FREEZE-ETCH STUDIES ON FISH SKELETAL MUSCLE · the cells myofibrils, T-tubules, the sarcotubular system and the nuclear envelope were investigated. Freeze-etch studies on muscle 539

J. Cell Sa. 6, 537-557 ('97o) 537Printed in Great Britain

FREEZE-ETCH STUDIES ON FISH

SKELETAL MUSCLE

W. S. BERTAUD, D. G. RAYNS AND F. O. SIMPSON

Physics and Engineering Laboratory, D.S.I.R., Lower Hutt, New Zealand,and The Electron Microscope Laboratories of the Pathology Department andthe Medical Research Council of New Zealand, and the Wellcome MedicalResearch Institute, Department of Medicine, University of Otago MedicalSchool, Dunedin, New Zealand

SUMMARY

The results of a freeze-etch study of skeletal muscle cells of a fish, the Black Molhe (Mol-lienesta sp ), correlated well with published descriptions of sectioned material. The arrays ofapertures of the T-tubules at the cell surface were clearly demonstrated. Numerous vesicles,communicating with the extracellular space, were also seen at the cell surface. The relation-ship of the T-tubules with the adjoining sarcotubular cisternae was studied; in transversefractures at Z-levels there was a tendency for pieces of T-tubule to adhere to the cisternae Noscalloping of the sarcotubular membrane was noted where it apposed the T-tubule, but someridges on its luminal surface were demonstrated at these regions; elsewhere the luminal surfaceof the sarcotubular membrane was densely covered with particles. In the myofibrils, a reversalof polarity of the structure of the thick filaments was suggested at the M-hne.

INTRODUCTION

The technique of freeze-etching for electron microscopy developed by Moor (Moor& Miihlethaler, 1963; Moor, 1964) has made it possible to examine the structuralfeatures of cell surfaces. For example the apertures of the transverse (T) tubules ofmammalian myocardial cells (Rayns, Simpson & Bertaud, 1967, 1968 a) and thecomplex surface features of mammalian skeletal muscle cells (Rayns, Simpson &Bertaud, 19686) are readily demonstrated.

It was decided to apply the technique also to the skeletal muscle cells of a fish,the Black Mollie (Mollienesia sp.), in which the walls of the T-tubules have beenshown (Franzini-Armstrong & Porter, 1964) to have direct continuity with the cellmembrane. This investigation has confirmed the relationship between T-tubules andcell membrane, has provided further information about the T-tubules and the sarco-tubular system, and has demonstrated some interesting points about the myofila-ments (Bertaud, Rayns & Simpson, 1968).

MATERIALS AND METHODS

Specimens of Black Molhe, about 4-5 cm long, were killed by a blow on the head. Smallpieces of body myotome were excised and soaked in 30 % glycerol in frog Ringer solutioncontaining 4 % glutaraldehyde or in the same solution containing no glutaraldehyde (see notepreceding Abbreviations on plates). The specimens were prepared for freeze-etching as pre-viously described (Rayns et al. 1968 a, b), and replicas were viewed in a Philips EM 200electron microscope.

538 W. S. Bertaud, D. G. Rayns and F. O. Simpson

RESULTS

The plasmalemma in fish skeletal muscle cells, as in other types of muscle cell(Rayns et al. 1967, 1968a, b), presented one of two appearances. In some instancesthe exposed membrane faces were characterized by depressions or pits (for example aindicated by arrow in Fig. 4) and in other instances (for example v indicated by arrowin Fig. 2) by numerous excrescences.

The identification of these characteristic membrane faces as outer (extracellular)and inner (cytoplasmic) surfaces depended first on the establishment of the shadowingdirection (Rayns et al. 1968a) and secondly on the recognition of the relationshipbetween the membranes and their surroundings in survey micrographs. When exam-ined in the electron microscope the greater part of a replica is seen to lie flat, but withvery numerous and conspicuous short rises and falls in fracture level. The generalreplica surface, that is, the 'flat' areas, is of medium or average shadowing density(g, e, Fig. 1 and mf, Fig. 3). Wherever an area is more heavily shadowed than average,it follows that this region is rising up above the general surface. Conversely, where asurface is less heavily shadowed than average, or totally lacks shadow, it follows thatsuch a region is tilted away from the source of shadowing, that is, is passing belowthe general surface. The degree of difference from the average shadowing density isindicative of the steepness of the rise or fall of the fracture level. It is well establishedthat fracture planes tend to follow membrane surfaces; thus most of the rises and fallsin fracture level occur along membrane surfaces.

