The ultrastructure of striated muscle from Limulus polyphemus

J. ULTRASTRUCTURERESEARCH5, 151-165 (1961)

151

The Ultrostructure of Strioted Muscle f r o m

Lirnulus polyphemus1, 2, GEORGE W. DE VILLAERANCAand DELBERT E. PHILPOTT

Department of Zoology, Smith College, Northampton, Massachusetts, and The Marine Biological Laboratories, Woods Hole, Massachusetts Received August 22, 1960 The fine structure of Limulus polyphemus skeletal muscle has been studied with the electron microscope. The striations were found to consist of A, I and Z bands only; no M lines, H zones, or other striations were observed. In addition mitochondria, sarcolemma, and sarcoplasmic reticulum were figured and described. The myofilament structure seemed to consist of large filaments about 160/~ in diameter in the A band, and small filaments, about 30 ~ in diameter, in the I band. Small filaments were sometimes observed in the A band when the specimen was prepared under tension. When no tension was applied during fixation, cross-bridging material and large filaments only were observed in the A band. An occasional hexagonal pattern of large filaments was also seen in this muscle. It was proposed that Limulus striated muscle represents a structural, biochemical, and physiological type intermediate between "classical" smooth muscle and striated muscle. It was proposed that a double filament structure might serve a function in stretch, rather than as a basis for contraction. A widely accepted theory of muscle contraction has been proposed which depends upon the existence of a double array of filaments (ll, 18). It is believed that the small, secondary filaments, extending from the Z band into the A band, slip past the larger, primary filaments of the A to give the shortened state. The double arrangement of filaments has been beautifully demonstrated by the electron microscopy of H. E. Huxley (16, 17). SjSstrand (33, 34, 35) and others (23) have not, however, found a double set of filaments in vertebrate striated muscle and raise the question as to what is the in vivo arrangement. The filamentous organization of invertebrate muscle is even less well known. Apparently a double filament pattern exists in certain insect muscle (11, 12, 17), although 1 Contribution number 237 from the Dept. of Zoology, Smith College. Work supported by a P.H.S. grant, A2647, from the Institute of Arthritis and Metabolic Diseases, Public Health Service. 3 A preliminary account of this work appeared in Biol. Bull. 117, 387 (1959).

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G E O R G E W . DE V I L L A F R A N C A A N D D E L B E R T E. P H I L P O T T

in other insects (4, 13, 14) cross-bridging, rather than secondary filaments, lies between the larger, primary filaments. Both smooth and striated muscle of molluscs contain two sets of filaments (12, 25); in smooth muscle of this group, however, the protein composition of the filaments is probably different from that of vertebrate myofilaments

(20, 21, 23). The structure of Limulus muscle was first described by Jordan in 1917 (19). He found no evidence of M lines or H zones in the central region of the A band. A similar striation pattern was also found in certain insect muscle (4, 9, 12, 26), making this muscle quite different from the vertebrate striated muscle which serves as the model for the sliding filament theory of contraction. Results of previous investigations on Limulus muscle point up other differences which are difficult to reconcile with the sliding filament hypothesis. In contradistinction to vertebrate muscle, A band removal results in extraction of actomyosin rather than myosin (7) and contraction of the fiber results in a shortening of the A more than the I band, rather than the I band alone (6). It seemed, therefore, that a reinvestigation of the structure of Limulus muscle, using electron microscopy with especial attention to the filament organization, might prove fruitful in extending to other striated muscle, or in revising, the sliding filament theory of muscle contraction.

