J. Nol. Biol. (1!17(i)103, 439-467

Structure of the Cross-striated Adductor Muscle of the Scallop HARRY M. MILLMAS~ Drpnrtruent of Biological A’cknce.~ Brock University At. Catharines Ontario, Can.adrc ANI) PAULINE M. HEXNEW Medical Besearch Council Muscle Biophysics Unit h’ing’s College, 262.9 Drury Lane, Lordon W.C.2, England

TIN structure of the cross-striated adductor muscle of the scallop has boc~~ studied by electron microscopy and X-ray diffraction using living rclaxed, glycerol-extracted (rigor), fixed and dried muscles. The thick filaments am arranged in a hexagonal lattice whose size varies with sarcomere length so as to a constant lattice volume. In the overlap region there are approximately maintain 12 thin filaments about each thick filament and these are arranged in a partially disordered lattice similar to that found in othrlr invcrt,obrate muscles, giving a thin-t,o-thick filament ratio in this rsgion of 6: 1. Tire thin filaments, which contain actin and tropom>-ositl. are about 1 pm lorig and the actin subunits arc arranged on a helix of pitch 2 k 38.5 nm. The thick filaments, which contain myosiu and pammyosin, are about 1.76 pm long and haves a backbone diameter of about 21 nm. \\:li’e propose that those filaments have a COW of paramvosin about 6 nm in diameter, around which the myosin molecules pack. In living relaxed muscle, the projecting myosin heads are symmetrically arranged. The data are consistent with a six-stranded helix, 0ach strand having a pitch of 290 run. Tl~o projections along the strands each correspond to the heads of one or t\vo myosin mol0cules and occur at alternating intervals of 13 and 16 nm. In rigor muscle these projections move away from the backhon0 and attach to thca thin filaments. planes of thick filaments art’ In both living and dried muscle, alternato st,aggered longitudinally relative to each other t)p about 7.2 nm. This gives rise to a body-centred orthorhombic lattice with a llnit ccl1 twice the volume of the basic filament lattice.

1. Introduction The larger part’ of the adductor muscle of the scallop is cross-striated and is used by the animal for rapidly closing the shell. By repeating this action and ejecting a stream of water, the animal can be propelled t’hrough the water. This muscle is almost unique in molluscs in that it is cross-striated, with band patterns very similar t,o t$host: f’ound in vt~rtc4nxtt~ striatrtl IIIIIS~~IIY (Harrsot~ & IA)w~. I !,N,). I II I’olltJ’iLSt~ t,c, t J‘r~wmt acIclros~ : DoJmrtmcni~ of Physics, Ullivcrsity Z!,

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many molluscan muscles which contract slowly and show the sustained state of cont)raction frequently referred to as “catch”, the striated scallop adductor has contractile properties and speeds much closer to those of frog skeletal muscle (Bozler, 1930; Millman, 1967; B. M. Millman, D. Gibson & D. Overy, manuscript in preparation). Such rapid contraction is essential to its function as a swimming muscle. In addition to the striated part of the muscle, the adductor also contains a smooth or “catch” portion, which serves to hold the shells closed for long periods of time (Marceau, 1909; Riiegg, 1961a). The thick filaments of t’he contractile apparatus of the striated adductor are of a uniform size with a diameter larger than that of vertebrate striated muscle filaments, and there are 10 to 12 thin filaments around each thick filament in the overlap region (Lowy & Hanson, 1962; Sanger & Szent-GyGrgyi, 1964; Lowy et al.? 1966). Like other molluscan muscles, the scallop striated adductor contains the protein A), though in small amounts paramyosin (formerly referred t’o as tropomyosin (Riiegg, 1961b; Hardwicke & Hanson, 1971; Cohen et al., 1971; Szent-GyGrgyi et al., 1973). It has recently been shown that the muscle contains little or no t,roponin and that its contractile activity is regulated by myosin in the thick filaments (SzentGyBrgyi et al., 1973). Despite the extensive biochemical and physiological work done on this muscle, little detailed structural information is available. In this paper we have undertaken a structural study using electron microscopy and X-ray diffraction. Diffraction patterns show that the muscle is highly organised and similar in many respects to other striated muscles. The major difference is in the structure of the thick filaments and particularly in the arrangement of the projections. We have developed a model for the thick filament structure which has been briefly described elsewhere (Millman & Bennett, p. 184a, Abstr. Biophys. Sot. Meeting, Columbus, Ohio! 1973).

2. Materials and Methods (a) Preparation Muscles

were

dissected

from

of specimens for X-ray diffraction

the striated

part of the adductor muscles of the scallop: Biological Laboratories in Plymouth, England, Research Associates, New Brunswick, Canada. The English animals were kept in cooled, aerated Plymouth sea water, the Canadian animals in “Instant Ocean” sea-water tanks. Since we could detect no difference in the microstructure of the muscles, results from both species are grouped together. X-ray diffraction patterns from living muscles were obtained from muscle strips (1 to 2 mm thick), which had been tied at both ends with soft cotton thread. These were placed in Perspex chambers with a small quantity of sea-water and kept at room temperature. Such preparations gave good X-ray diffraction patterns up to 24 h after dissection, particularly if antibiotics (5 pg cycloheximide/ml and 50 pp L-chloramphenicol/ml) were added to the sea-water. Glycerol-extracted muscles were prepared by 2 methods: that of Levine et al. (1972) or Baguet (1973). When using the latter method, we stored the muscles in a 50% rather than a 90% glycerol solution. Before use, the glycerol was removed from the muscle by soaking in solutions of successively decreasing glycerol concentration. For observation, the muscles were placed in a sealed chamber containing the appropriate equilibrating solution. Although both methods gave similar X-ray diffraction patterns, more consistent and detailed patterns were normally obtained from muscles prepared by Baguet’s method. Dried muscles were prepared by tying living muscle strips to Perspex rods, blotting them and air-drying for several hours before storing in a desiccator. Such preparations could be kept for several months without detectable deterioration.

