Printed in Sweden Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/1171-0063$02.00/0
Experimental Cell Research 117 (1978) 63-70
ISOLATION MYOBLAST
AND PURIFICATION NUCLEI CHICK
OF MYOTUBE
FROM CULTURES SKELETAL
AND
OF EMBRYONIC
MUSCLE
JOHN D. DAVID, ROBERT L. FREDRICKSON
and GARY R. PETERSON
University of Missouri, Columbia, MO 65201, USA
SUMMARY Methods are described for the preparation of purified myotubes from embryonic chick skeletal muscle cultures and the preparation of purified nuclei from both myotubes and myoblasts. Myotubes are released from the culture dish by digestion of their collagen substratum with collagenase, and purified by sucrose density gradient sedimentation. Nuclei are prepared from the isolated myotubes by controlled homogenization in Ca*+-free medium and sedimentation through 2.1 M sucrose. Nuclei are prepared from cultured myoblasts in a similar fashion, with the inclusion of the non-ionic detergent NP-40 in the homogenization medium and sedimentation through 2.4 M sucrose. Phase contrast microscopic examination showed that the nuclear preparations are free of visible cytoplasmic contamination, and are morphologically similar to nuclei observed in situ. Biochemical assays (protein/DNA and RNA/DNA ratios) confirm the purity of the nuclear preparations. Both nuclear preparations have been used to prepare purified chromatin which has spectral and chemical properties similar to those reported for chromatin purified directly from several chick tissues.
Although nuclei have been isolated from a wide variety of animal tissues and cultured cells [ 121,there have been very few reports of nuclear preparations from whole skeletal muscle [2, 9, 201, and no reports of nuclear isolation from cultured skeletal muscle cells. In addition, there is no reported method to separate the two basic cell types in a differentiating muscle cell culture, the single cells (myoblasts and tibroblasts), and the syncytial myotubes. This report describes (1) the isolation of the myotubular component of the developing cultures, free from significant single-cell contamination; (2) methods to isolate highly purified nuclei from chick skeletal muscle myoblasts, and from isolated myotubes. 5-781819
MATERIALS
AND METHODS
Materials Tissue culture dishes were obtained from Falcon Plastics, BioQuest, Cockeysville, Md. Reagents were obtained from the following sources: 6ulbecco’s Modified Eagle’s Medium and Horse Serum (Grand Island Biological Co., Grand Island, N.Y.); Collagenase (Code CLS or CLS III, EC 3.4.24.3) Worthington Biochemical Corp., Freehold, N.J.); Enzyme-g&de Tris (Trihydroxy methylamino methane) and Ultra Pure Sucrose (Schwarz&Iann Biochemicals, Orangeburg, N.Y.); NP-40 (Particle Data Labs, Evanston, Ill.); Harris-Lillie Hematoxylin Stain Solution (Fisher Scientific Pittsburgh, Pa); EDTA (Ethylenediaminetetraacetic acid) (Sigma Chemical Co., St Louis, MO). All other chemicals were reagent grade (Fisher Scientific, Pittsburgh, Pa).
Culture conditions Myoblasts from 12-day-old white leghorn chick embryo thigh muscle were obtained by a selective trypsinization and plating procedure (to be described in E.rp Cdl Rr., I I7 ( 19781
64
David et al.
detail in a later publication) which produces a population of cells 95 % myoblast and 5 % fibroblast in morphology and fusion-capability. The cells were plated on collagen-coated 150 mm* tissue culture dishes at 8x 108cells/plate in Dulbecco’s Modified Eagle’s Medium supplemented with 10% horse serum and 3 % chick embryo extract. Medium was changed on days 1 and 3.
Isolation
of myotubes
Four&v-old mates were rinsed once with Puck’s Saline d [ 141and once with buffer A (0.125 M sucrose, 10 mM Tris OH 7.5. 5 mM M&l,. 0.5 mM EDTA. 2 mM CaCl,,* 0.15 M KCl). T& milliliters of buffer A containing 0.01% collagenase were added to each plate and the plates rocked gently (Lab-Line platform rocker. 20” anaular disnlacement. 1 strokelsec) at 30°C until the cells-and mybtubes released from the plate. After the addition of 0.5 ml of 0.2 M EDTA (oH 9.0). the plates were rocked for an additional mint& ’ The cell-myotube suspension was pipetted up-anddown three times through a 10 ml pipet with a 2 mm oriface in order to disperse clumps of single cells and to free the myotubes of any attached single cells. This step completely freed the myotubes of attached single cells while doing little if any damage to the myotubes. The disnersed susnension (10 ml) was lavered on ton of a preformed discontinuous sucrose gradient of lb ml buffer B (0.25 M sucrose, 10 mM Tris pH 7.5, 10 mM MgC&, 5 mM EDTA, 0.15 M KCl) and 10 ml buffer C (0.32 M sucrose, 10 mM Tris nH 8.0. 5 mM MgCl,, 5 mM EDTA, 0.15 M KCl), both at s”C. The preparation was centrifuged at 16 g for 5 min at 5”C, the tubes rotated at 180” and centrifuged for an additional 5 min at 16g. This rotation is necessitated bv the tendency of large-myotubes to stick to the side walls of the glass centrifuge tubes at these low soeeds. Under these conditions; all but the smallest myotubes are found in the pellet, while the single cells remain in the upper l/3 of the tube.
Isolation
of myotube nuclei
The isolated myotubes were resuspended in a small volume of buffer C and homogenized 20 times in a Dounce homogenizer with the tight-fitting pestle. The homogenate was centrifuged at 100 g for 10 min. The pellet was resuspended in a small volume of buffer C, rehomogenized as above, and this second homogenate centrifuged at 100g for 10 min. The pellet, which contained unbroken myotube fragments and any single cells which contaminated the myotube preparation, was discarded. The supematants were combined and adjusted to 1.9 M in sucrose by the addition of 3.2 vol of buffer D (2.4 M sucrose, 10 mM Tris pH 8.0, 5 mM MgCl,, 5 mM EDTA, 0.15 M KCl). The crude nuclear suspension was dispersed in a Dounce homogenizer by 5 strokes of the loose-fitting pestle. The susnension was lavered over 1.5 ml of buffer E (2.1 M sucrose, 10 mM Tris pH 8.0, 5 mM MgCl,, 5 mM EDTA, 0.15 M KCl) and 1.5 ml of buffer D in a cellulose nitrate centrifuge tube. The nuclei were sedimented at 64000 I! for 90 min in the Beckman SW41 rotor. Clean nuclei collected at the buffer Dbuffer E interface. All operations were performed at 5°C.
