Keywords: Tobacco BY-2; Cell cycle synchronization; Aphidicolin;. Propyzamide. Introduction. Tobacco BY-2 suspension cultured cells (Nicotiana tabacum cv.
Protoplasma (1998) 202:232-236
PROTOR.ASMA 9 Springer-Verlag 1998 Printed in Austria
Optimizing conditions for tobacco BY-2 cell cycle synchronization
Rapid Communication A. L. Samuels*, J. Meehl, M. Lipe, and L. A. Staehelin Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado Received July 5, 1996 Accepted March 9, I998
Summary. Tobacco BY-2 cells have become a major tool in plant cell biological research, in part due to the availability of a cell cycle synchronization protocol. This method, pioneered by Nagata and coworkers, involves sequential treatments with aphidicolin (a DNA synthesis inhibitor) and propyzamide (a microtubule inhibitor which arrests mitosis). The effects of these inhibitors are reversible, allowing the cell culture to progress into M phase synchronously. However, attempts to reproduce high levels of synchrony with published protocols have not been uniformly successful. This paper describes critical parameters for cell cycle synchronization and documents the kinetics and variation typically found in using this protocol. Keywords: Tobacco BY-2; Cell cycle synchronization; Aphidicolin; Propyzamide.
Introduction Tobacco BY-2 suspension cultured cells (Nicotiana tabacum cv. Bright Yellow-2; Japan Tobacco, Inc.) are becoming an increasingly popular model as this cell line is fast growing, easily cultured and has the added advantage that protocols exist to synchronize the cell cycle (Nagata et al. 1992). Synchronization is achieved by reversible chemical inhibition of DNA synthesis (Nagata et al. 1982) or both DNA synthesis and microtubule assembly (Kakimoto and Shibaoka 1988). While successful examples of cell cycle synchronization have been reported (Asada et al. 1991, 1994; Yasuhara et al. 1993; Hasezawa et al. 1994; Shibaoka et al. 1996), this protocol is notoriously problematic. As noted by Nagata et al. (1992), "... *Correspondence and reprints: Department of Botany, University of British Columbia, Vancouver, B. C. V6T 1Z4, Canada.
one of us (T. N.) has been told rather often by other researchers that our protocol is not easy to reproduce". In the course of ultrastructural studies of cell plate formation in tobacco BY-2 cells (Samuels et al. 1995, Samuels and Staehelin 1996), we have defined parameters for successful synchronization. Material and methods Cell cycle synchronization Suspension cultured tobacco BY-2 cells were grown in Linsmaier and Skoog (1965) medium with minor modifications as described by Nagata etal. (1992) and subcultured every 7d. Aphidicolin (3 ~g/ml; Calbiochem or Fluka) was added to a 250 ml Erlenmeyer flask containing 95 ml of fresh culture medium and autoclaved. (Note: final volume will be 100 ml after addition of cells.) Stock solution of aphidicolin was made up in DMSO at 5 mg/ml and stored in aliquots at 4 ~ According to Kakimoto and Shibaoka (1988), DMSO at 0.1% and lower concentrations has no effect on the microtubules or cell division in tobacco BY-2 cells. To begin the synchronization, 5 ml of 6-7 d subcultured tobacco BY-2 cells were added to a 250 ml flask containing aphidicolin and to a control (no aphidicolin) culture. The cultures were grown for 24 h under standard culture conditions (Nagata et al. 1992). Cells were released from aphidicolin inhibition by washing with culture medium in an autoclaved Nalgene filter holder (Nalgene catalog nr. 300-4000; Rochester, N.Y.) with a 30 ~tm nylon mesh filter fitted into a 47 mm filter unit. This procedure seemed to be critical to achieve a sharp increase in mitotic index later. (Note: wash medium could be diluted 1:1 with sterile 3% sucrose without adverse effects.) Details of washing are the following. a. The contents of the aphidicolin-containing flask were emptied into the top of the Nalgene filter holder with one of its bottom ports removed, allowing the solution to drain through the filter into the lower compartment. The cells were collected on the nylon mesh but
