the fine structure of obliquely striated body wall … · by hanson. the myofibrils on opposite...

J. Cell Sci 7, 233-261 (1970) 233

Printed in Great Britain

THE FINE STRUCTURE OF OBLIQUELY

STRIATED BODY WALL MUSCLES IN THE

EARTHWORM, LUMBRICUS TERRESTRIS LINN.

P. J. MILL AND M. F. KNAPP

Department of Zoology, University of Leeds, England

SUMMARY

The fine structure of obliquely striated muscle fibres from the body wall of the earthwormhas been investigated. Certain details of the structure have been confirmed by cutting serialsections. The fibres contain both thick and thin myofilaments. The latter are attached to Z-matenal and the 2 types of myofilament are arranged in interdigitating arrays to give rise toA- and I-bands and an H-zone similar to those in cross-striated muscle. The A-bands containboth thick and thin myofilaments and the I-bands only thin myofilaments.

The Z-matenal is rod-shaped and these Z-rods, oriented perpendicular to the sarcolemma,are arranged in numerous parallel rows which run obliquely along the length of the fibre Aline drawn parallel to the longitudinal fibre axis through a Z-rod in one row passes through aZ-rod in the next row. A thin, sheet-like array of myofilaments lies between 2 such Z-rods,forming a single sarcomere containing an A-band and 2 I-bands. The flat surfaces of neigh-bouring sarcomeres are directly apposed to one another but, since the rows of Z-rods rundiagonally along the length of the fibre, each sarcomere is displaced longitudinally with respectto the next, so that the A- and I-bands follow an oblique course, instead of a transverse course asin cross-striated muscle. Because of the regular stagger of the sarcomeres A- and I-bands arecut alternately in transverse sections. Also the sarcomeres are very narrow and are seen asbands lying perpendicular to the sarcolemma.

In the A-band a variable number of thin myofilaments (up to 12) surrounds each thick one.Cross-links have been seen between the 2 types of filaments.

In longitudinal sections an appearance similar to that seen in cross-striated muscle is ob-tained in one plane (perpendicular to the sarcolemma). In the plane at right angles to this(parallel to the sarcolemma) the A- and I-bands are at an acute angle to the longitudinal fibreaxis. The thick myofilaments exhibit a banding of about 15 nm.

There is a system of transversely oriented tubules and peripheral vesicles with dyad-likestructures occurring at the juxtaposition between the peripheral vesicles and the sarcolemma.It is concluded that this system is sarcoplasmic reticulum, and it is compared with tubularsystems in other muscles.

Other cellular constituents are described, including a peripheral skeleton of fibnllar bundles.

INTRODUCTION

The interpretation of the fine structure of the body wall musculature of annelidshas in the past presented certain difficulties. Under the light microscope the musclefibres in the earthworm, Lumbricus terrestrts, have oblique striations, the angle betweenthe striations and the longitudinal axis of the fibre increasing from 50 in the relaxedmuscle to 300 in the contracted muscle (Hanson, 1957). These striations were notconsidered to be homologous with the transverse striations of striated muscle sensuostricto, and with the aid of the electron microscope were seen to result from the

234 P- J- Min and M- F- Knapp

presence of thick myofilaments packed into ribbon-shaped bundles called ' myofibrils'by Hanson. The myofibrils on opposite sides of a single muscle fibre slope in oppositedirections so that a diamond lattice-like arrangement is seen. This muscle was classi-fied as 'helical smooth muscle' by Hanson & Lowy (i960).

Working on 2 species of earthworm, Pheretima communissima and Eisenia foetidu,Kawaguti & Ikemoto (1957) described myofilaments in a sheet-like arrangement,alternating with electron-dense bodies which they called J-particles. In subsequentpapers Kawaguti & Ikemoto (1959) and Ikemoto (1963) demonstrated the presence of2 types of myofilaments in Eisenia foetida and showed that these are arranged in amanner similar to that of classical striated muscle; that is to say with the thin myo-filaments attached at one end to a J-particle and the thick ones lying between the thinones but not extending as far as the J-particles. The J-particles were shown to beaggregated into J-rods. They claimed that the thick myofilaments taper towards theirends and that each thin filament starts to one side of the centre of a thick one, runspast the end of the latter, and connects with a J-rod. However, in contrast to striatedmuscle, each row of myofilaments is displaced along the long axis of the muscle withrespect to the next row so that there is an oblique rather than a transverse banding.They initially classified this muscle as smooth, but later called it obliquely striated.They demonstrated a similar arrangement in the body wall muscle of the leechHirudo nipporda (Kawaguti & Ikemoto, 1958). In the earthworm, Ikemoto (1963)described a tubular system alternating with the J-rods. It is composed of vesiclesand tubules, which connect with large vesicles at the periphery of the fibre.

In contrast, Rohlich (1962), working on the leech Hirudo medicinalis, described onlyone type of myofilament and stated that it was uncertain whether the myofilamentsextended without interruption from one end of the fibre to the other. Straubesand &Kersting (1964) in an account of the body wall muscles of an earthworm, thought thatthe individual filaments extended the full length of the fibre. Like Kawaguti &Ikemoto (1959), they observed 2 types of element in the contractile system. The thinelements they called filaments and the thick ones fibrils. They argued that the fibrilsare not homogeneous, but are composed of thin filaments packed closely together,and that these contractile elements run the full length of the fibre and are anchoredin the region of specific zones of the plasmalemma. Furthermore, they interpretedthe alternating electron-dense bodies and tubules as part of the sarcoplasmic reticulum,the former being sections through walls of tubules. In a study of 5 different species ofleech, Pucci & Afzelius (1962) also refer to both of these structures as tubules, butdifferentiate between compact and hollow sarcotubules. Rohlich (1962) considered thehollow structures to be part of the sarcoplasmic reticulum and named the electron-dense bodies 'cross-filaments'.

