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 governs the assembly of sarcomeres in skeletal muscle in a manner analogous to programs for viral capsid assembly, we have defined the temporal and spatial distribution of 10 muscle-specific proteins in mononucleated myoblasts as a function of the time after terminal cell division. Single cells in mitosis were identified in monolayer cultures of embryonic chicken pectoralis, followed for selected time points (0-24 h postmitosis) by video time-lapse microscopy, and then fixed for immunofluorescence 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 only 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 these 7 myofibrillar proteins appears to be stochastic, in that many myoblasts were alpha-actinin+ or zeugmatin+ but MyHC- or titin- whereas others were troponin-I+ or MyHC+ but alpha-actinin- or alpha-actin-. In 15-h-old myoblasts, these contractile proteins were organized into nonstriated myofibrils (NSMFs). In contrast to striated myofibrils (SMFs), the NSMFs exhibited variable stoichiometries of the sarcomeric proteins and these were not organized into any consistent pattern. In this phase of maturation, two other changes occurred: (1) the microtubule network was reorganized into parallel bundles, driving the myoblasts into polarized, needle-shaped cells; and (2) the sarcolemma 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). The emergence of one, or a string of approximately 2 mu long sarcomeres, was 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 great majority of myoblasts stained positively for desmin and myofibrillar proteins prior to, rather than after, fusing to form myotubes. This sequential appearance of muscle-specific proteins in vitro fully recapitulates myofibrillar assembly steps in myoblasts of the myotome and limb bud in vivo, as well as in nonmuscle cells converted to myoblasts by MyoD. We suggest that this cell-autonomous myoblast differentiation program may be blocked at different control points in immortalized myogenic cell lines.

A sarcomeric alpha-actinin truncated at the carboxyl end induces the breakdown of stress fibers in PtK2 cells and the formation of nemaline-like bodies and breakdown of myofibrils in myotubes.

In many nonmuscle cells, nonsarcomeric alpha-actinin is distributed in the dense bodies of stress fibers, adhesion plaques, and adherens junctions. In striated muscle, a sarcomeric isoform of alpha-actinin (s-alpha-actinin) is found in the Z-bands of myofibrils and subsarcolemmal adhesion plaques. To understand the role(s) of the alpha-actinin isoforms in the assembly and maintenance of such cytoskeletal structures, full-length or truncated s-alpha-actinin cDNAs were expressed in PtK2 cells and in primary skeletal myogenic cells. We found the following. (i) In transfected PtK2 cells the truncated s-alpha-actinin was rapidly incorporated into preexisting dense bodies, adhesion plaques, and adherens junctions. With time these structures collapsed, and the affected cells detached from the substrate. (ii) In myotubes the truncated s-alpha-actinin was incorporated into nascent Z-bands. Many of these progressively hypertrophied, forming nemaline-like bodies. With time the affected myofibrils fragmented, and the myotubes detached from the substrate. (iii) In both cell types the truncated s-alpha-actinin was significantly more disruptive of the cytoskeletal structures than the full-length molecule. (iv) Pools of "over-expressed" full-length or truncated protein did not self-aggregate into homogeneous, amorphous complexes; rather the exogenous proteins selectively colocalized with the same cohort of cytoskeletal proteins with which the endogenous alpha-actinin normally associates. The similarity among the hypertrophied Z-bands in transfected myotubes, the nemaline bodies in patients with nemaline myopathies, and the streaming Z-bands seen in various muscle pathologies raises the possibility that the genetically determined nemaline bodies and the pathologically induced Z-band alterations may reflect primary and/or post-translational modifications of s-alpha-actinin.

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

Experiments are described supporting the proposition that the assembly of stress fibers in non-muscle cells and the assembly of myofibrils in cardiac cells share conserved mechanisms. Double staining with a battery of labeled antibodies against membrane-associated proteins, myofibrillar proteins, and stress fiber proteins reveals the following: (a) dissociated, cultured cardiac myocytes reconstitute intercalated discs consisting of adherens junctions (AJs) and desmosomes at sites of cell-cell contact and sub-sarcolemmal adhesion plaques (SAPs) at sites of cell-substrate contact; (b) each AJ or SAP associates proximally with a striated myofibril, and conversely every striated myofibril is capped at either end by an AJ or a SAP; (C) the invariant association between a given myofibril and its SAP is especially prominent at the earliest stages of myofibrillogenesis; nascent myofibrils are capped by oppositely oriented SAPs; (d) the insertion of nascent myofibrils into AJs or into SAPs invariably involves vinculin, alpha-actin, and sarcomeric alpha-actinin (s-alpha-actinin); (e) AJs are positive for A-CAM but negative for talin and integrin; SAPs lack A-CAM but are positive for talin and integrin; (f) in cardiac cells all alpha-actinin-containing structures invariably are positive for the sarcomeric isoform, alpha-actin and related sarcomeric proteins; they lack non-s-alpha-actinin, gamma-actin, and caldesmon; (g) in fibroblasts all alpha-actinin-containing structures are positive for the non-sarcomeric isoform, gamma-actin, and related non-sarcomeric proteins, including caldesmon; and (h) myocytes differ from all other types of adherent cultured cells in that they do not assemble authentic stress fibers; instead they assemble stress fiber-like structures of linearly aligned I-Z-I-like complexes consisting exclusively of sarcomeric proteins.

