Microtubule-dependent cell cycle regulation is implicated in the G₂ phase of Hydra cells

The relatively simple form and small number of different cell types of the freshwater coelenterate Hydra attenuata make it an attractive organism for studying differentiation and cellular interactions. Both the body wall and the tentacles of hydra are composed of an ectodermal and endodermal epithelium separated by an acellular mesogloea. Hydra nerve cells are derived from proliferating interstitial cells that migrate in the intercellular spaces to their respective destinations. Interstitial cells can also differentiate into nematocytes (stinging cells), which, in the tentacles, are situated within pockets of terminally differentiated ectodermal epitheliomuscular cells. They are anchored to their host cells and to the mesogloea by a complex configuration of junctions (Slautterback, 1967; Wood & Novak, 1982; Campbell, 1987). The epitheliomuscular cell together with its content of nematocytes and neurones is referred to as a ‘battery cell complex’ (Hyman, 1940; Hufnagel et al. 1985). Movements of prey against the sensory cnidocil of the nematocytes triggers nematocyte discharge followed by concerted tentacular reflexes, mouth opening and ingestion. Sensory neurones that have been activated by cellular components released from damaged prey tissue also appear to play an important role in the feeding response. Their high concentration and complex synaptic organization around the mouth area (Westfall & Kinnamon, 1984) suggest that they are particularly important for mouth movements.

Investigations into the differentiation of interstitial stem cells to neurones and nematocytes would be facilitated if their cell cycles could be synchronized. We therefore incubated these cells with various concentrations of the microtubule-depolymerizing agent nocodazole in an attempt to arrest their cell cycles in metaphase. This drug was chosen rather than the classical mitotic poison, colchicine, for two reasons. First, colchicine appears to have only a low affinity for Hydra tubulin (Campbell, 1976); and second, nocodazole has a high affinity for the tubulins of several taxonomically distant species. Surprisingly, we found that although cells were arrested only in metaphase at the lowest effective concentrations of nocodazole, at concentrations of 2nM-nocod-azole and above, most of the cells were arrested at a distinct point in the G₂ phase. On the basis of these results we devised an efficient cell cycle synchronization procedure that should prove useful in differentiation studies in Hydra and possibly in other primitive organisms. To determine whether Hydra microtubules were affected at these very low concentrations of nocodazole, they were visualized by immunofluorescent staining. We observed that spindle and stem cell cytoplasmic microtubules were indeed depolymerized by the concentrations of nocodazole used for cell cycle arrest. Our results strongly suggest, therefore, that the formation of a particular microtubule structure is required at a distinct point in G₂ before the cell cycle can progress any further.

Hydra attemiata were cultured in a medium consisting of mM-CaCl₂, 0 ·lmM-KCl, 0 ·lmM-MgCl₂, buffered with 0 ·5 mM-sodium phosphate, pH 7 ·6. They were fed daily between 9 and 10a.m. with nauplii of Artemia salina and washed 5 ·6 h after feeding. The water temperature was kept at 19°C (±2)°C.

Total protein was prepared by sonicating hydra in ice-cold sample buffer containing 4% SDS, 1 mM-CaCl₂, 10% glycerol, 10% 2-mercaptoethanol, 0 ·02% Bromophenol Blue, buffered with 125 mM-trisaminomethane ·HC1 at pH 6 ·8. After an incubation of 5 min at 0°C, the homogenate was heated to 95°C for 2 min and centrifuged for 10 min at 13 000 g. SDS-polyacryla-mide gel electrophoresis of the supernatant was carried out on a 12% polyacrylamide gel (Laemmli, 1970). Western blots were performed according to Towbin et al. (1979) with the monoclonal anti-tubulin antibodies DM1 A, DM IB (Amersham, Braunschweig, FRG) and YOL1/34 (Camón Labor Service, Wiesbaden, FRG) diluted 1:1000. Casein (1%, purified powder; Sigma, Munich, FRG) was used to block non-specific binding sites and was also included during incubations with the antibodies in phosphate-buffered saline (PBS: 140mM-NaCl, l ·0mM-sodium phosphate, pH 7 ·2). Bound antibodies were detected by alkaline phosphatase conjugated anti-mouse or antirat immunoglobulins (Sigma, Munich, FRG). Tubulin was prepared from pig brain by the method of Shelanski et al. (1973).

