Vol. 189, No. 3, 1992 December
BIOCHEMICAL
AND BIOPHYSICAL
FIBRONECTIN
RESEARCH COMMUNICATIONS
Pages 1429-1236
30. 1992
RECEPTORS ARE FUNCTIONAL
ON MITOTIC CHINESE HAMSTER
OVARY CELLS Pascal POMIES and Marc R. BLOCK Laboratoire d’Etude des Systemes Adhesifs Cellulaires, A.T.I.P.E. de I’URA 1178 du CNRS, Universite Joseph Fourier, BP 53X, F38041 GRENOBLE CEDEX-FRANCE Received
November
3,
1992
In this paper, evidence is provided indicating that the blockade of presynchronized CHO 15B cells in prometaphase by nocodazole is fully reversible and efficient enough to allow us to analyze the function of the integrin receptors. Flow cytometry analysis using a specific antibody raised against the fibronectin receptor, and binding studies of the radiolabeled fibronectin on the cell membrane, indicated a stable number of receptors at the cell surface during mitosis. Furthermore, in the mean time, only a slight increase in the Kd value of the fibronectin-receptor interaction was detected. A binding assay designed to test the affinity of the receptor for its extracellular ligand in an insoluble form was used. No difference was observed between mitotic and interphasic cells. Taken together, these results indicate that the rounding up of the cells observed during mitosis is not due to a loss of the receptor affinity for its extracellular ligand. c 1992ACadEmlC PrPss,Inc.
One of the most striking change during mitosis is the rounding up of the cultured cells accompanied by the disassembly of the microfilament bundles. It has been proposed that these morphological changes may be due to the disruption of the stress fibers: the dissociation from the microfilaments
of the phosphorylated caldesmon would result in a subsequent release of the
inhibition of gelsolin activity (1,2). Since the organization of stress fibers is under the control of the small GTP binding protein rho (3), one can imagine a regulation of the cell spreading along the cell cycle by this small G protein. Yet, the disruption of the actin network by the Clostridial toxins C2 or C3 or by the microinjection of recombinant p21 rho does not result in a fast disorganization of adhesion plaques (4, 5). Dramatic morphological changes are observed after 30 to 60 minutes (5), but the cells remain firmly attached to the substratum with a typical irregular shape quite different from what is observed during mitosis. Therefore, the disruption of the actin bundles may not be sufficient alone to account for the rapid detachment of the cells from the extracellular matrix during metaphase. Cell adhesion and spreading mostly occur through membrane receptors of the integrin family . Therefore an alternative explanation would be that the rounding of the cells during mitosis could be due to a loss of integrin affinity for their respective ligand after the phosphorylation of the cytoplasmic domain of these receptors (6). Such a modulation of activity has been well documented for the anbP3 platelet integrin and cxLp2 (LFA-1) integrin. It is due to a conformational change that can be detected immunologically
(7-13) or physically (14, 15). Whereas allh cytoplasmic domain
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controls the binding affinity of the extracellular segment of the platelet integrin (16), it is the p2 cytoplasmic domain that seems to play this role for LFA- 1 (17). Beside these well-studied models, modulations of the affinities of constitutively active integrins have been reported. For instance, the affinity of VLA (pl) integrins on T cells are rapidly and dramatically augmented after antigen stimulation, without any change of the level of expression (18). Similarly, inhibition of a5Pl and a6Pl activity during cell differentiation has been reported (19, 20). On CHO and HUVEC cells in culture, it has been demonstrated in an irl vim assay that the binding activities of the fibronectin and the vitronectin-fibrinogen receptors became dependent on cytosolic proteins and micromolar concentrations of intracellular calcium. These data suggest that calcium or calcium-dependent protein may play a key role in the control of integrin activity (21, 22). To address the question of a possible change in the integrin activity during mitosis, the affinity
of the fibronectin
receptor has been studied on CHO cells (clone 15B) blocked in
prometaphase. Here, we report that the fibronectin receptor normally present at the cell surface of CHO cells (the counterpart of the human a5Pl) is in a high affinity conformational state during mitosis. Our results indicate that the interaction between the integrin and the extracellular matrix is not the target of the biochemical mechanism underlying the rounding up of the cells during mitosis.
