Immunohistochemistry of chaetognath body wall muscles

Abstract. A light and electron immunohistochemical study was carried out on the body wall muscles of the chaetognath Sagitta friderici for the presence of a variety of contractile proteins (myosin, paramyosin, actin), regulatory proteins (tropomyosin, troponin), and structural proteins (α‐actinin, desmin, vimentin). The primary muscle (～80% of body wall volume) showed the characteristic structure of transversely striated muscles, and was comparable to that of insect asynchronous flight muscles. In addition, the body wall had a secondary muscle with a peculiar structure, displaying two sarcomere types (S1 and S2), which alternated along the myofibrils. S1 sarcomeres were similar to those in the slow striated fibers of many invertebrates. In contrast, S2 sarcomeres did not show a regular sarcomeric pattern, but instead exhibited parallel arrays of 2 filament types. The thickest filaments (～10–15 nm) were arranged to form lamellar structures, surrounded by the thinnest filaments (～6 nm). Immunoreactions to desmin and vimentin were negative in both muscle types. The primary muscle exhibited the classical distribution of muscle proteins: actin, tropomyosin, and troponin were detected along the thin filaments, whereas myosin and paramyosin were localized along the thick filaments; immunolabeling of α‐actinin was found at Z‐bands. Immunoreactions in the S1 sarcomeres of the secondary muscle were very similar to those found in the primary muscle. Interestingly, the S2 sarcomeres of this muscle were labeled with actin and tropomyosin antibodies, and presented no immunore‐actions to both myosin and paramyosin. α‐Actinin in the secondary muscle was only detected at the Z‐lines that separate S1 from S2. These findings suggest that S2 are not true sarcomeres. Although they contain actin and tropomyosin in their thinnest filaments, their thickest filaments do not show myosin or paramyosin, as the striated muscle thick myofilaments do. These peculiar S2 thick filaments might be an uncommon type of intermediate filament, which were labeled neither with desmin or vimentin antibodies.     

Desmin and vimentin coexist at the periphery of the myofibril Z disc.

Two-dimensional gel electrophoresis has revealed that vimentin, the predominant subunit of intermediate filaments in cells of mesenchymal origin, is a component of isolated skeletal myofibrils. It thus coexists in mature muscle fibers with desmin, the major subunit of muscle intermediate filaments. Antisera to desmin and vimentin, shown to be specific for their respective antigens by two-dimensional immunoautoradiography, have been used in immunofluorescence to demonstrate that vimentin has the same distribution as desmin in skeletal muscle. Both desmin and vimentin surround each myofibril Z disc and form honeycomb-like networks within each Z plane of the muscle fiber. This distribution is complementary to that of alpha-actinin within a given Z plane. Desmin and vimentin may thus be involved in maintaining the lateral registration of sarcomeres by transversely linking adjacent myofibrils at their Z discs. This linkage would support and integrate the fiber as a whole, and provide a molecular basis for the cross-striated appearance of skeletal muscle.     

The release of alanine by rat diaphragm muscle in vitro.

Z discs were isolated from Lethocerus flight muscle by removing the contractile proteins from myofibrils with a solution of high ionic strength. The protein composition of the Z discs was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the major proteins were alpha-actinin, actin and tropomyosin. Z lines were selectively removed from intact myofibrils by digestion with crude lipase and chymotrypsin, but not by purified lipase.     

Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle

The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length.     

Reduction of density and anisotropic distribution of intermediate filaments occur during avian skeletal myogenesis

Chicken skeletal muscle taken from embryos in ovo was examined by thin-section electron microscopy. Measurements of filament diameters reveal three nonoverlapping groups of filaments: thin (actin myofibrillar) filaments with mean diameters of 5.3 +/- 0.6 nm (S.D.), thick (myosin myofibrillar) filaments with mean diameters of 15 +/- 1.4 nm, and intermediate filaments with mean diameters of 9.3 +/- 0.9 nm. During muscle development these diameters do not change. By counting the number of filaments observed in the sarcoplasm at different stages, we find that the spatial density of intermediate filaments decreases during avian myogenesis in ovo, from 91 intermediate filaments/micron 2 at 6 days to 43 intermediate filaments/micron 2 at 17 days in ovo. Initially randomly arranged, some intermediate filaments become associated with Z discs, sarcoplasmic reticulum, nuclear membrane, and the sarcolemma between 6 and 10 days in ovo. These associated intermediate filaments course both parallel and transverse to myofibrils, forming lateral connections between myofibrillar Z discs and longitudinal connections from Z disc to Z disc within myofibrils. Intermediate filaments also appear to connect Z discs with the nuclear membrane. The intermediate filament associations persist through day 17 of development, after which the presence of cytoskeletal filaments is obscured by the densely packed myofibrils and membranes. Intermediate filament distribution becomes anisotropic during development. A greater proportion of intermediate filaments in the immediate perimyofibrillar area are oriented parallel to myofibrils than in other areas, so that the majority of the intermediate filaments nearest the myofibrils course parallel to them. The longitudinal intramyofibrillar intermediate filaments persist throughout development, as shown by their existence in KI-extracted adult myofibrils.     