A. L. Samuels et al.: Tobacco BY-2 cell cycle synchronization not allowed to dry. The port covers were then attached to the bottom ports to stop the flow of solution, 250 ml of wash medium was added to the top and the lid was capped back on. The time of this "aphidicolin release" point was noted and an aliquot of cells was sampled for mitotic index. b. To remove the aphidicolin-containing solution from the bottom receiver of the filter holder, a thin Tygon tube was fed through one of the bottom ports and the solution was removed by gentle application of a vacuum. The ports were capped and the filter holder with the suspended cells was agitated for 15 min on the culture shaker. c. At the end of the 15 rain, the wash solution was drained and replaced as above. In all, five changes of 250 ml each were made, to ensure complete removal of aphidicotin. After washing, the cells were resuspended in 95 ml of fresh medium and the lid was replaced. The wash solution was removed with the Tygon tube and the bottom ports capped. The lid was removed and the suspended cells were poured into a sterile 100 ml cylinder to check the volume before transferring to a 250 ml Erlenmeyer flask and returning the flask to the shaker. 3 h after aphidicolin release, propyzamide was added to a final concentration of 6 ,aM (propyzamide is also known as pronamide; it is the active ingredient in the commercial herbicide Kerb). Technicalgrade propyzamide was supplied by Rohm and Haas Co., Springhouse, Pa., U.S.A. Propyzamide was made up as a 10 mM stock solution in DMSO which has a shelf life of about 4 months. 5 h after propyzamide was added, cells were sampled for mitotic-index determination; the mitotic index at this point was a good indicator of the degree of synchronization at propyzamide removal. 6 h after adding propyzamide, the Nalgene filter holder was used to wash out the propyzamide. Three changes of 200 ml each were made; each wash solution was incubated for 10 min with shaking. The time at which the first propyzamide wash solution was added was noted and designated time zero, propyzamide release. At this time, samples were taken for a mitotic-index determination. After washing out the propyzamide, the cells were suspended in fresh medium and returned to the shaker. 30 min after propyzamide release (time 30) and every 30 min thereafter, samples for mitoticindex analysis were taken.
Histochemical staining methods The mitotic index was determined with DAPI (4',6-diamidino-2phenylindole; Molecular Probes, Eugene, Oreg., U.S.A.), which was prepared as a 0.5 mg/ml stock solution in acetone and used at a final concentration of 1 ug/ml. Sampling was done by removing 1 ml aliquots of cell suspension, cells were allowed to settle, then fixed in Carnoy's fixative (3 : 1 100% ethanol : glacial acetic acid). Cells were either stored in fixative for later analysis, or after at least 15 min in fixative, the cells were rinsed in growth medium and DAPI stained. The mitotic index was calculated as the number of cells in mitosis divided by the total number of cells counted (500 cells per time point). Callose deposits were visualized by staining with aniline blue. 1 ml aliquots of cell suspension were taken at 30 min intervals after propyzamide release and fixed in 0.25% glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2) for 30 min at room temperature or overnight at 4 ~ After two rinses in buffer, cells were resuspended in 0.1% aniline blue in 0.05 M phosphate buffer for staining. The cells were kept in the staining solution until they were viewed.
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Results and discussion The key to synchronization of the cell cycle in tobacco BY-2 cells is blocking DNA synthesis with the mycotoxin aphidicolin. This tetracyclic diterpenoid inhibits the plant DNA polymerase-c~ (Sala et al. 1980; reviewed by Spadari et al. 1982). The cells were cultured with aphidicolin for 24 h which allowed all of the cells to cycle into S phase where they were arrested; the average cell cycle time for the tobacco BY-2 cell line has been reported to be 13 h (Nagata et al. 1992) to 21 h (R. Cyr pers. comm.). At the end of the aphidicolin incubation, the mitotic index was 0-1% when the cells were examined with DAPI staining. The efficacy of aphidicolin varied widely among suppliers. Aphidicolin from Sigma, which was used at 5 ~tg/ml gave inconsistent synchronization results, while aphidicolin from Calbiochem and Fluka could be used at 3 ~tg/ml with superior results. Complete removal of the aphidicolin from the cell cultures was critically important in getting good synchronization. If the aphidicolin was not completely washed out, the mitotic index remained near zero. A Nalgene reusable autoclavable filter holder fitted with a 47 mm filter made from 30 ~tm nylon mesh was useful for allowing the cells maximum exposure to the wash solution. During the wash periods, the cultures were either agitated by hand, or returned to their shaker. Hand agitation sometimes resulted in large callose deposits (Fig. 1 g), suggesting injury to the cells. Therefore, returning the cultures to the shaker during the 15 rain wash period was the method of choice. The removal of aphidicolin allowed the cells to resume DNA synthesis and progress into M. During