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Determination of the mitotic index by microinjection of fluorescently labelled tubulin
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European Journal of Cell Biology 81, 169 ± 174 (2002, March) ¥ ¹ Urban & Fischer Verlag ¥ Jena http://www.urbanfischer.de/journals/ejcb
169
Determination of the mitotic index by microinjection of fluorescently labelled tubulin Sandra L. Schwindling2), Michael Faust2), Mathias Montenarh1) Medical Biochemistry and Molecular Biology, University of the Saarland, Homburg/Germany Received October 15, 2001 Received in revised version December 7, 2001 Accepted December 9, 2001
Microinjection ± fluorescently labelled tubulin ± immunofluorescence ± mitosis ± spindle The microneedle injection technique is one of the most established procedures for the introduction of proteins into living cells. To analyse injected proteins which are important in cell cycle progression it is often necessary to determine the mitotic index. Measuring the mitotic index after microinjection is complicated because only a limited number of cells of the whole cell population is microinjected. Therefore, we attempted to establish a new method to determine the mitotic index using microinjection of fluorescently labelled a/b-tubulin into mammalian cells which allows to monitor the injected cells simultaneously with the determination of the mitotic index. We demonstrated that fluorescently labelled tubulin incorporates efficiently into the mitotic spindle apparatus. Fluorescence remains stable for several hours which is sufficient to observe the progression of cells through the M-phase of the cell cycle. The determination of the mitotic index with the method presented here gave similar results to those determined using other methods. With this method also different stages of mitosis can be visualized by analysing various steps of spindle formation. Thus, this rapid method allows the monitoring of the injected cells after microneedle injection and simultaneously the determination of the mitotic index.
Introduction The process of cell division has fascinated scientists for more than 100 years. Most of what we know about cell cycle and cell division is the result of the study of cells in tissue culture. Our present knowledge about the cell cycle and mitosis stems from
1)
Professor Dr. Mathias Montenarh, Medical Biochemistry and Molecular Biology, University of the Saarland, Building 44, D-66424 Homburg/Germany, e-mail: [email protected], Fax: 49 6841 162 6027. 2) These authors contributed equally to this work.
observations of morphological changes and from the staining of proteins which are actively implicated in cell cycle regulation, morphological alterations or mitotic dynamics. More recently, proteins which are suspected to regulate cell cycle progression and cell division are introduced into living cells in order to study their contribution to the regulation of these processes. There are several techniques currently used for the introduction of proteins and also of nucleic acids into living cells namely transfection of DNA or microneedle injection of proteins or nucleic acids (Wang et al., 1982). Microinjection techniques have been used for over 40 years to introduce nucleic acids, viral proteins, antibodies and fluorescently labelled proteins (for review see (Wang et al., 1982)). The microinjection of fluorescently labelled actin or tubulin into mammalian tissue culture cells has demonstrated the general application of this approach to observe the contractile apparatus of living cells by showing the apparent incorporation of these dyes into the cytoskeleton of the living cells. The dynamics of this fluorescently labelled proteins in living cells allows to monitor cell cycle progression and in particular mitosis. In any population of cycling cells only a limited number of cells is in mitosis at the same time. The percentage of dividing cells is defined as the mitotic index which is commonly determined by various staining methods (staining of DNA, microtubules or mitosis-specific proteins) (Bunz et al., 1998; Gowdy et al., 1998; Wakefield et al., 2000), cell scoring techniques (determination of the DNA content by flow cytometry) (Bunz et al., 1998; Gowdy et al., 1998) or microscopic examination of the cell morphology (Clute and Pines, 1999). In mitosis chromosomes are condensed in a more compact form, and their morphological alterations allow distinguishing between interphase and mitosis. One established chromosome/DNA staining procedure is the labelling of chromosomes with DNA intercalating dyes (e. g. DAPI, Hoechst 33342) (Girard et al., 1992; Gabrielli et al., 1996; Bunz et al., 1998). Chromosome condensation is also associated with extensive phosphorylation of proteins which can be detected by mitosis-specific monoclonal MPM-2 (Davis et al., 1983) or 3F3/2 (Daum and Gorbsky, 1998) antibodies. Therefore, these antibodies are frequently used to determine the mitotic index (Escargueil et al., 2000). Another unique feature
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of mitosis is the formation of mitotic spindles from microtubules that will transport chromosomes into the daughter cells. This mitotic spindle formation can be detected by immunofluorescence analysis with a/b-tubulin-specific antibodies. Furthermore, with this method the identification of mitotic cells from late prophase up to cytokinesis is possible (Glover et al., 1998; Qian et al., 1999). An alternative approach to determine the mitotic index in a cell population is the use of fluorescenceactivated cell sorting (FACS) (Larsen et al., 1986). Mitotic cells are characterized by a rounded shape and a reduced cell surface contact. Therefore, mitotic cells can also be identified by microscopical examination of cell morphology. However, this method requires much expert knowledge in cell culture. To analyse the function of microinjected proteins, which are involved in the regulation of the G2/M transition or in mitosis the determination of the mitotic index is often necessary. In combination with microinjection of proteins all of the abovedescribed procedures have a lot of disadvantages. All staining methods to determine the mitotic index are associated with a loss of cells, due to fixation, permeabilisation, washing and staining procedures. Furthermore, between 200 and 500 cells are usually microinjected which is not enough to determine the mitotic index by FACS analysis because this method requires an average of 10 000 cells. Thus, FACS analysis is not an appropriate method to determine the mitotic index after microinjection. To overcome the disadvantages of the methods mentioned above we have established a new method to determine the mitotic index by using injected fluorescently labelled tubulin into mammalian cells. Fluorescently labelled tubulin incorporates into spindle microtubules, which can then be counted. The advantages of this ™one step technique∫ are the rapid discovery of the injected cells and the simultaneous determination of the mitotic index without subsequent immunohistochemical procedures.
(Eppendorf) and the Axiovert 100 microscope (Zeiss, Jena, Germany). After microinjection cells were incubated at 37 8C for different times. They were washed with PBS and fixed for 10 min at room temperature in 3.7% formaldehyde. After washing twice with phosphate-buffered saline (PBS), cells were briefly rinsed with water to remove salts, and finally coverslips were mounted with cells down on a drop of mounting medium and analysed under a fluorescence microscope.
