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Membr. Cell Biol., 1999, Vol.12 (6), pp. 895-905 Reprints available directly from the publisher Photocopying permitted by license only

© 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group Printed in Malaysia

Experimental Model for Studying the Primary Cilia in Tissue Culture Cells
I. B. Alieva, L. A. Gorgidze, Yu. A. Komarova, O. A. Chernobelskaya, and I. A. Vorobjev
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Vorobievy Gory, Moscow, 119899, Russia fax: (095) 939 3181; email: alieva@electron.genebee.msu.su (Received 13 October, 1997) In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tobulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia. (Received 13 October, 1997)

One of the functions of the centrosome in animal cells which has been described a long time ago is to form the primary (rudimentary) cilia [1, 2]. The primary cilium is a structure forming only on an active centriole; it is a continuation of the microtubules of the triplets at the distal end of the centriolar cylinder - the axonema enclosed by the membrane. Most

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vertebrate cells express the primary cilium at some times of their life history. Formation of the primary cilia is especially characteristic of embryonic cells of various tissues, as well as of cells in early postnatal development [3-6]. Many differentiated cells are known to have the primary cilium [7-13]. Only in a few cell types the active centriole never forms the primary cilium. These are various leukocytes, hepatocytes, adipocytes [14, 15]. The primary cilium is characteristic of various lines of cultured cells, especially those of fibroblasts. According to the literature data, cells with cilia are up to 87-88% (fibroblasts in primary culture [14]; lines of murine fibroblasts 3T6, 3T3, S3/13) [13, 16-19]. In epithelial cultures, the percentage of cilial cells is 30% (in PK culture cells [20], the primary culture of endothelial cells [12], in cells of PtK1 culture [15, 21, 22]). Information concerning the physiology of the primary cilium is rather fragmentary and incomplete. It pertains mainly to the early, descriptive period of electron-microscopy studies, because namely the method of electron microscopy made it possible to describe this structure in detail. Since that time, the primary cilia studies were limited by the high labor intensity of this technique. In the recent years, the primary cilium is studied by the method of immunofluorescence. The first works used standard antibodies against tubulin to stain microtubules [23]. However, this method could be used only for cells with a poorly developed network of cytoplasmic microtubules. Subsequent research used various poly- and monoclonal antibodies which specifically stained the primary cilium. Thus, the monoclonal antibodies against dethyrosylated -tubulin selectively stain the cilium in cells of various tissues [13, 24, 25]. To reduce the labor intensity, in this work we made an attempt (i) to choose antibodies which intensively stains the primary cilium in culture cells of various lines and (ii) to show that the use of these antibodies makes it possible not only to reveal primary cilia at the light-optical level but also to assess their number adequately. For this purpose, we counted the quantity of the primary cilia in culture cells of various lines simultaneously using immunofluorescent staining and electron microscopy. Then we studied the stability of expression of the primary cilium during passivation. And, finally, we made an attempt to develop a new approach to study the functions of the primary cilium, using cells synchronized by selection in mitosis. EXPERIMENTAL Cell cultures. Epithelial PK (pig kidney) cells were cultured at 37°C and 5% CO2 on medium 199 supplemented with 10% bovine serum and gentamicin. HeLa cells were grown at 37°C and 5% CO2 in medium 199 supplemented with 10% fetal calf serum and gentamicin. 3T3 culture cells, rat


