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Extra actin filaments at the periphery of skeletal muscle myofibrils
TISSUE AND CELL, 1991 23 (2) 191-197 0 1991 Longman Group UK Ltd.
F. BARD and C. FRANZINI-ARMSTRONG
EXTRA ACTIN OF SKELETAL
FILAMENTS AT THE PERIPHERY MUSCLE MYOFIBRILS
Keywords:
Skeletal
muscle,
myofibrils,
actin filaments
ABSTRACT. Myofibrils isolated from a variety of vertebrate muscle fibers have a set of peripheral filaments associated with the periphery of the Z line free to move away from the surface of the mvofibril. Decoration with myosin subfragment 1 shows that these are actin filaments. .
of the Z line, have also been identified by 1983; immunocytochemistry (Tokuyasu, Tokuyasu et al., 1983; Tokuyasu et al., 1985). On the basis of some of the above evidence, it was proposed that interaction between cytoskeletal network and myofibrils involves a specific attachment between intermediate filaments and the Z lines (Granger and Lazarides, 1978). However, direct demonstration of such an attachment has been difficult to obtain. Isolated myofibrils bear a set of peripherally associated filaments, and it was thought that these may be intermediate filaments (Wang and Ramirez Mitchell, 1983; Wang, 1984). We have re-examined this question by shadowing and replicating the surface of myofibrils isolated from a variety of muscle fibers and using antibodies and myosin Sl subfragment to label intermediate and actin filaments respectively. We find actin, but not intermediate filaments associated with the isolated myofibrils. Our results do not exclude that intermediate filaments are directly linked to the Z lines in the intact fiber, but do show that the only previous direct evidence in the favour of this hypothesis should be reconsidered.
Introduction The characteristic
cross-striation pattern of skeletal muscle fibers arises from the transverse alignment of sarcomeric striations of the myofibrils. This alignment seems to be maintained by an internal framework that links the myofibrils transversely to each other and to the sarcolemma (Lazarides, 1980; Pierobon-Bormioli, 1981). A longitudinal interfibrillar network that may connect Z line to Z line within the same myofibril has also been observed (Wang and Ramirez-Mitchell, 1983; Wang, 1984). The identities of the transverse and longitudinal networks are still the subject of some controversy. Intermediate filaments, mainly desmin filaments in adult muscle fibers, may contribute to the cytoskeletal framework (Granger and Lazarides, 1978; Lazarides and Granger, 1983). Intermediate filament proteins have been located at the periphery of the Z discs by immunohistochemistry (Bennet, et al., 1979; Granger and Lazarides, 1979; Holtzer, et al., 1982). Electron microscopy shows intermediate filaments forming circumferentially arranged bundles in the same location (Page, 1969; Bennet, et al., 1979). Longitudinally and transversely oriented intermediate filaments, some of them located at the level
Materials and Methods
Correspondence to: Dr. Clare Franzini-Armstrong, Department of Anatomy, University of Pennsylvania, Philadelphia, Pa. 191044018, USA.
