Activation of myosin synthesis in fusing and mononucleated myoblasts

The synthesis of the heavy chain subunit of myosin has been studied in breast muscle myoblasts from embryos of the Japanese quail, Coturnix coturnix japonica, during differentiation of these cells in culture. Specifically, these experiments were done to examine the roles of myoblast fusion and the regulation of myoblast cell division in the control of myosin heavy chain synthesis.The rates of myosin heavy chain synthesis have been quantitated in cultures of fusing myoblasts by measurement of the incorporation of radioactive leucine and valine precursors into myosin heavy chain, and simultaneous determination of the intracellular specific activities of these radioactive amino acids. These measurements demonstrate that, prior to fusion, dividing myoblasts synthesize little, if any, myosin heavy chain, but that during the period of myoblast fusion, myosin heavy chain synthesis becomes activated at least 50 to 100-fold. Myosin heavy chain synthesis was also measured in mononucleated myoblasts inhibited from fusing by the presence of EGTA in the culture medium. These experiments demonstrate that myosin synthesis can be activated in mononucleated myoblasts to reach rates similar to those attained in fused myoblasts. This activation occurs under conditions in which EGTA-inhibited myoblasts were induced to withdraw from the cell division cycle by reducing the concentrations of the serum and embryo extract components of the culture medium or by prior “conditioning” of standard growth medium.These experiments, therefore, establish that the activation of myosin synthesis in breast muscle myoblasts does not require fusion, but indicate that activation is co-ordinated with the withdrawal of myoblasts from the cell division cycle.     

Structural basis for the interaction of the myosin light chain Mlc1p with the myosin V Myo2p IQ motifs

Calmodulin, regulatory, and essential myosin light chain are evolutionary conserved proteins that, by binding to IQ motifs of target proteins, regulate essential intracellular processes among which are efficiency of secretory vesicles release at synapsis, intracellular signaling, and regulation of cell division. The yeast Saccharomyces cerevisiae calmodulin Cmd1 and the essential myosin light chain Mlc1p share the ability to interact with the class V myosin Myo2p and Myo4 and the class II myosin Myo1p. These myosins are required for vesicle, organelle, and mRNA transport, spindle orientation, and cytokinesis. We have used the budding yeast model system to study how calmodulin and essential myosin light chain selectively regulate class V myosin function. NMR structural analysis of uncomplexed Mlc1p and interaction studies with the first three IQ motifs of Myo2p show that the structural similarities between Mlc1p and the other members of the EF-hand superfamily of calmodulin-like proteins are mainly restricted to the C-lobe of these proteins. The N-lobe of Mlc1p presents a significantly compact and stable structure that is maintained both in the free and complexed states. The Mlc1p N-lobe interacts with the IQ motif in a manner that is regulated both by the IQ motifs sequence as well as by light chain structural features. These characteristic allows a distinctive interaction of Mlc1p with the first IQ motif of Myo2p when compared with calmodulin. This finding gives us a novel view of how calmodulin and essential light chain, through a differential binding to IQ1 of class V myosin motor, regulate this activity during vegetative growth and cytokinesis.     

A monoclonal antibody to the embryonic myosin heavy chain of rat skeletal muscle

A monoclonal antibody, 2B6, has been prepared against the embryonic myosin heavy chain of rat skeletal muscle. On solid phase radioimmunoassay, 2B6 shows specificity to myosin isozymes known to contain the embryonic myosin heavy chain and on immunoblots of denatured contractile proteins and on competitive radioimmunoassay, it reacts only with the myosin heavy chain of embryonic myosin and not with the myosin heavy chain of neonatal or adult fast and slow myosin isozymes or with other contractile or noncontractile proteins. This specificity is maintained with cat, dog, guinea pig, and human myosins, but not with chicken myosins. 2B6 was used to define which isozymes in the developing animal contained the embryonic myosin heavy chain and to characterize the changes in embryonic myosin heavy chain in fast versus slow muscles during development. Finally, 2B6 was used to demonstrate that thyroid hormone hastens the disappearance of embryonic myosin heavy chain during development, while hypothyroidism retards its decrease. This confirmed our previous conclusion that thyroid hormones orchestrate changes in isozymes during development.     

