally destroyed when the actin filaments were completely removed from the myosin filament lattice. Stretched semitendinosus muscles were therefore studied using a 35 cm camera (which takes about 6 h to record a pattern) since the object was to observe the presence or absence of the myosin layer-line pattern rather than observe the time course of any changes. When semitendinosus muscles were stretched within about six hours of dissection to sarcomere lengths about 3.7 pm so that the actin and myosin filaments no longer overlapped (Plate IV), the diffraction patterns were

STRUCTURAL

CHANGES

IN

MYOSIN

FILAMENTS

12.5

remarkably similar in general appearance to those of the rest-length muscles, showing both the myosin and the actin series of layer lines (Plate II). The relative intensity of the myosin and actin layer lines was less than in a rest length muscle, but this is probably because the muscles were stretched and photographed soon after dissection. Indeed, the large decrease in intensity of the meridional reflections is similar to the behaviour of reflections from sartorii stretched soon after dissection. Huxley (1972) observed that strong myosin patterns could be obtained from such stretched semitendmosus muscles by waiting 24 hours before or after stretching. These patterns support the conclusions drawn from the study of stretched sartorius muscles, namely that cross-bridges in the region of the myosin filament not overlapped by actin can, and do, give rise to the same set of layer lines as cross-bridges in the overlap region and that therefore the cross-bridges are arranged helically along the whole of the myosin filament. Three points about the patterns from stretched semitendinosus muscles are of interest and an interpretation is considered in the discussion. First, the meridional reflection at 128 A is seen more clearly than in patterns from live muscles at rest length, although it is not as strong as from muscles in rigor. Second, the spacings of the principal meridional reflections (72-l A and 1438 A) are greater than the spacings in a live muscle at rest length, but not as great as in rigor muscles (Tables 1 and 3). Third, the meridional reflections 86 and 110 A are very weak. (iii) Oscillating fnm8Ch It was suggested by Carlson (see the Discussion to Huxley, 1967) that the movement of cross-bridges when a muscle contracts may be brought about by shear forces between the actin and myosin filaments when the sarcomere undergoes small rapid fluctuations in length, as reported later by Larson et al. (1968). (But, Cleworth t Edman (1972) have been unable to detect any such “dithering” of sarcomere lengths when single fibres contract.) Short, rapid amplitude fluctuations similar to those found by Larson et al. (1968) were imposed on live, unstimulated muscles. The diffraction patterns of such muscles oscillating at lengths slightly larger than rest length with an amplitude of O-1 p (i.e. moving 0.1 pm either side of the mean value) and frequencies of l/3, 8 or 25 Hz were indistinguishable from the pattern from the resting muscle (Plate V). Muscles to be studied during oscillation at lengths greater than about 2.7 pm were first left at rest length for 24 hours after dissection and then photographed before and after being stretched : a muscle was used only if the relat,ive intensity of the act.in and myosin layer lines did not change upon stretch.! The diffraction patterns from these stretched muscles during oscillation were usually slightly weaker and less well ordered than when at rest at the mean stretched length, but since the actin and the myosin layer lines were affected similarly, it is thought that the result is an experimental artefact due perhaps to movement of the whole muscle, rather than due to molecular disorder of the filaments themselves. Therefore, in an inactivated muscle at least, a “dithering” of the sarcomere length has no effect on the ordering of the cross-bridges around the myosin filament. It is worthy of note that a muscle oscillating with an amplitude of O-1 pm and a frequency of 25 Hz will be shortening due to its own elasticity with a maximum velocity of over 15 pm/second which is about 7 L,lsecond and is close to the velocity of unloaded shortening of a muscle during contraction (Hill, 1938).

126

J. C. HASELGROVE

(iv) CcmclzGsionsfrom patterns of resting muscles The experiments on relaxed live muscles indicate that the ordered helical arrangement of cross-bridges along each myosin filament is probably an intrinsic feature of relaxed muscles, and that any changes are only temporary. The factors causing the changes that occur are not yet known. The actin layer lines in the diffraction patterns indicate that the helical arrangement of actin monomers within the filament is less affected by various treatments of the relaxed muscle than is the myosin filament structure. Since the diffraction pattern of live relaxed muscles does not depend on the sarcomere length of the muscle, then by studying the diffraction patterns of muscles contracting or going into rigor at different lengths one can determine which of the changes in the pattern are due to interaction of the myosin cross-bridges and actin filaments. The changes that are due to this interaction will be smaller at long sarcomere lengths where the number of actin-myosiu interactions is reduced, while changes independent of t,he interaction (i.e. charges in the non-overlapped crossbridges) will be the same at any sarcomere length. (c) Contracting

muscles

Diffraction patterns were recorded from sartorius muscles contracting isometrically at different lengths between 22 pm (where the actin filaments overlap all the crossbridges on the myosin filaments) and 3.0 pm (where the actin filaments overlap just less than half of the cross-bridges). Most muscles were studied at the longer lengths, and the results were compared with the results from rest-length muscles studied by Huxley & Brown (1967). The comparison showed that in the range of sarcomere lengths studied, the diffraction pattern of an isometrically contracting muscle is independent of the sarcomere length of the muscle: the myosin layer-line pattern is much weaker than for the resting muscles (Plate VI), the actin pattern about the same as in the resting pattern and the meridional reflections at 72 d and 143 A show a small but significant increase in spacing. (i) Myosin layer lines The change in intensity of the myosin layer lines when a muscle contracts was studied by comparing the intensity of the 429 d first layer line in the contracting muscle (I,) with intensity of the layer line in the same mucle at rest (Is). I, was taken as the mean of the value from the relaxed muscle before and after the contraction exposure. The mean value of the ratio IO/I, of muscles at rest length as found by Huxley and Brown is 31% & 6% (Table 2) and is the same as the value of 31 y0 f 8% S.D. found for muscles contracting at sarcomere lengths near 2.9 pm where only half the cross-bridges lie in the vicinity of actin filaments. It can be seen clearly from Figure 3 that for four (out of 20) of the muscles studied at the longer length, the myosin layer-line pattern decreased in intensity less than for the other muscles. Even at rest length (Huxley & Brown) there is a considerable scatter of results, and Huxley (1972) had noted a similar large variation in results from stretched semitendinosus muscles. It is experimentally very difficult to obtain diffraction patterns from stretched muscles during contraction, and the scatter of results may be attributed to variations between different specimens and experiments. Nevertheless the following two parameters which might affect the pattern were both investigated but neither of them showed any correlation with the decrease of the myosin layer line during contraction.