The following muscles were used: rabbit psoas; frog sartorius; chicken posterior latissimus dorsi (PLD); rat Sterno-mastoid; and crayfish fast abdominal flexor. The muscles
Received 6 March 1990. Revised 8 August 1990. 191
BARD AND FRANZINI-ARMSTRONG
192
were put into rigor by extracting for 4 days at 4°C in glycerol in a solution containing 1lOmM K sulfate, 4 mM sulfate, 20mM Iodoacetic acid, 20mM Tris, and 4mM EGTA (rabbit, rat) or 15 mM K phosphate, 100 mM K acetate, 5 mM K2 EGTA, pH 6.3 (frog, chicken, and crayfish). Rigor muscles were gently homogenized by three 1 set bursts of a Sorvall Omni Mixer (Omni Corp., Waterbury, CT) using the 5 ml vessel. The fibrils were washed three times in the rigor solution. a) For freeze drying, a drop of the fibril suspension was placed on a freshly split mica surface, fixed in 1% glutaraldehyde in 0.1 M cacodylate buffer for 10-15 min. and washed in 30% methanol or 70% ethanol. A thin layer of the alcohol solution was left on the mica, and specimens were frozen by immersion in freon 12 near freezing point. 70% ethanol (Isobe and Lemanski, 1986) gave more reliable results than 30% methanol and was used for most of the experiments. The fibrils were freeze-dried at -100 to -9O%C under a 10e6 Torr vacuum, for a period of time dependent upon the thickness of the water layer, usually l-2 hr. The specimens were rotary shadowed with platinum at 45” and replicated with carbon. The replicas were floated on water and collected on carboncoated or uncoated grids. b) Antibody and Sl decoration. Fibrils were placed on sheets of freshly cleaved mica and exposed to anti-desmin antibodies in 1: 10 and 1: 50 dilution in ‘cytoskeletal buffer’ (Small and Langanger, 1981), or to Sl (Sigma) at a concentration of 1.1 mg/ml in 0.17 M KCL and 16.7% glycerol for N-30’. After washing, the fibrils were fixed and freeze-dried as above. The antibodies were provided by Dr. H. Holtzer, and were specific for the adult chicken muscle used in this experiment. c) For thin sections, strips of muscle were fixed in 3% glutaraldehyde with 0.1% tannic acid in 0.1 M cacodylate buffer for 2-3 hr. Samples were washed in buffer, post-fixed in
2% 0~0~ in the same buffer for 30min, stained with 2% uranyl acetate in 50% ethanol for 30 min, and embedded. Observations were performed on JEOL 1OOBand Philips 410 electron microscopes. Results Single or small bundles of fibrils produce good replicas (Fig. 1). A and I bands, and M and Z lines are clearly identifiable, but the visibility of actin and myosin filaments varies from one preparation to the other. Remnants of the sarcoplasmic reticulum are associated with the M line and/or the Z line in some preparations. At the Z line level, the surface of the fibril is associated with a disordered network of peripheral filaments. These originate at the edges of the Z line, on either side, and run obliquely. Most of these filaments form a net above the I band and the lateral portions of the A band, few are more longitudinally oriented and reach to the center of the sarcomere. Some of the filaments seem to be associated with the membrane remnants over the M line. Most filaments are smooth and have a uniform diameter (10-12 nm), a few are larger and seem twisted. The pattern of peripheral filaments is the same in fibrils from different vertebrate muscles, regardless of whether they derive from muscle fibers that have the transverse tubule network and triads at the Z line (frog), or at the level of the A-I junction (chicken PLD, rat, rabbit). To determine the nature of the filaments, we labeled the myofibrils with either antidesmin antibodies or myosin subfragment Sl. Antibody labeling can be directly detected by shadowing techniques (Heuser and Kirschner, 1980). Immunolabeling was performed on fibrils from chicken muscle, since the antibodies, kindly provided by Dr. H. Holtzer, were raised against chicken desmin. The diameter of the peripheral filaments does not change with exposure to the antibody
Fig. 1. Freeze-dried, rotary shadowed myofibrils I bands and Z and M lines are marked. Peripheral the Z lines form a network around the I band and of membrane are associated with the fibrils at the
isolated from frog sartorius muscle. A and smooth filaments (arrows) originating from lateral portions of the A bands. Remnants level of the M line (arrowhead). x 66,000.
