The structure of the sarcomeric M band: localization of defined domains of myomesin, M-protein, and the 250-kD carboxy-terminal region of titin by immunoelectron microscopy.

Abstract

Abstract. The M band of vertebrate cross-striated myofibrils has remained an enigmatic structure. In addition to myosin thick filaments, two major structural proteins, myomesin and M-protein, have been localized to the M band. Also, titin is expected to be anchored in this structure. To begin to understand the molecular layout of these three proteins, a panel of 16 polyclonal and monoclonal antibodies directed against unique epitopes of defined sequence was assembled, and immunoelectron microscopy was used to locate the position of the epitopes at the sarcomere level. The results allow the localization and orientation of defined domains of titin, myomesin, and M-protein at high resolution. The 250-kD carboxy-terminal region of titin clearly enters the M band with the kinase domain situated ~52 nm from the central Ml-line. The positions of three additional epitopes are compatible with the view that the titin molecule reaches ~60 nm into the opposite sarcomere half. Myomesin also seems to bridge the central Ml-line and is oriented parallel to the long axis of the myofibril. The neighboring molecules are oriented in an antiparallel and staggered fashion. The amino-terminal portion of the protein, known to contain a myosin binding site, seems to adopt a specific three-dimensional arrangement. While myomesin is present in both slow and fast fibers, M-protein is restricted to fast fibers. It appears to be organized in a fundamentally different manner: the central portion of the polypeptide is around the Ml-line, while the terminal epitopes seem to be arranged along thick filaments. This orientation fits the conspicuously stronger M1-lines in fast twitch fibers. Obvious implications of this model are discussed.T HE stable and ordered arrangement of thick and thin filaments in sarcomeric muscle myofibrils arises from a complex cytoskeletal framework (for review see Small et al., 1992). Its major structures, the M bands and Z discs, seem to be interconnected by the giant protein titin (also called connectin). In the M band, the thick, hexagonal filament lattice appears to be stabilized by additional components. Some aspects of M band substructure have been deduced from negatively stained longitudinal cryosections (SjOstr6m and Squire, 1977). Thus, the M band appears as a zone of higher density, about 75 nm wide, which depending on fiber type and animal species consists of three to five major cross striations. These striations are thought to reflect bridging structures, called

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

“…According to observations on isolated filaments, myomesin and M-protein bind to the central zone of myosin filaments [16] and to the C-terminus of titin [17], indicating participation in the sarcomeric cytoskeleton. The approximate positions of titin, myomesin and M-protein in the sarcomere were determined by biochemical assays and EM epitope localization; these data led to a molecular model of the M-band [11,18,19]. In this model, M-protein molecules bridge the myosin filaments at the level of the central M1 line (see Box 1), which is consistent with previous EM observations [20,21].…”

“…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].…”

“…Similar to the PEVK fragment of titin, the EH-fragment is mostly unfolded and functions like an entropic spring [31] (Box 2). Phosphorylation sites (P, blue circles) [18,19] and interacting partners (identified above brackets) [11,18,30,69] are indicated.…”

Mechanoenzymatics of titin kinase

“…At the M-band, titin contains a serine/ threonine protein kinase domain (TK) (Fig. 1B) (3,4). TK is regulated in a dual autoinhibition mechanism by a C-terminal regulatory tail, blocking the ATP binding site, and tyrosine autoinhibition of the catalytic base by tyrosine-170 (5).…”

Myogenic Stage, Sarcomere Length, and Protease Activity Modulate Localization of Muscle-specific Calpain

“…During sarcomere assembly, connectin is considered a key molecule for integration of thin filament/Z-band precursors (I-Z-I bodies) and the thick filaments, which are independently assembled in growth tips of elongating myotubes. The N terminus of connectin is located in both I-Z-I body and mature Z, and the C terminus is in the M-line of the thick filament (12)(13)(14). A single molecule of connectin spans half of the sarcomere from Z to M to integrate the Z-band and the thick filaments, maintaining the location of thick filaments between the Z-bands.…”