- Email: [email protected]
Discontinuity of sarcoplasmic reticulum in the mid-sarcomere region in flight muscle of dragonflies
0040~8166/80/00560749
TISSUE & CELL 1980 12 (4) 749-759 Q 1980 Longman Group Ltd
MAGDA
lE02.W
de EGUILEOR, ROBERTO VALVASSORI and GIULIO LANZAVECCHIA
DISCONTINUITY OF SARCOPLASMIC RETICULUM IN THE MID-SARCOMERE IN FLIGHT MUSCLE OF DRAGONFLIES
REGION
ABSTRACT. The sarcoplasmic reticulum organization of dragonfly flight muscles is analyzed, with particular reference to the doubling existing at H-band level. This doubling could be explained as a consequence of a regular discontinuity in the sarcoplasmic reticulum covering myofibrils. In each sarcomere, two sleeves of the sarcoplasmic reticulum seem to overlap forming a telescopic system which can slide outside each other during the lengthening and shortening movements of the fiber.
sarcoplasmic reticulum. The fenestrated cisternae enveloping the myofibrils are not segmentally interrupted by the T-tubules, as happens in vertebrate striated muscles, where the cisternae are interrupted at every triadic junction. The only observation contrasting this general scheme is on the femoral muscles of Musca domestica (Pasquali-Ronchetti, 1970) where the T-tubules interrupt the sarcoplasmic reticulum cisternae. In this case, however, the T-tubule forms lateral vesicles connected with the terminal cisterna of the reticulum, forming a peculiar kind of triadic junction
Introduction THE
sarcoplasmic reticulum (SR) organization of the synchronous muscle fibers of insects has been described in detail in numerous papers (reviewed by Smith, 1966b; Franzini Armstrong, 1973 ; Elder, 1975 ; Miller, 1975). In all these fibers the sarcoplasmic reticulum is well developed and connects with the sarcolemmal infoldings (T-tubules), forming dyadic junctions. This situation, clearly observable in the indirect flight muscles of the dragonfly (Smith, 1961, 1966a), is characteristic of the synchronous flight muscles of other insects, and of many skeletal and visceral muscles of arthropods. In the Calliphora heart, Jensen (1977) described a series of fenestrated cisternae enveloping the sarcomeres at the A-band level. These cisternae are connected by a threedimensional system of anastomyzing tubules which form a net-work in the I-regions, crossing the Z-disks. According to Smith (1965) the synchronous insect muscle differs from the vertebrate striated muscle in its arrangement of the
Smith’s beautiful micrographs utilizing both ultrathin sections (Smith, 1966a) and the freeze-etching technique (Smith and Aldrich, 1971), clearly shows the continuity of the SR at the level of the T-tubules in flight muscles of the dragonfly. The same author, moreover, pointed out the existence of a typical doubling of the SR in the middle of the sarcomere (Smith, 1961); this doubling is visible in transverse and in longitudinal sections. Smith (1961) schematically represents his own interpretation of this peculiar pattern in Fig. 24 of his paper, where an independent belt-like cisterna lies outside the continuous SR layer, producing a groove perpendicular to the fiber axis on the surface of the adjacent mitochondrion. The problem of this SR doubling has been neglected in the
~ lstituto di Zoologia, Universita degli Studi, M&no. Mailing address: Giulio Lanzavecchia, Istituto di Zoologia, Via Celoria IO, 20133 M&no, Italy. Received 28 June 1979. Revised 30 July 1980. 749
750
DE
EGUILEOR,
later studies by Smith as well as other authors. With the aim of giving a better explanation of the structural and functional meaning of this arrangement, we must reconsider the problem of the three-dimensional organization of SR in flight muscles of dragonflies. Materials and Methods
Flight muscle bundles of adult dragonflies (Anux imperator and Aeshna mixta) were examined. Fixation and embedding were performed following the indications of Valvassori et al. (1978). Lengthening of the muscle was mechanically obtained after treatment with a relaxing medium according to Huxley (1963). The sections, obtained with an LKB Ultrotome III and stained with lead citrate, were observed in a Hitachi HU 11 ES electron microscope. Observations
It is useful to briefly summarize the main structural characteristics of the flight muscles of Odonata, carefully studied by Smith (1961, 1966a). In cross-sections, every fiber has a polygonal shape and a thin, central axis, where a longitudinal series of nuclei is placed (Fig. 1). The ribbon-like myofibrils are radially disposed and branch out into a Y-form towards the periphery. Continuous fenestrated cisternae, surrounding every myofibril length wise, regularly connect with T-tubules forming dyads; these tubules are flattened and typically beaded, running in grooves present on the mitochondrial surface (Smith and Aldrich, 1971), perpendicular to the main fiber axis. Long, wide and flattened
VALVASSORI
AND
LANZAVECCHIA
mitochondria separate the myofibrils along most of their periphery. The contractile system is constituted of myosin filaments deeply interdigitated with actin filaments; both types of filaments are organized in a hexagonal pattern, as in almost all the flight muscles of insects (Pringle, 1972). The sarcomere resting length is about 3 pm (2.6 pm for the A-band and 0.20 pm for every I-semiband); this length shows negligible variation (about 5 ‘A) during contraction. Sarcoplasmic
reticulum organization
As clearly shown by Smith (1966a), each fibril is surrounded by a sleeve-like fenestrated cisterna, which never fuses with that of adjacent fibrils. This arrangement, contrasting with that described for other muscles (in the vertebrate muscles, for instance), becomes evident in cross-sections when two fibrils are placed side by side in regions lacking mitochondria. In these regions, particularly wide in Odonata Zygoptera, the two myofibrils are generally divided by two independent cisternae. Cross-sections (or C-sections in Fig. 16). In cross-sections of fibers fixed in resting condition, or of fibers contracted in the presence of ATP and Ca2+, the H-zone (easily recognizable by the elliptical profiles of myosin filaments and by the absence of actin filaments is always surrounded by a double series of SR cisternae (Figs. 2-6). At the other levels of the sarcomere, on the contrary, the SR cisternae form a single layer. As previously indicated, in the regions lacking mitochondria, two cisternae of the SR are visible side by side (Fig. 2); when this condition is found at the H-band level, four parallel cisternae divide the adjacent myofibrils (Figs, 4, 5).