Some more recent work by Heumann & Zebe (1967) on Lumbricus terrestris and byRosenbluth (1968) on the polychaete Glycera largely supports the observations ofKawaguti & Ikemoto (1959) and Ikemoto (1963). Rosenbluth (1965, 1967) also analysedthe ultrastructure of the body wall muscle of the nematode Ascaris lumbricoides andconcluded that it also was obliquely striated. This is in support of some early light-microscope work by Plenk (1926). Both Heumann & Zebe (1967) and Rosenbluth

Structure of earthworm muscle 235

(1968) interpret the tubular system as sarcoplasmic reticulum. The tubular system inAscaris differs from that in annelids in that it is divided into 2 components, a trans-versely oriented T-system and vesicles of sarcoplasmic reticulum (Rosenbluth, 1965).The arrangement of the filaments in the translucent part of the adductor muscle ofthe oyster (Hanson & Lowy, 1961) is similar to that in annelids and nematodes. Inthis muscle the tubular system is non-existent, except for the presence of peripheralvesicles.

It was felt that, in view of the variations in the interpretation of obliquely striatedmuscle, a further detailed study was desirable, particularly of an earthworm. Thispaper provides additional evidence about their structure.

MATERIAL AND METHODS

In order to keep the body wall muscles in a relaxed condition whole specimens of Lumbricusterrestns Linn, were anaesthetized in 2-5 % ethanol, injected with 2-5 % glutaraldehyde(buffered at about pH 7-2 with o-i M sodium cacodylate), so as to stretch the body wall, andthen immersed in this fixative. After 5-10 min pieces of body wall were removed and placed infresh glutaraldehyde for a further hour. Then the preparations were washed in 0 1 M sodiumcacodylate, post-fixed in 1 % osmium tetroxide in veronal-acetate buffer (pH 7 4-7-6) for 1 h,washed in veronal-acetate buffer, dehydrated in graded ethanols and embedded in Epon. Thisprocedure was carried out at 15-16 CC.

Transverse and longitudinal sections of the muscle fibres were cut on a Huxley microtome(Cambridge Instrument Co. Ltd.), and an attempt was made to cut sections in various longi-tudinal planes at known orientations to the body wall. The sections were mounted on gridscoated with Formvar or carbon and examined in an AEI EM6B electron microscope. They werestained with uranyl acetate and lead citrate to improve the contrast.

RESULTS

General arrangement

There are 2 layers of muscle in the body wall of the earthworm: an inner longi-tudinal layer in which the muscle fibres are arranged in feather-like arrays (Fig. 2) andan outer circular layer. The fibres are several millimetres long and, in the longitudinalmuscle, may extend over 2 segments of the animal (Ogawa, 1939; Hanson, 1957). Intransverse sections most of the longitudinal muscle fibres are long and thin, measuringabout 4 /x,m by up to 45 /tm. On the other hand those of the circular muscle are morenearly oval or even circular (as also are some longitudinal fibres, particularly thosenear the junction between the 2 muscle layers). However, the fine structure of thesefunctionally antagonistic muscles is essentially the same.

Single muscle fibres occur at intervals within the connective tissue. These are quiteseparate from the main bundles of longitudinal and circular fibres in the two musclelayers. Their internal structure is essentially the same as that of other fibres. Some arelongitudinally oriented and situated either in the circular muscle layer or in the middleof the sheet of connective tissue which separates the 2 muscle layers. Other fibres aretransversely oriented and lie along the radial septae which support the longitudinalmuscle fibres.

236 P. J. Mill and M. F. Knapp

Contractile elements

Myofilaments. Transverse sections of the fibres show alternation of light and darkbands (Figs. 2, 3). In the more circular fibres these are arranged radially; in theflattened fibres they occur in 2 rows, perpendicular to the long sides. The bands onopposite sides of a fibre are often in line so that they appear to traverse the wholewidth. The bands are formed by the presence of 2 types of myofilament: thick oneswith a diameter between 20 and 60 nm and thin ones with a diameter of about 5 nra(Figs. 4, 5). Each dark band contains both thick and thin filaments, although acentral zone can usually be distinguished which is devoid of thin filaments. The lightbands contain only thin filaments and there is a zone down the centre of each bandcontaining both electron-dense and tubular elements. It will be shown later that thedark and light bands are analogous to the A- and I-bands respectively, of cross-striated muscle, despite the different appearance of the 2 types of muscle. Theelectron-dense elements correspond to the Z-lines. Since these are in the form of rodsthey will be called Z-rods.

The A-bands are normally between 0-13 and 0-40 /tm wide and contain from 2 to 6irregular rows of thick filaments which are more or less circular in cross-section.Those in the centre of the band have a larger diameter than those at the edge. Some-times the thick filaments in the centre of the A-band are elongated in the direction ofthe long axis of the band, but the circular appearance of the others indicates a trans-verse section (Figs. 6, 7, 20). The average distance between the surfaces of the thickfilaments is 30 nm, although it varies from 20 to 60 nm. The thick filaments appeargranular in cross-section. In some instances an outer dense granular ring can be dis-tinguished from a lighter core with a single dense granule in the centre (Fig. 4). Thearrangement of thin filaments within the A-band is variable. As many as 12 mayencircle a thick filament (Figs. 4, 5), but there are usually less than this and the arrayas a whole appears much less regular than in cross-striated muscle. Individual thinfilaments are often, but not always, shared by adjacent thick filaments. The centre ofthe band may be free of thin filaments and this leads to an incomplete encircling ofsome of the thick filaments and others with no thin filaments at all around them.Sometimes 'cross-links' can be seen between thick and thin filaments (Figs. 4, 5).

In the I-bands the thin filaments appear to be arranged at random, and are fairlyevenly spaced, the distance between the centres being 2-5-10 nm.

