Role of stress fiber-like structures in assembling nascent myofibrils in myosheets recovering from exposure to ethyl methanesulfonate.

Abstract

Abstract. When day 1 cultures of chick myogenic cells were exposed to the mutagenic alkylating agent ethyl methanesulfonate (EMS) for 3 d, 80% of the replicating cells were killed, but postmitotic myoblasts survived. The myoblasts fused to form unusual multinucleated "myosheets": extraordinarily wide, flattened structures that were devoid of myofibrils but displayed extensive, submembranous stress fiber-like structures (SFLS). Immunoblots of the myosheets indicated that the carcinogen blocked the synthesis and accumulation of the myofibrillar myosin isoforms but not that of the cytoplasmic myosin isoform.When removed from EMS, widely spaced nascent myofibrils gradually emerged in the myosheets after 3 d. Striking co-localization of fluorescent reagents that stained SFLS and those that specifically stained myofibrils was observed for the next 2 d, By both immunofluorescence and electron microscopy, individual nascent myofibrils appeared to be part of, or juxtaposed to, preexisting individual SFLS. By day 6, all SFLS had disappeared, and the definitive myofibrils were displaced from their submembranous site into the interior of the myosheet. Immunoblots from recovering myosheets demonstrated a temporal correlation between the appearance of the myofibrillar myosin isoforms and the assembly of thick filaments. The assembly of definitive myofibrils did not appear to involve desmin intermediate filaments, but a striking aggregation of sarcoplasmic reticulum elements was seen at the level of each I-Z-band. Our findings suggest that SFLS in the EMS myosheets function as early, transitory assembly sites for nascent myofibrils.EFINITIVE muscle cells selectively synthesize a complex set of contractile proteins that interact precisely to form the relatively invariant striated myofibril. Major myofibrillar components include muscle-specific isoforms such as myosin heavy and light chains, alpha-actin, tropomyosins, troponins, alpha-actinin, titin, nebulin, and M-band proteins (see articles cited in reference 52). These myofibrillar isoforms are synthesized and assembled into definitive sarcomeres arranged in tandem during the earliest stages of myofibrillogenesis in vivo and in vitro (2, 21, 33). With maturation there is an enormous increase in the numbers ofinterdigitating thick and thin filaments in each nascent myofibril, the total number of individual myofibrils, and the number of new sarcomeres, which apparently are added at the ends of the elongating myofibrils (1,14,31,33,36, 68).An intriguing aspect of myofibrillogenesis is that many of the muscle-specific isoforms correspond to a related set of proteins that are synthesized in virtually all cells, including presumptive myoblasts, chondroblasts, nerve cells, fibroblasts, and endothelial cells. In nonmuscle cells these constitutive contractile protein isoforms are not present in a stable arrangement but may interact to form stress fibers and/or microfilaments, depending upon the degree of cell spreading, motility, attachment to the substrate, and hemodynamic factors (2...

Precardiac mesoderm is specified during gastrulation in quail

“…Anti-LMM binds to A bands of myofibrils in all avian skeletal and cardiac muscle cells, but does not bind to smooth muscle or nonmuscle cells. By immunoblot anti-LMM recognizes only a 210 kD band from muscle extracts, but does not recognize proteins in nonmuscle cells or tissues (Antin et al, 1986).…”

The vinculin/sarcomeric-alpha-actinin/alpha-actin nexus in cultured cardiac myocytes

“…These bundles have been referred to as "stress fiber-like structures" or SFLS (2,21,29). However, our finding that no AJ or SAP was positive for non-s-o~-aednin prompted us to ask whether in fact cardiac cells assemble: (a) authentic stress fibers (e.g., bundles of microfilaments negative for sarcomeric, but positive for non-sarcomeric proteins, including caldesmon); (b) hybrid structures assembled from both sarcomeric and non-sarcomeric proteins; or (c) stress fiber-like structures consisting exclusively of sarcomeric proteins.…”

“…In brief, the conspicuous p-phaUoidin-positive bundles, which in cultured myocytes have been described as stress fiber-like structures (2,21,29,55), are likely to be bundles formed entirely of sarcomeric isoforms before their final remodeling into well-defined sarcomeres. When cultured cardiac cells assemble microscopically detectable F-actin bundles, they assemble o~-actin destined for myofibrils, not for authentic stress fibers.…”

Dynamics of actin and assembly of connectin (titin) during myofibrillogenesis in embryonic chick cardiac muscle cells in vitro

“…According to recent immunofluorescence studies (Dlugosz et al, 1984;Antin et al, 1986;Fiirst et al, 1989;Wang et al, 1988;Handel et al, 1989;Komiyama et al, 1990Komiyama et al, , 1992Schultheiss et al, 19901, it is generally agreed that proteins constituting I-Z-I brushes of myofibrils appear first and form nonstriated fibrils (NSFs), and that myosin is later incorporated into these preformed structures. However, controversies exist concerning the developmental relation of connectin (titin) with other sarcomeric proteins (Tokuyasu and Maher, 1987;Fiirst et al, 1989;Wang et al, 1988;Handel et al, 1989;Komiyama et al, 1990;Schultheiss et al, 1990).…”