STRUCTURAL

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127

FILAMENTS

TABLE 2

Changes in diffraction (A) Intensity

pattern

when muscles contract I contracting/1

chmgw

resting

Muscle length 2.25 pmt

Meridian Layer lines

0~66~0~10

143 12 429 from meridian) from meridian)

d contracting/d

(B) Spacing changes

2.9jO.l Meridiond

0.61~0.18 0*92-&0.22 0~31~0*08 (0.27$*0-07) 0.61hO.14 1:27-j=O.21 1.09*0.15

0.31&0.06

400 59 (l/180 A-l 59 (l/100 A-i

pm

2.9&0*1 pm

resting

Muscle length 2.2550.1 pm

> 3.7 pm$

reflections

143 Ad contracting/d resting before experiment 143 Ad contrecting/d resting after experiment 72 A d contracting/d resting before experiment 72 A d contracting/d resting after experiment Only one muscle w&s ever found footnote 11).

for which

1~011*0~004 1.009*0.006

cl contracting/d

1.010+0.006

1~003+0~005~~

1~011&0~006

1.006&-0.004

1.011*0~004 1~011*0~004

1.006 + 0.004 1.006&0.003

resting

was less than

1.000 (see

t Values of intensity changes at 2.26 F taken from Huxley 8s Brown (1967). $ Velues if the 4 snomalous points ere excluded from analysis. 3 Huxley’s 13 “best” non-overlap petterns. I/ Although the change is smaller than c (root-mean-square of deviation from mean) only one value of 13 w&s less than 1000.

(A) Time at which muscle was stretched. Some muscles were kept at rest for 24 hours before being stretched, and diffraction patterns taken to check that no change of the resting pattern had occurred upon stretching. (Muscles that had equilibrated for 24 hours before use in a contraction experiment fatigued far faster than fresh muscles and were more difficult to keep contracting with high tension for long periods.) The results from these muscles during contraction are shown in Figure 3 as filled circles where it can be seen that these carefully controlled muscles show the same variation of the ratio I,/I, as is shown by all the muscles. It is unlikely therefore that differences in treatment of the muscle for stretching give rise to differences in the behaviour of the diffraction pattern during contraction. (B) Mean tension generated by the muscles. Since the tension generated by the muscle during each contraction decreased during the period of X-ray exposure it is

128

J. C. HASELGROVE 100

.__-.__

-----+i--Ad

20

2.2

24

2.6

Sarcomere

3.2

length

(pm)

Pm. 3. Ratio of the intensity of the first myosin layer line from a muscle during isometric contra&ion (I,) and relaxed (ZR), plotted against the sarcomere length of the muscle. (0) No speoial preoautions taken when stretching muscle to experimental length. (0) Muscles which were left to rest for 24 hours before stretohing and for which the layer line intensity was not affected by stretching. ( n ) Mean value of Z,/ZR found by Huxley & Brown (1967). Solid line represents the standard deviation of their results.

possible that more and more of the fibres are remaining relaxed during each contraction, and that these relaxed fibres are giving a typical relaxed diffraction pattern with strong layer lines. Such a possibility was checked by comparing the ratio IJI, with the mean tension generated by the muscle during the exposure, expressed as a fraction of the maximum tension (P/T,). Although the muscles discussed above had values of ?‘t/To above 70%, Figure 4 shows the layer-lines intensity ratio I,/I, plotted against the mean tension p/T,, including data covering a large range of p/T,,. The data points show no correlation with the tension. Therefore it is concluded that the four very high values of I,/I, are anomalous results which cannot yet be explained, and that decreases in intensity of the myosin

0

L-I

20

40

60

00

100

% T/ To

FIG. 4. Intensity retio Z,/ZR of the myosin layer line plotted against the ratio F/T,, which is the average tension produced by the musole expressed as a freotion of the peak tension.

STRUCTURAL

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129

layer lines (and therefore the number of cross-bridges that move during contraction) do not depend on the number of cross-bridges that are adjacent to actin filaments. Since all the myosin layer lines decrease by the same fraction it can be concluded by the same arguments used earlier for stretched muscles, that the decrease results from many of the cross-bridges moving away from their resting positions until they no longer contribute to the layer-line pattern, while others remain close to their resting positions. No change in the spacing of the layer lines was found but the only layer line strong enough to measure at all accurately wm that at 429 A, and the accuracy of measurement of the spacing of this reflection is not thought to be better than 65%. (ii) Actin layer lines Little change was found in the intensity or spacings of the 59 A actin layer line when muscles contract. Measurements (Table 2) of the intensity at two positions along the layer line indicate that there is a slight increase in intensity and that the increase of the meridional end of the layer line (27% f 21% S.D.) may be greater than the increase further from the meridian (9% f 15% S.D.). Haselgrove (1970) and Parry & Squire (1973) have interpreted these slight increases as being due to cross-bridges attaching to actin in the contracting muscle, but the measured increases in intensity may well arise (at least in part) from better ordering of the layer line during contraction, and the very large standard deviations of the measurements allows little weight to be placed on the changes recorded. The “400 A” reflection (which lies further from the meridian than the principle myosin layer lines) decreases in intensity when the muscle contracts (Table 2) but the mean decrease to 61 o/ois less than the decrease to 319/o of the myosin layer lines. This is to be expected if the myosin component of the 400 A layer line decreases during contraction but the actin did not. No significant change in spacing of this layer line was found but the accuracy of measurement on the small films of such a diffuse layer line was very low. (iii) Meridional reJections Using the very fast 12 cm camera needed to photograph contracting muscles only four reflections are resolvable on the meridian, namely the 3rd, 4th, 5th and 6th orders of 429 A (with spacings of 143 A, 107 A, 86 A and 72 8). During contraction of stretched and unstretched muscles they all decrease significantly in intensity: the 86 A and 107 A reflections become so weak that accurate intensity or spacing measurements are not possible. As was found with the layer lines, the decrease in intensity of the 143 A and 72 A meridional reflections during contraction varied considerably from muscle to muscle : at sarcomere lengths where only about half of the cross-bridges are overlapped by actin, the mean decrease of the 143 A reflection to 61% (&lS% s.D.) and the 72 A re%ection to 92% (&220,/, S.D.) are both significantly less than the decrease to 31% of the layer lines, but the mean decrease of the 143 A reflection is the same as that of muscles contracting at rest length found by Huxley and Brown (I,/I, = 66% rt 10%). The reason that the 72 A reflection decreases less than the 143 A reflection may well be that the backbone of the filaments contributes signi%cantly to the meridional pattern (Szent-Gyorgyi et al., 1960). The spacings of the sharp meridional reflection at 143 A and 72 A were measured as accurately as possible, and to avoid errors of measurement of specimen-to-film distance the spacing of the reflections of each contracting muscle was compared with the 9

