Characterization of minicells (nuclei) obtained by cytochalasin enucleation

Experimental Cell Research 87 (1974) 365-311

CHARACTERIZATION

OF MINICELLS

BY CYTOCHALASIN T. EGE,l H. HAMBERG,

U. KRONDAHL,l

(NUCLEI)

OBTAINED

ENUCLEATION J. ERICSSON,2 and N. R. RINGERTZ’

IInstitute for Medical Cell Research and Genetics, Medical Nobel Institute, Karolinska Institutet, S-104 01 Stockholm 60, and ZDepartment of Pathology, Sabbatsberg Hospital, S-113 82 Stockholm, Sweden

SUMMARY By centrifuging monolayers of rat L6 myoblasts adhering to plastic discs in the presence of cytochalasin it is possible to enucleate close to 100 % of the cells. The nuclei drawn out of the cells are surrounded by a narrow rim of cytoplasm and an intact plasma membrane and therefore are ‘minicells’ stripped of most of the original cytoplasm. In addition to minicells, the pellet material on the bottom of the centrifuge tubes contains fragments of cytoplasm and occasional intact cells. The number of intact cells can be drastically reduced by precentrifugation of the monolayers prior to enucleation so as to remove loosely attached cells from the plastic discs. Biochemical analysis of minicell pellets show that at least 65-75 % of the original cytoplasm is removed during enucleation. Microinterferometric dry mass measurements on individual minicells where free cytoplasmic fragments (without nuclei) can be excluded indicate that at least 8Ck90% of the cytoplasm is removed. Most of the minicells are impermeable to trypan blue and are able for some time after enucleation to incorporate precursors into nucleic acids and proteins. Under normal tissue culture conditions the minicells fail, however, to regenerate a cytoplasm and to multiply. Instead the minicells undergo a gradual decrease in dry mass and most seem to be dead 36 h after enucleation. The minicells can, however, be rescued by fusion with enucleated cytoplasms and may therefore be used to reconstitute nucleated cells.

The cytochalasin enucleation technique [l, 21 DNA synthesis and form nucleoli [6]. represents a new approach to cell fractionaAlthough the chick nucleus prolongs the tion and may also be a valuable tool in survival of the cytoplasm somewhat [6] there somatic cell genetics. If for instance a new is as yet no indication that this type of renucleus can be introduced into the enucleated constituted cell divides so as to give rise cytoplasms so as to produce a viable ‘re- to a viable progeny. constituted’ cell this would open possibilities The present investigation was undertaken of analysing the role of nucleocytoplasmic in order to examine whether or not the interaction in the control of gene activity enucleation procedure developed by Prescott and cell differentiation as well as many other [2] could be used to prepare nuclei suitable problems. Attempts in this direction have for reconstitution experiments. Preliminary been made by fusing chick erythrocyte nuclei results [3, 71 showed that these nuclei are into enucleated cytoplasms [3-61. Chick surrounded by a narrow rim of cytoplasm erythrocyte nuclei introduced into enucleated and an intact plasma membrane which mouse L cell cytoplasms resume RNA and carries receptors for at least one virus capable Exptl Cell Res 87 (1974)

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of inducing cell fusion [S]. In the present work we will refer to this type of nuclear preparation as ‘minicells’ in order to distinguish these structures from detergentisolated nuclei which lack a plasma membrane and may be free from cytoplasm. The specific aim of the present study was to characterize the minicells with respect to cytoplasmic contamination apd viability. MATERIALS AND METHODS

considered to be dead or damaged. Because of the rounded shape of all cells at this stage it was not possible to determine with certainty whether the structures staining with trypan blue were damaged cells or minicells. However, since in most preparations the contamination with intact cells as judged by interferometry and long term survival was 0.3-3.0 % while the percentage of trypan blue stained structures was 5-30%, most of the damaged cells were clearly minicells. The viability was also tested by following the fate and dry mass changes of individual minicells and cells maintained in tissue culture as described in the Results section. The ability of minicells to incorporate precursors into nucleic acids and proteins represented another viability test and will be discussed below.