Isolation
of myoblast (single-cell)
nuclei
One-dav-old mates were rinsed once with Pucks saline G [14] and once with buffer F (10 mM Tris pH 8.0, 5 mM M&l,. 5 mM EDTA. 0.15 M KCl). Ten milliliters of b\ff& F were added to each plate and the cells removed with a rubber noliceman. The cells were concentrated by centrifugation at 600 g for 10 min and suspended in buffer G (0.25 M sucrose, 10 mM Tris oH 8.0, 5 mM MgCl*, 5 mM EDTA, 50 mM KCl). This suspension was homogenized 10 times in a Dounce homogenizer with the tight-fitting pestle. Although this resulted in the rupture of most of the cells, most of the nuclei were surrounded by cellular debris or had “cytoplasmic tags” attached. The non-ionic detergent NP-40 was added to 0.5 % final concentration and the preparation rehomogenized twenty times. This step dispersed the cellular debris and removed most of the “cytoplasmic tags”. The crude nuclear suspension was adiusted to 2.1 M in sucrose bv the addition of 6.15 vol of buffer H (2.4 M sucrose: 10 mM Tris pH 8.0, 5 mM M&l,. 5 mM EDTA. 50 mM KCl) and dispersed in-a Dounce homogenizer by 5 strokes of the loose-fittina oestle. The final susoension was lavered over 1.5 ml-buffer H and 1.5 ml buffer I (2.8 M sucrose, 10 mM Tris pH 8.0, 5 mM MgC&, 5 mM EDTA, 50 mM KCl) in a cellulose nitrate centrifuge tube. The nuclei were sedimented at 64 000 g for 90 min in the Beckman SW41 rotor. Clean nuclei collected at the buffer H-buffer I interface and in the buffer I laver. All operations were performed at 5°C.
Isolation of nuclei from whole skeletal muscle Thigh muscle from 12-day-old chick embryos was excised free of skin and bone in a chilled Petri dish containing Pucks saline G [ 141.The muscle was finely minced with scissors, rinsed three times with buffer C, and homogenized 20 times in buffer C with a Dounce homogenizer with the loose-fitting pestle at 5°C. The homogenate was then rehomogenized 20 times with the tight-fitting pestle. The remainder of the procedure is identical to that used to prepare myotube nuclei.
Chromatin
preparation
and purification
Isolated nuclei were diluted to 0.25 M sucrose with buffer G and pelleted by centrifugation at 64 000 g for 90 min. The pellet was rinsed gently with 10 mM Tris pH 8.0 and then resuspended in 1 ml of the same buffer. The nuclei were allowed to swell for 12-18 h. This preparation, termed “crude chromatin” was dispersed by vigorous homogenization in a Dounce tissue homogenizer fitted with a tight nestle. The dispersed crude chromatin was diluted with 3 vol of 10 mM Tris pH 8.0 and layered over 2 ml 1.7 M sucrose, 10 mM Tris pH 8.0 in a Beckman SW50.1 centrifuge tube. The interface was mixed gently and the chromatin sedimented at 58 000 g for 3 hy The clear gelatinous pellet was resuspended in 10 mM Tris pH 8.0 by sonication and termed “purified chromatin” [lo]. All operations were performed at 5°C.
Isolation of myotube and myoblast nuclei
Fig. 1. (a) Phase contrast photomicrograph of a rep-
resentative field of purified myotubes. Nuclei are slightly elongated and contain easily recognizable nucleoli. The myotubes are relatively fragile at this point and are often seen to “leak” fluid cytoplasm, although
65
nuclei rarely escape. Cross striations characteristic of myofilaments are visible at several points within the myotube. Some contaminating single cells remain. (b) As in (a) except myotube is smaller, less highly branched, and more elongated. Bar, 100pm.
DNA, RNA, and protein determinations DNA was determined using the paraldehyde-diphenylamine procedure [ 151.RNA was determined by the orcinol procedure [ 181.Protein was determined by the micro-Lowry method of Rutter [ 171.Alternatively, DNA and protein were determined spectrophotometrically [lo]. Results using both methods were not significantly different.
RESULTS
Isolation of multinucleate syncytia (myotubes) Phase contrast photomicrographs of resuspended myotube pellets are shown in fig. 1. These preparations were judged homoFixation and staining An aliquot of the myotube preparation was placed on geneous by visual observation. This was a clean glass slide and allowed to air-dry. The slide confirmed by monitoring the ratio of myowas immersed in fixative A (10% form01 in phosphatetube nuclei to single-cell nuclei in fixed and buffered saline) for 1 h followed by fixative B (acetic acid : methanol, 1 : 3) for 1 h. The slide was rinsed in stained preparations. Although some single water and immersed in Harris-Lillie hematoxylin stain solution for 15 min and developed with 1% am- cells do appear in the myotube preparation, monium hydroxide for 5 min. After rinsing with water at no time do they represent more than a coverslip was applied and the slide observed while 0.4% of the total nuclei counted. still wet. E.rp Cdl
Rc\
I17 lIY7Xl
66
David et al.
Fig. 2. (a), (b) Phase contrast photomicrographs of representative fields of isolated myotube nuclei collected from the buffer D-buffer E interface. Although the majority of the nuclei are intact and slightly elongated, some appear slightly damaged and/or crenated. All contain one or two visible nucleoli and none carry visible cytoplasmic “tags”. (c), (d) Phase contrast
photomicrographs of representative fields of isolated myoblast nuclei collected from the buffer H-buffer I interface. These nuclei are generally smoother in outline (less crenated) than isolated myotube nuclei. All contain one or two visible nucleoli and no visible cytoplasmic “tags”. Bar, 10 pm.
Using the procedure as described, single cells remain in the upper l/3 to l/2 of the sucrose gradient, small myotubes (10-20 nuclei) sediment into the bottom l/3 of the gradient but do not pellet, and the large myotubes can be recovered from the pellet. Some myotubes are disrupted by this treatment and the resultant debris is found in the upper layers of the gradient. If desired, the single cells and/or small myotubes can be recovered by differential centrifugation.
