J. Mol. Biol. (1976) 102, 325-332

A x i a l A r r a n g e m e n t o f C r o s s b r i d g e s i n T h i c k F i l a m e n t s o f

Vertebrate S k e l e t a l M u s c l e

ROOER CRAIG'~ AND GERALD OFFER

Department of Biophysics King's College, 26-29 D~'ury Lane, London WC2B 5RL, England

(Received 1 September 1975, and in revised form 15 December 1975)

Antibodies to subfragment-1 have been used to label glycerinated rabbi t psoas muscle in order to determine the axial arrangement of the crossbridges. I n the electron microscope, dense labelling is observed throughout the A-band except at the centre and near the ends. I t is concluded that (1), there is a region in the centre of the filament devoid of crossbridges as Huxley (1963) proposed; (2), this bare zone is about 1500 A wide; and (3), the crossbridges extend in a continuous array from the edge of the bare zone to the end of the filament except for a single gap between the second and third crossbridges from the end. The models of the thick filament proposed by Pepe (1967a,1971) and Squire (1973) are inconsistent with these observations.

1. Introduct ion

Although proposals have been made for the packing of myosin molecules in the thick filaments of vertebrate skeletal muscle, the detailed arrangement remains elusive.

From observations of negatively-stained thick filaments in the electron microscope, Huxley (1963) concluded that the myosin molecules in the two halves of a thick filament were packed in opposite directions, with their tails forming the shaft of the filament and their heads on the surface where they could function as crossbridges. He further concluded that the crossbridges occurred along the whole of the length of the filament except for a region at the centre called the bare zone where only tails were present. Because of the difficulty of preserving crossbridges for electron micro- scopy, a more precise description has not been possible using this technique. Huxley & Brown (1967) showed from their X-ray diffraction studies of muscle that the cross- bridges were arranged approximately on a hehx with an axial repeat of 429 4 and an axial translation of 143 A. However X-ray diffraction gives only the average value of the repeat over most of the filament length and does not straightforwardly reveal small perturbations or gaps in the repeat. Nevertheless, it has been generally assumed until recently, that the crossbridges in each half of the filament extend in an unbroken array with a 143 A axial spacing from the edge of the bare zone to the end of the filament.

This assumption has recently been challenged by Squire (1973) who proposed that there might be gaps in the 143 A sequence near the bare zone and also near the ends

t Present address: Rosenstiel Basic Medical Sciences Research Cen~er, Brandeis University, Waltham, Mass. 02154, U.S.A.

325

326

M- line

Bare zone

R. CRAIG AND G. OFFER

43011

IIII J J' i I I ~ (al

I s~

III I I ~,,i'' l'l' ',~~ (b)

Fro. 1. Comparison of (a) the axial dJstribution of crossbrldges in half a thick filament according to the model of Squire (1973) with two gaps of 429 A and four gaps of 286 A; and (b) the dis- tribution arrived at by combining our antibody labelling results with the freeze-sectioning results of SjSstrSm & Squire (unpublished results).

of the filament, while in the intervening region the sequence might be complete (Fig. l(a)).

As a test of Squire's suggestions, and of the model of Pepe (1967a,1971) which predicts no gaps, we have used the technique of ant ibody labelling to determine the axial distribution of the cross-bridges. The power of this technique has recently been demonstrated using antibodies to C-protein to reveal the axial position of C-protein in the thick filament (Offer, 1972; Craig, 1975; Craig & Offer, 1976; Pepe & Drucker, 1975). By using Fab fragments of this ant ibody prepared by papain digestion, the precision with which the antigenic sites can be located is enhanced; in the case of anti-C the axial extent of labelling is only about 100 A at each site (Craig & Offer, 1976). In this paper we have used papain fragments of antibodies to subfragment-1 to label the myosin heads of the thick filaments of rabbi t psoas fibres made permeable by glycerination. The bound ant ibody has been identified as a visible increase in staining density in the electron microscope. To be certain tha t it is the myosin heads which have been labelled and not some other myofibrillar component, we have taken great care tha t our ant ibody preparat ion is specific for subfragment-1.

We find tha t there is only a single gap in the crossbridge sequence near each end of the filament, in disagreement with the suggestions of Pepe and of Squire.

2. M a t e r i a l s a n d M e t h o d s

Column-purified myosin was prepared by the method of Offer et aL (1973). S-1 t was prepared from sucl~ myosin using soluble papain by the method of Margossian & Lowoy (1973).