195
EXTRA ACTIN FILAMENTS
(data not shown). Following exposure to Sl (Figs 2, 3), however, most if not all the peripheral filaments have an increased diameter and show the twisted rope-like structure characteristic of a surface view of decorated actin filaments (see Heuser and Cooke, 1983). Notice that in the same images (Figs 2, 3) and ‘constitutive’ thin filaments, i.e. those forming the myofibril, are also decorated. Clearly the peripheral filaments associated with the surface of isolated myofibrils are composed of actin. To determine whether the peripheral filaments are simply an artifact of mechanical disruption during myofibrii isolation, we examined thin sections of muscle extracted with the same procedure we used to obtain isolated fibrils. In extracted muscles, the periphery of the fibrils are more clearly visible than in fresh tissue, due to lack of soluble proteins. Figure 4 shows the periphery of fibrils in a rabbit muscle fixed in rigor, after extraction. Careful examination reveals individual filaments, with a diameter equal to that of actin filaments, floating at some distance from the surface of the myofibril, but attached to the Z lines. They resemble in size and disposition the peripheral actin filaments seen in isolated fib&. Once the muscle is in rigor, constitutive thin filaments should be ‘zippered’ by cross bridges to the myosin filament, wherever they overlap. The myofibrils in the intact muscle were not exposed to any mechanical stress, yet at their periphery we find segments of actin filaments which are not closely associated with myosin. We must conclude that these ‘extra’ filaments either cannot interact with myosin at least for some of their length, or do not have any cross bridges available for interaction. Invertebrate muscles have a different myofibrillar filament pattern (Hoyle, 1983), and it is interesting to examine whether their myofibrils also have ‘extra’ peripheral actin filaments. Myofibrils from crayfish muscle
differ significantly from the above description, however, since they have no filaments associated with the periphery of the myofibrils.
Discussion Ou; observations demonstrate that peripheral filaments associated with the surface of isolated myofibrils are composed of actin and are anchored to Z lines. The twisted appearance of the filaments and the formation of ‘networks’ are probably artifacts that occur during the homogenization and washing of the myofibrils. Considering that the muscle was in rigor before homogenization, it is surprising to find that some of the most peripheral filaments were sufficiently free to move away from an orderly alignment with the myosin filaments, to which they should be attached by crossbridges. One possibility is that the peripheral filaments that we observe are supernumerary, i.e., they exceed the number stated by the 2 : 1 thin : thick filament ratio of the vertebrate myofilament lattice. Having no cross bridges available, the extra filaments would not be constrained even when the myofibril is in rigor conditions. This is confirmed by the finding that the peripheral filaments do exist in the muscle prior to homogenization, as seen in thin sections. A second possibility is that some of the peripheral filaments can detach from the rigor cross bridges, perhaps because, being at the edge of the myofibril, they are interacting with less than three thick filaments. If the peripheral filaments are similar to the constitutive ones, they they should not reach the M line in fibrils where the sarcomere has an H zone. Yet we do find a few peripheral filaments at the level of the M line. This might indicate that the peripheral filaments are longer than the constitutive ones.
Figs. 2, 3. Sl decoration of myotirbils isolated from rabbit muscle. All filaments associated with the surface of the myofibril how the twisted rope-like structure characteristic of ‘decorated’ thin filaments (arrows, Fig. 2). The decoration is best visible in the stereomicrographs of Figure 3 (using a viewer) or in the inset of Figure 2. The filaments are composed of actin. Inset (Fig. 2) x 109,200.
1%
BARD AND FRANZINI-ARMSTRONG
Fig. 4. Longitudinal section of rabbit muscle tixed in rigor, after extraction in glycerol. Extra thin filaments anchored at the Z-line are located at the periphery of the myofibrils (arrow). These filaments are free of cross bridges. x 70,000.
This work does not solve the question of interaction between intermediate filaments and the myofibril. Although our results are negative in that regard, we cannot exclude that an existing interaction is disrupted either during extraction or homogenization. However, we should point out that thin section electron microscopy does not provide evidence for very close proximity of intermediate filaments to the Z line. At the edges of the Z line in some skeletal muscle fibers there are short filaments, which form a direct link from Z lines to adjacent membranes.
These iilaments do not have the appearance of intermediate filaments (Nunzi and Franzini-Armstrong, 1980). In developing cardiac muscle, high resolution labeling of desmin and vimentin filaments in deep etch images demonstrate no specific Z line attachment (Isobe et al., 1988). It would be interesting to known whether the ‘extra’ actin filaments are composed of a muscle actin isoform (alpha) or of a cytoplasmic isoform (beta or gamma). Recently, it has been shown that gamma actin is present in small amounts in adult vertebrate skeletal
EXTRA
ACTIN
197
FILAMENTS
muscle (Otey et al., 1988). This characteristically non myofibrillar actin could either be one of the components of the constitutive thin filaments or a component of the supernumerary actin filaments we observe.