130

J. C. HASELGROVE

spacings of that muscle at rest both before and after the contraction experiment. For muscles both at rest length and stretched to about half overlap length the spacing of the 143 A and 72 A meridional reflections from contracting muscles are about 1 y0 (-&O-5%) longer than the spacings from the relaxed muscles (Table 2) ; although the spacing change is small, no case was found in over 40 muscles where the spacings appeared to decrease during contraction. Taking the spacing of the relaxed muscle as 143.4 A the spacing of the contracting muscle is therefore 144.8 A and must have arisen by a small structural change of the myosin filaments.t Here too both reflections increase slightly in spacing when the muscle contracts by about 0.5% (Table 2) although the myosin filaments are withdrawn from the influence of the actin. The changes are slightly smaller than those of shorter muscles, possibly because slight changes in spacing had already occurred when the muscles were stretched: a similar effect is seen in the change in spacing when muscles pass into rigor at non-overlap lengths. (iv) Rigor-like rejlections It was hoped that the patterns of contracting muscles might have shown some of the layer lines which occur in rigor muscle when the myosin cross-bridges attach to the actin filaments. No such reflections could be seen in patterns from muscles at rest length although it is estimated that they would have been visible if they were present with about 25% of the intensity that they occur in rigor muscles. Therefore, if such layer lines were present they were very weak, and since the intensity of the X-ray reflection is proportional to the square of the number of diffraction units, then fewer than 50% of the cross-bridges in the contracting muscle can be instantaneously attached to the actin filaments in the rigor configuration. (d) Rigor muscles Diffraction patterns from muscles in rigor were studied, both at rest lengths where actin-myosin interaction can occur freely and at non-overlap lengths where actinmyosin interaction is prevented. As found by Huxley (1967) and Huxley & Brown (1967) the resting myosin layer-line pattern disappears completely at whatever length the muscle is put into rigor, and at rest length, new layer lines appear due to interaction of the myosin cross-bridges and the actin filament. As in contracting muscle, the reflections at 143 A and 72 A increase in spacing by 1 o/oat all sarcomere lengths. (i) Mu&es at rest length (A) Layer lines. The characteristics of the layer lines in rest length muscle have not yet been accounted for in detail although there is little doubt that they arise from the structure formed when cross-bridges attach to the actin filament (Huxley & Brown, 1967 ; Huxley, 1967). A visual examination of the pattern suggests initially that there are two series of reflections: one series lies close to the meridian and is sharp and intense (Plate VII) and has spacings which index approximately as orders of a repeat of 725 A. The other layer lines lie further from the meridian and are much weaker and more diffuse (like the outer parts of the layer lines at 59 and 51 A), so that accurate measurement of their spacings is impossible, but the values for their spacings that have been t H. E. Huxley kindly allowed me to measure the spacings of the 143 and 72 A reflections his X-ray patterns from muscles contraoting at non-overlap lengths (Huxley, 1972).

in

STRUCTURAL

CHANGES

IN

MYOSIN

FILAMENTS

131

TABLET Axial spmhg of bw-an& x-ray re~ectims from muscles in rigor recorded on a 35 cm camera (A) Satioriwr

(B) Semitendinoeus

muaclsa a.t rest length Layer

Meridian (4

Meridian (-‘Q

lines (4

366t 241.8

Layer

lines (A)

3663

22711 212 192 144.6 127.6

muaclea above 3.7 p

21711 186.4 144.6

191 144.8 128.7

186

120.25 109.8

110.8 101.8 87.8

72.3

72.3 69.6 69.06 60.96 Messurements

of meridional

reflections

69.1 61.1

accurate to &O-S%. Layer lines accurate to about + 1 ye.

t f: The layer lines are laid out in 2 columns to represent the distance from the meridian that the layer line appears (see Plate VII). s The ls,yer line at 120 il is so d&&se that it probably consists of 2 layer lines with spacings above and below 120 A. 11The reflections rtt 227 and 212 A are usally not resolved and appear &s one refleotion at 220 A. The great similarity of the meridional reflections at rest length and non-overlap length suggests that the reflection at 217 A might also be an unresolved doublet.

measured (Table 3) are consistent with the spacings expected from an actin-like helix which has a helix repeat of 725 A (Vibert et al., 1972). Indeed, the radial positions of these layer lines can be approximated by the diffraction pattern expected from spheres arranged in an a&in-like helix at a radius of 60 to 80 A, suggesting that they arise from cross-bridges attached to the actin %lament: a study is in progress to interpret the rigor diffraction patkern using more realistic models of the cross-bridges than simple spheres. Studies are in progress to interpret the pattern in detail and will be reported elsewhere. (B) ikteridional rejkctions. When a muscle passes into rigor the meridional reflections at 86 A, 91 A and IO7 A disappear, leaving the reflections at 143 A and 72 A as the most prominent meridional reflections (Plate VII). Because a similar change occurs when muscles contract, the rigor muscles were studied to see if here too (as during contraction) there is a change in spacing of the 143 and 72 A reflections. Fortunately, long exposure times were possible with the rigor muscles so a 35 cm camera was used, with a much better spatial resolution than with the 12 cm camera used for contracting muscles. The results from over 30 muscles (Table 4) shows that there is indeed a small but significant increase of about 1% of the spacing of the two principal refiections

132

J. C. HASELGROVE TABLE

Changes in spcing

of meridional

4 rejlections when muscles go into

iodoacetate-rigor at rest length

(4 143 72

d rigor/d resting live 1~0086&0~002t 1.011 10.003

(17)$ (14)

No case was found in which the spacing appeared to decrease at the onset of rigor. t Standard deviation. 3 No. of measurements.