Pecten maximue, obtained from the Marine and Placopecten naagellanicus from Marine

SCALLOP (h) I’reparatim

STRIATED

ADDUCTOR

of specimens

for

electron.

MUSCLE

441

,microscopy

All electron microscopy wa done with Pectin waaximus. Thin strips of muscle, about 1 mm thick, were excised from the adductor and tied to Perspex rods. These were then fixed in glutaraldehyde and/or formaldehyde, after which small pieces from the outside of t,he strips were post-fixed in osmium tetroxide. Several variations in the methods of fixation were tried, none of which was entirely satisfactory as judged by the final appearanre of sections in the electron microscope. Samples were monitored at various stagec of t,hc fixation procedure by X-ray diffraction to detect changes produced by the fixation. W’e found, however, that good preservation after fixation as implied by a reasonable X-ray diffraction pattern was not necessarily maintained after dehydration and embedding of t.hc sample. The method of fixation which gave the best appearance of thick and thin filaments in the electron microscope and which showed good longitudinal and lateral order was obtained by fixation in 4% formaldehyde in 10 mivr-cacodylate buffer (pH 7+), followed by 49/o glutaraldehyde in the same buffer and t,hen post-fixation in 1% osmium tetroxidr. The fixed muscles \ver(, dehydrated in ct,hanc)l or acetone and embedded in Araldite. Filament suspensions from the living muscles were prepared by the method of Hardwickc & Hanson (1971). The filaments were finally suspended in a relaxing medium containing 2 mM-EGTA, 5 m&r-MgCl,, 5 mM-ATP, 100 mM-KU, 1 m%f-dithiothreitol, 6.7 rn>!sodilrm phosphate (pH 7.0). (c) X-ray

diffraction,

Four types of low-angle X-ray cameras were used: double-mirror Franks cameras (Elliott & Worthington, 1963) ; a toroidal camera (A. Elliott, 1965) ; mirror-monochromator cameras of the type described by Huxley & Brown (1967) with specimen-to-film distances of either 20 or 40 cm; and a double crystal monochromator designed by Dr M. Spencer, with a specimen-to-film distance of about 7 cm. The double monochromator camera had the particular advantage of high intensity together with a sharp point focus (100 pm diameter), which enabled us to obtain good focal resolution in both meridional and equatorial directions at the same time. This was in contrast to the mirror-monochromator camera, which gave a line focus of dimensions about 100 pm x 500 pm. The X-ray generator used was either a fixed-anode type (Hilger and Watts, microfocus modal Y33) with a 100 pm line focus, or one with a rotating anode (Elliott Automation, model GX6) with a 100 or 180 pm focussing cup. The patterns were recorded on Kodirex X-ray film (Kodak Ltd). The most detailed axial patterns were obtained either with the mirror-monochromator cameras on the rotating anode generator, or the double-monochromator camera on the Hilger generator. Exposures ranged from 6 to 24 h. Most equatorial patterns were obtained using the Franks camera on the Hilger X-ray generat)or with exposures ranging from 1 to 5 11. (d) Electron

microscopy

Thin sections were cut on an LKB ultramicrotome. Sections took up stain rather slowly, but satisfactory contrast was obtained after 30 min in 2% phosphotungstic acid at room temperature and pH 7.0, 1.5 h in 5% many1 acetate at 6O”C, and 10 min in lead citrate (Reynolds, 1963). Negatively-stained filaments were prepared by placing a drop of the filament suspension on a carbon-coated specimen grid and washing off any excess with O+1o/o ammonium acetate. This also removed any phosphate buffer which might have precipitated with the uranyl stain. The filaments were negatively stained with 1 or 2% uranyl acetate. Specimens were examined in a Philips EM 200 electron microscope fitted with an anticontamination device cooled with liquid nitrogen. The microscope was calibrated using a grating replica with 2160 lines/mm, or using thr 8.6 nm lattice spacing in catalaso crystals (Wrigley, 1968). (e) Optical

di@action

Optical diffraction patterns were taken on a modified Lipson et al., 1971). The objects were transparancies on film of reverse micrographs, sandwiched in oil between two optical flats.

diffractometer (O’Brien contrast to the original

3. Results (a) G’eneral features of sectioned muscle The fibres of the cross-striated adductor of the scallop show an elongated shape in transverse sections (Fig. l(a)). Their average dimensions are approximately 1 pm x 10 pm. The filament array extends right across the fibre with the thick filaments lying on a hexagonal lattice. In longitudinal sections (Fig. 2(a)), the different densities of the interdigitating arrays of thick and thin filaments give rise to the characteristic appearance of a cross-striated muscle. At low magnification the H-zone cannot be distinguished from the overlap region because of the very high density of the thick filaments relative to the thin ones. Only at high magnification can individual thin filaments be seen. The lengths of both thick and thin filaments appear t,j be constant’ throughout the muscle. These and the observed sarcomere lengths arc similar to those of vertebrate striated muscle. The thick filament assembly or A-band does not show any structural feature which corresponds to the M-line in other striated muscles (Fig. 2(a)). The thick filaments have distinct bare zones and in neither longitudinal nor cross-sections is there any evidence of M-line bridges connecting the filaments. The thin filament assembly is held together at the Z-line by strongly staining amorphous material approximately 40 nm wide. The Z-line dots not show the rt>gular structurtb that is seen in vertebrate fast’ twitch muscles. III these two respects. the scallop muscle appears similar to frog slow muscle (Page, 1965). The degree of organization of thcl Z-line seems to be related to the alignment of both thick and thin filament arrays. When the Z-line has become disordered (probably because of poor fixation) t,hen the lateral register of both sets of filaments is poor. (b) Electro?b microscopy

of the jilament

lattice

The hexagonal arrangement of thick filaments seen in transverse sections (Fig. 1) has been confirmed from X-ray diffraction patterns of both living and fixed muscle. However, the interfilament distance seen in sections (40 to 45 nm) is much smaller than the value obtained from X-ray diffraction patterns of living muscle (60 nm at a sarcomere length of2.7 pm). This is probably caused by shrinkage during dehydration. In the overlap region, the thin filaments are approximately equidistant from the thick filaments (Fig. l(c)), but their detailed arrangement is not clear. Tn order to establish whether they occur at precise positions in the unit cell, the ratio of thin to thick filaments was found by counting the number of each type in relatively large areas of overlap regions in the electron micrographs. This ratio varied from 5: 1 to 6 : 1 with an average of 5.5 : 1. The number of thin filaments around each thick filament varied from 9 to 13 with an average of 11, consistent with the number previously reported by Sanger & 8zenLGy6rgi (1964). Tt is probable. however, that the number,