Fig. 1. Two adjacent myofibrils, sectioned at the H-band level (on the left) and at the A-band level (on the right) are separated by three layers of SR cisternae. x 63,000. Figs. 2, 3. Where two myofibrils come into contact (both at H-band level), four (or five) layers of SR cisternae are interposed. In the myofibril on the right, in Fig. 2, a ‘transition’ line separates two ordered patterns of thick filaments profiles. x 65,000. Fig. 4. Detail from cross-sectioned H-bands separated of SR cisternae are always recognizable. x 60,000.
by mitochondria.
Two layers
152
DE
EGUILEOR,
Sometimes, a myofibril taken at the H-band level is in contact with a myofibril from the A-band, due to a non-perfect alignment of the bands in adjacent myofibrils. In this particular case, three SR cisternae are interposed (Fig. 3). In fibers slightly elongated (about 10% with respect to the previously indicated conditions) each myofibril is surrounded by a single layer of SR in the H region as well as in other regions (Fig. 13). It is noteworthy that the length of the overlapping region of SR in the middle of the sarcomere, as it appears in the contracted fibers, corresponds quite precisely to the 10 % elongation of the sarcomere (Fig. 7). In stretched fibers (experimentally elongated up to 30x), the H and A regions are always surrounded by a single SR layer which appears fragmented or partially lacking in I and Z regions. This morphological condition appears more evident in longitudinal sections.
VALVASSORI
Longitudinal ribbon-like, inal sections to A and B
AND
LANZAVECCHIA
sections. As the myofibrils are two different kinds of longitud(specified in Fig. 16, and parallel planes) must be considered:
A sections. The myofibrils seem very narrow with interposed mitochondria. As indicated by Smith (1961) the latter show two indentations in every sarcomere (near the two A-band ends) where the flattened T-tubules lie. Another indentation, on the mitochondrial surface, is visible at the H-band level; here the SR cisternae surrounding the myofibril, fixed in resting or contracted conditions, become double (Figs. 7, 10). B sections. Only those sections enclosing the SR cisterna and the T-tubules in their thickness must be considered. In these cases, the myofibrils are tangentially sectioned and the SR cisterna appears frontally (Fig. 14). The flattened cisterna is typically fenestrated and,
Fig. 5. Sarcomere of a fiber 5 % contracted with respect to the resting condition. Sarcomere length, 2.85 pm; interdiadic distance (measured from center to center offlattened T-tubules), about 1.42 pm. In the middle of the sarcomere, the SR overlapping zone (about 0.3 pm width), is clearly recognizable. x 43,000. Fig. 6. Seven per cent elongated sarcomere with respect to the resting condition. The SR cisternae appear as continuous single rows of vesicles (a little overlap is visible on one side of the sarcomere, ,x ). Sarcomere length about 3.2 pm; interdiadic distance about 1.55 pm. x 43,000. Fig. 7. Sarcomeres 23 % elongated with respect to the rest condition. While the SR cisternae are continuous between the dyads, they appear broken or lacking in correspondence to I-bands and Z-lines. Sarcomere length about 3.7 pm; interdiadic distance about 1.55 pm. x 43,000. Fig. 8. Detail x 97,000.
of overlapping
zone as it appears
in fiber fixed in rest condition.
Figs. 9, 10. Detail of H- and Z-regions, respectively, in a fiber 10% elongated with respect to the resting condition. The SR cisterna appears as a continuous and single row of profiles. x 97,000. Fig. 11. In a cross-section of H-band region from a slightly (8 %) elongated SR cisternae form a single layer. x 60,000.
fiber, the
Fig. 12. Longitudinal B-section with a frontal view of the SR cisternae (sarcomere length, 3.1 pm; interdiadic distance about I.46 pm). The region of the dyads is clearly visible (7) and the overlapping region, at the H-level. appears as a darker zone (brackets). x 48,000. Fig. 13. Longitudinal B section of a 40% stretched 4.23 pm; interdiadic distance about 1.55 pm). While the diadic region, appears practically normal (without the middle), the cisternae in correspondence to the I-bands and torn up. x 36,000.
muscle (sarcomere length, SR cisterna, in the interoverlapping region in the Z-lines appear dramatically
DISCONTINUITY
OF
resting
SR
IN
30%
10%
condition
3.0
Fig.
MUSCLE
410
3.5
14. Interdiadic
a
distances
measured
in sarcomeres
SARCOMERE
of different
LENGTH Cl
lengths.
b
Fig. 15. Interpretation of the three-dimensional arrangement of SR at the H-band level: (a) the cisterna is continuous for the entire sarcomere length and it forms a double-fold at the H-band level; (b) two independent cisternae partially overlap at the H-band level. 48
756
DE
EGUILEOR,
a
VALVASSORI
AND
LANZAVECCHIA
b
Fig. 16. Three-dimensional reconstruction of the relationships between myofibrils, sarcoplasmic reticulum, T-system and mitochondria, in different conditions of elongation, approximately comparable to the situations shown in Figs. 7, 8 and 9: (a) resting condition,(b) 10% elongated fiber, Ic) 30 ‘A elongated fiber.
