Nascent Myofibrils Research Articles

The premyofibril: evidence for its role in myofibrillogenesis.

When cardiac muscle cells are isolated from embryonic chicks and grown in culture they attach to the substrate as spherical cells with disrupted myofibrils, and over several days in culture, they spread and extend lamellae. Based on antibody localizations of various cytoskeletal proteins within the spreading cardiomyocyte, three types of myofibrils have been identified: 1) fully formed mature myofibrils that are centrally positioned in the cell, 2) premyofibrils that are closest to the cell periphery, and 3) nascent myofibrils located between the premyofibrils and the mature myofibrils. Muscle-specific myosin is localized in the A-bands in the mature, contractile myofibrils, and along the nascent myofibrils in a continuous pattern, but it is absent from the premyofibrils. Antibodies to non-muscle isoforms of myosin IIB react with the premyofibrils at the cell periphery and with the nascent myofibrils, revealing short bands of myosin between closely spaced bands of alpha-actinin. In the areas where the nascent myofibrils border on the mature myofibrils, the bands of non-muscle myosin II reach lengths matching the lengths of the mature A-bands. With the exception of a small transition zone consisting of one myofibril, or sometimes several sarcomeres, bordering the nascent myofibrils, there is no reaction of these non-muscle myosin IIB antibodies with the mature myofibrils in spreading myocytes. C-protein is found only in the mature myofibrils, and its presence there may prevent co-polymerization of non-muscle and muscle myosins. Antibodies directed against the non-muscle myosin isoforms, IIA, do not stain the cardiomyocytes. In contrast to the cardiomyocytes, the fibroblasts in these cultures stain with antibodies to both non-muscle myosin IIA and IIB. The premyofibrils near the leading edge of the lamellae show no reaction with antibodies to either titin or zeugmatin, whereas the nascent myofibrils and mature myofibrils do. The spacings of the banded alpha-actinin staining range from 0.3 to 1.4 microns in the pre- and nascent myofibrils and reach full spacings (1.8-2.5 microns) in the mature myofibrils. Based on these observations, we propose a premyofibril model in which non-muscle myosin IIB, titin, and zeugmatin play key roles in myofibrillogenesis. This model proposes that pre- and nascent myofibrils are composed of minisarcomeres that increase in length, presumably by the concurrent elongation of actin filaments, the loss of the non-muscle myosin II filaments, the fusion of dense bodies or Z-bodies to form wide Z-bands, and the capture and alignment of muscle myosin II filaments to form the full spacings of mature myofibrils.

Incorporation of microinjected biotin-labelled actin into nascent myofibrils of cardiac myocytes: an immunoelectron microscopic study

Incorporation of microinjected biotin-labelled actin into nascent myofibrils of cultured cardiac muscle cells was investigated by immunogold electron microscopy. At the proximal parts of myofibrils, gold labelling was first found (at about 4 min after injection) around the A-band level. This observation suggests that polymerization of actin or the addition of newly-formed actin filaments occurs preferentially in association with myosin filaments to increase the myofibrillar girth. The distal terminals of developing myofibrils were also labelled at about 4 min after injection. This rapid incorporation of actin subunits at the myofibrillar ends suggests the continued reorganization and/or de novo formation of myofibrils at these positions. Along the extending direction of the myofibrillar terminals, gold particles were arranged in rows on the inner surface of the sarcolemma. These rows of particles continued to become longer with incubation. It appears that actin subunits are added at the membrane-associated ends of pre-existing actin filaments to increase the length of myofibrils.

Localization of CapZ during myofibrillogenesis in cultured chicken muscle.