Isolation of myotube nuclei Phase contrast photomicrographs of puritied myotube nuclei are shown in fig. 2. These nuclei banded at the buffer E-buffer D interface and were judged free of single cell, mitochondrial, myotubular, membraneous and other debris by extensive visual observation. There were no observable cytoplasmic “tags” attached to these nuclei. The “dirty” nuclei present all floated on buffer E and were easily re-
E.vp Cc//
Rr.\
117 (IY78)
Isolation
Table 1. Nucleic acid and protein whole skeletal musclea
content
of myotube and myoblast nuclei
of fractionated
myoblasts,
myotubes,
67 and
DNA Fraction Myobla& Homogenate Nuclei Purified chromatin Myotube* Homogenate Nuclei Purified chromatin Whole skeletal muscleC Nuclei Purified chromatin Whole skeletal muscled Homogenate Nuclei
(Pi?)
Percent of homogenate
Protein (pg)
Protein DNA
RNA DNA
262f54 82+22
100 31&8
2 16Of509 262+62
8.24k2.01 3.19+0.25
1.41*0.33 0.10+0.04
172+22
2.6lkO.03
0.084+0.02
7 680+710 213f 14
28.lk8.4 3.43kO.12
1.60+0.84 0.11+0.03
12lfl2
2.47f0.21
0.10~0.02
3.52kO.20
0.43+0.05
2.58kO.11
0.06~0.02
452+ 126 3.44kO.24
1.73kO.33 0.10~0.02
66_+18
25_+8
273f55 62f8.6
100 23f7
49*9.1
IS&3
a Results given as mean k S.D. b Average of 4 expts.
100 15
c Average of 2 expts. d As reported by Edelman et al. [2].
moved before the purified nuclei. Small is the DNA/RNA ratio which is 16-fold myotube fragments and unbroken single lower than that of the original homogenate, cells floated to the top of the tube and were comparable to values for nuclei isolated also easily removed. Purified nuclei ob- from whole skeletal muscle (table l), and served with phase contrast optics were in- also at the low end of the range reported for tact, generally oblong, and all had one or nuclei isolated from a variety of other cells two distinct nucleoli in an amorphous nu- and tissues [2, 211. We have recovered 18-30% of the DNA cleoplasm. They were morphologically indistinguishable from nuclei isolated directly in the purified nuclear fraction. Most of the remainder of the DNA is found in nuclei from chick embryo thigh muscle [2, 91. Some biochemical properties of the iso- associated with varying amounts of cellular lated myotube nuclei are shown in table 1. debris (mostly incompletely ruptured myoThe protein/DNA ratio of isolated myotube tubes). More extensive homogenization will nuclei agrees well with protein/DNA ratios release more nuclei, but also results in infor nuclei isolated from whole skeletal mus- creased numbers of visually damaged nucle as determined by Edelman [2] and our- clei. These homogenization conditions do selves (table l), and is at the low end of the not rupture single cells and thus all nuclei range reported for nuclei isolated from a recovered originated in myotubes. The poswide variety of other cells and tissues [2, sible loss of nuclear protein and RNA dur211. A further indication of chemical purity ing the isolation procedure has not been Exp Cell Res l/7(1978)
68
David et al.
Fig. 3. Abscissa: wavelength (nm); ordinate: ab-
sorbance. Absorbance profiles of “crude” and “purified” chromatin prepared from isolated myotube nuclei. 0- --0, “Crude” chromatin; X-X, “purified” chromatin. Absorbance profiles of chromatin prepared from myoblast nuclei and whole skeletal muscle nuclei are identical to those shown.
critically examined but our biochemical criteria suggest that such loss, if it occurs, is minimal.
the DNA in the purified nuclear fraction (buffer H-buffer I interface). Another 2030% of the nuclei had sedimented into the buffer I pad or pelleted at the bottom of the tube. These nuclei could also be recovered and were biochemically indistinguishable from those collected at the buffer H-buffer I interface.
Isolation of single-cell (myoblast + fibroblast) nuclei Phase contrast photomicrographs of purified myoblast nuclei are shown in fig: 2. These nuclei banded at the buffer H-buffer I interface and were judged free of visible Preparation of chromatin contaminants after extensive microscopic The purified nuclear fractions from both examination. Since the non-ionic detergent myotubes and myoblasts were used to preNP-40 is known to break most whole cells pare “purified” chromatin following standand solubilize most cytoplasmic compo- ard procedures. The spectral (fig. 3) and nents [6], it is not surprising that these ele- chemical (table 1) properties of the purified ments were not found as contaminants in chromatin are similar to those reported for the purified nuclear fraction. chromatin purified from a variety of tissues Nuclei which still contained attached and cells [5, 19,241, including several chick cytoplasmic “tags” floated on buffer H and tissues and 1l-day chick embryos. were removed by aspiration before the purified nuclei. The purified myoblast nuclei DISCUSSION were generally larger and more spherical than the myotube nuclei, but otherwise they In vitro myogenesis has been well characterized [3]. Although cultures of pure prewere morphologically indistinguishable from either myotube or whole muscle nuclei sumptive myoblasts are currently impossible to prepare, cell populations isolated (fig. 1 and references [2, 91). Biochemical from 1Zday chick embryo legs by differproperties of the isolated nuclei (table 1) attest to their high degree of purity and con- ential trypsinization and selective plating firm the visual analysis. The procedure re- procedures are over 90 % presumptive myosulted in the recovery of from 15-32 % of blasts-the remaining cells of the popuE.T/J Cdl
Rrs
117 (1978)
Isolation of myotube and myoblast nuclei
69