DISCONTINUITY
OF
SR IN
MUSCLE
in resting fibers, is clearly uninterrupted at both the T-tubules and Z-line levels. In the middle of the sarcomere (the region of the H-band), a belt-like darker zone is easily recognizable, transverse to the myofilament direction, where the hole frequency in the cisterna appears enhanced. This belt-like region lies in the same place where the SR cisternae overlap in the longitudinal A sections or in cross-sections. In about 10% elongated fibers, the myofibrils are surrounded by a single and continuous SR layer, apparently unbroken in correspondence with M and Z lines (Figs. 8, 11, 12). In stretched fibers, instead, the SR is still continuous in the central region of the sarcomere, between the two dyads, while it appears interrupted and sometimes absent in the I region (Figs. 9, 15). Two other points must be taken into consideration: (1) the mitochondria are in close longitudinal contact in resting contracted fibers, and detach from each other in elongated muscles; (2) the interdiadic distance increases by about the same amount as the length of the sarcomere, when the muscle is not elongated over 10%. When the fibers are stretched up to 30%, the interdiadic distance practically stops increasing. In dramatically stretched conditions, when SR is highly damaged and sometimes fragmented in the interdiadic regions as well, the distance between dyads appears irregularly increased. The interdiadic distances in sarcomeres of different length are plotted in Fig. 14. Serial cross-sections. An analysis of serial cross-sections through the H-band in unstretched fibers gives a reliable demonstration that the SR cisternae overlap, running parallel for a brief space without touching. However, the images are not a negative proof of the existence of some connection in the overlapping zone. In the transversal sections of the M-line the thick filaments appear solid and have elliptical profiles (Figs. 2, 4, 6); this fact seems to indicate the existence of a two-fold rotational symmetry of the filaments in this region ; the ellipses point in one of three different directions about 60” apart to the trigonal points of the hexagonal filament array. While a certain discrepancy in the various micrographs exists, the oval thick filament backbones are generally oriented in parallel series which cross each other as
751
illustrated in Fig. 4. This peculiar distribution markedly differs from that described for other insect muscles (Pringle, 1974; Squire, 1977) where the thick filament orientation is at random in the H-band. It differs also from that analyzed by Afzelius (1969) and by Halvarson and Afzelius (1969) in the arrowworm M-band, where the thick filaments have three non-equivalent symmetry axes, forming a bilaterally asymmetrical lattice; in the muscles of these animals, the thin and thick filament organization is identical to the one described for the insect flight muscles (Camatini and Lanzavecchia, 1966). According to Luther and Squire (1978) the myosin filament backbone in the M-band of vertebrate muscles, on the contrary, possesses a three-fold rotational symmetry. Discussion The analysis of cross and longitudinal sections (in the previously specified conditions) allows us to build a three-dimensional model for the SR organization in the dragonflies’ flight muscles. In the cross-sections and in the longitudinal A sections, the SR appears as linear rows of small vesicles encircling each myofibril; this aspect is due to the perforated structure of the cisternae, clearly resulting from the frontal observation of the longitudinal B-sections. Only in correspondence with the T-tubules are the fenestrae of the SR cisternae (where the dyads are formed) practically lacking as previously shown by Smith and Aldrich (1971). This situation is common to the one observed in other synchronous insect muscles. When the fibers are in rest conditions or contracted, the SR cisternae only form a two-fold covering around the myofibrils at the H-band level. This belt-like region disappears in 10% elongated fibers, but the overall SR morphology remains practically unchanged. In stretched fibers the SR cisternae appear normal (without the belt-like central region) between the two series of dyads, but are clearly broken, and largely absent in the I regions. In the places where the myofibrils contact (in resting muscles with no mitochondria interposed), one can see two layers of SR if the adjacent myofibrils are both sectioned at the A- or I-band levels. Three layers are visible if the cross-section cuts a myofibril in the A-band and the adjacent one
758
DE
EGUILEOR,
in the H-band. When both adjacent myofibrils are cross-sectioned in the H-band, they appear generally divided by four SR layers. This doubling of the SR layers in correspondence to the H-band always disappears in 10% elongated fibers which show in longitudinal (A) section a single and continuous SR layer lengthwise of the entire myofibril. In Fig. 15 two models are schematically drawn which can explain the situations previously described. In Fig. 15b the SR cisterna is considered continuous for the entire length of the sarcomere, with a double folding at the H-band level. A similar interpretation, however, leads to the existence of a threefold layer. In Fig. 15a, the SR cisternae surrounding each myofibril are considered segmentally interrupted at the H-band level of every sarcomere. This interpretation fits well with the images obtained both in longitudinal and cross-sectioned fibers, and can be considered acceptable. The ciscernae, in resting muscles, partially overlap, forming a kind of a telescopic system. When the fiber is elongated up to 10 %, the overlapping layers slide outside each other, giving the final appearance of a single continuous layer. A further stretching of the fiber causes a breakage of the SR layer around the Z-line while it remains unchanged in the A region. It is noteworthy that the interdiadic distance increases by the same amount as the sarcomere only when the fiber is not elongated over 10 %, a value similar to the width of the overlapping zone in contracted muscles. A possible explanation of these experimental data is the presence of some linkage anchoring the SR to the T-tubules and the Ttubules to mitochondria. The latter linkage can be morphologically justified by the presence of grooves on the mitochondrial surface while the linkage between SR and T-tubules could depend on the existence of feet and intermembranous large particles acting as functional coupling structures (Franzini Armstrong, 1976) and perhaps as anchoring elements (Franzini Armstrong, pers. comm.). Only when the muscles are stretched over