Actin filaments undergo dramatic changes in their organization during myofibrillogenesis. In mature skeletal muscle, both CapZ and the barbed end of the actin filaments are located at Z-discs. In vitro, CapZ binds the barbed end of actin filaments and prevents actin subunit addition and loss; CapZ also nucleates actin polymerization in vitro. Taken together, these properties suggest that CapZ may function to organize actin filaments during myofibrillogenesis. We report here that the amount of CapZ in myofibrils from adult chicken pectoral muscle is sufficient to "cap" each actin filament of the sacromere. Double immunofluorescence microscopy of skeletal muscle cells in culture was used to determine the spatial and temporal distributions of CapZ relative to actin, alpha-actinin, titin, and myosin during myofibrillogenesis. Of particular interest was the assembly of CapZ at nascent Z-discs in relation to the organization of actin filaments in nascent myofibrils. In myoblasts and young myotubes, CapZ was diffusely distributed in the cytoplasm. As myotubes matured, CapZ was initially observed in a uniform distribution along non-striated actin filaments called stress fiber-like structures (SFLS). CapZ was observed in a periodic pattern characteristic of mature Z-discs along the SFLS prior to the appearance of a striated staining pattern for actin. In older myotubes, when actin was observed in a pattern characteristic of I-bands, CapZ was distributed in a periodic pattern characteristic of mature Z-discs. The finding that CapZ was assembled at nascent Z-discs before actin was observed in a striated pattern is consistent with the hypothesis that CapZ directs the location and polarity of actin filaments during I-band formation in skeletal muscle cells. The assembly of CapZ at nascent Z-disc structures also was observed relative to the assembly of sarcomeric alpha-actinin, titin, and thick filaments. Titin and myosin were observed in structures having the organization of mature sarcomeres prior to the appearance of CapZ at nascent Z-discs. The distribution of CapZ and sarcomeric alpha-actinin in young myotubes was not coincident; in older myotubes, both CapZ and alpha-actinin were co-localized at Z-discs. In cardiac myocytes, CapZ was detected at Z-discs and was distributed in a punctate pattern throughout the cytoplasm. CapZ also was co-localized with A-CAM and vinculin at cell-cell junctions formed by the myocytes.

Association of titin and myosin heavy chain in developing skeletal muscle.

To understand molecular interactions that organize developing myofibrils, we examined the biosynthesis and interaction of titin and myosin heavy chain in cultures of developing muscle. Use of pulse-labeling, immunoprecipitation, and a reversible cross-linking procedure demonstrates that within minutes of synthesis, titin and myosin heavy chain can be chemically cross-linked into very large, detergent-resistant complexes retaining many features of intact myotubes. These complexes, predominantly of titin and myosin, occur very early in myofibrillogenesis as well as later. These data suggest that synthesis and assembly of titin and myosin are temporally and spatially coordinated in nascent myofibrils and support the hypothesis that titin molecules help to organize sarcomere formation.

The α5β1 integrin associates with a dystrophin-containing lattice during muscle development

The organization of the α 5 β 1 integrin on skeletal muscle was studied in culture and in sections from adult and embryonic tissue using monoclonal antibodies specific for the α 5 subunit. The α 5 β 1 integrin showed changes in organization and in the molecules with which it colocalizes. On early myoblasts, possessing a fibroblast-like morphology, the α 5 integrin organization was indistinguishable from that on fibroblasts; it was expressed prominently and localized in numerous focal contacts around the cell periphery. In bipolar myoblasts and early myotubes, the α 5 integrin was expressed only weakly and localized in a small number of focal contact-like structures. As myogenesis proceeded there was an apparent increase in integrin expression and a change in organization. In addition to the focal contact-like structures that persist throughout myogenesis in vitro, a dense lattice-like structure of integrin appeared. Fibrillar fibronectin, talin, and non-muscle α-actinin did not colocalize with the α 5 β 1 integrin in the lattice structure as they did in the focal contact-like structures. However, dystrophin, which displayed a diffuse distribution earlier, now colocalized with the α 5 β 1 integrin in the punctate lattice. Coincident with the registration of myofibrils into visible sarcomeres, the prominent dense, lattice structure disappeared leaving the focal contact-like structures as the only regions of organized α 5 β 1 integrin. Despite the presence of the β 1 integrin in neuromuscular or myotendinous junctions in vivo and on myotubes in vitro, the α 5 β 1 integrin was not present in either junction. These observations suggest that the α 5 β 1 integrin is involved in the adhesion of muscle to the extracellular matrix, the organization of the dystrophin-containing lattice, and the organization of nascent myofibrils which emanate from the focal contact- and stress fiber-like structures in muscle. Other integrins appear to anchor myofibrils at the myotendinous and neuromuscular junctions.