Acknowledgments
This work was supported by a grant from the MDA to the H.M. Watts Research Center. We are grateful to MS Denah Appelt for her expert help. We are grateful to John Armstrong for participating in this research.
Bennett, G. S., Fellini, S. A., Toyama, Y. and Holtzer, H. 1979. Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro. 1. Cell Biol., 82, 577-584. Granger, B. L. and Lazarides, E. 1978. The existence of an insoluble Z disc scaffold in chicken skeletal muscle. Cell, 15, 125.3-1268. Granger, B. L. and Lazarides, E. 1979. Desmin and vimentin coexist at the periphery of the myofibril Z disc. CeN, 18, 1053-1063. Heuser, J. E. and Cooke, R. 1983. Actin myosin interactions visualized by the quick freeze, deep-etch replica technique. .I. Mol. Biol., 169, 97-122. Heuser, J. E. and Kirschner, M. W. 1980. Filament organization revealed in platinum replicas of freeze-dried cytoskeleton. J. Cell Biol., 86, 212-234. Holtzer. H., Bennett, G. S., Tapscott, S. J., Croop, J. M. and Toyama, Y. 1982. Intermediate-size filament: changes in synthesis and distribution in cells of the myogenic and neurogenic lineages. Cold Spring Harbor Symp. Quant. Biol., 46,317-328. Hoyle, G. 1983. Mnxles and Their Neural Control. Wiley Intersciences, N.Y. Sons, Inc. Isobe, Y. and Lemanski, L. F. 1986. Localization of alpha actin in the cytoskeleton of cultured cardiomyocytes by platinum immunogold electron microscopy. J. Cell Eiol., 183, 126a. Isobe, Y., Heu, G. R. and Lemanski, L. F. 1988. Three dimensional distribution of desmin and vimentin in cultured hamster cardiac myocytes using the immunogold deep-etching replica technique. J. Cell Biol., 107, 473a. Lazarides, E. 1980. Intermediate filaments as mechanical integrators of cellular space. Nature, 283, 249256. Lazarides, E. and Granger, B. L. 1983. Transcytoplasmic integration in avian erythrocytes and striated muscle: the role of intermediate filaments. Modern Cell Biology, 2, 143-162. Nunzi, M. G. and Franzini-Armstrong, C. 1980. Trabecular network in adult skeletal muscle. J. Ultrmtr. Res., 73, 2126. Otey. C. A., Kalnoski, M. H. and Bulinksi, J. C. 1988. Immunolocalization of muscle and non muscle isoforms of actin in myogenic cells and adult skeletal muscle. CeU Motility and the Cytoskeleton, 9, 337-348. Page, S. G. 1969. Structure and some contractile properties of fast and slow muscle of the chicken. J. Physiol. 205, 131-145. Pierobon Bormioli, S. 1981. Transverse sarcomere tilamentous systems: ‘Z- and M-cables’. J. Muscle Res. Cell Motil , 2, 401-413. Small, J. V. and Langanger, G. 1981. Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks. J. Cell Biol., 91, 695-705. Tokuyasu, K. T. 1983. Visualization of longitudinally oriented intermediate filaments in frozen sections of chicken cardiac muscle by a new staining method. J. Cell Biol., 97, 562-565. Tokuyasu. K. T., Dutton, A. H. and Singer, S. J. 1983. Immunoelectron microscopic studies of desmin (skeleton) localization and intermediate filament organization in chicken skeletal muscle. J. Cell Biol., %, 1727-1735. Tokuyasu, K. T., Maher, P. A. and Singer, S. J. 1985. Distributions of vimentin and desmin in developing chick myotubes in viva. II Immunoelectron microscopic study. 1. Cell Biol., 100, 1157-1166. Wang, K. and Ramirez Mitchell, R. 1983. A network of transverse and longitudinal intermediate filaments is associated with sarcomeres of adult vertebrate skeletal muscle. J. Cell Biol., 96,562-570. Wang, K. 1984. Cytoskeletal matrix in striated muscle: the role of titin, nebulin, and intermediate filaments. Adu. hp. Med. Biol., 170, 285-305.