A series of about 170 transverse sections was cut over a distance of some 13 /im.From these it was found that the bands gradually changed their position with respectto the fibre as a whole. Thus, in Fig. 6A the outer end of A-band / / is part-way alongthe side between the asterisks,whereas in Fig. 6B, taken from a section later in theseries, it is at the corner near to the lower asterisk. Furthermore, this movement iscontinued in the same direction round the fibre so that the A-bands at the top move tothe right with respect to those at the bottom. For example A-band 2j is oppositeA-band 3 in Fig. 6A and opposite A-band 4 in Fig. 6B. Thus the bands are followinga diagonal course with respect to the longitudinal axis of the fibre. The lateral dis-placement is about 0-25 /(.m over a longitudinal distance of approximately 13 /tm of


the fibre. This gives an angle of i-o to 1-25° between the axis of the bands and thelongitudinal axis of the fibre in this extended preparation, which is of the same orderas that of the oblique striations observed in relaxed muscle under the light micro-scope by Hanson (1957). It is clear that the A- and I-bands are ribbon-shaped, withtheir inner edges near the centre of the fibre and their outer edges at the periphery,and that they follow a diagonal course along the length of the muscle fibre. The peri-pheral edges of the bands give rise to Hanson's alternating dark and light striations inpreparations of whole muscle fibres.

From the shape of the fibres in transverse section and from observations on thesesame serial sections it seems probable that the bands do not form a continuous helixfrom one end of the muscle fibre to the other. At the corners of the fibres the bandsbecome broken up into smaller masses. Thus A-bands IJ and 14 in Fig. 6A undergo aseries of changes and more or less lose their original identity by the section in Fig. 6B.This breaking up of the bands also occurs at the edges of the ribbon-shaped fibres.There is some rearrangement of the A-bands along the flat surfaces of the fibres.Thus they tend to branch along their course, the branches either disappearing orjoining up with other A-bands.

The course of individual thick myofilaments was followed through another serialseries of 26 sections over a distance of approximately 1-5 /mi. Figure 7A, B, C and Dshows sections 1, 8, 15 and 26 respectively. They demonstrate that in successivesections some thick myofilaments become smaller in diameter and are then lost (loss isindicated by the symbol | ->-) and other new ones appear ( + -*•). In A-band (1) all thethick myofilaments which are lost are on one side of the A-band and all those gainedare on the other side. Thus it is clear that the thick myofilaments do not extend thefull length of the fibre. Also, the 'rows' of myofilaments in the A-band are staggeredwith respect to one another. The diagonal slope of the A-band can be attributed, atleast in part, to this stagger. A-band (it) traverses the whole width of a muscle fibre.Here the loss or gain of myofilaments on one side of the fibre is on the oppositesides of the A-band to that on the other side of the fibre. This is consistent withHanson's (1957) observation that the slope of the striations is in opposite directionson opposite sides of the muscle fibre.

From the transverse sections it is clear that longitudinal sections cut in differentplanes will produce different pictures. The terminology of Rosenbluth (1965) will beadopted, based on the following axes: x for the transverse axis perpendicular to thelong transverse axis of the bands (parallel to the edge of the fibre); y for the transverseaxis parallel to the long transverse axis of the bands (perpendicular to the fibreedge); z for the longitudinal axis of the fibre. Apart from the transverse plane (xy)two other (longitudinal) planes are of particular importance for an interpretationof the fine structure: these are xz and yz. Although sections were cut in variouslongitudinal planes at different orientations to the body wall it was difficult toachieve constancy due to the curvature of the fibres.

The myofilaments are oriented approximately parallel to the longitudinal axis ofthe muscle fibre. In the xz plane the A- and I-bands run at a slight angle to the longi-tudinal fibre axis and the Z-rods are seen in cross-section, alternating with tubules


(Fig. 8). If the course of a single thick filament is followed in sections in this plane itcan be seen to start on one side of the A-band and end on the other. This is indicatedin Fig. 9 in which the 2 ends of a thick filament are marked. Thus the thick filaments,at least, run at an angle to the bands. Although in some instances individual thickfilaments can be followed most or all of the way from one side of an A-band to theother, it is more usual for them to be cut obliquely so that they run out of the section(Fig. 8). This is generally because the section is not quite parallel to the thick filaments,but the situation is. also complicated by the fact that the thick filaments may bendalong their length. The longest thick filaments measured were about 9-5 /un, but it isnot clear whether this is the typical length or whether there is some variation. Thethick myofilaments have an axial periodicity of about 15 nm (Fig. I IB) .

Sections in the yz plane pass through A- and I-bands alternately and a picture isobtained resembling that of longitudinal sections of cross-striated muscle (Figs. 11,12).However, apart from the difficulties caused by curvature of the fibres, the orientationrequired to obtain sections absolutely in this plane is extremely critical. A slightdifference in the angle of sectioning results in oblique sections through the sarcomeres.This is illustrated in Figs. 11 and 12. While the thick and thin myofilaments runapproximately parallel to the longitudinal axis of the fibre they are cut obliquely inthese sections. Thus the apparent sarcomere length as measured in these sections(5-2 and 5-6 /tm in Figs. 11 and 12 respectively) is less than the true sarcomere length.It has been noted that in transverse sections the diameter of the thick filaments is lessat the edge of the A-band than in the centre. This difference in diameter is also seenin longitudinal sections. In Fig. 11 A the thick myofilaments in the middle of theA-band are greater in width and taper towards each end of the band. Sections cut inlongitudinal planes other than xz and yz give more complicated arrangements in whichthe A- and I-bands are at various angles to the longitudinal axis of the fibre.

Z-rods. In transverse sections strips of electron-dense material occur in the middleof the I-bands (Fig. 14). They are about 60 nm wide but of variable length. In somecases a single strip may extend all the way from the periphery of the fibre to itscentre, but in others several strips form a discontinuous chain along the length of theI-band. In longitudinal sections in which the A- and I-bands are cut at or near thexz plane the electron-dense material is in the form of small oval masses which occurat regular intervals of 0-5-1-0 /im along the longitudinal axis of the I-band (Fig. 8).These oval dense structures measure 100-150 nm along the longitudinal axis of theI-band and are 40-50 nm wide. This material is obviously in the form of rods. Inlongitudinal sections in the yz plane the rods are cut along their length (resulting in anappearance similar to that obtained in transverse sections), extending from theperiphery of the fibre to its centre. In this plane, however, the rods are slightly widerthan in transverse sections, indicating an oval cross-section (Fig. 11). When sections arecut slightly oblique to the yz plane so as to pass through the longitudinal axis of theI-bands the rods are seen as a series of parallel transverse strips at regular intervals of0-5-1-0 /wn (Fig. 13).