The antiserum to column-purified rabbit myosin, elicited in a goat, was that used b y Offer (1976). Antibodies specific for S-1 were isolated from this antiserum by coupling 33 mg S-1 to 5 g diazotized p-aminobenzyl cellulose, mixing the product with 220 ml antiserum, washing out tmbound protein, eluting the bound antibodies at pH 2-7 to 2-9 and then neutralizing (Campbell et al., 1951 ; Pope, 1972). Papain fragments of the specific antibody were prepared as described by Craig & Offer (1976) using the optimal conditions of Putnam et al. (1962).

The antiserum to myosin gave only a single precipitin line with column-purified myosin or even crude myosin and the immune-precipitates contained only IgG and myosin (Offer,

t Abbreviation used: S-I, subfragment-1.

AXIAL ARRANGEMENT OF CROSSBRIDGES 327

1976). By immune-diffusion the antibodies to myosin in this serum were shown to be directed mainly against S-1 rather than myosin rod (Craig, 1975). Purification of the antibody by binding to S-1 coupled to p-aminobenzyl cellulose should have removed the small amount of antibodies to myosin rod as well as any antibodies to non-myosin com- ponents not detected by the tests already described.

Small bundles of glycerinated rabbit psoas fibres were labelled with antibodies and then prepared for electron microscopy as described by Craig & Offer (1976).

3. R e s u l t s

Figure 2(a) shows the labelling pattern obtained when glycerinated rabbit psoas muscle is treated with papain-digested anti S-1. When compared with controls treated with serum from a goat which had not been immunized against S-1 (see Craig & Offer, 1976), it is clear tha t the A-band has been heavily labelled but tha t there is no labelling elsewhere in the sarcomere.

The filling in of the H-zone is the most obvious feature: although the sarcomere is about 2.9/~m long and therefore has a long H-zone (0.8/xm), this is only just detect- able by its slightly lower density compared with the overlap region.

The strong labelling of the H-zone in our micrographs is also apparent from the clarity with which the unlabelled region, corresponding approximately to the bare zone, stands out in its centre. I t is particularly important to note the sharpness at the ends of the unlabelled region since it shows tha t the crossbridges in neighbouring filaments are in axial register. The length of the unlabelled zone is 1510 2~ ~- 10 A (mean and s.e.m, from 23 A-bands) assuming that the A-band is exactly 1.60/zm long (Page & Huxley, 1963).

I t is clear tha t the overlap region of the A-band has also been labelled because the whole of the A-band is much denser than in unlabelled specimens, and because the ends of the A-band are very sharply defined compared with the controls.

We have confirmed tha t both the H-zone and overlap zone have been labelled by looking at transverse sections where the antibody can be clearly seen in the inter- filament spaces. Unfortunately, even in our thinnest transverse sections, the antibody appears to be amorphously distributed around the thick filaments and gives no clue to the rotational symmetry of the crossbridge arrangement.

These observations of the labelling pattern in the electron microscope are con- sistent with the labelling patterns seen in the light microscope when fluorescently tagged anti S-1 was used to label myofibrils (Lowey & Steiner, 1972). They observed the strongest labelling in the H-zone but the overlap zone was also labelled. (However, Pepe (1967b) observed staining only in the H-zone when fluorescent antimyosin absorbed with light meromyosin was used for labelling.)

Although the labelling is uniformly dense throughout the crossbridge region of the H-zone and throughout most of the overlap zone (no periodic change in density is detectable), there is non-uniform labelling right at the ends of the A-band. This can be seen particularly well a t higher magnification (Fig. 2(b)). The labelling ends in a stripe, about 300 A wide, which is separated from the uniform labelling of the rest of the overlap zone by an unlabelled stripe about 150 A wide (arrow, Fig. 2(b)).

Beyond the stained stripe, which is taken to mark the end of the crossbridge array and thus the end of the A-band, is often seen a narrow fringe of faintly stained material continuing into the I-band. This may be due to thin filament material or possibly a non-myosin continuation of the thick filaments.

328 R . C R A I G A N D G. O F F E R

FIG. 2. Labelling of glycerinated rabbit psoas muscle with anti S-1. (a) Magrjfieation 11,200 x . An H-zone is marked. (b) Magnification 33,000 x . The arrow shows the gap in labelling at the end of an A-band.

A X I A L A R R A N G E M E N T OF C R O S S B R I D G E S 329

4. D i s c u s s i o n

The thick filament is a complicated structure made up of several proteins besides myosin (Craig, 1975; Craig & Offer, 1976; Pepe & Drucker, 1975). Consequently it is difficult to identify the structures responsible for particular features in electron mierographs of A-bands; they may be due to myosin heads, to myosin tails or to non-myosin components. This difficulty may be overcome by observing where specific antibodies are bound. Using antibody specific for S-1 we have been able to determine the axial distribution of myosin heads with a much greater degree of certainty.