F. BARD and C. FRANZINI-ARMSTRONG
EXTRA ACTIN OF SKELETAL
FILAMENTS AT THE PERIPHERY MUSCLE MYOFIBRILS
Keywords:
Skeletal
muscle,
myofibrils,
actin filaments
ABSTRACT. Myofibrils isolated from a variety of vertebrate muscle fibers have a set of peripheral filaments associated with the periphery of the Z line free to move away from the surface of the mvofibril. Decoration with myosin subfragment 1 shows that these are actin filaments. .
of the Z line, have also been identified by 1983; immunocytochemistry (Tokuyasu, Tokuyasu et al., 1983; Tokuyasu et al., 1985). On the basis of some of the above evidence, it was proposed that interaction between cytoskeletal network and myofibrils involves a specific attachment between intermediate filaments and the Z lines (Granger and Lazarides, 1978). However, direct demonstration of such an attachment has been difficult to obtain. Isolated myofibrils bear a set of peripherally associated filaments, and it was thought that these may be intermediate filaments (Wang and Ramirez Mitchell, 1983; Wang, 1984). We have re-examined this question by shadowing and replicating the surface of myofibrils isolated from a variety of muscle fibers and using antibodies and myosin Sl subfragment to label intermediate and actin filaments respectively. We find actin, but not intermediate filaments associated with the isolated myofibrils. Our results do not exclude that intermediate filaments are directly linked to the Z lines in the intact fiber, but do show that the only previous direct evidence in the favour of this hypothesis should be reconsidered.
Introduction The characteristic
cross-striation pattern of skeletal muscle fibers arises from the transverse alignment of sarcomeric striations of the myofibrils. This alignment seems to be maintained by an internal framework that links the myofibrils transversely to each other and to the sarcolemma (Lazarides, 1980; Pierobon-Bormioli, 1981). A longitudinal interfibrillar network that may connect Z line to Z line within the same myofibril has also been observed (Wang and Ramirez-Mitchell, 1983; Wang, 1984). The identities of the transverse and longitudinal networks are still the subject of some controversy. Intermediate filaments, mainly desmin filaments in adult muscle fibers, may contribute to the cytoskeletal framework (Granger and Lazarides, 1978; Lazarides and Granger, 1983). Intermediate filament proteins have been located at the periphery of the Z discs by immunohistochemistry (Bennet, et al., 1979; Granger and Lazarides, 1979; Holtzer, et al., 1982). Electron microscopy shows intermediate filaments forming circumferentially arranged bundles in the same location (Page, 1969; Bennet, et al., 1979). Longitudinally and transversely oriented intermediate filaments, some of them located at the level
Materials and Methods
Correspondence to: Dr. Clare Franzini-Armstrong, Department of Anatomy, University of Pennsylvania, Philadelphia, Pa. 191044018, USA.