Viewing replicas (e.g. Figs. 1, 3) along the direction of metal shadowing and usingthe above criteria, one can identify the surface pi as being adjacent to and rising upfrom the cytoplasm of a muscle cell. This surface is therefore the inner surface of thecell membrane. Conversely, the surface po (Fig. 3) is adjacent to and rises up towardsthe cytoplasm of another muscle cell. This surface is therefore the outer surface ofthe cell membrane. In addition, it may be seen that this surface and the surface po inFig. 1 rise up from extracellular material (e, Figs. 1, 3), confirming these areas asouter membrane surfaces.

It was found that inner surfaces (pi, Figs. 1, 3) usually bore a mixed population ofrounded excrescences about 70-90 nm in diameter, and crater-like structures ofvarious sizes, while outer surfaces {po, Figs. 1, 3) tended to be marked by numerousdepressions. With these facts and with evidence from sectioned material (Franzini-Armstrong & Porter, 1964) that vesicles are present on the cytoplasmic side of muscle-cell membranes, it seemed reasonable to use the presence of depressions or excres-cences as a means of identifying outer or inner cell membrane surfaces respectively,in cases where other evidence was lacking. At the surfaces of the cells several featureswere studied, including T-tubule apertures, vesicles, plaques and particles; withinthe cells myofibrils, T-tubules, the sarcotubular system and the nuclear envelopewere investigated.

Freeze-etch studies on muscle 539

T-tubule apertures

A number of round or ovoid features (a, Figs. 2-6) appeared on the plasma mem-brane in a rather irregular rectangular array, with a spacing of 1-2 /tm in the longitudi-nal direction of the muscle cell and usually a rather closer spacing in the transversedirection. Their number and the regularity of their arrangement varied from placeto place, and some areas of the plasmalemma (e.g. part of Fig. 3) had none.

On the outer surface of the membrane these features were seen as depressions100-200 nm in diameter (Figs. 3, 4 and also Fig. 1) and their appearance varied some-what, presumably depending on the presence or absence of material in their luminaand on their course in relation to the direction of viewing. Thus they were sometimesplugged by frozen contents (for example, Fig. 4, a, top right), and at other timesapparently closed by folds of membrane (for example, Fig. 4, lower depressions).Onthe inner surface of the membrane (Figs. 2, 5, 6) they appeared as craters or shortstumps 80-200 nm in diameter, and of variable height. It seems reasonable to identifythe depressions in the outer surface of the membrane as the apertures of the T-tubules,and the craters and stumps on the inner surface of the membrane as the remains ofthe T-tubules broken off near the cell surface.

Cell surface vesicles

Views of the outer surfaces of the cell membrane (Figs. 1, 4), revealed frequentdimples in the surface about 30-45 nm in diameter. These were interpreted as aper-tures of subsarcolemmal vesicles fused with the plasmalemma and opening to the extra-cellular space. Views of the inner surface of the cell membrane showed craters about30-54 nm in diameter and rounded excrescences about 70-90 nm in diameter. Theexcrescences were interpreted as complete vesicles fused with the plasmalemma, andthe craters as the apertures of vesicles, the sacs of which had been stripped awayfrom the membrane during fracturing.

The number of these vesicles varied in different areas; they were absent from someplaces (part of Fig. 4) and abundant in others (40-45 per square /.cm, Fig. 2).They usually seemed to be randomly distributed (for example, Figs. 3, 4, 6) but insome instances (Fig. 2) there appeared to be a tendency for them to lie in short rows.

Plaques

The surface of the cell membrane, seen from either the outside or the inside, oftenpresented a 'leather-grained' appearance (Figs. 4, 7), due to the presence of small'islands' or plaques. While the point was not specifically investigated in the presentstudy, this surface texture was similar to that seen in previous studies of other musclecells (Rayns et al. 1968 a, b). It has also been observed in artificial and other biologicalmembranes and interpreted as a variable partial splitting of the plasma membrane(Staehehn, 1968).