VALVASSORI
AND
LANZAVECCHIA
30% their length, these linkages between T-tubules and mitochondria sometimes break up, allowing an abnormal increase in the interdiadic distance. However, in these conditions a dramatic disarrangement of the entire muscular structure takes place, preventing any reliable morphological analysis. The changes in the myofibril organization during progressive elongation are schematically drawn in Fig. 16 (a, resting fiber; b, 10% elongated fiber; c, 30 % elongated fiber). The formation of a space between the two SR sleeves at the M-band level is prevented by rigid anchoring of the SR to the mitochondria by T-system. The entire reticulum can be greatly torn up only when the anchorage system is missing due to excessive lengthening of the fiber. For the time being it is impossible to adequately explain the functional meaning of such an organization. We can hypothesize, however, that the telescoping sleeves of the SR cisternae, able to passively slide one inside another, could reduce the viscous resistance of a system continually and quickly changing its length. During the morphogenesis of the dragonfly fibers (Valvassori et al., 1978) the SR cisternae gradually grow from the Z-line towards the middle of the sarcomere; in the early stages they appear as short, irregular rings, above every Z-line. These rings apparently connect at the H-band level immediately before metamorphosis, rarely giving rise to the doubling of the cisternae described in this paper, and always visible in the flying animals. The contraction frequencies, clearly related to the contraction speed, in synchronous flight muscles of the insects, according to Sotavalta (1947), is broadly variable. Studies are now in progress to solve the problem whether the doubling of SR cisternae in dragonflies’ flight muscles is a unique feature or is in relation to the contraction speed. Acknowledgement
This work has been supported grant no. 77.01659.04.115.3607.
by C.N.R.
DISCONTINUITY
OF
SR
IN
759
MUSCLE
References AFZELIUS,B. A.
1969. Symmetry and function of biological systems at the macromolecular level. In A. Engstrom and B. Strandberg (eds.), Nobel Symp., 11,415-421. Almqvist & Wiksell, Stockholm. CAMATINI, M. and LANZAVECCHIA, G. 1966. Osservazioni preliminari sull’ultrastruttura della muscolatura striata dei Chetognati. Atti Accad. Nar. Lincei, 41, 392-395. ELDER, H. Y. 1975. Muscle structure. In P. N. R. Usherwood, Insect Muscle, pp. l-75. Academic Press, London, New York and San Francisco. FRANZINI ARMSTRONG, C. 1973. Membranous systems in muscle fibers. In G. H. Bourne, The Structure and Function of Muscle, 2nd edn, Vol. 2, pp. 532-619. Academic Press, New York and London. FRANZINI ARMSTRONG,C. 1976. The comparative structure of intracellular junctions in striated muscle fibers. MDA
5th International
Conference,
Durango,
Cola.
HALVARSON, M. and AFZELIUS, B. A. 1969. Filament J. Ultrastruct.
organization
in the body muscles of the arrow-worm.
Res., 26, 289-295.
HUXLEY, H. E. 1963. Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. J. Mol. Biol., 7, 281-308. JENSEN, H. 1977. Ultrastructure of the myocardial cell and its membrane systems in the adult fly Calliphora erithrocephala (Insecta: Diptera). Cell Tim. Res., 180, 293-302. LUTHER, P. and SQUIRE,J. 1978. Three-dimensional structure of the vertebrate muscle M-region. J. Mol. Biol., 125, 3 13-324.
MILLER, T. A. 1975. Insect visceral muscle. In P. N. R. Usherwood, Insect Muscle, pp. 545-606. Academic Press, London, New York and San Francisco. PASQUALI RONCHETTI, I. 1970. The ultrastructural organization of femoral muscles in Musca domestica (Diptera). Tissue & Cell, 2, 339-354. PRINGLE, J. W. S. 1972. Arthropod muscle. In G. H. Bourne, The Structure and Function qfMu.wle, 2nd edn, Vol. I, pp. 491-541. Academic Press, New York and London. PRINCLE, J. W. S. 1974. Locomotion: flight. In M. Rockstein, The Physiology of Insecta, 2nd edn, Vol. 3, pp. 433-476. Academic Press, New York and London. SMITH, D. S. 1961. The organization of the flight muscle in a dragonfly, Aeshna sp. (Odonata). J biophys. biochem. Cytol., 11, 119-145. SMITH, D. S. 1965. The flight muscles of insects. Scient. Am., 212, 76-88. SMITH, D. S. 1966a. The organization of flight muscle fibers in the Odonata. J. Cell Biol., 28, 101-126. SMITH, D. S. 1966b. The organization and function of the sarcoplasmic reticulum and T-system of muscle cells. Progr. Biophys. Mol. Biol., 16, 107-142. SMITH, D. S. and ALDRICH, H. C. 1971. Membrane systems of freeze-etched striated muscle. Tissue & Cell, 3, 261-281. SOTAVAL~A,0. 1947. The flight-tone (wing-stroke frequency) of insects. Acta Em. Fem., 4, I-l 17. SQUIRE, J. M. 1977. The structure of insect thick filaments. In R. T. Tregear, Insect Flight Muscle, pp. 91-l 12. North-Holland, Amsterdam, New York and Oxford. VALVASSORI,R., DE EGUILEOR, M. and LANZAVECCHIA,G. 1978. Flight muscle differentiation in nymphs of a dragonfly Anax imperator. Tissue & Cell, 10, 167-178.