Cytoskeletal filaments in embryonic chick myocardial cells as revealed by the quick-freeze deep-etch method combined with immunocytochemistry

The three-dimensional organization of cytoskeletal filaments associated with the myofibrils and sarcolemma of the myocardial cells of early chick embryos was studied by the rapid-freeze deep-etch method combined with immunocytochemistry. In the endoplasmic region of saponin-treated myocardial cells, 12-14 nm filaments formed a loose network surrounding nascent myofibrils. These 12-14 nm filaments attached to the myofibrils and some of them converged into Z disc regions. In the non-junctional cytocortical region thinner 8-11 nm filaments composed a dense network just beneath the sarcolemma. In myofibril terminating regions at the sarcolemma, i.e., the fascia adherens, 3-5 nm cross-bridges were observed among the thin filaments. In Triton-permeabilized and myosin subfragment 1 (S1)- treated samples, subsarcolemmal 8-11 nm filaments proved to be S1-decorated actin filaments under which there was a loose network of S1-undecorated filaments. Subsarcolemmal S1-decorated actin filaments had mixed polarity and attached to the sarcolemma at one end. A loose network of S1-undecorated filaments among myofibrils in the endoplasmic region was revealed to consist of desmin-containing intermediate filaments after immuno-gold staining for desmin. These networks connecting myofibrils with sarcolemma were assumed to play an important role in integrating and transmitting the contractile force of individual myofibrils within early embryonic myocardial cells.

Studies on the role of microtubules in myofibrillogenesis

Go-localization of microtubnle (MT) and muscle myosin (MHC) myofibril immunofluoreseoneo in developing myotubes of chicken skeletal muscle cultures was observed by using double staining of tubulin and MHC indirect immunofluorescence. 12-o-tetradecanoyl-phorbol-13-acetate (TPA) selectively and reversibly blocks myofibrillogenesis and alters the morphology of myotubes into myosacs where MTs are present in radiating pattern. When the arrested myogenic cells recover and start myofibrillogenesis after released from TPA, prior to the emergence of myofibrils, the pro-existing MTs become bipolarly aligned eoineidontly with the tubular restoration of cell shape. Single nascent myofibrils overlapping with MTs extend into the base of growth tips where MTs go farther to the end of the tips. That MT might act as scaffold in guiding the bipolar elongation of the growing myofibrils was suggested. Taxol and eoleomid disturbed MT polymerization and disposition, and interfered with the normal spatial assembly of myofibrils in developing myotubos.

Three-dimensional electron microscopy of the sarcoplasmic reticulum and T-system in embryonic chick skeletal muscle cells in vitro

The formation of the sarcoplasmic reticulum (SR) and the transverse tubular system (T-system) in embryonic chick skeletal muscle cells in vitro was studied by either the critical point drying-physical rupturing or physical rupturing-freeze drying together with rotary shadowing. In these cells, two membranous systems were observed. One was composed of flattened sacs which were either isolated or were connected to each other with slender processes to form mostly longitudinally oriented strands. Initially, these sacs had small granules at their surface and were found mainly under the sarcolemma. Later, they became smooth at their surface, extending throughout the cytoplasm to form irregular and dense networks. At later phases, the networks tended to be disposed at right angle to nascent myofibrils, exhibiting a characteristic honeycomb appearance. From the similarities in thin section images, they were identified as developing SR.