Cells

Contamination with intact cells

The L6 line of rat myoblasts was used. The cells were cultured in Falcon plastic dishes on Eagle’s MEM supplemented with iO% calf serum, at low cell densities and with frequent subcultivations so as to keep myotube formation at a low level. For enucleation, cells were seeded on round plastic discs (0 2.5 cm).which had been punched out of Falcon tissue culture dishes. After seeding 3 x lo6 cells in a 90 mm dish containing 8 plastic discs a nearly confluent monolayer of cells formed in 12 h. These discs were then used for enucleation.

A convenient method for the routine testing of the number of intact cells contaminating the minicell preparation was to plate pellet material equivalent to 10 000 trypan blue negative cells suspended in Eagle’s MEM, 10 % calf serum in a 60 mm tissueculture dish and then to count 24 h later the number of cells which had been capable of flattening out and making a solid attachment to the plastic. The intact cells flatten out on the plastic and these cells become well attached. In pure populations of intact cells r 90 % attached well under the conditions used. Minicells, however, did not attach well and when the dishes with pellet material were washed with PBS the minicells came off the plastic surface. The remaining cells were counted and used as a measure of the number of intact cells in the pellet material. Usually the intact cells represented (3 % of the total number of cells plated. If incubated in tissue culture medium the remaining attached cells gave rise to cell clones. The assay used, therefore, gives a figure for the number of intact cells capable of prolonged cell proliferation. The term ‘intact cells’ also includes cells which have been stripped of a small (sublethal) amount of cytoplasm.

Enucleation procedure The discs with growing cells were transferred to plastic centrifuge tubes with the cell sides facing the bottom of the centrifuge tube. Each centrifuge tube contained 3-4 ml of phosphate-buffered saline (PBS) prewarmed to 37°C: To- fix the disc in the‘right position during centrifugation, a plastic plug with a diameter slightly less than the inner diameter of the centrifuge tube was nut on tot, of the disc. Loosely attached cells were-then removed by centrifuninn for 10 min at 10 000 rpm (12 000 .e). When la;ger quantities of enucleakd &ytoplas& were required, several plastic discs were piled on top of each other, separated only by 1 mm high plastic rings. The rotor used for the centrifugation (Sorvall SS 34 rotor) had been prewarmed to 37°C before the centrifugation was started. In order to enucleate, the plastic discs were transferred to new centrifuge tubes containing PBS, 10% calf serum and 7-15 ,ug/ml of cytochalasin B. The preparations were then centrifuged for 20 min at 37°C at the speeds indicated in the Results section. After enucleation, the slides were washed in PBS and then returned to normal growth medium, whereas the pellet material obtained-during the enucleation run was washed once in Eagle’s MEM and then kept at + 4°C until used.

Viability

test

The percentage of damaged cells was tested by diluting the pellet material 1 : 1 in a 0.4 % trypan blue solution in PBS. Cells which stained were Exptl

Cell Res 87 (1974)

Cytochemical methods For dry mass determinations on fixed material a sample of the pellet was allowed to air dry on a glass slide. The slide was then fixed in ethanol/ acetone (1: 1) and mounted in alvcerol. The drv mass was then determined by m&ointerferome&y as described bv Casoersson & Lomakka 191.For microspectrophoiometiic measurements oi - DNA and protein, air-dried and ethanol acetone fixed samples were stained by a combined Feulgen-Naphthol yellow method [lo, 111.All results obtained by quantitative cytochemistry were expressed in arbitrary units (AU). For autoradiography a sample of air-dried and ethanol acetone-fixed pellet material was used. The mass of individual cells was first determined by microinterferometry in order to clearly distinguish minicells from intact cells. The location of each cell measured was noted on a photographic map of the preparation so as to make it nossible to identifv the same cells in the autoradiograms. The coverglass was then removed and the slide was washed in distilled water

