Brain-associated glycoproteins expressed in bovine heart Purkinje fibres.

The Purkinje fibres of bovine heart were investigated immunohistochemically by use of monoclonal antibodies with specificity against the glycoproteins Thy-1 and Gp120, expressed in human brain. The existence and expression in bovine tissues (brain and thymus) of antigens displaying similar properties and immunochemical crossreactivity with monoclonal antibodies against the human antigens were confirmed. Both these antigens, as identified by use of anti Thy-1 and anti-Gp120 monoclonal antibodies were found to be associated with the membranes of the impulse conduction system. The presence of the antigens was seen in areas facing other conduction cells. No parts of the cells facing the basal membrane of the fibres were stained. The continuous staining between the cells was different from that of desmosome related proteins. These findings may have physiological and functional implications and are interesting in relation to recent evidences suggesting that the conduction tissue might be a derivative of the neural crest.

Myofibrillar M-band proteins in rat skeletal muscles during development.

The distribution of three myofibrillar M-band proteins, myomesin, M-protein and the muscle isoform of creatine kinase, was investigated with immunocytochemical techniques in skeletal muscles of embryonic, fetal, newborn and four-week-old rats. Furthermore, muscles of newborn rats were denervated and examined at four weeks of age. In embryos, myomesin was present in all myotome muscle fibres of the somites, whereas M-protein was detected only in a small proportion of the myotome muscle fibres and muscle creatine kinase was not detected at all. In fetal and newborn muscles, all fibres contained all three M-band proteins. At four weeks of age, when fibre types (type 1 or slow twitch fibres and type 2 or fast twitch fibres) were clearly discernable, the pattern was changed. Myomesin and muscle creatine kinase were still observed in all fibres, whereas M-protein was present only in type 2 fibres. On the other hand, in muscle fibres denervated at birth all three M-band proteins were still detected. Our results suggest 1) that during the initial stages of myofibrillogenesis expression and incorporation of myomesin into the M-band precede that of M-protein and muscle creatine kinase; 2) that expression and incorporation of all three M-band proteins during fetal development is nerve independent and non coordinated to the expression of different forms of myosin heavy chains, and 3) that the suppression of M-protein synthesis during postnatal development is nerve dependent and reflects the maturation of slow twitch motor units.

Fiber type-specific distribution of M-band proteins in chicken muscle.

The functions of two myofibrillar proteins, myomesin (Mr 185,000) and M-protein (Mr 165,000), associated with the M-band are as yet unknown. To extend our knowledge of these proteins, we have examined chicken striated muscles with fast and slow contractile properties, e.g., pectoralis major, PLD, ALD, medial adductor, and lateral adductor, to determine the expression and isoform composition of myomesin and M-protein in various muscles and fiber types. The high molecular weight M-band proteins were characterized and quantitated using monoclonal antibodies in immunoblotting and double-antibody sandwich ELISA. Fiber specificity was determined by immuno- and enzyme histochemistry. In addition to the previously reported Mr 195,000 and 190,000 isoforms of myomesin in heart [Grove et al. (1985): J Cell Biol 101:1431], the Mr 185,000 myomesin in skeletal muscles may represent different isoforms in fast and slow muscles on the basis of distinctive degradation patterns. M-protein has the same molecular weight in striated chicken muscles and degradation patterns indicate only one isoform. The low quantities of M-protein in slow muscles were shown to be due to the absence of M-protein in two of the generally recognized slow fiber types, types I and III. Thus, M-protein was present only in fast type II fibers, whereas myomesin was ubiquitous in all fiber types. Whatever the causal relationship, M-protein appears to function in fast motor units composed of type II fibers.

Muscle differentiation and the origin of muscle fiber diversity.

The biology of muscle development has gone full circle--from classical embryonic morphology to cell culture and now back to the embryo at the molecular and cellular levels. Recent advances in modern technology have made possible complex in vitro and in vivo biochemical and ultrastructural analyses of myogenesis. This article correlates theories regarding the events and regulatory factors involved in muscle differentiation and fiber-type formation based on findings from the model system of isolated muscle cells in culture with actual in vivo situations.

Noncoordinate expression of M band proteins in slow and fast embryonic chick muscles.

The expression of the myosin-associated M band proteins myomesin and M protein in differentiating muscle fibers in the anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) muscles during embryonic chicken development was examined by immunocytochemistry using monoclonal antibodies. Early in the embryonic development of both muscles, both myomesin and M protein are expressed in primary and secondary myotubes. However, beginning at 10 days in ovo, M protein is gradually suppressed first in primary, then in secondary, presumptive slow-tonic type 3 fibers. M protein is transiently suppressed in presumptive fast-twitch type 2 fibers derived from primary myotubes but continuously expressed in those derived from secondary myotubes. Thus, initially all myotubes have a common intrinsic M band composition with respect to myomesin and M protein, whereas at later stages the expression of M protein is fiber-type specific. Intrafusal spindle fibers, which are segregated from extrafusal fibers around 14 days in ovo, have a heterogeneous M band composition atypical of extrafusal fibers.