PRIMARY CILIA IN TISSUE CULTURE CELLS 897 kangaroo cells PTK1 and primary rat embryo fibroblasts (REF) were grown at 37°C and 5% CO2 in DMEM/F-12 HAM (Sigma, USA) supplemented with 10% fetal calf serum and gentamicin. Antibodies. Monoclonal antibodies C3B9 against acetylated tubulin were produced at the Laboratory of Prof. K. Gull (Manchester, UK). Monoclonal antibodies against y-tubulin were obtained at the Laboratory of I. A. Vorobjev and described earlier [26, 27]. FITC-conjugated secondary antibodies were from commercial (Sigma, USA). Immunofluorescent studies. For immunofluorescent analysis, the cells were fixed using two techniques: (i) in 4% formaldehyde solution for 30 min; (ii) in 1% glutaraldehyde solution (Merck, Germany) in phosphate buffer for 30 min followed by a triple treatment with a NaBH4 solution (2 mg/ml, 10 min each). In the latter case, prior to fixation the cells were lyzed in a mixture containing 1% Triton X-100 in microtubule-stabilizing conditions. The coverslips with the cells were taken out of the Petri dish, washed several times with phosphate buffered saline (pH 7.2) at 37°C and then lyzed for 15 min in a solution containing 50 mM imidazole (pH 6.8), 5 mM MgCl2,1 mM EGTA, 0.1 mM EDTA, 35% glycerol and 1% Triton X-10 (Sigma). The preparations were included into a 2.5% solution of 1,4-diazabicyclo[2.2.2]-octane (DABCO) (Sigma) on glycerol, examined under an Opton-3 photomicroscope (Opton, Germany) and photographed (film RF-3, Tasma, Russia). Electron microscopy. Cells were fixed with a 2.5% solution of glutaraldehyde (Merck, Germany) in phosphate buffer for 30 min or lyzed in a mixture containing Triton X-100, in microtubule-stabilizing conditions, and then fixed with 1% solution of glutaraldehyde (Merck) in phosphate buffer for 30 min [28]. Further treatment of the preparation for electron microscopy was carried out as described earlier [28]. Synchronization of cells. Mitotic cells were isolated by two techniques: (i) Mitotic cells of a rat fibroblast culture were accumulated by incubation of a monolayer culture in a medium containing nocodazole (0.1 (µg/ml) for 4-6 h. Then the metaphase cells were shaken off, collected into 2-ml Eppendorf tubes and centrifuged at 1000 rpm three times 3 min each; during centrifugation the cells were washed with the medium without nocodazole. The sedimented metaphase cells were plated in a drop of the medium onto coverslips. 30 min after (upon complete attachment of the cells to the cover-slips) 3 ml of the medium was added to the Petri dishes. (ii) Mitotic cells of the 3T3 fibroblast culture were collected by shaking off normal metaphase cells and plating them onto coverslips as above but without nocodazole. Thus, no mitostatic was used in this case and, by excluding its washout from the protocol, we reduced the time from the start of isolation to the time when cells were attached to coverslips.


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Figure 1. Immunpfluorescent micrographs of cultured cells stained with antibodies C3B9: a, 3T3 (the cilia are shown by the arrow); b, 3T3 (microtubule bundles of the mid-body are shown by the arrow); c, d, REF; e, PtK1 (a cilium is shown by the arrow), a and d, a primary cilium is stained more intensively than acetylated microtubules. Scale, 10 µm.

RESULTS AND DISCUSSION

Immunocytochemical imaging of the primary cilium in tissue culture cells. At present, studies of primary cilia are almost none. This is due not so much to the extreme labour intensity of the method of electron microscopy as to the absence of experimental approaches. This work is an attempt to develop a model for primary cilium studies by replacing the electron-microscopy analysis by a less labour-intensive technique. We used antibodies against acetylated tubulin which, according to the literature data, stain the primary cilium in cultured cells [13, 19]. Antibodies against acetylated tubulin stain the primary cilia (Fig. la, c-e), microtubules of the mid-body in cultured PK, HeLa, PtK1 3T3, REF cells (Fig. 1 b) and some cytoplasmic microtubules in part of the cells studied (Fig. la, d). In cells lacking primary cilia, depending on the stage of the cell cycle, these antibodies stain one or two dots in the perinuclear region (Figs. 2 and ). verify if these dots are centrioles, we used antibodies against -tubulin, which in interphase cells stain one or two dots corresponding to the position of the centrioles. Double staining showed that the dots revealed by antibodies against acetylated tubulin coincide with the dots revealed by


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Figure 2. Immunofluorescent staining of the telophases of 3T3 cells by antibodies C3B9: a, b, 1 h after plating mitotic cells. The active and inactive centrioles in the cells are at different distances from each other. Scale, 10 µm.

