FITC-labeled I-protein specifically binds to A-bands and/or Z-lines of glycerinated myofibrils of chicken breast muscle

Ion-exchange column-purified I-protein was labeled by fluorescein isothiocyanate (FITC) at an equimolar ratio. When FITC-labeled I-protein was reacted with glycerinated myofibrils of chicken breast muscle in a phosphate-buffered saline, fluorescence was observed at the A-band and/or the Z-line of the sarcomere. However, FITC-labeled I-protein did not stain freshly prepared myofibrils. When FITC-I-protein was reacted with a nitrocellulose paper sheet on which muscle proteins were blotted after SDS-polyacrylamide gel electrophoresis, some peptide bands, including connectin and nebulin, were fluorescent. These facts can explain why anti-I-protein antibodies stain the A-I junctional region of fresh myofibrils and A-bands and/or Z-lines of glycerinated myofibrils. It is very likely that I-protein is transferred from the A-I junctions of myofibrils and translocates to A-bands and Z-lines, where some components that can bind to I-protein are localized, as myofibrils are degraded during the glycerination.     

I-protein a new regulatory protein from vertebrate skeletal muscle. II. Localization.

Rabbit antiserum raised agains I-protein was used for immunofluorescent staining of chicken myofibrils. The FITC-conjugated anti-I-protein antibody stained A-band regions except at their middle regions. According to the conditions used, the myofibrils stained by their fluorescent antibody showed slightly different patterns, i.e., the nonstained regions in the center of the A-bands were wider. On fixing with glutaraldehyde, myofibrils were stained in the A-band regions except at their middle regions. Therefore, I-protein may be localized at A-bands except for the center.     

Myofibrillar interaction of blot immunoaffinity-purified antibodies against native titin as studied by direct immunofluorescence and immunogold staining

Titin (also called connectin), a major but so far highly elusive myofibrillar component in striated muscle was purified from glycerinated chicken breast muscle in its native state by use of a similar purification procedure as recently introduced for purification of native titin from rabbit psoas muscle. Low-angle rotary shadowing reveals highly convoluted, long and slender strands, sometimes more extended and with nodules, but also an aggregation into filamentous bundles and reticular networks. Antisera were raised against the purified native molecule and monospecific titin antibodies prepared by a rapid nitrocellulose blot immunoaffinity-purification procedure. Titin antibodies bound to the nitrocellulose immobilized native antigen were directly conjugated with fluorescein isothiocyanate. Titin specifity of purified antibodies was checked by immunoblotting. Direct immunofluorescence of glycerinated myofibrils revealed a uniform doublet staining pattern within the sarcomeres by labelling the region of the A-I junctions and some diffuse staining in the region of the myosin filaments. The same myofibrils examined by indirect immunoelectron microscopy revealed the gold particles highly concentrated at the A-I junctions with considerable labelling within the A-bands, except in their centers. Residual I-bands and Z-lines are free of label. In overstretched myofibrils immunogold staining labelled the gap filaments in the space between I- and A-bands. Isolated native thick filaments showed gold labelling of coiled superthin filaments at the ends of the thick filaments (end-filaments) and at their sides, respectively. The colloidal gold technique in combination with an affinity-purified titin antibody raised against the native molecule adds further evidence for the existence and distribution of an endosarcomeric superthin cytoskeletal filament lattice with titin as a major component.     

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.     

Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster

Twelve monoclonal antibodies have been raised against proteins in preparations of Z-disks isolated from Drosophila melanogaster flight muscle. The monoclonal antibodies that recognized Z-band components were identified by immunofluorescence microscopy of flight muscle myofibrils. These antibodies have identified three Z-disk antigens on immunoblots of myofibrillar proteins. Monoclonal antibodies alpha:1-4 recognize a 90-100-kD protein which we identify as alpha-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate alpha-actinins. Monoclonal antibodies P:1-4 bind to the high molecular mass protein, projectin, a component of connecting filaments that link the ends of thick filaments to the Z-band in insect asynchronous flight muscles. The anti-projectin antibodies also stain synchronous muscle, but, surprisingly, the epitopes here are within the A-bands, not between the A- and Z-bands, as in flight muscle. Monoclonal antibodies Z(210):1-4 recognize a 210-kD protein that has not been previously shown to be a Z-band structural component. A fourth antigen, resolved as a doublet (approximately 400/600 kD) on immunoblots of Drosophila fibrillar proteins, is detected by a cross reacting antibody, Z(400):2, raised against a protein in isolated honeybee Z-disks. On Lowicryl sections of asynchronous flight muscle, indirect immunogold staining has localized alpha-actinin and the 210-kD protein throughout the matrix of the Z-band, projectin between the Z- and A-bands, and the 400/600-kD components at the I-band/Z-band junction. Drosophila alpha-actinin, projectin, and the 400/600-kD components share some antigenic determinants with corresponding honeybee proteins, but no honeybee protein interacts with any of the Z(210) antibodies.     

Isoforms of C-protein in adult chicken skeletal muscle: detection with monoclonal antibodies

Monoclonal antibodies (McAbs) specific for the C-proteins of chicken pectoralis major and anterior latissimus dorsi (ALD) muscles have been produced and characterized. Antibody specificity was demonstrated by solid phase radioimmunoassay (RIA), immunoblots, and immunofluorescence cytochemistry. Both McAbs MF-1 (or MF-21) and ALD-66 bound to myofibrillar proteins of approximately 150,000 daltons; the former antibody reacted with pectoralis but not ALD myofibrils, whereas the latter recognized ALD but not pectoralis myofibrils. Chromatographic elution of the antigens from DEAE-Sephadex, and their distribution in the A-band, support the conclusion that both of these antibodies recognize variant isoforms of C-protein. Since both McAbs react with a protein of similar molecular weight in the A-band of all myofibrils of the posterior latissimus dorsi (PLD) muscle, we suggest that either another isoform of C-protein exists in the PLD muscle or both pectoralis and ALD-like isoforms coexist in the A-bands of PLD muscle.     

Comparison of M-line and other myofibril components during reversible phorbol ester treatment

The events occurring during phorbol ester mediated destruction of myofibrils in differentiated muscle cells were followed at the fluorescence and electron microscope levels using antibodies which bind troponin-T, a newly discovered 185 000 dalton M-line protein called myomesin and muscle type creatine kinase. The following series of events is proposed. Within one day of phorbol ester treatment, Z-bands and thin filaments, including troponin-T, are absent from many myofibrils resulting in the rapid loss of longitudinal and lateral alignment. A-bands become randomly oriented and clustered into ever smaller compartments within the rounding, myosac-like, multinucleated cells until after 3 days of treatment they too disappear. The M-line proteins are always present in existing A-bands. These results suggest that the Z-band and associated structures are responsible for the maintenance of alignment and the lateral register of myofibrils, whereas the M-line is responsible for the structural integrity of the A-band. When phorbol ester is removed, the cells revert to a myotube morphology and within 2 to 3 days are filled with myofibrils. A comparison of the appearance of troponin-T and the 185 000 dalton myomesin in the recovery period to their appearance during normal myofibrillogenesis reveals that these proteins are more temporally co-ordinated during myofibrillogenesis than in the phorbol ester experimental system.     

Localization of creatine kinase isoenzymes in myofibrils. II. Chicken heart muscle

Chicken heart muscle contains almost exclusively the BB isoenzyme of creatine kinase (CK), its myofibrils, moreover, lack an M-line. This tissue thus provides an interesting contrast to skeletal muscle, in which some of the MM-CK present as predominant CK isoenzyme is bound at the myofibrillar M-line. Approx. 2% of the total CK activity in a chicken heart homogenate remains bound to the myofibrillar fraction after repeated washing cycles; both the fraction and the absolute amount of CK bound are about threefold lower than in skeletal muscle. Almost all of the bound enzyme is located within the Z-line region of each sarcomere, as revealed by indirect fluorescent-antibody staining with antiserum against purified chicken BB-CK. After incubation with exogenous purified MM-CK, positive immunofluorescent staining for M- type CK at the H-region of heart myofibrils was observed, along with weaker fluorescence in the Z-line region. Chicken heart myofibrils may thus possess binding sites for both M and B forms of CK.     

Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken skeletal muscle

We studied the localization of desmin (skeletin), the major subunit of muscle-type intermediate filaments, by high resolution immunoelectron microscopy in adult chicken skeletal muscle. Immunoferritin labeling of ultrathin frozen sections of intact fixed sartorius muscle showed the presence of desmin between adjacent Z-bands and as strands peripheral to Z-bands, forming apparent connections between the Z-bands with adjacent sarcolemma, mitochondria, and nuclei. We observed no desmin labeling, however, in the vicinity of the T-tubules. In addition, intermediate filaments were morphologically discernible at the level of the Z-bands in plastic sections of glycerol-extracted muscle that had been infused with unlabeled antidesmin antibodies. Our results indicate that the desmin present in adult skeletal muscle, that had previously been detected by immunofluorescence light microscopy, is largely if not entirely in the form of intermediate filaments. The results provide evidence that these filaments serve to interconnect myofibrils at the level of their Z-bands, and to connect Z-bands with other specific structures and organelles in the myotube, but not with the T-tubule system.     

Monoclonal antibodies distinguish titins from heart and skeletal muscle

Murine monoclonal antibodies specific for titin have been elicited using a chicken heart muscle residue as antigen. The three antibodies T1, T3, and T4 recognize both bands of the titin doublet in immunoblot analysis on polypeptides from chicken breast muscle. In contrast, on chicken cardiac myofibrils two of the antibodies (T1, T4) react only with the upper band of the doublet indicating immunological differences between heart and skeletal muscle titin. This difference is even more pronounced for rat and mouse. Although all three antibodies react with skeletal muscle titin, T1 and T4 did not detect heart titin, whereas T3 reacts with this titin both in immunofluorescence microscopy and in immunoblots. Immunofluorescence microscopy of myofibrils and frozen tissues from a variety of vertebrates extends these results and shows that the three antibodies recognize different epitopes. All three titin antibodies decorate at the A-I junction of the myofibrils freshly prepared from chicken skeletal muscle and immunoelectron microscopy using native myosin filaments demonstrates that titin is present at the ends of the thick filaments. In chicken heart, however, antibodies T1 and T4 stain within the I-band rather than at the A-I junction. The three antibodies did not react with any of the nonmuscle tissues or permanent cell lines tested and do not decorate smooth muscle. In primary cultures of embryonic chicken skeletal muscle cells titin first appears as longitudinal striations in mononucleated myoblasts and later at the myofibrillar A-I junction of the myotubes.     

An antiactin antibody that distinguishes between cytoplasmic and skeletal muscle actins

We elicited antibodies in rabbits to actin purified from body wall muscle of the marine mollusc, Aplysia californica. We found that this antiactin has an unusual specificity: in addition to reacting with the immunogen, it recognizes cytoplasmic vertebrate actins but not myofibrillar actin. Radioimmunoassay showed little or no cross-reaction with actin purified from either chicken gizzard or rabbit skeletal muscle. Immunocytochemical studies with human fibroblasts and L6 myoblasts revealed intense staining of typical cytoplasmic cables. Myofibrils were not stained after treatment of human and frog skeletal muscle with the antibody, although the distribution of immunofluorescence suggested that cytoplasmic actin is associated with membrane systems in the muscle fiber. The antibody may therefore be especially suited for studying the localization of cytoplasmic actin in skeletal muscle cells even in the presence of a great excess of the myofibrillar form.     