G2, propyzamide was added to the cultures to inhibit spindle microtubule assembly and arrest the cells in prometaphase. Propyzamide acts as a reversible inhibitor of microtubule assembly in plant (but not animal) cells (Akashi et al. 1988). If the subject of the research is preprophase or prophase events, aphidicolin alone should be used, as propyzamide treatment disrupts microtubule assembly during preprophase, prophase, and prometaphase (e.g., Hasezawa et al. 1994). Repolymerization of spindle microtubules was observed within 15 min after removal of the drug from the culture (also reported by Akashi et al. 1988). Propyzamide was readily washed out of the cells in culture, using the same Nalgene filter holder equipped with a nylon mesh filter. The timing of experimental
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A.L. Samuels et al.: Tobacco BY-2 cell cycle synchronization
Fig. 1 a-g. DAPI stained tobacco BY-2 cell cultures following cell cycle synchronization, a Interphase cells' nuclei (In); bar: 10 ~tm. b Prophase cell (Pr) with thread-like condensing chromosomes; bar: 10 ~tm. c Prometaphase cells (PM) prior to removal of propyzamide; chromosomes are condensed but not aligned on the metaphase plate, d 30 rain after removal of propyzamide, metaphase cells (M) with re-formed spindles; chromosomes are aligned in the equatorial zone. e 60 rain after removal of propyzamide, cells in metaphase (M), anaphase (A), and telophase (T). f 90 min after propyzamide washout, telophase cells are abundant (T); bar for c-f: 50 ~m. g Aniline blue/DAPI double-stained cells with callose deposits (arrows) and cell plates (cp). Bar: 50 ~m
protocols became critical at this point, because the cells rapidly progressed through mitosis. A reliable technique for measurement of the mitotic index during this procedure was staining fixed cells 'with the fluorochrome DAPI (Fig. 1). Because the
ceils grew in clumps, this bright fluorescent stain was useful for accentuating the nuclei amongst the cells. Cells in various stages of mitosis were readily distinguished from interphase cells by the brightness of their condensed chromosomes (compare Fig. 1 a,
A. L. Samuelset al.: TobaccoBY-2 cell cycle synchronization
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60 [] prophase [] metaphase [] anaphase [] telophase
8 50 .~ 40 "~ 30 .~ 20
0
J control
0 30 60 90 120 time after release from propyzamide,rain
Fig. 2. Time course of tobacco BY-2 cells' progress throughmitosis following cell cycle synchronizationwith aphidicolin and propyzamide. Control samples were unsynchronizedcell cultures which were subculturedat the sametime as synchronizationprotocolswere initiated. Synchronizedcells were labeled at the time of removal of propyzamide and every 30 min thereafter; data expressed as mean with SE for 16 experiments
interphase nucleus, with b-g, mitotic arrays). Prophase cells were identified by their lack of nucleolus and condensing, thread-like chromosomes (Fig. 1 b). Cells arrested in prometaphase prior to propyzamide removal showed condensed chromosomes which were not aligned on the cell equator because the mitotic spindle had not yet re-formed (Fig. 1 c). 30 min after removal of propyzamide, the microtubules of the spindle had re-formed; the cultures were dominated by cells in metaphase, with the brightly condensed chromosomes in the plate-like equatorial array (Fig. 1 d). After 60 min, cells began to move into anaphase and telophase (Fig. 1 e), with cells in telophase dominating by 120 min (Fig. 1 f). To distinguish cells in telophase from those which had passed on into interphase, the condensation of the daughter nuclei was considered and the completion of the cell plate checked with phase-contrast bright-field microscopy. Perhaps the most intriguing aspect of the synchronization procedure was the pattern of variation in the cultures: cells in each clump tended to have similar responses to the synchronization procedure. Thus one clump had all cells in interphase, apparently unresponsive to the synchronization procedure, while an adjacent clump consisted of cells in mitosis only. To examine this variation and quantify the extent of synchronization, at least 500 DAPI-stained cells were counted for each time point over the course of mitosis (data from 16 experiments) (Fig. 2). In the earlier
