Myofibril Formation Research Articles

Myocyte differentiation generates nuclear invaginations traversed by myofibrils associating with sarcomeric protein mRNAs

Certain types of cell both in vivo and in vitro contain invaginated or convoluted nuclei. However, the mechanisms and functional significance of the deformation of the nuclear shape remain enigmatic. Recent studies have suggested that three types of cytoskeleton, microfilaments, microtubules and intermediate filaments, are involved in the formation of nuclear invaginations, depending upon cell type or conditions. Here, we show that undifferentiated mouse C2C12 skeletal muscle myoblasts had smoothsurfaced spherical or ellipsoidal nuclei, whereas prominent nuclear grooves and invaginations were formed in multinucleated myotubes during terminal differentiation. Conversion of mouse fibroblasts to myocytes by the transfection of MyoD also resulted in the formation of nuclear invaginations after differentiation. C2C12 cells prevented from differentiation did not have nuclear invaginations, but biochemically differentiated cells without cell fusion exhibited nuclear invaginations. Thus, biochemical differentiation is sufficient for the nuclear deformation. Although vimentin markedly decreased both in the biochemically and in the terminally differentiated cells, exogenous expression of vimentin in myotubes did not rescue nuclei from the deformation. On the other hand, non-striated premyofibrils consisting of sarcomeric actinmyosin filament bundles and cross-striated myofibrils traversed the grooves and invaginations. Time-lapse microscopy showed that the preformed myofibrillar structures cut horizontally into the nuclei. Prevention of myofibril formation retarded the generation of nuclear invaginations. These results indicate that the myofibrillar structures are, at least in part, responsible for the formation of nuclear grooves and invaginations in these myocytes. mRNA of sarcomeric proteins including myosin heavy chain and alpha-actin were frequently associated with the myofibrillar structures running along the nuclear grooves and invaginations. Consequently, the grooves and invaginations might function in efficient sarcomeric protein mRNA transport from the nucleus along the traversing myofibrillar structures for active myofibril formation.

The Role of Protein Kinase C in Cardiac Myocyte Attachment, Spreading and Myofibril Formation

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Inhibitors arrest myofibrillogenesis in skeletal muscle cells at early stages of assembly

A three-step model for myofibrillogenesis has been proposed for the formation of myofibrils [Rhee et al., 1994: Cell Motil. Cytoskeleton 28:1-24; Sanger et al., 2002: Adv. Exp. Med. 481:89-105]: premyofibril to nascent myofibril to mature myofibril. We have found two chemically related inhibitors that will arrest development at both the first and second step. Cultured quail embryonic skeletal myoblasts were treated with ethyl methane sulfonate (EMS) or 2-aminoethyl-methanesulfonate (MTSEA+). When the myoblasts fused in the presence of either of these compounds, myosheets rather than myotubes formed. Treated cells were fixed and immunostained against multiple proteins commonly found in muscle cells. Protein expression and localization throughout the myosheet were similar to that of developing myotube tips. Cells treated with high concentrations of EMS (10 mM) stained for non-muscle myosin II, sarcomeric alpha-actinin, and tropomyosin. No zeugmatin (Z-band region of titin) or muscle myosin II antibody staining was detected in fibers in this treatment group. These fibers are comparable to premyofibrils in control myotubes. At lower concentrations of EMS (7.5 to 5 mM), fibers that formed stained for muscle myosin II and titin as well as for non-muscle myosin IIB, sarcomeric alpha-actinin, and tropomyosin. Muscle myosin II was in an unbanded pattern. These fibers are comparable to nascent myofibrils observed during normal myofibrillogenesis. Similar effects to those obtained by treating cells with EMS were obtained when we treated cultured cells with MTSEA+ (5 mM) and stained them with sarcomeric alpha-actinin. MTSEA+ is chemically related to EMS, and is a well-known inhibitor of ryanodine receptors in skeletal muscle cells. Some abnormalities such as nemaline-like rods and other protein aggregates also appear within the myosheet during EMS and MTSEA+ treatment. Removal of these two inhibitors of myofibrillogenesis allows the premyofibrils and nascent myofibrils to form mature myofibrils.