Association of microinjected myosin and its subfragments with myofibrils in living muscle cells

Purified skeletal muscle myosin was labeled with iodoacetamidofluorescein and microinjected into cultured chick myotubes. The fluorescent myosin analogue became incorporated within 10- 15 min after injection, into either periodic (mean periodicity = 2.23 +/- 0.02 micron) bands or apparently continuous fibrillar structures. Comparison of rhodamine-labeled alpha-actinin with coinjected fluorescein-labeled myosin suggested that myosin fluorescence was localized at the A-bands of myofibrils. In addition, close examination of the fluorescent myosin bands indicated that they were composed of two fluorescent bars separated by a nonfluorescent line that corresponded to the H-zone. Once incorporated, the myosin underwent a relatively slow exchange along myofibrils as indicated by fluorescence recovery after photobleaching. Glycerinated myofibrils were able to bind fluorescent myosin in a similar pattern in the presence or absence of MgATP, indicating that actin-myosin interactions had little effect on this process. Fluorescent heavy meromyosin did not incorporate into myofibrillar structures after injection. Light meromyosin, however, associated with A-bands as did whole myosin. These results suggest that microinjected myosin, even with its relatively low solubility under the cytoplasmic ionic condition, is capable of association with physiological structures in living muscle cells. Additionally, the light meromyosin portion of the molecule appears to be mainly responsible for the incorporation.     

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.     

New aspects of obscurin in human striated muscles

Obscurin is a giant protein (700-800 kDa) present in both skeletal muscles and myocardium. According to animal studies, obscurin interacts with myofibrillar Z-discs during early muscle development, but is translocalised to be predominantly associated with the M-bands in mature muscles. The proposed function for obscurin is in the assembly and organisation of myosin into regular A-bands during formation of new sarcomeres. In the present study, the precise localisation of obscurin in developing and mature normal human striated muscle is presented for the first time. We show that obscurin surrounded myofibrils at the M-band level in both developing and mature human skeletal and heart muscles, which is partly at variance with that observed in animals. At maturity, obscurin also formed links between the peripheral myofibrils and the sarcolemma, and was a distinct component of the neuromuscular junctions. Obscurin should therefore be regarded as an additional component of the extrasarcomeric cytoskeleton. To test this function of obscurin, biopsies from subjects with exercise-induced delayed onset muscle soreness (DOMS) were examined. In these subjects, myofibrillar alterations related to sarcomerogenesis are observed. Our immunohistochemical analysis revealed that obscurin was never lacking in myofibrillar alterations, but was either preserved at the M-band level or diffusely spread over the sarcomeres. As myosin was absent in such areas but later reincorporated in the newly formed sarcomeres, our results support that obscurin also might play an important role in the formation and maintenance of A-bands.     

AMP and IMP dissociate actomyosin into actin and myosin

We investigated to determine why heating of squid muscle at 60 degrees C induced the liberation of actin from myofibrils. When a mixture of a myofibrillar fraction and a low-molecular sarcoplasmic fraction prepared from squid muscle was heated at 60 degrees C, actin liberation occurred. When a myofibrillar fraction was heated with ATP, AMP, or IMP, actin liberation occurred. Hence, AMP is perhaps one of the factors causing actin liberation in postmortem squid muscle. It was found that AMP and IMP reversibly dissociated actomyosin of chicken, bovine, and porcine skeletal muscles into actin and myosin on incubation at 0 degrees C at pH 7.2 in 0.2 M KCl. These results led us to conclude that AMP and IMP were the most responsible factors causing actin liberation from myofibrils in the heated muscle and causing reversible dissociation of actomyosin on storage of skeletal muscle at a low temperature. Hence, AMP and IMP are possible factors causing the resolution of rigor mortis in muscles.     