35 cci- 6

54-O W. S. Bertaud, D. G. Rayns and F. O. Simpson

Particles

Large numbers of randomly scattered particles, 9-10 nm in diameter, were presentin many areas on both the external (Fig. 4) and internal (Figs. 6, 7) surfaces of thecell membrane.

Myofibrils

The appearance of the myofibrils depended on the angle of fracture, and in thecase of transverse fractures, on the level within the sarcomere at which fracturingoccurred. Where the cells were fractured longitudinally, myofilaments were exposedin parallel arrays (Figs. 8-10), and glycogen granules could often be seen lying be-tween the myofibrils in proximity to the sarcotubules.

In transverse fractures through an A-band (mf, Fig. 1), the fractured ends of thickmyosin filaments were clearly evident, but in the I-band myofilaments were relativelyinconspicuous (Fig. 16). When the transverse fracture passed through a Z-region(Fig. 17, part of 16) then the regular quadratic Z-array was seen.

A particularly interesting picture was obtained when myofibrils were fracturedobliquely; the degree of prominence of the ends of the thick filaments was differenton the two sides of the M-line (Fig. 18).

Transverse (T) tubules

The apertures of the T-tubules at the cell surface have already been described. Inlongitudinally fractured cells (Figs. 8-11, 13), T-tubules could be seen flanked aboveand below by elements of the sarcotubular system. Continuity of the walls of theT-tubules with the cell membrane could be traced in some cases (Fig. n ) . Thecourse of the T-tubules was often slightly undulating, and the diameter of the tubulewas 60—100 nm. The cytoplasmic (tc) and luminal (tl) aspects of the T-tubule mem-brane could be identified (Figs. 8—11, 13-15); plaques (pi, Fig. 15), similar to thosealready described on the plasmalemma, were to be seen on both surfaces of the tubulemembranes but were perhaps more obvious on the cytoplasmic aspect. Both surfacesof the T-tubule membranes carried sparse populations of randomly scattered par-ticles 9-10 nm in diameter.

Corresponding views of T-tubules were obtained in transverse fractures at Z-levels(Figs. 16, 17). In such preparations, pieces of T-tubule membrane could sometimesbe seen apparently adhering to the underlying sarcotubule.

Sarcotubular systems

This showed the expected appearances, with dilated terminal cisternae abuttingon the T-tubules and with longitudinal branching channels lying around each sarco-mere, (Figs. 8, 13). The degree of branching into smaller channels was greatest inthe mid-sarcomere region. Continuity across the Z-line (i.e. around the T-tubules)was occasionally noted (Fig. 9).

The sarcotubular membrane could be seen from both the cytoplasmic (sc) andluminal (si) aspects; the cytoplasmic aspect was smooth, apart from occasional par-


tides, while the luminal aspect was densely covered with particles, 8-5-9 nm indiameter (Figs. 8-10, 13, 15).

Nature of contact between T-tubule and sarcotubule

Where the luminal surface of a sarcotubular cisterna was visible at the region ofcontact with a T-tubule, little ridges could be seen lying on this surface in the formof arcs (arrow, Fig. 15, also Figs. 11, 14). These ridges were of approximately similarwidth (10 nm) to the particles thickly covering the luminal aspect of the sarcotubule.Between the sarcotubule and the T-tubule, some elongated particles (ep, Fig. 15)approximately 10 nm in width were demonstrated, and in some instances these(Fig. 15) seemed to be aligned with the ridges on the luminal surface of the sarco-tubular membrane.

Nuclear envelope

The nuclear envelope revealed the usual double membrane arrangement withfrequent nuclear pores. The membrane (Fig. 12) in fact resembled the sarcotubularmembrane in that the nucleoplasmic surface was smooth apart from occasional par-ticles, while the luminal surface (i.e. seen from the space between the two layers ofnuclear membrane) was densely covered with particles 8-5-9 n m l n diameter.

