Tobacco BY-2 Cell Line as the “HeLa” Cell in the Cell Biology of Higher Plants

Tobacco BY-2 Cell Line as the “HeLa” Cell in the Cell Biology of Higher Plants Toshiyuki Nagata,’ Yasuyuki Nemoto,t and Seiichiro Hasezawas,

’

* Department of Biology, Faculty of Science, University of Tokyo, Tokyo

113,

Japan Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, Tokyo 184, Japan College of General Education, Seijo Junior College, Tokyo 157, Japan

*

1. Introduction

Although it was not until the introduction of established cell lines such as HeLa that molecular studies of animal viruses rapidly progressed, such cell lines have played an important role in the basic understanding of the molecular and cellular biology of mammalian cells, and many such examples can be observed in previous monographs (Willmer, 1965). More recently, understanding of the immortality of cell lines such as HeLa and L cells originating from cancer tissues helped elucidate the mechanism of tumorigenesis in animal cells (Baserga, 1985). On the other hand, no such plant cell lines can be studied from various aspects of interest, although the establishment of cell lines from plant tissues is relatively easy, and innumerable cell lines have been obtained from various tissues and species of higher plants. There are several cell lines, such as tobacco XD (Filner, 1965), soybean (Gamborg, 1970), Acer pseudoplatanus (Simpkins ef al., 1970), and Catharanthus roseus (Vinca rosea) (Misawa and Samejima, 1978), whose literature is extensive. However, the tobacco BY-2 (TBY-2) cell line, the subject of this chapter, has shown unique characteristics, exceptionally higher growth rates, and high homogeneity. As there may be some who have never heard of the TBY-2 cell line or its characteristics, details are described here, but at the onset some essential features are presented which we think are important. Although most people would recognize that a highly synchronous cell population is necessary for the study of plant cells, thus far most cell lines stated to have been synchronized had a mitotic index (MI) of 10-20% at

’

Present address: Department of Biology, Faculty of Science, University of Tokyo, Tokyo 113, Japan. Inremotional Review, of Cvrologv. Vol. I32

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Copyright Q 1992 by Academic Press. Inc. All nghts of reproduction in any form reserved.

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TOSHlYUKl NAGATA ET AL.

most. The serious question is about the remaining 80-90% of cells in the population. Nevertheless, such systems were traditionally called synchronized populations. The synchrony of the TBY-2 cell line, which we established, using aphidicolin, was 70-80% in terms of MI (Nagata et af., 1982). Thus, the basic understanding of the molecular and cellular biology of plant cells will probably progress by the use of this cell line, and the previous description will therefore have to be rewritten. Anyone who researches the cellular events of higher plants may have to use this cell line, or the question will be raised as to how a phenomenon that has been observed in other sources looks in TBY-2 cells. This statement may seem a bit overwritten, but after 9 years of establishing this experimental system, not afew people have noticed this point. This is the reason that this chapter has such an unusual title. Thus, we describe what has been done on this cell line, established almost 20 years ago, and what is going on in our laboratory. Supplementally, papers from Dr. H. Shibaoka’s laboratory are cited, as his group has successfully used our method.

II. Origin of TBY-2 Cells

The TBY-2 cell line was established from the callus induced on a seedling of Nicotiana tabacum L. cv. Bright Yellow 2 in the Central Research Institute of the Japan Tobacco and Salt Public Corporation (now the Tobacco Science Research Laboratory, Japan Tobacco, Inc.) (Kato et al., 1972).It has been propagated in the medium of Linsmaier and Skoog (1965) supplemented with sucrose and 2,4-dichlorophenoxyaceticacid (2,4-D). According to Kato et al. (1972), the TBY-2 cell line was the most proliferative among the examined materials of 40 species of Nicotiuna and three species of Populus, which suggests that this cultivar of tobacco may have some specific characteristics. Because of the higher growth rate and the absence of nicotine in the TBY-2 cell line, the Japan Tobacco and Salt Public Corporation developed a project to produce these cells on an industrial level to examine the possibility of using them as a raw material for cigarettes. After the stepwise increase of the size of culture vessel, first to 369 liters and subsequently to 1500 liters, and many technological improvements (Kato et al., 19761, the corporation built a pilot plant of a 20-kl culture tank, in which milder agitation was used to avoid mechanical damage to the cells, and the volumetric oxygen transfer coefficient was set at the rather lower value of 40-60 per hour in comparison to microorganisms. The possible contamination was prevented by using conventional polyvinyl alcohol filters in combination with membrane filters. During the successful operation of contin-

TOBACCO BY-2 CELL LINE

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uous culture for 66 days, a specific growth rate ( P ) of ~ 0.044 per hour (average generation time, 15.8 hr) was obtained. There were practically no problems in culturing the TBY-2 cells by long-term continuous culture on an industrial level. However, because of cost performance of the operation and some problems with the quality of the products (e.g., proteinous smell), the trial was stopped (Kato, 1982).These trials showed that there is no limitation for mass culture. Subsequently, the production of ubiquinone 10 (coenzyme Qlo),a drug for heart diseases, from cloned cells of the TBY-2 cell line was tried successfully, producing 10-fold more ubiquinone 10 than did tobacco leaves as a natural source (Ikeda er al., 1978). However, as the production of this drug by Pseudomonas and Rhodotorula is much higher than that of plant cells, the plant cells were replaced with the microorganisms (Misawa and Samejima, 1978). Further bioengineering aspects of TBY-2 cells, which are outside the scope of this review and are not described here, were studied intensively at the Japan Tobacco and Salt Public Corporation. Detailed results have been discussed by Kato et al. (1976). The TBY-2 cell line is available from our laboratory for scientific use under the assumption that investigators will underwrite the agreement required by Japan Tobacco, Inc., where this cell line was initiated. However, this does not necessarily mean that any TBY-2 cell line shows the characteristics described in this chapter. We must stress that one of the authors (T.N.) received this cell line in 1980 and established a method of high synchrony in 1982 by the use of aphidicolin, but such a high synchrony can be attained only when this cell line is properly propagated. This is probably because, during maintenance in our laboratory, some selection of actively dividing cells could have been added.