Hydra was macerated in 7% acetic acid and 7% glycerol (David, 1973) and the dissociated cells were fixed after 5 mm by adding 10 volumes of fresh 4% formaldehyde in PBS. The cells were concentrated by centrifugation at 1000g for 5 min and spread on gelatin-coated microscope slides. After drying, the slides were washed for 10 min in PBS. Alternatively, hydra were relaxed by gently adding 0’1 volume of a saturated aqueous solution of menthol to the culture medium. Two minutes later, medium was removed and 4% formaldehyde/PBS freshly prepared from paraformaldehyde was added. After fixation for 6h at room temperature, the hydra were washed for 5 mm in acetone/ethanol, 3:1 (v/v), for 10min in 10% Triton X-100 and overnight in PBS at 4°C. To obtain thin sections, fixed hydra were embedded in Tissue-Tek II (Miles, Naperville, USA) prior to 7 μm cryosectioning. Preparations of single cells, fixed animals or slides with thin sections were then treated as follows: non-specific binding was blocked by incubation for 30 min at room temperature in 1% casein in PBS. The monoclonal anti-tubulin antibodies DM1A, DM1B (Amersham, Braunschweig, FRG) and YOL1/34 (Camón Labor Service, Wiesbaden, FRG) diluted 1: 1000 in 1% casein in PBS were incubated with the samples at room temperature for 3h. After washing three times for 10 min in PBS, animals or slides were incubated for 1 h at room temperature with FITC-labelled anti-mouse or anti-rat IgX (Dakopatts, Hamburg, FRG) at a dilution of 1: 50 in 1% casein in PBS. After washing twice for 10 min in PBS, the preparations were counterstained for DNA by incubation for 10 min at room temperature in a solution of 0 ·5 mgl⁻¹’ HOECHST 33258 (Serva, Heidelberg, FRG) in PBS, and mounted in PBS/glycerol (1:2, v/v) containing 1 mgml⁻¹ o-phenylenediamine.

At 5-6 h after feeding, hydra were washed free of debris and incubated at a density of two animals per ml of medium with various concentrations of nocodazole (Janssen Biochemica, Beerse, FRG) diluted from a 40 μgml⁻¹stock solution in dimethyl sulphoxide. Controls were treated with equivalent dilutions of dimethyl sulphoxide only. The nocodazole was removed by washing three times with 20 volumes of culture medium.

After incubating hydra for 44 h with nocodazole, they were fixed in 4% formaldehyde in PBS for 30 min, stained for 30 min at room temperature in a solution of 0 ·5 mgl⁻¹ HOECHST 33258 and embedded in PBS/glycerol (1:2, v/v). Gastric regions were then screened for cells with condensed chromosomes using a fluorescence microscope.

Upper gastric portions of steady-state hydra were macerated and their dissociated cells were fixed on microscope slides as described by David (1973). The cells were then stained for 30min with a solution of 0 ·5 mgl⁻¹ HOECHST 33258 in PBS (Cowell & Franks, 1980) and mounted in this solution. The fluorescence of single interphase nuclei was determined with a ZEISS microscope photometer using the filters G365, FT395 and LP420. Cells were considered to be in the G₁ or S phase of the cell cycle when their nuclear fluorescence value was smaller than the threshold χ_TR = (2 × χ_2n) — S.D._4n, where χ_2n is the mean fluorescence value of 2n DNA nuclei (measured in nerve cells) and S.D. is the measurement error of the system. s.D.4,, is defined as the standard deviation of the mean value of the fluorescence values of foot mucous cells, since these cells are known to be arrested in Gz (Dübel et al. 1987). S.D.4,, was usually about 7 ·7% of the 4n value.