MATERIAL AND METHODS

L. polyphemus, the horseshoe crab, was obtained from Woods Hole. The animals were killed by exsanguination from severed legs. In most cases the dorsal muscle, extensors of the abdomen, was used, and either removed and glycerinated (6), or fixed in situ with 10 % formalin. Leg muscle, when used, was fixed in situ with 10 % formalin. After glycerination for 10 or more days in 50 % aqueous glycerol at -20°C, small pieces of muscle were teased from the glycerinated bundles and fixed with 1% osmic acid in 0.04 M KC1 plus 0.0067 M phosphate buffer at about 7.4. The KCl-phosphate buffer solution had been used in previous investigations for blending and washing muscle fibrils (6, 7); phase contrast microscope observations gave every indication of normal structure. Muscle fixed in situ was injected with 10% formalin in an artificial "serum" of 0.922 osmolar concentration buffered with phosphate at about pH 7.4. The artificial "serum" was prepared according to the concentrations of various salts found in Limulus plasma (29). After 15 minutes of formalin fixation, pieces of muscle were removed and washed with "serum"; some were examined with phase contrast to check on the structure and again appeared "normal." Fixation for varying lengths of time (45 minutes 1, 2, 4, and 21 hours) but usually, and unless otherwise specified, 1 hour with 1% osmic acid in the "serum" followed the preliminary formalin fixation. In some cases, and where indicated, the formalin- or glycerin-fixed muscle was placed on a rack and slight tension exerted on the muscle throughout osmic fixation, dehydration, and up to the point of embedding. After fixation in osmic acid, regardless of prior treatment, the material was washed and

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then dehydrated with isopropyl alcohol, and counter-stained with 2 % phosphotungstic acid in absolute isopropyl alcohol, for at least 1 hour and usually overnight. Embedding was either in a mixture of butyl-methyl methacrylate (3 : 1) after several previous changes, or in Vestopal W (31). In the latter case, after PTA staining, the material was transferred from isopropyl alcohol to acetone. Several changes of acetone followed, and the material was run up through increasing concentrations of Vestopal in acetone, to final embedding in Vestopal. Hardening for the methacrylate procedure was carried out at 60°C using prepolymerized methacrylate as the final mixture. Hardening for Vestopal was carried out at room temperature for 24 hours to allow complete impregnation, and then the material was incubated at 60°C for 24 hours to obtain complete hardness. Thin sections were cut on a special microtome (24) which had been modified to give a constant torque of the specimen wheel even at very low revolutions per minute. The sections were examined on carbon or Formwar under an RCA EMU 2B, modified by an external power supply to give magnifications up to 70,000 diameters. RESULTS GENERAL STRUCTURE

Electron micrographs of Limulus muscle at low magnifications (Fig. 1) confirm the cross-striated pattern described from fixed and stained material (13), and from fresh and glycerinated material (6) observed by light and phase microscopy. In contrast to vertebrate striated muscle, only three of the cross-striations are apparent; A, I and Z bands. The A band is readily distinguished from the I by the presence of large filaments in the former (Figs. 1 and 7), while the I contains only small filaments (Figs. 1, 4 and 7). The A band is manifestly of uniform density throughout its length when observed with the electron microscope; never has an M line or an H zone been encountered in the course of our three years of observation under a wide variety of conditions. It is of some importance to note that even in stretched Limulus muscle (muscle which had been stretched before fixation) an H zone does not appear. In Fig. 1 the sarcomere length is about 8.5 # which, assuming that the rest length of this muscle is 7.5 # (6), would correspond to a stretch of about 13 %. Thus, if small filaments were being pulled out of an A band, as would appear to be the case in stretched rabbit psoas muscle (17), one would certainly expect an H zone at this degree of stretch; that is, stretch which would yield a sarcomere length of 2.82 # and an H zone (5) in rabbit psoas does not give one in Limulus muscle. The Z band structure varies from one preparation to the next. In m a n y of our observations it appeared that filaments of the I band continued right through the Z band (Figs. 1, 4 and 7), much as reported in other investigations (8, 14, 28, 33, 34). Frequently in glycerinated material the Z band would appear granular in longitudinal section, or almost like a cross-section of small filaments. Occasionally the Z band was structureless with filaments anchored to its surface on both sides. It may well