At higher powers the rods can be seen to be made up of amorphous dense materialand thin filamentous 'threads'. The latter are cut transversely in transverse sections


(Fig. 5) and along their length in longitudinal sections (Fig. 13). It is not certainwhether the threads are continuations of the thin filaments or whether they are separatestructures to which the filaments are attached, as suggested by Ikemoto (1963). Which-ever is the case the Z-rods are thought to-serve as anchorage points for the thinfilaments and are, therefore, functionally analogous to the Z-disks or Z-lines in cross-striated muscle. Each Z-rod is oriented perpendicularly to the sarcolemma and extendsinto the middle of the I-band from near the periphery of the fibre to its centre.Occasionally Z-rods have been observed to pass out of the I-band at the periphery andto bend and continue parallel to the sarcolemma for a short distance. Sometimes severalsuch Z-rods have been seen to join together in this peripheral zone (Fig. 15). A diagramof the organization of the contractile elements is given in Fig. 1 (p. 242).

Tubule system

In addition to the elements of the contractile apparatus there is a tubule system inthe muscle fibre. Large vesicles occur at frequent intervals in the peripheral zone ofthe fibre immediately below the sarcolemma (Figs. 2, 3). The walls of the vesicles arejuxtaposed to the sarcolemma and the material between the 2 membranes is electron-dense (Fig. 16). This dense material is sometimes discontinuous, so that it appearsalmost septate. The space between the two membranes is approximately 10 nm wideand the total width of the apposed membranes together with the intervening space isapproximately 25 nm. Occasionally a thin line runs between the membranes (Fig. 16).Tubules arise from the vesicles and in transverse sections are seen to pass in the middleof the I-bands towards the centre of the fibre (Fig. 14). Occasionally a tubule can befollowed all the way from the peripheral vesicle to the centre of the fibre. However,discontinuous chains of pieces of tubule are more often seen with short lengths ofZ-rods between them.

In longitudinal sections near and through the xz plane cross-sections of tubulesoccur at regular intervals along the length of the I-band, alternating with the ovalcross-sections of the Z-rods. Occasionally a longitudinal tubule is seen linking two ofthese tubule sections (Fig. 8). Longitudinal tubules also form links between theperipheral vesicles. In the yz plane the tubules are at right angles to the fibre axis(Fig. 13) and extend from near the periphery of the fibre to its centre (as in transversesections). In the description of the Z-rods it was noted that in sections cut slightlyoblique to the yz plane, along the longitudinal axis of the I-band, the Z-rods appear asa series of parallel transverse strips of dense material. Alternating with these and parallelto them are less dense strips which are longitudinal sections of the tubules (Fig. 13).Like the Z-rods they occur at intervals of 0-5-1-0 /tm. The tubules are of variablediameter, approximately 50-150 nm deep (measured along the longitudinal axis of theI-band) and 20-50 nm wide. Their length is approximately equal to half the width ofthe fibre. The tubules are dilated in places to form small vesicles.

Other cellular constituents

Myofilaments occupy most of the muscle fibre, leaving only a narrow zone of sarco-plasm around the periphery. This widens in the region of the nucleus, which is


flattened between the sarcolemma and the myofilaments and elongated along thelongitudinal axis of the fibre (Fig. 10). Within the peripheral and perinuclear zones aremitochondria and granules (Fig. n ) . Very occasionally mitochondria are seen in thecentre of the fibres (Fig. 4) but this seems to be restricted to the prostomial and analregions of the body. The granules tend to aggregate into stellate masses which looklike glycogen. Some of these granules are scattered among the myofilaments (Figs. 11,13). Golgi bodies areoften found in the perinuclear zone. Small folds of the peripheralsarcoplasm are frequent and lie flat against the side of the muscle fibre (Fig. 10). Inaddition there are large sarcoplasmic folds or 'muscle tails' which may extend for aconsiderable distance and serve to attach the muscle fibre to the surrounding con-nective tissue or are associated with axonal terminals (Mill & Knapp, 1970).

Bundles of fibrils run through the peripheral sarcoplasm and also occur in the muscletails and in some instances they have been observed to run into the I-bands (Figs. 17,18, 19). Tangential sections through the peripheral zone indicate that these bundlespass between the large peripheral vesicles (Fig. 21). They are mainly oriented trans-versely, although some are longitudinal or even oblique. Extensions of the Z-rods intothis zone have been described above, but it is not known whether these connect withthe fibrillar bundles. The fibrillar bundles themselves are anchored to the surface atdesmosomes (Fig. 20) which interconnect neighbouring muscle fibres (Fig. 20) andalso connect muscle fibres and muscle tails to connective tissue (Fig. 6). The cellmembrane at the desmosome is distinct and straight and exhibits a much clearertrilaminate structure than the surrounding sarcolemma. On either side of the mem-brane there are narrow clear zones which are flanked by electron-dense plates (Fig. 20).The intracellular plate is usually thicker and more dense than the extracellular oneand the fibrillar bundles arise directly from it. When the attachment is betweenmuscle fibre and connective tissue there is another clear zone between the extra-cellular plate and the basement membrane; the latter may be slightly thickened in theregion of the desmosome. However, when the attachment is between 2 muscle fibres,fine fibrils arise from the extracellular dense plate and pass to the correspondingstructure of a desmosome on the other fibre (Fig. 20B). If these desmosomes are veryclose (about 20 nm apart) there is no further specialization (Fig. 7 A). If the gap isslightly greater, say in the region of 50 nm, a dense plate is found mid-way betweenthe 2 desmosomes. Finally, if the desmosomes are a considerable distance apart(more than 50 nm), a second dense plate is usually seen outside the first, with arelatively clear zone between them traversed by fibrils (Fig. 20).

DISCUSSION

Terminology

Because of the problems in the interpretation of the structure of obliquely striatedmuscles a variety of terms have been used by different authors to describe analogous oridentical structures (Table 1). Since the arrangement of the contractile elements in themuscle is basically similar to that of cross-striated muscle we suggest that the termsA-band, I-band, H-zone and Z-rod (the last being rod-shaped as distinct from the

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Fig. 1. For legend see opposite page.