Hydra were incubated for 24 h with various concentrations of nocodazole in an attempt to arrest proliferating cells in mitosis. To detect mitotic cells, the chromatin in whole hydra was stained with the DNA-specific fluorescent dye HOECHST 33258. Condensed chromosomes, indicating mitosis, were easily distinguished from the large round interphase nuclei of epithelial and interstitial cells and the small flattened nuclei of the nerve cells and nematocytes (Fig. 1A). At concentrations around 1 ·4 nM-nocodazole, a significant increase of cells with condensed chromosomes arranged in metaphase plates was visible in the gastric column (Fig. IB). To demonstrate the effects of different concentrations of nocodazole on this metaphase accumulation, we counted hydra having more than 10% of their large nuclei in metaphase within an area of the body column containing about 300-400 large nuclei. We chose 10% metaphases as an amount that could not be accounted for by normal rates of proliferation. The small nuclei of non-proliferating nerve cells and nematocytes were not counted. Every hydra contained more than 10% metaphase plates after an incubation with 1-4 nM-nocodazole. The number of hydra with more than 10% metaphases decreased, however, on increasing or decreasing the concentration of nocodazole from l ·4nM (Fig. 2). To determine the cell type responsible for the increase in metaphases, the mitotic index was calculated from cell counts of epithelial and interstitial cells in macerates of the gastral region after incubating hydra with 1 ·4 nM-nocodazole (Table 1). A comparison with the mitotic index of cells from control hydra showed that only interstitial cells had been arrested in mitosis.

The large decrease in the number of animals with cells arrested in metaphase on increasing the concentration of nocodazole above l ·4nM, as shown in Fig. 2, was surprising. The effect had not been observed in mammalian cells (DeBrabander et al. 1976; Zieveetal. 1980) or with other microtubule inhibitors. A hypothesis to explain this phenomenon is that with increasing concentrations of nocodazole, cells were arrested at another stage of the cell cycle, thus preventing them from entering mitosis. To test this hypothesis, we exposed hydra to 2 nM-nocodazole and measured the amount of DNA in the nuclei of interphase interstitial cells from macerates of the gastral region by fluorescence microphotometry. After a 24 h incubation in 2 nM-nocodazole, every interstitial cell was found to contain a 4n genome compared to the 2n genome of nerve cells (Fig. 3A,C), whereas untreated controls contained interstitial cells with DNA contents between 2n and 4n (Fig. 3B). It is apparent, therefore, that the interphase interstitial cells were arrested in the G₂ phase of the cell cycle. To investigate the concentration depen-dence of this G₂ arrest, the nuclear DNA content of interphase interstitial cells from hydra incubated for 24 h in various concentrations of nocodazole was determined. Interstitial cells with a DNA content significantly lower than 4n represent cells in the very short, if at all existent, G, phase or the S phase of the cell cycle (Campbell & David, 1974) and are referred as ‘cells in G₁/S’. The proportion of interphase interstitial cells in G₁/S was observed to decrease after incubating hydra with concentrations of nocodazole above 1 nM (Fig. 4). At concentrations above 2 nM-nocodazole, all of the interphase interstitial cells were arrested in G₂.

Incubations with 2 nM-nocodazole seemed promising for cell cycle synchronization. To choose an optimal incubation time, we measured the decrease with time of cells in G₁/S after incubation with 2 nM-nocodazole (Fig. 5). After 21 h, all of the cells had moved out of the G₁/S phase. We therefore continued to use our standard incubation time of 24 h that had been based on measurements of Hydra cell cycles (Campbell & David, 1974). To find out whether the cell cycle blocks were reversible, we determined the nuclear DNA contents of interphase interstitial cells after removal of nocodazole-Ten hours after washing, 75% of the interphase interstitial cells had entered G₁/S of the next cell cycle (Fig. 6). This was preceded by a wave of mitoses 7-8 h after removal of the G₂ block. The cells remained about 12 h in Gi/S and the average length of the cell cycle was about 35 h as indicated by the distance between the Gi/S peaks. This is about one and a half times longer than usual, probably due to the absence of food during the incubations with nocodazole. No additional waves of G₁/S cells were observed before the major peak in Fig. 6, indicating that the small proportion of cells arrested in metaphase were no longer viable.

