Glenn E. Morris

Cell fusion and differentiation in cultured chick muscle cells

Abstract A method for the quantitative estimation of myoblast fusion and the effects of variables in the culture conditions on the extent of fusion are described. The onset of fusion is delayed both by feeding and by lowering the initial cell density, but effects of alterations in sera and embryo extracts are evident only at later stages of differentiation. Using a sensitive fluorimetric assay for creatine phosphokinase, enzyme activity was determined throughout the culture period and related to the extent of cell fusion. The increase in activity during a 7-day culture period is biphasic, the first small increase (2–5 ×), which begins after 40–50 h in culture, being apparently closely related to cell fusion. There is a 48 h delay before the second, much larger (up to 25 ×) increase begins.

Isoenzymes of creatine phosphokinase during myogenesis in vitro

Abstract The dramatic increase in creatine phosphokinase activity which occurs in chick thigh muscle cell cultures after 4 days of culture was accompanied by the appearance of a new isoenzyme characteristic of adult muscle. Before this increase in enzyme activity, only the isoenzyme characteristic of brain and early embryonic muscle was detected.

Calcium and the control of muscle-specific creatine kinase accumulation during skeletal muscle differentiation in vitro.

Abstract A polyacrylamide gel separation method for creatine kinase (CPK) isoenzymes is described, and its use to determine muscle-specific CPK (M-CPK) levels in skeletal muscle cultures is illustrated. In cultures in which cell fusion has been prevented by very low Ca 2+ concentrations, the increases in M-CPK after 96 hr are similar to those in control cultures. Slightly higher concentrations of Ca 2+ , however, inhibit both cell fusion and M-CPK accumulation. As the calcium concentration is gradually increased further, cell fusion is permitted, followed, at even higher Ca 2+ levels, by M-CPK accumulation. These effects can be obtained both by adding EGTA to the culture medium and by using Ca 2+ -free culture medium and varying the Ca 2+ concentration directly. The latter method has the advantage that deleterious effects of EGTA on cell attachment and cell numbers do not occur, even at the lowest Ca 2+ concentrations. By revealing dramatic effects on CPK levels of small changes in external Ca 2+ concentrations, these observations may resolve conflicting data in the literature on the question of whether cell fusion is a prerequisite for muscle-specific protein synthesis. Possible mechanisms for the two effects of Ca 2+ on CPK specific activity (permissive at very low, but inhibitory at intermediate, concentrations) are considered, including membrane mediation, mediation by changes in ionized cytoplasmic Ca 2+ levels, and possible involvement of cyclic nucleotides.

Differential effects of calcium ion concentration on cell fusion, cell division and creatine kinase activity in muscle cell cultures

Abstract Cell fusion, cell number, soluble cell protein and creatine kinase activity have been measured simultaneously in chick muscle cell cultures exposed to various calcium ion concentrations for various periods of time, by adding either extra calcium chloride or the calcium-chelating agent, EGTA. Up to 0.75 mM EGTA cell fusion is not inhibited, but the specific activity of creatine kinase is reduced by 20–50%. Between 0.75 and 1.7 mM EGTA, cell fusion is gradually abolished and the increase in cell number prevented, but enzyme specific activity actually increases again and returns to control values. Adding extra Ca2+ produces small increases in cell fusion and soluble cell protein, but much greater increases in creatine kinase activity. EGTA stimulates thymidine incorporation into DNA at low concentrations and then inhibits again as its concentration is increased further. These effects of EGTA on cell division may be related to its effects on creatine kinase. The implications of these results are discussed in terms of current ideas about the inter-relationships between cell fusion, cell division and the accumulation of muscle proteins during differentiation. In particular they show that cell fusion is not essential for the attainment of normal levels of creatine kinase.

Two-dimensional gel analysis of nuclear proteins during muscle differentiation in vitro: I. Changes in nuclear protein content

Abstract Improved methods are described for the isolation of nuclei from skeletal muscle-cell cultures by lysis with Triton X-100 and centrifugation through 65% sucrose-5 mM MgCl 2 . Evidence is presented that levels of contamination by cytoplasmic and fibrillar proteins are acceptably low. Nuclear proteins were separated into saline-soluble and non-histone protein (NHP) fractions and analysed on two-dimensional gels stained with Coomassie Blue. 2D-gels of NHP and cytoplasmic proteins were compared and few proteins common to both fractions were found. Of three major cytoplasmic proteins, actin, tubulin and tropomyosin, only actin could be positively identified as a minor protein in NHP and tropomyosin was undetectable. An overall increase in nuclear NHP content (relative to DNA) occurred during the early period of cell differentiation in vitro (24–66 h of culture) and a corresponding increase in nuclear protein to DNA ratio was observed. There were differential changes between individual NHP, some major proteins showing little change, while others showed large increases. These changes may be related to increases in nuclear protein uptake which are reported to occur during differentiation and hormone-induced gene transcription in other cell types.