111. Growth and Cytological Characteristics TBY-2 cells are propagated in the modified medium of Linsmaier and Skoog (1965), in which KH2P04and thiamine HCl are increased to 370 and 1 mglliter, respectively, and sucrose and 2,4-D are supplemented to 3% and 0.2 mg/liter, respectively (Nagata et al., 1981). Every week 1-1.25 ml of stationary phase cells are transferred to 95 ml of the fresh medium and cultured on a rotary shaker at 130 rpm at 27°C in the dark. Under this condition cells grow as shown in Fig. I , and after 1 week initial cells p =

1 dx

- -,

x dt

where x represents cell density and t represents time (in hours).

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TOSHlYUKl NAGATA ET AL.

0

1

2

3

L

5

6

Time after transfer (days)

7

0

FIG. 1 Growth curve and mitotic indices of TBY-2 cells. Growth was monitored by counting the cell number per 1 ml at approximately 1-day intervals, and mitotic indices were determined at 12-hr intervals.

multiply 80- to 100-fold. Such a high growth rate of plant cells has not been reported elsewhere, so far as we are aware. MIS of 5 4 % were observed 1-4 days after transfer. Such a high growth rate is partly dependent on the phosphate content of the medium, the optimum value being 510 mghiter, which is 3-fold that of the original Murashige and Skoog (1962) medium. However, this phosphate in the culture medium was consumed by the third day of culture, while other major constituents, such as sucrose, nitrate, and sulfate, were still at 37%, 54%, and 57% of the initial concentration, respectively (Kato et al., 1977). Thus, the consumption of phosphate by this cell line is distinctive. Furthermore, a preliminary study of 31P nuclear magnetic resonance (31P-NMR)disclosed even more interesting features. According to the results with 31P-NMR,the cytoplasmic phosphate was discriminated from the vacuolar one because of the pH difference of both compartments; the cytoplasmic and vacuolar pHs of TBY-2 cells were 7.5 and 5.7, respectively (T. Nagata, unpublished observations). In fact, "P-NMR showed

TOBACCO BY-2 CELL LINE

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that when the stationary phase cells were transferred to the fresh medium, phosphate was rapidly accumulated in the cytoplasm and then was taken up into the vacuole, once the cytoplasmic phosphate pool was saturated. In the late log stage of culture, the accumulated phosphate in the vacuole was reutilized in the cytoplasm, as had been demonstrated in A. pseudoplatanus cultured cells by Rebeille er al. (1983), although the case of TBY-2 cells was more drastic. Apparently, the phosphate pool of TBY-2 cells was almost vacant at the stationary phase. During culture the vacuolated rod-shaped cells in the stationary phase became round cells enriched with cytoplasm, accompanied by an increase in specific gravity, when the cells are transferred to the fresh medium. The morphological changes in the organelles of plastids and mitochondria were most conspicuous when they were followed under an epifluorescence microscope after staining with the DNA-specific dye 4’6-diamidino-2phenylindole (DAPI). As shown in Fig. 2, a rapid increase in the fluorescence intensity of plastid nucleoids (DNA-protein complex) was observed soon after the stationary phase cells were transferred to the fresh medium, but after 2 days it decreased gradually. When the DNA content of plastid nucleoids was quantified with a supersensitive microspectrophotometer based on photon counting (Photonic Microscope System, Hamamatsu Photonics Co., Hamamatsu, Japan), it increased sharply after a lag of 6 hr and reached a maximum 28 hr after transfer, when it was approximately 13-fold the initial value (Fig. 3). To determine when plastid DNA synthesis occurred, autoradiography of the cells was performed after feeding [3H]thymidine. According to the labeling pattern of autoradiograms, cells were classified into four types. As shown in Fig. 4, in cells of the first and second types either the plastids (P+N-; Fig. 4A) or the nucleus (P-N+; Fig. 4C) was labeled, respectively. In the cells of the third type, both the plastids and the nucleus were labeled (P+N+;Fig. 4B), and in the last type neither the nucleus nor the plastids were labeled (P-N-; Fig. 4D).The proportions of these four labeling types of TBY-2 cells were followed during culture. The results (Table I) show that, during the first 24 hr of incubation, plastids were labeled in one-third of the cells (P+N-), while the nucleus alone was labeled (P-N+) in only 10% of the cells. The percentage of cells with labeled plastids (P+N- and P+N+)reached 83% on the first day and decreased to 12% on the second day (Table I). Thereafter, there was no labeling in the plastids, whereas, in contrast, the percentage of cells with labeled nucleus (P+N+ and P-N+) was 60% on the first and second days and then decreased gradually. The results of [3H]thymidine incorporation confirmed the preferential synthesis of plastid DNA during the first day of culture. Thereafter, plastid division was observed predominantly from the first to the second day of

FIG.2 Change in organelles in TBY-2 cells during culture. Cells harvested at day 0 (A and B), day 1 (C and D), day 2 ( E and F),

and day 4 (G and H)after transfer to fresh media were treated with cellulolytic enzymes to remove cell walls and examined under a fluorescence microscope after staining with DAPI according to Kuroiwa ef a / . (1981). Left and right rows show fluorescence and phase-contrast micrographs, respectively, of the same fields. N , Nucleus; single arrowhead, plastid; double arrowheads, mitochondrion. Bar = 10 pm.

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TOSHlYUKl NAGATA ET AL.

2

3

4

5

k x affer transfer bays)

6

7

FIG. 3 Change in DNA content per plastid (pt) nucleoid (see Fig. 2) during culture of TBY-2 cells. After staining with DAPI, DNA content of pt nucleoids was determined, using the Photonic Microscope System (Hamamatsu Photonics Co., Hamamatsu, Japan) with T4 phage as a standard. The content is expressed as multiples of T4 phage, using an arbitrary unit, T. At each time point DNA contents of at least 50 pt nucleoids were counted. Vertical bars represent the standard error.

culture, and the peak of the number of plastids per cell reached maximum at the second day of culture (Fig. 5 ) . The total amount of plastid DNA per cell can be calculated from the number of nucleoids per plastid, the DNA content per plastid nucleoid (Fig. 3), and the number of plastids per cell (Fig. 5). This DNA content, expressed as multiples of TCphage DNA ( T ) , can be converted to copy number of plastid DNA as a factor of 1.18T (Yasuda et al., 1988). The results (Table 11) show that the copy number of plastid DNA per cell rose from 1000to 11,000by the first day, representing an 11-fold increase within 24 hr. From the second day this number decreased gradually, to 1000 copies per cell on the seventh day. Thus, plastid DNA was synthesized almost exclusively during the first day of culture, and this DNA was distributed to daughter cells through successive cell divisions.