Nocodazole and other microtubule inhibitors are usually used at much higher concentrations than 2 nM to arrest cell cycles in mitosis. To determine, therefore, whether such low’ concentrations of nocodazole w’ere indeed affecting hydrA′s microtubules, they were visualized using the monoclonal antibody DM1 A. This anti-tubulin monoclonal antibody w>as selected from three commercially available tubulin monoclonal antibodies after testing their reaction against a Western blot of a Hydra lysate. DM1A reacted strongly with a band of the same molecular weight as pig brain tubulin and showed no cross-reactions with other proteins (Fig. 7). The microtubule distributions in hydra w’ere demonstrated by incubating cells or tissue with DM1A and a second fluorescent antibody. The specificity of DM1A for microtubules was checked using the monoclonal antibodies DM1B and YOL1/34, whose epitopes are known to be different from that of DM1A (Blose et al. 1984; Breitling & Little, 1986). They both stained the same structures as DM1 A. Furthermore, no immunofluorescent structures were visible after preincubation of DM1A with 20 μM pig brain tubulin.

Mitotic spindles, the cytoplasm of ectodermal epithelial cells, and the neuritic extensions of nerve cells were strongly fluorescent (Fig. 8A,B,E). In interstitial cells, the brightest immunofluorescence was seen in bridges connecting the typical 2ⁿ cell clusters (Fig. 8C). Nematocytes showed highly fluorescent structures in the cnidocil and in the conical part of the cell (Fig. 8D), in agreement w’ith the distribution of microtubules as observed by electron microscopy (Slautterback, 1963, 1967). The microtubule structures of single cells were also recognized in whole mounts and thin sections of hydra. Fig. 9 shows a whole mount of a hydra and a view of the body column. The basket-like arrangement of microtubules in nematocytes when viewed from the side as in Fig. 8D appeared as a ring when viewed from above. Mitotic spindles could also be observed in the body column of hydra as shown in Fig. 9B. Other microtubule structures, however, were difficult to identify due to the intense immunofluorescence of microtubules in epithelial cells. In contrast, terminally differentiated epithelial cells of tentacles showed no microtubule immunofluorescent structures (Fig. 10). It was therefore possible to observe the fluorescent microtubule structures of other tentacle cells more clearly. These included the intensely fluorescent basket-like structure surrounding the nematocyte, the small bristles radiating from the nematocytes representing the sensory cnidocils and the thread-like connections between nematocytes of the same and different battery cell complexes. From comparisons with ultrastructural and immunological investigations (Gnmmelik-huijzen, 1983; Westfall & Kinnamon, 1984; Yu et al. 1985; Yaross et al. 1986) these threadlike connections were identified as neurites. Microtubule immunofluor-urnescence was largely absent from the endoderm as shown in thin sections (Fig. 11). The more intense fluorescence of the tentacle ectoderm in sections was due to its relatively high concentration of nematocytes.

To investigate the effect of nocodazole on Hydra micro-tubules, microtubule immunofluorescence in thin sec-tions and whole mounts of animals was examined after incubation with various concentrations of nocodazole for 24h (Fig. 12). After incubation of hydra with 3-4nM-nocodazole, the microtubule immunofluorescence typical of nerves and of the interstitial and epithelial cells in the body wall was no longer visible. The cnidocil micro-tubules appeared to be more resistant. They were still visible, although reduced in number, after incubation with 10 nM-nocodazole. Nematocytes appeared to contain the most stable microtubule structures. After incubation with 34 nM-nocodazole, their typical microtubule struc-tures were beginning to disintegrate but were still visible in some cells. Cnidocil microtubule immunofluorescence had completely disappeared. After incubation with 100 nM-nocodazole, the typical basket-like microtubule immunofluorescence around nematocytes was destroyed and dispersed in the cytoplasm, surrounding the nemato-cyte nucleus. A summary of these observations is shown in Table 2.

