A mechanical dissociation method for preparation of muscle cell cultures

Tepperman, Katherine; Morris, Glenn E.; Essien, Francine B.; Heywood, Stuart M.

doi:10.1002/jcp.1040860313

Cited by 41 publications

(13 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This was accomplished by VBSO4 precipitation in the presence of calf brain tubulin. Because muscle cultures that have not been fed for [20][21][22][23][24] hr synthesize protein at a slower rate than do freshly fed cultures (21), "unfed" cultures were utilized in all subsequent experiments. To further decrease translational incorporation, the pulse period was shortened to 15 min and the radioactive label was added directly to the medium of the unfed culture plate.…”

Section: Resultsmentioning

confidence: 99%

Intact microtubules are required for rapid turnover of carboxyl-terminal tyrosine of alpha-tubulin in cell cultures.

Thompson¹,

Deanin²,

Gordon³

1979

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

View full text Add to dashboard Cite

In cultured muscle cells the carboxyl-terminal tyrosine of a-tubulin was shown to exchange rapidly with free tyrosine. The rapid turnover of this residue was dependent upon the presence of intact microtubules. Half-life determinations were made by two methods: (i) the cells were pulse-labeled in hypertonic medium, in which the major tyrosine incorporation was post-translational, and then chased with isotonic medium; and (ii) the cells were pulsed and chased in isotonic medium, and the post-translational component of the radioactivity of purified a-tubulin was calculated. Both methods yielded a half-life of 37 min or less for the terminal tyrosine residue, whereas the half-life of tubulin itself was shown to be greater than 48 hr. A rapid assembly and disassembly of several filamentous intracellular structures is coordinated with changes in cellular function and growth (1-3). The mechanisms that regulate these processes are not known, but attention has been focused on post-translational events because, at least in the case of actin and tubulin, de novo protein synthesis is not an essential aspect of this dynamic process (4,5).In this laboratory we have been studying a specific posttranslational modification of tubulin that may be involved in the control of tubulin utilization in vivo. Tubulin:tyrosine ligase (TTLase) catalyzes the addition of single units of tyrosine (or phenylalanine) to the carboxyl terminus of a-tubulin (6). TTLase activity is widely distributed in avian and mammalian tissues (7). The specific activity of the TTLase has been shown to undergo marked changes during the development of brain, liver, muscle, and sensory ganglia of the chicken, the highest activities occurring in the embryo (8, 9). The periods of high TTLase activity in brain, sensory ganglia, and muscle are temporally related to development of cellular asymmetries. Although the biochemical reaction that is catalyzed by TTLase has been characterized in vitro (10) and shown to occur in vivo (11), the significance of the modification of tubulin and the high TTLase levels to cellular function and embryonic development is not yet established. Rodriguez and Borisy (12) have recently reported that the degree of tyrosination of a-tubulin varies as a function of brain development in a manner that correlates with the changes in TTLase activity. We have speculated that the rate of turnover of the terminal tyrosine residue might be of more significance to cellular function than the absolute degree of tubulin tyrosination (8).In this paper, we examine the rate of turnover of the carboxyl-terminal tyrosine in primary cultures of chicken breast muscle, which maintain high levels of TTLase (unpublished observations). Estimates were obtained from two different types of pulse-chase experiments. The first utilized hypertonic culture medium to inhibit translational incorporation of tyrosine by blocking the formation of initiation complexes (13). Cells Npulsed with [3H]tyrosine in hypertonic medium selectively incorporate the label at the carbo...

show abstract

Section: Resultsmentioning

confidence: 99%

Intact microtubules are required for rapid turnover of carboxyl-terminal tyrosine of alpha-tubulin in cell cultures.

Thompson¹,

Deanin²,

Gordon³

1979

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

View full text Add to dashboard Cite

show abstract

“…Myoblasts were obtained from 12-day chicken embryonic breast muscle as described (24). Muscle cells were plated in 100-mm culture dishes at an initial density of 6 x 106 cells per dish.…”

Section: Methodsmentioning

confidence: 99%

Cytoplasmic utilization of liposome-encapsulated myosin heavy chain messenger ribonucleoprotein particles. During muscle cell differentiation.