Rapid degradation of newly synthesized tubulin in lithium-treated sensory neurons.

When cultured chick sensory neurons were labeled with [35S]methionine for 1 h or longer in the presence of 5-25 mM LiCl, we found a dose-dependent reduction in the level of radiolabeled tubulin, to one third of control levels, with no noticeable effect on other proteins. The magnitude of this response was identical after a 1-h or 72-h preincubation in 25 mM LiCl and returned to control values within 1 h after removal of LiCl. Short (5-min) pulse-chase experiments revealed that tubulin synthesis was not affected by Li+, but that newly synthesized tubulin was rapidly degraded, such that 50% of the labeled beta-tubulin was lost within 5 min. There was no enhanced degradation of tubulin present before exposure to Li+. Addition of LiCl at various times before and after a 10-min pulse suggested that tubulin becomes completely refractory to Li(+)-induced degradation within 10 min after translation. Although Li+ treatment resulted in a decrease in the fraction of extant tubulin present in the unassembled form, the Li(+)-induced degradation of nascent tubulin is not a consequence of shifts in assembly state, because colcemid or taxol treatment did not lead to rapid degradation of newly synthesized tubulin, and neither drug altered the response to Li+. We suggest that Li+ interferes with the correct folding of tubulin polypeptides, exposing sites, normally hidden, to the action of a protease(s).

Lithium chloride inhibits the phosphorylation of newly synthesized neurofilament protein, NF-M, in cultured chick sensory neurons.

The middle and high molecular weight members of the neurofilament triplet, NF-M and NF-H, undergo extensive posttranslational polyphosphorylation, a process requiring 24 h or more for completion. We have investigated ways of perturbing this process in intact cells and have found that phosphorylation of newly synthesized NF-M in cultured chick sensory neurons is inhibited by Li+. [35S]Methionine pulse-chase experiments were carried out with pure neuronal cultures, and the phosphorylation of newly synthesized NF-M was monitored by following the accompanying change, with chase time, in apparent size and charge of the polypeptide. Addition of LiCl to the medium inhibited this mobility shift in a dose-dependent manner over concentrations between 2 and 25 mM. Incorporation of 32P into NF-M, as well as NF-H, was also inhibited, whereas incorporation into the low molecular weight neurofilament protein, beta-tubulin, and total protein was unaffected. Protein synthesis was not altered. Exposure to 25 mM LiCl for up to 72 h was not toxic, and the inhibition of NF-M phosphorylation was completely reversible. When 25 mM Li+ was added after NF-M had become partially phosphorylated, further progression was blocked, but there was no net dephosphorylation or degradation of NF-M. Additional experiments suggest that this action of Li+ is probably not due to effects on second messenger levels or to effects on tubulin metabolism and assembly state presented in our accompanying article, but rather to interference by Li+ itself, with the phosphorylation of NF-M and NF-H by specific neurofilament kinase(s).

Slow posttranslational modification of a neurofilament protein.

The synthesis and subsequent modification of neurofilament (NF) polypeptides has been examined in pulse-chase experiments, using cultured chick spinal cord neurons. Fluorography of the [35S]methionine-labeled cytoskeletal proteins, after separation by two-dimensional gel electrophoresis, revealed that (a) the mid-size chicken NF protein, NF-M160, is synthesized as a smaller and more basic precursor, NF-M130; (b) beginning approximately 8 h after translation, NF-M130 slowly and continuously becomes larger and more acidic, attaining the size and charge of NF-M160 16 or more h later, and undergoing no further change in mobility for many days thereafter; and (c) in contrast, the low molecular weight NF protein, NF-L, is synthesized as such, and undergoes no subsequent change in apparent size or charge. Additional experiments provided evidence that the conversion of NF-M130 to NF-M160 is due, at least in part, to phosphorylation: (a) Incubation of similar cultures in 32PO4 resulted in incorporation into NF-M160 and transitional forms, but not into NF-M130. (b) An antiserum to NF-M160 was found by immunoblot analysis to bind strongly to untreated NF-M160, but poorly to phosphatase-treated NF-M160, and not at all to NF-M130. It has already been demonstrated (Bennett, G. S., S. J. Tapscott, C. DiLullo, and H. Holtzer, 1984, Brain Res., 304:291-302) that this anti-NF-M160 fails to stain the soma of motor neurons in sections of chick spinal cord, but detects an increasing gradient of immunoreactivity in the proximal axons. These results, together with the known kinetics of axoplasmic transport of NF, suggest that the mid-size chicken NF protein is synthesized as NF-M130 and is extensively modified, at least in part by phosphorylation, to become NF-M160 during transport along proximal neurites. Once maximally modified, NF-M160 undergoes no further net change during transport along distal neurites.