Materials and methods
Microinjected tubulin-rhodamine incorporates into the microtubules of living cells
Cell culture
To verify whether injected tubulin-rhodamine can be used as a marker for the determination of the mitotic index, we first analysed the incorporation of dye-labelled tubulin into the cytoskeleton of cos-1 cells. Tubulin-rhodamine was diluted and microinjected into the cytoplasm of cos-1 cells, which were grown on Cellocate coverslips (Eppendorf) for 48 h. On average 200 ± 300 cells were injected in one session. The tubulin-rhodamine-injected cells were incubated at 37 8C in a humidified 5% CO2 atmosphere for 1 h to allow incorporation of labelled tubulin into the cytoskeleton. Cells were washed with PBS at room temperature, to avoid tubulin depolymerisation. Then, cells were fixed with 3.7% formaldehyde and prepared for fluorescence microscopy. In Figure 1A it is shown that labelled tubulin incorporates into interphase arrays and into the bright centrosomal focus of cos-1 cells. For comparison Figure 1B shows an immunofluorescence analysis with monoclonal anti-a-tubulin antibodies. Staining is quite similar to the one shown in Figure 1A. As a further control we investigated whether labelled tubulin was incorporated in the microtubules or only spread diffusely within the cytoplasm. To address this problem we microinjected tubulin-rhodamine with or without the cytoskeleton-affecting drug nocodazole (200 mg/ml; Sigma). Nocodazole inhibits the assembly of newly formed microtubules and breaks down pre-existing microtubules into free tubulin subunits (De Brabander et al., 1976). We found
cos-1 cells were maintained in Dulbecco×s modified Eagle×s medium (DMEM) supplemented with 10% foetal calf serum (FCS) at 37 8C in a 5% CO2 atmosphere. For the culture of SAOS ts p53 V138 ± 8 cells (kindly provided by Dr. Klaus Roemer, Homburg) the medium contained 200 mg/ml G418. To obtain a G2/M arrest, the SAOS ts p53 V138 ± 8 cells were shifted to 31 8C for 48 h.
Microinjection For microinjection experiments, cells were grown to 50% ± 70% confluence on Cellocate coverslips with 55 mm grid size (Eppendorf, Hamburg, Germany) in 60-mm Petri dishes. Coverslips were transferred to fresh medium, containing 25 mM Hepes buffer (pH 7.8) shortly before injection. After injection, coverslips were placed into fresh medium containing 10% FCS. Cellocate coverslips were pre-coated with poly-L-lysine (Sigma, Taufkirchen, Germany) or collagen (Becton Dickinson, New Jersey, USA) as described by the manufacturer. For the microinjection experiments, we used rhodamine-conjugated bovine brain tubulin (10 mg/ml, tubulin-rhodamine, Molecular Probes, Leiden, The Netherlands). This reagent is supplied in 10-ml aliquots and should be rapidly thawed, divided into 1 ± 2-ml aliquots, rapidly frozen in liquid nitrogen and stored at 70 8C. Prior to use, the aliquots were thawed, diluted in the respective solution and centrifuged for 30 min at 90 000 g at 4 8C. The supernatant was used for the experiments. Samples should be kept strictly on ice until use, because tubulin will polymerise at room temperature and clog the micropipette. For microinjection we used Femtotips II, the Transjector 5246, the Micromanipulator 5171
Immunofluorescence For immunofluorescence, cells were grown on coverslips without grids in 100-mm Petri dishes up to 50% ± 70% confluence. We used the mouse monoclonal MPM-2 antibody (mitotic protein monoclonal # 2, Biomol, Hamburg, Germany) and the mouse monoclonal anti-a-tubulin antibody (Sigma). Cells were rinsed with PBS and fixed in 3.7% formaldehyde in PBS for 10 min at room temperature. Cells were washed 3 10 min with PBS and permeabilised with 0.5% Triton X-100 for 10 min. Cells were washed again 3 10 min with PBS and incubated in PBS 10% bovine serum albumin (BSA) at room temperature for 10 min to block non-specific protein binding. Cells were incubated with the primary antibody (MPM2 or anti-a-tubulin) for 1 h at room temperature or at 37 8C for 30 min. After three washes with PBS containing 0.1% Tween 20 at room temperature, cells were incubated with an appropriate FITC- or TRITC-conjugated anti-mouse antibody (# 488 or # 564, Molecular Probes) at room temperature for at least 30 min. Cells were washed again under the same conditions, rinsed briefly and the coverslips were mounted on a drop of mounting medium and analysed under a fluorescence microscope.
DAPI staining
After fixation, cells were incubated with 50 ml DAPI (4',6-Diamidino-2phenylindol, 0.1 mg/ml) per coverslip at 37 8C for 15 min, and then washed twice with PBS and once with water. The coverslips were mounted on a drop of mounting medium and the cells were analysed under a fluorescence microscope.
Results
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Fig. 1. Incorporation of tubulin-rhodamine into microtubules of living cells. A) Rhodamine-conjugated bovine brain tubulin was microinjected into cos-1 cells. After incubation for 1 h at 37 8C in a 5% CO2 atmosphere cells were washed with phosphate-buffered saline (PBS) and fixed for 10 min at room temperature in 3.7% formaldehyde. After washing twice with PBS cells were briefly rinsed with water to remove salts, and coverslips were mounted with cells down on a drop of mounting medium and analysed under a fluorescence microscope. A1) Rhodamine-labelled tubulin incorporated into interphase arrays and bright centrosomal foci, which are prominent during interphase. A2) Corresponding phase-contrast micrograph. B) Cells were fixed in 3.7% formaldehyde and permeabilised with Triton X-100. The mouse monoclonal anti-a-tubulin antibody was diluted 1 : 1000. As second antibody we used TRITC-conjugated goat anti-mouse antibodies. B1) Interphase arrays and the bright centrosomal foci. B2) Corresponding phase-contrast micrograph. C) Labelled tubulin was injected with (right panel) or without (left panel) the cytoskeleton-affecting drug nocodazole (200 mg/ml, Sigma).
that labelled tubulin only polymerized and incorporated into the microtubules in the absence of nocodazole (Fig. 1C). Thus, we showed that the injected tubulin- rhodamine was indeed incorporated into the microtubules of living cells.