(a) General conclusions about the distribution of crossbridges

The whole of the A-band was labelled with anti S-1 except for a zone in the centre and a gap near the ends. From this appearance important conclusions can be made about the distribution of crossbridges. First, as proposed by Huxley (1963) there is a zone in the centre of the A-band which is completely free of crossbridges. Second, because the labelling distal to the bare zone was continuous except at the ends of the A-band we irder tha t the erossbridges are closely spaced with no large gaps throughout this region. However, the gap in labelling near the ends of the A-bands shows tha t there is a gap separating the main array of the erossbridges from those at the end.

How far apart could neighbouring erossbridges be without an obvious gap appearing in the ant ibody labelling pattern? I f the myosin heads occupied an axial extent of about 110 A (which would be expected if myosin heads 150 ,~ long were tilted 45 ~ with respect to the filament axis (Moore et al., 1970), and the at tached antibody added a further 30 A to the width (Craig & Offer, 1976), then no obvious gap would occur in the labelling pat tern if the crossbridges were separated by 140 A or less. But an interval of 200/~ or more between crossbridges would certainly be expected to be visible as a gap in the labelling. We can therefore conclude that from the bare zone right out to the observed gap near the ends of the A-band the crossbridges are spaced at intervals of less than 200 A. Although this has been generally assumed from the 143 A axial spacing deduced from X-ray diffraction data (Huxley & Brown, 1967) it has not before been directly demonstrated.

The width of the only observed gap in labelling was about 150 A. Allowing for the axial extent of the erossbridges and the attached antibody this would imply an interval of about 300/~ between the crossbridges at this point.

The antibody labelled stripe at the ends of the A-band was about 300 A wide. This would suggest the presence of two rows of erossbridges about 150 A apart at the ends of the A-band.

(b) Length of the bare zone

The antibody-free zone at the centre of the A-band is sharply defined and its extent {1510 A • 10 A) can therefore be measured accurately. However its length may be slightly different from the length of the bare zone because the attached antibody molecules may add to the axial extent of the erossbridges. Such an effect was notice- able when we used papain-digested anti-C to label the C-protein stripes in the A-band; their axial extent increased from about 70/~ to about 100/~ (Craig & Offer, 1976). We therefore assume tha t 30 A is a reasonable estimate for the amount which the

330 R. CRAIG AND G. OFFER

bound antibody might add to the axial extent of the crossbridges. On this assumption the length of the bare zone (defined as the distance between the proximal edges of the first crossbridges in the two halves of a filament) is 1540 A. The error in the measurement is probably about 50/~ (to allow for the uncertainty in the length of the A-band on which the measurement is based). This value for the length of the bare zone is in good agreement with the value of 1490 A ~: 20 /~ measured from A-segments stained with ammonium molybdate (Craig, 1975). The length of the bare zone has been previously estimated to be 1500 to 2000 /~ from individual thick filaments (Huxley, 1963), and 1200 to 1500 A (Huxley, 1966) and approximately 1700 A (Pepe, 1967a) from sections. The variations in these previous estimates suggest tha t the rows of crossbridges bordering the bare zone have not always been correctly identified. The use of specific antibodies has allowed us to place a reliable and fairly precise value on the length of the bare zone.

The length of the bare zone measured here is consistent with the suggestion of Harrison et al. (1971) that antiparallel overlaps of 1300/~ between myosin tails of length 1450 A are involved ~n the bare zone. The bale zone in Squire's (1973) thick filament model is based on the results of Harrison et al. (1971) and is therefore con- sistent with our measurements. The maximum antiparaUel overlap in Pepe's (1967a) model is 860 ~, producing a bare zone 2000 /~ long. This is inconsistent with our results.

(c) Comparison of the antibody labelling results with other observations of the A-band

Our ability to identify the positions of myosin heads is of great help in interpreting previous observations of the A-band. Although myosin heads are not adequately preserved in normal sections, a gap in density about 200 A wide is seen at the ends of the A-band (see for example Fig. 3 of Huxley, 1967). A similar gap was observed in shadowed myofibrils (Rozsa et al., 1950). Since these gaps both occur in a position similar to the gap we have observed in antibody labelled muscle, we suggest tha t these previous observations were due to the missing row of myosin heads at this position. In A-segments and myofibrils stained with ammonium molybdate, a set of stripes with a 143/t~ repeat was observed in the A-band but there was a gap of approximately 2 • 143 A near the ends of the A-band separating the last two stripes from the main array (Craig, 1975). The assignment of these stripes to myosin heads is supported by the observations reported here.