FIG. 4 (A-D) Autoradiograms of four types of TBY-2 cells with different labeling patterns. Cells were fed [3H]thymidine at I-day intervals and labeled for 24 hr. The locations of the nuclei were determined under a fluorescence microscope after staining with DAPI. Cells were classified on autoradiograms into four types, according to their labeling pattern [note that labeled plastids showed clusters of silver grains (A and B)]: (A) only the plastids labeled (P+N-); (B) both the plastids and the nucleus labeled (P+N+);(C) only the nucleus labeled (P-N+); (D) neither the plastids nor the nucleus labeled (P-N-). Bar = 10 pm.

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TOSHlYUKl NAGATA ET AL.

TABLE I Changes in the Proportion of Cells with Four Types of Labeling Patterns during Culture of TBY-2 Cells'

Incubation period (%) Labeling pattern P+NP+N+ P-N+ P-N-

0-1 days

1-2 days

2-3 days

3-4 days

4-5 days

5-6 days

33.6 49.9 10.8 5.7

5.2 7.0 52.9 34.9

0 0.6 28.3 71.1

0 0 14.5 85.5

0 0 8.4 91.6

0 0 0.7 99.3

6-7 days

0 0 0 100

Cells were incubated with [3H]thymidine for 24 hr at daily intervals, and autoradiograms were prepared. Cells on the autoradiograms were classified into four types (shown in Fig. 4). At least 500 cells were counted for each period.

Cannon et al. (1985) reported that the copy number of plastid DNA per photoautotrophic tobacco cell was 3-fold that of heterotrophic cells, but no such difference was found between similar cells of soybean (Cannon et al., 1986). This apparent discrepancy might have resulted from the incorrect assumption that plastid DNA per cell does not change much, but as Yasuda et al. (1988) showed, this is not the case. The question may be

100

.L 1

2

3

4

5

6

7

qme after transfer (days) FIG. 5 Change in the number of plastids per cell during culture of TBY-2 cells. Plastid numbers were determined under a fluorescence microscope equipped with phase-contrast optics, after staining with DAPI. Dumbbell-shaped plastids, suggesting plastid division, were frequently observed at 1-2 days of culture. For each point at least 200 cells were counted. Vertical bars represent the standard error.

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TOBACCO BY-2 CELL LINE TABLE II Change in the Copy Number of Plastid DNA per Cell during Culture of TBY-2 Cellsa

Day of culture

T value per plastid nucleoid PIastid nucleoids per plastid Plastids per cell T value of plastid DNA per cell Plastid DNA copies per cell

0

I

2

3

4

5

6

2.8 6.6 46 850 lo00

39.7 3.9 60 9290 10,960

17.6 4.2 92.5 6840 8070

14.9 3.8 56 3170 3740

6.1 3.7 44 993 1170

3.1 5.1 53 838 989

3.2 5. I 54 881 1040

a The T values of plastid DNA per cell were calculated from the observed numbers of plastid nucleoids per plastid, the Tvalues of plastid nucleoids (Fig. 3), and the number of plastids per cell (Fig. 5 ) . These T values were converted to copy numbers of plastid DNA according to the equation copy number = 1.18T.

raised, then, as to whether TBY-2 cells are a peculiar example. A recent report by Takio and Nagata (1990) showed that, essentially according to the same method described above, plastid DNA was preferentially synthesized in the photomixotrophic cell culture of a moss, Barbula unguiculata, which had a higher growth rate, showed active photosynthesis under illumination, and retained well-developed chloroplasts (Takio and Nagata, 1990). From these results it can be said that the preferential synthesis of plastid DNA in TBY-2 cells has reflected faithfully the dynamics of the plastid DNA synthesis of plant cells. It is interesting that, even in the intact seedlings of wheat, the synthesis of plastid DNA is preceding that of chromosomal DNA (Miyamura et al., 1990). Mitochondria1 DNA of TBY-2 cells has been analyzed by Sato et al. (1991). Quantitative fluorescence microscopy of mitochondrial nucleoids stained with DAPI revealed that each mitochondria retained mitochondrial DNA in the size range of 120-200 kbp, while the deduction from the restriction enzyme analyses shows that the size is approximately 270 kbp, as shown in other papers (Sparks and Dale, 1980). This discrepancy suggests that each mitochondrion might not contain the whole sequence of mitochondrial DNA. On the other hand, the copy number dynamics of mitochondrial DNA do not keep pace with chromosomal DNA, and the synthesis of mitochondrial DNA localized between days 1 and 3 after transfer to the fresh medium (Sato et a / . , 1991). This information elucidates the replication dynamics of plant mitochondria. Thus, as a model material TBY-2 cells are suitable for studying the biochemistry and molecular biology of plant organelles.

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TOSHlYUKl NAGATA ET AL.

IV. Studies of Subcellular Organelles To study the biochemistry and molecular biology of organelles, the isolation of such subcellular organelles is necessary, in intact condition if possible. It is not easy to isolate organelles from plant cells, as these cells are covered with thick cell walls. The preparation of protoplasts from TBY-2 cells, from which the isolation of organelles is easy, has been established, and a detailed procedure has been published (Nagata, 1987a,b), so only essential points are described here. Treatment of 3-dayold TBY-2 cells with 0.1% Pectolyase Y-23 and 1% Cellulase YC (both from Seishin Pharmaceutical Co., Nihonbashi-Koamicho, Tokyo) at 30°C for 50-60 min liberated protoplasts efficiently. The disruption of protoplasts suspended in lysis buffer [17% sucrose, 20 mM Tris-HC1 (pH 7.6), 0.5 mM EDTA, 1.2 mM spermidine, 7 mM mercaptoethanol, and 0.4 mM phenylmethylsulfonyl fluoride] can be easily accomplished by passage of the cells through 20-pm mesh placed in the apparatus, which was constructed to give a constant pressure of 0.5 kglcm’ to the filter to treat a large amount of material (approximately 1 liter) (Fig. 6). After removing the nuclei the plastid fraction was further purified on the stepwise sucrose density gradient. The plastids recovered from the interface of 40-60% sucrose retained intact plastid nucleoids under an epifluorescence microscope after staining with DAPI (Fig. 7A-C), and an electron-microscopic view showed the presence of matrix retaining higher electron opacity (Fig. 7D,E). When Nonidet P-40 was added to the plastid suspension (final concentration of 1%)and the suspension was stirred for 10 min, plastids were disrupted to release plastid nucleoids (Fig. 8).

