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 Wine 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 .Myofibrils, the organelles that form the contractile apparatus of cross-striated muscle cells, show distinct structures perpendicular to the thick and thin filaments. The Z-line structure that defines single sarcomeres anchors thin filaments and probably giant proteins such as titin (1) as well. Several possible functions for the other prominent transverse structure, the M line, have been proposed (2-8). More and more Z-and M-line protein components have been detected, isolated, and characterized, which has revealed an ever increasing complexity ofthese structures. The existence of at least three structural elements has been postulated for the M-line region (9), where high resolution electron microscopy reveals a minimal number of five transverse striations and a so called M filament, which parallels the myosin filaments in the M region (2, 10-13) .To date, two proteins have definitely been shown to be associated with the M line; one is MM-CK (4, 14, 15) and the other is a 165,000-dalton (165-kd) protein (3,(16)(17)(18) . MM-CK has been assigned to substriations M4 and M4' (19, 20), while the sublocalization ofthe high molecular weight M-line protein is still unclear . However, from several observations in 518 BARBARA KAY GROVE, VERENA KURER, CHRISTIAN LEHNER, THOMAS C. DOETSCHMAN, JEAN-CLAUDE PERRIARD, and HANS M . EPPENBERGER Institute for Cell Biology, Swiss Federal Institute of Technology, CH-8093 Zürich, Switzerland our laboratory and in other laboratories as well, it has been concluded that additional protein components must be located with...

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.Recently we have reported on a new 185-kD protein in the M-band of skeletal muscle myofibrils (1). This brings the number of positively identified M-band proteins to three, the other two being MM-CK (2, 3) and M-protein (4). To avoid multiple nomenclature, it has been agreed to call the 165-kD M-band protein "M-protein" and the recently identified 185-kD M-band protein "myomesin" (1, 5).Several lines of evidence indicate the functional significance of the M-band which transverses the center of the sarcomere. M-band structural components appear to provide a scaffolding for the alignment and orientation of myosin thick fdaments within myofibrils (6-10). Furthermore, there is evidence that the M-band, as well as the Z-disc, are cytoskeletal attachment points and that they assist in maintaining intermyofibril register under the stress of lateral tension imposed on muscle fibers during contraction (11-13).Of the known M-band proteins, the substructural localization and function of only one has been elucidated. MM-CK, associated with the M4, 4' substriations (14,15), is present in sufficient quantity to regenerate in isolated chicken myofibrils the ATP required for contraction (16). Attempts have been made to ascertain the roles of other M-band components in

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.

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 compositon 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 ofmyomesin in heart [Grove et aL (1985):

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