Proteolytic activity of proteasome on myofibrillar structures

The physiologic function of proteasome remains unclear. Evidence suggests a role in degradation of ubiquitin-protein conjugates, MHC antigen presentation, and some specificity of substrate within certain cell types. To explore further the properties of proteasome we have examined its effect on a well defined structure, the myofibril. We find that despite its large size (20S) proteasome is able to degrade myofibrils and intact, permeabilized muscle fibrils. The proteins degraded showed some specificity because actin, myosin and desmin were degraded faster than -actinin, troponin T and tropomyosin. Changes in ultrastructure were slow and included a general loss of structure with Z and I bands effected before the M band and costameres.     

The synthesis and deployment of filamin in chicken skeletal muscle

During myogenesis in vitro the actin-binding protein filamin is present in myoblasts and early fused cells and is associated with α-actinin-containing filament bundles, as judged by double immunofluorescence using antibodies specific for these two proteins. Approximately one day after cell fusion, yet before the development of a-actinin-containing Z line striations, filamin disappears from the cells. Later in myogenesis, several days after the appearance of α-actinin-containing Z line striations, filamin reappears and accumulates in the cells. Double immunofluorescence with antibodies to filamin and vimentin (or desmin) reveals that the newly appearing filamin localizes now to the myofibril Z line and is visible there shortly before vimentin or desmin becomes associated with the Z line. Immunofluorescent localization of filamin in isolated chicken skeletal myofibrils and Z disc sheets indicates that filamin has the same distribution as desmin and vimentin; it surrounds each myofibril Z disc and forms honeycomb-like networks within each Z plane of the muscle fiber. Filamin may thus be involved in the transition of desmin and vimentin to the Z disc. Analysis of whole-cell extracts by SDS-polyacrylamide gel electrophoresis and by immunoautoradiography shows that filamin is present in myoblasts and in myotubes early after cell fusion. Concomitant with the absence of filamin fluorescence during the subsequent few days of myogenesis, the quantity of filamin is markedly reduced. During this time, metabolic pulse-labeling with ³⁵S-methionine reveals that the synthetic rate of filamin is also markedly reduced. As filamin fluorescence appears at the Z line, the quantity of filamin and its synthetic rate both increase. The removal of filamin from the cells suggests that filamin either may not be required, or may actually interfere with a necessary process, during the early stages of sarcomere morphogenesis. These results also indicate that the periphery of the Z disc is assembled in at least two distinct steps during myogenesis.     

Polysome structure in sea urchin eggs and embryos: an electron microscopic analysis

Primary cultures of cardiac myocytes from newborn normal and genetically cardiomyopathic (strain UM-X7.1) hamsters were analyzed by electron microscopy and immunofluorescent staining for myosin, actin, tropomyosin, and alpha-actinin. Antibody staining of these contractile proteins demonstrates that both normal and cardiomyopathic (CM) myocytes contain prominent myofibrils after 3 days in culture, although the CM myofibrils are disarrayed and not aligned as those in normal cells. The disarray becomes even more pronounced in CM cells after 5 days in culture. The immunofluorescent staining patterns of individual myofibrils in normal and CM cells were similar for myosin, actin, and tropomyosin. However, alpha-actinin staining reveals that the CM myofibrils have abnormally wide and irregularly shaped Z bands. Electron microscopy confirms the irregular Z-band appearance as well as the myofibril disarray. Thus, CM cardiomyocytes clearly show an aberrant pattern of myofibril structure and organization in culture.     

Linear dichroism of acrylodan-labeled tropomyosin and myosin subfragment 1 bound to actin in myofibrils.