Myofibrillar protein turnover. Synthesis of protein-bound 3-methylhistidine, actin, myosin heavy chain and aldolase in rat skeletal muscle in the fed and starved states.

The turnover of 3-methylhistidine (N tau-methylhistidine) and in some cases actin, myosin heavy chain and aldolase in skeletal muscle was measured in a number of experiments in growing and adult rats in the fed and overnight-starved states. In growing fed rats in three separate experiments, measurements of the methylation rate of protein-bound 3-methylhistidine by either [14C]- or [3H]-methyl-labelled S-adenosylmethionine show that 3-methylhistidine synthesis is slower than the overall rate of protein synthesis indicated by [14C]tyrosine incorporation. Values ranged from 36 to 51%. However, in one experiment with rapidly growing young fed rats, acute measurements over 1 h showed that 3-methylhistidine synthesis could be increased to the same rate as the overall rate. After overnight starvation in these rats, the steady-state synthesis rate of 3-methylhistidine was 38.8% of the overall rate. This was a similar value to that in adult non-growing rats, in which measurements of the relative labelling of 3-methylhistidine and histidine after a single injection of [14C]histidine indicated that 3-methylhistidine synthesis was 37% of the overall rate in the fed or overnight-starved state. According to measurements of actin, myosin heavy-chain and aldolase synthesis in the over-night-starved state with young rats, with a variety of precursors, slow turnover of 3-methylhistidine results from the specific slow turnover of actin, since turnover rates of myosin heavy chain, mixed protein and aldolase were 2.5, 3 and 3.4 times faster respectively. However, in the fed state synthesis rates of actin were increased disproportionately to give similar rates for all proteins. These results show that (a) 3-methylhistidine turnover in muscle is less than half the overall rate in both young and adult rats, (b) slow 3-methylhistidine turnover reflects the specifically slow turnover of actin compared with myosin heavy chain and other muscle proteins, and (c) during growth the synthesis rate of actin is particularly sensitive to the nutritional state and can be increased to a similar rate to that of other proteins.     

Metabolism of myosin heavy chain in steady-state chick skeletal muscle cultures.

Synthesis, accumulation and breakdown of the 200000-mol.wt. heavy subunit of myosin were analysed over an 11 day period in muscle cell cultures isolated from the leg muscle of 12-day chick embryos. Muscle cells accumulated myosin heavy chain rapidly from days 2 to 5 and maintained a maximum, constant myosin-heavy-chain concentration between days 7 and 11. Myosin-heavy-chain content and breakdown rate were compared in steady-state muscle cultures grown either in the presence of an optimum batch of horse serum (control) or in the presence of horse serum that had been pre-selected for its ability to inhibit several-fold the rate of synthesis of myosin heavy chain (inhibitory). The quantity of myosin heavy chain in the inhibited cultures was decreased in direct proportion to the decrease in the rate of synthesis of myosin heavy chain; however, the half-lives of myosin heavy chain (control, 17.7h; inhibitory, 17.0h) were virtually identical. In contrast, the absolute rate of breakdown of myosin heavy chain, expressed as molecules/min per nucleus, was approx. 5-fold lower in the inhibited cultures (4.3 X 10(3) molecules/min per nucleus) than in the control cultures (21.7 X 10(3) molecules/min per nucleus). Thus, inhibition of myosin-heavy-chain synthesis in this case was accompanied by diminished myosin-heavy-chain concentration and absolute breakdown rate at the altered steady state, but relative myosin-heavy-chain breakdown rates were unchanged.     

