Rabbit skeletal muscle glycogen. A morphological and biochemical study of glycogen beta-particles isolated by the precipitation-centrifugation method.

Glycogen in its particulate beta-form is localized in the sarcoplasm close to the sarcoplasmic reticulum. Some particles are in close contact with the membranes, on the outer side of the vesicles. The mild technique of differential precipitation-centrifugation has been adapted to the preparation of glycogen from adult skeletal muscle. A preliminary low-speed centrifugation which eliminates the contractile protein structures and the cell debris is followed by a high-speed centrifugation which produces pellets containing glycogen mixed with smooth-walled vesicles, the glycogen-sarcovesicular fraction. The glycogen obtained after treatment of this fraction with deoxycholate and two washings contains 3% protein. A similar protein content contaminates glycogen banded in a linear sucrose gradient. The glycogen-sarcovesicular fraction and the purified glycogen have been examined, under the electron microscope, in sections of fixed and embedded material or with the negative staining technique. The glycogen beta-particles in negatively stained preparations have an average diameter of 39.4 mmicro. The largest particles present irregular outlines, suggesting the presence of conglomerated subunits, about 20 mmicro in diameter. These subunits seem to fall apart under the influence of concentrated potassium hydroxide. The mean sedimentation coefficients calculated for infinite dilution vary from 115 to 135S. The spectrophotometric analysis of the glycogen-iodine complex indicates the presence of long end-chains in the molecule.

The alteration of myosin isoform compartmentation in specific mutants of Caenorhabditis elegans.

Myosin isoforms A and B are located at the surface of the central and polar regions, respectively, of thick filaments in body muscle cells of Caenorhabditis elegans, whereas paramyosin and a distinct core structure comprise the backbones of these filaments. Thick filaments and related structures were isolated from nematode mutants that have altered thick filament protein compositions. These mutant filaments and their complexes with specific antibodies were studied by electron microscopy to determine the distribution of the two myosins. The compartmentation of the two myosin isoforms in body wall muscle thick filaments depends not only upon the intrinsic properties of the myosins but their interactions with other components such as paramyosin and their relative quantities determined by synthesis.

Externalization of an endogenous chicken muscle lectin with in vivo development.

Chicken-lactose-lectin-I (CLL-I), an endogenous lectin that is developmentally regulated in embryonic muscle, was localized by immunohistochemical techniques in tissue samples taken at various stages of in vivo development and in primary muscle cultures. Lectin, which was diffusely distributed in myoblasts, became localized in myotubes in a distribution similar to that of the sarcoplasmic reticulum and T tubules. Later in development, lectin was predominantly extracellular. This sequence suggests that externalization may have occurred by migration in the T tubules, which are continuous with the extracellular space, although alternative explanations are possible. Only traces of lectin were found in the adult. These studies did not reveal the function of CLL-I in muscle development. However, we infer that it acts by organizing complementary glycoconjugates in the intracellular tubular network, on the muscle surface, and/or in extracellular materials.

Development of muscle fiber specialization in the rat hindlimb.

The appearance of fast and slow fiber types in the distal hindlimb of the rat was investigated using affinity-purified antibodies specific to adult fast and slow myosins, two-dimensional electrophoresis of myosin light chains, and electron microscope examination of developing muscle cells. As others have noted, muscle histogenesis is not synchronous; rather, a series of muscle fiber generations occurs, each generation forming along the walls of the previous generation. At the onset of myotube formation on the 15th d of gestation, the antimyosin antibodies do not distinguish among fibers. All fibers react strongly with antibody to fast myosin but not with antibody to slow myosin. The initiation of fiber type differentiation can be detected in the 17-d fetus by a gradual increase in the binding of antibody to slow myosin in the primary, but not the secondary, generation myotubes. Moreover, neuromuscular contacts at this crucial time are infrequent, primitive, and restricted predominantly, but not exclusively, to the primary generation cells, the same cells which begin to bind large amounts of antislow myosin at this time. With maturation, the primary generation cells decrease their binding of antifast myosin and become type I fibers. Secondary generation cells are initially all primitive type II fibers. In future fast muscles the secondary generation cells remain type II, while in future slow muscles most of the secondary generation cells eventually change to type I over a prolonged postnatal period. We conclude that the temporal sequence of muscle development is fundamentally important in determining the genetic expression of individual muscle cells.