DISCUSSION

As was to be expected from the study of sectioned Black Mollie muscle (Franzini-Armstrong & Porter, 1964), a regular array of T-tubule apertures was demonstratedon the cell membrane by the freeze-etch method. The frequency of regular, unequi-vocal T-tubule apertures was somewhat lower than expected from sectioned materialbut the areas of membrane lacking T-tubule apertures (for example, Fig. 3) may inpart correspond to places where there is an accumulation of glycogen or a group ofmitochondria in the subsarcolemmal region (Franzini-Armstrong & Porter, 1964).Since continuity between T-tubules and plasmalemma is known to be a particularlylabile feature of fish muscle cells (Franzini-Armstrong & Porter, 1964) it is possiblethat the glycerination treatment caused discontinuities in some instances.

The fish skeletal muscle cell membranes in the present study are similar to mam-malian myocardial cell membranes (Rayns et al. 1967, 1968 a) in revealing areas witha regular distribution of T-tubule apertures and also in presenting a surface array ofsmall plaques and particles. The fish skeletal muscle cell membrane differs from thatof mammalian myocardial cells in the far greater number of vesicles present on themembrane and in this respect resembles superficially mammalian skeletal musclecells (Rayns et al. 19686), but the vesicles in the latter were less regular in shape, weredistributed to some extent according to the sarcomere pattern, and their apertureswere apparently indistinguishable from those of the T-tubules. The vesicles shownin the present material presumably correspond to the 'caveolae' noted in tangentialsections by Franzini-Armstrong & Porter (1964).

It has been of interest to compare the structure of the cell contents, as demon-35-2


strated by freeze-etching, with that shown by Franzini-Armstrong & Porter. Therelationship of the walls of the T-tubules with the sarcolemma, the distribution ofT-tubules and sarcotubules, the shape of the myofibrils, the array of myofilaments atthe various levels of the sarcomere and the distribution of glycogen granules are allvirtually identical in the two studies. In the present study, the T-tubules appear to be alittle wider and the sarcotubules perhaps a little less dilated, but this probably dependson the details of specimen preparation. Franzini-Armstrong & Porter (1964) foundcytoplasmic strands, originating from neighbouring cells, lying within the T-tubules;nothing similar could be identified in the present study but it is possible that suchidentifications would be technically difficult.

The relationship of T-tubules to sarcotubular cisternae is a point of particularinterest. Franzini-Armstrong & Porter (1964) showed that in fish muscle the mem-branes of the two types of tubule lie very close together and that the sarcotubular mem-brane at such points is scalloped; similar appearances have been described in rat skele-tal muscle (Walker & Schrodt, 1966). In the present study the close relationshipbetween the T-tubule and the adjacent sarcotubular cisternae is certainly confirmed,but no 'scalloping' of the sarcotubular membrane at the line of contact could beobserved. The little arcuate ridges which were noted on the luminal surface of thesarcotubule at its zones of contact with the T-tubule appeared to bear a relationshipboth to the elongated particles between the T-tubules and sarcotubular cisternae andto the particulars material lining the lumen of the cisternae. Franzini-Armstrong(1968) has demonstrated the presence of rows of minute cross-bridges at the interfacesbetween sarcotubules and T-tubule membranes in frog striated muscle and it maybe that the elongated particles described in the present study are an expression of asimilar system in fish muscle. The information available at present is insufficient,however, to support such a conclusion or to allow of further speculation concerningthe nature of the particles and arcuate ridges.

The similarity of the inner and outer aspects of the nuclear membrane to theluminal and cytoplasmic aspects of the sarcotubular membranes was to be expected,as the nuclear membrane is known to be continuous with the sarcotubular system(Watson, 1955; Franzini-Armstrong & Porter, 1964).

The general appearance of myofilaments in transversely fractured muscle cells hasbeen described for mouse cardiac muscle (Moor, Ruska & Ruska, 1964) and forguinea-pig cardiac and skeletal muscle (Rayns et al. 1968 a, b). However, oblique frac-tures have in the present study shown a phenomenon of considerable interest, namelya difference in the appearance of the thick filaments on either side of the M-line.This has been discussed in detad elsewhere (Bertaud et al. 1968) but in brief it appearsto indicate a reversal at the M-line of the polarity of the myosin molecules whichmake up the thick filaments. It thus confirms the findings of Huxley (1963) whodemonstrated that individual myosin molecules consist of a 'head' and a 'tail' about140-200 nm in length, and that the tails are orientated towards the M-hnes. In termsof the freeze-etch findings, it seems likely that during the fracturing process the tailsof the molecules lying across the point of fracture will tend to follow the 'heads'.


