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

Abstract

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 M-band: an elastic web that crosslinks thick filaments in the center of the sarcomere

“…By contrast, myomesin which was first discovered as a copurifying component in M-protein preparations [12,13], is found in all types of vertebrate striated muscle investigated so far [25] as well as in the first sarcomeres assembled in the developing heart [3]. The importance of myomesin is further supported by the observation that myomesin seems to be expressed at a fixed ratio to myosin in different muscle types [26].…”

“…The C-termini of titin molecules also overlap there [11] (Box 1). In addition, the M-band contains two principal candidates for the role of M-bridges, originally named myomesin [12,13] (myomesin-1 in RefSeq database) and M-protein [14] lines missing), whereas the slowest fibers have a four-line pattern (M1 line missing), and fibers of intermediate speed have variations of a fiveline pattern [23,65,66]. Accordingly, the M-band appearance changes during differentiation of slow fibers of the rat [23] and correlates with the beating rate in the heart of different species [21].…”

Mammalian Formin Fhod3 Regulates Actin Assembly and Sarcomere Organization in Striated Muscles

“…We next investigated the localization of Fhod3 with respect to the M-line, located at the center of a sarcomere. Double staining for Fhod3 and the sarcomeric M-line protein myomesin (16) revealed that the M-line was localized in a narrower gap of Fhod3 bands (Fig. 1C).…”

Intracompartmental sorting of essential myosin light chains: Molecular dissection and in vivo monitoring by epitope tagging

“…The monoclonal antibody anti-a-sm-1, recognizing a-smooth muscle actin and described elsewhere (Skalli et al, 1986), as well as the IgM monoclonal antibody My-21 recognizing the 20 kd myosin LC, were purchased from Bio Makor. The monoclonal antibody 84 recognizing the M band protein myomesin was raised in our laboratory (Grove et al, 1984 equipped with a Bio-Rad MRC 600 laser scanning unit was used. The image processing software will be described elsewhere.…”