N

FIG. 6 Schematic presentation of an apparatus to disrupt protoplasts suspended in a large volume. Intact organelles are recovered from the suspension of broken protoplasts (BPI. Released organelles can be purified by sucrose gradient centrifugation. AD, Auto-dispenser; FH, filter holder; IB, ice bath; IL, inlet tube; NM, nylon mesh; OL, outlet tube; OV, one-way valve; PG, pressure gauge; PP, suspension of protoplasts.

TOBACCO BY-2 CELL LINE

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FIG. 7 Isolated proplastids. The same optical field of proplastids was observed under fluorescence and phase-contrast microscopes after staining with DAPI. (A) Fluorescence microscopy. (B) Fluorescence and phase-contrast microscopy. (C) Phase-contrast microscopy. (D and E) Isolated proplastids were observed under the electron microscope. Arrows show plastid nucleoids. Bars = 5 wm.

Biochemical study revealed that plastid nucleoid is the complex of plastid DNA and four species of proteins, whose molecular masses are 69, 31, 30, and 14 kDa, respectively (Nemoto et al., 1988). As these four proteins were liberated from the plastid nucleoids at different NaCl concentrations, their binding affinities should be different. It is also interesting that, when plastid DNA mixed with these four proteins was dialyzed, plastid nucleoids were reformed and looked very much like those freshly isolated from plastids under fluorescence microscopy. Since the released proteins from plastid nucleoids were different from those of chloroplast nucleoids, the true function of these proteins remains to be elucidated. Since the cytological study described in the previous section revealed that plastid DNA was synthesized preferentially, Takeda et al. (1992)tried to elucidate its replication mechanism. Copies of plastid DNA are multiple in plant cells and replicate autonomously, but their genomes are preserved

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TOSHlYUKl NAGATA ET AL.

FIG. 8 (A) Isolated plastid nucleoids were observed under the fluorescence microscope after staining with DAPI. The same optical field was observed under (B)fluorescence and phasecontrast microscopes and (C) the phase-contrast microscope. (Dand E) The same specimens were observed under the electron microscope. Bars = 5 pm.

and succeed to descendant generations. It is intriguing to study the regulation mechanism of copy number dynamics of plastid DNA, as it is not known in higher plants. The first step to elucidate this mechanism should be to determine the replication origins of plastid DNA. There were reports by Kolodner and Tewari (1975) describing two replication origins located in the opposite strands, using the pea according to examination of the plastid DNA with electron microscopy. However, the D-loop strand observed by electron microscopy is not decisive, as it is very difficult to discriminate true replication origin from other possible artifacts (e.g., the denaturation loop, the R loop by transcription, and the displacement loop by recombination or replication). Other technical difficulties, such as length out of standard deviation of the expected value and binding of proteins to DNA, might accompany such artifacts. Thus, the replication origin of higher plant cells is an open question. On the other hand, the use of synchronously replicating plastid DNA was a key to determining the replication origin in Chlamydomonas reinhardtii (Waddel et al., 1984; Wang et al., 1984) and Euglena gracilis

TOBACCO BY-2 CELL LINE

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(Koller and Delius, 1982; Ravel-Chapuis et al., 1982). Thus, the application of preferential replication of plastid DNA in TBY-2 cells after transfer to the fresh medium should be a candidate to determine the replication origin of plastid DNA. When protoplasts prepared from the stationary phase were transferred to the fresh medium, the preferential incorporation of [3H]thymidine into plastids was observed. As the replication origin could be recovered in the replication fork fraction, such fraction was concentrated by benzoylated naphthoylated diethylaminoethyl (DEAE) cellulose column chromatography after treatment with restriction endonucleases (Hay and DePamphilis, 1982).After the single-strand gaps and tails were filled with a Klenow fragment of Escherichia coli DNA polymerase I, newly synthesized DNA was labeled by the addition of [cx-~’P]~CTP according to the primer extension. When the plastid DNA was digested with several restriction endonucleases and hybridized with the labeled probes, the labels were observed only in specific sites, located in the 23 S rRNA region specified in the inverted repeats of the sequence data of tobacco plastid DNA (Shinozaki et al., 1986).On the other hand, the observation of plastid DNA by electron microscopy revealed D loops in this specific region, which coincided with the fraction labeled specifically in the replication fork fraction. Therefore, Takeda et NI. (1992) concluded that they could determine the replication origin of tobacco plastid DNA, the first such discovery in higher plants. It is worth mentioning that the replication origin of the plastid DNA of higher plants was determined by use of the TBY-2 cell line.

V. Synchronization of Cells The reproduction of cells is composed of the replication of genetic materials and the successive distribution of genetic materials as well as cell components to two daughter cells, and this recurrent progression of the cell cycle is a fundamental subject in cell biology. Although the first recognition of the cell cycle that comprises G I , S, G2, and M was proposed in the study of plant cells (Howard and Pelc, 1951), understanding of molecular events of the cell cycle in plant cells is far behind that of animal cells and that of Saccharnmytes cereuisiue (Baserga, 1985).Although the essential mechanism of the cell cycle should be common among eukaryotic cells, identification of the cdc-2 gene and the protein p34cd‘-2has recently been described in plant material (John et al., 1989; Feiler and Jacobs, 1990). This is due mainly to the lack of a suitable cell synchronization system in plant cells. The study of the cell cycle of plant cells is also intriguing, however, as

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TOSHlYUKl NAGATA ET AL.

