JOURNAL OF ULTRASTRUCTURE RESEARCH 58, 1 5 5 - 1 6 1 (1977)

Morphological Studies on the Enucleation of Colchicine-Treated L-929 Cells

JERRY W . SHAY AND MIKE A . CLARK

Department of Cell Biology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235

Received June 2, 1976, and in revised form, July 30, 1976

Trea tmen t of mammal ian cells tha t are growing in monolayer culture with 2 t~g/ml of colchicine for 48 h r induces f ragmentat ion of the nucleus, a process termed micronucleation. If these t reated cells are centrifuged in medium containing 10 tLg/ml of cytochalasin B, the individual karyomeres are removed in a single strand. Once removed, each karyomere and its associated cytoplasm has been termed a "microcell" [Ege and Ringertz (1974)Exp. Cell Res. 87, 378] or '~microkaryoplast" [Shay and Clark (1975) 33rd Annu. Proc. Electron Microscopy Soc. Amer., p. 306]. Each microkaryoplast contains a small amount of decondensed chromatin surrounded by a nuclear envelope, a th in band of cytoplasm containing ribosomes and mito- chondria and limited by an in tac t plasma membrane. This procedure provides a means of obtaining par t of the genome of a cell packaged in such a way tha t allows its introduction into another cell without damage and may provide useful information for the study of nuclear- cytoplasmic interactions.

Phillips and Phillips (8) reported that treatment of mammalian cell cultures growing in monolayer with the drug col- chicine, at a concentration of 5 × 10 -5 M for 16-36 hr, not only interfered with the cell's ability to divide, but also resulted in a fragmentation of the nucleus, a process called micronucleation (3). In another area of research, Carter (2) reported that treatment of mammalian cells growing in monolayer with the mold metabolite cyto- chalasin B (CB) caused rapid and dramatic morphologic changes, including nuclear extrusion and, in a small number of cells, spontaneous enucleation. Since Carter's discovery, methods have been developed for obtaining large numbers of enucleated cells [cytoplasts (•3)] and nuclei [kary- oplasts (13, 14) = minicells (3-6)] by us- ing mild centrifugal force during CB treat- ment (10, 20). Previous reports have de- scribed the stucture and behavior of cyto- plasts (7, 13, 14, 19) and karyoplasts (13- 15, 19), and these components have been successfully recombined using inactivated Sendai virus to form viable reconstructed cells (4, 5, 16).

Recently, Ege and Ringertz (3) and Shay

Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

and Clark (12) reported that by first in- ducing micronucleation with colchicine, they could subsequently remove the kary- omeres by centrifugation in the presence of CB. These structures once removed have been termed ¢~microcells" (3) or "microkar- yoplasts" (12). The term "microkary- oplasts" is preferred, as these isolated structures are not viable cells, and, thus, the term '~microcell" may be misleading. In addition, the term "microkaryoplast" is descriptive of the morphology observed and is consistent with the nomenclature originally established for complete nuclei removed with CB and centrifugation [i,e., karyoplasts (•3)]. In this paper, the proc- ess of enucleating micronucleated L-929 cells and the fine structure of the micro- karyoplasts resulting from this procedure are described. Such microkaryoplasts are capable of Sendai virus-induced fusion to other cells (6) or portions of cells and may provide useful information for the study of nuclear-cytoplasmic interactions.

MATERIALS AND METHODS

L-929 mouse cells used in these experiments were grown in Dulbecco's modified Eagle's medium con-

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taining 10% fetal calf serum (Flow Labs, California) and 1% penicillin (10,000 tL/ml)-streptomycin (10,000 mcg/ml) (Flow Labs, California). Cells to be col- chicine treated were initially plated at 4 × 105 cubated at 37°C in 5% CO2 for 24 hr. A colchicine stock solution (J. T. Baker Chemical Co., New Jer- sey) was then added to the growth medium to pro- duce a final concentration of 2 t~g/ml and, after 48 hr additional incubation, greater than 75% of the cells were micronucleated.

A stock solution of cytochalasin B (Aldrich Chem- ical Co., Inc., Wisconsin) was prepared by dissolving 1 mg of CB in 1 ml of 95% ethanol. This was in turn dissolved in Dulbecco's medium without serum to give a final CB concentration of 10 tLg/ml. The enu- cleation procedure involved first pouring off the growth medium and colchicine and then completely filling the flask with CB medium. The flasks were then placed in special acrylic holders (17) and spun at 6000g for 20 min in a Sorval GSA rotor prewarmed to 37°C. This procedure resulted in greater than 95% enucleation with a minimal amount of cytoplasmic and whole cell contamination in the pellet. The mi- crokaryoplasts plus contaminants were then resus- pended in complete medium for 1 hr and allowed to attach to glass coverslips prior to preparation for scanning electron microscopy using the procedures previously described by Porter et al. (9).