Myofibrillogenesis in skeletal muscle cells in the presence of taxol.

We address the controversy of whether mature myofibrils can form in the presence of taxol, a microtubule-stabilizing compound. Previous electron microscopic studies reported the absence of actin filaments and Z-bands in taxol-treated myocytes [Antin et al., 1981: J Cell Biol 90:300-308; Toyoma et al., 1982: Proc Natl Acad Sci USA 79:6556-6560]. Quail skeletal myoblasts were isolated from 10-day-old embryos and grown in the presence or absence of taxol. Taxol inhibited the formation of multinucleated elongated myotubes. Myocytes cultured in the continual presence of taxol progressed from rounded to stellate shapes. Groups of myocytes that were clustered together after the isolation procedure fused in the presence of taxol but did not form elongated myotubes. Actin filaments and actin-binding proteins were detected with several different fluorescent probes in all myofibrils that formed in the presence of taxol. The Z-bands contained both alpha-actinin and titin, and the typical arrays of A-Bands were always associated with actin filaments in the myofibrils. Myofibril formation was followed by fixing cells each day in culture and staining with probes for actin, muscle-specific alpha-actinin, myosin II, nebulin, troponin, tropomyosin, and non-muscle myosin II. Small linear aggregates of alpha-actinin or Z-bodies, premyofibrils, were detected at the edges of the myocytes and in the arms of the taxol-treated cells and were always associated with actin filaments. Non-muscle myosin II was detected at the edges of the taxol-treated cells. Removal of the taxol drug led to the cells assuming a normal compact elongated shape. During the recovery process, additional myofibrils formed at the spreading edges of these elongated and thicker myotubes. Staining of these taxol-recovering cells with specific fluorescent reagents reveals three different classes of actin fibers. These results are consistent with a model of myofibrillogenesis that involves the transition of premyofibrils to mature myofibrils.

Targeted disruption of the murine Bin1/Amphiphysin II gene does not disable endocytosis but results in embryonic cardiomyopathy with aberrant myofibril formation.

The mammalian Bin1/Amphiphysin II gene encodes an assortment of alternatively spliced adapter proteins that exhibit markedly divergent expression and subcellular localization profiles. Bin1 proteins have been implicated in a variety of different cellular processes, including endocytosis, actin cytoskeletal organization, transcription, and stress responses. To gain insight into the physiological functions of the Bin1 gene, we have disrupted it by homologous recombination in the mouse. Bin1 loss had no discernible impact on either endocytosis or phagocytosis in mouse embryo-derived fibroblasts and macrophages, respectively. Similarly, actin cytoskeletal organization, proliferation, and apoptosis in embryo fibroblasts were all unaffected by Bin1 loss. In vivo, however, Bin1 loss resulted in perinatal lethality. Bin1 has been reported to affect muscle cell differentiation and T-tubule formation. No striking histological abnormalities were evident in skeletal muscle of Bin1 null embryos, but severe ventricular cardiomyopathy was observed in these embryos. Ultrastructurally, myofibrils in ventricular cardiomyocytes of Bin1 null embryos were severely disorganized. These results define a developmentally critical role for the Bin1 gene in cardiac muscle development.

Sheep heart RNA stimulates myofibril formation and beating in cardiac mutant axolotl hearts in organ culture.