Flu. 1. Transverse sections of scallop striated adductor muscle, fixed in 4% glutaraldehyde in 15 mix-cacodylate buffer followed by 4% formaldehyde in the same buffer and post-fixation in osmium. (a) A low magnification view showing the elongated profile of t,he muscle fibres and the appearance of the filaments at different regions in the sarcomere. B, the bare zone; H, the part of the H-zone where the thick filaments show projections; 0, the region where the thick and thin filament,3 overlap; I, the I-band. Magnification, 26,000x. (b) (c) and (d) High magnification ( 110,000 x ) view of different regions of the sarcomere: (b) the bare zone; (c) the overlap region showing the filament lattice (the arrangement of thiu and thick fllament~s); (d) the beginning of t,he 1 region where the thick filamnnts have a decreasing diameter.

Fm. 2. Longitudinal sections of scallop striatcsd adduotor musolo. (a) Muscle fixed as in Fig. 1. Magnification, 36,000 x . (b) Part of the A-band in a longitudinal section. The muscle was fixed in 4% glutaraldehyde in half-strength sea-water buffered with 10 m&f-oaoodylate, followed by 2% formaldehyde in the same medium and post-fixed with osmium. Magnification, 54,000 x . iVote the banding which can be sren in some of tho t,hiok filaments (arrow) corresponding t,r) a regular wpeat of aboutj 14 nm. (0) Opt,ioal diffraction patt,nm of (b).

SCALLOP

STRIATED

ADDUCTOR

44R

MUSCLE

of thin filaments counted is an underestimate, since t,hese filaments do not appea,r to be as well-preserved as the thick filaments, nor their positions as well defined. The optical diffraction patterns from transverse sections in the overlap region showed only a few discrete reflect’ions from the hexagonal lattice, usually the 1 ,O, 1 ,I and 2.0 reflections. In addition we sometimes saw a diffuse ring of intensity at a posit’ion between three and five times the distance of the I ,O reflection from the origin. This ring of intensity was also present in diffraction patterns from masks punched with holes in the positions of the thin filaments seen in the sections, indicating that thtb thin filaments are partly ordered with respect to one a,nother wit’h an average sepamtion corresponding to the radial spa,cing of the ring (i .P. 10 to 15 nm). (c) Electroth microscopy of the thin ji1amen~t.s WV did not’ observe details of the thin filament structure in sections, but negatively stained preparations of the thin filaments exhibited the typical double-stranded appearance of F-actin (Hanson & Lowy. 1963; Hanson, 1967). The ends of the thin filament assembly were not sharply defined because: of disorder in the Z-line, and in order to estimate the length of the thin filaments it’ was necessary to measure individual filament lengths, with a resultant loss of precision. The filament lengths as measured from the end of a filament in the overla,p region to the centre of the Z-line were rather variable, but gave an average value of 14 pm (Table 1). In vertcbratc TABLE

~)ivw~rsion.s

of the $lamenJts

of scallop electron

1

striated

adrbucfor

nr us&

CIS determined

from

micrographs

Thin Jilaments: Length (longitudinal sections) Thick fclnmentn: Length (longitudinal soct,ions) Length of hare zone (longitudinal sections) (negatively stained) Width of bare zone (transverse sections) (negatively stained) Width of backbone (transverse sections) (negat~ively stained) Thin: thick filament rat,ir> in the overlal~ region

17 11111 0 = 2 21 nm 3 : 5.5: 1 (rang:, 5: 1 to 6: 1).

(54) (8%

t 0, Standard deviation. f Sumbor of moasuramcnts.

striated muscle the thin filament length can be measured more accurately for two reasons : the transverse register of the thin filament assembly is bett’er and the I-band shows stripes every 38.5 nm identified with the location of troponin (Ohtsuki et al., 1967). The scallop muscle does not exhibit these stripes, which is consistent with the finding that there is little or no troponin or other analogous prot,ein in this muscle (Szent-GpGrgyi et al., 1973). (d) Electron microscopy

of the thick jilaments

: sectioned

preparations

The dimensions of the thick filaments, measured on both sectioned and negatively stained material, are shown in Table 1. The length of the thick filament is difficult to measure. ln longitudinal sections the thick filament’s, like t’he thin, are not) in