there are several specific features of plant cells. Some specific features are the presence of the preprophase band (PPB), cortical microtubules (MTs), and phragmoplasts (Lloyd, 1987). The relationship between cortical MTs and the orientation of cellulose microfibrils is also intriguing. In considering these events we tried to establish high cell synchrony in plants, and finally TBY-2 cells were synchronized using aphidicolin and an MI of approximately 70-80% was recorded (Nagata et al., 1982). Aphidicolin is a drug purified from culture filtrates of a fungus, Cephalosporium aphidicolia, and is reported to be a specific inhibitor for DNA polymerase a (Ikegami et al., 1978). Before this work the highest cell synchrony was found in Haplopappus gracilis cell cultures, using hydroxyurea as described by Eriksson (1969, in which a 35% MI was recorded. Although the chromosome abberation was frequently observed by hydroxyurea treatment, such anomaly was rare in aphidicolin treatment. This is probably because hydroxyurea was used at the millimolar level and aphidicolin was used at the micromolar level. In this section we describe the basic protocol and some specific details on the synchronization method of TBY-2 cells. Although, for a review article, such a methodological description may be unusual, it is necessary, as one of us (T.N.) has been told rather often by other researchers that our protocol is not easy to reproduce. An important point is that the growth of TBY-2 cells should be fast; cells must multiply 70- to 100-fold in 1 week, as described in Section 111, which implies that this protocol could be applied to other plant cells, if they grow fast. Under this condition high synchrony should be attained easily. In fact, several laboratories, including that of H. Shibaoka of Osaka University, have successfully introduced this synchro-

;

o

o

l 75

Time after release [houid FIG.9 Change in the mitotic index ofTBY-2 cells after release from aphidicolin treatment. S. Gz, M , and G , represent the middle of these phases of the cell cycle.

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TOBACCO BY-2 CELL LINE

nization protocol to their subjects. Ten milliliters of stationary phase cells is transferred to 95 ml of the fresh medium that contains aphidicolin (5 mg/liter), which was dissolved in pure dimethyl sulfoxide at a concentration of 5 mg/ml and kept refrigerated until use. After a 24-hr incubation on the rotary shaker at 130 rpm at 27"C, the drug was removed from the culture medium by washing with 1 liter of the fresh medium. Upon washing the cell suspension was poured into a cylindrical funnel with a fused-in fritted glass filter (17 G2 Hario glass, Tokyo), in which the flow rate of the washing medium was controlled with a Hoffman clamp placed on a silicon tube connected to the bottom of the funnel. The whole system could be easily sterilized by autoclaving. A total washing time of approximately 15 min was suitable for obtaining higher synchrony, and the cells were subsequently resuspended in the same volume of the fresh medium. When we followed the change in MI, which was determined under a microscope after staining with lactopropionic orcein according to Eriksson (1965) or with DAPI according to Yasuda et al. (1988), the first and second peaks were located 10 and 23 hr, respectively, after the release from aphidicolin, which implies that one generation time was estimated to be approximately 13 hr. The duration of cells in the M phase was calculated to be 2 hr from the first peak (Fig. 9). The length of the S phase was determined by autoradiography and by microspectrophotometry, using propidium iodide. Figure 10 shows that the percentage of tritium-labeled nuclei increased immediately after the release from aphidicolin, and essen-

"6

3

6

9

firm after release lhows)

!

FIG. 10 [3H]Thymidine incorporation into TBY-2 cells after release from aphidicolin treatment. Cells were incubated for 20 min with [3H]thymidine at the indicated times, and the percentage of cells with labeled nuclei was determined by autoradiography. The locations of the nuclei were determined by fluorescence microscopy after staining with DAPI.

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TOSHlYUKl NAGATA ET AL.

4°C %

E

c

5u

i3

2°C 0

3

6

9

12

Time after release (hours) FIG. 11 Change in the DNA content of TBY-2 cells after release from aphidicolin treatment. DNA content was determined by quantitative fluorescence microscopy after staining with propidium iodide. The 2°C and 4°C levels were calculated as the average of the values of 500 cells.

tially all cells incorporated [3H]thymidine into their nuclei during the subsequent 5-hr incubation. Fluorescence microspectroscopy after staining with propidium iodide also showed that the DNA content per cell doubled during the first 5-hr incubation (Fig. 11). Thus, the length of the S phase was estimated to be 5 hr. As seen in Fig. 9, G2 and G I are estimated to be 4 and 2.5 hr, respectively. This generation time of 13 hr is the shortest reported for plant cell suspension cultures (Gould, 1984), but this value could become much shorter, as the growth speed of TBY-2 cells is still gradually increasing. Although the cell synchrony of TBY-2 cells by aphidicolin treatment is exceptionally high, it is still insufficient, especially for the transition from the M phase to the G, phase of the cell cycle, as the extent of synchrony is gradually decreasing from the moment of release from aphidicolin treatment. To get much higher synchrony, the synchronized TBY-2 cells obtained after aphidicolin treatment were further treated with propyzamide, an antitubulin drug (Akashi et a / . , 1988), to induce mitotic arrest (Kakimot0 and Shibaoka, 1988). Propyzamide (1.6 mg/liter) was added to TBY-2 cells 6 hr after the release from aphidicolin treatment. When propyzamide was removed from the medium after 4 hr of treatment, mitotic arrest was released, the progression of the cell cycle restarted, and nearly 80-90% MI was observed soon afterward. As the effect of this drug was almost reversible, which is in sharp contrast to colchicine, the cell cycle progression was immediately recovered.