TISSUE & CELL 1980 12 (4) 749-759 Q 1980 Longman Group Ltd
MAGDA
lE02.W
de EGUILEOR, ROBERTO VALVASSORI and GIULIO LANZAVECCHIA
DISCONTINUITY OF SARCOPLASMIC RETICULUM IN THE MID-SARCOMERE IN FLIGHT MUSCLE OF DRAGONFLIES
REGION
ABSTRACT. The sarcoplasmic reticulum organization of dragonfly flight muscles is analyzed, with particular reference to the doubling existing at H-band level. This doubling could be explained as a consequence of a regular discontinuity in the sarcoplasmic reticulum covering myofibrils. In each sarcomere, two sleeves of the sarcoplasmic reticulum seem to overlap forming a telescopic system which can slide outside each other during the lengthening and shortening movements of the fiber.
sarcoplasmic reticulum. The fenestrated cisternae enveloping the myofibrils are not segmentally interrupted by the T-tubules, as happens in vertebrate striated muscles, where the cisternae are interrupted at every triadic junction. The only observation contrasting this general scheme is on the femoral muscles of Musca domestica (Pasquali-Ronchetti, 1970) where the T-tubules interrupt the sarcoplasmic reticulum cisternae. In this case, however, the T-tubule forms lateral vesicles connected with the terminal cisterna of the reticulum, forming a peculiar kind of triadic junction
Introduction THE
sarcoplasmic reticulum (SR) organization of the synchronous muscle fibers of insects has been described in detail in numerous papers (reviewed by Smith, 1966b; Franzini Armstrong, 1973 ; Elder, 1975 ; Miller, 1975). In all these fibers the sarcoplasmic reticulum is well developed and connects with the sarcolemmal infoldings (T-tubules), forming dyadic junctions. This situation, clearly observable in the indirect flight muscles of the dragonfly (Smith, 1961, 1966a), is characteristic of the synchronous flight muscles of other insects, and of many skeletal and visceral muscles of arthropods. In the Calliphora heart, Jensen (1977) described a series of fenestrated cisternae enveloping the sarcomeres at the A-band level. These cisternae are connected by a threedimensional system of anastomyzing tubules which form a net-work in the I-regions, crossing the Z-disks. According to Smith (1965) the synchronous insect muscle differs from the vertebrate striated muscle in its arrangement of the
Smith’s beautiful micrographs utilizing both ultrathin sections (Smith, 1966a) and the freeze-etching technique (Smith and Aldrich, 1971), clearly shows the continuity of the SR at the level of the T-tubules in flight muscles of the dragonfly. The same author, moreover, pointed out the existence of a typical doubling of the SR in the middle of the sarcomere (Smith, 1961); this doubling is visible in transverse and in longitudinal sections. Smith (1961) schematically represents his own interpretation of this peculiar pattern in Fig. 24 of his paper, where an independent belt-like cisterna lies outside the continuous SR layer, producing a groove perpendicular to the fiber axis on the surface of the adjacent mitochondrion. The problem of this SR doubling has been neglected in the
~ lstituto di Zoologia, Universita degli Studi, M&no. Mailing address: Giulio Lanzavecchia, Istituto di Zoologia, Via Celoria IO, 20133 M&no, Italy. Received 28 June 1979. Revised 30 July 1980. 749
750
DE
EGUILEOR,
later studies by Smith as well as other authors. With the aim of giving a better explanation of the structural and functional meaning of this arrangement, we must reconsider the problem of the three-dimensional organization of SR in flight muscles of dragonflies. Materials and Methods
Flight muscle bundles of adult dragonflies (Anux imperator and Aeshna mixta) were examined. Fixation and embedding were performed following the indications of Valvassori et al. (1978). Lengthening of the muscle was mechanically obtained after treatment with a relaxing medium according to Huxley (1963). The sections, obtained with an LKB Ultrotome III and stained with lead citrate, were observed in a Hitachi HU 11 ES electron microscope. Observations
It is useful to briefly summarize the main structural characteristics of the flight muscles of Odonata, carefully studied by Smith (1961, 1966a). In cross-sections, every fiber has a polygonal shape and a thin, central axis, where a longitudinal series of nuclei is placed (Fig. 1). The ribbon-like myofibrils are radially disposed and branch out into a Y-form towards the periphery. Continuous fenestrated cisternae, surrounding every myofibril length wise, regularly connect with T-tubules forming dyads; these tubules are flattened and typically beaded, running in grooves present on the mitochondrial surface (Smith and Aldrich, 1971), perpendicular to the main fiber axis. Long, wide and flattened
VALVASSORI
AND
LANZAVECCHIA
mitochondria separate the myofibrils along most of their periphery. The contractile system is constituted of myosin filaments deeply interdigitated with actin filaments; both types of filaments are organized in a hexagonal pattern, as in almost all the flight muscles of insects (Pringle, 1972). The sarcomere resting length is about 3 pm (2.6 pm for the A-band and 0.20 pm for every I-semiband); this length shows negligible variation (about 5 ‘A) during contraction. Sarcoplasmic
reticulum organization
As clearly shown by Smith (1966a), each fibril is surrounded by a sleeve-like fenestrated cisterna, which never fuses with that of adjacent fibrils. This arrangement, contrasting with that described for other muscles (in the vertebrate muscles, for instance), becomes evident in cross-sections when two fibrils are placed side by side in regions lacking mitochondria. In these regions, particularly wide in Odonata Zygoptera, the two myofibrils are generally divided by two independent cisternae. Cross-sections (or C-sections in Fig. 16). In cross-sections of fibers fixed in resting condition, or of fibers contracted in the presence of ATP and Ca2+, the H-zone (easily recognizable by the elliptical profiles of myosin filaments and by the absence of actin filaments is always surrounded by a double series of SR cisternae (Figs. 2-6). At the other levels of the sarcomere, on the contrary, the SR cisternae form a single layer. As previously indicated, in the regions lacking mitochondria, two cisternae of the SR are visible side by side (Fig. 2); when this condition is found at the H-band level, four parallel cisternae divide the adjacent myofibrils (Figs, 4, 5).