Stability of tubulin-rhodamine at different times after microinjection To study biological processes such as mitosis it is important that the mitotic marker (in this case labelled tubulin) has to be stable over several hours because the length of mitosis can vary in different cell types. In order to analyse how long labelled tubulin can be detected, fluorescently labelled tubulin was microinjected into cos-1 cells and then cells were incubated for different times at 37 8C. The total amount of tubulin in a fibroblast cell is about 10 pg. On average the volume of injected labelled tubulin was 0.3 pl with a concentration of 10 pg/pl. Dilution experiments revealed that this concentration is
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Fig. 2. Stability of tubulin-rhodamine. Rhodamine-conjugated bovine brain tubulin was microinjected in cos-1 cells. Cells were incubated for different time intervals (30 min ± 5 h) at 37 8C in a 5% CO2 atmosphere. Then they were washed with PBS and fixed for 10 min at room temperature in 3.7% formaldehyde. After washing twice with PBS cells were briefly washed with water to remove salts, and coverslips were mounted with cells down on a drop of mounting medium and analysed under a fluorescence microscope. The figure shows the labelled cells as well as the phase-contrast micrographs. Arrows point to cells, which were not injected.
sufficient for the detection of rhodamine-labelled tubulin by fluorescence microscopy (data not shown). Figure 2 shows that after 30 min and until 5 h injected labelled tubulin could be detected in the cells. The time period of about 5 hours is usually sufficient to study mitosis in living cells. As further shown in Fig. 2 microinjected labelled tubulin served also as a control for successful microinjection of cells (arrows point to cells which were not injected).
Determination of the mitotic index by ™spindle counting∫ and MPM-2 immunofluorescence Having established the optimal conditions for the incorporation of fluorescently labelled tubulin into microtubules we were now able to address the question of the determination of the mitotic index by counting mitotic spindles. To test whether this
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method gives the same results as the MPM-2-based method described for the determination of the mitotic index, we used a cell line (SAOS ts p53 V138 ± 8), which can be arrested in G2/M phase of the cell cycle in a temperature-dependent manner. Cells were grown on coverslips at 37 8C or, to arrest cells in G2/ M phase, for 2 days at 31 8C. Immunofluorescence analyses were performed with an anti-a-tubulin antibody as well as with the MPM-2 antibody. The MPM-2 antibody recognizes mitosisspecific phosphorylation sites that are phosphorylated at the beginning of the G2/M transition and dephosphorylated by the end of mitosis (Engle et al., 1988). The double immunofluorescence analysis in Figure 3A shows that cells that possess mitotic spindles (anti-a-tubulin immunofluorescence) were also detected by the MPM-2 antibodies (arrows point to the mitotic cells). In addition the detection of a-tubulin can be used for the identification of cytokinesis (marked by an asterisk). For a statistic evaluation 1000 cells were counted and the mitotic index was determined by comparison of the ™a-tubulin spindle counting method∫ and the staining of MPM-2-positive cells. The results are shown in Figure 3B. Normal proliferating SAOS ts p53 V138 ± 8 cells (37 8C) had a mitotic index of 5.4% (mitotic spindle-positive cells) or 5.1% (MPM-2-positive cells). After induction of the G2/M arrest the mitotic index decreased to 1.1% (mitotic spindle-positive cells) or 0.9% (MPM-2-positive cells). Thus, with these two different methods we obtained
Fig. 3. Determination of the mitotic index. A) SAOS ts p53 V138 ± 8 cells were fixed in 3.7% formaldehyde, permeabilised with Triton X100 and incubated with mouse monoclonal anti-a-tubulin antibody and the mouse monoclonal MPM-2 antibody in a 1 : 1000 dilution. As second antibody we used either TRITC- or FITC-conjugated goat antimouse antibodies. The double immunofluorescence shows that cells that possess mitotic spindles (anti-a-tubulin immunofluorescence) were also detected by the MPM-2 antibodies (arrows point to the mitotic cells). An asterisk marks cytokinesis. B) SAOS ts p53 V138 ± 8 cells were incubated for 2 days at 37 8C or 31 8C (cells arrested in G2/M). After double immunofluorescence analysis with a mouse monoclonal anti-a-tubulin antibody and the mouse monoclonal MPM-2 antibody 1000 cells were counted and the mitotic index was determined by comparison of the a-tubulin spindle counting method and the staining of MPM-2-positive cells.
almost similar results for the mitotic index and, therefore, counting of the mitotic spindles is an appropriate method to determine the mitotic index.
Determination of the mitotic index after microinjection of tubulin-rhodamine To further analyse whether microinjection of labelled tubulin followed by counting of the mitotic spindles might be suitable to determine the mitotic index, we incubated SAOS ts p53 V138 ± 8 cells for 2 days at 37 8C or at 31 8C, then tubulin-rhodamine was microinjected and mitotic spindles were counted. Subsequently, chromosomal staining with DAPI was performed. Figure 4A shows injected cells grown at 37 8C or at 31 8C. For a statistic evaluation 400 injected cells were analysed and the mitotic spindles were counted (Figure 4B). In normal proliferating SAOS ts p53 V138 ± 8 cells (37 8C) we found 5.3% mitotic spindle-positive cells. After induction of the G2/M arrest the number of mitotic spindle positive cells decreased to 1.3%.
Fig. 4. Determination of the mitotic index after microinjection of tubulin-rhodamine. SAOS ts p53 V138 ± 8 cells were incubated for 2 days at 37 8C or 31 8C (cells arrested in G2/M). After microinjection of tubulin-rhodamine, cells were incubated for 1 h at 37 8C or 31 8C in a 5% CO2 atmosphere. They were washed with PBS and fixed for 10 min at room temperature in 3.7% formaldehyde. After fixation, cells were incubated with 50 ml DAPI per coverslip at 37 8C for 15 min, and then washed twice with PBS and once with water. The coverslips were mounted on a drop of mounting medium, and the cells were analysed under a fluorescence microscope. A) Microinjected tubulin-rhodamine incorporated into microtubules (left panel), DAPI staining of injected cells (middle panel) and corresponding phase-contrast micrographs (right panel). The mitotic index of microinjected cells was determined by counting the spindles (B).
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These results were comparable to the mitotic index determined by immunochemical procedures (Figure 3B). Thus, we demonstrated that this method to determine the mitotic index is suitable and gives similar results as with other methods.
