Activation of myosin synthesis in fusing and mononucleated myoblasts

Emerson, Charles P.; Beckner, Suzanne K.

doi:10.1016/0022-2836(75)90238-7

Cited by 197 publications

(44 citation statements)

References 26 publications

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“…However, they concluded that the development of these enzyme activities in both M3A and L6 cells was dependent on cell density and not on fusion. Somite myoblasts synthesize myosin prior to fusion (43), and in other experiments one or more characteristics of fused muscle was detected in single myoblasts in which fusion was reversibly inhibited by Ca2+ deprivation (7,9) or treatment with phospholipase C (35 …”

Section: Discussionmentioning

confidence: 97%

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Loss of growth control and differentiation in the fu-1 variant of the L8 line of rat myoblasts.

Kaufman¹,

Parks²

1977

Proc. Natl. Acad. Sci. U.S.A.

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A nonfusing variant, fu-1, of the L8 line of rat myoblasts was isolated and characterized with respect to its growth in vitro and developmental properties. Comparative analyses of density-dependent inhibition of growth, serum requirements, cell adhesiveness, colony formation-in soft agar, and hexose transport in L8 and fu-1 cells support the conclusion that the fu-1 cells are transformed. In addition, fu-1, but not Le, cells promote the development of tumors in athymic nude mice. fu-1 cells also do not make increased levels o creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) or myosin and they express an endogenous type-C virus. Both L" and fu-1 cells express myokinase (ATP:AMP phosphotransferase, EC 2.7.4.3) activities in single cells. In contrast to fu-1 cells, the parent L8 line has increased creatine kinase and myosin after fusion and spontaneously contracts; expression of an endogenous virus could not be detected in these cells. These results suggest that loss of the ability to differentiate normally is associated with the loss of the normal control of cell division of myoblasts grown in vitro and in vivo.Myoblasts, the precursors of highly differentiated skeletal muscle, proliferate in cell culture as single cells. Upon reaching a critical stage in their development, the myoblasts withdraw from the cell cycle and fuse into multinucleate cells (myotubes) (1)(2)(3)(4) of rat myoblasts (fu-i) that is defective in fusion from the myogenic L8 line. Analysis of several parameters that are usually attributed to transformed cells in vitro suggests that the nonfusing variant fu-i cells are transformed when compared to the parent L8 cells. In addition, only fu-l cells will form tumors in athymic mice. We have also found that the fu-i cells express an endogenous C-type virus; the L8 cells do not express this virus.MATERIALS AND METHODS Cell Culture Conditions. The L8 cells, kindly provided by D. Yaffe, were cloned and maintained by serial passage. Cloned cells were retrieved from frozen stocks as needed. fu-i cells were selected from the cloned L8 line as described in the text. All cells were grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% horse serum (ISI), 0.5% chick embryo extract, penicillin at 100 units/ml, and streptomycin at 100 ,ug/ml, at 370 and in a 10% CO2 atmosphere. Stock cultures of 2 X 105 cells were seeded in 10 ml of medium and grown on 100-mm Falcon tissue culture dishes coated with 0.1% gelatin. To maintain the myogenic capacity of L8 cells, it is essential to subculture the cells before they become confluent. Cells were routinely harvested by detachment with 0.05% trypsin in Ca2+,Mg2+-free Earle's solution. In experimental cultures, 3 ml of medium containing 1 to 2 X 105 cells were seeded in 50-mm dishes; medium was changed every day or as indicated. The cells were found free of mycoplasma.Uptake of 2-Deoxyglucose. Cells (2 X 105) were plated in 3 ml of medium on 50-mm dishes coated with gelatin. To determine uptake of 2-deoxyglucose (dGlc), the cel...

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Section: Discussionmentioning

confidence: 97%

“…Fusion into myotubes usually takes place after withdrawal of the myoblasts from the cell cycle (7,14,15), perhaps as the 'result of the expression or activation of new information during the immediately preceding round of replication (2,14). Cells that fuse to form myotubes have depleted levels of DNA polymerase and cease replication (16,17).…”

mentioning

confidence: 99%

Loss of growth control and differentiation in the fu-1 variant of the L8 line of rat myoblasts.

Kaufman¹,

Parks²

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…Calcium ion is required for myoblast fusion (49); cultures treated with a low-calcium medium (49) or with the Ca §247 EGTA (37), do not fuse. There have been several recent reports that fusion can be selectively blocked by calcium depletion from the medium without any apparent inhibition of the synthesis of several major components that mark the course of muscle differentiation, i.e., the appearance of muscle proteins such as the acetylcholine receptor (36), acetylcholinesterase, and adenylate cyclase (41), contractile proteins (16,19,34,57), creatine phosphokinase activity (31,55,56,62) and the elevation of cAMP levels (62). (b) BUdR treatment.…”

Section: Discussionmentioning

confidence: 99%

Cell-to-cell communication and myogenesis.

