SEQUENTIAL APPEARANCE OF MUSCLE-SPECIFIC PROTEINS IN MYOBLASTS AS A FUNCTION OF TIME AFTER CELL-DIVISION - EVIDENCE FOR A CONSERVED MYOBLAST DIFFERENTIATION PROGRAM IN SKELETAL-MUSCLE

Based on the assumption that a conserved differentiation program gover ns the assembly of sarcomeres in skeletal muscle in a manner analogous to programs for viral capsid assembly, we have defined the temporal a nd spatial distribution of 10 muscle-specific proteins in mononucleate d myoblasts as a function of the time after terminal cell division. Si ngle cells in mitosis were identified in monolayer cultures of embryon ic chicken pectoralis, followed for selected time points (0-24 h h pos tmitosis) by video time-lapse microscopy, and then fixed for immunoflu orescence staining. For convenience, the myoblasts were termed x-h-old to define their age relative to their mitotic ''birthdate.'' All 6 h myoblasts that emerged in a mitogen-rich medium were desmin(+) but onl y 50% were positive for a alpha-actin, troponin-I, alpha-actinin, MyHC , zeugmatin, titin, or nebulin. By 15 h postmitosis, approximately 80% were positive for all of the above proteins. The up-regulation of the se 7 myofibrillar proteins appears to be stochastic, in that many myob lasts were alpha-actinin(+) or zeugmatin(+) but MyHC(-) or titin(-) wh ereas others were troponin-I+ or MyHC(+) but alpha-actinin(-) or alpha -actin(-). In 15-h-old myoblasts, these contractile proteins were orga nized into nonstriated myofibrils (NSMFs). In contrast to striated myo fibrils (SMFs), the NSMFs exhibited variable stoichiometries of the sa rcomeric proteins and these were not organized into any consistent pat tern. In this phase of maturation, two other changes occurred: (1) the microtubule network was reorganized into parallel bundles, driving th e myoblasts into polarized, needle-shaped cells; and (2) the sarcolemm a became fusion-competent. A transition from NSMFs to SMFs took place between 15 and 24 h (or later) postmitosis and was correlated with the late appearance of myomesin, and particularly, MyBP-C (C protein). Th e emergence of one, or a string of similar to 2 mu long sarcomeres, wa s invariably characterized by the localization of myomesin and MyBP-C to their mature positions in the developing A-bands. The latter group of A-band proteins may be rate-limiting in the assembly program. The g reat majority of myoblasts stained positively for desmin and myofibril lar proteins prior to, rather than after, fusing to form myotubes. Thi s sequential appearance of muscle-specific proteins in vitro fully rec apitulates myofibrillar assembly steps in myoblasts of the myotome and limb bud in vivo, as well as in nonmuscle cells converted to myoblast s by MyoD. We suggest that this cell-autonomous myoblast differentiati on program may be blocked at different control points in immortalized myogenic cell lines. (C) 1994 Wiley-Liss, Inc.