when a muscle passes into rigor, so that the cross-bridge spacing in a rigor muscle is 144.6 A. Within the experimental errors, the increase in spacing is the same as that which occurs when a muscle contracts, and since the changes in intensity of other meridional reflections are so similar during contraction and in rigor it seems that the same, or very similar structural changes are occurring. Weaker reflections are also visible on the meridian of rigor muscles, at, spacings of 101~8,110~8,127~5, 192,212 and 227 A. All these reflections and those at 144 and 72 A can be indexed on a long repeat of 2315 A which is about, 1 o/ogreater than the repeat of 2295 A in live muscle. 2315 A is about twice the spacing of 1150 .k common to all myosin structures (Squire, 1973) and is also six tjmes the tropomyosin-troponin repeat of 385 8. It is thus difficult to assign all reflections unambiguously to either the actin or the myosin structures, but some of the reflections can be assigned, and the two at 127 and 192 A are thought to arise from actin for the following reasons. (i) They index directly as the 2nd and 3rd order of the troponin repeat, of 385 8. (ii) In some patterns of muscles in glycerol rigor at non-overlap the whole pattern is sometimes very disordered; in these patterns the 143 A reflection is distinctly arced while the 127 ..&reflection is still relatively straight, indicating that the 127 A and 143 A reflections come from different structures. The presence of the 127 A reflections in patterns from such disordered muscles shows that the reflection arises from the individual Laments themselves rather than from a lattice arrangement of actin filaments as suggested by Miller & Tregear (1973) for insect muscle. Since no myosin cross-bridges are attached in the non-overlap muscle the change in intensity of the 127 A reflection is probably generated by the activation of the thin filament in a similar way to the changes in the actin layer lines (Haselgrove, 1972). (iii) Muscles which were put into normal rigor by leaving them to die in an oxygen-free Ringer solution gave diffraction patterns which were the same as those from iodoacetate rigor muscles except that the 127 A and 192 A reflections were often much weaker relative to the 143 A reflection. The intensity of the 192 A reflection followed that of the 127 d reflection indicating that they both arise from the same source; which is thought not to be myosin because the 143 A was still strong in patterns where the 127 and 192 A reflections were barely visible. The reason for these changes in intensit,y of the 127 and 192 A reflections is unknown but work is in progress to see if they are associated with changes in the 385 A meridional reflection, and to find their origin. (iv) Reflections at 192 A and 172 di have been seen in patterns from oriented gels of thin filaments (Hanson et al., 1972).

STRUCTURAL

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FILAMENTS

133

It is important to note the similarities between the frog striated muscle in rigor and insect muscle in rigor (see Miller & Tregear, 1972). The X-ray diffraction patterns both from live and from rigor insect muscles have reflections at 1445 A and 72.2 A which are the same in spacings as for the rigor frog muscle but 1% greater than the relaxed frog muscle. Furthermore, the insect X-ray pattern also shows a reflection at 128 A and a meridional reflection at about 193 A is seen in patterns of insect (lethocerus) flight muscle taken by H. E. Huxley (although Roger (1973) says that the reflection is absent from patterns from lethocerus flight muscles). There is no report of a reflection at 220 A in the X-ray patterns from insect flight muscle, but Reedy (1967,1968) has obtained light diffraction patterns from electron micrographs of insect muscle in rigor which show reflections at spacings of 228 A, 190 A, 143 A and 127 A, all of which are seen in the X-ray patterns of frog rigor muscles. Thus there is remarkable similarity between the meridional diffraction pattern of insect muscle and rigor frog muscle which probably reflects an underlying similarity of the filament structures. (ii) Muscles stretched beyond overlap length Muscles which were stretched to non-overlap lengths so that actin-myosin interaction was prevented (Plate IV) and then treated by techniques known to induce rigor in rest length muscles, underwent changes similar to that of muscles at rest length viz., they became white, opaque and rigid, while the X-ray diffraction pattern contained no sign of the resting myosin layer-line pattern. Therefore, these stretched muscles were considered to be in a state of rigor but with no cross-bridges attached to actin, although rigor is usually thought of as being that state in which the crossbridges are all attached to actin to form an extensively cross-linked and rigid structure. By comparing the diffraction patterns from muscles in rigor at rest length and at non-overlap lengths it is possible to determine which features of the pattern depend on the interaction of the myosin and actin, and which features are independent of such interaction. The most striking feature of the X-ray diffraction pattern from these stretched rigor muscles is that the resting cross-bridge layer-line pattern has completely disappeared (Plate VII) showing that large movements of the cross-bridges have occurred, but no new series of layer lines have appeared as they do in rest length muscles. It can be seen therefore that the initial movement of the cross-bridges from their resting positions when a muscle passes into rigor is not due to the influence of the actin filaments since the movement occurs whether or not actin filaments are present between the myosin filaments. On the other hand the final positions of the cross-bridges are affected by the presence of actin filaments for at non-overlap lengths the crossbridges are unable to interact with actin, and this apparently results in their occupying random positions without forming a new regular structure. At rest length, actinmyosin interaction is possible and the cross-bridges attach to the actin filaments to form a regular a&in-like structure, which, as suggested by Huxley & Brown (1967) gives rise to a series of new layer lines. The two actin layer lines at 59 and 51 A are also present in the patterns from muscles in rigor at non-overlap lengths, and as would be expected if no cross-bridges attach to actin, the radial positions of the layer lines are no different than for live muscles. The meridional pattern from a non-overlap muscle in rigor is the same as the meridional pattern from a rest length muscle in rigor. The reflections at 86 A, 91 A

134

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and 107 A in the live pattern have disappeared completely and reflections are now visible at spacings of 217, 191, 144.8, 128.7, 109.8 and 72.3; that at 144.8 is by far the strongest. No reflection was seen at 101 A as from rest length muscles but t,his is probably because the reflection is very weak. Careful measurements of the spacings of the 144 A and 72 A reflections show that they are the same as for sartorius muscles in rigor at rest length, i.e. 1% larger than live muscles at rest length. (An analysis of the percentage increase in spacings of these two reflections when a live, stretched muscle passes into rigor, shows a smaller increase (0.6% and 0.2%) than occurs at rest length, but this is because the spacing had already increased very slightly when the live muscles were stretched soon after dissection.) The differences between the meridional patterns of live and rigor stretched muscles indicates that, as at rest length, there has been a change of conformation of the myosin structure, while the similarity of the meridional pattern from rigor muscles at rest length and non-overlap indicates that the rigor structure of the backbone is the same at both lengths. This rigor-type structure is therefore independent of the ability of the actin and myosin filaments to interact.