Cells were fixed in a 3% glutaraldehyde solution buffer with 50% Puck's saline G (pH 7.2) and 0.05 M cacodylate at 37°C for 45 min. Cells were then post- fixed with a 1% osmium tetroxide solution for 60 min, then rapidly dehydrated in a graded acetone series, and finally dried by the critical point method. The specimens were sputter-coated with gold/pal- ladium in a Technics Hummer II and then observed in a JEOLCO U-3 or Cambridge S-4 scanning elec- tron microscope. For transmission electron micros- copy, the cells were fixed in a 3% glutaraldehyde solution in 0.05 M cacodylate buffer at a final pH of 7.2. Cells were fixed for 1 hr and then washed for 1 hr in four 15-min changes of cacodylate buffer. Cells were postfixed in a 1% solution of osmium tetroxide for 1 hr and then dehydrated in a graded alcohol series to propylene oxide and finally embedded in Epon 812.

OBSERVATIONS

Scanning Electron Microscopy

The surface morphology of L-929 cells varies wi th each phase of the cell cycle and is influenced by the cell densi ty in the culture flask (11). In un t rea ted L-929 cul- tures at a densi ty of 3 × 104 cells/cm 2, a stellate morphology is most f requent ly ob- served (Fig. 1). The cells are flat, except

dur ing mitosis, and they have occasional marg ina l ruffles and microvilli dis t r ibuted over the cell surface. When these cells are t rea ted wi th 2 t~g/ml of colchicine for 48 hr, the nucleus f ragments into a n u m b e r of ka ryomeres and dramat ic changes occur in the surface morphology (Fig. 2). As the cells lose the i r polar orientat ion, they be- come more rounded and the surface be- comes populated wi th different sized, blebs (Fig. 2). W h e n these micronucleated cells are centr ifuged in the presence of CB for 10 min and then fixed, it is possible to observe the results of par t ia l microenucleat ion (Fig. 5). Observat ions in the scanning elec- t ron microscope indicate tha t the kary- omeres are removed in a single cytoplas- mic strand, as opposed to ka ryomeres

b e i n g removed separately in m a n y cyto- plasmic s t r a n d s . The bulges in the strands, as revealed in the l ight micro- scope, are Feulgen positive and correspond to the karyomeres . If centr i fugat ion is con- t inued, the s t rands f requent ly reach 300 tLm in l eng th and 0.5 tLm in d iameter and eventual ly break. When the pellet result- ing from the centr i fugat ion is resuspended in complete medium, the microkaryoplasts, cytoplasts, and whole cel l con taminan t s will a t tach to a glass substrate wi th in 1 h r (Fig. 6), The microkaryoplas ts are usual ly spherical in shape and f requent ly c lump together. I f the pellet is allowed to recover for 12 hr, the drug- t rea ted whole cells and cytoplasmic con taminants f lat ten out, but the microkaryoplas ts re ta in their spheri- cal shape. Microkaryoplasts exclude try- pan blue and va ry in size. The smallest microkaryoplas ts are approximate ly 1.0 tLm in d iameter for this cell type.

Transmission Electron Microscopy

The fine s t ructure of un t rea ted L-929 cells tha t were trypsinized and resus- pended for 1 hr in complete med ium and then fixed for t ransmiss ion electron mi- croscopy is shown in Fig. 3. This micro- graph shows tha t the controls fixed in this

FIa. 1. Scanning electron micrograph of unt rea ted L-929 cells frequently reveal ing a f lat tened stellate morphology, x 1400.

Fro. 2. After 48 hr incubat ion in 2 ~g/ml of colchicine, dramat ic surface changes occur. × 1500. FIG. 3. Transmission electron micrograph of L-929 control cell. × 6800. Fro. 4. L-929 cell after colchicine t rea tment . Karyomeres are of various sizes. The small dark s ta in ing

structures in the cytoplasm are of unknown identity, but they are also observed occasionally in control cells where they resemble lysosomes. × 7500.