In the Mexican axolotl, Ambystoma mexicanum, recessive mutant gene c, when homozygous, results in a failure of the heart to form sarcomeric myofibrils and contract normally. Previous studies have shown that purified RNA from normal anterior endoderm or from medium conditioned with anterior endoderm/pre-cardiac mesoderm has the capacity to rescue mutant hearts in organ culture. In the present study, RNA extracted from adult sheep heart was tested for its capacity to promote differentiation in the mutant axolotl hearts. Mutant hearts cultured in the presence of the sheep heart RNA in Steinberg's solution for 48 h displayed rhythmic contractions. Ultrastructural studies showed that the rescued mutant axolotl ventricular myocardial cells contained myofibrils of normal morphology. Mutant hearts cultured in Steinberg's solution alone did not beat throughout their lengths and myofibrils were not observable in the ventricles. Confocal microscopy confirmed the increase of Tropomyosin expression and formation of myofibrils in mutant hearts treated by sheep heart RNA. Thus, sheep heart RNA promotes myofibrillogenesis and the development of contractile function in embryonic cardiac mutant axolotl hearts.

Drosophila paramyosin is important for myoblast fusion and essential for myofibril formation.

Paramyosin is a major structural protein of thick filaments in invertebrate muscles. Coiled-coil dimers of paramyosin form a paracrystalline core of these filaments, and the motor protein myosin is arranged on the core surface. To investigate the function of paramyosin in myofibril assembly and muscle contraction, we functionally disrupted the Drosophila melanogaster paramyosin gene by mobilizing a P element located in its promoter region. Homozygous paramyosin mutants die at the late embryo stage. Mutants display defects in both myoblast fusion and in myofibril assembly in embryonic body wall muscles. Mutant embryos have an abnormal body wall muscle fiber pattern arising from defects in myoblast fusion. In addition, sarcomeric units do not assemble properly and muscle contractility is impaired. We confirmed that these defects are paramyosin-specific by rescuing the homozygous paramyosin mutant to adulthood with a paramyosin transgene. Antibody analysis of normal embryos demonstrated that paramyosin accumulates as a cytoplasmic protein in early embryo development before assembling into thick filaments. We conclude that paramyosin plays an unexpected role in myoblast fusion and is important for myofibril assembly and muscle contraction.

Texture and microstructure properties of frozen chicken breasts pretreated with salt and phosphate solutions

This study investigated the effects of 10% NaCl, trisodium phosphate (TSP), sodium tripolyphosphate (STPP), and tetrapotassium pyrophosphate (TKPP) treatments on textural and microstructural properties of chicken breasts during 10 mo of frozen storage at -20 C. Fresh chicken breasts were treated for 10 min with 10% NaCl and various phosphate solutions, including TSP, STPP, and TKPP, and stored in a -20 C freezer for 10 mo. Frozen chicken breasts were completely thawed at 4 C and oven-baked at 177 C for 20 min. Shear force, drip loss, and cooking loss were measured. In addition, ice crystal formation and structure changes of frozen chicken breasts during storage were evaluated using transmission electron microscopy (TEM). Treating chicken breasts with 10% TSP and STPP solution significantly reduced drip and cooking losses as well as minimized ice crystal formation and freeze-induced shrinkage of myofibrils. No significant texture toughening was observed in frozen chicken breasts regardless of treatments. These results suggest that the perceived quality losses of frozen chicken breast were not associated with texture toughening. The water-binding ability of chicken meat was the most important factor in maintaining the quality of chicken breast during extended frozen storage, which can be accomplished by treating chicken breasts with 10% TSP and STPP solutions before frozen storage.

Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis.

