MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes ( ABSTRACTShortly after their birth, postmitotic mononucleated myoblasts in myotomes, limb buds, and conventional muscle cultures elongate and assemble a cohort of myofibrillar proteins into definitively striated myofibrils. MyoD induces a number of immortalized and/or transformed nonmuscle cells to express desmin and several myofibrillar proteins and to fuse into myosacs. We now report that MyoD converts normal dermal fibroblasts, chondroblasts, gizzard smooth muscle, and pigmented retinal epithelial cells into elongated postmitotic mononucleated striated myoblasts. The sarcomeric localization of antibodies to desmin, a-actinin, titin, troponin-I, a-actin, myosin heavy chain, and myomesin in these converted myoblasts are indistinguishable from in vivo and in vitro normal myoblasts. Converted myoblasts fuse into typical anisodiametric multinucleated myotubes that often contract spontaneously. Conversion and subsequent expression of the skeletal myogenic program are autonomous events, occurring in four nonmuscle microenvironments consisting of different combinations of foreign extracellular matrix molecules. Early events associated with conversion by MyoD involve (i) withdrawal from the cell cycle, (ii) down-regulation of the subverted cell's ongoing differentiation program, and (iii) initiation of desmin synthesis in presumptive myoblasts and dramatic redistribution of microtubules and desmin intermediate filaments in postmitotic myoblasts.

A new class of filaments intermediate in diameter between actin and myosin filaments has been demonstrated in skeletal muscle cells cultured from chick embryos. These filaments, which account for the majority of free filaments, average 100 A in diameter. They may run for more than 2 in a single section and can be distinguished in size and appearance from the thick and thin filaments assembled into myofibrils. The 100-A filaments are seen scattered throughout the sarcoplasm at all stages of development and show no obvious association with the myofibrils. The 100-A filaments are particularly conspicuous in myotubes fragmented by the mitotic inhibitors, colchicine and Colcemid. In addition, filaments similar in size and appearance to those found in myotubes are present in fibroblasts, chondrocytes, and proliferating mononucleated myoblasts. The 100-A filaments are present in cells arrested in metaphase by mitotic inhibitors. Definitive thick (about 150 A) or thin (about 60 A) myofilaments are not found in skeletal myogenic cells arrested in metaphase. Myogenic cells arrested in metaphase do not bind fluorescein-labeled antibody directed against myosin or actin. For these reasons, it is concluded that not all "thin" filaments in myogenic cells are uniquely associated with myogenesis.

The parasitic protozoan Toxoplasma gondii has been examined with the electron microscope in order to study the fine structure and the formation of the membranes surrounding the cell . The study of the ultrastructure of the membranes covering the parasite shows the existence of a three-membraned complex . Only the outer membrane is considered to be the plasma membrane ; the two membranes below it form an inseparable whole of changeable molecular architecture (modifications in appearance depending on the methods of fixation, local differentiation) . During reproduction, which takes place by fission or more often by endogeny, the membranes of the daughter individuals are formed from the membranes of the parent . At first the middle and inner membranes of the parent extend, separating the cytoplasm of the daughter cells from that of the parent . The three-membrane complex of the endozoites is completed at the time of their liberation ; the external membrane of the parent covers the leaving endozoites ; thus, the plasma membrane of the daughter cells derives also from that of the parent . These findings on the origin and role of limiting membranes during reproduction differ entirely from those described so far for other cells .

The topographical relationship between stress fiber-like structures (SFLS) and nascent myofibrils was examined in cultured chick cardiac myocytes by immunofluorescence microscopy. Antibodies against muscle- specific light meromyosin (anti-LMM) and desmin were used to distinguish cardiac myocytes from fibroblastic cells. By various combinations of staining with rhodamine-labeled phalloidin, anti-LMM, and antibodies against chick brain myosin and smooth muscle alpha- actinin, we observed the following relationships between transitory SFLS and nascent and mature myofibrils: (a) more SFLS were present in immature than mature myocytes; (b) in immature myocytes a single fluorescent fiber would stain as a SFLS distally and as a striated myofibril proximally, towards the center of the cell; (c) in regions of a myocyte not yet penetrated by the elongating myofibrils, SFLS were abundant; and (d) in regions of a myocyte with numerous mature myofibrils, SFLS had totally disappeared. Spontaneously contracting striated myofibrils with definitive Z-band regions were present long before anti-desmin localized in the I-Z-band region and long before morphologically recognizable structures periodically link Z-bands to the sarcolemma. These results suggest a transient one-on-one relationship between individual SFLS and newly emerging individual nascent myofibrils. Based on these and other relevant data, a complex, multistage molecular model is presented for myofibrillar assembly and maturation. Lastly, it is of considerable theoretical interest to note that mature cardiac myocytes, like mature skeletal myotubes, lack readily detectable stress fibers.

The distribution of intermediate filament (IF) subunits during maturation of skeletal myotubes in vitro was examined by immunofluorescence, using antibodies against two different types of chick IF subunits: (a) 58-kdalton subunits of fibroblasts (anti-58K), and (b) 55-kdalton subunits of smooth muscle (anti-55K) . Anti-58K bound to a filament network in replicating presumptive myoblasts and fibroblasts, as well as in immature myotubes. The distribution in immature myotubes was in longitudinal filaments throughout the cytoplasm . With maturation, staining of myotubes by anti-58K diminished and eventually disappeared . Anti-55K selectively stained myotubes, and the fluorescence localization underwent a drastic change in distribution with maturation-from dense, longitudinal filaments in immature myotubes to a cross-striated distribution in mature myotubes that was associated with the I-Z region of myofibrils . However, the emergence of a cross-striated anti-55K pattern did not coincide temporally with the emergence of striated myofibrils, but occurred over a period of days thereafter. KEY WORDS intermediate filaments myogenesis " muscle differentiation immunofluorescenceReports from this laboratory and from others have demonstrated that the intermediate or 100-A filament (IF) subunits from different cell types and organisms are not all identical, and can be distinguished immunologically and biochemically (I,2,9, 11a, IIb,21,29) . We demonstrated that antibodies against the 58-kdalton chick fibroblast IF subunit (anti-58K) stained filaments in many different kinds of cultured chick cells, including fibroblasts, chondroblasts, melanocytes, neurons, glia, cardiac and smooth muscle myoblasts, and both replicating presumptive skeletal myoblasts and postmitotic myoblasts and myotubes. Contrastingly, antibodies against the 55-kdalton chick smooth muscle IF subunit (anti-55K) were highly selective and stained only definitive smooth, cardiac, and skeletal muscle cells. Replicating pre-J . CELL BIOLOGY