lation have a tibroblast morphology and most highly differentiated chick skeletal may either remain biochemically and mor- muscle cultures. Recently, however, a phologically “undifferentiated” or initiate method has been reported which effectively chondroitin sulfate synthesis typical of ma- accomplishes this goal by killing all replicatture chondrocytes. The functional element ing cells (almost all of the single-cell popuof differentiated muscle, the non-dividing lation) with cytosine arabinoside [23]. Our multinucleate cell (the myotube) is formed method, while not as simply performed, has by cytoplasmic fusion of mononucleated the advantages that it removes all single precursor cells (the post-mitotic myo- cells (not just the replicating ones), is faster blasts). (cytosine arabinoside requires 2 days’ inFusion, under appropriate conditions, in- cubation to be fully effective), and does not volves 60-80% of the cells, never reaching require that the myotube nuclei be sub100% even when the culture is initiated jected to unusual chemical treatment (cytowith a pure clone of myogenic cells. Once sine arabinoside will, of necessity, affect multinucleate cells appear, they rapidly DNA repair processes in even non-replicatform elongate myotubes. Fusion of 80% of ing nuclei). the cells can occur within 12 h, and is acOur nuclear isolation and purification companied by a cessation of DNA synthe- procedures were originally based on the sis and the first evidence of accumulation earlier work of Edelman [2] on the isolation of muscle-specific proteins [22]. Thus a dif- of rat skeletal muscle nuclei and numerous ferentiated muscle cell culture consists of reports of the utilization of non-ionic detertwo main cellular elements: (1) the multi- gents for the isolation of nuclei from single nucleate muscle fibers formed by fusion cells [6-g, 13, 161.We found, as did Edelof the mononucleated myoblasts; and (2) man, that although preincubation of myothe remaining mononucleated cells which tubes and cells in a Ca2+-containing buffer include postmitotic myoblasts, replicat- is advantageous, yields of clean nuclei are drastically reduced if the actual homoing presumptive myoblasts, and replicating mononucleated cells which are operation- genization buffer contains Ca2+. Furthermore, there is a critical balance between the ally indistinguishable from fibroblasts [4]. Our interest in the isolation of nuclei from Mg2+ and EDTA concentrations that canmyotubes and myoblasts results from our not be substantially exceeded in either diinvestigations into the role of nuclear non- rection. The lowered ionic strength of the histone proteins and chromatin structure in myoblast nuclear isolation buffers results in the regulation of chick skeletal muscle myo- cleaner nuclei that show a reduced tengenesis. The first mandatory step for an dency to clump. Most procedures for the isolation of nuanalysis of either nuclear proteins or chromatin structure is the preparation of highly clei employ a low speed (700 g, 10 min) .purified nuclei free of cytoplasmic and centrifugation step to obtain a crude nuclear pellet free of many cytoplasmic ormembrane contamination. When we initiated these efforts no meth- ganelles. In our hands, this step resulted in ruptured and badly clumped nuclei which ods were available to isolate the multinucleate syncytia (myotubes) from the re- could not be dissociated without further numaining undifferentiated single cells (myo- clear rupture. Therefore we proceeded blasts and fibroblasts) present in even the directly from homogenization to floatation Exp Cell Res 117 (1978)
70
David et al.
in high-density sucrose. Likewise, in both the isolation of myotube and myoblast nuclei, the sucrose pad is chosen to result in the collection of the nuclei at a sucrose interface rather than as a pellet since the isolated nuclei are quite fragile and, once pelleted, are very difficult to resuspend without considerable nuclear lysis. Our criteria for nuclear purity are both visual and chemical. All nuclear preparations are free from visible contamination by single cells, myotube fragments, plasma membrane, cytoplasmic tags, or various free cytoplasmic constituents (mitochondria, myofilaments, membraneous structures). Furthermore, the protein/DNA and RNA/DNA ratios (which are routinely used by many laboratories to determine nuclear purity) indicate a very low or non-existant cytoplasmic contamination. The purified nuclei are morphologically similar (at the light microscope level) to nuclei isolated from whole skeletal muscle tissue and nuclei observed in situ in fixed, sectioned, muscle tissue. Chromatin is generally prepared from purified nuclei by nuclear lysis in hypotonic buffer, followed by limited mechanical shear and sucrose density gradient sedimentation [lo]. The latter step, resulting in a clear gelatinous chromatin pellet, is designed to remove nuclear membranes and ribonucleoprotein particles normally present in the “crude” chromatin preparation [ 1, 111.Our results indicate that nuclei prepared by the procedure described in this communication are entirely suitable for the preparation of purified chromatin by standard techniques. This research was supported, in part, by grants from the Muscular Dystrophy Association of America, the Missouri Heart Association, and the Research Council of the Graduate School, University of Missouri, Columbia (Biomedical Research Support Grant RR07053. NIH). Exp Cell Res 117 (1978)
REFERENCES 1. Bhorjee, J S & Pederson, R, Biochemistry 12 ( 1973)2766. 2. Edelman, J C, Edelman, P M, Knigge, K M & Schwartz, I L, J cell bio127 (1%5) 365. 3. Holtzer, H, Sanger, J W, Ishikawa, H & Strachs, K, Cold Spring Harbor symp quant biol 37 (1972) 549. 4. Holtzer, H, Strachs, K, Biehl, J, Sonlys, A P & Ishikawa, H, Science 188(1975) 943. 5. Huang, R C C & Huang, P C, J mol bio139 (1969) 365. 6. Lerner, R A, Meinke, W & Goldstein, D A, Proc natl acad sci US 68 (1971) 1212. 7. Levy, R, Levy, S, Rosenberg, S A & Simpson, R T. Biochemistrv 12 (1973) 224. 8. Magliozzi, J, Pttro: D,‘Lin, C, Ortman, R & Dounce. A L. EXD cell res 67 (1971) 111. 9. Marchok, A C &Wolff, J A, Biochim biophys acta 155(1%8) 378. 10. Marushige, K & Bonner, J, J mol biol 15 (1966) 160. 11. Marushige, K, Brutlage, D & Bonner, J, Biochemistry 7 (1968) 3149. 12. Pederson, T, Cell biology, biological handbooks (ed P L Altmann & D D Katz) vol. 1, p. 364. Federation of American societies for experimental biology, Bethesda, Md (1976). 13. Penman, S, J mol biol 17 (1966) 117. 14. Puck, T T, Cieciura, S J & Robinson, A, J exp med 108(1958) 945. 15. Richards, G M, Anal biochem 57 (1974) 369. 16. Rovera, G & Baserga, R, J cell physiol 77 (1971) 201. 17. Rutter, W J, Methods in developmental biology (ed F H Wilt & N K Wessells) p. 671. Thomas Y Crowell, New York (1%7). 18. Schneider, W C, Methods in enzymology (ed S P Colowick & N 0 Kaplan), vol. 3, p. 680. Academic Press, New York (1967). 19. Seligy, V & Miyagi, M, Exp cell res 58 (1969) 27. 20. Silakova, A I, Polishchuk, S N t Konoplitskaya, K L, Tsitologiia 12 (1970) 53. 21. Stein, G S & Stein, J L, Cell biology, biological handbooks (ed P L Altmann & D D Katz) vol. 1, p. 379. Federation of American societies for experimental biology, Bethesda, Md (1976). 22. Stockdale, F E & Holtzer, H, Exp cell res 24 (l%l) 508. 23. Trotter, J A & Nameroff, M, Dev biol 49 (1976) 548. 24. Vidali, G, Cell biology, biological handbooks (ed P L Altmann & D D Katz) vol. 1, p. 393. Federation of American societies for experimental biology, Bethesda, Md (1976).