The authors wish to thank Miss J. M. Ledingham, Mr L. Adamson, Mr R. VV. Thomsonand Mrs S O'Kane for skilful technical assistance. The Medical Research Council of NewZealand provided financial assistance for visits by D. G R. to Lower Hutt.

REFERENCES

BERTAUD, W. S., RAYNS, D G. & SIMPSON, F. O. (1968). Myofilaments in frozen-etchedmuscle. Nature, Lond. 220, 381-382.

FRANZINI-ARMSTRONG, C. (1968). The tnadic junction in frog muscle fibres. J. Cell Biol. 39,6A.

FRANZINI-ARMSTRONG, C. & PORTER, K. R. (1964). Sarcolemmal imaginations constituting theT-system in fish muscle fibres. J. Cell Biol. 22, 675-696.

HUXLEY, H E. (1963). Electron microscope studies on the structure of natural and syntheticprotein filaments from striated muscle. J. molec. Biol. 7, 281-308.

MOOR, H (1964). Die Gefrier-fixation lebender Zellen und lhre Anwendung in der Elek-tronenmikroskopie. Z Zellforsch. mikrosk. Anat. 62, 546—580.

MOOR, H. & MCHLETHALER, K (1963). Fine structure in frozen-etched yeast cells. J. CellBiol. 17, 609-628.

MOOR, H., RUSKA, C. & RUSKA, H. (1964). Elektronenmikroskopische Darstellung tienscherZellen mit der Gerfneratztechnik. Z Zellforsch. mikrosk. Anat. 62, 581-601.

RAYNS, D. G., SIMPSON, F. O. & BERTAUD, W. S. (1967). Transverse tubule apertures inmammalian myocardial cells, surface array. Science, N.Y. 156, 656-657.

RAYNS, D. G., SIMPSON, F. O. & BERTAUD, W. S. (1968a). Surface features of striated musclecells. I. Guinea-pig cardiac muscle. J. Cell Set. 3, 467-474.

RAYNS, D. G., SIMPSON, F. O. & BERTAUD, W. S. (19686). Surface features of striated musclecells. II. Guinea-pig skeletal muscle. J. Cell Set. 3, 475-482

STAEHELIN, L. A. (1968). The interpretation of freeze-etched artificial and biological mem-branes. J. Ultrastruct. Res. 22, 326-347

WALKER, S. M. & SCHRODT, G. R (1966). Connections between the T-system and sarcoplasmicreticulum. Anat. Rec. 155, 1-10.

WATSON, M. L. (1955). The nuclear envelope. Its structure and relation to cytoplasmicmembranes. J. bwphys. biochem Cytol. 1, 257-270.

(Received 2 June 1969)


ABBREVIATIONS ON PLATES

aceep

gMmfni

nonpPPiPi

aperture (or stump) of T-tubulecollagen fibrilsextracellular iceelongated particles at T-tubule/

sarcotubule interfaceglycogen particlesM-linemyofilamentsinner nuclear membraneouter nuclear membranenuclear poreplasmalemmainner aspect of plasmalemmaplaques on plasmalemma

popsPtsc

si

tc

tl

V

Z

outer aspect of plasmalemmapennuclear spaceparticles on the plasmalemmacytoplasmic surface of sarcotubular

membraneluminal surface of sarcotubular

membranecytoplasmic surface of transverse

tubular membraneluminal surface of transverse tubular

membranevesicleZ-band

Figs. 2 and 7-17 are of tissue treated with glutaraldehyde in glycerol-Ringersolution prior to freezing. Figs. 1, 3-6 and 18 are of tissue pre-treated with glycerol-Ringer solution only. The double-headed arrows indicate the direction of metalshadowing.