Minicells obtained by cytochalasin enucleation

367

Table 1. Enucleation frequency and yield of minicells Enucleation conditions Speed rpm (8)

Cytochalasin cont. (m/ml) I

7000 (6000)

10

10000 (12000)

1: 15 7 10 15 7 10 15

14000 (23 500) 18000 (39000)

15

Disc

Pellet material MiniYield of cells cells x lo3 ( %) 20 6 18 185 235 230 415 365 340 355 610 494

55 58 12 73 71 56 86 90 8-l 74 93 72

Dead cells’ (%I

Intact cells ( %I

30

15 12 43 3 10 13 0.6 0.5 1.9 3.6 0.6 0.3

E 24 19 31 13 9 11 22 2;

Enucleation ( %) 2 : 55 II IO 91 98 98 97 100 98

a Mainly minicells. and ethanol to remove the glycerol and in 5 % cold TCA for 5 min to remove non-incorporated radioactive precursors. The preparations were then rinsed in cold running tap water, air-dried and covered by Kodak AR 10 emulsion for autoradiography according to standard procedures.

Chemical methods The amount of DNA and protein in detergentisolated nuclei and in minicell pellets was determined by the method of Burton [12] and Lowry et al. [13], respectively. Detergent isolation of nuclei was carried out as described by Goto & Ringertz [14]. Determinations of enzyme activities of minicells and intact cells was carried out on freshly prepared material. The samples were sonicated and the enzyme activities were determined on the supernatant obtained after centrifugation. Lactic dehydrogenase(LDH), NADPH cytochrome c reductase and succinic acid dehydrogenase (SDH) were determined according to standard methods [15-171.

Electron microscopical methods Pellet material obtained after enucleation was washed in a small amount of PBS and fixed in 2 or 4% purified cacodylate-buffered glutaraldehyde containing sucrose (0.1 M cacodylate buffer; 0.1 M sucrose) for 24 h at +4”C. After fixation was completed, the suspension was sucked up with a pipette which was placed over a Millipore filter. A negative pressure of 20 mm Hg was then applied. When the pellet material had settled on the filter, the whole filter was immersed in 2 % s-collidine-buffered 0~0, (pH 7.4) and the material was post-fixed in this solution for 1 h at +4”C. Dehydration was performed in a graded series of ethanol solutions of increasing

concentration, and propylene oxide in which the filter dissolved completely. The thin film of naked cell material left behind was then embedded in Epon. Thin sections were stained with lead citrate and were studied in a Siemens Elmiskop I electron microscope.

RESULTS Composition of minicell preparations In the enucleation procedure of Prescott et al. [2] the enucleated cells remain attached to a plastic disc, whereas the nuclei removed can be collected from the bottom of the centrifuge tube at the end of the centrifugation run. In the present experiments a small proportion of the cells remaining on the disc were not enucleated, and some intact cells became detached from the plastic surface during the enucleation procedure and were found contaminating the minicell preparation. Furthermore, cytochalasin treatment and centrifugation also produced small cytoplasmic fragments which were found together with minicells on the bottom of the centrifuge tube. In order to work out optimal conditions for the preparation of large numbers of minicells stripped of most of the Exptl Cell Res 87 (1974)

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Fig. 1. Microphotographs

of Giemsa-stained preparations of (a) normal L 6 rat myoblasts; (6) enucleated L 6 cells; (c) L 6 minicells. The small, rounded structures in the background surrounding the 6 minicells are cytoplasmic fragments.