Fig. 1. Two adjacent myofibrils, sectioned at the H-band level (on the left) and at the A-band level (on the right) are separated by three layers of SR cisternae. x 63,000. Figs. 2, 3. Where two myofibrils come into contact (both at H-band level), four (or five) layers of SR cisternae are interposed. In the myofibril on the right, in Fig. 2, a ‘transition’ line separates two ordered patterns of thick filaments profiles. x 65,000. Fig. 4. Detail from cross-sectioned H-bands separated of SR cisternae are always recognizable. x 60,000.
by mitochondria.
Two layers
152
DE
EGUILEOR,
Sometimes, a myofibril taken at the H-band level is in contact with a myofibril from the A-band, due to a non-perfect alignment of the bands in adjacent myofibrils. In this particular case, three SR cisternae are interposed (Fig. 3). In fibers slightly elongated (about 10% with respect to the previously indicated conditions) each myofibril is surrounded by a single layer of SR in the H region as well as in other regions (Fig. 13). It is noteworthy that the length of the overlapping region of SR in the middle of the sarcomere, as it appears in the contracted fibers, corresponds quite precisely to the 10 % elongation of the sarcomere (Fig. 7). In stretched fibers (experimentally elongated up to 30x), the H and A regions are always surrounded by a single SR layer which appears fragmented or partially lacking in I and Z regions. This morphological condition appears more evident in longitudinal sections.
VALVASSORI
Longitudinal ribbon-like, inal sections to A and B
AND
LANZAVECCHIA
sections. As the myofibrils are two different kinds of longitud(specified in Fig. 16, and parallel planes) must be considered:
A sections. The myofibrils seem very narrow with interposed mitochondria. As indicated by Smith (1961) the latter show two indentations in every sarcomere (near the two A-band ends) where the flattened T-tubules lie. Another indentation, on the mitochondrial surface, is visible at the H-band level; here the SR cisternae surrounding the myofibril, fixed in resting or contracted conditions, become double (Figs. 7, 10). B sections. Only those sections enclosing the SR cisterna and the T-tubules in their thickness must be considered. In these cases, the myofibrils are tangentially sectioned and the SR cisterna appears frontally (Fig. 14). The flattened cisterna is typically fenestrated and,
Fig. 5. Sarcomere of a fiber 5 % contracted with respect to the resting condition. Sarcomere length, 2.85 pm; interdiadic distance (measured from center to center offlattened T-tubules), about 1.42 pm. In the middle of the sarcomere, the SR overlapping zone (about 0.3 pm width), is clearly recognizable. x 43,000. Fig. 6. Seven per cent elongated sarcomere with respect to the resting condition. The SR cisternae appear as continuous single rows of vesicles (a little overlap is visible on one side of the sarcomere, ,x ). Sarcomere length about 3.2 pm; interdiadic distance about 1.55 pm. x 43,000. Fig. 7. Sarcomeres 23 % elongated with respect to the rest condition. While the SR cisternae are continuous between the dyads, they appear broken or lacking in correspondence to I-bands and Z-lines. Sarcomere length about 3.7 pm; interdiadic distance about 1.55 pm. x 43,000. Fig. 8. Detail x 97,000.
of overlapping
zone as it appears
in fiber fixed in rest condition.
Figs. 9, 10. Detail of H- and Z-regions, respectively, in a fiber 10% elongated with respect to the resting condition. The SR cisterna appears as a continuous and single row of profiles. x 97,000. Fig. 11. In a cross-section of H-band region from a slightly (8 %) elongated SR cisternae form a single layer. x 60,000.
fiber, the
Fig. 12. Longitudinal B-section with a frontal view of the SR cisternae (sarcomere length, 3.1 pm; interdiadic distance about I.46 pm). The region of the dyads is clearly visible (7) and the overlapping region, at the H-level. appears as a darker zone (brackets). x 48,000. Fig. 13. Longitudinal B section of a 40% stretched 4.23 pm; interdiadic distance about 1.55 pm). While the diadic region, appears practically normal (without the middle), the cisternae in correspondence to the I-bands and torn up. x 36,000.
muscle (sarcomere length, SR cisterna, in the interoverlapping region in the Z-lines appear dramatically
DISCONTINUITY
OF
resting
SR
IN
30%
10%
condition
3.0
Fig.
MUSCLE
410
3.5
14. Interdiadic
a
distances
measured
in sarcomeres
SARCOMERE
of different
LENGTH Cl
lengths.
b
Fig. 15. Interpretation of the three-dimensional arrangement of SR at the H-band level: (a) the cisterna is continuous for the entire sarcomere length and it forms a double-fold at the H-band level; (b) two independent cisternae partially overlap at the H-band level. 48
756
DE
EGUILEOR,
a
VALVASSORI
AND
LANZAVECCHIA
b
Fig. 16. Three-dimensional reconstruction of the relationships between myofibrils, sarcoplasmic reticulum, T-system and mitochondria, in different conditions of elongation, approximately comparable to the situations shown in Figs. 7, 8 and 9: (a) resting condition,(b) 10% elongated fiber, Ic) 30 ‘A elongated fiber.