Immunocytochemical studies of spectrin in hamster cardiac tissue

The spectrins are a family of cytoskeletal-membrane proteins that have a wide tissue distribution. In the present study, we employed polyclonal antibodies made against mammalian and avian erythroid spectrins as well as mammalian brain spectrin to assess their presence and distributions in the mammalian heart. Western blot analyses revealed that all three antibodies were specific for a 240,000 molecular weight alpha-spectrin subunit found in hamster erythrocyte ghost homogenates, whole hamster heart, and isolated hamster cardiac myofibril homogenates. Spectrin staining was absent from the Triton X-100-extracted supernatant fraction of myofibril preparations, suggesting that the protein is linked to the myofibril precipitate after exposure to the detergent. Frozen, unfixed, 2-microns-thick; sections of adult. Syrian golden hamster cardiac tissue exhibited strong immunofluorescent staining of intercalated discs and Z-bands using all three antibodies. In addition, the mammalian erythroid spectrin antibodies showed staining of the sarcolemma, and in cross section, revealed a delicate internal network of staining that appears to surround individual myofibrils. This may be T-tubule-associated staining. Myofibrils isolated from cardiac myocytes using Triton X-100 show positive Z-band staining using all three antibodies. Double staining with Texas Red-labeled monoclonal desmin and FITC-labeled polyclonal spectrin antibodies revealed that both stained the myofibrillar Z-line regions. These results demonstrate that spectrin is closely associated with the membranes, myofibrils, and intermediate filaments in the mammalian heart.     

Striated myofibrils in anti-myosin stained, isolated chicken gizzard smooth muscle cells.

Highly purified chicken gizzard myosin was used to induce antibody production in rabbits. The IgG fraction was separated from the antisera and coupled to fluorescein isothiocyanate (FITC). Specific antibody (AGM) was isolated from the IgG fraction by affinity purification. Comparisons of the specificity of IgG and AGM for chicken smooth muscle myosin revealed a much greater specificity by AGM. Staining with IgG led to an apparent cross-reactivity with guinea pig smooth muscles which was not seen with AGM staining. Therefore, staining of cells for localization of myosin was performed with AGM. Isolated cells were obtained from chicken gizzards either by collagenase digestion or by agitation of glycerinated pieces. Stained cells and cell fragments revealed the presence of myofibrils as structural units with diameters of about 1.0 micrometer. Stained myofibrils occasionally displayed regular banding patterns with a repeating period of about 1.5 +/- 0.2 micrometer. The presence of banded myofibrils in non-cultured cells shows that the organization of the contractile material is similar to that previously reported for cultured cells by Gr?schel-Stewart.     

Localization of paratropomyosin in skeletal muscle myofibrils and its translocation during postmortem storage of muscles

Using polyclonal antibodies against paratropomyosin, which is believed to modify the actin-myosin interaction in postrigor skeletal muscles, we studied the localization of paratropomyosin in chicken breast muscle myofibrils. Intact myofibrils stained with fluorescent antibodies showed that paratropomyosin was exclusively located at the A-I junction region of sarcomeres. In stretched myofibrils (3.7 micron in sarcomere length), the approximate width of the fluorescent stripes and their relation to the A band remained constant. Removal of the A band from myofibrils led to loss of stainability. During postmortem storage of muscles, on the other hand, paratropomyosin was translocated from its original position at the A-I junction region onto thin filaments. The translocation of paratropomyosin was successfully induced with a calcium ion concentration of 10(-4) M in the presence of protease inhibitors. We therefore conclude that in postrigor muscles, paratropomyosin is released from the A-I junction region following the increase in the sarcoplasmic calcium ion concentration to 10(-4) M, and then binds to thin filaments, which results in weakening of rigor linkages formed between actin and myosin.     

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.     

AMP and IMP Dissociate Actomyosin into Actin and Myosin

We investigated to determine why heating of squid muscle at 60 °C induced the liberation of actin from myofibrils. When a mixture of a myofibrillar fraction and a low-molecular sarcoplasmic fraction prepared from squid muscle was heated at 60 °C, actin liberation occurred. When a myofibrillar fraction was heated with ATP, AMP, or IMP, actin liberation occurred. Hence, AMP is perhaps one of the factors causing actin liberation in postmortem squid muscle. It was found that AMP and IMP reversibly dissociated actomyosin of chicken, bovine, and porcine skeletal muscles into actin and myosin on incubation at 0 °C at pH 7.2 in 0.2 M KCl. These results led us to conclude that AMP and IMP were the most responsible factors causing actin liberation from myofibrils in the heated muscle and causing reversible dissociation of actomyosin on storage of skeletal muscle at a low temperature. Hence, AMP and IMP are possible factors causing the resolution of rigor mortis in muscles.