Evidence for regulation of lamellipodial and tail contraction of glycerinated chicken embryonic fibroblasts by myosin light chain kinase

Permeabilized cell models of muscle and nonmuscle cells have proven useful for examining the regulation of actin, myosin, and other cytoskeletal proteins during cell contraction. Upon addition of Ca2+ and ATP, glycerinated chick embryonic skin fibroblasts retract their tails and lamellipodia. Ca2+-independent contractions are obtained by preincubation of cell models in Ca2+ ATP gamma S, followed by EGTA and ATP addition, or by addition of trypsin-treated myosin light chain kinase that no longer requires Ca2+ for reactivation. By pretreating cells before glycerination with colchicine, it is possible to study lamellipodial contraction independent of tail contraction. Similar responses to ATP gamma S pretreatment and unregulated myosin light chain kinase are observed in cells that only contain lamellipodia. SDS-PAGE electrophoresis of glycerinated fibroblasts incubated in ATP gamma 35S and Ca2+ shows that only two major proteins are thiophosphorylated, and that one of them, a band that comigrates with the 20K MW light chain of myosin, is thiophosphorylated in a Ca2+-dependent manner. Since the rate of tail contraction is several-fold faster after Ca2+ and ATP gamma S pretreatment or incubation in excess myosin light chain kinase, myosin light chain phosphorylation may be a rate-limiting step during contraction.     

Messenger RNA affinity column fractionation of eukaryotic initiation factor and the translation of myosin messenger RNA.

Both myosin mRNA (26 S) and globin mRNA (9 S) have been bound to activated Sepharose 4B. The affinity of initiation factors derived from native 40 S ribosomal subunits from embryonic chick muscle for these messengers has been determined. Although both messengers bind the major components of the muscle factor preparation with the same affinity, some differences are noted in the minor components. There is an enrichment of components which bind myosin mRNA with a high affinity when the 15–18 S initiation factor complex is prepared from initiating 40 S ribosomal subunits found on myosin synthesizing polysomes rather than from total cellular factor preparations. The proteins which have a high binding affinity to myosin mRNA also have a discriminating effect when added to a wheat germ system containing myosin and globin mRNA. This is demonstrated by the fact that the synthesis of myosin heavy chain is specifically stimulated and the number of ribosomes found on myosin mRNA increase five to seven-fold; whereas neither the synthesis of globin nor the number of ribosomes associated with globin mRNA is increased. The components of an impure reticulocyte eukaryotic initiation factor 3 prepared in a similar manner as the muscle factor, do not bind myosin mRNA with the same high affinity, and these fractions separated on the myosin mRNA affinity column did not show a discriminatory effect. These results suggest that specific components of muscle 15–18 S initiation factor preparations have a higher binding affinity for myosin mRNA than globin mRNA and that these proteins may be those factors previously reported to be present which discriminate between mRNAs.     

Structural and phylogenetic analysis of the chicken ventricular myosin heavy chain rod

Summary We have isolated and characterized five overlapping clones that encompass 3.2 kb and encode a part of the short subfragment 2, the hinge, and the light meromyosin regions of the myosin heavy chain rod as well as 143 bp of the 3 untranslated portion of the mRNA. Northern blot analysis showed expression of this mRNA mainly in ventricular muscle of the adult chicken heart, with trace levels detected in the atrium. Transient expression was seen in skeletal muscle during development and in regenerating skeletal muscle following freeze injury. To our knowledge, this is the first report of an avian ventricular myosin heavy chain sequence. Phylogenetic analysis indicated that this isoform is a distant homolog of other ventricular and skeletal muscle myosin heavy chains and represents a distinct member of the multigene family of sarcomeric myosin heavy chains. The ventricular myosin heavy chain of the chicken is either paralogous to its counterpart in other vertebrates or has diverged at a significantly higher rate.Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, IL60637, USA     

Developmental consequences of the lack of myosin heavy chain in Dictyostelium discoideum