cytoplasm and minimally contaminated with intact cells, rat L6 myoblasts were enucleated with different doses of cytochalasin and different speeds of centrifugation. The pellet material was scored for the total yield of cells and the percentages of dead cells, intact minicells and intact cells. Non-nucleated cytoplasmic fragments were not included in the analysis at this stage. The dead cells consisted almost exclusively of damaged minicells which stained with trypan blue. Minicells and intact cells did not stain with trypan blue. Because the distinction between minicells and intact cells immediately after enucleation was not clear, pellet material equivalent to 10 000 trypan blue negative cells was seeded on new plastic dishes. The number of intact cells was then scored 24 h later by counting the number of well attached cells (see Material and Methods). The plastic discs used for enucleation were examined for the percentage of enucleated and nucleated cells by fixing and staining the preparations with Giemsa. Data summarized in table 1 show that at 7 000 rpm (6 000 g) only l-2% of the L cells were enucleated, Exptl Cell Res 87 (1974)

whereas at speeds of 14 000 rpm (23 500 g) and higher 90-100 % of the cells remaining on the slide lost their nuclei (fig. lb). Within the concentration range of 7-15 pug cytochalasin/ml there was no significant difference in the enucleation frequency. At 7 000 rpm some minicells could be obtained but these were heavily contaminated with intact cells (12-43 %) and dead cells (30-45 %). At 14 000 and 18 000 rpm, however, the yield of minicells was maximal. The pellet material then contained 72-90 Y0undamaged minicells, 9-27 % dead cells (mainly dead minicells) and 0.3-3.6 % intact cells. The light microscopic appearance of intact L6 cells and of minicells is illustrated in fig. 1 a, c. Ultrastructure of minicell fractions and intact cells Intact L6 myoblasts were large with abundant cytoplasm forming numerous villous and blunt projections of varying height and size (fig. 2). The nuclei showed an irregular shape with frequent deep indentations; they contained moderate amounts of condensed heterochromatin and nucleoli. The cytoplasm

Minicells obtained by cytochalasin enucleation

369

Fig. 2. Part of L 6 cell with abundant cytoplasm containing various cytoplasmic organelles. Note the indented nucleus (N) and small microvillous projections on the surface ( x 14 000). Fig. 3. Minicell with indented nucleus surrounded by a small rim of cytoplasm composed of ground substance with numerous free ribosomes ( x 14 000). Exptl Cell Res 87 (1974)

370 T. Ege et al.

Fig. 4. (a) Large cytoplasmic fragment containing rough endoplasmic reticulum, mitochondria, numerous vesicular and vacuolar structures and two lipid droplets (L). Note microvillous projections on the surface ( x 25 000); (b) cytoplasmic fragment containing dilated rough endoplasmic reticulum, free ribosomes and small lipid droplets (L) ( x 11 000); (c) small cytoplasmic fragment surrounded by a distinct plasma membrane and containing free ribosomes, two vesicular structures and a mitochondrion ( x 33 000).

Exptl Cell Res 87 (1974)

Minicells obtained by cytochalasin enucleation of the myoblasts was rich in rough-surfaced endoplasmic reticulum; mitochondria and lysosomes were present in moderate numbers; the Golgi regions were fairly large; some cells showed presence of lipid droplets, either single or clustered; and the ground cytoplasm was characterized by a moderately electron-dense matrix in which numerous free ribosomes and occasional microtubules and microfilaments were dispersed. The minicell fraction was made up of minicells, fragments of cytoplasm lacking nuclei and occasional intact L6 cells. The minicells (fig. 3) had a rounded, roughly spherical shape with a thin rim of cytoplasm surrounding a nucleus showing a fine structure inseparable from that observed in intact L6 myoblasts (see above). The cells were bordered by a distinct plasma membrane which had a smooth or slightly wavy outline; microvillous or blunt projections were lacking. The rim of cytoplasm was dominated by matrix substance and free ribosomes. Occasionally, vesicular or cisternal elements of rough endoplasmic reticulum, mitochondria and lysosomes were encountered. Severely damaged, apparently non-viable cells were also observed. The cytoplasmic fragments (fig. 4) were all bordered by a distinct plasma membrane but otherwise showed a highly variable appearance. Most of these fragments were smaller than the minicells. Some fragments consisted only of cytoplasmic ground substance-often containing abundant free ribosomes. Others showed presence of variable amounts of rough endoplasmic reticulum, mitochondria, lysosomes and lipid droplets. The outline of the fragments was either smooth or irregular with occurrence of microvilli and blunt projections. The appearance of the cytoplasmic fragments was followed in multiple adjacent (serial) sections. Nuclei or nuclear fragments were never