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be that in the latter case the apparent lack of structure could be a result of glycerination before osmium fixation. Continuity of the Z band from one myofibril to the next was never clearly demonstrated in our preparations. In fact, a straight Z line running across a major portion of a fiber was rather a rare occurrence: more frequent was the zig zag pattern shown in Fig. 1. At the lower mangifications of phase contrast (6), however, the Z bands of adjacent myofibrils appeared most commonly in register. The Z band does, we believe, connect to the sarcolemma of the muscle fiber (Fig. 1). Frequent observations in which the membrane is thrown into folds, similar to those figured by Jordan (19), clearly demonstrate that the indentations of the membrane, or the residual attachments of the membrane, always occur at the Z-band level. This is indicated in Fig. 1, although it is by no means as pronounced as in some of our micrographs. Moreover, at the level of the Z bands, there seems to be a thickening of this material into a plate which is extremely electron dense. Sometimes this thickening occurs as a "sole" to a foot-like extension of the sarcolemma. The outer covering of each muscle fiber again shows a variability of structure with variations in fixation and from preparation to preparation. In preparations which we considered to show the best fixation because filament packing was more regular, and vesicular, mitochondrial, and surface membranes were clear, the structure separating two muscle cells appeared to be composed of from four to six membranes (Figs. 1 and 2). The inner membrane, or inner two membranes, are probably comparable to the sarcolemma as described by Robertson (30). The innermost membrane of the sarcolemma may develop into tubes, transverse to the myofilament axis, which penetrate into the sarcoplasm of the fiber (Fig. 2) and perhaps into the myofibrils (Fig. 10). This has been observed in almost all preparations showing the muscle membranes. These tubes may well be continuous with the sarcoplasmic reticulum. Caveolae (2) have also been observed along the membrane lying in the sarcoplasm (Figs. 2 and 10). Frequently, and particularly in glycerinated material, the structure between two muscle cells appears homogeneous, although a delimiting membrane can be made out next to each cell's sarcoplasm. Although fixation was not directed toward their study, other structures frequently encountered were numerous vesicles existing between the myofibrils (Figs. 1, 3, FIG. 1. Fresh-fixed Limulus muscle embedded in Vestopal. No tension applied during fixation. Bands marked A, I, and Z; S, sarcolemma and muscle membranes; s.r., sarcoplasmic reticulum; m, mitochondria. Arrow marks the electron-dense sarcolemma-Z band junction. × 5720. Fla. 2. Cross-section of Limulus muscle fresh-fixed, without tension, and embedded in Vestopal. Abbreviations as in Fig. 1. Note the tubule, marked by an arrow, given off by the muscle membranes. × 41,250. FIG. 3. As in Fig. 2. The myofibril, marked by the letters my, appears almost completely surrounded by a tubule of the sarcoplasmic reticulum, x 61,600.

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and 9). These, in all probability, are part of a sarcoplasmic reticulum network (1, 9,

27, 28) which is, apparently, quite extensive in this animal. There appears to be clumping of small vesicles in the I-Z region and between Z's of adjacent myofibrils. Larger and more numerous vesicles separate myofibrils and completely encircle them at the A-band level (Figs. 1 and 3). It is probably this network, of a different refractive index from the myofibrils, which occasionally gives the impression of an H zone in whole, teased fibers viewed under phase contrast (6). Fig. 3 shows one myofibril almost continuously surrounded by this tubular system. Apparently the majority of the tubules runs transverse to the major muscle and myofilament axis, although occasional tubules may run parallel to the major axis (Figs. 3 and 9). Mitochondria are not too numerous in this muscle. They occur on each side of the Z bands (Fig. 1) and just below the sarcolemma (Fig. 2). It may well be that only one mitochondrion occurs at the Z band (Fig. 4). At different sectioning levels one mitochondrion could give the impression of two separate mitochondria. The typical internal tubular structure is readily apparent (Fig. 4). Nuclei are large and encountered at the surface of the muscle fibers (Fig. 10). MYOFILAMENTS