Plasminogen activator in mammalian skeletal muscle: characteristics of effect of denervation on urokinase-like and tissue activator.

Analyses were made of the fibrinolytic, plasminogen-activating system in skeletal muscle to determine if a regulating influence of the nerve could be detected on these enzymes. Young male mice underwent right sciatic neurectomy. Extracts were prepared from denervated muscle at 2-17 d after axotomy and compared with controls. Using a cascade-style biochemical assay (Rånby, M., B. Norrman, and P. Wallén, 1982, Thromb. Res., 27:743-748) we found that low levels of plasminogen activator (PA) were present in adult, innervated mouse muscle, but that denervation resulted in a marked time-dependent increase in enzyme activity. Qualitative separation showed an eightfold increase in urokinase-like PA with moderate elevation of tissue PA activity after 10 d. Fibrin zymography (Granelli-Piperno, A., and E. Reich, 1978, J. Exp. Med., 148:223-234) revealed clear zones of lysis corresponding to molecular masses of 48 kD for urokinase-like PA and 75 kD for tissue PA, consistent with the molecular masses found for these enzymes in other tissues of the mouse (Danø, K., P. A. Andreasen, J. Grøndahl-Hansen, P. Kristensen, L. S. Nielsen, and L. Skriver, 1985, Adv. Cancer Res., 44:139-266). In other studies we have shown that PA-activated plasmin readily attacks critical adhesive basement membrane molecules. The present results indicate that enzymes involved in plasminogen activation, particularly urokinase-like PA, rapidly increase after axotomy, suggesting they may have a role early in muscle denervation. Similar alterations in PA activity might underlie the elimination of polyneuronal innervation during mammalian muscle development. Certain neuromuscular diseases may also involve activation of these enzymes, resulting in degradation of basement membrane zone components and, therefore, warrant further study.

Isolation, characterization, and localization of the spanning protein from skeletal muscle triads.

A monoclonal antibody has been developed against the putative junctional protein or spanning protein (SP) from skeletal muscle triads. By immuno-affinity chromatography, we have purified this protein. The native protein has a molecular mass of 630-800 kD, as determined by gel filtration and rate zonal centrifugation. Within the limits of the methods used, the basic unit of the SP appears to be a dimer. In electron micrographs, it is shown to exhibit a circular profile with a diameter of approximately 100 A. In thin section analysis, the protein is frequently observed as parallel tracks of electron-dense particles bordering a translucent core. We suggest that the basic unit of the junctional structure is a dimer of 300-kD subunits and that four such entities constitute the intact SP. The purified protein has been used to develop polyclonal antibodies. By immunoelectron microscopy using immunogold probes, the SP has been localized to the junctional gap of the triad. By attaching the SP to an affinity resin, three proteins have been identified as forming associations with the SP. The Mrs of the proteins are 150, 62, and 38 kD; the 62-kD protein is calsequestrin.

Multiple actins in Drosophila melanogaster.

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.

Improved procedures for immunoferritin labeling of ultrathin frozen sections.

In employing fixed frozen ultrathin sections as substrates for immunoferritin labeling of intracellular antigens, we have found that conventional glutaraldehyde fixation sometimes permits very little specific staining of the sections, either because it inactivates certain protein antigens, or because it renders them inaccessible to the antibody stains. We have developed several fixation procedures that are chemically milder and allow a uniform but less extensive cross-linking of the specimen. With these procedures and precautions in the handling of the more fragile frozen sections, excellent structural preservation and specific immunoferritin labeling has been achieved with several systems.

Stereological analysis of mammalian skeletal muscle. I. Soleus muscle of the adult guinea pig.