Kalderon¹,

Epstein²,

Gilula³

1977

The Journal of Cell Biology

105

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Cell-to-cell communication was characterized in prefusion chick embryo myoblast cultures, and it was determined that the prefusion myoblasts can interact via gap junctions, ionic coupling, and metabolic coupling. The biological relevance of this communication was supported by the detection of gap junctions between myoblasts in embryonic muscle. Communication was also examined in fusionarrested cultures to determine its potential relationship to fusion competency. In cultures that were fusion arrested by treatment with either 1.8 mM ethyleneglycolbis-(fl-aminoethyl ether)N,N'-tetraacetic acid (EGTA), 3.3 x 10 -6 M 5-bromodeoxyuridine (BUdR), or 1 /~g/ml cycloheximide (CHX), both gap junctions and ionic coupling were present. Therefore, it is possible to conclude that cell communication is not a sufficient property by itself, to generate fusion between myoblasts. The potential role of communication in myogenesis is discussed with respect to these observations. KEY WORDS cell-to-cell communication junctions 9 ionic coupling metabolic coupling myoblast fusion myogenesis gapThe differentiation of skeletal muscle in vivo and in vitro (in culture) is accompanied by the fusion of mononucleated muscle cells (myoblasts) into multinucleated mature muscle fibers (58). This fusion event, in culture, is preceded by a period of cell multiplication and cellular interaction. During this time, the cells are closely apposed and aligned in multicellular strings; this stage of differentiation has been referred to as the "prefusion lag period" (61). Under standard culture conditions this lag period is quite constant, and only cells which have completed their prefusion changes participate in the formation of muitinucleated fibers (59). The fusion process that follows the lag period has been previously regarded as a critical event in myogenesis. However, at present, it appears that it is just one of the important events that contribute to the formation of a differentiated muscle fiber (1, 12. 16, 19, 26, 31, 34, 36, 41, 54, 55, 56, 57, 62). The actual fusion process requires a direct physical interaction of the plasma membranes from adjacent cells, and this had led to a series of investigations of the cell surface elements that may be involved in regulating fusion (5,14,20). Other studies have suggested that fusion is not necessarily regulated by cell surface components; it may, in fact, be under the direct control of intracellular elements (62). Recently, it has been reported that a specific type of cell contact, the gap junction (30), and low-resistance pathways (6) are present between muscle cells during amphibian myotome development in vivo. In both studies, it was suggested that gap junctional communication between the muscle cells is related to the efficient transfer of excitatory stimuli from somite to somite, and not to cell fusion. Data from studies on embryonic rat and chick skeletal muscle cells in culture indicate the possible existence of ionic coupling (measured indirectly) and gap junctionlike structures betwee...

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“…However, previous studies have shown that myoblast fusion and expression of muscle-specific genes can be uncoupled by the use of inhibitors (10,12,22) or as a result of mutations (1,23). Therefore, we looked for musclespecific gene expression in tdM5i-QECs by staining cells with antibodies to muscle-specific proteins.…”

Section: Skv-qecsmentioning

confidence: 99%

Transformation-defective v-ski induces MyoD and myogenin expression but not myotube formation.

1991

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The ski oncogene induces muscle differentiation in otherwise nonmyogenic quail embryo cells (C. Colmenares and E. Stavnezer, Cell 59:293-303, 1989). Here we report that v-ski induces both MyoD and myogenin expression, suggesting that activation of these muscle regulatory genes may be a critical step in ski-induced myogenesis. We also describe a transformation-defective mutant of v-ski (tdM5i) that fails to induce myotube formation, although it induces the expression of many muscle-specific genes, including the MyoD and myogenin genes. Therefore, if activation of MyoD and myogenin expression is a necessary component of the myogenic program triggered by ski, it is clearly insufficient to account for complete muscle differentiation.

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Activation of myosin synthesis in fusing and mononucleated myoblasts

Cited by 197 publications

References 26 publications

Loss of growth control and differentiation in the fu-1 variant of the L8 line of rat myoblasts.

Loss of growth control and differentiation in the fu-1 variant of the L8 line of rat myoblasts.

Cell-to-cell communication and myogenesis.

Transformation-defective v-ski induces MyoD and myogenin expression but not myotube formation.

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