Calreticulin (crt) is an ubiquitously expressed and multifunctional Ca2+-binding protein that regulates diverse vital cell functions, including Ca2+ storage in the ER and protein folding. Calreticulin deficiency in mice is lethal in utero due to defects in heart development and function. Herein, we used crt − / − embryonic stem (ES) cells differentiated in vitro into cardiac cells to investigate the molecular mechanisms underlying heart failure of knockout embryos. After 8 d of differentiation, beating areas were prominent in ES-derived wild-type (wt) embryoid bodies (EBs), but not in ES-derived crt − / − EBs, despite normal expression levels of cardiac transcription factors. Crt − / − EBs exhibited a severe decrease in expression and a lack of phosphorylation of ventricular myosin light chain 2 (MLC2v), resulting in an impaired organization of myofibrils. Crt − / − phenotype could be recreated in wt cells by chelating extracellular or cytoplasmic Ca2+ with EGTA or BAPTA, or by inhibiting Ca2+/calmodulin-dependent kinases (CaMKs). An imposed ionomycin-triggered cystolic-free Ca2+ concentration ([Ca2+]c) elevation restored the expression, phosphorylation, and insertion of MLC2v into sarcomeric structures and in turn the myofibrillogenesis. The transcription factor myocyte enhancer factor C2 failed to accumulate into nuclei of crt − / − cardiac cells in the absence of ionomycin-triggered [Ca2+]c increase. We conclude that the absence of calreticulin interferes with myofibril formation. Most importantly, calreticulin deficiency revealed the importance of a Ca2+-dependent checkpoint critical for early events during cardiac myofibrillogenesis.

Targeting and functional role of N-RAP, a nebulin-related LIM protein, during myofibril assembly in cultured chick cardiomyocytes.

Targeting and functional effects of N-RAP domains were studied by expression as GFP-tagged fusion proteins in cultured embryonic chick cardiomyocytes. GFP-tagged N-RAP was targeted to myofibril precursors, myofibril ends and cell contacts, expression patterns that are similar to endogenous N-RAP. The GFP-tagged N-RAP LIM domain (GFP-N-RAP-LIM) was targeted to the membrane in cells with myofibril precursors and cell-cell contacts. The GFP-tagged super repeats (N-RAP-SR) and the GFP-tagged domain normally found in between the super repeats and the LIM domain (N-RAP-IB) were each observed at sites of myofibril assembly, incorporating into myofibril precursors in a manner similar to full length N-RAP. However, unlike full-length N-RAP, N-RAP-SR and N-RAP-IB were also found in mature myofibrils, associating with the sarcomeric actin filaments and the Z-lines, respectively. N-RAP-IB was also colocalized with alpha-actinin at cell contacts. Each of the N-RAP constructs could inhibit the formation of mature myofibrils in cultured cardiomyocytes, with the effects of N-RAP-SR and N-RAP-IB depending on the time of transfection. The results show that each region of N-RAP is crucial for myofibril assembly. Combining the targeting and functional effects of N-RAP domains with information in the literature, we propose a new model for initiation of myofibrillogenesis.

Zipper Nonmuscle Myosin-II Functions Downstream of PS2 Integrin in Drosophila Myogenesis and Is Necessary for Myofibril Formation

Nonmuscle myosin-II is a key motor protein that drives cell shape change and cell movement. Here, we analyze the function of nonmuscle myosin-II during Drosophila embryonic myogenesis. We find that nonmuscle myosin-II and the adhesion molecule, PS2 integrin, colocalize at the developing muscle termini. In the paradigm emerging from cultured fibroblasts, nonmuscle actomyosin-II contractility, mediated by the small GTPase Rho, is required to cluster integrins at focal adhesions. In direct opposition to this model, we find that neither nonmuscle myosin-II nor RhoA appear to function in PS2 clustering. Instead, PS2 integrin is required for the maintenance of nonmuscle myosin-II localization and we show that the cytoplasmic tail of the βPS integrin subunit is capable of mediating this PS2 integrin function. We show that embryos that lack zygotic expression of nonmuscle myosin-II fail to form striated myofibrils. In keeping with this, we demonstrate that a PS2 mutant that specifically disrupts myofibril formation is unable to mediate proper localization of nonmuscle myosin-II at the muscle termini. In contrast, embryos that lack RhoA function do generate striated muscles. Finally, we find that nonmuscle myosin-II localizes to the Z-line in mature larval muscle. We suggest that nonmuscle myosin-II functions at the muscle termini and the Z-line as an actin crosslinker and acts to maintain the structural integrity of the sarcomere.