A quantitative analysis of the volumes, surface areas, and dimensions of the ultrastructural components in the soleus muscle fibers of the guinea pig was made by using point counting methods of stereology. Muscle fibers have structural orientation (anisotropy) and have spatial gradients of the structures within the fiber; therefore the standard stereological methods were modified where necessary. The entire analysis was repeated at two section orientations to test the modifications and identical results obtained from both. The volume of lipid droplets was 0.20 +/- 0.06% (mean +/- standard error, n = 5 animals) and the nuclei volume was 0.86 +/- 0.20% of the fiber volume. The total mitochondrial volume was 4.85 +/- 0.66% of the fiber volume with about one-third being found in an annulus within 1 microm of the sarcolemma. The mitochondrial volume in the remaining core of the fiber was 3.6 +/- 0.4%. The T system has a volume of 0.14 +/- 0.01% and a surface area of 0.064 +/- 0.005 microm(2)/microm(3) of the fiber volume. The surface area of the sarcolemma is 0.116 +/- 0.013 microm(2)/microm(3) which is twice the T system surface area. The volume of the entire sarcoplasmic reticulum is 3.52 +/- 0.33% and the surface area is 0.97 +/- 0.09 microm(2)/microm(3). The sarcoplasmic reticulum is composed of the terminal cisternae whose volume is 1.04 +/- 0.19% and surface area is 0.24 +/- 0.05 microm(2)/microm(3). The tubules of the sarcoplasmic reticulum in the I band and A band have volumes of 1.97 +/- 0.24% and 0.51 +/- 0.08%, and the surface areas of the I and A band reticulum are 0.56 +/- 0.07 microm(2)/microm(3) and 0.16 +/- 0.04 microm(2)/microm(3), respectively. The Z line width, myofibril and fiber diameters were measured.

A new 185,000-dalton skeletal muscle protein detected by monoclonal antibodies.

The M line, which transverses the center of the thick filament region of skeletal muscle sarcomeres, appears to be a complex array of multiple structural elements. To date, two proteins have definitely been shown to be associated with the M line. They are MM-CK, localized in the M 4,4' substriations, and a 165,000-dalton (164 kd) protein, referred to as both M-protein and myomesin. Here we report the positive identification of a third M-line protein of 185 kd. In the course of making monoclonal antibodies (mAbs) against a 165-kd fraction, we also obtained mAbs that bound to the M line of isolated myofibrils as detected by indirect immunofluorescence, but recognized a protein band of 185 kd in immunoblotting experiments with either the original immunogen or low ionic strength myofibril extracts as antigenic targets. The evidence that the 185- and 165-kd proteins are distinct protein species is based on the separation of the two proteins into discrete peaks by ion exchange chromatography, the distinctive patterns of their degradation products, and non-cross-reactivity of any of seven mAbs. These mAbs recognize three unique antigenic determinants on the 185-kd molecule and at least two and probably four sites on the 165-kd molecule as determined from competitive binding and immunofluorescence experiments. To resolve the problem of multiple nomenclature for the 165-kd protein, the 185-kd protein will be referred to as myomesin and the 165-kd protein as M-protein.

Expression of cell-adhesion molecules in embryonic induction. I. Morphogenesis of nestling feathers.

The potential relationship of cell adhesion to embryonic induction during feather formation was examined by immunohistochemical analysis of the spatiotemporal distribution of three cell-adhesion molecules (CAMs), neural CAM (N-CAM), liver CAM (L-CAM), and neuron-glia CAM (Ng-CAM), and of substrate molecules (laminin and fibronectin) in embryonic chicken skin. The N-CAM found at sites of embryonic induction in the feather was found to be similar to brain N-CAM as judged by immuno-cross-reactivity, migratory position in PAGE, and the presence of embryonic to adult conversion. In contrast to the N-CAM found in the brain, however, only one polypeptide of Mr 140,000 was seen. N-CAM-positive dermal condensations were distributed periodically under L-CAM-positive feather placodes at those sites where basement membranes are known to be disrupted. After initiation of induction, L-CAM-positive placode cells became transiently N-CAM-positive. N-CAM was asymmetrically concentrated in the dorsal region of the feather bud, while fibronectin was concentrated in the ventral region. During feather follicle formation, N-CAM was expressed in the dermal papilla and was closely apposed to the L-CAM-positive papillar ectoderm, while the dermal papilla showed no evidence of laminin or fibronectin. The collar epithelium was both N-CAM- and L-CAM-positive. During the formation of the feather filament, N-CAM appeared periodically and asymmetrically on basilar cells located in the valleys between adjacent barb ridges. In contrast to the two primary CAMs, Ng-CAM was found only on nerves supplying the feather and the skin. These studies indicate that at each site of induction during feather morphogenesis, a general pattern is repeated in which an epithelial structure linked by L-CAM is confronted with periodically propagating condensations of cells linked by N-CAM.