Having found that some Hydra microtubule structures were indeed affected by very low concentrations of nocodazole, we made a more detailed investigation of the effect of 2 nM-nocodazole, the concentration used to synchronize interstitial cells. The results are shown in Fig. 13. The microtubule immunofluorescence of the body of hydra was largely accounted for by nematocytes; immunofluorescence from interstitial and epithelial cells had disappeared (cf. Fig. 9). Nerve cell microtubules had also largely disappeared from the body column, but they were still visible in the extremities. The neuritic network of the peduncle, for example, was still fairly intact in several animals (Fig. 13B), as were many neurones, probably sensory neurones, at the tip of the hypostome (Fig. 13C). Tentacular nerve processes were also unaffected (Fig. 13C). Fluorescent labelling of both microtubules and DNA showed that cells of the body wall that were arrested in metaphase lacked mitotic spindles (Fig. 13D,E). It appears, therefore, that the metaphase arrest was indeed due to the inhibition of microtubule assembly.

During incubations with 2 nM-nocodazole, hydra were observed to contract their tentacles partially and shorten their bodies significantly (Fig. 14B). At higher concentrations of nocodazole, the hydra appeared swollen, probably due to an inability to open their mouths.

Animals incubated with up to 8 nM-nocodazole, but not 10 nM-nocodazole, were able to resume feeding after several days of reconvalescence and grow. To investigate the effect of nocodazole concentrations known to destroy neurite microtubules on the feeding response, animals were incubated with various concentrations of nocodazole for 24 h and then assayed for feeding reactions by adding shrimps or shrimp extract. The moderately contracted tentacles of hydra treated with 2 nM-nocodazole were still able to respond to feeding; nematocyte discharge, tentacle movements and mouth movements still occurred (Table 3). However, whereas tentacular movements ceased completely after incubating hydra with 4 nM-nocodazole, mouth movements and nematocyte discharge were not affected. It appears, therefore, that intact microtubule structures in neurites are not required for mouth movements and nematocyte discharge.

It is interesting to note that the term ‘microtubules’ was used for the first time 25 years ago by Slautterback (1963) in his description of these organelles in the interstitial cells and nematocytes of hydra. In agreement with his ultrastructural investigations (Slautterback, 1963, 1967) our immunofluorescence procedure using a tubulin monoclonal antibody showed a particularly high concentration of microtubules in the conical part of the dropshaped nematocyte. We were also able to observe mitotic spindles and the microtubule distribution in single cells, thin sections and whole mounts.

Furthermore, the neuritic network connecting nematocytes and neurones in Hydra tentacles was clearly visible due to the absence of microtubule immunofluorescence in the epithelial cells. Similar neuritic connections have also been observed using a monoclonal antibody to a protein found mainly in neurones (Yu et al. 1985). The absence of microtubules in tentacular epithelial cells is correlated with a stage at which these cells are terminally arrested in the G2 phase of the cell cycle (Dübel et al. 1987). This lack of tubulin expression may therefore be useful as a differentiation marker for ectodermal epithelial cells of the tentacles. A simplified diagram of tentacular and body wall sections that emphasize the distribution of microtubules is shown in Fig. 15.

Differences in the sensitivity of Hydra microtubules to nocodazole could be due to associated proteins, post-translational modifications or function-specific tubulins. The number of different tubulin isotypes in Hydra is unknown, and although sequence data on over 60 tubulin subunits are now available (for review, see Little & Seehaus, 1988), tubulins from the evolutionarily important Phyla of coelenterates have not been analysed. It is known, however, that Hydra tubulins, unlike other tubulins, do not appear to bind colchicine with high affinity. Microtubules were still visible in Hydra cells after incubation with 10 mM-colchicine (Campbell, 1976). In contrast, it is clear from our present work that one or more Hydra tubulins have an extremely high affinity for nocodazole. Spindles and stem cell microtubules, for example, disappeared at only 0-8-1 nM-nocodazole. These concentrations of nocodazole are much lower than those required for the disruption of mammalian microtubules. For example, 130nM-nocod-azole is required for the efficient arrest of mitosis in several mammalian cell lines (DeBrabander et al. 1976; Zieve et al. 1980) and 7 /tM-nocodazole is required to inhibit mammalian brain microtubule assembly by 50% (Kilmartin, 1981). Similar concentrations of nocodazole are also necessary for the disruption of yeast microtubules (Kilmartin, 1981).