Received February 2 1, 1978 Revised version received May 26, 1978 Accepted May 29, 1978
Experimental Cell Research 117 (1978) 63-70
ISOLATION MYOBLAST
AND PURIFICATION NUCLEI CHICK
OF MYOTUBE
FROM CULTURES SKELETAL
AND
OF EMBRYONIC
MUSCLE
JOHN D. DAVID, ROBERT L. FREDRICKSON
and GARY R. PETERSON
University of Missouri, Columbia, MO 65201, USA
SUMMARY Methods are described for the preparation of purified myotubes from embryonic chick skeletal muscle cultures and the preparation of purified nuclei from both myotubes and myoblasts. Myotubes are released from the culture dish by digestion of their collagen substratum with collagenase, and purified by sucrose density gradient sedimentation. Nuclei are prepared from the isolated myotubes by controlled homogenization in Ca*+-free medium and sedimentation through 2.1 M sucrose. Nuclei are prepared from cultured myoblasts in a similar fashion, with the inclusion of the non-ionic detergent NP-40 in the homogenization medium and sedimentation through 2.4 M sucrose. Phase contrast microscopic examination showed that the nuclear preparations are free of visible cytoplasmic contamination, and are morphologically similar to nuclei observed in situ. Biochemical assays (protein/DNA and RNA/DNA ratios) confirm the purity of the nuclear preparations. Both nuclear preparations have been used to prepare purified chromatin which has spectral and chemical properties similar to those reported for chromatin purified directly from several chick tissues.
Although nuclei have been isolated from a wide variety of animal tissues and cultured cells [ 121,there have been very few reports of nuclear preparations from whole skeletal muscle [2, 9, 201, and no reports of nuclear isolation from cultured skeletal muscle cells. In addition, there is no reported method to separate the two basic cell types in a differentiating muscle cell culture, the single cells (myoblasts and tibroblasts), and the syncytial myotubes. This report describes (1) the isolation of the myotubular component of the developing cultures, free from significant single-cell contamination; (2) methods to isolate highly purified nuclei from chick skeletal muscle myoblasts, and from isolated myotubes. 5-781819
MATERIALS
AND METHODS
Materials Tissue culture dishes were obtained from Falcon Plastics, BioQuest, Cockeysville, Md. Reagents were obtained from the following sources: 6ulbecco’s Modified Eagle’s Medium and Horse Serum (Grand Island Biological Co., Grand Island, N.Y.); Collagenase (Code CLS or CLS III, EC 3.4.24.3) Worthington Biochemical Corp., Freehold, N.J.); Enzyme-g&de Tris (Trihydroxy methylamino methane) and Ultra Pure Sucrose (Schwarz&Iann Biochemicals, Orangeburg, N.Y.); NP-40 (Particle Data Labs, Evanston, Ill.); Harris-Lillie Hematoxylin Stain Solution (Fisher Scientific Pittsburgh, Pa); EDTA (Ethylenediaminetetraacetic acid) (Sigma Chemical Co., St Louis, MO). All other chemicals were reagent grade (Fisher Scientific, Pittsburgh, Pa).
Culture conditions Myoblasts from 12-day-old white leghorn chick embryo thigh muscle were obtained by a selective trypsinization and plating procedure (to be described in E.rp Cdl Rr., I I7 ( 19781
64
David et al.
detail in a later publication) which produces a population of cells 95 % myoblast and 5 % fibroblast in morphology and fusion-capability. The cells were plated on collagen-coated 150 mm* tissue culture dishes at 8x 108cells/plate in Dulbecco’s Modified Eagle’s Medium supplemented with 10% horse serum and 3 % chick embryo extract. Medium was changed on days 1 and 3.
Isolation
of myotubes
Four&v-old mates were rinsed once with Puck’s Saline d [ 141and once with buffer A (0.125 M sucrose, 10 mM Tris OH 7.5. 5 mM M&l,. 0.5 mM EDTA. 2 mM CaCl,,* 0.15 M KCl). T& milliliters of buffer A containing 0.01% collagenase were added to each plate and the plates rocked gently (Lab-Line platform rocker. 20” anaular disnlacement. 1 strokelsec) at 30°C until the cells-and mybtubes released from the plate. After the addition of 0.5 ml of 0.2 M EDTA (oH 9.0). the plates were rocked for an additional mint& ’ The cell-myotube suspension was pipetted up-anddown three times through a 10 ml pipet with a 2 mm oriface in order to disperse clumps of single cells and to free the myotubes of any attached single cells. This step completely freed the myotubes of attached single cells while doing little if any damage to the myotubes. The disnersed susnension (10 ml) was lavered on ton of a preformed discontinuous sucrose gradient of lb ml buffer B (0.25 M sucrose, 10 mM Tris pH 7.5, 10 mM MgC&, 5 mM EDTA, 0.15 M KCl) and 10 ml buffer C (0.32 M sucrose, 10 mM Tris nH 8.0. 5 mM MgCl,, 5 mM EDTA, 0.15 M KCl), both at s”C. The preparation was centrifuged at 16 g for 5 min at 5”C, the tubes rotated at 180” and centrifuged for an additional 5 min at 16g. This rotation is necessitated bv the tendency of large-myotubes to stick to the side walls of the glass centrifuge tubes at these low soeeds. Under these conditions; all but the smallest myotubes are found in the pellet, while the single cells remain in the upper l/3 of the tube.
Isolation
of myotube nuclei
The isolated myotubes were resuspended in a small volume of buffer C and homogenized 20 times in a Dounce homogenizer with the tight-fitting pestle. The homogenate was centrifuged at 100 g for 10 min. The pellet was resuspended in a small volume of buffer C, rehomogenized as above, and this second homogenate centrifuged at 100g for 10 min. The pellet, which contained unbroken myotube fragments and any single cells which contaminated the myotube preparation, was discarded. The supematants were combined and adjusted to 1.9 M in sucrose by the addition of 3.2 vol of buffer D (2.4 M sucrose, 10 mM Tris pH 8.0, 5 mM MgCl,, 5 mM EDTA, 0.15 M KCl). The crude nuclear suspension was dispersed in a Dounce homogenizer by 5 strokes of the loose-fitting pestle. The susnension was lavered over 1.5 ml of buffer E (2.1 M sucrose, 10 mM Tris pH 8.0, 5 mM MgCl,, 5 mM EDTA, 0.15 M KCl) and 1.5 ml of buffer D in a cellulose nitrate centrifuge tube. The nuclei were sedimented at 64000 I! for 90 min in the Beckman SW41 rotor. Clean nuclei collected at the buffer Dbuffer E interface. All operations were performed at 5°C.