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strict register across the fibre (again probably because of poor fixation) so t,hat the A-band does not have sharply defined ends and its lengt,h cannot be accurately estimated (Fig. 2(a)). In addition, the filaments t’aper at t,he ends and become SO thin that they cannot always be distinguished from thin filaments lying behind t,hem. Measurements of individual filaments gave a Iengt)h of I.76 pm (Table I), although because of filament taper, t,he backbone has a t hickncss great,er t’han 10 urn only over a length of 1.6 pm. The length of the lighter-stained bare zone is aboub 120 nm and the diameter of the filament in this region is approximately 22 nm, greater than the backbone diameter in any other part of the filament. In transverse sections the bare zones are approximately circular but occasionally seem to be polygonal (Fig. l(b)). From the end of the bare zone over a short distance at the beginning of the cross-bridge region, the backbone diameter decreases rapidly to 17 nm (cf. Fig. l(h) and (c)). It then appears to be constant for much of the filament length before tapering to zero over the final 0.3 pm (Fig. l(d)). Some other muscles which have thick filaments of approximately the same diameter as the scallop filaments (e.g. insect muscle: Auber & Couteaux, 1963) show in crosssection a non-staining area in the centre of these filaments. In our micrographs, no such non-staining region was seen either in the bare zone or close to it (Fig. l(b)). Such a region was occasionally seen in other parts of the filament (Fig. l(c)). but this was probably a fixation or staining artifact, since such “cores” were not seen with all fixation procedures. Of course, this does not rule out the presence of a cor(b of prot’ein other than myosin which has staining properties similar to myosin. In longitudinal sections, substructure can occasionally be seen in the crossbridge region of the thick filament. Dark lines are sometimes seen running across the filaments with an average axial separation of 14 nm (Fig. 2(b)). Optical diffraction confirms the presence of a periodic structure in the thick filaments. The pattern from a section (Fig. 2(c)) shows only meridional reflections corresponding to a basic prriodicity of 14.5 nm and its higher orders at 7.2 and 4.8 nm. No reflections were seen which could be related to a helical arrangement, of the projections or to a repeat,ing structure in the thin filaments. Nor was t)here any evidence for a longer periodicity in the thick filament such as the 43-nm repeat seen in sections of vertebrate striated muscle and attributed to extra prot,cin components (Huxley, 1967: Offer! 1972). Therefore, in sections, the substructure seen in the thick filaments is probably related to the packing of paramyosin and myosin rods in t,he filament shaft to give a 14.5 nm periodicity. (e) Electron microscopy

of the thick filaments : negatively

stained preparations

An electron micrograph of a filament preparation from the scallop cross-striated adductor muscle is shown in Figure 3. The thick filaments show many of the features that are seen in sections. They have a well-defined bare zone of length 120 nm and diameter 24 nm (Table 1). Except in the bare zone, the projecting myosin heads make it difficult to measure the backbone diameter, but about 0.1 pm away from the end of the bare zone it is approximat)ely 21 nm (Table 1). It was impossible to 34 pm), the 1 .l reflection is not seen, but it, appears at sarromere lengt.hs l&w this value and hrcomes more intense t,hc shorter the sarcomere. A strong but diffuse equatorial reflection wa,s regularly seen at about 13 nm (range from 11 to 16 nm) (Fig. 6(b)). T1It: sharpness and position of the reflection matched the sharpness and position in the radial tlitwtion of the actin reflection on

FIG. 0. X-ray diffraction patterns from scallop striated adductor muscle. (a) Living musclr+ using the mirror-monochromat,or camera with specimen-to-film distance of 20.8 cm. Sarcomerr length, 2.6 pm. (b) Living muscle pattern using double monochromator with specimen-to-film rliat,anco of 7.4 cm. Sarcomoro length, 3.6 pm. Tho contra1 insert is the back film from the same IIX~OSUPI?. (c) As (a), but glycerol-cstracted muscle, with specimen-to-film distance of 20.3 cln and srtrcomnro length --: 2.3 pm. ((I) .As (a). hut, air-tlrivtl muscle ant1 >l specimen-t,o-film distalwc: fus(qr,F:

45!)

tlifiicult,y in makiug these measurements since they involve small differences betweeu diffuse reflections (see Huxley & Brown (1967) for a detailed discussion of this point). The fact that in both fixed and rigor muscle patt’erns, where a first layer line could be seen, the direct measurement gave a lower value for the half-pitch than the difference method suggests that there may be a systematic error involved. In addition, dissimilarities between the intensity distributions along the layer lines in patterns from relaxed and rigor muscles may also induce such errors. Therefore. although we cannot discount the possibility of small differences in the actin symmetry between resting and rigor muscle, we consider that the best value for the actin half-pitch is the average of all our measurement’s, namely 38.5 nm. This value is greater than those obtained for vertebrate striated muscle (379 nm, Huxley $ Brown, 1967), and for a series of molluscan smooth muscles (35.6 to 37.3 nm, Lowy & Vibert, 1967), hut similar to those for insect flight muscle (38.5 nm, Miller & Tregear, 1972) and for horseshoe crab muscle (38.3 nm, Wray et al.: 1971). In view of the difficulty in determining this paramct’er accurately, we doubt that these differences are significant. Of t’he other thin filament proteins, the scallop muscle contains tropomyosin (SzentGyorgyi et al., 1973). but troponin seems t,o be largely absent. It does not, appear on sodium dodecyl sulphate gels in amounts comparable to vertebrate skeletal muscle (Bzent-Gyorgyi et al.. 1973; Bennett & Millman, unpublished results) although Dr Kendrick-Jones (personal communication) has detected small but variable quantities. In our electron micrographs we have seen no trace of the 38 nm periodicit! seen in electron micrographs of the I-band of vertebrate striated muscle and at’tributrd to t,roponin by Ohtsuki et al. (1967). It, was therefore puzzling to see in our x-ray patterns a series of sharp meridional reflections, orders of about 38 nm, which appeared to be similar to but somewhat weaker than those orders of about 38 nm attributed t)o troponin in vertebrate muscle (Rome, 1972; Rome et al., 1973). However, troponiu has a 38 nm periodicity only because it specificallv binds to tropomyosin which lies in the long pitch grooves of the actin filaments (O’Brien et al., 1971). Tropomyo+in is presumably packed in the same way in the scallop muscle. Our results therefore suggest that this series of reflections can arise \vhen tropomyosin alone is present, as in the scallop, although it is enhanced by the presence of troponin as in the thin filaments of vertebrate muscle. The equatorial reflection at about 13 nm which we have associated with the thin filaments (Results, section (g)) indicates that some type of order exists between them. The increased sharpness of the reflection at longer sarcomere lengths in conjunrt’ion with an increased sampling on the 5.9 and 5.1 nm actin layer lines at, ;t similar radial spacing suggest that in the I-band the actin filaments may form a limited lattice of period about 13 nm. The lattice is probably similar to that seen in other invertebrate and vertebrate muscles (Lowy & Vibert’, 1967; Elliot’t & Lowy, 1968 ; Rome, 1972) and associated with the I-band or regions lacking thick filaments. The observation of this reflection at shorter sarcomere lengths, and its strength in this muscle as compared t,o vertebrate striated muscle, suggests that an I-band thin filament lattice is not the only possible contributor to this reflection. It is quite likely that the thin filaments lying in between the thick filaments are aligned relative to one another with short-range order. This is supported by optical diffraction patterns obtained from masks corresponding to the thin filament positions in electron micro,graplrs of transverse sectioiis (Results. section (1,)). which show a diffuse ring at