DISCONTINUITY
OF
SR IN
MUSCLE
in resting fibers, is clearly uninterrupted at both the T-tubules and Z-line levels. In the middle of the sarcomere (the region of the H-band), a belt-like darker zone is easily recognizable, transverse to the myofilament direction, where the hole frequency in the cisterna appears enhanced. This belt-like region lies in the same place where the SR cisternae overlap in the longitudinal A sections or in cross-sections. In about 10% elongated fibers, the myofibrils are surrounded by a single and continuous SR layer, apparently unbroken in correspondence with M and Z lines (Figs. 8, 11, 12). In stretched fibers, instead, the SR is still continuous in the central region of the sarcomere, between the two dyads, while it appears interrupted and sometimes absent in the I region (Figs. 9, 15). Two other points must be taken into consideration: (1) the mitochondria are in close longitudinal contact in resting contracted fibers, and detach from each other in elongated muscles; (2) the interdiadic distance increases by about the same amount as the length of the sarcomere, when the muscle is not elongated over 10%. When the fibers are stretched up to 30%, the interdiadic distance practically stops increasing. In dramatically stretched conditions, when SR is highly damaged and sometimes fragmented in the interdiadic regions as well, the distance between dyads appears irregularly increased. The interdiadic distances in sarcomeres of different length are plotted in Fig. 14. Serial cross-sections. An analysis of serial cross-sections through the H-band in unstretched fibers gives a reliable demonstration that the SR cisternae overlap, running parallel for a brief space without touching. However, the images are not a negative proof of the existence of some connection in the overlapping zone. In the transversal sections of the M-line the thick filaments appear solid and have elliptical profiles (Figs. 2, 4, 6); this fact seems to indicate the existence of a two-fold rotational symmetry of the filaments in this region ; the ellipses point in one of three different directions about 60” apart to the trigonal points of the hexagonal filament array. While a certain discrepancy in the various micrographs exists, the oval thick filament backbones are generally oriented in parallel series which cross each other as
751
illustrated in Fig. 4. This peculiar distribution markedly differs from that described for other insect muscles (Pringle, 1974; Squire, 1977) where the thick filament orientation is at random in the H-band. It differs also from that analyzed by Afzelius (1969) and by Halvarson and Afzelius (1969) in the arrowworm M-band, where the thick filaments have three non-equivalent symmetry axes, forming a bilaterally asymmetrical lattice; in the muscles of these animals, the thin and thick filament organization is identical to the one described for the insect flight muscles (Camatini and Lanzavecchia, 1966). According to Luther and Squire (1978) the myosin filament backbone in the M-band of vertebrate muscles, on the contrary, possesses a three-fold rotational symmetry. Discussion The analysis of cross and longitudinal sections (in the previously specified conditions) allows us to build a three-dimensional model for the SR organization in the dragonflies’ flight muscles. In the cross-sections and in the longitudinal A sections, the SR appears as linear rows of small vesicles encircling each myofibril; this aspect is due to the perforated structure of the cisternae, clearly resulting from the frontal observation of the longitudinal B-sections. Only in correspondence with the T-tubules are the fenestrae of the SR cisternae (where the dyads are formed) practically lacking as previously shown by Smith and Aldrich (1971). This situation is common to the one observed in other synchronous insect muscles. When the fibers are in rest conditions or contracted, the SR cisternae only form a two-fold covering around the myofibrils at the H-band level. This belt-like region disappears in 10% elongated fibers, but the overall SR morphology remains practically unchanged. In stretched fibers the SR cisternae appear normal (without the belt-like central region) between the two series of dyads, but are clearly broken, and largely absent in the I regions. In the places where the myofibrils contact (in resting muscles with no mitochondria interposed), one can see two layers of SR if the adjacent myofibrils are both sectioned at the A- or I-band levels. Three layers are visible if the cross-section cuts a myofibril in the A-band and the adjacent one
758
DE
EGUILEOR,
in the H-band. When both adjacent myofibrils are cross-sectioned in the H-band, they appear generally divided by four SR layers. This doubling of the SR layers in correspondence to the H-band always disappears in 10% elongated fibers which show in longitudinal (A) section a single and continuous SR layer lengthwise of the entire myofibril. In Fig. 15 two models are schematically drawn which can explain the situations previously described. In Fig. 15b the SR cisterna is considered continuous for the entire length of the sarcomere, with a double folding at the H-band level. A similar interpretation, however, leads to the existence of a threefold layer. In Fig. 15a, the SR cisternae surrounding each myofibril are considered segmentally interrupted at the H-band level of every sarcomere. This interpretation fits well with the images obtained both in longitudinal and cross-sectioned fibers, and can be considered acceptable. The ciscernae, in resting muscles, partially overlap, forming a kind of a telescopic system. When the fiber is elongated up to 10 %, the overlapping layers slide outside each other, giving the final appearance of a single continuous layer. A further stretching of the fiber causes a breakage of the SR layer around the Z-line while it remains unchanged in the A region. It is noteworthy that the interdiadic distance increases by the same amount as the sarcomere only when the fiber is not elongated over 10 %, a value similar to the width of the overlapping zone in contracted muscles. A possible explanation of these experimental data is the presence of some linkage anchoring the SR to the T-tubules and the Ttubules to mitochondria. The latter linkage can be morphologically justified by the presence of grooves on the mitochondrial surface while the linkage between SR and T-tubules could depend on the existence of feet and intermembranous large particles acting as functional coupling structures (Franzini Armstrong, 1976) and perhaps as anchoring elements (Franzini Armstrong, pers. comm.). Only when the muscles are stretched over