Myofibrillar M-band structure and composition of physiologically defined rat motor units.

The isometric contraction time of 19 fast and slow rat motor units in the soleus and the anterior tibial muscles were recorded. The motor unit fibers, subsequently distinguished by glycogen depletion, were histochemically differentiated into fiber types and analyzed immunohistochemically for high molecular weight M-band proteins, as well as ultrastructurally for M-band fine structure, Z-disc width, and volume density of mitochondria. All fibers belonging to slow-twitch motor units in both the anterior tibialis and soleus muscles were histochemically classed as type 1. They lacked the Mr 165,000 M-protein, showed ultrastructurally a four-line M-band pattern, and had broad Z-discs, whereas the volume density of the mitochondria varied considerably. Muscle fibers belonging to the fast-twitch motor units were histochemically classed as types 2A and 2B in anterior tibialis and type 2A in soleus. They contained a three- or a five-line M-band pattern and medium-to-thin Z-discs in the anterior tibialis and a five-line M-band pattern and broad Z-discs in the soleus. Furthermore, the volume density of mitochondria showed considerable variation within and in between soleus and anterior tibialis type 2 fibers. As the differences in M-band composition and structure between fiber types overrode the intragroup variability in contraction times of slow and fast units within and between the two muscles, it is concluded that the M-band composition and structure is fundamentally related to whether the fiber is innervated by a slow or fast motor neuron, whereas other parameters such as contraction time, Z-disc width, and mitochondrial content of fibers of fast and slow units are relative and vary between muscles. Thus the M-band appearance can be used as a reliable marker to distinguish between fibers of slow- and fast-twitch motor units in rat leg muscles.

Myomesin and M protein: differential expression in embryonic fibers during pectoral muscle development.

By applying immunocytochemistry using monoclonal antibodies, we found that the myofibrillar M band of both presumptive type-I and -II fibers in the pectoralis major muscle of chickens contains two high-molecular-weight proteins, i.e., myomesin (Mr, 185,000) and M protein (Mr, 165,000), early in embryonic development (7 days in ovo), even though adult type-I fibers lack M protein. The developmental expression of M protein is unusual in that, from 10 to 14 days in ovo, it is gradually suppressed not only in presumptive type-I fibers but also in presumptive type-II fibers formed from primary-generation myotubes. This latter suppression is transient, as M protein is expressed in all adult type-II fibers derived from both the primary- and second-generation myotubes. Myomesin, on the other hand, is continuously expressed in all myotubes throughout development. This finding shows that myomesin and M protein expression is regulated independently in different myotube populations, and that the suppression of M protein in primary-generation myotubes accounts for the delayed accumulation of M protein during development, as previously revealed by biochemical analysis. Presumptive type-I fibers, which form in the deep portion of the muscle, become concentrated in a narrow band known as the red strip.

Cryoultramicrotomy and immunocytochemistry in the analysis of muscle fine structure.

Cryoultramicrotomy, which avoids the use of harsh fixation procedures, deleterious dehydration and plastic embedding can be combined with immunocytochemistry to determine the ultrastructural localization of cellular proteins. Our attempts to use the cryosectioning technique in combination with immunolabelling to bridge the gap between light and electron microscopic analysis of muscle morphology have enabled us to obtain new information on fibre typing at the ultrastructural level. Furthermore, we have obtained a marked improvement in the resolution of myofibrillar structures by using semithin cryosections for fluorescence microscopy. Data are also presented on correlated light and electron microscope immunocytochemistry of myocardial intermediate filaments confirming the presence of longitudinally oriented intermediate filaments of desmin in the region of the intercalated discs of mammalian cardiac myocytes, whereas elsewhere in the myocyte the bulk of intermediate filaments of desmin is concentrated in the intermyofibrillar space at the level of the Z disc.

Myomesin and M-protein: expression of two M-band proteins in pectoral muscle and heart during development.

The expression of the myofibrillar M-band proteins myomesin and M-protein was studied in chicken pectoral muscle and heart during differentiation using monoclonal antibodies in a double-antibody sandwich enzyme-linked immunosorbent assay, immunoblotting, and immunocytochemistry. In presumptive pectoral muscle, myomesin accumulated first, increasing from 2% of the adult concentration at day 7 to 70% by day 16 in ovo. M-protein accumulation lagged 6-7 d behind that of myomesin attaining only 40% of the adult concentration in ovo. The molecular masses of myomesin (185 kD) and M-protein (165 kD) remained constant during embryogenesis. In cultured myogenic cells the accumulation and M-band localization of myomesin preceded that of M-protein by 1.5 d. Chicken heart was shown, in addition to M-protein, to contain unique isoforms of myomesin. In hearts of 6 d embryos, a 195-kD myomesin isoform was the major species; throughout development, however, a transition to a mixture of 195 and 190 kD was observed, the latter being the major species in the adult tissue. During heart differentiation the initial accumulation of myomesin again preceded that of M-protein, albeit on an earlier time scale than in pectoral muscle with M-protein reaching adult proportions first.