4. Discussion (a) Movement of cross-bridges The data presented here show that the diffraction patterns from muscles contracting isometrically at sarcomere lengths between 2.0 and 3.0 pm are independent of the sarcomere length of the muscle. Compared with the pattern from resting muscles the most obvious feature of the “contracting” pattern is the large uniform decrease of all the myosin layer lines indicating that a large proportion of the cross-bridges have moved significant distances from their resting positions. At sarcomere lengths of about 3.0 pm, fewer than half of the cross-bridges are in the region of overlap of actin and myosin filaments and are able to interact with actin, yet the patterns from such stretched muscles are identical with those from rest length muscles (where all crossbridges can interact with actin), so the same proportion of cross-bridges move in each case. The control patterns from stretched muscles at rest indicate that the crossbridges in a resting muscle are ordered similarly in the H-zone and the overlap region, so it is evident that the initial movement of cross-bridges away from their resting positions is not dependent on the presence of actin. Huxley (1972) succeeded in obtaining diffraction patterns from muscles contracting at sarcomere lengths above 3.7 pm so that no cross-bridges should have been able to interact with actin, and showed that even at such long sarcomere lengths the movement of individual cross-bridges is not directly dependent on the influence of actin. The same effect occurs when muscles pass into rigor. The diffraction patterns from muscles in rigor at sarcomere lengths of 2.2 and 3.7 pm (full overlap and zero overlap of the filaments) confirms and extends the findings of Huxley & Brown (1967) and Huxley (1967) that at any length the cross-bridges have all moved from this resting position, and as with contracting muscle, it is evident that such movement is independent of the presence of actm in the immediate vicinity of the moving cross-bridges. The data thus show that the initial movement of the cross-bridges occurring when a muscle contracts or passes into rigor is independent of a direct interaction with actin, but the cause of this movement still has to be found. Two mechanisms seem possible. In the first, each cross-bridge is affected individually by some change in the sarcoplasm when the

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muscle contracts or passes into rigor. In the second mechanism (Haselgrove, 1970) a change in the backbone of the filament allows or causes the cross-bridges to move: such a mechanism involving the backbone of the filament might involve co-operative interactions between myosin molecules. Huxley (1972) has discussed this problem briefly and points out that there is no biochemical evidence that myosin on its own detects activation in these muscles, and he considers a mechanism in which interaction of a few myosin cross-bridges with actin filaments will cause all the other cross-bridges to move too. In spite of this lack of biochemical evidence, the X-ray patterns indicate that there is a distinct change in the myosin filament backbone upon activation even when the actin filaments are apparently completely removed by stretching (see Plates IV and VII). Therefore this change in the filament structure will be considered further. (b) Con&~ratior&

change in the myosin jikzment

Consider first the evidence that the change in the backbone of the myosin filament is associated with the movement of cross-bridges. During isometric contraction at any sarcomere length between 2-O and 3.0 pm, the decrease in intensity of the layer lines is accompanied by an increase by 1% in the spacings of the reflections at 143 A and 72 A. A similar, although smaller, change occurs in muscles at non-overlap lengths. When a muscle passes into rigor at any length the resting layer-line pattern disappears completely, and the meridional pattern changes, as far as can be seen in the same way as it does during contraction: viz. the reflections at 107 A and 86 A disappear, while the main reflections at 143 A and 72 A increase in spacing by 1%. Finally, a kind of half-change occurs when semitendinosus muscles are stretched soon after dissection to non-overlap lengths, for here a slight decrease in the intensity of the layer lines is accompanied by a decrease on the intensity of the reilections at 86 and 107 A, and the 143 A and 72 A reflections increase in spacing by only about O-5%. Thus, the decrease in the intensity of the layer-line pattern and the changes in spacing of the meridional reflections occur together. Before considering the form of the change it is necessary to confirm that the very small increase in the axial spacing is part of this structural change and not just an artefact of the experiment. This 1 o/oincrease in spacing is not an elastic strain of the filaments when under tension during contraction because the same increase is seen in muscles in rigor, where the filaments are not under tension. The increase in length is also seen when muscles are stretched to non-overlap lengths and then put into rigor, showing that the increase is not caused by interaction between the actin and myosin &laments. Rome (1972a,b) has recently suggested that the increase in spacing may be accounted for by a change in the interference of the diffraction from the backbone and the cross-bridges. This seems unlikely to be the case here because the measurements of the diffraction patterns (Tables 2 and 4) show that the movements of the 72 A and 143 A reflection are the same percentage of the initial spacing, indicating a change in length of the diffracting periodicity; whereas a change in sampling would be expected to show the same absolute movement of each reflection in reciprocal space (a movement corresponding to 1% of the 143 A spacing would be only 0.5% of the 72 A reflection). When describing the diffraction patterns earlier it was tacitly assumed that the increase in the spacing of the cross-bridges is brought about by a change in the periodicity of the myosin filament backbone rather than from an interaction of the cross-bridges with actin; this assumption is justified by the pattern from

136

J. C. HASELGROVE

non-overlap semitendinosus muscles in rigor, for here the cross-bridges are unable to interact with non-myosin structures so the axial repeat of the whole myosin filament structure must be controlled by the backbone. It is thus concluded that the extensive movement of cross-bridges when a muscle contracts or passes into rigor is accompanied by a specific structural change in the backbone of the myosin filaments such that the axial repeat of the filament structure increases by about 1%. Unfortunately the X-ray data alone cannot be used to deduce the precise form of the changes taking place, but it is possible to speculate on these changes by comparing the X-ray patterns from insect and vertebrate striated muscles: the meridional patterns from insect flight muscle (Miller & Tregear, 1972) and from rigor vertebrate striated muscle are so similar that the myosin filament structures in these two muscles are probably basically the same. Note that they are not identical structures because the insect muscle myosin filament is thought to be six-stranded and the vertebrate structure three-stranded (Squire, 1973; Tregear & Squire, 1973). The surface lattice of the insect myosin filament is the same in relaxed and in rigor muscle, and is shown in Figure 5(a). If, as the X-ray data indicates, the activated vertebrate ,.....

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FIG. 6. Surface lattices of cross-bridges on the myosin filaments in insect muscle and vertebrate striated muscle. (a) Insect flight muscle (Squire, 1973). The lattice repeats after 8 subunit periods. (b) lattice for vertebrate striated muscle in rigor. The lattice is indentical with that ., Pronosed * in (a) but only half as wide. (c) Relaxed vertebrate striated musole (Squire, 1973). The axial repeat is 1% less than that of (b). The dotted line lies approximately in the direction of the line which does not change length on the surface of the filament when changing from the relaxed to the activated lattice.

myosin has the same basic structure as the insect muscle, then the vertebrate surface lattice would be as shown in Figure 5(b) (which is just half of the lattice in Fig. 5(a)). But the surface lattice for the relaxed vertebrate muscle is slightly different (Squire, 1973) and has a 1% smaller axial spacing than the other lattice; it is shown to the same scale in Figure 5(c). It can be seen how similar are the surface lattices of the relaxed and active forms of the vertebrate myosin filament and how little movement of the lattice points is needed to convert one to the other. (A convenient way of considering the necessary movement is to think of the myosin filament as being a many stranded rope which is being twisted; when the strands wind up slightly the change in