Gene activation during muscle differentiation and the role of nonhistone chromosomal protein phosphorylation

Abstract The phosphorylation of nuclear proteins, in a standard assay with nuclei isolated from chick muscle cell cultures, increases steadily with time in culture. This increase is shown to reflect increased nuclear protein kinase activity. Most of the phosphorylated protein is chromatin bound and so are the enzymes responsible for the phosphorylation. Analysis of chromosomal proteins on hydroxylapatite columns shows that 32P is incorporated almost entirely into nonhistone proteins, while the specific radioactivity of the histones is low and does not increase during cell culture. Experiments in which cells were grown at different initial densities and, consequently, underwent cell fusion at different times show that phosphorylation is dependent neither on cell density nor on the degree of cell fusion. Chick thigh fibroblasts, prepared by repeated subculture of fibroblasts from muscle cell preparations to eliminate myogenic cells, do not show the increase in phosphorylation with culture time, suggesting that the changes may be specific for primary myoblast cultures. These changes are not shown by thigh muscle nuclei isolated during development in the whole embryo. In fact, nuclear protein phosphorylation activity decreases rapidly in ovo with increasing embryonic age. Possible roles for phosphorylation of nonhistone chromosomal proteins in gene activation during myogenesis are discussed in the light of these observations.

A monoclonal antibody against the skeletal muscle enzyme, creatine kinase.

Creatine kinase (CK) is often used as an index of differentiation in cultures of embryonic muscle cells [l] and its presence in serum is also used to monitor muscle damage in disease [2,3]. Immunological assays of CK can be made specific for any one of the CK isoenzymes and polyclonal rabbit antisera have been used for the study of muscle-specific CK (MM-CK) accumulation and distribution in differentiating myoblast cultures [4,5]. A monoclonal antibody (McAb) for these assays would have the added advantages of providing an unlimited supply of antibody with defined properties which can be selected for during McAb preparation. review), McAb CK-JOE does appear to be suitable for measuring MM-CK levels in muscle cell cultures.

Biosynthesis of muscle-specific creatine kinase during differentiation in vitro

Measurements of creatine phosphokinase (CPK) activity have been used for some time as an index of differentiation in muscle-cell cultures [l-4 ] . The developmental transition from embryonic BB isoenzyme, through the MB intermediate, to the mature muscle-specific MM form suggests that the increase in enzyme activity during myogenesis involves new gene expression [5,6] . However, we have previously emphasized the limitations on the use of total CPK activity as an index of differentiation, since quite large increases in embryonic BB-form activity also occur in cell culture [7] . Even measurements of musclespecific CPK, by specific activity [7] or immunological methods [8], are limited as an index of new gene expression by the fact that they can only provide data on enzyme accumulation rather than rates of enzyme synthesis. In this report, we present some experiments on the isolation of newly-synthesized CPK after incubation of cells with a radioactive amino acid as the basis for a method of determining the rate of CPK synthesis during differentiation. No measurements of rates of CPK synthesis in muscle cell cultures by amino acid incorporation studies have so far been reported, although, unlike the extensively studied myosin heavy chain 19-121 , CPK is relatively easily resolved into its embryonic and mature components. The results provide further evidence that increases in musclespecific CPK activity during differentiation involve new enzyme synthesis, but do not reveal a similar involvement of new CPK synthesis in the increases in embryonic form activity reported-in early cultures [7] . The ratio of the rates of synthesis of

RNA synthesis and the stimulation of insulin biosynthesis by glucose

Glucose stimulates both incorporation of amino acids into insulin [ 1, 21 and incoproration of label from erotic acid into RNA of rat islets of Langerhans [3] . We have shown that glucose, in the physiological concentration range, stimulates incorporation of amino acids into a proinsulincontaining protein fraction from isolated rat islets with a considerable degree of specificity and have interpreted these results as indicating increased proinsulin biosynthesis in response to glucose [4] . In this report we describe experiments in which actinomycin D was used to investigate the relationship between RNA synthesis and the stimulation of proinsulin synthesis by glucose. Actinomycin D, added before glucose, inhibited amino acid incorporation but a stimulatory effect of glucose was still present, suggesting that the glucose stimulation of proinsulin synthesis is not mediated by newly synthesized RNA. Actinomycin D, added after the glucose stimulation, caused an additional stimulation of protein synthesis. Actinomycin D reduced the rate of secretion of insulin when added before glucose but had no immediate effect when added after glucose. The possibility that the drug is affecting other processes directly, as well as RNA synthesis, is considered.

Two-dimensional gel analysis of nuclear proteins during muscle differentiation in vitro: II. Changes in protein phosphorylation

Abstract Isolated nuclei from chick skeletal muscle cell cultures at different stages of differentiation were labelled in vitro with γ-[ 32 P]ATP. Nuclear proteins were separated into saline-soluble and non-histone (NHP) fractions and analysed by two-dimensional gel electrophoresis and autoradiography. Relatively few proteins (10–12) were phosphorylated to any extent and most of these corresponded to minor, or even undetectable, components on stained gels. 32 P incorporation into most of these proteins increased between 24 and 40 h and decreased sharply between 40 and 66 h, the changes being more marked in some phosphoproteins than in others. Pulse-chase data suggest that these overall changes in phosphorylation reflect changes in protein kinase, rather than protein phosphatase activities. One highly phosphorylated acidic protein (mol. wt 10 000 D; pI 4.2) which was undetectable on stained gels, showed a continuous increase in 32 P incorporation between 24 and 66 h in both saline-soluble and NHP fractions. This protein may be the muscle counterpart of a phosphorylated nuclear protein partially characterized in other cell types. Both nuclear protein phosphorylation and low molecular weight non-histone proteins have been particularly implicated in activation of gene transcription in other systems, although we have no direct evidence, as yet, to connect them with control of myoblast growth and differentiation.