(c) The surfufuce lnttice

of the fhickj1amerl.t

The detailed layer-line pattern obtained hy X-ray diffraction from living relaxed muscle has been ascribed t’o the helical arrangement of myosin heads projecting from the thick filament (Results, section (h)). Alt the layer lines index on an axial spacing of 145 nm. The presence of a st’rong 14.5 nm meridional reflection and a strong 3rd layer line determines the basic lattice on which the helicall~v arranged projections lie. This lattice is shown in Figure 9(a). The horizontal rows of projections are spaced 145 1

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FIG. 9. The t,hicli filament surface lattice in scallop striated atlductor muscle. The circles indicat,e the positions of scat,tering ccntres on the filament surface, assuming that the surface of the filament, has been spread on a plane. If the filament, backbone diameter is 21 nm, the base of the projections in any circumferential line will be separated by about 11 nm. The filled circles illustrate one of the long helices of pitch 290 nm (twice the unit cell length). (a) Lattice with rows of scattering centres spaced 14.5 nm along the filament, axis. (b) Latt,icr for living muscle in which alternate rows of scattering centres am displaced by 1.5 nm. For qt,ical tliffract,ion patterns from those lattices, SW Fig. 10 (b) and (d), respectively.

at 14.5 nm and give rise to the meridional reflection; the diagonal strands, of which three cross the vertical in one whole repeat of 145 nm, are the source of the strong 3rd layer line. At the intersections of these two sets of lines lie the projections, though note that one projection is not necessarily equivalent to one myosin molecule. The number (IV) of strands which constitute the structure is the same as bhe number of project’ions every 14.5 nm and is not uniquely determined by this data. In Figure 9 we have shown N = 6 for reasons discussed later. Figure 10(a) and (b) shows a punched mask of this helix and its optical diffraction pattern. The pattern shows meridional reflections at 14.5 nm and higher orders, and layer lines with axial spacings corresponding to the 3rd. 7th, 13t’h. et’c. orders of 145 nm, all of which correspond to

SCALLOP

STHIATE

1) .4 1) I)I’(‘TOK

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461

lG(:. 10. (a) and (c) Photographs of optical diffraction masks crmstructe(l from holes punche(l in ,,payuc film at positions corresponding t,o helical projections of t,he t,hick filament latt,ices shown in Fig. R(a) and (b), respectively. (e) Optical diffraction mask producc?d photographically from a series of projections like (c), alternately staggered by about, 7.2 nm and with centres at randomly select,ed lateral separations between 30 and 60 nm. (b), (d) and (f) Optical diffraction patterns of (a), (c) and (e), respectively. (b) Note t.he meridional r&actions corresponding to integral orders of 14.5 nm. (d) A pattern similar to (b) but with additional reflections corresponding to the 31~1 and 5th orders of 29 nm. (f) A simdar pattern to (d) but with let,tice sampling along the reflections and a clear splitt,ing of the 14.5 nm meridional reflection.

wfleations seen in the X-ray diffraction paWrn s of t,htl living muscle (see Table 2 and Pig. 7). 111addition t’o these reflections, our model must! also account for meridional reflections at 9.7 and 543 nm, the 3rd and 5th orders of 29 nm. It is not likely t,hat these rcflcctions. parbicularly the one at 9.7 nm, are from other parts of the muscle system or from additional proteins, since they are alwags (and only) present in patterns showing t*he other thick filament layer lines. They are absent, in patterns from gly’t~r”l-extracted, dried and fixed muscles. The occurrence trf these reflections suggvsts t,hat, the: axial repeat in these thick filaments is not Il.5 nm hut 29 nm. This bvould 1~: the CAW if evrt~y second ring of projections was displaced axially by a small amount. Lf the displacement, is about 1.5 nm, such that the projections OCCUI

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>I.