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FIG. 5. Colchicine-treated cell spun in cytochalasin B for 10 min, then fixed, x 1300.

manner are usually mononuclear and have a round form with many surface cyto- plasmic projections. Prolonged colchicine treatment of L-929 cells prior to trypsina- tion and fixation results in fragmentation of the nucleus, referred to as micronuclea- tion (Fig. 4). Approximately 15% of the cells remain mononuclear with little ap- parent change, while another 10% remain in mitosis with condensed chromosomes. The remaining 75%, however, become mi- cronucleated. The number of karyomeres, as observed in the light microscope, range from 2-40/ce11, with the average contain- ing 12-18/cell. A thin section of the pellet (Fig. 7) shows possible whole cell contami- nants which may be distinguished from the microkaryoplasts by their larger size and a larger relative proportion of cyto- plasm. The microkaryoplasts vary in size and usually contain one karyomere (Figs. 9, 10). These vary from 1.0 tLm in diameter with the largest being approximately 8.0 tLm. Whole cells are usually 10.0 t~m or greater, so it is possible that the larger structures such as in Fig. 10 may in fact be equivalent to a karyoplast (i.e., intact nu- cleus with a reduced amount of cyto- plasm). This would be expected, as 15% of

the cells remain mononuclear even after prolonged colchicine treatment. Occasion- ally, microkaryoplasts have been observed by serial sectioning to contain several kar- yomeres (Fig. 8). Each karyomere is sur- rounded by a nuclear envelope containing nuclear pores (Figs. 8, 9, 10), except for those that remained in mitosis with con- densed chromosomes during the enuclea- tion procedure (Fig. 11). Within the cyto- plasm of the microkaryoplasts, mitochon- dria, ribosomes, and fragments of endo- plasmic reticulum are observed (Fig. 8, 9, 10). However, centrioles, microtubules, and microfilaments have not been ob- served. Microkaryoplasts do not resyn- thesize the missing cytoplasm, and within 18-24 hr, most become highly vacuolated, detach from the substrate, and quickly de- generate.

DISCUSSION

One of the more promising uses of this microenucleation technique has recently been demonstrated by Ege et al. (6). They fused a part of the genome (microkary- oplast) to a whole cell with the ult imate aim being the ability to study the genome

Q

FIG. 6. Enucleation pel le t consisting of microkaryoplasts, cytoplasts and whole cell contaminants. × 2500.

FIG. 7. Enucleation pellet consisting of cytoplasts, whole cell contaminants, and various sizes of micro- karyoplasts. [Serial sectioning has confirmed that micrographs depicted here are representative.] × 4000.

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i : ili4? ~ i ~!Y

Fias. 8-11. Microkaryoplasts vary considerably in size and structure. The microkayoplast in Fig. 8, as confirmed by serial sectioning, contains more t h a n one karyomere. As revealed in Figs. 9 and 10, however, most microkaryoplastg contain only one karyomere but still vary from 1.0 to 8.0/zm in diameter . Occassion- ally, a s tructure wi th condensed chromosomes is observed (Fig. 11), bu t these as well as the s t ructures in Figs. 8, 9, and 10 do not resynthesize the missing components and usually degenerate wi th in 24 hr. x 10 000.

ENUCLEATION OF COLCHICINE-TREATED CELLS 161

activity of foreign chromosomes in host cells. In our laboratory, we are currently trying to determine whether or not the chromatin in microkaryoplasts is incorpo- rated into the whole cell genome and if microkaryoplast proteins are synthesized and translated. In order to have more con- trol over such fusion studies, it will first be necessary to remove whole cells and cyto- plasmic contaminants from the microkary- oplasts. It would also be useful to separate the various sizes of microkaryoplasts. Ex- periments using Ficoll and serum gra- dients are currently in progress as a means to remove whole cells and cytoplas- mic contaminants and to isolate microkar- yoplasts consisting of only 1 or 2 chromo- somes. In addition, we are investigating the usefulness of a fluorescent cell sorter (Coulter) for separating microkaryoplasts of various sizes.

Most colchicine-induced micronucleated cells die within 72 hr of continuous treat- ment, and this proces~s is nonreversible by diluting out the colchicine. This may be because colchicine apparently denatures tubulin irreversibly (18). Experiments are currently in progress using Podophyllo- toxin, a drug that causes dissociation of microtubules in a similar manner to col- chicine but without denaturing tubulin (1). Preliminary experiments indicate that micronucleation can be induced using this drug and that it may then be feasible to spin such micronucleated cells for a short time and remove a segment of the cyto- plasmic strand containing a few kary- omeres and then allow the cell to recover, resulting in a cell with fewer than normal chromosomes.

The obvious advantages of the micro- enucleation-fusion procedures are that one transfers only a small number of donor chromosomes into a recipient cell and that the direction of chromosome segregation is determined by the choice of the donor. Since it may be possible to transfer only one chromosome, gene mapping may be greatly simplified. Such studies will hope- fully yield useful information on nuclear-

cytoplasmic interactions and may provide new methods for gene mapping.

This research was supported by grants from the Muscular Dystrophy Association, Inc., general re- search support grant 5-SO1-RR-05426-123, Division of Research Facilities and Resources, National In- stitutes of Health, and Cancer Center Support Grant (NIH 1P01 CA 17065-02). The technical as- sistance of Ms. Rhonda Porterfield is appreciated.

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