Glutamate uptake by a stimulated insect nerve muscle preparation.

Recent reports suggest that glutamate may be the excitatory neuromuscular transmitter in insects. In this study, glutamate uptake by isolated cockroach nerve muscle preparations was investigated by means of chemical and electron microscope radioautographic techniques. We found that the preparation had a high affinity for glutamate and that nerve stimulation enhanced glutamate uptake. Chemical studies showed that the average tissue concentration of glutamate bound during a 1 hr incubation period in 10(-5)M glutamate-(3)H after nerve stimulation was 2.8 x 10(-5)M. Less than 1% of the radioactivity was present in the perchloric acid-precipitated protein fraction. Using electron microscope radioautography, we observed that sheath cells showed the highest glutamate concentration of all cellular compartments. Uptake was greater at neuromuscular junctions than in other regions of the tissue. The data suggest a possible mechanism for transmitter inactivation and protection of synapses from high blood glutamate.

Distribution of fast myosin heavy chain isoforms in thick filaments of developing chicken pectoral muscle.

Colloidal gold-conjugated monoclonal antibodies were prepared to stage-specific fast myosin heavy chain (MHC) isoforms of developing chicken pectoralis major (PM). Native thick filaments from different stages of development were reacted with these antibodies and examined in the electron microscope to determine their myosin isoform composition. Filaments prepared from 12-d embryo, 10-d chick, and 1-yr chicken muscle specifically reacted with the embryonic (EB165), neonatal (2E9), and adult (AB8) antimyosin gold-conjugated monoclonal antibodies, respectively. The myosin isoform composition was more complex in thick filaments from stages of pectoral muscle where more than one isoform was simultaneously expressed. In 19-d embryo muscle where both embryonic and neonatal isoforms were present, three classes of filaments were found. One class of filaments reacted only with the embryonic antibody, a second class reacted only with the neonatal-specific antibody, and a third class of filaments were decorated by both antibodies. Similar results were obtained with filaments prepared from 44-d chicken PM where the neonatal and adult fast MHCs were expressed. These observations demonstrate that two myosin isoforms can exist in an individual thick filament in vivo. Immunoelectron microscopy was also used to determine the specific distribution of different fast MHC isoforms within individual filaments from different stages of development. The anti-embryonic and anti-adult antibodies uniformly decorated both homogeneous and heterogeneous thick filaments. The neonatal specific antibody uniformly decorated homogeneous filaments; however, it preferentially decorated the center of heterogeneous filaments. These observations suggest that neonatal MHC may play a specific role in fibrillogenesis.

Sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation.

The expression of cytoplasmic beta-actin and cardiac, skeletal, and smooth muscle alpha-actins during early avian cardiogenesis was analyzed by in situ hybridization with mRNA-specific single-stranded DNA probes. The cytoplasmic beta-actin gene was ubiquitously expressed in the early chicken embryo. In contrast, the alpha-actin genes were sequentially activated in avian cardiac tissue during the early stages of heart tube formation. The accumulation of large quantities of smooth muscle alpha-actin transcripts in epimyocardial cells preceded the expression of the sarcomeric alpha-actin genes. The accumulation of skeletal alpha-actin mRNAs in the developing heart lagged behind that of cardiac alpha-actin by several embryonic stages. At Hamburger-Hamilton stage 12, the smooth muscle alpha-actin gene was selectively down-regulated in the heart such that only the conus, which subsequently participates in the formation of the vascular trunks, continued to express this gene. This modulation in smooth muscle alpha-actin gene expression correlated with the beginning of coexpression of sarcomeric alpha-actin transcripts in the epimyocardium and the onset of circulation in the embryo. The specific expression of the vascular smooth muscle alpha-actin gene marks the onset of differentiation of cardiac cells and represents the first demonstration of coexpression of both smooth muscle and striated alpha-actin genes within myogenic cells.