STRUCTURAL

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137

relative azimuth of lattice points is accompanied by a small change in the axial spacing. The myosin molecules may lie along the imaginary strands although it is not necessary that they do so.) The change in the vertebrate myosin structure necessary to convert the filament from one form to the other is so small that it is easy to conceive of such a change occurring when a muscle contracts or relaxes. It is not necessary to assume that the insect structure is six-stranded and the vertebrate muscle three-stranded and indeed the basic similarity of the insect and vertebrate myosin structures was first proposed (Haselgrove, 1970) on the basis of the then current two-stranded models for insect and vertebrate muscles. The similarity of lattices is a function of the basic unit cell rather than the number of strands of the lattice. It thus seems possible that the surface lattice of the myosin filament in an activated or rigor vertebrate muscle is the same as that in insect muscle and that relaxation of the vertebrate muscle is accompanied by a change in this lattice. (c) Myosin-linked regulation system What is then the functional role of this change in conformation of the backbone of the myosin filament! Because this change is always associated with the movement of cross-bridges to and from their positions in the relaxed muscle it is instructive to consider the movements of cross-bridges. In relaxed muscle the cross-bridges are all held close to the myosin filament in such specific positions that they give a helical diffraction pattern and are held so rigidly that even rapid axial oscillations fail to cause a noticeable movement. But during contraction, and at the onset of rigor, the cross-bridges are able to move considerable distances from their resting positions so that they can interact with available actin monomers (Haselgrove & Huxley, 1973). Indeed present theories of muscle contraction require that the cross-bridges operate cyclically, and when detached from actin can move presumably at random until able to re-attach to another actin monomer. It has already been shown that the initial movement of cross-bridges from their firmly bound resting positions is not due to interaction with the actin filament, but this movement must have some cause. It seems likely therefore that the changes observed in the backbone of the filament from the relaxed to the activated backbone structure allows or even causes the cross-bridges to move. The above discussion, while answering one question has posed this second one. If the contraction mechanism is designed so that cross-bridges can move relatively freely when not attached to actin, and if the interaction is controlled only by the tropomyosin-troponin system on the thin filaments, then why do the cross-bridges move away from the actin filaments and take up such specific positions in the relaxed muscle? Why do the cross-bridges not remain near the actin filaments ready to interact at the next possible opportunity! The answer to this question may be that the muscle contains both an actin-linked and a myosin-linked control system because the a&in-linked system is not eficient enough. Heat measurements of frog sartorius muscle indicate that the live muscle is able to suppress the ATPase activity of the active muscle by about 1000 times, which is about 50 times as great as the suppression of rabbit actomyosin in vitro by the actin control system. (Unfortunately heat and biochemical measurements have not yet been made on the same muscle species. But B&r&y (1967) reports that the a&n-activated ATPase rates at 20°C in frog sartorius muscle is 17 pmol P,/g muscle per second which is about 95 mol ATP/mol myosin per second and ia of the same order as the value of 30 mol ATP/mol myosin per second for

138

J. C. HASELGROVE

rabbit skeletal myosin, so it is not unreasonable to compare the heat rates of frog muscle with the biochemical ATPase rates of rabbit myosin.) Comparison of the heat and biochemical measurements can be made by taking the myosin content of muscle to be about 8% of the wet weight, the mass of the myosin molecule to be 450,000 daltons and assuming that all the heat measured arises from the hydrolysis of ATP by myosin with 11 kcal heat produced for each mole ATP split (Woledge, 1972). Then the heat rate of isometrically contracting frog sartorius muscle is about 40 meal/g per second (Hill, 1965) and corresponds to a turnover rate of ATP of 20 mol ATP/mol myosin per second, which is in reasonable agreement with the value of 30 mol ATP/mol myosin per second for uninhibited rabbit actomyosin in vitro (Eisenberg L Moos, 1970). The heat rate of resting frog sartorius muscle of O-043 meal/g per second (Clinch, 1968) corresponds to a turnover rate of 0.02 mol ATP/mol myosin per second which is the value found for the rate of mg/ATP hydrolysis by myosin in vitro at 20°C (Lymn & Taylor, 1970), and is about 1000 times less than that of the fully active muscle. However, suppression of the actomyosin ATPase using the troponin-tropomyosin system in biochemical studies is much less efficient : it has not yet been possible to suppress the ATPase rate of actomyosin in vitro by more than 20 times (Bremel et al., 1972), so the living muscle is able to suppress the ATPase activity by 50 times more than can be achieved at present in vitro. This discrepancy leads to the conclusion that a control system may be operating in addition to the actin linked system. Such a system could operate by physically binding the cross-bridges to the myosin filament so that interaction between the active sites on the myosin and active filaments is physically prevented. It is worthwhile speculating further that the changes in myosin filament structure discussed above are associated with a myosin-control mechanism. Lehman et al. (1972) have shown that many different muscles exhibit both actin and myosin-linked regulation and find that in general, in vitro inhibition of systems which are only actin-linked systems, is less effective than the myosin-linked or dual systems. Kendrick-Jones (1974) also finds that the DTNB light chain of vertebrate striated myosin will regulate scallop myosin, suggesting that it may have a functional role in the regulation of vertebrate striated myosin ATPase. Biochemical studies have not yet shown directly the existence of a myosin-linked regulation system in vertebrate striated muscle nor proved that the DTNB light chain does have this functional role. The reason may well be that the myosin-linked regulation operates only on the intact filament structure and that in vitro studies have not yet duplicated the correct conditions. (d) Two state model for th.e myosin Jilament The preceding discussion leads to the conclusion that the myosin filament vertebrate striated muscle can exist in one or other of two different states.

of

(i) Relaxed The backbone of the filament has a structure based on the lattice shown in Figure 5(c) . As a result of this backbone configuration the cross-bridges are held firmly in well defined lattice positions close to the myosin filaments and are physically prevented from interacting with actin. The X-ray pattern from a relaxed filament shows a series of reflections on the meridian, the most prominent of which have spacings of 143.4, 110.7, 107.2, 91.0, 85.9 and 71.6 A. The helical ordering of the cross-bridges gives rise to a series of layer lines indexing on 429 A.

Active

Active

(6) Rigor. Non-overl8p

Some active

(4) Rigor rest-length

Relaxed.