The early expression of myofibrillar proteins in round postmitotic myoblasts of embryonic skeletal muscle

Previous reports on skeletal muscle myogenesis have shown that postmitotic spindle-shaped myoblasts express muscle-specific proteins, some of which are organized into nascent myofibrils. However, we show that, in skeletal muscle cultures derived from 12-day chick embryos, by 6 h after plating the predominant mononucleated cell type that expresses muscle-specific proteins is a round cell. These round myoblasts appear to precede spindle-shaped myoblasts in development, since the latter are more abundant in later cultures and contain larger amounts of muscle proteins and more highly organized myofibrils. By double immunofluorescence microscopy using antibodies specific for the muscle proteins titin, myosin heavy chain (MHC) and zeugmatin we find that 18 h after plating approximately 20% of the round myoblasts that are titin-positive are negative for myofibrillar MHC and zeugmatin. On the other hand, all spindle-shaped myocytes that are positive for titin are also positive for myofibrillar MHC and zeugmatin. These results suggest that titin expression precedes that of myofibrillar MHC and zeugmatin in the non-synchronized round myoblasts, and is consistent with earlier suggestions that titin may function as an initial organizer of myofibrillar proteins during myogenesis. Immunofluorescence data indicate that the earliest localization of the myofibrillar proteins titin, MHC, zeugmatin and alpha-actinin in the round myoblasts is surrounding the nucleus with no immunofluorescent labeling of the cytoplasm or near the plasma membrane. Furthermore, pairwise double immunofluorescence experiments show that these four myofibrillar proteins are all co-localized, at the light-microscopic level of resolution, in irregular patterns that may appear in either a punctate or a basket-like distribution. These labeling patterns around the nucleus are resistant to extraction with Triton X-100, suggesting that the proteins are associated in a stable array. These Triton X-100-resistant assemblies in round myoblasts appear to be composed solely of structural myofibrillar proteins, since the non-structural myofibrillar protein creatine kinase (CK) does not colocalize with the other myofibrillar proteins. These results indicate that in early myoblasts myofibrillar proteins form stable pre-myofibrillar assemblies surrounding the nucleus, and raise the possibility that these initial assemblies may play an organizing role during subsequent early stages of myofibrillogenesis.

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes.

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown.

Skeletal muscle myofibrillogenesis as revealed with a monoclonal antibody to titin in combination with detection of the α- and γ-isoforms of actin

The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogenic cultures and fluorescent-antibody staining. Efforts were made to compare the distribution and temporal sequence of incorporation of titin relative to that of the α- and γ-isoforms of actin. The present observations suggested the following sequence of titin assembly: (1) newly synthesized titin molecules are distributed in a diffuse pattern throughout the sarcoplasm, (2) the titin molecules gradually associate with α- and γ-actin-positive stress fiber-like structures (SFLS), (3) groups of titin molecules begin to segregate on the SFLS, and (4) titin molecules align in a mature doublet configuration in the sarcomeres of nascent myofibrils. Titin assembly on the SFLS often appeared prior to the onset of either α- or γ-actin periodicity on nascent myofibrils; the latter result suggested a role for titin in sarcomeric organization. Actin distribution on SFLS and its periodicity on nascent myofibrils was usually identical between the α- and γ-isoforms. This suggested that γ-actin participated in myofibrillogenesis in a manner indistinguishable from that of α-actin. The transition seen from continuous actin staining of SFLS to the I-band staining pattern of mature myofibrils is discussed in relation to the corresponding reorganization of actin filaments and the molecular associations that this would entail.

Myofibril assembly is linked with vinculin, α‐actinin, and cell‐substrate contacts in embryonic cardiac myocytes in vitro

The relationship of nascent myofibrils with the accumulation of adhesion plaque proteins and the formation of focal cell contacts was studied in embryonic chick cardiac myocytes in vitro. The cultures were double-stained with various combinations of the specific antiactin drug phalloidin and antibodies against vinculin, alpha-actinin, connectin (titin), myosin heavy chain, fibronectin, and desmin and examined under fluorescence and interference reflection microscopy. In the areas of myofibril assembly, vinculin and alpha-actinin plaques were formed at the ventral sarcolemmae. These areas overlapped with the sites of cell-to-substrate focal contacts and extracellular fibronectin. Because the myofibrils always ran in a straight line between these sites, polarized lines appeared to be generated within the cells in response to their physical (e.g., stress) and/or biochemical environment (e.g., adhesion plaque proteins). The possible presence of other factors cannot be ruled out for the proper alignment of myofibrils. As soon as myofibrils came to span between these adhesion sites, they exhibited typically mature cross-striated characteristics. Thus, the formation of these inferred lines has some relation to, or is in fact necessary for, the maturation of myofibrils, in addition to the directional arrangement of sarcomeric proteins. Additionally, synthesis and distribution of myosin and connectin were tightly linked during early developmental (premyofibril and myofibril) stages. The spatial deployment of desmin was not coupled with vinculin. Thus, connectin and desmin do not appear to form the initial scaffold of sarcomeres.

Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. III. Generation of fasciae adherentes and costameres.

To study whether the first myofibrils are separate from or firmly bound to the myocytic cell membranes, whole mount preparations of 6-12-somite-stage chick embryonic hearts were examined by fluorescence microscopy after double labeling with antibodies to vinculin (fluorescein-conjugated) and rhodamine-phalloidin, or with antibodies to titin (rhodamine-conjugated) and nitrobenz-oxadiazole-phallacidin. When a small number of myofibrils appeared for the first time at the nine somite stage, most of them were already bound to the cell membranes through zonulae adherentes, fasciae adherentes, or costameres. In the outer of the two myocardial cell layers, in which the myocytes were closely in contact with each other along polygonal boundaries, fasciae adherentes and costameres developed at the boundaries, apparently by conversion of preexisting zonulae adherentes. On the other hand, in the inner cell layer, in which myocytes were more loosely associated with each other, both costameres and fasciae adherentes appeared to develop de novo, the former in association with the inner surface of the myocardial wall and the latter at the intercellular boundaries. The myofibrillar tracks in the inner layer followed long and smooth courses and were as a whole aligned in the circumferential direction of the tubular heart wall from the earliest stage of myofibril formation. Those in the outer layer were arranged in a pattern of two- or three-dimensional networks in the 9-10 somite stage, although many myofibrils were also circumferentially directed. The fact that the majority of the first myofibrils were already bound to the cell membranes in a directed manner suggests that myocytes at the earliest stage of myofibril formation are endowed with spatial information that directs the organization of nascent myofibrils. It is proposed that the myocyte cell membranes perform an essential role in cardiac myofibrillogenesis.

Visualization and Identification of Cytoskeletal Filaments in Embryonic Chick Heart by the Quick-Freeze Deep-Etch Method Combined with Immunocytochemistry

Cytoskeletal filaments in myocardial cells of chick embryo (stage 18-20, day 3) were studied by immunocytochemical and rapid-freeze deep-etch methods. A three-dimensional network of cytoplasmic filaments surrounding nascent myofibrils was visualized in saponin-treated myocardial cells. The major part of the network was composed of 12 to 14 nm filaments in platinum replicas. To identify the filaments, the myocardial cells were permeabilized with Triton X-100 and treated with myosin subfragment-1 (S1) for actin or immunogold-labeled antibody for desmin. A large number of filaments in myofibrils and a few cytoplasmic filaments were decorated with S1. A loose network surrounding the myofibrils was not decorated with S1 but with gold particles. This finding means that the majority of filaments occupying the interfibrillar space were desmin-containing filaments.

Three-dimensional immunogold localization of alpha-actinin within the cytoskeletal networks of cultured cardiac muscle and nonmuscle cells.

The ultrastructural distribution of alpha-actinin was studied in cultured hamster heart cells by immunogold replica electron microscopy. This technique enabled us to localize alpha-actinin within the cytoskeletal networks at high resolution and in three dimensions. Colloidal gold, indicating the presence of alpha-actinin, was localized on the Z bands of nascent myofibrils in myocytes and on stress fiber bundles in nonmuscle cells. alpha-Actinin staining was also seen on stellate foci, where cytoskeletal filaments converged along the inner myocyte cell membranes. Intermediate filaments were associated with Z bands of myofibrils, stress fibers, and subplasmalemmal actin networks at the specific points where alpha-actinin was localized on these structures. Heavy meromyosin treatment prior to immunostaining confirmed that the thin filaments contained actin. These results suggest that alpha-actinin serves to interlink these various cytoskeletal elements. In addition, this protein may be involved in the initial phases of filament organization during myofibrillogenesis along the inner surface of the myocyte plasma membrane.

Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. II. Generation of alpha-actinin dots within titin spots at the time of the first myofibril formation.

In whole mount preparations of the 9 somite stage chick embryonic hearts that were immunofluorescently double labeled for titin and alpha-actinin, presumptive myofibrils were recognized as rows of several periodically aligned titin spots. Within these titin spots, smaller alpha-actinin dots were observed. These periodical arrangements of titin spots and alpha-actinin dots were not found in the 7 somite stage hearts. In wide myofibrils in the 10 somite stage hearts, the alpha-actinin dots and titin spots simultaneously became 'lines.' To study the ultrastructural features of the titin-positive regions in the 6-9 somite stage hearts, the thoracic portions of the embryos were immunofluorescently labeled for titin and embedded in resin. Ultrathin sections were mounted on electron microscopic grids and examined in immunofluorescence optics. The titin-positive regions thus identified were then examined in the electron microscope. No readily discernable specific ultrastructural features were found in titin-positive regions of the 6 somite stage cardiac primodia. Examination of the sections of the 9 somite stage hearts, on the other hand, revealed the occasional presence of small dense bodies, Z bodies, in the titin-positive regions. These observations strongly suggest that these Z bodies are the ultrastructural counterparts of the alpha-actinin dots seen by immunofluorescence optics and that they are formed nearly at the time of the formation of the first myofibrils. In some of the nascent myofibrils the Z bodies were found to be considerably narrower than the myofibrils, implying that the Z bodies are required not for the assembly of myofibrils per se but for their stabilization. Immunofluorescent labeling for titin and alpha-actinin revealed that the length of the shortest sarcomeres in the first myofibrils is approximately 1.5 micron, approximately the width of the A bands of mature myofibrils. The possibility that the A bands might define the initial length of nascent sarcomeres was indicated.

Role of stress fiber-like structures in assembling nascent myofibrils in myosheets recovering from exposure to ethyl methanesulfonate.

When day 1 cultures of chick myogenic cells were exposed to the mutagenic alkylating agent ethyl methanesulfonate (EMS) for 3 d, 80% of the replicating cells were killed, but postmitotic myoblasts survived. The myoblasts fused to form unusual multinucleated "myosheets": extraordinarily wide, flattened structures that were devoid of myofibrils but displayed extensive, submembranous stress fiber-like structures (SFLS). Immunoblots of the myosheets indicated that the carcinogen blocked the synthesis and accumulation of the myofibrillar myosin isoforms but not that of the cytoplasmic myosin isoform. When removed from EMS, widely spaced nascent myofibrils gradually emerged in the myosheets after 3 d. Striking co-localization of fluorescent reagents that stained SFLS and those that specifically stained myofibrils was observed for the next 2 d. By both immunofluorescence and electron microscopy, individual nascent myofibrils appeared to be part of, or juxtaposed to, preexisting individual SFLS. By day 6, all SFLS had disappeared, and the definitive myofibrils were displaced from their submembranous site into the interior of the myosheet. Immunoblots from recovering myosheets demonstrated a temporal correlation between the appearance of the myofibrillar myosin isoforms and the assembly of thick filaments. The assembly of definitive myofibrils did not appear to involve desmin intermediate filaments, but a striking aggregation of sarcoplasmic reticulum elements was seen at the level of each I-Z-band. Our findings suggest that SFLS in the EMS myosheets function as early, transitory assembly sites for nascent myofibrils.

Development of myofibrils in the gizzard of chicken embryos. Intracellular distribution of structural proteins and development of contractility.