Detailed observations of A-band structure have also been made by applying the technique of freeze-sectioning to muscle (SjSstrSm & Squire, unpublished results). In negatively stained specimens, structural detail is well preserved along the whole length of the A-band (this has not yet proved possible with A-segments). A sequence of stain- excluding stripes about 143 A apart is observed starting about 800 A from the centre of the A-band, and continuing to the end, with a single gap between the second and third stripes from the end. This pattern of stripes is entirely consistent with our labell- ing results ff we assume tha t each stripe represents a row of myosin heads.

SjSstrSm & Squire (unpublished data) have alternatively suggested that some of the stripes near the bare zone and the ends of the filament might represent either non-myosin proteins or staining of the thick filament backbone, rather than myosin heads. Their suggestion was made because the repeat of the stripes is slightly distorted in these regions and it was considered tha t the crossbridges should lie on an exact repeat, and

AXIAL ARRANGEMENT OF CROSSBRIDGES 331

because the stripes are of variable intensity. As stated before, we conclude tha t a row of crossbridges lies on every one of the stripes on the approximately 1 4 3 / l repeat observed by SjSstr6m & Squire (unpublished results). The slight distortion of the repeat can be most simply explained by changes in the myosin packing near the bare zone and near the ends of the filament (Craig & Offer, 1976; Craig, manuscript in preparation). The variation in intensity of the stripes is most simply accounted for by superposition of non-myosin or backbone staining on crossbridge stripes. A simple explanation along these lines is offered by Craig (manuscript in preparation) to account for the variable intensity of the crossbridge stripes near the bare zone seen in A-segments.

From our results alone the interval between rows of crossbridges cannot be accur- ately determined nor can we count the number of rows. :From the results of Sj5strSm & Squire (unpublished) alone the identification of the rows of crossbridges is uncertain. But, combining the two sets of results and assuming that there is no difference in filament structure in the two types of muscle, we can conclude (Fig. l(b)) (1) tha t the myosin crossbridges in each half of the filament occur on an approximate 143 A sequence but there is a gap on the third row in from the end; (2) tha t the number of occupied rows is 49 which would imply a thick filament length of 1.57/~m. This means tha t the thick filament contains 196, 294 or 392 myosin molecules according to whether there are two, three or four crossbridges at each axial position (Huxley & Brown, 1967; Tregear & Squire, 1973; Squire, 1973; Potter , 1974; Morimoto & Harrington, 1974).

(d) Comparison of the observed distribution with previous models

Squire (1973) suggested two closely-related models for the packing of myosin molecules in the bare zone which produced one gap of 429 -~ and either one or two gaps of 286 A in the sequence of crossbridges bordering the bare zone in each haft of the thick filament. He also suggested that the packing of myosin molecules at the ends of the filament might resemble that in the bare zone producing a similar pat tern of gaps. The axial distribution of crossbridges corresponding to his model with the most gaps is illustrated in Figure l(a). None of these proposals agree with the dis- tribution of crossbridges we have observed, and we conclude that these packing schemes are wrong t . Our present results of course do not conflict with Squire's model for the arrangement of myosin molecules in the rest of the filament (the region of constant packing) although elsewhere we have argued against it (Craig & Offer, 1976).

The 143 A array in Sqnire's model is generated by a combination of staggers of 430 A and 720 A between myosin tails. The gaps occur because these two interactions are used to build up the crossbridge array systematically near the bare zone and the ends of the filaments. Since the gaps he predicted are not observed, it appears tha t 430 A and 720 A staggers do not act in this way. This weakens the argument tha t these staggers are the basis of all myosin-myosin interactions. Since the last two cross- bridges are separated by 143 A, it seems possible tha t this may represent an important stagger interaction.

In the model of Pepe (1967a,1971) there are no gaps in the 143 A sequence. His model is therefore also inconsistent with the gap near each end of the filament indi- cated by our antibody labelling results.

:Further and much needed information on the arrangement of crossbridges in the thick filament might be obtainable by using Fab fragments directed against a smaller

t Squire (1973) ment ions tha t one or two var ia t ions of these schemes are poss ib le bu t no detai ls are g iven .

332 R. CRAIG AND G. OFFER

portion of the myosin heads than S-l, for example to alkali or I )TNB light chains. This might enable all the rows of antibody-labelled crossbridges to be resolved.

We are indebted to Dr M. SjSstrSm and Dr J. M. Squire for showing us their results prior to publication. We thank Mr R. Starr and Mr A. Outen for gifts of colllmn-purified myosin, Mr H. Baker for help in preparing subfragment-1, Dr A. Elliott, Dr E. O'Brien and Miss P. Bennett for their help in the preparation of the manuscript and Mr Z. Gabor for photographic assistance. R. Craig acknowledges t h e receipt of a Commonwealth scholarship. This work was in part fulfilment by R. Craig of the degree of Doctor of Philosophy in the University of London.

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