Determination of different mitotic phases The method described so far might be also useful to determine different phases of mitosis such as late pro-, meta-, ana-, telophase and cytokinesis. This could be of interest for analysing proteins that are involved in regulating the transition of the different mitotic phases. In order to test this assumption cos-1 cells were grown on Cellocate coverslips (Eppendorf) for 48 h and microinjected with rhodamine-labelled tubulin. Figure 5 presents cells in different mitotic phases such as metaphase, anaphase and telophase/cytokinesis (arrows). Microtubules are visualized by the incorporation of fluorescent tubulin-rhodamine; chromosomes are stained with DAPI. In metaphase (Fig. 5A) almost all the microtubules are located within the spindle and the chromosomes have moved to the equatorial plate. In anaphase (Fig. 5B) the sister chromatids have separated from each other and moved toward the poles. The distance between the centrosomes has increased and besides the spindle fibres new arrays of cytoplasmic fluorescent microtubules appear at both spindle poles. In telophase (lower part of Fig. 5C) chromosomes have finished their migration to the poles, the chromosomes are less condensed. The spindle fibres disintegrate and the cleavage furrow is formed. In cytokinesis (Fig. 5C, arrows) chromosomes return to their interphase morphology and the separation of the parent cell into two daughter cells is almost finished.
Fig. 5. Determination of different mitosis phases. Rhodamine-conjugated bovine brain tubulin was microinjected in SAOS ts p53 V138 ± 8 cells. After microinjection cells were incubated for 1 h at 37 8C in a 5% CO2-atmosphere. Then they were washed with PBS and fixed for 10 min at room temperature in 3.7% formaldehyde. After fixation, cells were incubated with 50 ml DAPI per coverslip at 37 8C for 15 min, and then washed twice with PBS and once with water. The coverslips were mounted on a drop of mounting medium, and the cells were analysed under a fluorescence microscope. There are different mitotic phases such as metaphase (A), anaphase (B), telophase (C) and cytokinesis (arrows, C). The upper figures show the incorporation of injected tubulin-rhodamine into different mitotic structures of microtubules. The figures below show the corresponding DAPI staining of the chromosomes.
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Discussion Expression of foreign genes in mammalian cells has been used over the past 40 years to elucidate the functions of these genes and their products. An alternative way to directly study the functions of proteins is the microinjection of these proteins into living cells by monitoring changes in cell morphology, cell cycle progression and cell division. To evaluate cell division the mitotic index, i. e. the number of cells in mitosis in relation to the whole cell population, is calculated. There are several commonly used methods to determine the mitotic index such as staining of DNA, microtubules or mitosis-specific proteins, determination of the DNA content by flow cytometry or microscopic examination of the cell morphology (Bunz et al., 1998; Gowdy et al., 1998; Clute and Pines, 1999; Wakefield et al., 2000). Recently, the establishing of a cell line permanently expressing GFP-a-tubulin was reported (Rusan et al., 2001). The availability of these cells provides a simple, easily manipulated system to examine microtubule behaviour in mammalian cells. With these cells cell cycle progression should be easily monitored. However, in combination with microinjection of proteins these cells have a great disadvantage because only a limited number of cells is usually microinjected and therefore it is often complicated to identify the microinjected cells. Therefore, we attempted to find a system, which allows monitoring simultaneously the microinjected cells as well as to count the spindle fibres in order to calculate the mitotic index. We used fluorescently labelled tubulin for the microinjection experiments and we showed that it rapidly incorporated into the cytoskeleton of the injected cells. Using different buffers for the microinjection as well as different cell types showed the versatility of this method (data not shown). Cells need different time intervals to progress through mitosis and therefore it was important to show that the fluorescent tubulin remains stable in the microtubules over several hours. This stability is high enough and exceeds the time, which is usually required for cells to pass through mitosis (Israels and Israels, 2001). The mitotic index which was determined with this method is quite similar to the one that was obtained by labelling of cells with a mitosis-specific antibody, namely MPM-2 (Davis et al., 1983) as well as by using anti-tubulin antibodies. However, these latter methods require additional staining and washing steps and usually go along with a loss of cells. FACS analysis for the determination of cell cycle progression and mitosis (Larsen et al., 1986) is not suitable for microinjected cells because FACS analysis requires a minimum of 10 000 cells, and microinjection experiments are usually performed with 300 ± 400 cells. With the method presented here microinjected fluorescently labelled tubulin can be used as a positive marker for successful injection and after incorporation into spindle microtubules as a marker for mitotic cells. Another interesting virtue of the presented method is the possibility to identify different phases of mitosis such as late pro-, meta-, ana-, telophase and cytokinesis. Acknowledgement. This study was supported by grant Mo309/11-3 and project B 4 of SFB 399 from Deutsche Forschungsgemeinschaft and from Fonds der Chemischen Industrie.
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References Bunz, F., Dutriaux, A., Lengauer, C., Waldman, T., Zhou, S., Brown, J. P., Sedivy, J. M., Kinzler, K. W., Vogelstein, B. (1998): Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282, 1497 ± 1501. Clute, P., Pines, J. (1999): Temporal and spatial control of cyclin B1 destruction in metaphase. Nature Cell Biol. 1, 82 ± 87. Daum, J. R., Gorbsky, G. J. (1998): Casein kinase II catalyses a mitotic phosphorylation on threonine 1342 of human DNA topoisomerase IIa which is recognized by the 3F3/2 phosphoepitope antibody. J. Biol. Chem. 273, 30622 ± 30629. Davis, F. M., Tsao, T. Y., Fowler, S. K., Rao, P. N. (1983): Monoclonal antibodies to mitotic cells. Proc. Natl. Acad. Sci. USA 80, 2926 ± 2930. De Brabander, M., Van de Veire, R., Aerts, F., Geuens, S., Hoebeke, J. (1976): A new culture model facilitating rapid quantitative testing of mitotic spindle inhibition in mammalian cells. J. Natl. Cancer Inst. 56, 357 ± 363. Engle, D. B., Doonan, J. H., Morris, N. R. (1988): Cell-cycle modulation of MPM-2-specific spindle pole body phosphorylation in Aspergillus nidulans. Cell Motil. Cytoskeleton 10, 434 ± 437. Escargueil, A. E., Plisov, S. Y., Filhol, O., Cochet, C., Larsen, A. K. (2000): Mitotic phosphorylation of DNA topoisomerase II a by protein kinase CK2 creates the MPM-2 phosphoepitope on Ser-1469. J. Biol. Chem. 275, 34710 ± 34718. Gabrielli, B. G., De Souza, C. P. C., Tonks, I. D., Clark, J. M., Hayward, N. K., Ellem, K. A. O. (1996): Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells. J. Cell Sci. 109, 1081 ± 1093.