Active. Some relaxed

stretched

(2) Relaxed

Relaxed

Active

Relaxed

(3) Contracting: all sarcomere lengths

rest-length

(1) Relaxed

-

State of muscle

72.3

72.3

144.6

144.8

72-3

144.8

376

429 (very weak)

429 (weaker than (1))

72.1

None

439

143.8

72.3

71.6

Layer-line specings A

patterns

429

107, 86

107, 86

Principel meridional reflections A

jeutures of diffraction

5

71.6

143.4

144.8

143.4

of principal

St&e of myosin 6l8ments

Summary

TABLE

of model

of model

New 18yer lines arise because cross-bridges take actin-like structure Close to actin filclments

No layer-lines because myosin filement in active stete. Crossbridges in random positions because interaction with actin prevented

Most fibres active and giving sctive pattern. Some fibres (about 30%) give resting pattern

Some myosin filaments in active stctte. Spacings of principal reflections and intensity of layer lines between that of active and relaxed muscle

Basic assumption

Basic assumption

Comments

Half-way between actin and myosin

Free to move at random away from myosin Close to myosin

Close to myosin

Radi81 position of cross-bridges (from equatori patterns)

and their interpretation

140

J. C. HASELGROVE

(ii) A&&ted The backbone of the filament in the activated muscle has a structure different from that of the “relaxed” filament (and may be similar to the structure in Fig. 5(b)). As a result of this backbone structure the cross-bridges are not held close to the myosin filament but are free to move at random while joined to the filament backbone only by the S, rod portion of the molecule (Pepe, 1967; Huxley & Brown, 1967; Huxley, 1969) and are free to interact with adjacent actin filaments. The ATPase of the myosin will be activated by actin and as in in vitro studies, the level of interaction will depend on the extent to which the actin filaments are activated or relaxed. The X-ray pattern consists of meridional reflections indexing on 2315 A (1 o/o more than in the relaxed state). The principal meridional reflections have spacings of 144.8, 72.3 A and the reflections near 86 and 110 A (seen in the relaxed pattern) are either very weak or absent. Because the cross-bridges can move relatively freely, no layer lines are seen. (e) Interpretation

of X-ray

diffraction

patterns

The myosin X-ray reflections visible from muscle in different conditions are summarized in Table 5 and can be interpreted as follows on this two-state model for the myosin filament. (i) Relaxed muscle: rest-length The myosin filaments are all in the relaxed state so the diffraction pattern is that of the relaxed filaments with meridional reflections indexing on 2295 A, the strongest of which has a spacing of 143.4 A. Layer lines are seen which index on a spacing of 429 A. The cross-bridges are all held sufficiently firmly in place that they do not become disordered when the muscle undergoes rapid oscillatory length changes. The proximity of the cross-bridges to the myosin filaments gives rise to an equatorial pattern with an intensity ratio I 1, O/I 1, 1 at any sarcomere length higher than for contracting or rigor muscles. A few fibres may contain myosin in the activa,ted state with the cross-bridges free to make contact with the actin filaments. Slight interaction between these free cross-bridges and inhibited actin filaments may well account for the “filamentary resting tension” found by Hill (1968). (If on the other hand Hill’s filamentary resting tension were due to interaction of the actin with myosin filaments in the relaxed state, then the 1 o/o increase in length of the myosin filaments on activation would give rise to a slight drop in tension-the latency relaxation-as first suggested by Hill (1938).) (ii) Relaxed muscles stretched The X-ray patterns can be simply explained if it is assumed that in some way as yet unknown, stretching the muscle causes the transition of many of the myosin filaments from the relaxed to the activated state causing the X-ray pattern to be composed of a relaxed pattern from some fibres and an activated pattern from others: on stretching the muscle the layer-line pattern becomes weaker, the main meridional reflection appears to have increased in spacing to 143.8 A due to two overlapping reflections at 143,4 and 144.8 A. (The reflection at 128 A is visible weakly on the meridian and indicates a change in the actin filaments too.) Shortening of muscle again or letting it stand at the stretched length causes the activated myosin filaments to return to the relaxed state with a parallel change in the diffraction pattern, but allowing the muscle

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141

to stand before stretching prevents the change occurring when the muscle is stretched. It is tempting to think that the change in state of the myosin filaments may be related to the resting tension of the muscle because the tension certainly increases when the muscle is stretched, becoming significant at about 2.7 pm-the sarcomere lengths at which the diffraction patterns started to change. Furthermore the resting tension decreases both when the muscle shortens after stretching and when it is held stretched for a period: observations while doing the experiments indicated that less force was necessary to stretch muscles left at rest-length for 24 hours before stretching than for freshly dissected muscles. Perhaps the resting tension affects the sarcoplasmic reticulum to allow minute quantities of calcium into the sarcoplasm (enough to activate the myosin but not the actin). The increase in the rate of heat production in stretched muscles (the Feng effect, Feng, 1932; Clinch, 1968) may be due to myosin interacting slightly with unactivated actin. (iii) ContrQcting ntusclscle At any sarcomere length, stimulation of the muscle results both in the activation of the actin filaments and also in a change of state of the myosin filaments from the relaxed state to the activated state. The X-ray diffraction pattern of a fully active contracting muscle at any sarcomere length is thus typical of the activated myosin filaments with no layer lines visible and the main meridional reflections at 144.8 and 72.3 A. The resting type layer lines observed with about 3076 of the intensity of resting muscle may arise from myosin filaments which have not been activated. (The reason for an average about 30% of the filaments remaining inactivated cannot yet be explained.) The cross-bridges of the activated filaments are free to move at random between the actin and myosin filaments, and if few of them are instantaneously attached to actin, the space-time average position of the cross-bridges from the activated filaments will be half-way between the actin and myosin filaments, as found from the equatorial patterns (Haselgrove & Huxley, 1973). Alternatively, if all fibres are active and 45% of the cross-bridges move out near the actin filaments (Haselgrove & Huxley, 1973) while the other 55% remain close to their resting position, then the layer-line intensity of the contracting muscle will be about 30% (= 5$/100), as is observed. In either case, sufficiently few of the cross-bridges are instantaneously attached to the actin filaments even in a rest-length muscle during contraction that no rigor-like layer lines are seen (Huxley, 1972). (iv) Rigor muscles In rigor muscles all the myosin filaments have changed to the activated state allowing the cross-bridges to move and make the maximum possible number of connections with the actin filaments. The meridian of the X-ray pattern from rigor muscles at any length is therefore characteristic of the activated myosin filament pattern with the reflections all indexing on 2315 and the principal reflections having spacings of 1449 and 72.3 A. No “resting” layer lines are seen. At rest length suficient cross-bridges are attached to the actin filaments to give rise to the series of layer lines indexing on an actin-like structure. The cross-bridges being close to the actin filaments give an equatorial diffraction pattern in which the I,1 reflection is more intense than the 1,0 reflection. At longer sarcomere lengths, few cross-bridges can make contact with actin and at non-overlap length none is able to do so. Therefore at non-overlap