Assembly of thick, thin, and titin filaments in chick precardiac explants.

De novo cardiac myofibril assembly has been difficult to study due to the lack of available cell culture models that clearly and accurately reflect heart muscle development in vivo. However, within precardiac chick embryo explants, premyocardial cells differentiate and commence beating in a temporal pattern that corresponds closely with myocyte differentiation in the embryo. Immunofluorescence staining of explants followed by confocal microscopy revealed that distinct stages of cardiac myofibril assembly, ranging from the earliest detection of sarcomeric proteins to the late appearance of mature myofibrils, were consistently recognized in precardiac cultures. Assembly events involved in the early formation of sarcomeres were clearly visualized and accurately reflected observations described by others during chick heart muscle development. Specifically, the early colocalization of alpha-actinin and titin dots was observed near the cell periphery representing I-Z-I-like complex formation. Myosin-containing thick filaments assembled independently of actin-containing thin filaments and appeared centered within sarcomeres when titin was also linearly aligned at or near cell borders. An N-terminal epitope of titin was detected earlier than a C-terminal epitope; however, both epitopes were observed to alternate near the cell periphery concomitant with the earliest formation of myofibrils. Although vascular actin was detected within cells during early assembly stages, cardiac actin predominated as the major actin isoform in mature thin filaments. Well-aligned thin filaments were also observed in the absence of organized staining for tropomodulin at thin filament pointed ends, suggesting that tropomodulin is not required to define thin filament lengths. Based on these findings, we conclude that the use of the avian precardiac explant system accurately allows for direct investigation of the mechanisms regulating de novo cardiac myofibrillogenesis.

Characterization of heterokaryons between skeletal myoblasts and somatic cells formed by fusion with HVJ (Sendai virus); effects on myogenic differentiation.

In skeletal myogenic differentiation, myoblasts fuse with myogenic cells spontaneously, but do not fuse with non-myogenic cells either in vivo or in vitro, suggesting that the fusion of myoblasts with non-myogenic cells is unsuitable for differentiation. To understand the inevitability of the fusion among myoblasts, we prepared heterokaryons in crosses between quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and rodent non-myogenic cells, such as tumor cells, fibroblasts, or neurogenic cells by HVJ (Sendai virus) and examined how myogenic differentiation was influenced in the prepared heterokaryons, focusing on myogenin expression and myofibril formation as markers of differentiation. When presumptive QM-RSV cells were fused with non-myogenic cells by HVJ and induced to differentiate, both myogenin expression and myofibril formation were suppressed. When myotubes of QM-RSV cells that had already expressed myogenin and formed myofibrils were fused with non-myogenic cells, both myogenin and myofibrils disappeared. Especially, fibrous structures of myofibrils were significantly lost and dots or aggregations of F-actin were formed within 24 hr after formation of heterokaryons. However, the fusion of presumptive or differentiated QM-RSV cells with rodent myoblasts did not disturb myogenin expression or myofibril formation. These results suggest that mutual fusion of myoblasts is indispensable for normal myogenic differentiation irrespective of the species, and that some factors inhibiting myogenic differentiation exist in the cytoplasm of non-myogenic cells, but not in myoblasts.

Myofibrillogenesis and formation of cell contacts mediate the localization of N‐RAP in cultured chick cardiomyocytes

The expression of N-RAP was investigated in immuofluorescently stained embryonic chick cardiomyocyte cultures. After 1 day in culture, the cardiomyocytes were spherical and N-RAP, titin, α-actinin, and vinculin were all diffusely distributed. As the cardiomyocytes spread and formed myofibrils and cell contacts, N-RAP became localized to distinct areas in the cells. During myofibrillogenesis, N-RAP was found concentrated in premyofibrils. As the premyofibrils transformed into bundles of mature myofibrils, N-RAP became concentrated at the longitundal ends of the cells, and was not found in the mature sarcomeres. At sites of cell-cell contacts, N-RAP was localized to the cell junction even in cells without any significant myofibril formation. As the cell-cell contacts became more extensive and formed structures resembling the intercalated disks found in hearts, N-RAP became even more specifically concentrated at these junctions. The results show that myofibrillogenesis and cell contact formation can each independently target N-RAP to the longitudinal ends of cardiomyocytes. Cell Motil. Cytoskeleton 47:63–76, 2000. Published 2000 Wiley-Liss, Inc.