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Girard, F., Strausfeld, U., Cavadore, J.-C., Russell, P., Fernandez, A. (1992): cdc25 is a nuclear protein expressed constitutively throughout the cell cycle in nontransformed mammalian cells. J. Cell Biol. 118, 785 ± 794. Glover, D. M., Hagan, I. M., Tavares, A. A. M. (1998): Polo-like kinases: a team that plays throughout mitosis. Genes Dev. 12, 3777 ± 3787. Gowdy, P. M., Anderson, H. J., Roberge, M. (1998): Entry into mitosis without Cdc2 kinase activation. J. Cell Sci. 111, 3401 ± 3410. Israels, E. D., Israels, L. G. (2001): The cell cycle. Stem cells 19, 88 ± 91. Larsen, J. K., Munch-Petersen, B., Christiansen, J., Jorgensen, K. (1986): Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde. Cytometry 7, 54 ± 63. Qian, Y.-W., Erikson, E., Maller, J. L. (1999): Mitotic effects of a constitutively active mutant of the Xenopus Polo-like kinase Plx1. Mol. Cell. Biol. 19, 8625 ± 8632. Rusan, N. M., Fagerstrom, C. J., Yvon, A.-M. C., Wadsworth, P. (2001): Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-a tubulin. Mol. Biol. Cell 12, 971 ± 980. Wakefield, J. G., Huang, J.-Y., Raff, J. W. (2000): Centrosomes have a role in regulating the destruction of cyclin B in early Drosophila embryos. Curr. Biol. 10, 1367 ± 1370. Wang, K., Feramisco, J. R., Ash, J. F. (1982): Fluorescent localisation of contractile proteins in tissue culture cells. Methods Enzymol. 85, 514 ± 562.
European Journal of Cell Biology 81, 169 ± 174 (2002, March) ¥ ¹ Urban & Fischer Verlag ¥ Jena http://www.urbanfischer.de/journals/ejcb
169
Determination of the mitotic index by microinjection of fluorescently labelled tubulin Sandra L. Schwindling2), Michael Faust2), Mathias Montenarh1) Medical Biochemistry and Molecular Biology, University of the Saarland, Homburg/Germany Received October 15, 2001 Received in revised version December 7, 2001 Accepted December 9, 2001
Microinjection ± fluorescently labelled tubulin ± immunofluorescence ± mitosis ± spindle The microneedle injection technique is one of the most established procedures for the introduction of proteins into living cells. To analyse injected proteins which are important in cell cycle progression it is often necessary to determine the mitotic index. Measuring the mitotic index after microinjection is complicated because only a limited number of cells of the whole cell population is microinjected. Therefore, we attempted to establish a new method to determine the mitotic index using microinjection of fluorescently labelled a/b-tubulin into mammalian cells which allows to monitor the injected cells simultaneously with the determination of the mitotic index. We demonstrated that fluorescently labelled tubulin incorporates efficiently into the mitotic spindle apparatus. Fluorescence remains stable for several hours which is sufficient to observe the progression of cells through the M-phase of the cell cycle. The determination of the mitotic index with the method presented here gave similar results to those determined using other methods. With this method also different stages of mitosis can be visualized by analysing various steps of spindle formation. Thus, this rapid method allows the monitoring of the injected cells after microneedle injection and simultaneously the determination of the mitotic index.
Introduction The process of cell division has fascinated scientists for more than 100 years. Most of what we know about cell cycle and cell division is the result of the study of cells in tissue culture. Our present knowledge about the cell cycle and mitosis stems from
1)
Professor Dr. Mathias Montenarh, Medical Biochemistry and Molecular Biology, University of the Saarland, Building 44, D-66424 Homburg/Germany, e-mail: [email protected], Fax: 49 6841 162 6027. 2) These authors contributed equally to this work.
observations of morphological changes and from the staining of proteins which are actively implicated in cell cycle regulation, morphological alterations or mitotic dynamics. More recently, proteins which are suspected to regulate cell cycle progression and cell division are introduced into living cells in order to study their contribution to the regulation of these processes. There are several techniques currently used for the introduction of proteins and also of nucleic acids into living cells namely transfection of DNA or microneedle injection of proteins or nucleic acids (Wang et al., 1982). Microinjection techniques have been used for over 40 years to introduce nucleic acids, viral proteins, antibodies and fluorescently labelled proteins (for review see (Wang et al., 1982)). The microinjection of fluorescently labelled actin or tubulin into mammalian tissue culture cells has demonstrated the general application of this approach to observe the contractile apparatus of living cells by showing the apparent incorporation of these dyes into the cytoskeleton of the living cells. The dynamics of this fluorescently labelled proteins in living cells allows to monitor cell cycle progression and in particular mitosis. In any population of cycling cells only a limited number of cells is in mitosis at the same time. The percentage of dividing cells is defined as the mitotic index which is commonly determined by various staining methods (staining of DNA, microtubules or mitosis-specific proteins) (Bunz et al., 1998; Gowdy et al., 1998; Wakefield et al., 2000), cell scoring techniques (determination of the DNA content by flow cytometry) (Bunz et al., 1998; Gowdy et al., 1998) or microscopic examination of the cell morphology (Clute and Pines, 1999). In mitosis chromosomes are condensed in a more compact form, and their morphological alterations allow distinguishing between interphase and mitosis. One established chromosome/DNA staining procedure is the labelling of chromosomes with DNA intercalating dyes (e. g. DAPI, Hoechst 33342) (Girard et al., 1992; Gabrielli et al., 1996; Bunz et al., 1998). Chromosome condensation is also associated with extensive phosphorylation of proteins which can be detected by mitosis-specific monoclonal MPM-2 (Davis et al., 1983) or 3F3/2 (Daum and Gorbsky, 1998) antibodies. Therefore, these antibodies are frequently used to determine the mitotic index (Escargueil et al., 2000). Another unique feature
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of mitosis is the formation of mitotic spindles from microtubules that will transport chromosomes into the daughter cells. This mitotic spindle formation can be detected by immunofluorescence analysis with a/b-tubulin-specific antibodies. Furthermore, with this method the identification of mitotic cells from late prophase up to cytokinesis is possible (Glover et al., 1998; Qian et al., 1999). An alternative approach to determine the mitotic index in a cell population is the use of fluorescenceactivated cell sorting (FACS) (Larsen et al., 1986). Mitotic cells are characterized by a rounded shape and a reduced cell surface contact. Therefore, mitotic cells can also be identified by microscopical examination of cell morphology. However, this method requires much expert knowledge in cell culture. To analyse the function of microinjected proteins, which are involved in the regulation of the G2/M transition or in mitosis the determination of the mitotic index is often necessary. In combination with microinjection of proteins all of the abovedescribed procedures have a lot of disadvantages. All staining methods to determine the mitotic index are associated with a loss of cells, due to fixation, permeabilisation, washing and staining procedures. Furthermore, between 200 and 500 cells are usually microinjected which is not enough to determine the mitotic index by FACS analysis because this method requires an average of 10 000 cells. Thus, FACS analysis is not an appropriate method to determine the mitotic index after microinjection. To overcome the disadvantages of the methods mentioned above we have established a new method to determine the mitotic index by using injected fluorescently labelled tubulin into mammalian cells. Fluorescently labelled tubulin incorporates into spindle microtubules, which can then be counted. The advantages of this ™one step technique∫ are the rapid discovery of the injected cells and the simultaneous determination of the mitotic index without subsequent immunohistochemical procedures.