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lengths the diffraction pattern is typical of the activated filament but shows no actin-like layer lines because the cross-bridges do not label the actin filaments. (f) Activation

and relaxation

of the myosin filament

The transition between these two states is thought to be controlled only by the ionic conditions prevailing during contraction or at the onset of rigor. Since contraction is thought to be controlled only by the effect of calcium in the presence of ATP, while rigor is induced by the removal of ATP whether or not calcium is present, it is possible that the change in the myosin filament may be caused by different mechanisms during contraction and rigor. In rigor, the work on muscles at non-overlap lengths shows clearly that the change is not the result of act&myosin interactions, but it is not yet possible to say whether the removal of ATP from the sarcoplasm is the sole cause. The situation in contracting muscle has been discussed by Huxley (1972) who points out that he could not be certain that actin-myosin interactions had been completely abolished in his experiments. Therefore, a mechanism by which interaction of some cross-bridges with actin causes the change in the backbone cannot be ruled out, although the work with rigor muscles shows that actin is not necessary. Rome (19’72b) has studied in some detail the relaxation of glycerinated rabbit psoas muscles. She found that it is possible to relax the muscle well as judged from the criteria of tension and stiffness, and that the myosin cross-bridges move back from the actin filaments to the region of the myosin filaments. But the layer-line pattern from the relaxed muscles is much weaker than from the live muscles indicating that the myosin filament structure had not completely returned to the form of the living relaxed muscle. Thus, although such relaxed glycerinated preparations may be suitable as models for live muscles in many cases, they should not be considered as models suitable for studying the myosin filament structure until preparations can be obtained which give myosin layer lines as intense as from a living relaxed muscle. REFERENCES B&Any, M. (1967). J. Gen. Physiol. 50 (6), part 2, 197-218. Bremel, R. D., Murray, J. M. & Weber, A. (1972). Cold Spring Harbor Symp. Quant. Biol. 37, 267-275. Cleworth, D. & Edman, K. A. P. (1972). J. Physiol. 227, 1-17. Clinch, N. F. (1968). J. Phytiol. 196, 397-414. Ebashi, S. & Endo, M. (1968). Prog. Biophys. and Mol. BioZ. 18, 123-183. Eisenberg, E. & Moos, C. (1970). J. Biol. Chem. 245, 2451-2456. Feng, T. P. (1932). J. Physiol. 74, 441-454. Hanson, J. & Lowy, J. (1963). J. Mol. BioZ. 6, 46-60. Hanson, J. & Huxley, H. E. (1955). Symp. Sot. Ezp. BioZ. 9, 228-264. Hanson, J., Lednev, V., O’Brien, E. J. & Bennett, P. M. (1972). Cold Spring HarborSymp. Quunt. BioZ. 37, 311-318. Hmelgrove, J. C. (1970). Ph.D. Thesis, University of Cambridge. Ha&grove, J. C. (1972). Cold Spring Harbor Symp. Quant. BioZ. 37, 341-352. Haselgrove, J. C. & Huxley, H. E. (1973). J. Mol. BioZ. 77, 549-568. Hill, A. V. (1938). Proc. Roy. Sot. ser. B, 126, 136-195. Hill, A. V. (1965). Trails and Trials in PhysioZogy. Edward Arnold, London. Hill, D. K. (1968). J. Phyaiol. 199, 637-684. Huxley, H. E. (1967). J. Gen. Physiol. 50, (6), part 2, 71-83. Huxley, H. E. (1969). Science, 164, 1356-1366. Huxley, H. E. (1971). Biochem. J. 125, 85. Huxley, H. E. (1972). Cold Sp&g Harbor Symp. Quant. BioZ. 37, 361-376.

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Huxley, H. E. & Brown, W. (1967). J. Mol. BioZ. 30, 383-434. Kendrick-Jones, J. (1974). Nature (London), 249, 631-634. Larson, R. E., Kushmerick, M. J., Haynes, D. H. & Davies, R. E. (1968). Biophys. J. 8, MA4. Lehman, W., Kendrick-Jones, J. & Szent-Gyorgyi, A. G. (1972). ColdSpring Harbor Symp. Quant. Biol. 37, 319-330. Lymn, R. ‘IV. & Taylor, E. W. (1970). Biochemistry, 9, 2975-2983. Miller, A. & Tregear, R. T. (1972). J. Mol. BioZ. 70, 85104. Offer, G. (1972). Cold Spring Harbor Symp. Exp. Biol. 37, 87-93. Page, S. G. & Huxley, H. E. (1963). J. Cell BioZ. 19, 369-383. Parry, D. A. D. & Squire, J. M. (1973). J. Mol. BioZ. 75, 33-55. Pepe, F. A. (1967). J. Mol. Biol. 27, 227-236. Reedy, M. K. (1967). Am. Zoologist, 7, 465-481. Reedy, M. K. (1968). J. Mol. BioZ. 31, 155176. Roger, C. D. (1973). Ph.D. Thesis, University of Oxford. Rome, E. (1972a). Cold Spring Harbor Symp. Quant. BioZ. 37, 331-339. Rome, E. (1972b). J. Mol. BioZ. 65, 331-345. Rome, E. (1973). Nature New BioZ. 244, 152-154. Selby, C. C. & Bear, R. S. (1956). J. Biophys. Biochem. CytoZ. 2, 71-85. Squire, J. M. (1971). Nature (London), 233, 457-462. Squire, J. M. (1972). J. Mol. BioZ. 72, 125-138. Squire, J. M. (1973). J. Mol. BioZ. 77, 291-323. Starr, R. & Offer, G. (1971). FEBS Letters, 15, 49-44. Szent-Gyorgyi, A. G., Cohen, C. & Philpott, D. E. (1960). J. Mol. BioZ. 2, 1333142. Tregear, R. T. & Squire, J. M. (1973). J. Mol. BioZ. 77, 279-290. Vibert, I’. J., Haselgrove, J. C., Lowy, J. & Poulson, F. R. (1972). J. Mol. BioZ. 71, 757-767. Woledgc, R. C. (1972). Cold Spring Harbor Symp. Quaat. Biol. 37, 629-634. Wooster, IV,:. A. (1964). Acta CrystaZZogr. 17, 878-882.