Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin.

Abstract

Abstract. Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were 1.9-2.4 um long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 ~m apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at ~day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 ~m sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 #m. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 #m sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.M YOFIBRIL formation in embryonic vertebrate skeletal and cardiac cells has been examined in a number of different ways. Biochemical data have shown there is coordinate synthesis of actin, myosin, tropomyosin, and alpha-actinin during vertebrate myogenesis (6). Immunocytochemical and electron microscopic data suggest that thick and thin filaments are formed at the same time in the cytoplasm and subsequently become organized into sarcomeric arrays (13, 18). The assumption in these studies (13,18) is that the filaments are the s...

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“…A few similar observations have been made for other cytoskeletal proteins, including actin (Eppenberger-Eberhardt et al, 1990) and MHC isoforms (Eppenberger et al, 1988). Interestingly, attempts to reproduce the sorting of endogenous proteins by introducing labeled proteins, e.g., a-actinins of different sources, into cultured cells (McKenna et al, 1985;Sanger et al, 1986) have hitherto failed. Success was reported, however, by Schafer and Perriard (1988), who showed by microinjecting synthetic RNAs coding for two isoforms of creatine kinase (B-CK and M-CK) and chimeras thereof that the M-CK targeting to the myofibrillar M line appeared to be directed by isoform-specific characteristics embedded mainly in the C-terminal half of the M-CK isoprotein structure.…”

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