(Eppendorf) and the Axiovert 100 microscope (Zeiss, Jena, Germany). After microinjection cells were incubated at 37 8C for different times. They were washed with PBS and fixed for 10 min at room temperature in 3.7% formaldehyde. After washing twice with phosphate-buffered saline (PBS), cells were briefly rinsed with water to remove salts, and finally coverslips were mounted with cells down on a drop of mounting medium and analysed under a fluorescence microscope.
Materials and methods
Microinjected tubulin-rhodamine incorporates into the microtubules of living cells
Cell culture
To verify whether injected tubulin-rhodamine can be used as a marker for the determination of the mitotic index, we first analysed the incorporation of dye-labelled tubulin into the cytoskeleton of cos-1 cells. Tubulin-rhodamine was diluted and microinjected into the cytoplasm of cos-1 cells, which were grown on Cellocate coverslips (Eppendorf) for 48 h. On average 200 ± 300 cells were injected in one session. The tubulin-rhodamine-injected cells were incubated at 37 8C in a humidified 5% CO2 atmosphere for 1 h to allow incorporation of labelled tubulin into the cytoskeleton. Cells were washed with PBS at room temperature, to avoid tubulin depolymerisation. Then, cells were fixed with 3.7% formaldehyde and prepared for fluorescence microscopy. In Figure 1A it is shown that labelled tubulin incorporates into interphase arrays and into the bright centrosomal focus of cos-1 cells. For comparison Figure 1B shows an immunofluorescence analysis with monoclonal anti-a-tubulin antibodies. Staining is quite similar to the one shown in Figure 1A. As a further control we investigated whether labelled tubulin was incorporated in the microtubules or only spread diffusely within the cytoplasm. To address this problem we microinjected tubulin-rhodamine with or without the cytoskeleton-affecting drug nocodazole (200 mg/ml; Sigma). Nocodazole inhibits the assembly of newly formed microtubules and breaks down pre-existing microtubules into free tubulin subunits (De Brabander et al., 1976). We found
cos-1 cells were maintained in Dulbecco×s modified Eagle×s medium (DMEM) supplemented with 10% foetal calf serum (FCS) at 37 8C in a 5% CO2 atmosphere. For the culture of SAOS ts p53 V138 ± 8 cells (kindly provided by Dr. Klaus Roemer, Homburg) the medium contained 200 mg/ml G418. To obtain a G2/M arrest, the SAOS ts p53 V138 ± 8 cells were shifted to 31 8C for 48 h.
Microinjection For microinjection experiments, cells were grown to 50% ± 70% confluence on Cellocate coverslips with 55 mm grid size (Eppendorf, Hamburg, Germany) in 60-mm Petri dishes. Coverslips were transferred to fresh medium, containing 25 mM Hepes buffer (pH 7.8) shortly before injection. After injection, coverslips were placed into fresh medium containing 10% FCS. Cellocate coverslips were pre-coated with poly-L-lysine (Sigma, Taufkirchen, Germany) or collagen (Becton Dickinson, New Jersey, USA) as described by the manufacturer. For the microinjection experiments, we used rhodamine-conjugated bovine brain tubulin (10 mg/ml, tubulin-rhodamine, Molecular Probes, Leiden, The Netherlands). This reagent is supplied in 10-ml aliquots and should be rapidly thawed, divided into 1 ± 2-ml aliquots, rapidly frozen in liquid nitrogen and stored at 70 8C. Prior to use, the aliquots were thawed, diluted in the respective solution and centrifuged for 30 min at 90 000 g at 4 8C. The supernatant was used for the experiments. Samples should be kept strictly on ice until use, because tubulin will polymerise at room temperature and clog the micropipette. For microinjection we used Femtotips II, the Transjector 5246, the Micromanipulator 5171
Immunofluorescence For immunofluorescence, cells were grown on coverslips without grids in 100-mm Petri dishes up to 50% ± 70% confluence. We used the mouse monoclonal MPM-2 antibody (mitotic protein monoclonal # 2, Biomol, Hamburg, Germany) and the mouse monoclonal anti-a-tubulin antibody (Sigma). Cells were rinsed with PBS and fixed in 3.7% formaldehyde in PBS for 10 min at room temperature. Cells were washed 3 10 min with PBS and permeabilised with 0.5% Triton X-100 for 10 min. Cells were washed again 3 10 min with PBS and incubated in PBS 10% bovine serum albumin (BSA) at room temperature for 10 min to block non-specific protein binding. Cells were incubated with the primary antibody (MPM2 or anti-a-tubulin) for 1 h at room temperature or at 37 8C for 30 min. After three washes with PBS containing 0.1% Tween 20 at room temperature, cells were incubated with an appropriate FITC- or TRITC-conjugated anti-mouse antibody (# 488 or # 564, Molecular Probes) at room temperature for at least 30 min. Cells were washed again under the same conditions, rinsed briefly and the coverslips were mounted on a drop of mounting medium and analysed under a fluorescence microscope.