Table 2. Biochemical properties preparations

Expt

Parameter

1 1

DNA Protein LDH NADPH cytochrome c reductase SDH

2 3 4

371

of minicell

DetergentMinicells isolated in %of nuclei in 9’ intact cells of intact cZlls 93.8 42.9 22.6

90.0 22.2

36.4 35.0

encountered in these sections, thus indicating that the observed structures did not represent grazing sections of intact cells (with nuclei). Chemical analysis of minicell fractions In order to obtain an estimate of the average amount of cytoplasm removed from the intact cell during minicell preparation, this preparation was analyzed for DNA, protein and three enzymes which are believed to be cytoplasmic (see [18] for references). One of these enzymes, lactic dehydrogenase (LDH) occurs in the soluble fraction of the cytoplasm whereas the other two, NADPH cytochrome c reductase and succinic dehydrogenase (SDH), are endoplasmic reticulum and mitochondrial bound enzymes, respectively. Nuclei were also isolated from intact cells by a conventional detergent procedure in order to estimate how much of the total cell protein should be considered to be nuclear. The results summarized in table 2 show that the minicell fraction and the detergent isolated nuclei had DNA contents which came very close to that of the intact cells. The protein content calculated per minicell represented approx. 42 % of the total cell protein, whereas detergent isolated nuclei represented 22 % of the total cell protein. The fact that these two determinations differed by a value equiExptl Cell Res 87 (1974)

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Table 3. Microinterferometric dry mass determinations on individual minicells and enucleated cells. Arbitrary units (AU)

Intact cells Enucleated cells Detergent isolated nuclei Minicells Cytoplasmic fragments

Dry mass in AU mean + S.E.M.

in % of intact cells

15.47 kO.83 8.72 iO.66 3.09iO.12 4.34 kO.41 0.49 F0.06

100 56 20 28 3

associated with the minicell preparation. However, from both the electron microscope studies and cytochemical measurements on minicells and enucleated cytoplasms to be discussed below, it was clear that part of the cytoplasmic material found in the pellet was made up of cytoplasmic fragments which were not directly associated with the minicells. Cytochemical measurements were therefore made on individual minicells. Dry mass determinations on individual minicells and enucleated cytoplasms

valent to 20 % of the total cell protein made it possible to estimate the maximum amount of cytoplasmic protein in the average minicell. Expressed in relation to the maximum amount of cytoplasmic protein (value for intact cells minus the value for detergent isolated nuclei) this figure indicates that 75 % of the original cytoplasm had been removed during enucleation. The average minicell according to this calculation retained a maximum of 25 % of the original cytoplasm. If instead the calculations were based on enzyme activities in the pellet material it appeared that 65-77 % of the cytoplasm had been removed and that 23-35 % of the original cytoplasm was

Table 3 summarizes microinterferometric dry mass determinations on intact cells, enucleated cytoplasms, minicells and cytoplasmic fragments found in the pellet after enucleation. The intact cells were subjected to cytochalasin treatment but were not centrifuged. In order to be able to measure the enucleated cells, the respective cytoplasms had to be removed from the plastic discs by mild trypsinization at the end of the centrifugation, after which they were seeded onto glass slides suitable for microinterferometry. The minicells and contaminating cytoplasmic fragments were collected from the bottom of the centrifuge tubes and

Table 4. Effect of speed and cytochalasin dose on the dry mass (arbitrary units) of minicells and enucleated cytoplasms Enucleation conditions

Minicells

Enucleated cells

Speed 07-d

Cytochal. cont. pg/ml

Mean

+ S.E.M.