VALVASSORI
AND
LANZAVECCHIA
30% their length, these linkages between T-tubules and mitochondria sometimes break up, allowing an abnormal increase in the interdiadic distance. However, in these conditions a dramatic disarrangement of the entire muscular structure takes place, preventing any reliable morphological analysis. The changes in the myofibril organization during progressive elongation are schematically drawn in Fig. 16 (a, resting fiber; b, 10% elongated fiber; c, 30 % elongated fiber). The formation of a space between the two SR sleeves at the M-band level is prevented by rigid anchoring of the SR to the mitochondria by T-system. The entire reticulum can be greatly torn up only when the anchorage system is missing due to excessive lengthening of the fiber. For the time being it is impossible to adequately explain the functional meaning of such an organization. We can hypothesize, however, that the telescoping sleeves of the SR cisternae, able to passively slide one inside another, could reduce the viscous resistance of a system continually and quickly changing its length. During the morphogenesis of the dragonfly fibers (Valvassori et al., 1978) the SR cisternae gradually grow from the Z-line towards the middle of the sarcomere; in the early stages they appear as short, irregular rings, above every Z-line. These rings apparently connect at the H-band level immediately before metamorphosis, rarely giving rise to the doubling of the cisternae described in this paper, and always visible in the flying animals. The contraction frequencies, clearly related to the contraction speed, in synchronous flight muscles of the insects, according to Sotavalta (1947), is broadly variable. Studies are now in progress to solve the problem whether the doubling of SR cisternae in dragonflies’ flight muscles is a unique feature or is in relation to the contraction speed. Acknowledgement
This work has been supported grant no. 77.01659.04.115.3607.
by C.N.R.
DISCONTINUITY
OF
SR
IN
759
MUSCLE
References AFZELIUS,B. A.
1969. Symmetry and function of biological systems at the macromolecular level. In A. Engstrom and B. Strandberg (eds.), Nobel Symp., 11,415-421. Almqvist & Wiksell, Stockholm. CAMATINI, M. and LANZAVECCHIA, G. 1966. Osservazioni preliminari sull’ultrastruttura della muscolatura striata dei Chetognati. Atti Accad. Nar. Lincei, 41, 392-395. ELDER, H. Y. 1975. Muscle structure. In P. N. R. Usherwood, Insect Muscle, pp. l-75. Academic Press, London, New York and San Francisco. FRANZINI ARMSTRONG, C. 1973. Membranous systems in muscle fibers. In G. H. Bourne, The Structure and Function of Muscle, 2nd edn, Vol. 2, pp. 532-619. Academic Press, New York and London. FRANZINI ARMSTRONG,C. 1976. The comparative structure of intracellular junctions in striated muscle fibers. MDA
5th International
Conference,
Durango,
Cola.
HALVARSON, M. and AFZELIUS, B. A. 1969. Filament J. Ultrastruct.
organization
in the body muscles of the arrow-worm.
Res., 26, 289-295.
HUXLEY, H. E. 1963. Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. J. Mol. Biol., 7, 281-308. JENSEN, H. 1977. Ultrastructure of the myocardial cell and its membrane systems in the adult fly Calliphora erithrocephala (Insecta: Diptera). Cell Tim. Res., 180, 293-302. LUTHER, P. and SQUIRE,J. 1978. Three-dimensional structure of the vertebrate muscle M-region. J. Mol. Biol., 125, 3 13-324.
MILLER, T. A. 1975. Insect visceral muscle. In P. N. R. Usherwood, Insect Muscle, pp. 545-606. Academic Press, London, New York and San Francisco. PASQUALI RONCHETTI, I. 1970. The ultrastructural organization of femoral muscles in Musca domestica (Diptera). Tissue & Cell, 2, 339-354. PRINGLE, J. W. S. 1972. Arthropod muscle. In G. H. Bourne, The Structure and Function qfMu.wle, 2nd edn, Vol. I, pp. 491-541. Academic Press, New York and London. PRINCLE, J. W. S. 1974. Locomotion: flight. In M. Rockstein, The Physiology of Insecta, 2nd edn, Vol. 3, pp. 433-476. Academic Press, New York and London. SMITH, D. S. 1961. The organization of the flight muscle in a dragonfly, Aeshna sp. (Odonata). J biophys. biochem. Cytol., 11, 119-145. SMITH, D. S. 1965. The flight muscles of insects. Scient. Am., 212, 76-88. SMITH, D. S. 1966a. The organization of flight muscle fibers in the Odonata. J. Cell Biol., 28, 101-126. SMITH, D. S. 1966b. The organization and function of the sarcoplasmic reticulum and T-system of muscle cells. Progr. Biophys. Mol. Biol., 16, 107-142. SMITH, D. S. and ALDRICH, H. C. 1971. Membrane systems of freeze-etched striated muscle. Tissue & Cell, 3, 261-281. SOTAVAL~A,0. 1947. The flight-tone (wing-stroke frequency) of insects. Acta Em. Fem., 4, I-l 17. SQUIRE, J. M. 1977. The structure of insect thick filaments. In R. T. Tregear, Insect Flight Muscle, pp. 91-l 12. North-Holland, Amsterdam, New York and Oxford. VALVASSORI,R., DE EGUILEOR, M. and LANZAVECCHIA,G. 1978. Flight muscle differentiation in nymphs of a dragonfly Anax imperator. Tissue & Cell, 10, 167-178.