Isolation
of myoblast (single-cell)
nuclei
One-dav-old mates were rinsed once with Pucks saline G [14] and once with buffer F (10 mM Tris pH 8.0, 5 mM M&l,. 5 mM EDTA. 0.15 M KCl). Ten milliliters of b\ff& F were added to each plate and the cells removed with a rubber noliceman. The cells were concentrated by centrifugation at 600 g for 10 min and suspended in buffer G (0.25 M sucrose, 10 mM Tris oH 8.0, 5 mM MgCl*, 5 mM EDTA, 50 mM KCl). This suspension was homogenized 10 times in a Dounce homogenizer with the tight-fitting pestle. Although this resulted in the rupture of most of the cells, most of the nuclei were surrounded by cellular debris or had “cytoplasmic tags” attached. The non-ionic detergent NP-40 was added to 0.5 % final concentration and the preparation rehomogenized twenty times. This step dispersed the cellular debris and removed most of the “cytoplasmic tags”. The crude nuclear suspension was adiusted to 2.1 M in sucrose bv the addition of 6.15 vol of buffer H (2.4 M sucrose: 10 mM Tris pH 8.0, 5 mM M&l,. 5 mM EDTA. 50 mM KCl) and dispersed in-a Dounce homogenizer by 5 strokes of the loose-fittina oestle. The final susoension was lavered over 1.5 ml-buffer H and 1.5 ml buffer I (2.8 M sucrose, 10 mM Tris pH 8.0, 5 mM MgC&, 5 mM EDTA, 50 mM KCl) in a cellulose nitrate centrifuge tube. The nuclei were sedimented at 64 000 g for 90 min in the Beckman SW41 rotor. Clean nuclei collected at the buffer H-buffer I interface and in the buffer I laver. All operations were performed at 5°C.
Isolation of nuclei from whole skeletal muscle Thigh muscle from 12-day-old chick embryos was excised free of skin and bone in a chilled Petri dish containing Pucks saline G [ 141.The muscle was finely minced with scissors, rinsed three times with buffer C, and homogenized 20 times in buffer C with a Dounce homogenizer with the loose-fitting pestle at 5°C. The homogenate was then rehomogenized 20 times with the tight-fitting pestle. The remainder of the procedure is identical to that used to prepare myotube nuclei.
Chromatin
preparation
and purification
Isolated nuclei were diluted to 0.25 M sucrose with buffer G and pelleted by centrifugation at 64 000 g for 90 min. The pellet was rinsed gently with 10 mM Tris pH 8.0 and then resuspended in 1 ml of the same buffer. The nuclei were allowed to swell for 12-18 h. This preparation, termed “crude chromatin” was dispersed by vigorous homogenization in a Dounce tissue homogenizer fitted with a tight nestle. The dispersed crude chromatin was diluted with 3 vol of 10 mM Tris pH 8.0 and layered over 2 ml 1.7 M sucrose, 10 mM Tris pH 8.0 in a Beckman SW50.1 centrifuge tube. The interface was mixed gently and the chromatin sedimented at 58 000 g for 3 hy The clear gelatinous pellet was resuspended in 10 mM Tris pH 8.0 by sonication and termed “purified chromatin” [lo]. All operations were performed at 5°C.
Isolation of myotube and myoblast nuclei
Fig. 1. (a) Phase contrast photomicrograph of a rep-
resentative field of purified myotubes. Nuclei are slightly elongated and contain easily recognizable nucleoli. The myotubes are relatively fragile at this point and are often seen to “leak” fluid cytoplasm, although
65
nuclei rarely escape. Cross striations characteristic of myofilaments are visible at several points within the myotube. Some contaminating single cells remain. (b) As in (a) except myotube is smaller, less highly branched, and more elongated. Bar, 100pm.
DNA, RNA, and protein determinations DNA was determined using the paraldehyde-diphenylamine procedure [ 151.RNA was determined by the orcinol procedure [ 181.Protein was determined by the micro-Lowry method of Rutter [ 171.Alternatively, DNA and protein were determined spectrophotometrically [lo]. Results using both methods were not significantly different.
RESULTS
Isolation of multinucleate syncytia (myotubes) Phase contrast photomicrographs of resuspended myotube pellets are shown in fig. 1. These preparations were judged homoFixation and staining An aliquot of the myotube preparation was placed on geneous by visual observation. This was a clean glass slide and allowed to air-dry. The slide confirmed by monitoring the ratio of myowas immersed in fixative A (10% form01 in phosphatetube nuclei to single-cell nuclei in fixed and buffered saline) for 1 h followed by fixative B (acetic acid : methanol, 1 : 3) for 1 h. The slide was rinsed in stained preparations. Although some single water and immersed in Harris-Lillie hematoxylin stain solution for 15 min and developed with 1% am- cells do appear in the myotube preparation, monium hydroxide for 5 min. After rinsing with water at no time do they represent more than a coverslip was applied and the slide observed while 0.4% of the total nuclei counted. still wet. E.rp Cdl
Rc\
I17 lIY7Xl
66
David et al.
Fig. 2. (a), (b) Phase contrast photomicrographs of representative fields of isolated myotube nuclei collected from the buffer D-buffer E interface. Although the majority of the nuclei are intact and slightly elongated, some appear slightly damaged and/or crenated. All contain one or two visible nucleoli and none carry visible cytoplasmic “tags”. (c), (d) Phase contrast
photomicrographs of representative fields of isolated myoblast nuclei collected from the buffer H-buffer I interface. These nuclei are generally smoother in outline (less crenated) than isolated myotube nuclei. All contain one or two visible nucleoli and no visible cytoplasmic “tags”. Bar, 10 pm.
Using the procedure as described, single cells remain in the upper l/3 to l/2 of the sucrose gradient, small myotubes (10-20 nuclei) sediment into the bottom l/3 of the gradient but do not pellet, and the large myotubes can be recovered from the pellet. Some myotubes are disrupted by this treatment and the resultant debris is found in the upper layers of the gradient. If desired, the single cells and/or small myotubes can be recovered by differential centrifugation.