(n)

Mean

18000

7 10 15

4.8 4.1 4.3

kO.3 kO.2 +0.2

(30)

6.9 7.6

Exptl Cell Res 87 (1974)

fS.E.M.

+ 0.4 20.6

(n)

Minicells obtained by cytochalasin enucleation sedimented onto glass slides. For comparison, nuclei were also isolated by conventional detergent procedure and plated on glass slides for microinterferometric measurements. From table 3 it can be concluded that the minicells prepared from L6 cells had a dry mass which represented 28 yOof the dry mass of the intact cell. Detergent isolated nuclei on the other hand had a dry mass which represented 20 % of the dry mass of the intact cell. Minicells plus enucleated cytoplasms together accounted for 84 y0 of the total dry mass of the intact cell. If those cytoplasmic fragments in the pellet which were large enough to be measured in the interferometer (3.2 fragments/minicell) were included it was possible to account for 93 % of the dry mass of the intact cells. Measurements of the dry mass of minicells obtained at different centrifugation speeds and at varying cytochalasin doses showed a surprisingly uniform dry mass (table 4). Minicells obtained at 10 000 rpm had a higher dry mass than those prepared at 14 000 and 18 000 rpm. At the higher speeds the minicells had a mean dry mass of approx. 4.5 units as compared with 6.7 units at 10 000 rpm (table 4). The cytoplasms remaining on the discs after centrifugation at 10 000 rpm had a dry mass of approx. 12 dry mass units. This value decreased to about 9 units at 14 000 rpm and 7 units at 18 000 rpm. The variation in dry mass of individual minicells, intact cells and detergent isolated nuclei is illustrated in fig. 5. As can be seen by comparing the histogram for intact cells (fig. 5a) and that of minicells (fig. 5b), the largest minicells had a dry mass similar to that of the smallest L6 cells. As a whole, however, the dry mass distribution of the minicells was distinctly different from that of intact cells but similar to that of detergentisolated nuclei (fig. 5~). In order to analyse

li

313

a

10

./ IL ,iJ&a

li

b c

11 5 L

dry mass(AU); ordinate: no. of cells (nuclei). Histograms of microinterferometric dry mass determinations on (a) intact L6 rat myoblasts; (b) minicells prepared from L6 cells; (c) detergentisolated nuclei from L6 cells.

Fig. 5. Abscissa:

whether the heaviest of the minicells were in fact intact small cells or whether they were large minicells derived from cells with large nuclei, the same individual cells were measured for DNA and total protein, using microspectrophotometry at two wavelengths after staining with Feulgen-naphthol yellow [lo, 111. Fig. 6 shows the DNA and total protein contents of individual minicells and individual intact L6 cells. The nuclei of the minicells had DNA values characteristic of Gl, S and G2 nuclei and the different stages of the replication cycle were represented in the same frequencies as they occurred in the intact cells. Minicells containing G2 nuclei contained approximately twice as much protein as the G 1 minicells and had a protein content similar to that of the smallest of the intact cells. The latter, however, had Gl nuclei. It is clear therefore that the minicell population is well defined and that the preparation analysed contained very few intact cells. Exptl Cell Res 87 (1974)

374

T. Ege et al.

moo-

* x 2500*

x *

.

Fig. 6. Abscissn: Feulgen-DNA values (AU); ordinnte: naphthol yellow binding protein (AU). Plot of microspectrophotometric determinations of total protein and DNA on ( x ) intact L6 cells; (0) minicell fraction. The preparations were stained with Feulgen and naphthol yellow.