HENNETI

alternately at 13 and 16 nm intervals along the filament (Fig. 9(h)), the lowanglt~ diffraction pattern is very similar to that of the simple helix with a 14.5 nm subunit repeat except for the presence of extra meridionals at odd orders of 29 nm (Figurcl 10(c) and (d)). The first order is weak but the 3rd (9.7 nm) and 5th (5% nm) are nt,rong. We have chosen a 1.5 nm displa,cement to maximize t,he 9.7 and minimize the 29 nm meridional reflections. Although the other low-angle layer lines are unchanged, different layer lines would appear at higher angles. As predicted by this model, the layer line at 6.73 nm (Table 2) indexes more closely to the 22nd order of 145 nm rather than the 23rd order, as would be expected on the simpler model. It is not clear what may cause this alternating displacement’ of projections. Szent-GyGrgyi et al. (1973) not,ed t’hat light meromyosin from scallop striated adductor muscle forms paracrystals with a 58 nm repeat (2 x 29 nm) rather bhan the 43 nm or 14.3 nm repeat observed in paracrystals from other light meromyosins. This suggests that there may be a unique packing of myosin in t’he backbone of the scallop thick filaments. The above discussion is independent’ of the number (N) of helical strands which constitute the structure. It is necessary, however. to know this number in order to define the surface lattice exactly and to compare it with that deduced for other thick filaments. We can obtain an estimate for N from the radial position (R) of the maximum intensity along the layer lines. To a first approximation, the low-angle pattern can be thought of as coming from point scatterers lying somewhere between the edge of the filament backbone and the nearest thin filament, i.e. at a radius (r) from the thick filament axis between 10.5 and 26 nm. In this case, the intensity along the 3rd layer line (and the 7th. 13th and 17t’h layer lines as well) varies as the square of a Bessel function of order N. Assuming no lattice sampling along t,he layer line, the intensity is maximal at a specific value of 2n~H (the argument of the Bessel function) which depends on N (see Table 5). We observed a maximum at about R = l/14 nm-l (Table 2) with no lattice sampling. Allowing for the fact that the projections must have a finite size, and thus their cenbres must lie at least 2.5 nm from the filament surfaces, these values of r and R give a possible range for N of 5 to 8 (see Table 5). We can probably eliminate 8 as the number of projections every 14.5 nm. At long sarcomere lengths such projections would lie very close to the thin filaments. When the muscle passed into rigor there would be very little movement possible as the crossbridge attached to the t’hin filament and t,he observed change in the relative intensities of the equatorial reflections could not occur. A similar calculation for the number of helical strands can be carried out using the measurement)s made on optical diffraction patterns from negatively stained filaments. Here no lattice sampling can occur and the positions of the myosin heads can be TABLE

Arguments

5

(2nrR) and radial positions (r) for the ,first maximum Bessel function of order n

of rr.

SC’ALLOP

STItlATED

ADl~U(“~OB

MUR(‘LE

4(i:i

more accurately defined. In these patterns, however, the radial position of the 3rd layer line maximum is more variable because of the distortions induced in the filament by the preparative procedure. The results obtained give a range for N from 4 to 8. consistent with the X-ray data. The number of projections every 14.5 nm is probably therefore between 5 and 7. An independent estimate of r the distance of the projections from the filament axis can be obtained from the radial positions of the subsidiary maxima along the 14.5 nm layer line (Fig. 7, Table 2). The intensity distribution along t’his layer line should follow a zero order Bessel function. The first subsidiary maximum could not be used for this purpose because its radial position may be influenced by superlattice sampling (see Results, section (h)). The second subsidiary maximum, however, is far enough from the meridian so as not to be affected. Its position (l/14*3 nm-l) corresponds to that of the second subsidiary maximum of a zero order Bessel function arising from helically arranged scattering centres at a radius of 16 nm. For scattering centres at this radius, the radial position of the 46.4 nm la’yer line reflection is most consistent with its being a 6th-order Bessel function (Table 5). Our data therefore lead us to conclude that t,he number of helically arranged projections every 145 nm along the scallop thick filaments is six and that these projections lie with their centres of mass 16 to 17 nm from the filament axis. Wray et al. (1975) have recently used X-ray diffraction to study the crossbridge configuration of several invertebrate muscles. They calculated that a thick filament with 6- or ‘i-fold rotational symmet,ry and bridges centred at 17 nm was consistent with data from Placopecten magellanicus, in agreement with our results. Squire (1972,1973) has proposed a general model for muscle thick filament,s in which one myosin molecule is associat’ed with each point on the surface lattice. If in t,ht: scallop muscle each projection corresponds to a single myosin molecule, our model is similar to the specific model proposed by Squire for insect flight muscle. except that in the insect muscle there is no shift of alternate rows of projections. Elliott (1974) from his observations on paramyosin-containing filaments from molluscan smooth muscles has suggested that more than one myosin molecule may be present at each node on the surface of these filaments. If t’his is true for scallop striat’ed muscle filaments, each helical projection would consist of more than one myosin molecule. Knowing the ratio of actin to myosin in the muscle and the filament dimensions. and using a calculation similar to that performed by Tregear $ Squire (1973) for vertebrate striated muscle, we can estimate the number of myosin molecules every 14.5 nm. Szent-Gyorgyi et al. (1973) have estimated the weight rat,io of actin to myosin heavy chains in the scallop striated muscle by gel electrophoresis in the presence of sodium dodecyl sulphate and their figures give a ratio of 1:2.23. They also determined the molecular weight of these myosin heavy chains as 186,000. Using our measurements of filament lengths, bare zone length and thick-to-thin filament ratio (see Table l), a molecular weight for act’in of 41,800 (Elzinga d ul., 1973) and an axial period for actin of 2.7 nm. \ve obtained a value of nine for the number of myosin molecules per 14.5 nm. There are, however, uncertainties in the measurement of thick filament length and thick-to-thin filament ratio, which together with possible errors in some of the biochemical data make this figure rather imprecise. Thus. at this time. we cannot assign a specific number of myosin molecules to each projection of the scallop thick filaments. but only conclude that there are vitllw ont or t\vo tn~v)sin tllolt~t+lllt~s ~~ssotkttd wit11 twt*II of tlrth six pmjt~ctions.