Z-disk) should be used. The tubules and vesicles are not obviously analogous witheither the transverse or the longitudinal tubular systems of cross-striated muscle andwill, therefore, be referred to as sarcoplasmic reticulum for the present.

Arrangement of the contractile elements

From the results of this study it is apparent that the Z-rods project from thesurface to the centre of the muscle fibre. Since all Z-rods are at right angles to thesarcolemma, neighbouring rods are parallel with one another. They are arranged inrows, but the rods within a row (for example, xx-xd in Fig. 1) are not immediatelybelow one another. Instead they are displaced slightly to one side so that each rowruns diagonally with respect to the longitudinal axis of the fibre. In extended musclethe angle between the rows and the longitudinal fibre axis is between i° and 20; inrelaxed muscle it is 5° (Hanson, 1957). In Fig. 1 this angle is exaggerated for clarity.There are many rows of Z-rods, all parallel to one another.

As a result of this diagonal arrangement of the rods and of their spacing a sectionapproximately parallel to the longitudinal axis of the fibre (drawn parallel to it inFig. 1) will cut through rods from consecutive rows (for example, x^y^). The myo-filaments are arranged between such consecutive Z-rods on the same longitudinalaxis. A row of thin myofilaments is attached to each one and these extend towards theother. Their length is less than half the distance between the rods, in extendedmuscle at least, and so a gap is left in the centre. There is a single row of thick fila-ments and these lie between and parallel to the thin ones, but do not extend as far as theZ-rods. This array of myofilaments is clearly similar to that in the sarcomere of cross-striated muscle and contains an A-band, 2 I-bands and an H-zone.

The sarcomeres differ from those in cross-striated muscle in one important respect.In cross-striated muscle the Z-hne material is in the form of a disk and so a singlesarcomere is columnar; whereas in this muscle each sarcomere is very thin in onedimension, since the Z-material is rod-shaped. Also, since the ends of the sarcomeres(i.e. the Z-rods) are obliquely staggered, any transverse section through the fibre mustcut through different regions of numerous sarcomeres. Since the stagger is regular thisproduces the alternation of A- and I-bands seen in transverse sections. As a furtherconsequence of the narrowness of the sarcomere only sections which are exactly parallel

Fig. 1. A stereograph representing a block from an obliquely striated earthworm musclefibre. X, Y and Z are the three different perpendicular axes from which the transverse(xy) and longitudinal (xz, ys) planes are derived. The angle of the stnations in thexz plane has been exaggerated for clarity. The block has been cut back at an angle fromthe yz plane so that the cut passes through one of the rows of Z-rods (solid horizontallines) and transverse tubules of the sarcoplasmic reticulum. In the xz plane sectionsthrough the Z-rods and tubules are shown as solid and open ovals respectively. Thethick vertical lines represent thick myofilaments and the thin vertical lines thinmyofilaments. The latter are attached to the Z-rods. In the xy plane sections throughthick and thin myofilaments are shown as large and small dots respectively. Folds inthe sarcolemma (pf) and mitochondria (m) in the peripheral zone (pz) are alsoshown.

16-2

244 p- J- Mil1 and M- F- Knapp

to the longitudinal axis of the sarcomere and exactly in the yz plane will produce apicture similar to that observed in cross-striated muscle. In this paper the term'sarcomere' is used in a different way from that in which it is used by Rosenbluth(1965, 1967, 1968), who refers to the oblique series of sarcomeres which extends alongthe length of the fibre as a single sarcomere. The longest thick myofilaments that havebeen measured are approximately 9-5 /im and it is suggested on the basis of this thatthe sarcomere length must be at least 10 fim.

This interpretation of the arrangement of myofilaments within the obliquelystriated muscle of L. terrestrts is in many respects similar to that of Hanson & Lowy(1961) for the oyster, Crassostrea angulata, of Ikemoto (1963) for the earthworm,Eisenia foetida, of Heumann & Zebe (1967) for L. terrestris, and of Rosenbluth for thenematode Ascaris (1965) and the polychaete Glycera (1968), but contrasts with that ofStraubesand & Kersting (1964) for an earthworm. Straubesand & Kersting suggestthat the thick myofilaments are composed of thin ones, but the present results andthose of the other authors quoted indicate that they are separate components.

In Lumbrtcus we have found the normal diameter mid-way along the thick filamentsto be 50 nm, although Heumann & Zebe (1967) recorded a value of only 30-35 nm asthe maximum diameter. In Eisenia the maximum diameter is only 20 nm (Ikemoto,1963). The array of myofilaments in Eisenia is very regular according to Ikemoto. Heobserved 6 thin filaments around each thick one, whereas in Lumbricus as many as12 may be seen (Figs. 4, 5). Furthermore, in the I-band of Eisenia the thin filamentsmake an angle of about ioc to the longitudinal fibre axis, whereas in Lumbricus they areapproximately parallel to this axis.

The suggestion that the thin myofilaments are anchored within the Z-rods is inagreement with the interpretation of Ikemoto (1963), Rosenbluth (1965) and Heumann& Zebe (1967), but whether they end in the Z-rods or pass through is less clear.Ikemoto suggests that the thin myofilaments are attached to slightly thicker J-filaments within the Z-rods (J-rods of Ikemoto). It is true that the filaments within theZ-rods appear to be more electron-dense than the myofilaments in the I-band (Fig. 13),but this could equally be attributed to the presence of some cementing substance.