The A band is composed of large filaments which run continuously from one edge of the A band to the other (Figs. 1, 5, 6, 7 and 8). The filaments are approximately 160 A in diameter, with a space of 180 A between filaments measured after fresh fixation, Vestopal embedding, and in cross-section. The dimensions determined after glycerination, methacrylate embedding, and from cross-section are approximately 160/~ in diameter (average), with a space of approximately 100 A between filaments (Table I). The interspace values for filaments after glycerination are probably low owing to shrinkage caused by the glycerin procedure. In preparing glycerinated material for the electron microscope it is difficult to decide which is better; osmotic shock (i.e., plunging the fibers directly into fixative from 50 % glycerol), or slow equilibration with the fixative. It happened that shock was used on the glycerinated material used in constructing Table I. For the reasons suggested here, undoubtedly the best preservation was given by fresh fixation in the serum fixative. An hexagonal arrangement of large filaments may occasionally be observed in both glycerinated and fresh-fixed muscle in cross-section (Figs. 3 and 9), although most FIG. 4. Fresh-fixed muscle, fixed and dehydrated under tension; Vestopal embedded. The cristae mitochondriales in the mitochondria may be seen. The mitochondria seem pinched at the level of theZ bands. × 37,100. F16.5. Fresh-fixedmuscle,fixedand dehydrated under tension and embeddedin methacrylate. Section through center of an A band. × 289,000.

L

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GEORGE

W. DE VILLAFRANCA

AND DELBERT

E. P H I L P O T T

TABLE I DIAMETER

OF T H E L A R G E F I L A M E N T S

OF T H E m B A N D A N D T H E S P A C E B E T W E E N F I L A M E N T S

All measurements were made directly on the electron microscope plates of cross-sectioned Limulus muscle, with a dissecting scope, using a Bausch and Lomb screw micrometer. Ten different filament and interspace measurements were made on each exposure and averaged. Method of fixation

Glyc.-wash Glyc.-wash Fresh-serum Fresh-serum Fresh-serum

Tension during fixation

No Yes No Yes Yes

Plastic used for embed,

Methacry. Methacry. Vestopal Vestopal Methacry.

N o of expos, meas.

14 12 8 5 3

Range in average

122-158 132-227 153-203 136-165 156-216

Average ill. diam. in ,~

S.D. ~:

Average interspace (,~)

M.D. --

138 192 170 155 204

9 25 17 15 36

79 127 201 155 208

14 45 17 19 52

frequently the more regular pattern observed was diamond-shaped. In some preparations material with a diameter of about 30 • exists between the large filaments in the A band region (Figs. 3 and 9). This material m a y very well be secondary filaments but we were never able to observe a regular arrangement of the small filaments a r o u n d the larger filaments: that is, six small filaments, or for that matter, any consistent n u m b e r of small filaments a r o u n d one large filament. In some cases (Fig. 9) as m a n y as five small filaments could be made out in the space between three large filaments. The n u m b e r apparently varies, and seems to increase in the regions which m a y be interpreted as I - I junctions (Fig. 9 and also Fig. 7). This would suggest that if these small filaments are continuous with the small filaments of the I band, they would either end abruptly in the A band or coalesce to make the large filaments. In some cross-sections the fine filaments appear as cross-bridging material (Figs. 2 and 11), while in other cross-sections no filaments are apparent. There is merely h o m o g e n e o u s interspace material. After a period of three years, during which we have varied specimen preparation to include both fresh and glycerin fixation, with and without tension, different plastics, etc., we are able to place our observations of longitudinal sections of horseshoe crab muscle into two groups. First (illustrated by Figs. 6 and 8), and most frequent of our observations, are those in which only large filaments (160 A) with cross-bridging material between can be seen in the A band. Here there is no evidence of small, secondary filaments; one m a y observe only large filaments together with smaller material which is considered cross-bridging. In Fig. 8 the plane of sectioning is n o t consistently at one level of the filaments, and it m a y be seen that where a large filaFro. 6. Glycerinated muscle fixed without tension and embedded in methacrylate. × 100,500. Fio. 7. Prepared as in Fig. 5. Note the filamentous structure of the Z band, and the large number of secondary filaments between the larger filaments. The arrows indicate secondary filaments running through the center of the A band. × 63,900.