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.

Intermediate filaments in muscle and epithelial cells of nematodes.

Current concepts of the developmentally controlled multigene family of intermediate filament (IF) proteins expect the origin of their complexity in evolutionary precursors preceding all vertebrate classes. Among invertebrates, however, firm ultrastructural as well as molecular documentation of IFs is restricted to some giant axons and to epithelia of a few molluscs and annelids. As Ascaris lumbricoides is easily dissected into clean tissues, IF expression in this large nematode was analyzed by electron microscopic and biochemical procedures and a monoclonal antibody reacting with all mammalian IF proteins. We document for the first time the presence of IFs in muscle cells of an invertebrate. They occur in three muscle types (irregular striated pharynx muscle, obliquely striated body muscle, uterus smooth muscle). IFs are also found in the epithelia studied (syncytial epidermis, intestine, ovary, testis). Immunoblots on muscles, pharynx, intestine, uterus, and epidermis identify a pair of polypeptides (with apparent molecular masses of 71 and 63 kD) as IF constituents. In vitro reconstitution of filaments was obtained with the proteins purified from body muscle. In the small nematode Caenorhabditis elegans IF proteins are so far found only in the massive desmosome-anchored tonofilament bundles which traverse a special epithelial cell type, the marginal cells of the pharynx. We speculate that IFs may occur in most but perhaps not all invertebrates and that they may not occur in all cells in large amounts. As electron micrographs of the epidermis of a planarian--a member of the Platyhelminthes--reveal IFs, the evolutionary origin of this cytoplasmic structure can be expected either among the lowest metazoa or already in some unicellular eukaryotes.

Cytoplasmic myosin from Drosophila melanogaster.

Myosin is identified and purified from three different established Drosophila melanogaster cell lines (Schneider's lines 2 and 3 and Kc). Purification entails lysis in a low salt, sucrose buffer that contains ATP, chromatography on DEAE-cellulose, precipitation with actin in the absence of ATP, gel filtration in a discontinuous KI-KCl buffer system, and hydroxylapatite chromatography. Yield of pure cytoplasmic myosin is 5-10%. This protein is identified as myosin by its cross-reactivity with two monoclonal antibodies against human platelet myosin, the molecular weight of its heavy chain, its two light chains, its behavior on gel filtration, its ATP-dependent affinity for actin, its characteristic ATPase activity, its molecular morphology as demonstrated by platinum shadowing, and its ability to form bipolar filaments. The molecular weight of the cytoplasmic myosin's light chains and peptide mapping and immunochemical analysis of its heavy chains demonstrate that this myosin, purified from Drosophila cell lines, is distinct from Drosophila muscle myosin. Two-dimensional thin layer maps of complete proteolytic digests of iodinated muscle and cytoplasmic myosin heavy chains demonstrate that, while the two myosins have some tryptic and alpha-chymotryptic peptides in common, most peptides migrate with unique mobility. One-dimensional peptide maps of SDS PAGE purified myosin heavy chain confirm these structural data. Polyclonal antiserum raised and reacted against Drosophila myosin isolated from cell lines cross-reacts only weakly with Drosophila muscle myosin isolated from the thoraces of adult Drosophila. Polyclonal antiserum raised against Drosophila muscle myosin behaves in a reciprocal fashion. Taken together our data suggest that the myosin purified from Drosophila cell lines is a bona fide cytoplasmic myosin and is very likely the product of a different myosin gene than the muscle myosin heavy chain gene that has been previously identified and characterized.