Viability of minicells The viability of minicells was studied by trypan blue penetration tests, by examining the ability of the minicells to incorporate labelled precursors and by measuring the dry mass changes of individual minicells for two days after enucleation. For routine purposes trypan blue staining was used as a rapid means of scoring the proportion of dead or damaged cells. In experiments with a high frequency of enucleation the minicell pellet usually contained 10-30~ cells which stained directly with trypan blue (table 1). When minicells were washed free from cytochalasin and incubated in tissue culture medium the frequency of trypan blue positive minicells increased so that all Exptl Cell Res 87 (1974)

minicells were trypan blue positive 2-3 days after enucleation. The minicell preparations also contained a small minority (0.3-3.0 %) of intact cells which excluded trypan blue and were able to attach and undergo cell division. In some experiments the minicells were washed free from cytochalasin and then incubated with radioactive amino acids or nucleic acid precursors. In the experiment shown in fig. 7 individual minicells were identified and measured for dry mass before autoradiography. One hour pulse-labelling with 3H-leucine and 3H-uridine resulted in labelling of 58 and 88 % respectively of the minicells whereas a 12 h incubation with 3H-thymidine only labelled 45 % of the minicells. In the experiment shown in

Minicells obtained by cytochalasin enucleation fig. 7, 12% of the minicells were dead or damaged as judged by the trypan blue test. The contamination with intact cells (defined as cells capable of long-term survival) was < 1 %. In fig. 7 the non-incorporating minicells are represented by the light bars, whereas the labelled minicells are shown by black bars. From this graph it is clear that minicells which failed to incorporate uridine are smaller than average (fig. 7~). Also with respect to 3H-leucine incorporation (fig. 7a) there is a higher frequency of non-incorporators among minicells with a low dry mass than among larger minicells. The same is true for 3H-thymidine incorporation: among the smallest minicells there are practically no nuclei capable of DNA synthesis (fig. 7b). The viability of minicells was also examined by direct microscopic observations on living minicells in tissue culture. In one experiment minicells and contaminating intact cells were incubated in a small chamber which could be inserted directly into a scanning and integrating interferometer. Individual cells were

375

ZOO-

loo.

<

rb io 30 io sb-time in culture (hours); ordinate: ” dry mass in % of initial dry mass. Microinterferometric dry mass determinations on 13 minicells and 7 cells in the same culture chamber. The same individual cells and minicells were measured at different time points after enucleation. The points plotted are mean values; 0, intact L6 cells; A, L6 minicells.

Fig. 8. Abscissa:

then identified and their positions marked out on a photographic map. By interferometry the dry mass of thirteen minicells and seven intact cells was measured at different times after enucleation. During measurements, the temperature was maintained at approx. 37°C by blowing a jet of warm air onto the incubation chamber, and between measurements the chamber was kept in a 37°C incubator. As illustrated in fig. 8, the mass of the intact cells increased and two cells also divided during the observation period. The minicells on the other hand underwent a gradual decrease in dry mass. Measurements were stopped after 2 days when the minicells were clearly dead and undergoing lysis. Among the minicells which had not been singled out for measurements there were some which died sooner or later than the ones examined.

Fig. 7. Abscissa: dry mass (AU); ordinate: no. of cells.

Histograms showing the freauencies of I. labelled and q,-unlabelled n&icells in different dry mass classes, after (a) 1 h muse with SH-leucine (1 ~Ciiml): (b) 12 h pulse with 3fi-thymidine (0.1 ,&i/mi); (c) 1 h pulse with 3H-uridine (1 ,&i/ml). Labelling was carried out immediately after the minicells had been washed free from cytochalasin and transferred to normal culture medium.

DISCUSSION The present results show that the nuclei removed from rat L6 cells during enucleation are surrounded by a narrow rim of cytoplasm and an intact plasma membrane. It therefore Exptl Cell Res 87 (1974)

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T. Ege et al.