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.

Regulation of membrane transport sites for amino acids in myogenic cells. A differentiation dependent phenomenon.

Myoblasts from 12-day chick embryos in cell culture transport the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB) two to three-fold more rapidly than multinucleated myotubes which form from them. This decrease in transport is due to a relative decrease in the number of transport sites per unit area of cell surface suggesting a compositional change in the plasma membrane during myogenesis. In studies reported here, AIB transport was monitored throughout myogenesis and correlated with other aspects of differentiation. During myogenesis the number of amino acid transport sites remains constant per myotube nucleus. As myogenesis proceeds, there is a marked increase in cellular protein and cell surface without a commensurate increase in amino acid transport sites. The net consequence of the surface area change is fewer amino acid transport sites per unit area of myotube membrane surface. The decrease in membrane transport sites for AIB per unit area of membrane is not a result of length of time in culture per se, medium depletion, or cell density, but is a result of differentiation, since blocking myoblast fusion by deprivation of calcium delays the decrease in AIB transport sites per unit cell surface area while reversal of the calcium deprivation block is accompanied by a rapid decrease in the number of AIB transport sites per unit cell surface area. Thus, the decrease in AIB transport sites is an aspect of differentiation which accompanies the marked elaboration of surface membrane during myogenesis.

Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody.

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate antigenic changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion, myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitatively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.

The role of ribosomal ribonucleic acid in the structure and function of mammalian brain ribosomes.

In order to resolve the functional role of intact rRNA in polypeptide chain elongation mouse brain ribosomes were treated with dilute pancreatic or T(1) RNAase (ribonuclease). After RNAase treatment, several physical-chemical properties as well as the functional activity of the ribosomes were measured. RNAase treatment resulted in the extensive hydrolysis of both 18S and 28S rRNA; however, the sedimentation properties of mono-ribosomes were unaltered and more than 90% of the relatively low-molecular-weight RNA fragments remained associated with ribosome particles. Analysis of the ability of RNAase-treated ribosomes to participate in cell-free protein synthesis showed that ribosomes with less than 2% intact rRNA retained more than 85% of their activity in polyphenylalanine incorporation. Proof that the incorporation of phenylalanine by ribosomes with hydrolysed rRNA actually represented active translocation was obtained by the effective inhibition of incorporation by diphtheria toxin. In addition, the oligopeptide products of protein synthesis could be identified by BD (benzoylated diethylaminoethyl)-cellulose column chromatography. Analysis of the size distribution of oligopeptides synthesized by normal and RNAase-treated ribosomes showed no significant differences which indicated that there was no change in the proportion of ribosomes engaged in protein synthesis. Thus strong RNA-protein and protein-protein interactions must serve to maintain the functional integrity of ribosomes even when the rRNA is extensively degraded. The ability of the enzyme-treated ribosomes to efficiently incorporate amino acids clearly demonstrated that ;intact' rRNA is not required for protein-synthetic activity.

Thermostability of mammalian brain ribosomes and the effects of nucleoside triphosphates on their heat-sensitivity.

Mammalian brain ribosomes were found to be heat-labile. On preincubation of the ribosomes at 37 degrees C, their ability to participate in polypeptide-synthesis reactions was substantially diminished. Despite the sensitivity of ribosomal protein synthesis to heat-inactivation, preincubation resulted in no significant alterations in ribosomal sedimentation profiles or changes in the integrity of the ribosomal RNA. The thermolability of brain ribosomes was shown to be associated with their inability to bind both template RNA and aminoacyl-tRNA. Similar experiments with brain ribosomal subunits demonstrated that the small (40S) subunit was more sensitive to heat-inactivation than the large (60S) subunit. The presence of ATP (1mm) protected ribosomes from thermal inactivation, although this protection was shown to be temporary. The protection appeared to be specific to nucleoside triphosphates, since GTP and UTP also stabilized ribosomes to thermal denaturation whereas nucleoside diphosphates (ADP) and nucleoside monophosphates (AMP and cyclic AMP) did not alter ribosomal sensitivity to heat. Although 1mm concentrations of nucleoside triphosphates protected ribosomes from heat-inactivation, the presence of higher concentrations resulted in complete inactivation of ribosomal activity.