stages of synchronization, the degree of synchrony was higher: at the time of propyzamide washout, the majority of the cells (54% + 4%, mean and SE) were in prometaphase and 30 min later metaphase cells still predominated (47% + 4%). At the later time points, there was a mixture of phases of mitosis present, e.g., 90 min after propyzamide removal, there were cells in metaphase (24% + 2%), anaphase (14% + 1%), and telophase (21% + 3%). For each time point, the total mitotic index (sum of all phases, mean 61% + 13%) did not change drastically. This suggests that although a large wave of cells progress through mitosis as expected, there were also subpopulations of cells which progressed through mitosis with different kinetics, e.g., those still in metaphase after 120 min. At later time points (~150 min), the mitotic index declined steadily (data not shown). The relative abundance of cells in different phases of mitosis also reflected the length of time that it takes the phase to occur. For example, anaphase was always underrepresented when compared to metaphase and telophase; anaphase is the shortest phase of mitosis. The inherent variability in this technique remains enigmatic. The greatest variation was in the degree of synchronization, whereas for the majority of cells, the timing of the mitotic events following propyzamide removal was predictable. Certainly, healthy wellmaintained cultures are essential. But even in synchronizations from these active cultures, the degree of synchronization could range between a total mitotic index of 42-81%. Each suspension culture may contain subpopulations of cells with slightly different phenotypes. In the case of response to aphidicolin, some cells may have stopped actively dividing. Nagata etal. (1992) described [3H]thymidine autoradiography in which tobacco BY-2 cells were incubated in [3H]thymidine for 24 h, then classified according to whether they had synthesized DNA into their plastids or nuclei. Their results showed that 1-2 days after subculturing, an average of 40% of the cells had no label in the nuclei. These results are consistent with our finding that, on average, 40% of the cells in our populations were not responsive to aphidicolin inhibition of DNA synthesis. Understanding the basic mechanisms of this protocol is important for integrating synchronization into well designed experiments. While the synchronization is not perfect, it represents an approximately 10-fold enrichment of mitotic cells compared to unsynchronized control samples.
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Acknowledgements This project was supported by USDA grant no. 96-35304-3710 and a Canadian NSERC post-doctoral fellowship to ALS, also thanks to Kristan Donelly, Heidi Chail, and Jim D'Antonio for staying late to help with synchronizations. Thanks to Richard Cyr for input and advice.
References Akashi T, Izumi K, Nagano M, Enomoto E, Mizuno K, Shibaoka H (1988) Effects of propyzamide on tobacco cell microtubules in vivo and in vitro. Plant Cell Physiol 29:1053-1062 Asada T, Shibaoka H (1994) Isolation of polypeptides with microtubule-translocating activity from phragmoplasts of tobacco BY2 cells. J Cell Sci 107:2249-2257 Sonobe S, Shiboaka H (1991) Microtubule translocation in the cytokinetic apparatus of cultured tobacco cells. Nature 350: 238-241 Hasezawa S, Sano T, Nagata T (1994) Oblique cell plate formation in tobacco BY-2 ceils originates in double preprophase bands. J Plant Res 107:355-359 Kakimoto T, Shibaoka H (1988) Cytoskeletal ultrastructure of phragmoplast-nuclei complexes isolated from cultured tobacco cells. Protoplasma Suppl 2:95-103 Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100-127 -
A.L. Samuels et al.: Tobacco BY-2 cell cycle synchronization Nagata T, Okada K, Takebe I (1982) Mitotic protoplasts and their infection with tobacco mosaic virus RNA encapsulated in liposomes. Plant Cell Rep 1:250-252 - Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the "HeLa" cell in the cell biology of higher plants. Int Rev Cytol 132:1-30 Sala F, Parisi B, Burroni D, Amileni AR, Pedrali-Noy G, Spadari S (1980) Specific and reversible inhibition by aphidicolin of the alike DNA polymerase of plant cells. FEBS Lett 117:93-98 Samuels AL, Staehelin LA (1996) Caffeine inhibits cell plate formation by disrupting membrane reorganization just after the vesicle fusion step. Protoplasma 195:144-155 Giddings TH Jr, Staehelin LA (1995) Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants. J Cell Biol 130:1345-1357 Shibaoka H, Asada T, Yamamoto S, Sonobe S (1996) The use of model systems prepared from tobacco BY-2 cells for studies of the plant cytoskeleton. J Microsc 181:145-152 Spadari S, Sala F, Pedrali-Noy G (1982) Aphidicolin: a specific inhibitor of nuclear DNA replication in eukaryotes. Trends Biochem Sci 62:29-32 Yasuhara H, Sonobe S, Shibaoka H (1993) Effects of taxol on the development of the cell plate and of the phragmoplast in tobacco BY-2 cells. Plant Cell Physiol 34:21-29
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