Figure 3. Double immunofluorescent staining of the centrosome in an interphase HeLa cell: a, by antibodies C3B9; b, by polyclonal antibodies to tubulin; c, phase contrast. Scale, 10 µm.

antibodies against -tubulin (Fig. 3). Thus, antibodies against acetylated tubulin do reveal centrioles. In all five cultures studied, visual differentiation of the primary cilium and cytoplasmic acetylated microtubules presented no problem. The cilium was stained more intensively than individual microtubules, its stain was


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Figure 4. infrastructure of short cilia (shown by arrows) in two 3T3 cells: a, a cilium shaped as a "stump", the axonema of the cilium is in an embryonic state (the active centriole contains pericentriolar satellites and is in contact with the membrane cyst); b, a short cilium (the active centriole with the pericentriolar satellites and the pronounced cilial axonema). A, active centriole; I, inactive centriole. Scale, 0.2 µm.

homogeneous along the entire length, the axonema of the cilium was shorter than the bundles of acetylated microtubules in the cytoplasm. The length of the cilium and its curvature varied both depending on the type of culture studied (in REF cells it was significantly longer than in cells of other lines) and between individual cells in one culture (Fig. la, c-e). Studies of cells of the above lines by electron microscopy gave similar results. The cilium on ultrathin sections looked as a small "stump" (Fig. 4a) and could be either short (Fig. 4b) or rather long and twisted (data not shown). As seen in Table 1, in cells of both epithelial nature (PK, PtK1) and in fibroblasts (REF, 3T3) the primary cilium occurs rather frequently: from 1/5 to 1/3 of the cells studied contained the cilium. Only HeLa cells have almost no cilia. To determine if the fluorescent method makes it possible to assess the number of cilia in cultured cells adequately, we counted simultaneously the number of cilia in cell preparations stained with antibodies and in electronmicroscopy specimens of cells taken from the same passage (Table 1). Our doubts were based on electron-microscopy data on cells with very short cilia, the length of whose axonema did not exceed half of that for the centriolar cylinder (Fig. 4a). As it follows from Table 1, the results obtained by the immunochemical method virtually coincide with those obtained by electron microscopy. Expression of the primary cilium in cell passivation. In the course of the experiments with the culture of 3T3 cells which were obtained (as indicated


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above) frozen, the cells started to express the cihum not immediately alter unfreezing. In the first two passages, no cells with the primary cilium were
Table 1. Comparative frequency of occurrence of the primary cilium as revealed by immunocytochemistry and electron microscopy in cells of various lines (per 100 and 50 cells, respectively). Cell line Frequency of occurrence (%) according to the data of immunocytochemistry PK REF 3T3 PtK1 HeLa 28 31 21 26 2 electron microscopy 28 32 22 2

recorded. Only in the third passage single cells first had the primary cilium. In the following passages, starting from the fourth, expression of the primary cilium was more significant (Table 2). In passages 4-9, expression was stabilized, and the percentage of cilia reached the maximum (19-23%) obtained in all the experiments. Further on, the percentage of cells with the primary cilium decreased and was stable in 10-12 passages (12-16%).
Table 2. Frequency of occurrence of the primary cilium as revealed by immunocytochemistry in cultured 3T3 cells depending on the number of passage, starting from the time of unfreezing (per 100 cells). Number of cells with the Passage No Number of cells with Passage No primary cilium, % the primary cilium, % 1 2 3 4 8 0 0 Single cells 21 19 9 10 11 12 23 12 15 16

Thus, expression of the primary cilium is not constant over several passages, it is stable for a short time which is, evidently, individual for each particular cell culture. This presents additional problems in studies of its physiology, as it requires constant control of expression in the course of experiments. A model for studies of the primary cilium in tissue culture cells. In the centrosome of mitotic cells the primary cilium is always absent [29, 21]; therefore, the completion of mitosis can be the starting point for experiments