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~i~ ~i~ ji~~~ i ~

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~~i!~!~i~, ~

~i~ J

160

GEORGE W. DE VILLAFRANCA AND DELBERT E. PHILPOTT

ment passes out of the plane of section there is neither any indication of a small, secondary filament, nor are there any small filaments between the widely dispersed filaments (Figs. 6 and 8). Thus, if there were small secondary filaments running parallel to the major axis, they should either appear in the wide clear area between adjacent large filaments, or they should appear when the space between the large filaments becomes wider owing to the fact that one large filament has passed from the section plane. In Fig. 6, the cross-bridging material seemingly forms a spiral structure winding around the larger filaments. The second group of observations was made after the suggestion of Dr. H. E. Huxley that the muscle be fixed and dehydrated under slight tension in order to keep the filaments lined up. When this was done it became fairly common, but not presumptive, to find good secondary filaments running between the larger filaments (Fig. 7). There is, however, considerable difference between the organization of these secondary filaments and the organization described for vertebrate striated muscle (17). As mentioned before, variable numbers of the secondary filaments exist between the primary filaments (Fig. 7). Sometimes at higher magnifications (Fig. 5) there is so much material that it is difficult to make out discrete filaments. A second, and extremely important, difference is that secondary filaments run through the center of the A band: the region forming the H zone in vertebrate striated muscle, and lacking in secondary filaments (17). This would explain the lack of an H band in this muscle. It should be noted, parenthetically, that we have never encountered a region of pure large filaments in cross-section: one always finds some kind of material between the large filaments. Thus, a large number of observations, on the one hand, where no secondary filaments were observed, plus the fact that occasionally, and so far only, when muscle is subjected to tension one may see secondary filaments, makes it impossible at this time to state unequivocally whether secondary filaments do or do not exist in this muscle. Furthermore, it is also true that, even when tension is applied, one does not always obtain secondary filaments under conditions of sectioning and observation where one would expect to see them. It is difficult to assess the exact conditions necessary for observing secondary filaments and whether or not they exist in the living, physiologic muscle. It may well be that cross-bridging material in the form of a series of " H ' s " spread between parallel large filaments may be pulled into line by tension applied during fixation and dehydration. This would appear as a secondary filament at the resolutions used here. Or the converse situation might hold true: that is, unless tension is maintained, the delicate structure of the secondary filaments ~s lost and cross-bridges are observed. The I band is easily distinguished from the A band by its lack of large filaments. Occasionally, however, one or two large filaments have been observed in the i band

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FIG. 8. Prepared as described in Fig. 6. x 145,300. FIG. 9. Cross-section of muscle which was fresh-fixed with tension; embedded in Vestopal. Note the large number of secondary filaments between the larger filaments, x 124,200.