The following muscles were used: rabbit psoas; frog sartorius; chicken posterior latissimus dorsi (PLD); rat Sterno-mastoid; and crayfish fast abdominal flexor. The muscles
Received 6 March 1990. Revised 8 August 1990. 191
BARD AND FRANZINI-ARMSTRONG
192
were put into rigor by extracting for 4 days at 4°C in glycerol in a solution containing 1lOmM K sulfate, 4 mM sulfate, 20mM Iodoacetic acid, 20mM Tris, and 4mM EGTA (rabbit, rat) or 15 mM K phosphate, 100 mM K acetate, 5 mM K2 EGTA, pH 6.3 (frog, chicken, and crayfish). Rigor muscles were gently homogenized by three 1 set bursts of a Sorvall Omni Mixer (Omni Corp., Waterbury, CT) using the 5 ml vessel. The fibrils were washed three times in the rigor solution. a) For freeze drying, a drop of the fibril suspension was placed on a freshly split mica surface, fixed in 1% glutaraldehyde in 0.1 M cacodylate buffer for 10-15 min. and washed in 30% methanol or 70% ethanol. A thin layer of the alcohol solution was left on the mica, and specimens were frozen by immersion in freon 12 near freezing point. 70% ethanol (Isobe and Lemanski, 1986) gave more reliable results than 30% methanol and was used for most of the experiments. The fibrils were freeze-dried at -100 to -9O%C under a 10e6 Torr vacuum, for a period of time dependent upon the thickness of the water layer, usually l-2 hr. The specimens were rotary shadowed with platinum at 45” and replicated with carbon. The replicas were floated on water and collected on carboncoated or uncoated grids. b) Antibody and Sl decoration. Fibrils were placed on sheets of freshly cleaved mica and exposed to anti-desmin antibodies in 1: 10 and 1: 50 dilution in ‘cytoskeletal buffer’ (Small and Langanger, 1981), or to Sl (Sigma) at a concentration of 1.1 mg/ml in 0.17 M KCL and 16.7% glycerol for N-30’. After washing, the fibrils were fixed and freeze-dried as above. The antibodies were provided by Dr. H. Holtzer, and were specific for the adult chicken muscle used in this experiment. c) For thin sections, strips of muscle were fixed in 3% glutaraldehyde with 0.1% tannic acid in 0.1 M cacodylate buffer for 2-3 hr. Samples were washed in buffer, post-fixed in
2% 0~0~ in the same buffer for 30min, stained with 2% uranyl acetate in 50% ethanol for 30 min, and embedded. Observations were performed on JEOL 1OOBand Philips 410 electron microscopes. Results Single or small bundles of fibrils produce good replicas (Fig. 1). A and I bands, and M and Z lines are clearly identifiable, but the visibility of actin and myosin filaments varies from one preparation to the other. Remnants of the sarcoplasmic reticulum are associated with the M line and/or the Z line in some preparations. At the Z line level, the surface of the fibril is associated with a disordered network of peripheral filaments. These originate at the edges of the Z line, on either side, and run obliquely. Most of these filaments form a net above the I band and the lateral portions of the A band, few are more longitudinally oriented and reach to the center of the sarcomere. Some of the filaments seem to be associated with the membrane remnants over the M line. Most filaments are smooth and have a uniform diameter (10-12 nm), a few are larger and seem twisted. The pattern of peripheral filaments is the same in fibrils from different vertebrate muscles, regardless of whether they derive from muscle fibers that have the transverse tubule network and triads at the Z line (frog), or at the level of the A-I junction (chicken PLD, rat, rabbit). To determine the nature of the filaments, we labeled the myofibrils with either antidesmin antibodies or myosin subfragment Sl. Antibody labeling can be directly detected by shadowing techniques (Heuser and Kirschner, 1980). Immunolabeling was performed on fibrils from chicken muscle, since the antibodies, kindly provided by Dr. H. Holtzer, were raised against chicken desmin. The diameter of the peripheral filaments does not change with exposure to the antibody
Fig. 1. Freeze-dried, rotary shadowed myofibrils I bands and Z and M lines are marked. Peripheral the Z lines form a network around the I band and of membrane are associated with the fibrils at the
isolated from frog sartorius muscle. A and smooth filaments (arrows) originating from lateral portions of the A bands. Remnants level of the M line (arrowhead). x 66,000.