Two different Dictyostelium discoideum cell lines that lack myosin heavy chain protein (MHC A) have been previously described. One cell line (mhcA) was created by antisense RNA inactivation of the endogenous mRNA and the other (HMM) by insertional mutagenesis of the endogenous myosin gene. The two cell lines show similar developmental defects; they are delayed in aggregation and become arrested at the mound stage. However, when cells that lack myosin heavy chain are mixed with wild-type cells, some of the mutant cells are capable of completing development to form mature spores. The pattern of expression of a number of developmentally regulated genes has been examined in both mutant cell lines. Although morphogenesis becomes aberrant before aggregation is completed, all of the markers that we have examined are expressed normally. These include genes expressed prior to aggregation as well as prespore genes expressed later in development. It appears that the signals necessary for cell-type differentiation are generated in the aborted structures formed by cells lacking MHC A. The mhcA cells have negligible amounts of MHC A protein while the HMM cells express normal amounts of a fragment of the myosin heavy chain protein similar to heavy meromyosin (HMM). The expression of myosin light chain was examined in these two cell lines. HMM cells accumulate normal amounts of the 18,000-D light chain, while the amount of light chain in mhcA cells is dramatically reduced. It is likely that the light chains assemble normally with the HMM fragment in HMM cells, while in cells lacking myosin heavy chain (mhcA) the light chains are unstable.     

Regulation of muscle gene expression: The accumulation of messenger RNAs coding for muscle-specific proteins during myogenesis in a mouse cell line

The expression of RNA sequences coding for myofibrillar proteins has been followed during terminal differentiation in a mouse skeletal muscle cell line. Cloned complementary DNA probes hybridizing with the actins, skeletal muscle α-actin, myosin heavy chain and the myosin alkali light chains were employed in Northern blotting experiments with total cellular poly (A)-containing RNA extracted from the cultures at different times after plating. At the same times, parallel cultures were pulse-labelled with [³⁵S]methionine and the pattern of newly synthesized proteins was analysed by two-dimensional gel electrophoresis. Synthesis of skeletal muscle α-actin and of the myosin alkali light chains (LC_lemb, LC₁, LC₃) was not detectable in dividing myoblast cultures. From the onset of cell fusion, the synthesis of myosin heavy chain, LC_lemb and α-actin increases with similar kinetics. Synthesis of LC₃ (and trace amounts of LC_1F) is detectable and subsequently increases at later stages of myotube formation. The corresponding messenger RNAs coding for myosin heavy chain and skeletal muscle α-actin are first detectable immediately before the initiation of myofibrillar protein synthesis. mRNAs coding for the non-muscle actins are accumulated in myoblasts and diminish after cell fusion. Comparisons between muscle mRNAs depend on the relative sensitivities of the different probes, reflecting mainly their homology with the isoform of the actin or myosin multigene family expressed. Quantitative analysis of Northern blots gives an estimated increase in skeletal muscle α-actin mRNA, with an homologous probe, of at least 130-fold with a minimum level of detection of 40 to 80 molecules per cell. Accumulation of this species and of the myosin heavy chain mRNA follows similar kinetics. mRNA coding for LC₃, the principal myosin light chain detected with the probe, appears to accumulate to a lesser extent initially, paralleling synthesis of the corresponding protein. These results using cloned probes demonstrate a close temporal correlation between muscle mRNA accumulation and protein synthesis during terminal myogenesis in this muscle line.     

Changes in myosin and myosin light chain kinase during myogenesis

Myosins and myosin light chain kinases have been isolated from a cloned line of myoblasts (L5/A10) as this cell line undergoes differentiation toward adult muscle. At least three myosin isozymes were obtained during this developmental process. Initially a nonmuscle type of myosin was found in the myoblasts. The molecular weights of the myoblast light chains were 20 000 and 15 000. Myosin isolated from early myotubes had light chains with molecular weights of 20 000 and 19 500. Myosin isolated from myotubes which contained sarcomeres had light chains with molecular weights of 23 000, 18 500, and 16 000. This last myosin was similar in light chain complement to adult rat thigh muscle. Two forms of the myosin light chain kinase activity were detected: a calcium-independent kinase in the myoblasts and a calcium-dependent kinase in the myotubes with sarcomeres. No myosin light chain kinase activity was detected in the early myotubes.     