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on the expression of the primary cilium. We assumed that, by accumulating mitotic cells (REF or 3T3), it would be possible to follow the dynamics of primary cilia in cultured cells and study the sequence of morphological changes in the cilia formed. For this purpose, we obtained preparations of synchronized mitotic cells in two ways. In the first series of experiments, mitotic cells of primary REF were obtained after their preliminary accumulation in culture using low concentrations of nocodazole (0.1 µg/ml). Sporadic telophase cells are observed in the preparation 1 h after plating mitotic cells on coverslips. The mid-body was not stained by antibodies against acetylated tubulin but bundles of microtubules were well seen in the junction between daughter cells (Fig. 1b). The primary cilium was not found in any cells analyzed. The first cilia emerged in the preparation 2 h after plating the cells on coverslips; the number of cells with the primary cilium was 5%. Further on, the percentage of cells having the primary cilium increased to reach 9% in 4 h. Besides single metaphase cells, the preparations were found to have groups of interphase cells which, apparently, broke loose to make a kind of islets during the shake-off. In the first experiments their number was sufficient to enable a cilium count. The percentage of cilia in these cells proved to coincide with that for control cells, which can indicate that the protocol used to isolate mitotic cells has no effect on the expression of the primary cilium. Thus, in the first series of experiments we determined the time when the primary cilia emerge upon cessation of mitosis. However, mitotic cells were then isolated using nocodazole which, due to its depolymerizing effect on microtubules, could have an effect on the rate of cilium formation. To verify this suggestion, we used the culture of 3T3 fibroblasts, mitotic cells of which are normally round-shaped and are easily shaken off the substrate. In another series of experiments, already 40 min after shaking off mitotic cells and plating them on a new coverslip we observed single telophases in the preparation; the daughter cells are connected by the mid-body (data not shown). The mid-body was not stained, either, by antibodies against acetylated tubulin in 3T3 cells, but bundles of microtubules in the zone of junction between daughter cells were well seen (Fig. 1b). No primary cilium analyzed 40 min to 1 h after the stain was found in any of the cells. The proportion of the cells which had the primary cilium was 7.5% 2 h after the cells were plated. Later on, the cilia became more, and 4 h after plating their percentage almost reached the normal level (Table 3). Most pairs of sister cells had only one primary cilium. However, in some cases the primary cilium was formed in both sister cells in 3-4 h. Thus, isolation of mitotic cells without a mitostatic appears to be a more suitable procedure for the purposes associated with the physiology of the primary cilium, because in this case its expression reaches the control level faster (Table 3).


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Table 3. Frequency of occurrence of the primary cilium as revealed by immunocytochemistry in preparations of synchronized mitotic cells from the time of plating the cells and during subsequent cultivation (per 30 cells). Frequency of occurrence of the (1 -4 h from Frequency of occurrence Cell line the primary cilium (%)time of plating) of the primary cilium (%) in intact cells of the same passage 1h 2h 3h 4h REF 3T3 0 0 5 7.5 1 2.5 9 18 31 19

CONCLUSION To out knowledge, to date there are no papers on the primary cilium carried out by immunocytochemical techniques where the frequency of occurrence is counted. Apparently, the authors are not certain of the reliability of such counts and rely on electron-microscopy data. One of the possible reasons for the data of immunocytochemistry to be underestimated due to the existence of primary cilia with the very short axonema was examined in this work. The frequency of occurrence of the primary cilia in immunocytochemical staining and from the data of electron microscopy coincide. Thus, in cultured cells the use of antibodies against acetylated tubulin is justified. Another concern known from personal communications was unstable expression of the primary cilium over the generations. This concern found proof in our studies. The percentage of fibroblasts carrying the cilium varied within broad limits over 12 passages. Thus, in any effects the results of an experiment can be compared with the control only within the same passage. In four out of five cultures studied the primary cilium occurs in 1/3 of the interphase cells. The exception were only HeLa cells in which the primary cilium was extremely rare, which can be due to the high degree of their transformability. In cells of the epithelioid lines the cilium is significantly shorter than in fibroblasts. In cultured fibroblasts the cilia are absent in mitosis, they occur 1 h after the cells exit mitosis and in 4 h their frequency of occurrence is almost the same as in non-synchronous cells. Similar observations were made earlier in in vivo experiments on mouse embryo enterocytes, where the cilia are present in Gl, S and G2 periods according to the data of electron microscopy [30]. Summing up the results obtained in vitro and in vivo, we conclude that our data confirm the suggestion that in cells the cilia can exist at all stages of the cell cycle, except mitosis and the earliest interphase [29].


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The authors are grateful to B. Lange and K. Gull for provided antibodies C3B9 and R. E. Uzbekov for fruitful discussion of this work. The work was supported by the Russian Foundation for Basic Research (grants Nos 96-04-50935 and 95-04-12703), the Civilian Research and Development Foundation (grant No 168100) and the Ministry of Science and Technical Policy (Project UNIKOL). REFERENCES
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