The network of fibrillar bundles in the peripheral zone probably functions as anintracellular skeleton. It has been noted that the fibrillar bundles sometimes branchand send an arm into an I-band. Also, in the I-bands some dense strips have been seenwhich are wider than the Z-rods and closely resemble the peripheral bundles. Incontrast, Z-rods occasionally pass from the I-band into the peripheral zone and con-tinue parallel to the sarcolemma; in some cases several such rods may be joined toone another. Whether the Z-rods are attached to the fibrillar bundles and the variousobservations described above are thus different examples of this interconnexion is notknown. In any event this phenomenon is seen only occasionally, so any interconnexionsmust be relatively rare. In the series of sections that were cut to follow the course of themyofilaments none was seen. However, intermittent attachment of Z-rods to theperipheral skeleton could provide anchorage points for the contractile elements. InAscaris the intracellular skeleton is more extensive than in the earthworm, withfibrillar bundles which interconnect the dense bodies to which thin myofilaments are


attached (Rosenbluth, 1965). Rosenbluth (1967) has suggested that the function ofthese bundles may be to transmit tension to the surrounding connective tissue. Theycould possibly serve a similar function in the earthworm. However, if the 2 systemsare not interconnected our observations would indicate that at intervals the fibrillarbundles pass into the interior of the fibre to provide a more extensive skeletal structure.

Chemical nature of the myofilaments

The thin myofilaments are similar in diameter (5 nm) to the actin filaments incross-striated muscle. Godeaux (1954) prepared actomyosin from earthworm bodywall muscles, and Hanson & Lowy (1963) demonstrated the presence of F-actin in thethin filaments in Lumbricus longitudinal muscle fibres. The thick myofilaments on theother hand are much thicker than the myosin filaments of cross-striated muscle.They have an axial periodicity of about 15 nm (Hanson & Lowy (1957) and thispaper), which is similar to that of paramyosin, but also to the sub-unit repeat patternof myosin filaments. Tropomyosin has been identified in earthworm muscle (Kominz,Saad & Laki, 1957) and tropomyosin A (paramyosin) in the white part of the oysteradductor muscle (Hanson et al. 1957). In addition there is evidence for the presence ofsome myosin in earthworm muscle (Maruyama & Kominz, 1959). From the availableevidence it seems probable that the thick myofilaments in earthworm muscle areprimarily composed of paramyosin, but that myosin may also be present.

The sarcoplasmtc reticulum

The tubule system in Lumbricus muscles (Heumann & Zebe (1967) and this paper) issimilar to that in other annelid obliquely striated muscles which have been investigated,namely Eisenia (Ikemoto, 1963), leech (Pucci & Afzelius, 1962; Rohlich, 1962) andGlycera (Rosenbluth, 1968). Table 2 summarizes the types of tubular and vesicularsystems found in these and other muscles. In the cross-striated vertebrate skeletal,arthropod skeletal and insect visceral muscles and in the obliquely striated body wallmuscle of Ascarts there are 2 tubule systems. One of these is transversely oriented(T-system) and is formed by invaginations of the sarcolemma, and is thereforeextracellular; the other is apparently entirely intracellular and may be longitudinallyoriented or consist of flattened vesicles. The intracellular system is generally referredto as the sarcoplasmic reticulum. The membranes of the 2 tubular systems are closelyapposed in certain places.

In some cross-striated vertebrate slow muscles, in the obliquely striated translucentpart of the oyster adductor muscle and in obliquely striated annelid body wall muscleonly the intracellular system is found. Although the tubule system in annelids re-sembles the T-system in its orientation, the authors agree with previous workers thatsince it is intracellular it should be referred to as sarcoplasmic reticulum. This isfurther supported by the fact that the membrane surrounding the tubule system inannelids is unilaminar, as is the membrane of the sarcoplasmic reticulum in othermuscles. In contrast, that surrounding the T-system is continuous with the sarcolemmaand, like the latter, is trilaminar. In the obliquely striated muscles of annelids and theoyster modification of the membranes resembling dyads occurs at the juxtaposition of

Tab

le 2

. T

he T

-sys

tem

and

sar

copl

asm

ic r

etic

ulum

in

a se

lect

ion

of m

uscl

e fi

bres

Mus

cle

T-s

yste

m(e

xtra

cellu

lar)

Sarc

opla

smic

ret

icul

um(i

ntra

cellu

lar)

Con

tact

bet

wee

nT

-sys

tem

and

sarc

opla

smic

ret

icul

umR

efer

ence

Cro

ss-s

tria

ted

Ver

tebr

ate

Mos

t sk

elet

al

Gar

ter

snak

e sl

owIn

vert

ebra

teIn

sect

flig

htIn

sect

vis

cera

l

Obl

ique

ly s

tria

ted

Asc

aris

(N

emat

oda)

Ann

elid

s

Oys

ter

(tra

nslu

cent

part

of

addu

ctor

)

Tra

nsve

rse

tubu

les

Tra

nsve

rse

tubu

les

Tra

nsve

rse

tubu

les

Tra

nsve

rse

tubu

les

Lon

gitu

dina

l sh

eets

or

tubu

les

Dep

lete

d ve

sicl

es

Lon

gitu

dina

l tu

bule

sFl

atte

ned

vesi

cles

Flat

tene

d ve

sicl

esiP

erip

hera

l ve

sicl

es^

-! an

d tr

ansv

erse

ly j

-[

orie

nted

tub

ules

JPe

riph

eral

ves

icle

s

* Se

e te

xt

Tri

ads

Dya

dsD

yads

Dya

ds a

nd t

riad

s

Dya

ds*

at s

urfa

ce

Dya

ds*

at s

urfa

ce

j Hux

ley

(196

4);

I Pa

ge (

1965

)H

ess

(196

5)

Smith

(19

61)

Smit

h, G

upta

& S

mith

(196

6)

Ros

enbl

uth

(196

5)(H

eum

ann

& Z

ebe

(196

7)-

Ros

enbl

uth

(196

8)[P

rese

nt p

aper

Han

son

& L

owy

(196

1)

t


the peripheral vesicles with the sarcolemma at the surface of the cell, and it is sug-gested that the term 'dyad' should be extended to include these structures, sinceT-system tubules are extracellular.

REFERENCES

GODEAUX, J. (1954). Recherches electrophor^tiques sur les prot&nes musculaires du Lombrie.Bull. Acad. r. Belg. Cl. Set. 5th ser. 40, 948-961.

HANSON, J. (1957). The structure of the smooth muscle fibres in the body wall of the earth-worm. J. biophys. biocliem. Cytol. 3, 111-121.