DAPI staining
After fixation, cells were incubated with 50 ml DAPI (4',6-Diamidino-2phenylindol, 0.1 mg/ml) per coverslip at 37 8C for 15 min, and then washed twice with PBS and once with water. The coverslips were mounted on a drop of mounting medium and the cells were analysed under a fluorescence microscope.
Results
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Fig. 1. Incorporation of tubulin-rhodamine into microtubules of living cells. A) Rhodamine-conjugated bovine brain tubulin was microinjected into cos-1 cells. After incubation for 1 h at 37 8C in a 5% CO2 atmosphere cells were washed with phosphate-buffered saline (PBS) and fixed for 10 min at room temperature in 3.7% formaldehyde. After washing twice with PBS cells were briefly rinsed with water to remove salts, and coverslips were mounted with cells down on a drop of mounting medium and analysed under a fluorescence microscope. A1) Rhodamine-labelled tubulin incorporated into interphase arrays and bright centrosomal foci, which are prominent during interphase. A2) Corresponding phase-contrast micrograph. B) Cells were fixed in 3.7% formaldehyde and permeabilised with Triton X-100. The mouse monoclonal anti-a-tubulin antibody was diluted 1 : 1000. As second antibody we used TRITC-conjugated goat anti-mouse antibodies. B1) Interphase arrays and the bright centrosomal foci. B2) Corresponding phase-contrast micrograph. C) Labelled tubulin was injected with (right panel) or without (left panel) the cytoskeleton-affecting drug nocodazole (200 mg/ml, Sigma).
that labelled tubulin only polymerized and incorporated into the microtubules in the absence of nocodazole (Fig. 1C). Thus, we showed that the injected tubulin- rhodamine was indeed incorporated into the microtubules of living cells.
Stability of tubulin-rhodamine at different times after microinjection To study biological processes such as mitosis it is important that the mitotic marker (in this case labelled tubulin) has to be stable over several hours because the length of mitosis can vary in different cell types. In order to analyse how long labelled tubulin can be detected, fluorescently labelled tubulin was microinjected into cos-1 cells and then cells were incubated for different times at 37 8C. The total amount of tubulin in a fibroblast cell is about 10 pg. On average the volume of injected labelled tubulin was 0.3 pl with a concentration of 10 pg/pl. Dilution experiments revealed that this concentration is
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Fig. 2. Stability of tubulin-rhodamine. Rhodamine-conjugated bovine brain tubulin was microinjected in cos-1 cells. Cells were incubated for different time intervals (30 min ± 5 h) at 37 8C in a 5% CO2 atmosphere. Then they were washed with PBS and fixed for 10 min at room temperature in 3.7% formaldehyde. After washing twice with PBS cells were briefly washed with water to remove salts, and coverslips were mounted with cells down on a drop of mounting medium and analysed under a fluorescence microscope. The figure shows the labelled cells as well as the phase-contrast micrographs. Arrows point to cells, which were not injected.
sufficient for the detection of rhodamine-labelled tubulin by fluorescence microscopy (data not shown). Figure 2 shows that after 30 min and until 5 h injected labelled tubulin could be detected in the cells. The time period of about 5 hours is usually sufficient to study mitosis in living cells. As further shown in Fig. 2 microinjected labelled tubulin served also as a control for successful microinjection of cells (arrows point to cells which were not injected).
Determination of the mitotic index by ™spindle counting∫ and MPM-2 immunofluorescence Having established the optimal conditions for the incorporation of fluorescently labelled tubulin into microtubules we were now able to address the question of the determination of the mitotic index by counting mitotic spindles. To test whether this
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method gives the same results as the MPM-2-based method described for the determination of the mitotic index, we used a cell line (SAOS ts p53 V138 ± 8), which can be arrested in G2/M phase of the cell cycle in a temperature-dependent manner. Cells were grown on coverslips at 37 8C or, to arrest cells in G2/ M phase, for 2 days at 31 8C. Immunofluorescence analyses were performed with an anti-a-tubulin antibody as well as with the MPM-2 antibody. The MPM-2 antibody recognizes mitosisspecific phosphorylation sites that are phosphorylated at the beginning of the G2/M transition and dephosphorylated by the end of mitosis (Engle et al., 1988). The double immunofluorescence analysis in Figure 3A shows that cells that possess mitotic spindles (anti-a-tubulin immunofluorescence) were also detected by the MPM-2 antibodies (arrows point to the mitotic cells). In addition the detection of a-tubulin can be used for the identification of cytokinesis (marked by an asterisk). For a statistic evaluation 1000 cells were counted and the mitotic index was determined by comparison of the ™a-tubulin spindle counting method∫ and the staining of MPM-2-positive cells. The results are shown in Figure 3B. Normal proliferating SAOS ts p53 V138 ± 8 cells (37 8C) had a mitotic index of 5.4% (mitotic spindle-positive cells) or 5.1% (MPM-2-positive cells). After induction of the G2/M arrest the mitotic index decreased to 1.1% (mitotic spindle-positive cells) or 0.9% (MPM-2-positive cells). Thus, with these two different methods we obtained
Fig. 3. Determination of the mitotic index. A) SAOS ts p53 V138 ± 8 cells were fixed in 3.7% formaldehyde, permeabilised with Triton X100 and incubated with mouse monoclonal anti-a-tubulin antibody and the mouse monoclonal MPM-2 antibody in a 1 : 1000 dilution. As second antibody we used either TRITC- or FITC-conjugated goat antimouse antibodies. The double immunofluorescence shows that cells that possess mitotic spindles (anti-a-tubulin immunofluorescence) were also detected by the MPM-2 antibodies (arrows point to the mitotic cells). An asterisk marks cytokinesis. B) SAOS ts p53 V138 ± 8 cells were incubated for 2 days at 37 8C or 31 8C (cells arrested in G2/M). After double immunofluorescence analysis with a mouse monoclonal anti-a-tubulin antibody and the mouse monoclonal MPM-2 antibody 1000 cells were counted and the mitotic index was determined by comparison of the a-tubulin spindle counting method and the staining of MPM-2-positive cells.
almost similar results for the mitotic index and, therefore, counting of the mitotic spindles is an appropriate method to determine the mitotic index.