Molecular forms of N-CAM and its RNA in developing and denervated skeletal muscle.

The neural cell adhesion molecule (N-CAM) is present in both embryonic and perinatal muscle, but its distribution changes as myoblasts form myotubes and axons establish synapses (Covault, J., and J. R. Sanes, 1986, J. Cell Biol., 102:716-730). Levels of N-CAM decline postnatally but increase when adult muscle is denervated or paralyzed (Covault, J., and J. R. Sanes, 1985, Proc. Natl. Acad. Sci. USA., 82:4544-4548). To determine the molecular forms of N-CAM and N-CAM-related RNA during these different periods we used immunoblotting and nucleic acid hybridization techniques to analyze N-CAM and its RNA in developing, cultured, adult, and denervated adult muscle. As muscles develop, the extent of sialylation of muscle N-CAM decreases, and a 140-kD desialo form of N-CAM (generated by neuraminidase treatment) is replaced by a 125-kD form. This change in the apparent molecular weight of desialo N-CAM is paralleled by a change in N-CAM RNA: early embryonic muscles express a 6.7-kb RNA species which hybridizes with N-CAM cDNA, whereas in neonatal muscle this form is largely replaced by 5.2- and 2.9-kb species. Similar transitions in the desialo form of N-CAM, but not in extent of sialylation, accompany differentiation in primary cultures of embryonic muscle and in cultures of the clonal muscle cell lines C2 and BC3H-1. Both in vivo and in vitro, a 140-kD desialo form of N-CAM and a 6.7-kb N-CAM RNA are apparently associated with myoblasts, whereas a 125-kD desialo form and 5.2- and 2.9-kb RNAs are associated with myotubes and myofibers. After denervation of adult muscle, a approximately 12-15-fold increase in the levels of N-CAM is accompanied by a approximately 30-50-fold increase in N-CAM RNA, suggesting that N-CAM expression is regulated at a pretranslational level. Forms of N-CAM and its RNA in denervated muscle are similar to those seen in perinatal myofibers.

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.

Posttranscriptional control of embryonic rat skeletal muscle protein synthesis. Control at the level of translation by endogenous RNA.

The onset of muscle cell differentiation is associated with increased transcription of muscle-specific mRNA. Studies from this laboratory using 19-d embryonic rat skeletal muscle, suggest that additional, posttranscriptional controls regulate maturation of muscle tissue via a quantitative effect upon translation, and that the regulatory component may reside within the poly A- RNA pool (Nathanson, M.A., E.W. Bush, and C. Vanderburg. 1986. J. Biol. Chem. 261:1477-1486). To further characterize muscle cell translational control, embryonic and adult total RNA were separated into oligo(dT)cellulose-bound (poly A+) and -unbound (poly A-) pools. Unbound material was subjected to agarose gel electrophoresis to resolve constituents of varying molecular size and mechanically cut into five fractions. Material of each fraction was electroeluted and recovered by precipitation. Equivalent loads of total RNA from 19-20-d embryonic rat skeletal muscle exhibited a 40% translational inhibition in comparison to its adult counterpart. Inhibition was not due to decreased message abundance because embryonic, as well as adult muscle, contained equivalent proportions of poly A+ mRNA. An inhibition assay, based upon the translatability of adult RNA and its inhibition by embryonic poly A- RNA, confirmed that inhibition was associated with a 160-2,000-nt poly A- fraction. Studies on the chemical composition of this fraction confirmed its RNA composition, the absence of ribonucleoprotein, and that its activity was absent from similarly fractionated adult RNA. Rescue of inhibition could be accomplished by addition of extra lysate or mRNA; however, smaller proportions of lysate were required, suggesting a strong interaction of inhibitor and components of the translational apparatus. Additional studies demonstrated that the inhibitor acted at the level of initiation, in a dose-dependent fashion. The present studies confirm the existence of translational control in skeletal muscle and suggest that it operates at the embryonic to adult transition. A model of muscle cell differentiation, based upon transcriptional control at the myoblast level, followed by translational regulation at the level of the postmitotic myoblast and/or myotube, is proposed.