Coordinate regulation of contractile protein synthesis during myoblast differentiation

The synthesis of contractile proteins has been studied during the differentiation of quail skeletal muscle myoblasts in culture. Myoblast differentiation was synchronized by transferring secondary cultures of rapidly dividing myoblasts into medium lacking cell division-promoting factors. Cultures at various stages of differentiation were then pulse-labeled with 35S-methionine, and cell extracts were resolved by electrophoresis on two-dimensional gels. Incorporation into specific proteins was quantitated by autoradiography and fluorography using a scanning densitometer. Contractile proteins synthesized by muscle cultures were identified by their co-electrophoresis on two-dimensional gels with contracile proteins purified from quail breast muscle. Our results show that the synthesis of myosin heavy chain, two myosin light chains, two subunits of troponin and two subunits of tropomyosin is first detected at the time of myoblast fusion and then rapidly increase at least 500 fold to maximum rates which remain constant in muscle fibers. Both the kinetics of activation and the molar rates of synthesis of these contractile proteins are virtually identical. Muscle-specific actin (alpha) synthesis also increases at the time of myoblast fusion, but this actin (alpha) is synthesized at 3 times the rate of other contractile proteins. The synthesis of 30 other muscle cell proteins was quantitated, and most of these are shown to follow different patterns of regulation. From these results, we conclude that the contractile proteins are regulated coordinately during myoblast differentiation.     

Post-transcriptional regulation of ribosome accumulation during myoblast differentiation

The synthesis, accumulation and stability of rRNA were examined in embryonic quail myoblasts differentiating in cell culture. Quail myoblasts initially divide rapidly in culture, and accumulate 28S and 18S rRNA and ribosomes at a rate which maintains a constant ribosome content during cell division. After these myoblasts fuse, cell division ceases and ribosomes accumulate in fibers, but at a reduced rate which is only one fourth that in dividing myoblasts. Measurements of rRNA stability by 3H-methyl-methionine pulse-chase analysis show that 28S and 18S rRNA formed by fibers turn over with half-lives of 45 hr, and rRNA formed by myoblasts remains stable until fusion and then also turns over in fibers. Turnover of rRNA in fibers accounts for only half the reduction in ribosome accumulation following myoblast fusion. Measurements of the incorporation of 3H-adenosine into rRNA and ATP pools show that the rates of synthesis of rRNA precursor do not decrease after myoblast fuse, but half the rRNA molecules synthesized by fibers are degraded during processing. Degradation of rRNA during processing reduces the rate of formation of 28S and 18S rRNA, and together with rRNA turnover quantitatively accounts for the reduced rate of ribosome accumulation in fibers.     

Chromatin protein kinases and phosphoproteins during myoblast growth and differentiation

A photolabile derivative of α-bungarotoxin which binds specifically to $\text{Torpedo}\text{californica}$ acetylcholine receptor has been used to investigate the topography of the membrane associated protein. It is shown that the toxin can be crosslinked to a polypeptide of 40,000 daltons, to which it is known to bind, and in addition to another polypeptide of 65,000 daltons which is a major constituent of the membrane. The results substantiate the notion that this nicotinic acetylcholine receptor is composed of different polypeptides and that some of these interact with each other or are in close proximity on the exterior surface of the post-synaptic membrane.     

Stability of ribosomal protein mRNA and translational feedback regulation in Escherichia coli

Summary It was previously observed that the stability of ribosomal protein (r-protein) mRNA in Escherichia coli decreases under the conditions where its translation is feedback inhibited by repressor r-protein. We have now demonstrated that the stability of mRNA for r-proteins S13, S11 and S4 increases in a strain carrying a mutation in the gene for S4, a translational repressor regulating these r-proteins. The results confirm the previous observations that translational repression increases the decay rate of r-protein mRNA, and in addition, show that the half-life of S13-S4 r-protein mRNA in cells growing under ordinary conditions is significantly shorter than its inherent stability would predict, due to the operation of translational feedback regulation.     

Coordinate accumulation of contractile protein mRNAs during myoblast differentiation.

The synthesis of contractile protein mRNAs has been studied during the differentiation of quail skeletal muscle myoblasts in culture. Eight contractile protein mRNAs were identified by translation of total cellular RNA isolated from differentiated myofibers in wheat germ and reticulocyte lysates. Products of the translation systems were fractionated by two-dimensional gel electrophoresis, and incorporation of [³⁵S]methionine into individual contractile proteins was quantitated by computerized densitometry of autoradiograms. These translation assay systems were used to quantitate levels of contractile protein mRNAs in cultures of myoblasts undergoing highly synchronous differentiation. Our results show that dividing myoblasts contain very little, if any, translatable contractile protein mRNA. The mRNAs coding for myosin heavy chain, the musclespecific actin, three myosin light chains, two tropomyosin subunits, and one troponin subunit begin to coordinately accumulate at fusion, when contractile protein synthesis is activated. Their levels increase 50- to 200-fold during the next 30 hr, paralleling increases in the rates of contractile protein synthesis. These results indicate that the contractile protein mRNAs accumulate coordinately during myoblast differentiation and that contractile protein synthesis is regulated by changes in the levels of these mRNAs.     

Down regulation of ribosomal protein mRNAs during neuronal differentiation of human NTERA2 cells

We have analysed the expression of 32 ribosomal protein (RP) mRNAs during retinoic acid induced neuronal differentiation of human NTERA2 cells. Except for a new S27 variant (S27v), all were down regulated both in selectively replated differentiated neurons and the most differentiated continuous cultures, i.e., non-replated cultures. However, the expression profiles of the individual RP mRNAs were different, most (L3, L7, L8, L10, L13, L23a, L27a, L36a, L39, P0, S2, S3, S3a, S4X, S6, S9, S12, S13, S16, S19, S20, S23, and S27a) exhibited a constant down regulation, whereas a few were either initially constant (L11, L32, S8, and S11) or up regulated (L6, L15, L17, L31, and S27y) and then down regulated. The expression of S27v remained elevated in the most differentiated continuous cultures but was down regulated in replated differentiated neurons. The down regulation of RP mRNAs was variable: the expression levels in differentiated replated neurons were between 10% (S3) and 90% (S11) of the levels in undifferentiated cells. The ratio between rRNA and RP mRNA changed during the differentiation; in differentiated neurons there were, on average, about half the number of RP mRNAs per rRNA as compared to undifferentiated cells. The expression profiles of a few translation-related proteins were also determined. EF1alpha1, EF1beta1, and EF1delta were down regulated, whereas the expression of the neuron and muscle specific EF1alpha2 increased. The reduction in the expression of RP mRNAs was coordinated with a reduction in the expression level of the proliferation marker PCNA. The expression levels of most RP mRNAs were lower in purified differentiated post-mitotic neurons than in the most differentiated continuous cultures, despite similar levels of PCNA, suggesting that both the differentiation state and the proliferative status of the cells affect the expression of RP mRNAs.