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VI. Changes in Cytoskeletons during the Cell Cycle Progression By using this highly synchronized cell popoulation, Hasezawa er al. (I991) and Hasezawa and Nagata (1991) followed characteristic features, along with the progression of the cell cycle of plant cells. Among other aspects they focused on the change in the cytoskeletons, as the orientation of cellulose microfibrils, which are main components of cell walls, are believed to be determined by MTs (Newcomb, 1969).Furthermore, as shown in stomata1 guard mother cell differentiation, the unequal cell division by septum formation initiates the direction of differentiation. As the septum formation is processed by a subcellular structure of phragmoplast, which is a complex of MTs, microfilaments (MFs), and some other components, it may be said that cytoskeletons determine the direction of differentiation of plant cells. Although previous works have clarified to some extent the role of cytoskeletons during the cell cycle (Lloyd, 1987), various important problems remain to be resolved, especially relating to the transitions between different cell cycle stages. Meanwhile, the substantial progress of the staining methods of cytoskeletons, especially of MFs, allowed observation of the detailed changes in the cytoskeletons. Although MFs were very labile to conventional fixative treatment (e.g., glutaraldehyde) and could not be seen under a fluorescence microscope, this difficulty was circumvented with the pretreatment of specimens with a buffer containing tropomyosin and rn-maleimidobenzoyl N-hydroxysuccinimide ester, a protein-stabilizing agent (Kakimoto and Shibaoka, 1987; Sonobe and Shibaoka, 1989). Thus, the triple staining with indirect fluorescent antibody to MTs, rhodamine-labeled phalloidin to MFs, and DAPI to chromosomes revealed the interrerlationship among these structures. Soon after the release from aphidicolin treatment, when the S phase began, the cytoplasmic MTs extending from perinuclear regions that had not been observed at G I reached cell membranes and were clearly observed, and the MTs were mostly overlapped with MFs (Fig. 12A and B). Accompanying this cytoskeletal change, the nucleus moved nearly to the center of the cells (Fig. 12C) and its position was supported by cytoplasmic strands consisting of MTs and MFs. At the G2 phase the formation of the PPB was most conspicuous. The PPB was connected to perinuclear MTs and partly overlapped with MFs, whose location was broader than that of the MTs (Fig. 12D-F). When a compact girdle-shaped PPB was observed, the structures, in which spindles were apparently organizing, were observed at the two pole positions. The spindle seemed to be produced at the expense of the PPB. By the time

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of mitosis, the cortical MTs disappeared completely and the location of MTs coincided with a spindle, while MFs seemed to surround the spindle and were not observed inside it (Fig. 12G-I). Toward the end of mitosis, a phragmoplast is formed, a specific structure in plant cells which is responsible for cell plate formation. MFs as well as MTs were major components of the phragmoplast (Fig. 12J-L). During the transition between cytokinesis and the G I phase, characterized as the presence of disintegrating phragmoplasts, short MTs were transiently observed on the surface of a nucleus (Fig. 12M, N, and Q), and then the MTs developed toward the direction of cell plates and elongated along the inner surface of the cell membrane to the distal end from the location of the forming cell plate. As a consequence a gradient of the distribution of MTs was observed transiently. In the G I phase no MTs were observed in the perinuclear region (Fig. 12P), and most of MTs were observed as more or less ordered cortical MTs (Hasezawa and Nagata, 1991). Thus, Hasezawa et al. (1991) and Hasezawa and Nagata (1991) followed the sequence of change in the cytoskeletons during TBY-2 cell cycle progression. This search revealed several novel features which had not been previously observed. First, the decisive moment was observed at which cortical MTs originate from the perinuclear surface. However, the passage through this moment was extremely short, and the origin of the MTs at the perinuclear surface was observed as a rare event, even after synchrony by aphidicolin treatment. In fact, as the landmark of the transition of the border of M / G I , they observed the coexistence of disintegrating phragmoplast and short perinuclear MTs (Fig. 12M) at a frequency of 0.08% (?0.03%) in the total population. To avoid certain ambiguities, the sequential synchronization method of aphidicolin and propyzamide was introduced to induce higher synchrony during progression from the M phase to the G I phase (described in Section V). Three hours after the release from propyzamide treatment, the frequency of the coexistence of disintegrating phragmoplast and short perinuclear MTs increased to 1.2% (?0.2%), while at this stage 52.2% (? 1.12%) of cells were located at the border of M/GI. If the total passage through

FIG. 12 The changes in microtubules (MTs) and microfilaments (MFs) during progression of the synchronized TBY-2 cell cycle with aphidicolin. The changes in MTs, MFs, and chromosomes were followed by a triple staining with fluorescent antibodies against MTs, rhodamine phalloidin, and DAPI, respectively. (A-C) S phase; (D-F) G2 phase; (G-I) M phase; (J-L) late M phase; (M-0) M E I ; (P-R) G I . (A, D, G, J, M, and P) Fluorescent antibodies against MTs; (B, E, H , K , N , and Q ) rhodamine phalloidin staining; (C, F, I, L, 0, and R) DAPI staining. A preprophase band is clearly seen in (D). A phragmoplast is clearly seen in (J). An arrow in (M)shows a disintegrating phragmoplast. while short MTs are observed in the perinuclear region of two daughter cells. Bars = 20 pm.

FIG. 12 (continued)

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M/GI is assumed to be approximately 1 hr, the passage through the right moment of the coexistence of disintegrating phragmoplast and short perinuclear MTs is only 2-3 min. It is important that even such a rare event could be interpreted according to the sequential synchronization method. Thus far, there have been two suppositions on the origin of cortical MTs. Earlier, Gunning’s (1980) school proposed, from electron-microscopic surveys, that the nucleating sites for MT arrays are located along cell edges. More recently, Clayton et (11. (1985) proposed, after staining with an anticentrosomal immune serum 5051 from human cells, that the nuclear envelope serves as the initiation site for the earliest MTs and subsequently ordered cortical arrays are formed. However, there was also an argument against the latter statement that the autoimmune antibody did not show any specificity (Wick, 1989). Thus, the significance of the observation by Hasezawa and Nagata (1991) is that they showed directly that short MTs initiate at the nuclear envelope and develop somewhat later to cortical MTs, but the short MTs in the perinuclear region were transiently observed and were not observed in the later stage. Since MTs have been shown to develop from the perinuclear region during the formation of PPB, a spindle, and a phragmoplast (Lloyd, 1987; Hasezawa et al., 1991; Katsuta et al., 1990), it can be generalized that there are MT organization centers on the perinuclear regions. However, this does not exclude the possibility that MTs are organized elsewhere than in the perinuclear region. Second, the phragmoplast has been characterized from the synchronized TBY-2 cells by the sequential synchronization method. Although the phragmoplast is from previous morphological studies (Lloyd, 1987), considered a plant-specific structure, its function remains to be elucidated. To study its function, it is desirable that the phragmoplast be isolated from the cells and be analyzed biochemically as well as structurally. However, as the phragmoplast forms only briefly (approximately 20-30 min) during each cell cycle, it was impossible to separate this structure according to the traditional synchronization methods. Only the highly synchronized cell system could realize this requirement. When the synchronized cells, with the sequential treatment with aphidiColin and propyzamide, were treated appropriately with cellulolytic enzymes (as described in Section IV), the protoplasts at the highest MI were obtained (Kakimoto and Shibaoka, 1988). When the enzyme treatment is started 30 min before termination of the propyzamide treatment, protoplasts are prepared 30 min after the release from this treatment. As these protoplasts formed phragmoplast synchronously, the simple disruption of the protoplasts gave phragmoplast-rich fractions, which could now serve for biochemical analysis. In fact, this fraction showed a highly intact phragmoplast under electron microscopy. The incubation of this fraction