Muscle contraction can be activated by the binding of myosin heads to the thin filament, which appears to result in thin filament structural changes. In vitro studies of reconstituted muscle thin filaments have shown changes in tropomyosin-actin geometry associated with the binding of myosin subfragment 1 to actin. Further information about these structural changes was obtained with fluorescence-detected linear dichroism of tropomyosin, which was labeled at Cys 190 with acrylodan and incorporated into oriented ghost myofibrils. The fluorescence from three sarcomeres of the fibril was collected with the high numerical aperture objective of a microscope and the dichroic ratio, R (0/90 degrees), for excitation parallel/perpendicular to the fibril, was obtained, which gave the average probe dipole polar angle, Theta. For both acrylodan-labeled tropomyosin bound to actin in fibrils and in Mg2+ paracrystals, Theta congruent to 52 degrees +/- 1.0 degrees, allowing for a small degree of orientational disorder. Binding of myosin subfragment 1 to actin in fibrils did not change Theta; i.e., the orientation of the rigidly bound probe on tropomyosin did not change relative to the actin axis. These data indicate that myosin subfragment 1 binding to actin does not appreciably perturb the structure of tropomyosin near the probe and suggest that the geometry changes are such as to maintain the parallel orientation of the tropomyosin and actin axes, a finding consistent with models of muscle regulation. Data are also presented for effects of MgADP on the orientation of labeled myosin subfragment 1 bound to actin in myofibrils.     

Gelsolin sensitivity of microfilaments as a marker for muscle differentiation

The ability of porcine smooth muscle gelsolin to sever actin filaments was used to study alterations in the organization of F-actin containing structures during skeletal myogenesis. In permeabilized fibroblasts and unfused myoblasts, gelsolin induced complete degradation of the actin cytoskeleton. After fusion of myoblasts to multinucleated myotubes, gelsolin removed a substantial amount of actin, revealing fibers with a sarcomere-like arrangement of gelsolin-insensitive actin. These fibrils were much thinner and had shorter sarcomeres than fully differentiated myofibrils. The proportion of gelsolin-resistant fibrils increased during differentiation, resulting in almost complete inertness of mature myofibrils. Fibrils isolated from adult muscle were also found nearly resistant to gelsolin. Extraction of tropomyosin and myosin in buffer of high ionic strength prior to gelsolin treatment reestablished the susceptibility to the severing protein, both in myotubes and isolated myofibrils. Only small remnants of phalloidin-stainable material were retained. We therefore conclude that during myotube differentiation either an increased interaction of actin with actin-binding proteins (e.g., myosin and tropomyosin), or the assembly of muscle-specific isoforms of these proteins protect the filaments against degradation by actin severing proteins.     

Activation of the Adenosine Triphosphatase of Limulus polyphemus Actomyosin by Tropomyosin

Purified actin does not stimulate the adenosine triphosphatase (ATPase) activity of Limulus myosin greatly. The ATPase activity of such reconstituted preparations is only about one-fourth the ATPase of myofibrils or of natural actomyosin. Actin preparations containing tropomyosin, however, activate Limulus myosin fully. Both the tropomyosin and the actin preparations appear to be pure when tested by different techniques. Tropomyosin combines with actin but not with myosin and full activation is reached at a tropomyosin-to-actin ratio likely to be present in muscle. Tropomyosin and actin of several different animals stimulate the ATPase of Limulus myosin. Tropomyosin, however, is not required for the ATPases of scallop and rabbit myosin which are fully activated by pure actin alone. Evidence is presented that Limulus myosin, in the presence of ATP at low ionic strength, has a higher affinity for actin modified by tropomyosin than for pure actin.     

Incorporation of fluorescently labeled actin and tropomyosin into muscle cells

The two major proteins in the I-bands of skeletal muscle, actin and tropomyosin, were each labeled with fluorescent dyes and microinjected into cultured cardiac myocytes and skeletal muscle myotubes. Actin was incorporated along the entire length of the I-band in both types of muscle cells. In the myotubes, the incorporation was uniform, whereas in cardiac myocytes twice as much actin was incorporated in the Z-bands as in any other area of the I-band. Labeled tropomyosin that had been prepared from skeletal or smooth muscle was incorporated in a doublet in the I-band with an absence of incorporation in the Z-band. Tropomyosin prepared from brain was incorporated in a similar pattern in the I-bands of cardiac myocytes but was not incorporated in myotubes. These results in living muscle cells contrast with the patterns obtained when labeled actin and tropomyosin are added to isolated myofibrils. Labeled tropomyosins do not bind to any region of the isolated myofibrils, and labeled actin binds to A-bands. Thus, only living skeletal and cardiac muscle cells incorporate exogenous actin and tropomyosin in patterns expected from their known myofibrillar localization. These experiments demonstrate that in contrast to the isolated myofibrils, myofibrils in living cells are dynamic structures that are able to exchange actin and tropomyosin molecules for corresponding labeled molecules. The known overlap of actin filaments in cardiac Z-bands but not in skeletal muscle Z-bands accounts for the different patterns of actin incorporation in these cells. The ability of cardiac myocytes and non-muscle cells but not skeletal myotubes to incorporate brain tropomyosin may reflect differences in the relative actin-binding affinities of non-muscle tropomyosin and the respective native tropomyosins. The implications of these results for myofibrillogenesis are presented.     

Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A- band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha- actinin and, if actin is added subsequently, the exogenous alpha- actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.     

Degradation of rat cardiac myofibrils and myofibrillar proteins by a myosin-cleaving protease.

The degradation of rat cardiac myofibrils and their constituent proteins with a myosin-cleaving protease was studied. Electrophoretograms of the digestion products of myofibrils showed that myosin,M-protein, C-protein, and troponin were degraded, but actin and tropomyosin were not. Degradation of these constituents resulted in losses of the Mg2+-ATPase activity and its Ca2+-sensitivity of myofibrils. Incubation of myofibrils with the protease induced the release of alpha-actinin without degradation. Susceptibilities of myosin, actin, troponin, and alpha-actinin purified from rat and pig hearts to the protease were essentially identical to those of the assembled forms in myofibrils. Although the purified tropomyosin was readily degraded into five fragments with the protease, the tropomyosin assembled in myofibrils and actin-tropomyosin complex were insusceptible to the protease. Digestion of myosin in the filamentous state with the protease resulted in the disappearance of myosin heavy chain and light chain 2, producing two fragments having molecular weights of 130,000 and 94,000 which originated from the degradation of heavy chain. The Ca2+- and EDTA-ATPase activities of the degradation products remained unchanged during incubation for 22 h. The actin-activated ATPase activity of myosin was reduced by 30% during incubation for 6 h, and recovered to the original level on adding actin to give a ratio of actin to myosin of 2:1. The pH optima for degradation of myosin in the soluble and filamentous states were 8.5 and 7.0, respectively. The results indicate that cardiac myosin in the filamentous state was more readily degraded with the protease than the myosin in the soluble state.     

Caldesmon is necessary for maintaining the actin and intermediate filaments in cultured bladder smooth muscle cells

Caldesmon (CaD), a component of microfilaments in all cells and thin filaments in smooth muscle cells, is known to bind to actin, tropomyosin, calmodulin, and myosin and to inhibit actin-activated ATP hydrolysis by smooth muscle myosin. Thus, it is believed to regulate smooth muscle contraction, cell motility and the cytoskeletal structure. Using bladder smooth muscle cell cultures and RNA interference (RNAi) technique, we show that the organization of actin into microfilaments in the cytoskeleton is diminished by siRNA-mediated CaD silencing. CaD silencing significantly decreased the amount of polymerized actin (F-actin), but the expression of actin was not altered. Additionally, we find that CaD is associated with 10 nm intermediate-sized filaments (IF) and in vitro binding assay reveals that it binds to vimentin and desmin proteins. Assembly of vimentin and desmin into IF is also affected by CaD silencing, although their expression is not significantly altered when CaD is silenced. Electronmicroscopic analyses of the siRNA-treated cells showed the presence of myosin filaments and a few surrounding actin filaments, but the distribution of microfilament bundles was sparse. Interestingly, the decrease in CaD expression had no effect on tubulin expression and distribution of microtubules in these cells. These results demonstrate that CaD is necessary for the maintenance of actin microfilaments and intermediate-sized filaments in the cytoskeletal structure. This finding raises the possibility that the cytoskeletal structure in smooth muscle is affected when CaD expression is altered, as in smooth muscle de-differentiation and hypertrophy seen in certain pathological conditions.     

Movement of smooth muscle tropomyosin by myosin heads.

It has been proposed that during the activation of muscle contraction the initial binding of myosin heads to the actin thin filament contributes to switching on the thin filament and that this might involve the movement of actin-bound tropomyosin. The movement of smooth muscle tropomyosin on actin was investigated in this work by measuring the change in distance between specific residues on tropomyosin and actin by fluorescence resonance energy transfer (FRET) as a function of myosin head binding to actin. An energy transfer acceptor was attached to Cys374 of actin and a donor to the tropomyosin heterodimer at either Cys36 of the beta-chain or Cys190 of the alpha-chain. FRET changed for the donor at both positions of tropomyosin upon addition of skeletal or smooth muscle myosin heads, indicating a movement of the whole tropomyosin molecule. The changes in FRET were hyperbolic and saturated at about one head per seven actin subunits, indicating that each head cooperatively affects several tropomyosin molecules, presumably via tropomyosin's end-to-end interaction. ATP, which dissociates myosin from actin, completely reversed the changes in FRET induced by heads, whereas in the presence of ADP the effect of heads was the same as in its absence. The results indicate that myosin with and without ADP, intermediates in the myosin ATPase hydrolytic pathway, are effective regulators of tropomyosin position, which might play a role in the regulation of smooth muscle contraction.     