Alteration of cardiac myofibrillogenesis by liposome-mediated delivery of exogenous proteins and nucleic acids into whole embryonic hearts.

A precise organization of contractile proteins is essential for contraction of heart muscle. Without a necessary stoichiometry of proteins, beating is not possible. Disruption of this organization can be seen in diseases such as familial hypertrophic cardiomyopathy and also in acquired diseases. In addition, isoform diversity may affect contractile properties in such functional adaptations as cardiac hypertrophy. The Mexican axolotl provides an uncommon model in which to examine specific proteins involved with myofibril formation in the heart. Cardiac mutant embryos lack organized myofibrils and have altered expression of contractile proteins. In order to replicate the disruption of myofibril formation seen in mutant hearts, we have developed procedures for the introduction of contractile protein antibodies into normal hearts. Oligonucleotides specific to axolotl tropomyosin isoforms (ATmC-1 and ATmC-3), were also successfully introduced into the normal hearts. The antisense ATmC-3 oligonucleotide disrupted myofibril formation and beating, while the sense strands did not. A fluorescein-tagged sense oligonucleotide clearly showed that the oligonucleotide is introduced within the cells of the intact hearts. In contrast, ATmC-1 anti-sense oligonucleotide did not cause a disruption of the myofibrillar organization. Specifically, tropomyosin expression can be disrupted in normal hearts with a lack of organized myofibrils. In a broader approach, these procedures for whole hearts are important for studying myofibril formation in normal hearts at the DNA, RNA, and/or protein levels and can complement the studies of the cardiac mutant phenotype. All of these tools taken together present a powerful approach to the elucidation of myofibrillogenesis and show that embryonic heart cells can incorporate a wide variety of molecules with cationic liposomes.

Three-Dimensional Structure of a Vertebrate Muscle Z-band: Implications for Titin and α-Actinin Binding

The Z-band in vertebrate striated muscles, mainly comprising actin filaments, α-actinin, and titin, serves to organise the antiparallel actin filament arrays in adjacent sarcomeres and to transmit tension between sarcomeres during activation. Different Z-band thicknesses, formed from different numbers of zigzag crosslinking layers and found in different fibre types, are thought to be associated with the number of repetitive N-terminal sequence domains of titin. In order to understand myofibril formation it is necessary to correlate the ultrastructures and sequences of the actin filaments, titin, and α-actinin in characteristic Z-bands. Here electron micrographs of the intermediate width, basketweave Z-band of plaice fin muscle have been subject to a novel 3D reconstruction process. The reconstruction shows that antiparallel actin filaments overlap in the Z-band by about 22–25 nm. There are three levels of Z-links (probably α-actinin) in which at each level two nearly diametrically opposed links join an actin filament to two of its antiparallel neighbours. One set of links is centrally located in the Z-band and there are flanking levels orthogonal to this. A 3D model of the observed structure shows how Z-bands of different widths may be formed and it provides insights into the structural arrangements of titin and α-actinin in the Z-band. The model shows that the two observed symmetries in different Z-bands, c2 and p121, may be attributed respectively to whether the number of Z-link levels is odd or even.

Assembly of myofibrils in cardiac muscle cells.