Isolation of myotube nuclei Phase contrast photomicrographs of puritied myotube nuclei are shown in fig. 2. These nuclei banded at the buffer E-buffer D interface and were judged free of single cell, mitochondrial, myotubular, membraneous and other debris by extensive visual observation. There were no observable cytoplasmic “tags” attached to these nuclei. The “dirty” nuclei present all floated on buffer E and were easily re-
E.vp Cc//
Rr.\
117 (IY78)
Isolation
Table 1. Nucleic acid and protein whole skeletal musclea
content
of myotube and myoblast nuclei
of fractionated
myoblasts,
myotubes,
67 and
DNA Fraction Myobla& Homogenate Nuclei Purified chromatin Myotube* Homogenate Nuclei Purified chromatin Whole skeletal muscleC Nuclei Purified chromatin Whole skeletal muscled Homogenate Nuclei
(Pi?)
Percent of homogenate
Protein (pg)
Protein DNA
RNA DNA
262f54 82+22
100 31&8
2 16Of509 262+62
8.24k2.01 3.19+0.25
1.41*0.33 0.10+0.04
172+22
2.6lkO.03
0.084+0.02
7 680+710 213f 14
28.lk8.4 3.43kO.12
1.60+0.84 0.11+0.03
12lfl2
2.47f0.21
0.10~0.02
3.52kO.20
0.43+0.05
2.58kO.11
0.06~0.02
452+ 126 3.44kO.24
1.73kO.33 0.10~0.02
66_+18
25_+8
273f55 62f8.6
100 23f7
49*9.1
IS&3
a Results given as mean k S.D. b Average of 4 expts.
100 15
c Average of 2 expts. d As reported by Edelman et al. [2].
moved before the purified nuclei. Small is the DNA/RNA ratio which is 16-fold myotube fragments and unbroken single lower than that of the original homogenate, cells floated to the top of the tube and were comparable to values for nuclei isolated also easily removed. Purified nuclei ob- from whole skeletal muscle (table l), and served with phase contrast optics were in- also at the low end of the range reported for tact, generally oblong, and all had one or nuclei isolated from a variety of other cells two distinct nucleoli in an amorphous nu- and tissues [2, 211. We have recovered 18-30% of the DNA cleoplasm. They were morphologically indistinguishable from nuclei isolated directly in the purified nuclear fraction. Most of the remainder of the DNA is found in nuclei from chick embryo thigh muscle [2, 91. Some biochemical properties of the iso- associated with varying amounts of cellular lated myotube nuclei are shown in table 1. debris (mostly incompletely ruptured myoThe protein/DNA ratio of isolated myotube tubes). More extensive homogenization will nuclei agrees well with protein/DNA ratios release more nuclei, but also results in infor nuclei isolated from whole skeletal mus- creased numbers of visually damaged nucle as determined by Edelman [2] and our- clei. These homogenization conditions do selves (table l), and is at the low end of the not rupture single cells and thus all nuclei range reported for nuclei isolated from a recovered originated in myotubes. The poswide variety of other cells and tissues [2, sible loss of nuclear protein and RNA dur211. A further indication of chemical purity ing the isolation procedure has not been Exp Cell Res l/7(1978)
68
David et al.
Fig. 3. Abscissa: wavelength (nm); ordinate: ab-
sorbance. Absorbance profiles of “crude” and “purified” chromatin prepared from isolated myotube nuclei. 0- --0, “Crude” chromatin; X-X, “purified” chromatin. Absorbance profiles of chromatin prepared from myoblast nuclei and whole skeletal muscle nuclei are identical to those shown.
critically examined but our biochemical criteria suggest that such loss, if it occurs, is minimal.
the DNA in the purified nuclear fraction (buffer H-buffer I interface). Another 2030% of the nuclei had sedimented into the buffer I pad or pelleted at the bottom of the tube. These nuclei could also be recovered and were biochemically indistinguishable from those collected at the buffer H-buffer I interface.
Isolation of single-cell (myoblast + fibroblast) nuclei Phase contrast photomicrographs of purified myoblast nuclei are shown in fig: 2. These nuclei banded at the buffer H-buffer I interface and were judged free of visible Preparation of chromatin contaminants after extensive microscopic The purified nuclear fractions from both examination. Since the non-ionic detergent myotubes and myoblasts were used to preNP-40 is known to break most whole cells pare “purified” chromatin following standand solubilize most cytoplasmic compo- ard procedures. The spectral (fig. 3) and nents [6], it is not surprising that these ele- chemical (table 1) properties of the purified ments were not found as contaminants in chromatin are similar to those reported for the purified nuclear fraction. chromatin purified from a variety of tissues Nuclei which still contained attached and cells [5, 19,241, including several chick cytoplasmic “tags” floated on buffer H and tissues and 1l-day chick embryos. were removed by aspiration before the purified nuclei. The purified myoblast nuclei DISCUSSION were generally larger and more spherical than the myotube nuclei, but otherwise they In vitro myogenesis has been well characterized [3]. Although cultures of pure prewere morphologically indistinguishable from either myotube or whole muscle nuclei sumptive myoblasts are currently impossible to prepare, cell populations isolated (fig. 1 and references [2, 91). Biochemical from 1Zday chick embryo legs by differproperties of the isolated nuclei (table 1) attest to their high degree of purity and con- ential trypsinization and selective plating firm the visual analysis. The procedure re- procedures are over 90 % presumptive myosulted in the recovery of from 15-32 % of blasts-the remaining cells of the popuE.T/J Cdl
Rrs
117 (1978)
Isolation of myotube and myoblast nuclei
69
lation have a tibroblast morphology and most highly differentiated chick skeletal may either remain biochemically and mor- muscle cultures. Recently, however, a phologically “undifferentiated” or initiate method has been reported which effectively chondroitin sulfate synthesis typical of ma- accomplishes this goal by killing all replicatture chondrocytes. The functional element ing cells (almost all of the single-cell popuof differentiated muscle, the non-dividing lation) with cytosine arabinoside [23]. Our multinucleate cell (the myotube) is formed method, while not as simply performed, has by cytoplasmic fusion of mononucleated the advantages that it removes all single precursor cells (the post-mitotic myo- cells (not just the replicating ones), is faster blasts). (cytosine arabinoside requires 2 days’ inFusion, under appropriate conditions, in- cubation to be fully effective), and does not volves 60-80% of the cells, never reaching require that the myotube nuclei be sub100% even when the culture is initiated jected to unusual chemical treatment (cytowith a pure clone of myogenic cells. Once sine arabinoside will, of necessity, affect multinucleate cells appear, they rapidly DNA repair processes in even non-replicatform elongate myotubes. Fusion of 80% of ing nuclei). the cells can occur within 12 h, and is acOur nuclear isolation and purification companied by a cessation of DNA synthe- procedures were originally based on the sis and the first evidence of accumulation earlier work of Edelman [2] on the isolation of muscle-specific proteins [22]. Thus a dif- of rat skeletal muscle nuclei and numerous ferentiated muscle cell culture consists of reports of the utilization of non-ionic detertwo main cellular elements: (1) the multi- gents for the isolation of nuclei from single nucleate muscle fibers formed by fusion cells [6-g, 13, 161.We found, as did Edelof the mononucleated myoblasts; and (2) man, that although preincubation of myothe remaining mononucleated cells which tubes and cells in a Ca2+-containing buffer include postmitotic myoblasts, replicat- is advantageous, yields of clean nuclei are drastically reduced if the actual homoing presumptive myoblasts, and replicating mononucleated cells which are operation- genization buffer contains Ca2+. Furthermore, there is a critical balance between the ally indistinguishable from fibroblasts [4]. Our interest in the isolation of nuclei from Mg2+ and EDTA concentrations that canmyotubes and myoblasts results from our not be substantially exceeded in either diinvestigations into the role of nuclear non- rection. The lowered ionic strength of the histone proteins and chromatin structure in myoblast nuclear isolation buffers results in the regulation of chick skeletal muscle myo- cleaner nuclei that show a reduced tengenesis. The first mandatory step for an dency to clump. Most procedures for the isolation of nuanalysis of either nuclear proteins or chromatin structure is the preparation of highly clei employ a low speed (700 g, 10 min) .purified nuclei free of cytoplasmic and centrifugation step to obtain a crude nuclear pellet free of many cytoplasmic ormembrane contamination. When we initiated these efforts no meth- ganelles. In our hands, this step resulted in ruptured and badly clumped nuclei which ods were available to isolate the multinucleate syncytia (myotubes) from the re- could not be dissociated without further numaining undifferentiated single cells (myo- clear rupture. Therefore we proceeded blasts and fibroblasts) present in even the directly from homogenization to floatation Exp Cell Res 117 (1978)
70
David et al.