Determination of the mitotic index after microinjection of tubulin-rhodamine To further analyse whether microinjection of labelled tubulin followed by counting of the mitotic spindles might be suitable to determine the mitotic index, we incubated SAOS ts p53 V138 ± 8 cells for 2 days at 37 8C or at 31 8C, then tubulin-rhodamine was microinjected and mitotic spindles were counted. Subsequently, chromosomal staining with DAPI was performed. Figure 4A shows injected cells grown at 37 8C or at 31 8C. For a statistic evaluation 400 injected cells were analysed and the mitotic spindles were counted (Figure 4B). In normal proliferating SAOS ts p53 V138 ± 8 cells (37 8C) we found 5.3% mitotic spindle-positive cells. After induction of the G2/M arrest the number of mitotic spindle positive cells decreased to 1.3%.
Fig. 4. Determination of the mitotic index after microinjection of tubulin-rhodamine. SAOS ts p53 V138 ± 8 cells were incubated for 2 days at 37 8C or 31 8C (cells arrested in G2/M). After microinjection of tubulin-rhodamine, cells were incubated for 1 h at 37 8C or 31 8C in a 5% CO2 atmosphere. They were washed with PBS and fixed for 10 min at room temperature in 3.7% formaldehyde. After fixation, cells were incubated with 50 ml DAPI per coverslip at 37 8C for 15 min, and then washed twice with PBS and once with water. The coverslips were mounted on a drop of mounting medium, and the cells were analysed under a fluorescence microscope. A) Microinjected tubulin-rhodamine incorporated into microtubules (left panel), DAPI staining of injected cells (middle panel) and corresponding phase-contrast micrographs (right panel). The mitotic index of microinjected cells was determined by counting the spindles (B).
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These results were comparable to the mitotic index determined by immunochemical procedures (Figure 3B). Thus, we demonstrated that this method to determine the mitotic index is suitable and gives similar results as with other methods.
Determination of different mitotic phases The method described so far might be also useful to determine different phases of mitosis such as late pro-, meta-, ana-, telophase and cytokinesis. This could be of interest for analysing proteins that are involved in regulating the transition of the different mitotic phases. In order to test this assumption cos-1 cells were grown on Cellocate coverslips (Eppendorf) for 48 h and microinjected with rhodamine-labelled tubulin. Figure 5 presents cells in different mitotic phases such as metaphase, anaphase and telophase/cytokinesis (arrows). Microtubules are visualized by the incorporation of fluorescent tubulin-rhodamine; chromosomes are stained with DAPI. In metaphase (Fig. 5A) almost all the microtubules are located within the spindle and the chromosomes have moved to the equatorial plate. In anaphase (Fig. 5B) the sister chromatids have separated from each other and moved toward the poles. The distance between the centrosomes has increased and besides the spindle fibres new arrays of cytoplasmic fluorescent microtubules appear at both spindle poles. In telophase (lower part of Fig. 5C) chromosomes have finished their migration to the poles, the chromosomes are less condensed. The spindle fibres disintegrate and the cleavage furrow is formed. In cytokinesis (Fig. 5C, arrows) chromosomes return to their interphase morphology and the separation of the parent cell into two daughter cells is almost finished.
Fig. 5. Determination of different mitosis phases. Rhodamine-conjugated bovine brain tubulin was microinjected in SAOS ts p53 V138 ± 8 cells. After microinjection cells were incubated for 1 h at 37 8C in a 5% CO2-atmosphere. Then they were washed with PBS and fixed for 10 min at room temperature in 3.7% formaldehyde. After fixation, cells were incubated with 50 ml DAPI per coverslip at 37 8C for 15 min, and then washed twice with PBS and once with water. The coverslips were mounted on a drop of mounting medium, and the cells were analysed under a fluorescence microscope. There are different mitotic phases such as metaphase (A), anaphase (B), telophase (C) and cytokinesis (arrows, C). The upper figures show the incorporation of injected tubulin-rhodamine into different mitotic structures of microtubules. The figures below show the corresponding DAPI staining of the chromosomes.
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Discussion Expression of foreign genes in mammalian cells has been used over the past 40 years to elucidate the functions of these genes and their products. An alternative way to directly study the functions of proteins is the microinjection of these proteins into living cells by monitoring changes in cell morphology, cell cycle progression and cell division. To evaluate cell division the mitotic index, i. e. the number of cells in mitosis in relation to the whole cell population, is calculated. There are several commonly used methods to determine the mitotic index such as staining of DNA, microtubules or mitosis-specific proteins, determination of the DNA content by flow cytometry or microscopic examination of the cell morphology (Bunz et al., 1998; Gowdy et al., 1998; Clute and Pines, 1999; Wakefield et al., 2000). Recently, the establishing of a cell line permanently expressing GFP-a-tubulin was reported (Rusan et al., 2001). The availability of these cells provides a simple, easily manipulated system to examine microtubule behaviour in mammalian cells. With these cells cell cycle progression should be easily monitored. However, in combination with microinjection of proteins these cells have a great disadvantage because only a limited number of cells is usually microinjected and therefore it is often complicated to identify the microinjected cells. Therefore, we attempted to find a system, which allows monitoring simultaneously the microinjected cells as well as to count the spindle fibres in order to calculate the mitotic index. We used fluorescently labelled tubulin for the microinjection experiments and we showed that it rapidly incorporated into the cytoskeleton of the injected cells. Using different buffers for the microinjection as well as different cell types showed the versatility of this method (data not shown). Cells need different time intervals to progress through mitosis and therefore it was important to show that the fluorescent tubulin remains stable in the microtubules over several hours. This stability is high enough and exceeds the time, which is usually required for cells to pass through mitosis (Israels and Israels, 2001). The mitotic index which was determined with this method is quite similar to the one that was obtained by labelling of cells with a mitosis-specific antibody, namely MPM-2 (Davis et al., 1983) as well as by using anti-tubulin antibodies. However, these latter methods require additional staining and washing steps and usually go along with a loss of cells. FACS analysis for the determination of cell cycle progression and mitosis (Larsen et al., 1986) is not suitable for microinjected cells because FACS analysis requires a minimum of 10 000 cells, and microinjection experiments are usually performed with 300 ± 400 cells. With the method presented here microinjected fluorescently labelled tubulin can be used as a positive marker for successful injection and after incorporation into spindle microtubules as a marker for mitotic cells. Another interesting virtue of the presented method is the possibility to identify different phases of mitosis such as late pro-, meta-, ana-, telophase and cytokinesis. Acknowledgement. This study was supported by grant Mo309/11-3 and project B 4 of SFB 399 from Deutsche Forschungsgemeinschaft and from Fonds der Chemischen Industrie.
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