Havaranis¹,

Heywood²

1981

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

View full text Add to dashboard Cite

Myosin heavy chain messenger ribonucleoprotein particles (MHC mRNPs) have been isolated. Characterization of the RNA components revealed an mRNA of approximately the same size as tobacco mosaic virus RNA and three low molecular weight components. The protein consists of 9-10 major bands ranging in molecular weight between 22,000 and 130,000. The messenger contained in these mRNPs was found to direct the synthesis ofboth fast-muscle and slow-muscle MHC in a cell-free system. When MHC [3H]mRNPs were encapsulated into liposomes and subsequently delivered to myoblasts and myotubes, the mRNPs were taken up by the cells at both stages ofdifferentiation. However, the MHC [3H]mRNPs taken up by the myoblasts remained as free cytoplasmic particles (80-120S), whereas in myotubes the incorporated mRNP RNA was associated with polysomes. The results indicate that MHC mRNPs contain a repressor molecule(s) and that myotubes possess a mechanism for activating these mRNPs that is absent from myoblasts.The importance of both transcriptional and posttranscriptional controls on gene expression in eukaryotes is well established (1, 2). The existence of posttranscriptional controls has been demonstrated at mRNA processing, differential utilization of mRNA, and mRNA half life (3)(4)(5)(6)(7)(8). In addition, cytoplasmic messenger ribonucleoprotein particles (mRNPs) have been found to be present in a large variety of cell types (7)(8)(9)(10)(11)(12)(13)(14).Several studies have demonstrated that cytoplasmic mRNPs can be translated in heterologous cell-free systems (15)(16)(17) (14,23).In this report, MHC mRNP is isolated under conditions that favor the retention of its native properties. This mRNP is subsequently inserted into muscle cells by utilizing liposomes, to study its function in developing muscle. Large unilamellar vesicles (LUV) were formed to encapsulate and deliver MHC mRNPs into cells. LUV-encapsulated MHC mRNPs are incorporated by both myoblasts and myotubes; however, they are differentially utilized at these two stages of muscle cell development. MATERIALS AND METHODSMyoblasts were obtained from 12-day chicken embryonic breast muscle as described (24). Muscle cells were plated in 100-mm culture dishes at an initial density of 6 x 106 cells per dish. Cultures were incubated as described (22). Fresh medium was added to the cultures 24 hr after plating and again 2 hr prior to the addition of liposomes. DNA was determined by the procedure of Kissane and.Robins (25).Muscle cell cultures were labeled by the addition of 40 ,uCi(1 Ci = 3.7 x 1010 becquerels) of [3H]uridine 10 hr prior to harvesting. The cells were collected at 42 hr, when 30-40% of the cells were fused. Cells were lysed in buffer containing cycloheximide as described (23). The muscle cell lysate was layered on 11-ml 10-40% sucrose density gradients and centrifuged at 40,000 rpm in an IEC SB 283 rotor for 60 min. The fraction sedimenting at 80-120S was collected and pelleted by centrifugation at 200,000 X g for 3 hr. The mRNP-ribosome pellets were resuspe...

show abstract

“…Breast muscle cell cultures, obtained from 12-d chick embryos, were prepared as previously described (23) . Briefly, pectoralis muscle was mechanically dissociated and the cells were plated at an initial density of 6 x 106/ 100-mm culture dish.…”

Section: Methodsmentioning

confidence: 99%

Encapsulation of "core" eIF3, regulatory components of eIF3 and mRNA into liposomes, and their subsequent uptake into myogenic cells in culture.

O’Loughlin¹,

Lehr²,

Havaranis³

et al. 1981

The Journal of Cell Biology

View full text Add to dashboard Cite

Eukaryotic initiation factor 3 (eIF3), encapsulated in liposomes, is taken up by chick muscle cells in culture. The exogenously supplied factor (isolated from 14-d embryonic muscle) rapidly associates with 40S ribosomal subunits and particles sedimenting at 80-1205 (the known sedimentation value of myosin heavy chain [MHC] mRNPs) . In addition, exogenously supplied eIF3 has a specific stimulatory effect on myofibrillar protein synthesis. This stimulation is most apparent at the onset of cell fusion and after the accumulation of MHC-mRNPs. As previously reported (8), total eIF3 can be fractionated on an MHC-mRNA affinity column into a "core" eIF3 and a high affinity component (HAF) which dictates the discriminatory activity of core eIF3 . Liposome-encapsulated core eIF3 delivered to cells is found predominantly in 40S ribosomal subunits and gives only a slight stimulation of total protein synthesis. When 3 H-MHC-mRNA, preincubated with HAF, is introduced into myoblasts via liposomes, the mRNA is found in heavy polysomes. On the other hand, when the messenger alone or with core eIF3 is taken up by the cells, it is found only on small polysomes. Similar experiments, using viral RNA with the HAF, show no increase in the size class of polysomes. These results mimic the differences observed between myoblast and myotube utilization of MHC-mRNA previously observed (17) . These results demonstrate the mRNA discriminatory activity of specific proteins associated with muscle eIF3 and suggest that these proteins play a role in mRNA activation and translation during muscle differentiation .

show abstract

A mechanical dissociation method for preparation of muscle cell cultures

Cited by 41 publications

References 11 publications

Intact microtubules are required for rapid turnover of carboxyl-terminal tyrosine of alpha-tubulin in cell cultures.

Intact microtubules are required for rapid turnover of carboxyl-terminal tyrosine of alpha-tubulin in cell cultures.

Cytoplasmic utilization of liposome-encapsulated myosin heavy chain messenger ribonucleoprotein particles. During muscle cell differentiation.

Encapsulation of "core" eIF3, regulatory components of eIF3 and mRNA into liposomes, and their subsequent uptake into myogenic cells in culture.

Contact Info

Product

Resources

About