in high-density sucrose. Likewise, in both the isolation of myotube and myoblast nuclei, the sucrose pad is chosen to result in the collection of the nuclei at a sucrose interface rather than as a pellet since the isolated nuclei are quite fragile and, once pelleted, are very difficult to resuspend without considerable nuclear lysis. Our criteria for nuclear purity are both visual and chemical. All nuclear preparations are free from visible contamination by single cells, myotube fragments, plasma membrane, cytoplasmic tags, or various free cytoplasmic constituents (mitochondria, myofilaments, membraneous structures). Furthermore, the protein/DNA and RNA/DNA ratios (which are routinely used by many laboratories to determine nuclear purity) indicate a very low or non-existant cytoplasmic contamination. The purified nuclei are morphologically similar (at the light microscope level) to nuclei isolated from whole skeletal muscle tissue and nuclei observed in situ in fixed, sectioned, muscle tissue. Chromatin is generally prepared from purified nuclei by nuclear lysis in hypotonic buffer, followed by limited mechanical shear and sucrose density gradient sedimentation [lo]. The latter step, resulting in a clear gelatinous chromatin pellet, is designed to remove nuclear membranes and ribonucleoprotein particles normally present in the “crude” chromatin preparation [ 1, 111.Our results indicate that nuclei prepared by the procedure described in this communication are entirely suitable for the preparation of purified chromatin by standard techniques. This research was supported, in part, by grants from the Muscular Dystrophy Association of America, the Missouri Heart Association, and the Research Council of the Graduate School, University of Missouri, Columbia (Biomedical Research Support Grant RR07053. NIH). Exp Cell Res 117 (1978)
REFERENCES 1. Bhorjee, J S & Pederson, R, Biochemistry 12 ( 1973)2766. 2. Edelman, J C, Edelman, P M, Knigge, K M & Schwartz, I L, J cell bio127 (1%5) 365. 3. Holtzer, H, Sanger, J W, Ishikawa, H & Strachs, K, Cold Spring Harbor symp quant biol 37 (1972) 549. 4. Holtzer, H, Strachs, K, Biehl, J, Sonlys, A P & Ishikawa, H, Science 188(1975) 943. 5. Huang, R C C & Huang, P C, J mol bio139 (1969) 365. 6. Lerner, R A, Meinke, W & Goldstein, D A, Proc natl acad sci US 68 (1971) 1212. 7. Levy, R, Levy, S, Rosenberg, S A & Simpson, R T. Biochemistrv 12 (1973) 224. 8. Magliozzi, J, Pttro: D,‘Lin, C, Ortman, R & Dounce. A L. EXD cell res 67 (1971) 111. 9. Marchok, A C &Wolff, J A, Biochim biophys acta 155(1%8) 378. 10. Marushige, K & Bonner, J, J mol biol 15 (1966) 160. 11. Marushige, K, Brutlage, D & Bonner, J, Biochemistry 7 (1968) 3149. 12. Pederson, T, Cell biology, biological handbooks (ed P L Altmann & D D Katz) vol. 1, p. 364. Federation of American societies for experimental biology, Bethesda, Md (1976). 13. Penman, S, J mol biol 17 (1966) 117. 14. Puck, T T, Cieciura, S J & Robinson, A, J exp med 108(1958) 945. 15. Richards, G M, Anal biochem 57 (1974) 369. 16. Rovera, G & Baserga, R, J cell physiol 77 (1971) 201. 17. Rutter, W J, Methods in developmental biology (ed F H Wilt & N K Wessells) p. 671. Thomas Y Crowell, New York (1%7). 18. Schneider, W C, Methods in enzymology (ed S P Colowick & N 0 Kaplan), vol. 3, p. 680. Academic Press, New York (1967). 19. Seligy, V & Miyagi, M, Exp cell res 58 (1969) 27. 20. Silakova, A I, Polishchuk, S N t Konoplitskaya, K L, Tsitologiia 12 (1970) 53. 21. Stein, G S & Stein, J L, Cell biology, biological handbooks (ed P L Altmann & D D Katz) vol. 1, p. 379. Federation of American societies for experimental biology, Bethesda, Md (1976). 22. Stockdale, F E & Holtzer, H, Exp cell res 24 (l%l) 508. 23. Trotter, J A & Nameroff, M, Dev biol 49 (1976) 548. 24. Vidali, G, Cell biology, biological handbooks (ed P L Altmann & D D Katz) vol. 1, p. 393. Federation of American societies for experimental biology, Bethesda, Md (1976).
Received February 2 1, 1978 Revised version received May 26, 1978 Accepted May 29, 1978