Transient expression of a neurofilament protein by replicating neuroepithelial cells of the embryonic chick brain.

An immunohistochemical survey was carried out on frozen sections of the early embryonic chick brain between 1 and 6 days of incubation, with antisera to the three neurofilament proteins (NF-L, NF-M, NF-H). Large numbers of replicating neuroepithelial cells were found to express one of these proteins, NF-M, generations before the existence of any postmitotic neuroblasts (Days 1-2 1/2 of incubation). NF-L and NF-H could not be detected. Not all primordial brain regions contained NF-M-positive cells, but in those that did, every cell was positive. These regions included the dorsal forebrain, optic vesicles, and dorsal hindbrain, but not the dorsal midbrain. All cells in all regions of the cephalic neural tube contained vimentin, whether or not they also contained NF-M. This NF-M expression was transient in the sense that later generations of these NF-M-positive neuroepithelial cells became NF-M negative, before finally giving rise to some descendents that ultimately express all three NF proteins. This transient NF-M expression was found in certain other cells of early embryos, including cardiac myoblasts. The identity of the component in these early neural and nonneural tissues, that bound the antibody, was demonstrated to be identical to adult brain NF-M by one- and two-dimensional immunoblots. These findings demonstrate an unusual kind of biochemical heterogeneity among neuroepithelial cells, and they are relevant to considerations regarding lineage analysis and lineage "markers" in the vertebrate central nervous system.

Expression of a neurofilament protein by the precursors of a subpopulation of ventral spinal cord neurons.

The expression of neurofilament proteins (NF-H, NF-M, and NF-L) in replicating neuroepithelial cells and postmitotic neuroblasts in the embryonic chick trunk neural tube was examined by immunohistochemistry. Anti-NF-M, in particular, resulted in bright staining of some mitotic cells, which were found to be strictly localized to a midventral and an extreme dorsal position in the neural tube. Those in the midventral position were observed with greatest frequency during Days 3 and 4 of incubation and became increasingly rare thereafter. During the same period of time, and in the same small ventral region, NF-M-positive interphase cells, presumably migrating postmitotic neuroblasts, were also present. In contrast, NF-L-positive mitotic cells were rarely seen. NF-L-positive migrating and differentiating neuroblasts were observed throughout the ventral half of the neural tube except in the midventral area containing NF-M-positive mitotic cells and NF-M-positive migrating neuroblasts. These results, together with known temporal and spatial patterns of neurogenesis in the spinal cord, suggest that the expression of NF-L and NF-M, in the form recognized by our antibodies, may not be initiated coordinately, or even in the same sequence, in different types of neuroblasts, and that only the immediate precursors of a specific subpopulation of ventral spinal cord neurons begin expressing NF-M in the terminal cell cycle. In addition, the NF-M-positive mitotic cells, when observed in anaphase and telophase, had NF-M-positive material associated with both emerging daughter cells and the migrating neuroblasts were frequently found in closely associated pairs, consistent with the suggestion that these precursor cells undergo a symmetrical terminal division to yield two daughter postmitotic neuroblasts.

Differential binding of antibodies against the neurofilament triplet proteins in different avian neurons.

Using monospecific antisera against each of the three chicken neurofilament (NF) proteins, NF70, NF160 and NF180, the distribution of each of these proteins in several types of neurons was examined by immunohistochemistry. Striking differences were observed in the relative staining by the three antibodies when the soma of different types of neurons were compared, and also when the soma of some neurons were compared with their axons. Both the soma and axons of dorsal root sensory neurons were brightly stained by each of the antisera. The soma of spinal cord ventral horn neurons, however, were stained only by A-NF70 and A-NF180, not by A-NF160. The axons of these neurons were uniformly stained by A-NF70 and A-NF180, while only gradually becoming NF160-positive over the first several hundred microns. The lack of staining by A-NF160 was also observed in many neuronal soma in cultures of dissociated spinal cord cells. The soma and dendrites of adult cerebellar Purkinje cells were weakly stained by A-NF70 and A-NF180 and not at all by A-NF160, but both A-NF70 and A-NF180 yielded prominent staining of immature Purkinje cells and dendrites. These results suggest that the three NF proteins may be unequally distributed within the soma and processes of different types of neurons and/or may be subject to regionally selective modification.