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with [3H] UDP-glucose resulted in incorporation of the radioactivity into the central region of phragmoplasts, while [3H] UDP-xylose was incorporated into Golgi vesicles during electron-microscopic autoradiography (H. Shibaoka, personal communication). On the other hand, Asada et al. (1991) treated the protoplasts, which had been synchronized by the sequential synchronization procedure, with glycerin. Although the glycerinated model cells lost cell cycle progression, the phragmoplast could translocate MTs toward the minus ends concomitantly, with tubulin polymerization at the plus ends located in the equatorial region. The translocation could be visualized by fluorescence microscopy, when the cells were treated with tubulins labeled with dichlorotriazonil aminofluorescein. The translocation was induced effectively by GTP, but less effectively by ATP. Thus, it has been shown that the equatorial region of phragmoplasts seems to be associated with a mechanochemical enzyme that generates the force for MT translocation by hydrolyzing GTP. Thus, the study of the isolated phragmoplasts could contribute to understanding the biochemical and molecular features of plant phragmoplasts. Another characteristic of the cell cycle events of the plant cell is the migration of the nucleus from the periphery to the center of the cell, while in the GI phase the cell nucleus is located in the periphery. This migration has been examined in details by Katsuta et al. (1990), using the TBY-2 synchronized system. Although the nucleus was tethered with cytoplasmic strands consisting of both MTs and MFs, this migration was insensitive to cytochalasin D, an anti-MF drug, but sensitive to propyzamide, an anti-MT drug (Akashi et al., 1988), which suggests that the role of MTs is more important than that of MFs in this migration. Furthermore, the disruption of MTs by propyzamide prevented the formation of PPB-like MFs as well as PPB, while the disruption of MFs by cytochalasin D did not prevent the formation of PPB. Thus, the migration of the nucleus in the premitotic stage is primarily responsible for MTs, which could play a role such as a scaffold, and along with this structure the network of MFs is formed. On the other hand, in later stages, the MFs support the mitotic spindle as well as phragmoplasts, when the cytoplasmic MTs disappeared completely. Similarly, Katsuta and Shibaoka (1988) observed that when protoplasts from TBY-2 cells were cultured in the elongation medium as described by Hasezawa and Syono (1983), the nucleus became located in the central position of the cell and was supported only by MFs. In these cells the disruption of MFs by cytochalasin D brought about the translocation of the nucleus to the cell periphery. As the cells in this culture condition can be interpreted as G I phase in the cell cycle, the migration of the nucleus in GI phase should be controlled by MFs.

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Although thus far we have seen drastic structural changes in cytoskeletons during the cell cycle progression of TBY-2 cells, a subsequent intriguing question should be what is the control mechanism of such drastic change in the cytoskeletons. According to the accumulated information from studies using animal cells as well as yeast, phosphorylation plays an important role during cell cycle progression. The level of phosphorylation should be controlled by protein kinases as well as protein phosphases. Although such information is poor in plant cells, some drugs (e.g., okadaic acid and calyculin A) could be useful for such analysis; both of these drugs have been shown to be specific inhibitors for protein phosphatases I and IIa (Haystead et al., 1989; Kipreros and Wang, 1990). In fact, the addition of these drugs to each cell cycle stage of the synchronized TBY-2 cells blocked the progression of each cell cycle stage to the next. Two noticeable changes were observed: (1) the treatment of G 2cells with these drugs did not form a PPB at all, and (2) upon the addition of one of these drugs at G2/M, the progression of mitosis was accelerated significantly. Furthermore, progression of the cell cycle was severely blocked at the border of MIGl (S. Hasezawa, S. Kawasaki, and T. Nagata, unpublished observations). Although this observation is still indirect, phosphorylation seems to play an important role during the cell cycle progression of plant cells as well, and the drastic spatial changes of MTs during the cell cycle as a plant-specific phenomenon have been regulated by phosphorylation.

VII. Cytological Investigation of Cell Elongation Although elongation growth is a plant-specific phenomenon and there is a mass of literature at the tissue and organ levels (Wareing and Phillips, 1981), there has been a paucity of information on the cellular level. When protoplasts prepared from TBY-2 cells were cultured in the FMS medium (Hasezawa and Syono, 1983)supplemented with I-naphthalene acetic acid (0.1 mglliter) and 6-benzyl aminopurine (1 mglliter), conspicuous elongation of TBY-2 cells was observed, while in the medium supplemented with 0.2 mglliter of 2,4-D, active division was observed. In TBY-2 cells elongation growth can be separated from division growth by changing combinations of plant hormones. Thus, the regulation of the cell elongation by a combination of auxin and cytokinin is intriguing for analyzing the elongation mechanism of plant cells under this condition. Elongation of cultured protoplasts started from 2 days of culture, and after 1 week reached nearly 400 pm. When the orientation and development of MTs and MFs were

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followed in cultured protoplasts, MTs and MFs were oriented in random directions just after the culture, then became perpendicular to the long axis after 2 days of culture. Thus, the direction of orientation of MTs and MFs went parallel with the elongation of cells (Hasezawa et al., 1988,19891,but the causal relationship between cell elongation and the orientation of MTs remain to be determined.