HANSON, J. & LOWY, J. (1957). Structure of smooth muscles Nature, Lond. 180, 906-907.HANSON, J. & LOWY, J. ( I 960). Structure and function of the contractile apparatus in the muscles

of invertebrate animals. In The Structure and Function of Muscle, vol. 1 (ed. G. H. Bourne),pp. 265—335. London: Academic Press.

HANSON, J. & LOWY, J. (1961). The structure of the muscle fibres in the translucent part of theadductor of the oyster, Crassostrea angulata. Proc. R. Soc. B 154, 173-196.

HANSON, J. & LOWY, J. (1963). The structure of F-actin and of actin filaments isolated frommuscle. J molec. Biol. 6, 46-60.

HANSON, J , LOWY, J., HUXLEY, H. E., BAILEY, K , KAY, C. M. & RUEGG, J. C. (1957). Structure

of molluscan tropomyosin. Nature, Lond. 180, 1134-1135.HESS, A. (1965). The sarcoplasmic reticulum, the T-system and the motor terminals of slow

and twitch muscle fibres in the garter snake. J. Cell Biol. 26, 467—476.HEUMANN, H.-G. & ZEBE, E. (1967). Uber Feinbau und Funktionweise der Fasern aus dem

Hautmuskel8chlauch des Regenwurms, Lumbncus terrestns L. Z. Zellforscli. mikrosk. Anat.78, 131-150.

HUXLEY, H. E (1964). Evidence for continuity between the central elements of triads and extra-cellular space in frog sartonus muscle. Nature, Lond. 202, 1067-1071.

IKEMOTO, N. (1963). Further studies in electron microscopic structures of the oblique-striatedmuscle of the earthworm, Eisenia foctida. Biol. J. Okayama Univ. 9, 81-126.

KAWAGUTI, S. & IKEMOTO, N. (1957). Electron microscopy on the smooth muscle from thebody wall of the earthworms, Pheretima commumssima and Euenia for.tida. Biol. J. OkayamaUniv. 3, 223-238.

KAWACUTI, S. & IKEMOTO, N. (1958). Electron microscopy on the smooth muscle of the leech,Hirudo nipponia. Biol. J. Okayama Univ. 4, 79—91.

KAWAGUTI, S. & IKEMOTO, N. (1959). Electron microscopic patterns of earthworm muscle inrelaxation and contraction induced by glycerol and adenosinetnphosphate. Biol. J. OkayamaUniv. 5, 57-87.

KOMINZ, D. R., SAAD, F. & LAKI, K. (1957). Vertebrate and invertebrate tropomyosins.Nature, Lond. 179, 206-207.

MARUYAMA, K. & KOMINZ, D. R. (1959). Earthworm myosin. Z. vergl. Physiol. 42, 17-19.MILL, P. J. & KNAPP, M. F. (1970). Neuromuscular junctions in the body wall muscles of the

earthworm, Lumbricus terrestris Linn. J. Cell Set. 7, 263-271.OGAWA, F. (1939). The nervous system of the earthworm (Pheretima commumsstma) in different

ages. Sci. Rep. Tdhoku Umv , Ser IV, 13, 395-488.PAGE, S. G. (1965). A comparison of the fine structure of frog slow and twitch muscle fibres.

J. Cell Biol. 26, 477-497-PLENK, H. (1926). Beitrage zur Histologie der Muskelfasern von Hirudo und Lumbricus nebst

Benchtigungen zu meinen Untersuchungen uber den Bau der Ascaris- und Mollusken-muskelfasern. Z. mikrosk.-anat. Forsch 4, 163-202.

Pucci, I & AFZELIUS, B. A. (1962). An electron microscope study of sarcotubules and relatedstructures in the leech muscle. ,7. Ultrastruct. Res. 7, 210-224.

R5HLICH, P. (1962). The fine structure of the muscle fibre of the leech, Hirudo medicinalis.J. Ultrastruct. Res. 7, 399-408.

ROSENBLUTH, J. (1965). Ultrastructural organisation of obliquely striated muscle fibres inAscaris hmibricotdes. J. Cell Biol. 25, 495-515.


ROSENBLUTH, J. (1967). Obliquely striated muscle. I, II. Contraction mechanism of Ascarisbody muscle. J. Cell Biol. 34, 15-33.

ROSENBLUTH, J. (1968). Obliquely striated muscle. IV. Sarcoplasmic reticulum, contractileapparatus, and endomysium of the body muscle of a polychaete, Glycera, in relation to itsspeed. J. Cell Biol. 36, 245-259.

SMITH, D. S. (1961). The structure of insect fibrillar flight muscle. J. biophys. biocliem. Cytol.(Suppl.) 10, 123-158.

SMITH, D. S., GUPTA, B. L. & SMITH, U. (1966). The organization and myofilament array ofinsect visceral muscles. J. Cell Sci. 1, 49-57.

STRAUBESAND, J. & KERSTING, K. H. (1964). Feinbau und Organisation der Muskelzellen desRegenwurms. Z. Zellforsch. mikrosk. Anat. 62, 416-442.

{Received 17 September 1969—Revised 27 January 1970)

ABBREVIATIONS ON PLATESmt muscle tail11 nucleuspf peripheral fold in the sarcolemmapz peripheral zone of sarcoplasms connective tissue septumt tubule of sarcoplasmic reticulumv peripheral vesicle of sarcoplasmic reticulumZ Z-rod

The scale on all the micrographs is equivalent to 1 /tm.

Fig. 2. Low-power transverse section of ribbon-shaped longitudinal muscle fibres,attached to a connective tissue septum is). Because of the curvature of the fibres a singlefibre may be cut transversely in some places (arrows with open circle) and obliquely inothers (arrows with solid circle), x 5500.Fig. 3. Enlargement of the enclosed region in Fig. 2. Contractile elements occupymost of the volume of muscle fibre and are arranged to give alternating A-bands {A)and I-bands (/). Within the I-bands are Z-rods (Z) and tubular elements of thesarcoplasmic reticulum it). There is a narrow peripheral zone of sarcoplasm (ps) con-taining vesicles of the sarcoplasmic reticulum (v). Note that the central region of thetop fibre and the right half of the bottom fibre are cut in transverse section, x 17000.