How do myofibrils assemble in cardiac muscle cells? When does titin first assemble into myofibrils? What is the role of titin in the formation of myofibrils in cardiac muscle cells? This chapter reviews when titin is first detected in cultured cardiomyocytes that have been freshly isolated from embryonic avian hearts. Our results support a model for myofibrillogenesis that involves three stages of assembly: premyofibrils, nascent myofibrils and mature myofibrils. Titin and muscle thick filaments were first detected associated with the nascent myofibrils. The Z-band targeting site for titin is localized in the N-terminus of titin. This region of titin binds alpha-actinin and less avidly vinculin. Thus the N-terminus of titin via its binding to alpha-actinin, and vinculin could also help mediate the costameric attachment of the Z-bands of mature myofibrils to the nearest cell surfaces.

Microinjection of fluorescently labeled alpha-actinin into living cells.

The introduction of microinjection of trace amounts of a fluorescently labeled protein (,) into cultured cells leads to its incorporation into the cell’s pool of endogenous protein. Provided the microinjected protein has retained the properties of the native protein, it will become incorporated into the same structures as the endogenous protein and will serve, therefore, as a marker of the native protein’s distribution in the cell. This allows the changes in a protein’s localization to be followed in live cells in response to normal functions such as movement () or division () or formation of structures like stress fibers or myofibrils (, , , , ). Responses of the protein to inhibitors of cell function (,), or to interactions of the injected cell with other cells such as bacteria, can also be analyzed within a single live cell ().

Dynamic distribution of an antigen involved in the differentiation of avian myoblasts: II. Possible association of beta1 integrin with myofibril organization.

Previous studies have shown that a monoclonal antibody, H-145, inhibits myotube formation of quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) [Hyodo and Kim, 1994: Exp. Cell Res. 212:120-131]. The antigen recognized by H-145 (H-145 antigen), which is a glycoprotein with a molecular mass of about 116 kDa, is related to a step immediately before myoblast fusion. To determine the functional significance of H-145 antigen, we examined its dynamic state during myogenic differentiation of QM-RSV cells. H-145 antigen showed a unique and discrete distribution. In immature myotubes immediately after myoblast fusion, many ring-like structures of H-145 antigen appeared on the ventral surface of the cells, encircling the actin dots detected simultaneously by immunofluorescence and interference reflection microscopy. The core of the ring-like structures was filled with the termini of actin bundles, mainly formed by alpha-actin. Other cytoskeletal-associated proteins, such as vinculin and alpha-actinin, were also associated with these structures. The ring-like structures of H-145 antigen were observed only during a restricted period when myoblasts fused actively, suggesting their relationships to myotube formation and an early stage of myofibril formation. With maturation of the myotubes, most of the H-145 antigen became redistributed in linear arrays on the apical cell surface and was probably associated with the termini of actin bundles to organize myofibrils, suggesting that the antigen was also related to maturation of myotubes. Experiments using monoclonal antibodies against chick beta1 integrin showed that H-145 antigen is beta1 integrin or a very closely related derivation. Thus H-145 antigen (beta1 integrin) is possibly involved in both myoblast fusion and the myofibril organization in myotubes.

Myofibrillogenesis and formation of cell contacts mediate the localization of N-RAP in cultured chick cardiomyocytes.

The expression of N-RAP was investigated in immuofluorescently stained embryonic chick cardiomyocyte cultures. After 1 day in culture, the cardiomyocytes were spherical and N-RAP, titin, alpha-actinin, and vinculin were all diffusely distributed. As the cardiomyocytes spread and formed myofibrils and cell contacts, N-RAP became localized to distinct areas in the cells. During myofibrillogenesis, N-RAP was found concentrated in premyofibrils. As the premyofibrils transformed into bundles of mature myofibrils, N-RAP became concentrated at the longitundal ends of the cells, and was not found in the mature sarcomeres. At sites of cell-cell contacts, N-RAP was localized to the cell junction even in cells without any significant myofibril formation. As the cell-cell contacts became more extensive and formed structures resembling the intercalated disks found in hearts, N-RAP became even more specifically concentrated at these junctions. The results show that myofibrillogenesis and cell contact formation can each independently target N-RAP to the longitudinal ends of cardiomyocytes.