Novel myosin heavy chain kinase involved in disassembly of myosin II filaments and efficient cleavage in mitotic dictyostelium cells

We have cloned a full-length cDNA encoding a novel myosin II heavy chain kinase (mhckC) from Dictyostelium. Like other members of the myosin heavy chain kinase family, the mhckC gene product, MHCK C, has a kinase domain in its N-terminal half and six WD repeats in the C-terminal half. GFP-MHCK C fusion protein localized to the cortex of interphase cells, to the cleavage furrow of mitotic cells, and to the posterior of migrating cells. These distributions of GFP-MHCK C always corresponded with that of myosin II filaments and were not observed in myosin II-null cells, where GFP-MHCK C was diffusely distributed in the cytoplasm. Thus, localization of MHCK C seems to be myosin II-dependent. Cells lacking the mhckC gene exhibited excessive aggregation of myosin II filaments in the cleavage furrows and in the posteriors of the daughter cells once cleavage was complete. The cleavage process of these cells took longer than that of wild-type cells. Taken together, these findings suggest MHCK C drives the disassembly of myosin II filaments for efficient cytokinesis and recycling of myosin II that occurs during cytokinesis.     

Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain

Dictyostelium amebae have been engineered by homologous recombination of a truncated copy of the myosin heavy chain gene (heavy meromyosin (HMM) cells) and by transformation with a vector encoding an antisense RNA to myosin heavy chain mRNA (mhcA cells) so that they lack native myosin heavy chain protein. In the former case, cells synthesize only the heavy meromyosin portion of the protein and in the latter case they synthesize negligible amounts of the protein. Surprisingly, it was demonstrated that both cell lines are viable and motile. In order to compare the motility of these cells with normal cells, the newly developed computer-assisted Dynamic Morphology System (DMS) was employed. The results demonstrate that the average HMM or mhcA ameba moves at a rate of translocation less than half that of normal cells. It is rounder and less polar than a normal cell, and exhibits a rate of cytoplasmic expansion and contraction roughly half that of normal cells. In a spatial gradient of cAMP, the average ameba of HMM or mhcA exhibits a chemotactic index of +0.10 or less, compared to the chemotactic index of +0.50 exhibited by normal cells. Finally, the initial area, rate of expansion, and final area of pseudopods are roughly half that of normal cells. The five fastest HMM amebae (out of 35 analyzed in detail) moved at an average rate of translocation equal to that of normal amebae, and exhibited an average chemotactic index of +0.34. In addition, the average rate of cytoplasmic flow in fast HMM cells was equal to that of the average normal ameba. However, fast HMM amebae still exhibited the same defects in pseudopod formation that were exhibited by the entire HMM cell population. These results suggest that myosin heavy chain is involved in the "fine tuning" and efficiency of pseudopod formation, but is not essential for the basic behavior of pseudopod expansion.     

Turnover of myosin heavy and light chains in cultured embryonic chick cardiac and skeletal muscle

The relative rates of synthesis and breakdown of myosin heavy and light chains were studied in primary cell cultures of embryonic chick cardiac and skeletal muscle. Measurements were made after 4 days in culture, at which time both skeletal and cardiac cultures were differentiated and contracted spontaneously. Following a 4-hr pulse of radioactive leucine, myosin and its heavy and light chains were extracted to 90% or greater purity and the specific activities of the proteins were determined. In cardiac muscle, myosin heavy chains were synthesized approximately 1.6 times the rate of myosin light chains, and in skeletal muscle, heavy chains were synthesized at approximately 1.4 times the rate of light chains. Relative rates of degradation of muscle proteins were determined using a dual-isotope technique. In general, the soluble and myofibrillar proteins of both types of muscle had decay rates proportional to their molecular weights (larger proteins generally had higher decay rates) based on analyses utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A notable exception to this general rule was myosin heavy chains, which had decay rates only slightly higher than the myosin light chains. Direct measurements on purified proteins indicated that the heavy chains of myosin were turning over at a slightly greater rate (approximately 20%) than the myosin light chains in both cardiac and skeletal muscle. The reasons for the apparent discrepancy between these measurements of myosin heavy and light chain synthesis and degradation are discussed.