AddsJbglgoIin

A-banddesmosomedense stripfibrillar bundleglycogenGolgi bodyI-bandmitochondrion


Figs. 4, 5. Transverse sections of muscle fibres. The large and small dots are sectionsthrough thick and thin myofilaments, respectively. Thick filaments are completely(•—>) or incompletely (O—>) encircled by thin filaments. Cross-links between thickand thin myofilaments can be seen.

Fig. 4. A fibre from the anal segment. The thick myofilaments appear granular,x 57000.

Fig. 5. A circular muscle fibre. The thick myofilaments appear to be composed of anouter granular ring surrounding a lighter core with single dense granule in the centre,x 60000.

Structure of earthworm muscle


Fig. 6. Transverse sections of a longitudinal muscle fibre, A, Section 60 and B,section 155 from a series of about 170 sections. These two sections are approximately8 jim apart. The A-bands have been numbered from 1 to 28 and a few of these numbershave been entered on the figure. The side of the fibre between the asterisks (^) is thatreferred to in the text. A detailed explanation of the series appears in the text, x 6500.Fig. 7. Transverse sections of longitudinal muscle fibres, A, B, C, and D are sections 1, 8,15 and 26, respectively, of a series cut over a distance of approximately 1-5 fim. Arrowswith a minus sign ( 2 ) indicate thick myofilaments which disappear between thesection on which they are marked and the section in the following part of the figure,and arrows with a plus sign ( t ) indicate myofilaments which have appeared since theprevious part of the figure. The symbols O 9 D • mark 4 thick myofilaments whichare present in each of the 4 sections. A more detailed explanation is given in the text,x 31000.

254 p- J- Mil1 and M- F- Knapp

Fig. 8. A longitudinal section, in the xz plane, of a muscle fibre from the prostomium.Thick and thin myofilaments lie approximately parallel to the longitudinal fibre axis(<->). A- and I-bands are at a slight angle to this axis. Sections through Z-rods (Z) andtransverse tubules of the sarcoplasmic reticulum (i) are seen in the I-band. A longi-tudinal tubule can be seen connecting 2 of the transverse tubules (—>). x 35000.Fig. 9. A longitudinal section in the xz plane of a circular muscle fibre. The black arrowsindicate each end of a thick myofilament which extends from one side of the A-bandto the other side. This myofilament is 9-5 /tm long. The centre of the I-band on eachside of the A-band is marked by white arrow-heads, x 14000.Fig. 10. A longitudinal section of a circular muscle fibre showing the perinuclear zonewith a nucleus, Golgi body and glycogen granules. Folds in the peripheral sarcoplasmare also seen, x 15000.


Fig. 11 A. A longitudinal section in the yz plane of a circular muscle fibre, showing onesarcomere. This section is not quite parallel to the longitudinal axis of the sarcomere.Thick and thin myofilaments run approximately parallel to the longitudinal axis of thefibre. An A-band and 2 I-bands can be seen between 2 transversely oriented Z-rods.The thick myofilaments taper towards each end of the A-band. The apparent sarcomerelength is 52 /ttn. x 23 000. Fig. 11 B. An enlargement of the region marked in Fig. 11 Ato show the paramyosin-like banding of the thick myofilaments. x 52000.

Fig. 12. A longitudinal section in theysr plane of a circular muscle fibre. This section isnot quite parallel to the longitudinal fibre axis. The apparent sarcomere length is 5-6 fim.Some of the thick myofilaments extend most of the way from one end of the A-band tothe other. The arrow indicates a branching transverse tubule, x 20000.

Fig. 13. This section of a longitudinal muscle fibre is parallel to the longitudinal axis ofthe I-band. Transversely oriented Z-rods and tubules of the sarcoplasmic reticulumalternate. Within the Z-rods dense ' threads' can be seen which are continuous with thethin myofilaments. Between these threads some amorphous electron-dense materialoccurs. Note the small glycogen granules scattered among the myofilaments and2 star-like aggregations of glycogen granules, x 50000.


C E L 7


Fig. 14. A transverse section of a circular muscle fibre showing transverse tubules ofthe sarcoplasmic reticulum arising from the peripheral vesicles (small arrows). One ofthe Z-rods (large arrow) extends from the periphery to the centre of the fibre, x 28000.Fig. 15. A transverse section of a longitudinal muscle fibre showing Z-rods extendingfrom the I-band into the peripheral zone. Two neighbouring Z-rods are connected toeach other, x 19000.Fig. 16. A peripheral vesicle of the sarcoplasmic reticulum showing the specializationin the region where the vesicular membrane is closely apposed to the sarcolemma.x 75000.

Figs. 17, 18. Transverse sections of circular muscle fibres showing fibrillar bundlesin the peripheral zone. A branch of one bundle (Fig. 18) extends into an I-band. Ineach figure a dense strip, which is similar in appearance to the peripheral fibrillarbundles, is seen within an I-band. It is not known whether these strips are unusuallylarge Z-rods or whether they are internal extensions of the peripheral fibrillar bundles,x 19000.

Fig. 19. Muscle fibres which have been cut obliquely. In. the lower fibre a peripheralfibrillar bundle extends for a considerable distance. Another fibrillar bundle can be seenin the muscle tail attached to the upper fibre, x 14000.


Fig. 20 A. A transverse section of longitudinal muscle fibres showing desmosomes.In 2 instances (outlined) they join two muscle fibres to one another and in the thirdthey connect a muscle fibre to the muscle tail of another fibre. Peripheral fibrillarbundles arise from the desmosomes and in this figure one of these bundles interconnects2 desmosomes. x 19000. The insets, Figs. 20B, 20c, are higher power electron micro-graphs of the desmosome complexes indicated in Fig. 20A. x 39000.Fig. 21. A tangential section through the peripheral zone of a muscle fibre showingfibrillar bundles passing between the peripheral vesicles of the sarcoplasmic reticulum ;t indicates a tubule connecting two vesicles, x 25000.


v-v-^. • V.., •••;v.'v-v-v. «;":.:;-.v:«

the fine structure of obliquely striated body wall … · by hanson. the myofibrils on opposite...

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