195
EXTRA ACTIN FILAMENTS
(data not shown). Following exposure to Sl (Figs 2, 3), however, most if not all the peripheral filaments have an increased diameter and show the twisted rope-like structure characteristic of a surface view of decorated actin filaments (see Heuser and Cooke, 1983). Notice that in the same images (Figs 2, 3) and ‘constitutive’ thin filaments, i.e. those forming the myofibril, are also decorated. Clearly the peripheral filaments associated with the surface of isolated myofibrils are composed of actin. To determine whether the peripheral filaments are simply an artifact of mechanical disruption during myofibrii isolation, we examined thin sections of muscle extracted with the same procedure we used to obtain isolated fibrils. In extracted muscles, the periphery of the fibrils are more clearly visible than in fresh tissue, due to lack of soluble proteins. Figure 4 shows the periphery of fibrils in a rabbit muscle fixed in rigor, after extraction. Careful examination reveals individual filaments, with a diameter equal to that of actin filaments, floating at some distance from the surface of the myofibril, but attached to the Z lines. They resemble in size and disposition the peripheral actin filaments seen in isolated fib&. Once the muscle is in rigor, constitutive thin filaments should be ‘zippered’ by cross bridges to the myosin filament, wherever they overlap. The myofibrils in the intact muscle were not exposed to any mechanical stress, yet at their periphery we find segments of actin filaments which are not closely associated with myosin. We must conclude that these ‘extra’ filaments either cannot interact with myosin at least for some of their length, or do not have any cross bridges available for interaction. Invertebrate muscles have a different myofibrillar filament pattern (Hoyle, 1983), and it is interesting to examine whether their myofibrils also have ‘extra’ peripheral actin filaments. Myofibrils from crayfish muscle
differ significantly from the above description, however, since they have no filaments associated with the periphery of the myofibrils.
Discussion Ou; observations demonstrate that peripheral filaments associated with the surface of isolated myofibrils are composed of actin and are anchored to Z lines. The twisted appearance of the filaments and the formation of ‘networks’ are probably artifacts that occur during the homogenization and washing of the myofibrils. Considering that the muscle was in rigor before homogenization, it is surprising to find that some of the most peripheral filaments were sufficiently free to move away from an orderly alignment with the myosin filaments, to which they should be attached by crossbridges. One possibility is that the peripheral filaments that we observe are supernumerary, i.e., they exceed the number stated by the 2 : 1 thin : thick filament ratio of the vertebrate myofilament lattice. Having no cross bridges available, the extra filaments would not be constrained even when the myofibril is in rigor conditions. This is confirmed by the finding that the peripheral filaments do exist in the muscle prior to homogenization, as seen in thin sections. A second possibility is that some of the peripheral filaments can detach from the rigor cross bridges, perhaps because, being at the edge of the myofibril, they are interacting with less than three thick filaments. If the peripheral filaments are similar to the constitutive ones, they they should not reach the M line in fibrils where the sarcomere has an H zone. Yet we do find a few peripheral filaments at the level of the M line. This might indicate that the peripheral filaments are longer than the constitutive ones.
Figs. 2, 3. Sl decoration of myotirbils isolated from rabbit muscle. All filaments associated with the surface of the myofibril how the twisted rope-like structure characteristic of ‘decorated’ thin filaments (arrows, Fig. 2). The decoration is best visible in the stereomicrographs of Figure 3 (using a viewer) or in the inset of Figure 2. The filaments are composed of actin. Inset (Fig. 2) x 109,200.
1%
BARD AND FRANZINI-ARMSTRONG
Fig. 4. Longitudinal section of rabbit muscle tixed in rigor, after extraction in glycerol. Extra thin filaments anchored at the Z-line are located at the periphery of the myofibrils (arrow). These filaments are free of cross bridges. x 70,000.
This work does not solve the question of interaction between intermediate filaments and the myofibril. Although our results are negative in that regard, we cannot exclude that an existing interaction is disrupted either during extraction or homogenization. However, we should point out that thin section electron microscopy does not provide evidence for very close proximity of intermediate filaments to the Z line. At the edges of the Z line in some skeletal muscle fibers there are short filaments, which form a direct link from Z lines to adjacent membranes.
These iilaments do not have the appearance of intermediate filaments (Nunzi and Franzini-Armstrong, 1980). In developing cardiac muscle, high resolution labeling of desmin and vimentin filaments in deep etch images demonstrate no specific Z line attachment (Isobe et al., 1988). It would be interesting to known whether the ‘extra’ actin filaments are composed of a muscle actin isoform (alpha) or of a cytoplasmic isoform (beta or gamma). Recently, it has been shown that gamma actin is present in small amounts in adult vertebrate skeletal
EXTRA
ACTIN
197
FILAMENTS
muscle (Otey et al., 1988). This characteristically non myofibrillar actin could either be one of the components of the constitutive thin filaments or a component of the supernumerary actin filaments we observe.