Fig. 1. Transverse fracture through a muscle cell (lower right). After traversingmyofilaments (mf) and granular material (g, probably glycogen), the line of fracturepasses up the inner aspect of the plasmalemma (pi) of the same cell. Small excrescencesand crater-like structures are seen on this membrane surface; the excrescences repre-sent whole vesicles, while the craters probably represent the remains of vesicles brokenoff near the membrane. At the top of the figure, the outer surface of the plasmalemma(po) of another cell is seen; the shadowing shows the structures in the surface to bepits, and these are interpreted as the apertures of vesicles. One larger pit, a, is probablythe aperture of a T-tubule. Collagen fibrils (c) can be seen in the extracellular spacee. x 35000.

Fig. 2. Inner surface of plasmalemma. The long axis of the cell probably lies acrossthe page Two rows of large crater-like structures (alt at) are interpreted as the re-mains of T-tubules, broken off near the plasmalemma. Numerous smaller excrescen-ces and craters, representing vesicles, are also seen. A few very small particles arealso present on the plasmalemma. x 28000.



Fig. 3. Outer surface of plasmalemma po and contents (mf, upper left) of one cell,and inner surface of plasmalemma pi and contents (mf, lower right) of a neighbouringcell. Vesicle apertures are sparse over most of the outer surface of the plasmalemma inthis replica, except towards the top of the figure Many vesicles are present on theinner surface of plasmalemma of the lower cell. Several T-tubule apertures (a) arepresent in the plasmalemma towards the top of the figure, x 9000

Fig. 4 Higher-power view of the T-tubule apertures a seen at the upper part ofFig. 3. They are seen as pits, some of which are blocked with granular material andothers not (also see text). Smaller, randomly scattered pits representing vesicle aper-tures, and tiny particles on the plasmalemmal surface, are also visible, x 34000.

Freeze-etch studies on muscle S47


Fig. 5. Area of inner surface of plasmalemma(pi)- The long axis of the cell coincidesapproximately with the vertical line of the page. Rows of structures rising up fromthe surface are seen (a), each casting a negative shadow; they represent stumps ofT-tubules, x 10000.

Fig. 6. Higher-power view of part of Fig. 5, showing detail of the rows of T-tubulestumps (a); the stumps contain granular material, presumably frozen extracellularfluid. The diameter of the stump seems to depend partly on how close to the plasma-lemma the tubule has been broken. Many vesicles (v) are present and also numerousparticles (pt). x 28600.

Fig. 7 Detail of inner plasmalemmal surface, revealing numerous small craters(the necks of fractured vesicles), a few intact vesicles (v) scattered tiny particles (pt)and the ' leather-grained' texture of the membrane, due to the presence of numerousraised plaques (pi), x 54000.

Freeze-etch studies on muscle 54$


Fig. 8. Radial longitudinal fracture near a cell surface. Two triads are seen in faceview and also finer elements of the sarcotubular system away from the triads. Thetransverse tubules tl, tc are seen following slightly undulating courses at right anglesto the plasmalemma (p). Following the same course at a constant distance aboveand below the transverse tubules are broad elements of the sarcotubular systemsi, sc. The transverse tubular membranes reveal mainly the luminal surfaces {tl),but also some cytoplasmic surfaces (tc). The sarcotubular membranes also showmainly the luminal surfaces (si) although some cytoplasmic surfaces (sc) are seen. Simi-lar appearances of the finer longitudinal elements of the sarcotubules are clearlyevident. Longitudinally running myofilaments (mf)are visible and also groups of glyco-gen particles (g) x 58000.

Freeze-etch studies on muscle S3*'


Fig. 9. Longitudinal fracture showing a triad in which there is continuity of thesarcotubular system (sF) across the level of the transverse tubule (tl). x 47000.

Fig. 10. Radial longitudinal fracture showing a triad Adjacent to the cell membrane(p), all the elements of the triad are seen as the luminal surfaces of the componentmembranes si, tl. x 47 000.

Fig. 11. Radial longitudinal fracture showing another triad. The transverse tubulereveals the cytoplasmic surface (tc) which is continuous with the plasmalemma (p).One sarcotubular cisterna is seen. In the region of contact between the sarcotubuleand the T-tubule there is a series of small ridges (see also Fig. 15). x 47200.