4fi-l

IS. 31. Jlll,l,MAN

ASI)

(d) Thp j&mud

1’. 11. 1~1’:NNJ~‘I”l’

hackbow

The thick filaments from scallop striated muscle arc similar in lengt,h to those from v&&rate skeletal muscle and show a similar bare zone and baper at the ends. ‘I%(%> are: however, much thicker than t,he v&c hrate filaments. Our measuwnwnts of backbone diameter from sections in the crossbridge region ( 17 nm) is presumal)ly smaller than the value in living muscle. because of shrinkage during preparation. I:sinp X-ray diffraction, Elliott & Lrnvy (lQ70) sho\ved that the intermolwula,r distanw in paramyosin filaments from the smooth oyster adduct,or muscle drrrcased by about, lf;“,, on dehydrat,ion. This factor would give a value of 20 nm for the diameter of th(t filament backbone in living muscle, similar to that which KC observed in neg:tt,ivt~ly stained preparations where there is probably little shrinkage in the plane of the grid. Tt has been shown for other molluscan muscles, such a,s the obliquely striated and smooth adductors of the ogst,er, t,hat the thick fila,ments of these muscles contain a core of the protein paramyosin (Lowy & Hanson, 1962 : Hardwicke & Hanson. 1971: Szent-Gyargyi ef al., 1971). The scallop Ariated adductor also wntains paranlyosin. though in much smaller amounts than the ot.hw molluscan muscles. and it is reasonable to suppose that t’his protein a,lso occupies th(l thick filamc~nt cores in ttw scallop muscle. Certain features of the X-ray diffract’ion pattern at,tri huted to the scallop thick filaments are indicative of paramyosin-containing filaments. Tht~ sub.q 14.5 nm not, 14.3 nm as found in vertebrate muscle. unit repeat in the living muscle 1. This 14.5 nm periodicit,y is charact,eriatic of paramyosin musclw (Elliott. 1967 : Mil1ma.n & Elliott: 1972). Also. the 4.8 nm meridional reflection in both thr X-ra! diffraction pattern of whole muscle and the optical diffraction patbwn f’rom single as was found for filaments is greatly enhanced after fixation with glut,araldrhyde. muscle (Millman. unparamyosin muscles (Miller. 1968) but not for wrtebrat~e published results). These results, coming from fixed a.nd dried muscles where the myosin projections are disordered, indicate that the paramyosin in scallop muscle is present in the backbone of the thick filament. We thu CIconclude that’. as in other molluscan muscles, paramyosin forms tlrr core of t’llca scallop t,hick filaments around which the mposin rods are packed. The diameter of the paramyosin cow can be estimated from t,he amount, of this protein present, and the molecular lengths of paramyosin (129 nm. Cohen et OZ.: 1971) and the “t.ail” of scallop nlyosin (150 nm, Elliot,t et al.. 1976). One paramyosin molecule t)herefore can span nine repeats of 14.5 mn and the myosin “tail” can occupy ten such repeats. Assuming that, both paramyosin and m,wsin occupy the same cross-sectional area. and given t,hat the paramyosin in the muscle is 510 of the myosin heavy-chain component (Szent-Gyargyi et al., 1973), this amount of paramyosin would fill a core of diamet,er 6 nm in a filament, 21 rim in diameter. (c) The thick jilan/ent

superlatflce

In X-ray diffraction patterns from living and dried muscle, the 14.5 nm reflection is split across the meridian. The radial position of the resulting reflections corresponds t,o a, spacing about twice tha,t of bhe 1,O equatorial reflection. In patterns from dried muscle. the 7.2 nm meridional is not split, but the 4% nm reflection, although diffuse. is sufficiently broad as to suggwt) thnt it too is split. In t,he living muscle it is not possible to tell which, if 11tly. of tjtic ot,hclt. rnwidiond. s are split. only that th 7.2 rim one a,nd the extra one at 9.6 nm appear not to b(,. These results can be explained

s(‘AI~T,O

t’ S’I’R~ATEl~

XDDI’(‘TOR

III I’SC’LF:

Ifi.

I,!- a rq&~r axial displacement of neighhouring tilxments rc:lat,ivr to one anot,lw. This displacement must hc clost~ to 7.2 nm in t,hfb dried mua&. but may I)fb slightI>. greater in the living muscle, perhap, q related t,o t,hc small shift) in the position of projrctions every 29 nm. This axial displacement is a common feature of closely stained preparations (see Results. aligned pairs of filaments seen in negatively section (f)). The simplest thick filament arrangement consistent with the data, is the l)ody-wntred lattice shohvn in Figure S(h). which Ilas a unit cell \\ith twice the area of the basic hexagonal lattiw. .Uthough t#hr superlattice effects are seen in tlw meridional reflections, the othrl layer lines in patterns from resting muscle show no sign of lattice sampling, which suggests some kind of lat,tice disorder. The most, prohahle explanation is that the, filaments are regularly staggered relative to one another. as in Figure 8(h). hut that t,hcir azimuthal orientation is random. This is illustrated in Figure 10(e) and (f). \vhiclj show a projection of the filaments in the 1.1 plane of the superlattice and thtl optical diffraction pattern ohtained from a mask of this projection. Alternate filaments are displaced axially ty 7.2 nm. Each filament, has the helical spmmetr~ dcscrilr~etl in se&ion (c), ahore. hut they appear different because they are project,etl at random orientation. The optical diffraction pattern from this mask show only thtb Il..5 nm meridional clearly split (Fig. IO(f)). A similar split,ting of the 14.5 nm meridional reflection has been observed in t,hr> flight muscle from the blowfly (G. F. Elliott, 1965) and in horseshoe crab musclr (Wray et ccl., 1974). In t,he insect, muscle, the 7.2 nm reflection was also split but the 4.8 nm reflection was not,. This result has heen explained by an axial shift of a,d,jacent thick filaments by 4.8 nm t)o give a hexagonal superlattice with sides 1.7 times t hosch of’ the basic hexagonal latt’ice (Elliot, Millman & Worthington, unpublished rwults). In the> horseshoe crab, the split meridional is ohserved in rigor hut not, in order is mediat,ed 1)~. wlasc~~ mus&. suggestin, 0‘ that in this case the interfilamt~nt iwtin tilaments. 1II vwtt~hr,~tr striated muscle. the clear samplin g along the la,yer line+ depends on an exact orientation of the filament projections in t’he superlattice (Huxley 19,Brown. 196i). This muscle has M-line scaffolding to hold the filaments in the correct orientation. WC Ilaw not, observed such M-line bridges in the scallop muscle, although the M-liw may have been destroyed by our preparative procedures. Alternative]>-. othw wnst,rairits. such as charge effects. ma,v kwp the filaments in a superlattiw. (f)