seems appropriate to refer to these structures as minicells rather than nuclei. In the present experiments we have characterized the minicells with respect to cytoplasmic content, overall chemical composition, ultrastructure, ability to regenerate a cytoplasm and general viability. Biochemical determinations of protein in minicell pellets indicated that at least 75 Y0 of the original cytoplasm had been removed and determinations of LDH, NADPH cytochrome c reductase and succinic dehydrogenase suggested a figure of 65-75 %. Electronmicroscopical and light microscopical examination revealed, however, that the minicell preparations were contaminated by a few intact cells and by cytoplasmic fragments. The biochemical estimates of the amount of cytoplasm which is removed during enucleation are therefore minimal estimates. Microinterferometric determinations of the dry mass of individual minicells, detergent isolated nuclei and intact cells were performed in order to obtain more accurate estimates of the amount of cytoplasm surrounding the minicell nucleus. The L6 minicells had a dry mass which was approx. 40% greater than that of detergent-isolated nuclei. This difference may be due to cytoplasm and/or to a greater loss of protein from detergentisolated nuclei than from the minicells. However, even if all of the difference in dry mass between the minicells and the detergentisolated nuclei is cytoplasmic, this value only corresponds to 10% of the original amount of cytoplasm in the intact cell. The number of cytoplasmic fragments in the pellet material appeared to increase with increased speeds of centrifugation. In agreement with this was the observation that the mean dry mass of the cytoplasms remaining of the discs decreased by almost 50% as the speed of centrifugation was increased from 7 000 to 18 000 rpm. Furthermore the sum Exptl Cell Res 87 (1974)

of the dry mass of minicells and cytoplasms obtained at 14 000 and 18 000 rpm was somewhat less than that of the intact cells. Another aim of the present work has been to analyse the viability of the minicells. Our data show that it is possible to prepare relatively homogeneous populations of minicells where less than 27 % (in the best experiments less than 10YO) of the minicells are damaged or dead as judged by a dye exclusion test. Immediately after enucleation the minicells are capable of incorporating 3H-uridine and 3H-leucine and a considerable proportion of minicells also incorporate 3H-thymidine. Similar results were recently reported by Prescott & Kirkpatrick [19]. The nonincorporating minicells have a very low dry mass and therefore may be the ones which have been most efficiently stripped of cytoplasm. If incubated under standard tissue culture conditions the minicells undergo a progressive decrease in dry mass and ultimately die. One might have expected that the minicells should vary with respect to the amount of cytoplasm retained and that possibly those cells which had only lost a small amount of cytoplasm would have been able to regenerate cytoplasm and go on dividing. Instead we find that the minicells constitute a fairly well defined group where practically all minicells have lost most of the original cytoplasm. Although the largest minicells have protein contents equal to the smallest of the intact cells there is no true overlapping since the large minicells have large G2 nuclei and little cytoplasm whereas the small intact cells have small G 1 nuclei and considerable amounts of cytoplasm. It is possible therefore to prepare relatively homogeneous populations of minicells where the majority exclude trypan blue and are capable of incorporating 3H-uridine. The fact that minicells fail to regenerate a

Minicells obtained by cytochalasin enucleation cytoplasm and ultimately die is of interest from the viewpoint of cell physiology. More interesting, however, is the fact that minicells can be induced to fuse with enucleated cytoplasms [8] so as to give rise to reconstituted cells. By combining minicells prepared from one type of cell with a cytoplasm from another cell it may be possible to study the role of nucleocytoplasmic interactions in the regulation of gene expression and cell differentiation. In a succeeding paper [20] we also report how the enucleation technique can be used to produce nucleated structures which are even smaller than minicells. These structures, which we have named microcells are prepared by enucleation of cells which have been induced to undergo micronucleation. The nuclei of the smallest of the microcells have a DNA content equivalent to l-2 chromosomes. The practical interest of the microcells lies in the fact that they can be fused with other cells [20]. The microcells therefore offer the possibility of introducing a small part of the genome of one cell into another cell and therefore may be of great value in somatic cell genetics. The authors wish to thank G. Blomgren, G. Jacobson and M. Lundstrom for valuable assistance and Dr G. Auer and Dr A. Zetterberg for help with the Naphthol Yellow staining method. The investigation was supported by grants from the Swedish Cancer Society.

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