Fig. 12. Replica of parts of a nuclear envelope (outer membrane, no; inner mem-brane, m), as seen from within the nucleus The surface facing the perinuclear space,i.e. the luminal surface, has a dense population of particles, the surface adjacent tothe nucleoplasm has relatively few. Many nuclear pores are seen, x 34000.

Fig. 13 Radial longitudinal fracture showing a triad and part of the sarcotubularnetwork in face view. The transverse tubular membrane reveals its cytoplasmic sur-face (tc) and its luminal surface {tl). Both these surfaces possess ovoid plaques (p[)and occasional small particles. The sarcotubular system also reveals both surfaces ofits membranes. The cytoplasmic face (sc) appears to be smooth, lacking plaques andcarrying few small particles. In contrast, the luminal face (si) is densely coveredwith particles obscuring the underlying surface. This holds true for both the broadtriad elements and the finer elements away from the triad. Arrays of glycogen par-ticles C?) can be seen adjacent to some of the sarcotubules. Some broken endsof myofilaments (mf) are visible, x 54000.


554 W- <5. Bertaud, D. G. Rayns and F. O. Simpson

Fig. 14. Radial longitudinal fracture of a cell revealing a considerable length of onetriad. The myofibrillar axis runs horizontally. The transverse tubule is seen as theluminal surface of the membrane (tl) The adjacent sarcotubular cisternae both revealthe luminal surfaces of the membrane (si) and one, the cytoplasmic surface (sc) also.Adjacent to the triad, myofilaments(mf) and glycogen particles(g)are shown, x 51 000

Fig 15. Radial longitudinal fracture showing details of a triad. On the luminal surfaceof the left sarcotubule (si) are numerous fine ridges (arrow) These lie at right anglesto the course of the tubules and occur in the regions of apposition of the sarcotubuleand the transverse tubule (tc, tl). Elongated particles (ep) lying between the 2 tubulessometimes appear to be aligned with the ridges within the sarcotubule. The myo-fibrillar axis runs horizontally, x 78000.


C E L 6


Fig. 16. Transverse fracture of a cell demonstrating tubules of 3 adjacent horizontaltriad systems, 1, 2 and 3. These are described in turn from the bottom to the topof the micrograph. Triad 1 appears firstly as the luminal surface of the transversetubule (tl), then as the smooth cytoplasmic surface of the underlying sarcotubule (sc)and finally as the particle-covered luminal surface of this sarcotubule (si). Triad 2is seen mainly as the cytoplasmic surface of the sarcotubule (sc) with islands of trans-verse tubule seen as the luminal surface (tl) upon it. At the top is a short zone of thissarcotubule revealing the luminal surface (s/)- Triad 3 is seen firstly as the luminalsurface of the transverse tubule (t[) clearly overlying the cytoplasmic surface of a sarco-tubule (sc). It then continues upwards revealing the luminal surface of this sarcotubule(si). The fracture plane passes mainly through I-band regions but a small area show-ing the quadratic array of myofilaments in the Z-bands is seen in the lower part ofthe figure (Z). The plane of the Z-band rises gradually out of the fracture plane fromthe lower left to the upper right of the micrograph, x 40000.

Fig. 17. Transverse fracture of a cell at the level of a Z-band The quadratic arrayof thin filaments is clearly visible (Z), flanked by elements of the transverse tubules(tl, tc) of adjacent triads, x 40000.

Fig. 18. Oblique fracture of a cell passing through 2 successive Z-bands (Z) andincorporating parts of 3 myofibrils (1, 2, 3) revealing broken ends of myofilaments(mf). The triads have been fractured across and the constituent tubules either pulledout slightly, revealing depressions in the replica, or fractured above the general sur-face, producing raised stumps. A hollow is seen in the upper half of the figure wherea sarcotubule reveals its luminal surface (si). Two stumps are seen in the lower partof the figure where 2 transverse tubules (tc) have been broken off above the generalsurface. A distinct difference in appearance of the thick filaments (mf) either side ofthe M-line (M) is clearly shown, x 23000.


Z

tl\

sc;

ti-s/

f

V tc

: ' >

36-2

Documents

FREEZE-ETCH STUDIES ON FISH SKELETAL MUSCLE · the cells myofibrils, T-tubules, the sarcotubular system and the nuclear envelope were investigated. Freeze-etch studies on muscle 539