VIII. Gene Delivery into Cells

Delivery of genes into the protoplasts from TBY-2 cells mediated by liposomes has been introduced only briefly and has been described previously in detail by one of us (Nagata, 1987a,b). Liposomes of phosphatidylserine and cholesterol encapsulating tobacco mosaic virus (TMV) RNA were prepared essentially according to the reversed-phase evaporation vesicle method (Szoka and Papahadjopoulos, 1978)and were mixed with TBY-2 protoplasts. Subsequently, 12.5% polyethylene glycol (PEG) 1540 or 10% polyvinyl alcohol (PVA) was added to the mixture of liposomes and protoplasts. After an incubation of 10 min (PEG) or 15 min (PVA) at room temperature, the PEG or PVA was removed by washing with a solution of 0.05 M glycine buffer (pH 10.5), 0.05 M CaC12,and 0.4 M mannitol. The protoplasts were then cultured for 24 hr in the modified Linsmaier and Skoog (1965) medium (described in Section 111), supplemented with 0.4 M mannitol and 1% sucrose. In this system the delivery of intact and functional TMV RNA could be assessed by staining with a fluorescent antibody raised against TMV. Under optimal conditions the functional RNA was introduced to 80% of the protoplasts. It should be noted that liposomes were taken up endocytotically by protoplasts by electron microscopy, although delivery has been performed during treatment, using a combination of PEG and PVA with high pH-high calcium buffer. The treatment of protoplasts in this way was originally developed for the fusion of protoplasts (Nagata, 1987a,b). Electroporation of genes into protoplasts from TBY-2 cells has been successfully performed, but, since details have been described by one of us (Nagata, 1989) in a previous volume in this series, only essential points are described here. After protoplasts suspended in a medium consisting of 5 mM 2-(N-morpholino)ethanesulfonicacid buffer (pH 5 . 8 ) , 70 mM KCl, and 0.4 M mannitol were mixed with DNA or RNA, an electric impulse was released from a condensor (100 FF), to which electricity had been stored from a 300 V power supply. Upon a simple impulse genes were introduced to inore than 90% of the protoplasts. Delivery into protoplasts of four cell cycle stages from highly synchronized TBY-2 cells was also

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feasible (Nagata et al., 1987). Stable transformants were obtained according to the procedure described above, when neomycin phosphotransferase I1 was used as a selectable marker (Okada e f al., 1986). The gene delivery into TBY-2 cells could be performed by aid of Agrobacterium tumefaciens at high frequency. An (1985) showed that when the tobacco NT-I cell line, a sibling of TBY-2, was cocultivated simply with A . tumefuciens, harboring a binary vector of pGA473 and pTi Bo542, kanamycin-resistant clones were obtained maximally at a frequency of 50%. pGA473 retains a kanamycin-resistant neomycin phosphotransferase I1 gene as a selectable marker in plants. This frequency was dependent on the culture stage and was highest after 3-4 days of subculture. Concurrently, the induction of VirD, VirB, and VirE genes in the Ti plasmid were observed maximally 1, 2, and 2-3 days after transfer, respectively. According to recent understanding of the transformation mechanism of plant cells by the Ti plasmid, first Vir genes are induced by the plant exudates; subsequently, by the action of Vir genes, the T strand will be excised from the Ti plasmid. The T strand, combined with DNA-binding proteins, is transferred to the plant cells and integrated into plant genomes by recombination (Zambryski et al., 1989). This story is one of the most attractive in the recent development of plant molecular biology. During the elucidation of this mechanism, tobacco NT-I (TBY-2) played an important role. As cited by An (1985), Vir genes were induced by cocultivation with tobacco cells. At the time of cocultivation Stachel et al. (1986) noticed that a low-molecular-weight heat-stable plant metabolite exudated from plant cells into the culture medium and induced the expression of Vir genes. This metabolite was soon identified as acetosyringone and related phenolics (Stachel et al., 1985; Bolton et al., 1986).

IX. Supplementary Remarks As we have described, TBY-2 cells grow fast and can be synchronized to a high degree, and thus are suitable for studying the cellular and molecular biologies of plant cells. Such a cell line is highly necessary, as information on plant cells at the cellular level is still much less than that on other eukaryotes of animal cells and yeast. Another important point is that molecular and subcellular studies are easily feasible, as mass culture of TBY-2 cells is readily available. However, there is a laborious point, as this material must be continuously cultured in a proper condition to obtain a high growth rate and high synchrony. This is due to the fact that practical freeze preservation method has not been developed for plant cells. Conse-

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quently, this material could accumulate possible point mutations during long-term culture, and so it may not be appropriate for study of the genome structure of plant cells. Thus far, the generation of whole plants from this material has not been found and although one of us (T.N.) is still trying to develop such a method, such efforts seem to be in vain. This problem could be overcome by fusion with other cells retaining regeneration ability, as Maliga et al. (1977) have shown. Even if we consider such drawbacks of this cell line, there is no doubt that this material is most suitable for basic study of the cellular and molecular biologies of plant cells. This material could probably be combined with Arabidopsis rhaliana, which has been shown to be suitable for genetic study, as this material has small genome (Meyerowitz and Pruitt, 1985). Finally, we can supply this material to anyone who would like to use it to increase understanding of the basic characteristics of plant cells.

X. Summary

TBY-2 derived from the seedlings of N . tabacum L. cv. Bright Yellow 2 grows fast and multiplies 80- to 100-fold in 1 week. After the stationary phase cells of TBY-2 were transferred to a medium containing aphidicolin for 24 hr and then released from treatment, high synchrony was obtained starting from the S phase. The subsequent arrest of cells at metaphase with propizamide and the release from this treatment offered higher synchrony, starting from the M phase. Using this synchrony system, the change in the cell cycle progression of TBY-2 cells successfully followed the change in cytoskeletons. Another important point is that one could do biochemical and molecular biological studies on this material, since mass culture of this material is readily feasible, although, so far, cell cycle events have been studies of only the morphological aspects. Such an example is the biochemical study of a phragmoplast, a plant-specific structure, which has not been separated by conventional methods. Acknowledgments We would like to express our deepest thanks to Takashi Matsumoto, Japan Tabacco, Inc., who ailowed one of us (T.N.) to use this cell line initially, and Nobutaka Takahashi, University of Tokyo, who made great efforts to help us with the purchase of this cell line. Thanks are also due to Hiroh Shibaoka of Osaka University, who allowed us to see several unpublished articles by his group and gave us helpful comments in preparing this manuscript. This research was supported in part by grants from both the Ministry of Education, Science and Culture of Japan and the Ministry of Agriculture, Forestry and Fisheries of Japan (to T . N . ) .

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