Concllbsbons

‘l’hrb scallop croswtriated adductor muscle is one of a very few striated molluscnn musc~lrs. Its sarcomere structure is similar to that of vertebrate slow striated muscle titws. although the structure of its contractile apparatus differs from that of vertcbhratr muscle in that the thick filaments have a larger diameter and there are approximately I:! thin filaments about each thick filament. As in other molluscan muscles. thfl striated adductor contains little or no troponin and the regulation of cont)raction is associabed with the thick filaments (Szent-Gyiirgyi et al., 1973). Despite the fact that thf: muscle contains paramyosin. albeit’ in small amounts, its physiological properties art similar to those of vertebrat-e striated muscle with contraction times matching those of frog sartorius muscle and it exhibits none of t)hc “cat’ch” properties of &her molluscan muscles (Millman, 1967). Sf~vcwl prohlcms remain to IN%solved c~oncfwCng thcb structurc~ of this muscle. \Tc

466

n. Iv. MTI~I,Jf.\N .ANI) I’. 31. Ii~NSli:‘r’l

do not, JY+ know how pwramyosin is packed in t81wthick tilamwts nor ho\\. it,s parking is related to the packing of myosin. What, is the cause of the alt,ernat,e staggering of projections along the thick filaments which gives rise to the 29 nm axial periodicity ! What specific structures in the thin filaments produce the series of meridional reflections at. orders of about 38 nm-tropomyosin itself or a distortion of the actin helix-and is this relat,ed to regulation in this muscle: These problems will bc c%xplored in future work. We would like to dedicate this paper t,o t.hc memory of Professor Jean Hanson, ~IIO did much original electron microscopy on the cross-striated adductor, and who cncomaged and inspired us throughout the experimentation. She took as much excitement from t,he results as we did ourselves. Her comments. criticisms and ideas Ira,vta added considerably to the substance of this research. We would also like to thank Drs A. Elliott, E. J. O’Brien and G. Offer for helpful discussion, Mrs D. Terry and Mrs W. Pettyan for technical assistance and Mr Z. Gabor for photographic assistance. One of us (B.M.M.) is grateful to t,he National Research Council of Canada for a Travel Fellowship and for operat.ing grant support,. REFERENCES April, E. W., Brandt, P. W. & Elliott, G. F. (1971). J. Cell. Biol. 51, 72 82. Auber, J. & Couteaux, R. (1963). J. Microscopic, 2, 309-324. Baguet, F. (1973). Pjliigers Arch. Ges. Physiol. 340, 19-34. Bozler, E. (1930). 2. Vergl. Physiol. 12, 579-602. Cohen, C., Szent-GyGrgyi, A. G. & Kendrick-Jones. .J. (1971). J. Mol. Biol. 56, 223-237. Elliott, A. (1965). J. Sci. In&. 42, 312-316. Elliott, A. (1967). In Symp~ium on Fibrous Proteins, p. 115, Butterworths, Australia. Elliott, A. (1974). Proc. Roy. Sot. ser. B, 186, 53-66. Elliott, A. & Lowy, J. (1970). J. Mol. Biol. 53, 181-203. Elliott, A., Offer, G. & Burridge, K. (1976). 1’roc. Roy. Sot. ser. R, 193, 45 53. Elliott, G. F. (1965). J. Mol. Biol. 13, 956--k%. Elliott, G. B. & Lowy, J. (1968). n’ature (London), 219, 156-157. Elliott, G. F. & Worthington, C. H. (1963). ,J. Ultrastruct. Res. 9, 166-170. Elliott, G. F., Lowy, J. & Worthington, C. K. (1963). J. Mol. Biol. 6, 295-305. Elzinga, M., Collins, J. H., Kuehl, W. M. & Adelstein, R. 8. ( 1973). Proc. Nat. ~lcarl. Sci., ti.S.A. 70, 2687-2691. Hagopian, M. (1966). J. Cell Biol. 28, 545. 562. Hanson, J. (1967). Nature [London), 213, 353-356. Hanson, J. & Lowy, J. (1960). In Structure and Function of &IuscZe (Bourne. (:. H., ed.), vol. 1, p. 265, Academic Press, New- York. Hanson, J. & Lowy, J. (1963). J. Mol. Biol. 6, 46-60. E. .J. & Bennett. P. M. (1972). Cold Spring Harbor Hanson, J ., Lednev, V., O’Brien, Symp. &ant. Biol. 37, 311-318. Hardwicke, P. M. D. & Hanson, J. (1971). J. Mol. Riol. 59, 50% 516. Huxley, H. E. (1953). Proc. Roy. Sot. ser. B, 141, 59-62. Huxley, H. E. (1967). J. Gem. PhysioZ. 50 (suppl.), 71-83. Huxley, H. E. (1968). .I. Mol. Biol. 37, 507-520. Huxley, H. E. & Brown, W. (1967). J. Mol. Biol. 30, 383-434. Lednev, V. V. (1974). Biojizika, 19, 116-~121. Levine, R. J. C., Dewey, M. M. & Villafranca, G. W. (1972). J. Cell Biol. 55, 221-235. Lowy, J. & Hanson, J. (1962). Physiol. Rev., 42, 34-47. Lowy, J. & Vibert, P. J. (1967). Il’ature (London), 215, 1254-1255. Lowry, ,J., Hanson, J., Elliott, G. F., Millman, B. M. & McDonough, M. FV. (1966). In Principles of Biomolecular Organization (Wolsterholm, G. E. W. & O’Connor, M.. ods), p. 229, Churchill, London. Marceau, F. (1909). Arch. 2001. Exp. Gin. sect. 5, 2, 29S469.

S.. J. S.. Vihert, I?. .J. & Cohen, C. (1975). Nature (London), 257, 561. 564. \Vriyl(l)-. K. G. (1968). .J. Cltrastrztct. Res. 24, 454GWa.