The intracellular distributions of major muscle proteins, myosin, actin, tropomyosin, alpha-actinin, and desmin, in smooth muscle cells of chicken gizzard at various stages of embryogenesis were investigated by immunofluorescence-labeling of enzyme-dispersed cells cultured up to three hours. These muscle proteins, except some part of myosin, were organized into fibrous structures as soon as synthesis and accumulation of proteins started. As for myosin, a considerable amount of it was dispersed in soluble cytoplasm as well. On the other hand, Ca++-dependent contractility was detected with detergent-extracted myoblasts and glycerinated tissue from embryos older than 7 days. Although the nascent myofibrils bear a resemblance to "stress fibers," the former could be distinguished from the latter by their high stability in dispersed, spherical cells. The above findings, therefore, show that the synthesis of contractile proteins is followed by immediate assembly of them into functional myofibrils without undergoing any intermediate structure. Based on these findings, the mechanism of myofibril formation in developing smooth muscle cells is discussed.

Observations of microfilament bundles in living cells microinjected with fluorescently labelled contractile proteins.

Fluorescently labelled contractile proteins (alpha-actinin and filamin) were used to study the dynamic nature of three types of microfilament bundles: myofibrils, stress fibres and polygonal networks. Cultured muscle and non-muscle cells that were microinjected with fluorescent alpha-actinin rapidly incorporated the labelled protein into Z-bands, stress fibre densities and the polygonal foci. Living, injected cells were then observed for varying periods of time, and changes in orientation and periodicity of the myofibrils, stress fibres and polygonal networks were recorded. Permeabilized cells were also reacted with fluorescently labelled proteins and with contractile protein antibodies in order to analyse further the changes taking place in the myofibrils and stress fibres. In both living cardiac myocytes and living skeletal muscle myotubes, contractile myofibrils were present in the same cell with non-contractile nascent myofibrils. The periodicities of small Z-bodies in the nascent non-contractile myofibrils were shorter than the Z-band spacings in the contractile myofibrils, yet both types of myofibrils contained muscle myosin. Over a period of 24 h, a nascent myofibril in a living, microinjected myotube was observed to grow from Z-body spacings of 0.9-1.3 micron to full sarcomere spacings (2.3 microns). During the same time, nascent myofibrils appeared de novo and Z-band alignment became more ordered in the fully formed myofibrils. Stress fibres were not observed to undergo the predictable type of growth seen in myofibrils, but stress fibre periodicities did change in some fibres; some shortened while others lengthened. The orientation of fibres shifted in cytoplasm of both mobile cells and stationary cells. Attachment plaques and foci also changed position and in some cases subdivided and/or disappeared. Models of stress fibres and polygonal networks are presented that suggest that the changes in the periodicities of the dense bodies in stress fibres and the distances between polygonal foci are related to the movement of the interdigitating actin and myosin filaments.

Novel thick filament protein of chicken pectoralis muscle: the 86 kd protein: II. Distribution and localization

Antibodies specific for the novel 86 kd protein purified from chicken pectoralis myofibrils stained by indirect immunofluorescence the middle third of each half A-band of isolated myofibrils and myotubes. Pectoralis muscle 86 kd protein, like pectoralis C-protein, displayed a fibre-type specific distribution by being restricted to fast twitch fibres and absent in slow tonic and heart muscle fibres. This was demonstrated by immunoblotting experiments with tissue extracts and by immunofluorescence labelling of cryosections. In primary cell cultures prepared from embryonic chicken breast muscle, 86 kd protein, C-protein and myomesin were all detected in post-mitotic myoblasts where fluorescence was found in a cross-striated pattern along strands of nascent myofibrils. Fluorescence due to the 86 kd protein was restricted to myofibrils within myotubes and no significant labelling of the sarcoplasm was evident. Glycerinated fast twitch muscle fibres, after incubation with antibodies to 86 kd protein, revealed in each half of the A-band nine distinctly labelled stripes, spaced about 43 nm apart. Simultaneous incubation of fibres with antibodies against 86 kd protein and C-protein showed a co-localization of the seven C-protein stripes (stripes 5 to 11), with seven stripes of 86 kd protein. The two additional stripes (stripes 3 and 4) labelled by anti-86 kd antibody continued towards the M-band at the same periodicity from the last C-protein stripe (stripe 5). Thus, partial co-localization of two different thick filament proteins is demonstrated and the identity of transverse stripes at positions 3 and 4 attributed in part to the presence of the new 86 kd protein.