Accumulation of myosin, actin, tropomyosin, and alpha-actinin in cultured muscles cells.

In an effort to understand the conditions that promote the assembly of myofibrillar proteins in muscle cells, the temporal sequence of accumulation of four myofibrillar proteins, actin, myosin, tropomyosin, and α-actinin, was monitored during the period of de novo assembly of myofibrils in differentiating muscle cells. Isotope dilution experiments indicated that all four proteins were accumulated simultaneously. Therefore, assembly of myofibrils may be occurring in the presence of a full complement of myofibrillar proteins.     

Development of myofibrils in the gizzard of chicken embryos

Summary The intracellular distributions of major muscle proteins, myosin, actin, tropomyosin, -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.     

Myogenesis in the mouse embryo: differential onset of expression of myogenic proteins and the involvement of titin in myofibril assembly

Antibodies to muscle-specific proteins were used in immunofluorescence to monitor the development of skeletal muscle during mouse embryogenesis. At gestation day (g.d.) 9 a single layer of vimentin filament containing cells in the myotome domain of cervical somites begins to stain positively for myogenic proteins. The muscle-specific proteins are expressed in a specific order between g.d. 9 and 9.5. Desmin is detected first, then titin, then the muscle specific actin and myosin heavy chains, and finally nebulin. At g.d. 9.5 fibrous desmin structures are already present, while for the other myogenic proteins no structure can be detected. Some prefusion myoblasts display at g.d. 11 and 12 tiny and immature myofibrils. These reveal a periodic pattern of myosin, nebulin, and those titin epitopes known to occur at and close to the Z line. In contrast titin epitopes, which are present in mature myofibrils along the A band and at the A-I junction, are still randomly distributed. We propose, that the Z line connected structures and the A bands (myosin filaments) assemble independently, and that the known interaction of the I-Z-I brushes with the A bands occurs at a later developmental stage. After fusion of myoblasts to myotubes at g.d. 13 and 14 all titin epitopes show the myofibrillar banding pattern. The predominantly longitudinal orientation of desmin filaments seen in myoblasts and in early myotubes is transformed at g.d. 17 and 18 to distinct Z line connected striations. Vimentin, still present together with desmin in the myoblasts, is lost from the myotubes. Our results indicate that the putative elastic titin filaments act as integrators during skeletal muscle development. Some developmental aspects of eye and limb muscles are also described.     

Incorporation of fluorescently labeled contractile proteins into freshly isolated living adult cardiac myocytes.

When fluorescently labeled contractile proteins are injected into embryonic muscle cells, they become incorporated into the cells' myofibrils. In order to determine if this exchange of proteins is unique to the embryonic stage of development, we isolated adult cardiac myocytes and microinjected them with fluorescently labeled actin, myosin light chains, alpha-actinin, and vinculin. Each of these proteins was incorporated into the adult cardiomyocytes and was colocalized with the cells' native proteins, despite the fact that the labeled proteins were prepared from noncardiac tissues. Within 10 min of injection, alpha-actinin was incorporated into Z-bands surrounding the site of injection. Similarly, 30 sec after injection, actin was incorporated into the entire I-bands at the site of injection. Following a 3-h incubation, increased actin fluorescence was noted at the intercalated disc. Vinculin exchange was seen in the intercalated discs, as well as in the Z-bands throughout the cells. Myosin light chains required 4-6 h after injection to become incorporated into the A-bands of the adult muscle. Nonspecific proteins, such as fluorescent BSA, showed no association with the myofibrils or the former intercalated discs. When adult cells were maintained in culture for 10 days, they retain the ability to incorporate these contractile proteins into their myofibrils. T-tubules and the sarcoplasmic reticulum could be detected in periodic arrays in the freshly isolated cells using the membrane dye WW781 and DiOC6[3], respectively. In conclusion, the myofibrils in adult, as in embryonic, muscle cells are dynamic structures, permitting isoform transitions without dismantling of the myofibrils.