Acknowledgments
This work was supported by a grant from the MDA to the H.M. Watts Research Center. We are grateful to MS Denah Appelt for her expert help. We are grateful to John Armstrong for participating in this research.
Bennett, G. S., Fellini, S. A., Toyama, Y. and Holtzer, H. 1979. Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro. 1. Cell Biol., 82, 577-584. Granger, B. L. and Lazarides, E. 1978. The existence of an insoluble Z disc scaffold in chicken skeletal muscle. Cell, 15, 125.3-1268. Granger, B. L. and Lazarides, E. 1979. Desmin and vimentin coexist at the periphery of the myofibril Z disc. CeN, 18, 1053-1063. Heuser, J. E. and Cooke, R. 1983. Actin myosin interactions visualized by the quick freeze, deep-etch replica technique. .I. Mol. Biol., 169, 97-122. Heuser, J. E. and Kirschner, M. W. 1980. Filament organization revealed in platinum replicas of freeze-dried cytoskeleton. J. Cell Biol., 86, 212-234. Holtzer. H., Bennett, G. S., Tapscott, S. J., Croop, J. M. and Toyama, Y. 1982. Intermediate-size filament: changes in synthesis and distribution in cells of the myogenic and neurogenic lineages. Cold Spring Harbor Symp. Quant. Biol., 46,317-328. Hoyle, G. 1983. Mnxles and Their Neural Control. Wiley Intersciences, N.Y. Sons, Inc. Isobe, Y. and Lemanski, L. F. 1986. Localization of alpha actin in the cytoskeleton of cultured cardiomyocytes by platinum immunogold electron microscopy. J. Cell Eiol., 183, 126a. Isobe, Y., Heu, G. R. and Lemanski, L. F. 1988. Three dimensional distribution of desmin and vimentin in cultured hamster cardiac myocytes using the immunogold deep-etching replica technique. J. Cell Biol., 107, 473a. Lazarides, E. 1980. Intermediate filaments as mechanical integrators of cellular space. Nature, 283, 249256. Lazarides, E. and Granger, B. L. 1983. Transcytoplasmic integration in avian erythrocytes and striated muscle: the role of intermediate filaments. Modern Cell Biology, 2, 143-162. Nunzi, M. G. and Franzini-Armstrong, C. 1980. Trabecular network in adult skeletal muscle. J. Ultrmtr. Res., 73, 2126. Otey. C. A., Kalnoski, M. H. and Bulinksi, J. C. 1988. Immunolocalization of muscle and non muscle isoforms of actin in myogenic cells and adult skeletal muscle. CeU Motility and the Cytoskeleton, 9, 337-348. Page, S. G. 1969. Structure and some contractile properties of fast and slow muscle of the chicken. J. Physiol. 205, 131-145. Pierobon Bormioli, S. 1981. Transverse sarcomere tilamentous systems: ‘Z- and M-cables’. J. Muscle Res. Cell Motil , 2, 401-413. Small, J. V. and Langanger, G. 1981. Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks. J. Cell Biol., 91, 695-705. Tokuyasu, K. T. 1983. Visualization of longitudinally oriented intermediate filaments in frozen sections of chicken cardiac muscle by a new staining method. J. Cell Biol., 97, 562-565. Tokuyasu. K. T., Dutton, A. H. and Singer, S. J. 1983. Immunoelectron microscopic studies of desmin (skeleton) localization and intermediate filament organization in chicken skeletal muscle. J. Cell Biol., %, 1727-1735. Tokuyasu, K. T., Maher, P. A. and Singer, S. J. 1985. Distributions of vimentin and desmin in developing chick myotubes in viva. II Immunoelectron microscopic study. 1. Cell Biol., 100, 1157-1166. Wang, K. and Ramirez Mitchell, R. 1983. A network of transverse and longitudinal intermediate filaments is associated with sarcomeres of adult vertebrate skeletal muscle. J. Cell Biol., 96,562-570. Wang, K. 1984. Cytoskeletal matrix in striated muscle: the role of titin, nebulin, and intermediate filaments. Adu. hp. Med. Biol., 170, 285-305.