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which may have resulted from a clumping of the small filaments. The precise organization of the I band is difficult to ascertain. In Figs. 4 and 7 the I band seems to be composed of an unorganized network of small filamentous material. Even when tension is applied (Fig. 7) the material remains in the form of a network. The connection between the large filaments of the A band and the I band filaments, if such a connection exists, is not at all clear, although it would appear, in certain observations, that the large filaments unravel to form small filaments as suggested recently (35). DISCUSSION The striated, skeletal muscle of a primitive, generalized arthropod, L. polyphemus, seems to reflect just those qualities. Its fine structure seems to place it somewhere between single-filamented smooth muscle (generally considered to be primitive), and the highly specialized, double-filamented striated muscle of vertebrates, pecten (25), and some insects (12, 17). The sarcomeres are long and uninterrupted either by M lines (see also 19, 6), comparable to certain insects (Coleoptera (4) and Orthoptera (9)), or by H zones. If one assumes that the secondary filaments of the horseshoe crab muscle are artifacts due to tension applied during fixation, this striated muscle then has, in c o m m o n with "classical" smooth muscle (12), only one set of filaments (3, 32) although they are considerably larger than the filaments of smooth muscle. Also in common with smooth muscle, but, perhaps, related to a single filament structure, is the finding that myosin extraction methods applied to Limulus muscle always resulted in extraction of the actomyosin complex (7), and unpublished results on uterine muscle of the rabbit. Lastly, Limulus striated muscle lies somewhere between smooth muscle and vertebrate striated muscle in speed of contraction (15). The question of whether or not there are two sets of filaments in Limulus muscle cannot, however, be answered categorically and unequivocally at this time. The possibility that the secondary filaments observed under certain conditions are artifactual has been dealt with elsewhere in this paper. If the two filaments observed are real and exist in the living muscle, their role must differ in Limulus and in other striated muscle (11, 16, 17, 18), because the large filaments probably contain both actin and myosin (7), and because the secondary filaments extend through the center of the A band, and possibly all the way through the A band, without interruption by an H zone. Since there is no H zone, it is difficult to imagine sliding small filaments without

Fro. 10. Muscle flesh-fixed under tension and embedded in Vestopal. Note the tubular and vesicular marked by arrows arising from the sarcolemma. A nucleus with its double membrane is located in the upper left. x 91,000. FIo. 11. Muscle fresh-fixed, but without tension applied, and embedded in Vestopal. Specimen supported on the grid by the Vestopal alone. × 103,000.

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GEORGE W. DE VILLAFRANCA AND DELBERT E. PHILPO]TT

some contraction or piling up on their part. It is conceivable that the small filaments could be contractile, as postulated in clam muscle (25), but this is rendered unlikely by the extraction experiments (7) and by the fact that the A band shortens more than the I from rest length to equilibrium length (6). In this muscle the secondary filaments might serve to bind the larger filaments together and to hold them to the Z bands. Further, they might also serve an elastic function (6); in the I band they appear to form a network which could be oriented to tension, giving the I band a springy quality. The contractile element in Lirnulus muscle thus would appear to be the large filaments which are probably composed of actomyosin. The muscle would be, in effect, a single-filament-type muscle. It is rather difficult to conceive of a uniform theory of muscle contraction which could be applied to single-filament as well as to double-filament muscle. Since it has not been established whether or not actin is restricted to the I band of vertebrate muscle (10, 21, 23), it is possible to postulate an actomyosin contractile system in the filament overlap area of the A band (vertebrate), in the A band (Limulus), or in the entire smooth muscle cell, which would pull on the Z bands via small filaments arranged in parallel (vertebrate), parallel and series (Limulus), or on the cell membranes directly in smooth muscle. This would have the advantage of unity of contraction mechanism: actomyosin threads, smooth muscle, Lirnulus muscle, and vertebratetype striated muscle would all have the same mechanism on the protein level (contraction of actomyosin), but would differ on the connections between the contractile proteins and the structure to which the contractile force is applied. The chief disadvantage to such a postulate is that it tells us nothing about the actin-myosin interactions which provide the contractile force and thus, unfortunately, we are no better off than before. The authors believe that it might be constructive to consider an alternative view to the function of two sets of filaments (6). Striated muscle connected to a skeleton structure (with an exception in the case of insect visceral muscle (16)) is liable to be pulled on abruptly by antagonistic muscle action. If the contractile elements were hooked directly to membranes, as in smooth muscle, without the intervention of some yielding element (a sliding secondary filament in vertebrate muscle; the I network in Limulus muscle) the contractile protein structure might be torn by a sudden contraction of the antagonist. The two sets of filaments would, therefore, serve a stretching rather than contracting function. The authors wish to acknowledge with gratitude the helpful discussions of this problem which they have had with Dr. A. G. Szent-Gy6rgyi. This and the preceding paper (6) are dedicated to the memory of the late Suzanne Crane de Villafranca, whose love, understanding, and good fellowship aided the authors beyond measure.

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