Significant amounts of hydra interstitial cells were arrested in metaphase after incubation with 1 nM-nocod-azole. Interphase cells in G₁/S and G₂ were present in approximately equal amounts. After incubation with 2 nM-nocodazole, however, only a minor proportion of the interstitial cells were in metaphase and all of the interphase interstitial cells had been arrested in G₂. At these extremely low concentrations of nocodazole, the observed effects can only be due to very specific reactions. The observed correlation of cell cycle arrest with microtubule disassembly of spindle and cytoplasmic microtubules strongly indicates that nocodazole is reacting specifically with tubulin. In mammalian cells, the lack of G₂ arrest may possibly be due to the much lower sensitivity of their microtubules to nocodazole.

Removal of the 2 nM-nocodazole block led to a wave of mitoses after 7-8 h followed about 2h later by the entry of 75% of the interphase interstitial cells in G₁/S of the next cell cycle. The synchronous passage of interstitial cells into G₁/S indicates that most of them were arrested at a similar point in G₂. The length of G₂ in interstitial cells has been shown to vary between 4 and 22 h, and mitosis lasts about 1 h (Campbell & David, 1974). It appears, therefore, that the lapse of time between the G₂ arrest point and mitosis is much less variable than the length of G₂ itself.

An extra small wave of cells in G₁/S preceding the major peak was not observed, indicating that cells arrested in metaphase had not been able to recover. This is in agreement with the finding that mammalian cells arrested for longer periods of time in metaphase with nocodazole lose their ability to return to interphase (Zieve et al. 1980). It is probably fortuitous, therefore, that nocodazole blocks most of the interstitial cell cycles in G₂, since they can be held in this phase for relatively long periods without adverse effects. To our knowledge this is the first report of cell cycle synchronization by arrest.

The steps following G₂ arrest and leading to mitosis seem to be dependent on the assembly of a microtubuledependent structure. Without it the series of events leading to nuclear membrane breakdown and chromosome condensation are blocked. A possible candidate might resemble the novel dot-like structure recently identified by tubulin immunofluorescence that appeared 60 min before metaphase in nuclei of the slime mould Physarumpolycephalum (Paul et al. 1987). The assembly of this or another microtubule structure involved in Physarum cell cycle regulation might start even earlier, however, since nocodazole applied not later than 2h before mitosis delayed chromosome condensation and prevented the mitosis-specific increase of thymidine kinase activity (McClory & Coote, 1987).

Unlike colchicine, nocodazole has been shown to have a high affinity for the microtubules of taxonomically distant species. The use of this mitotic inhibitor and tubulin antibodies might therefore be expected to be equally useful for studying microtubule-dependent functions of other primitive organisms. The synchronization of rapidly dividing stem cells in Hydra and possibly other organisms should also provide a useful starting point for answering questions concerning their differentiation. However, perhaps the most intriguing questions raised by our results are what is the nature of the putative microtubule structure in the G₂ phase and how does it regulate the progress of the cell cycle towards mitosis?

We are very grateful for the support of Professor H. Chica Schaller in whose laboratory this work was carried out and for helpful discussions during the preparations of this manuscript. We also thank the members of this laboratory, especially Sabine Hoffmeister, for their encouragement. Our thanks are also due to Frank Breitling for stimulating discussions, to Elke Bach for cultivating Hydra, to Michael Stôhr for the use of his fluorescence microscope photometer and to Fnederike Schmitt for typing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 317).