EXPERIMENTAL
PROCEDURES
Cell culture and synchronization. CHO 15B cells were grown on plates in the minimum essential medium with alpha modification (a MEM) without nucleoside and supplemented with 7.5% fetal calf serum (v:v) at 37’C in a humidified 5% CO2 / 95% air atmosphere. Cells were harvested with a Hank’s Balanced Salt Solution (HBSS) supplemented with 1mM EDTA and 0.05% trypsin (w:v). Mitotic cells were obtained according to (23). CHO 15B cells (3.105 per ml) were incubated in aMEM containing 5 mM thymidine. After 16 hours, cells were washed with HBSS and the incubation was carried out for another 4 hours in fresh cxMEM. Then 35 rig/ml of nocodazole (Sigma) dissolved in DMSO was added. The incubation was continued for 3 hours. At the end of this period approximately 70% of the cells were blocked in prometaphase and mitotic cells were halvested by gently tapping the dishes without any proteases. A mitotic index of 95% was obtained on the harvested cells, as disclosed by the staining of the chromosomes with a 1 PM Hoechst 33258 solution in HBSS, for 45 minutes at room temperature and in the dark. Cell adhesion experiments. Experiments were performed in bacterial plastic dishes coated with 0.25 mg/ml fibronectin in 2M urea and PBS for 2 hours at 37°C. Using this material, no block with bovine serum albumin was required. Mitotic cells were pelleted at 4”C, resuspended in a MEM with 7.5% (v:v) fetal calf serum and incubated at 37’C on fibronectin coated petri dishes with or without 35 @ml nocodazole. After 150 minutes the cells in suspension were harvested, washed once and resuspended in fresh a MEM. At different times cells were observed under phase contrast and photographed using a Diaphot inverted microscope (Nikon) and counted. The number of spreaded cells in a constant area (0,03 mm?) was estimated. All assays were run in triplicate. Flow cytometry. Mitotic and non mitotic CHO 15B cells were harvested, pelleted at 4’C. They were resuspended and fixed in HBSS supplemented with 0.1% paraformaldehyde for 15 min at 20°C as described in (10). Nocodazole (35 rig/ml) was present or not for mitotic or control cells respectively. The fixed cells were pelleted, resuspended in HBSS and incubated with the antifibronectin receptor monoclonal antibody PBl (24), or with a control antibody for 90 min at 4°C. After two washes, cells were incubated for 30 min with fluorescein isothiocyanate (FITC)conjugated goat anti-mouse IgG (Bio-Rad Laboratories). Then, 5000 cells were analyzed on a FACScan (Becton Dickinson) equiped with Argon laser (x=488 nm). Fibronectin purification and radiolabeling. Fibronectin was purified by affinity chromatography from bovine plasma according to (25). Radiolabeling of fibronectin with carrier free [125]Iodine (Amersham) was performed using 1,3,4,6-tetrachloro-3a,6a diphenyl glycouril (Sigma) according to (26). Elimination of free iodine was achieved by desalting onto a lOm1 Biogel P6-DG column (Bio-Rad Laboratories). 1430
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Soluble fibronectin binding assays.CHO 15B cells were pelleted, washed two times with HBSS and 25 m M Hepes, 5% BSA (w:v), 150 m M NaCI. They were resuspended in this later medium supplemented with 1 m M MgCl2 and used in our binding assays. Fibronectin binding on mitotic or non mitotic CHO 15B cells in suspension was carried out as described in (27), with 35 rig/ml nocodazole for mitotic cells. Insoluble fibronectin binding assays.Polystyrene latex beads LB-30 (Sigma) with an average diameter of 3.0 pm were used for these binding assays.The beads were precoated with 0.1 mg/ml of fibronectin for 2 hours and subsequently blocked with bovine serum albumin at the concentration of 50 mg/ml for 18 hours. For the controls, beads incubated only with bovine serum albumin (50 mg/ml) or with HBSS for 20 hours were used. All the incubations were carried out at room temperature. Immediately before use, the beads were submitted to a sonication for 1s in order to disperse the bead aggregates. Binding assays with mitotic and non mitotic CHO 15B cells were made at room temperature for 30 minutes. After two washes in HBSS lo6 cells/ml were incubated with 20.106 beads/ml in 25 m M Hepes pH 7.2, 5% BSA (w:v), 150 m M NaCl, 1 m M MgC12. With mitotic cells, 35 @ml nocodazole was also added in the binding buffer. At the end of the incubation time, a drop of the cell suspension was withdrawn and observed under phase contrast using a FxA microscope equiped with a plan 20X/ph (na=0.5) objective lens.
RESULTS To be significant, any experiment performed with nocodazole must fulfill the following conditions: i) the cells must stay in prometaphase during the whole time course of the experiment; ii) the cells should maintain a physiological state, i.e., their viability must be preserved, and the cell cycle must immediately resume upon the removal of the drug. Long telm exposure of the cells to nocodazole results in an efficient blockade of the cells in mitosis. However, it impairs the reversibility of this process and shar@y reduces the viability of the cells. The procedure described in (23) relies on the presynchronization of the cells in S phase by a thymidine block. Then. a short exposure to nocodazole at low concentration is sufficient to allow the cells to reach prometaphase. Under these experimental conditions, the cells exit from metaphase within 10 min. after the removal of nocodazole. Using this procedure we have obtained a population of round CHO 15B cells with a mitotic index of 95% determined according to the staining of the DNA with Hoechst 33258. The spreading of these cells onto a fibronectin coat was analyzed and correlated to the mitotic index. Fig. 1 shows that upon the removal of nocodazole, more that 90% of the cells have exited from mitosis and were spread on fibronectin within less than 100 min. at 37’C. This indicates the excellent viability of our biological material at the begining of the experiment. Conversely, when the concentration of 35 rig/ml of nocodazole was maintained in a MEM, 72 to 78% of the cells remained unattached after 2.5 hours and were still mitotic as disclosed by the Hoechst staining. The other cells were attached and spread on the fibronectin coated petri dishes and the observation ot their DNA content revealed that they were no longer in mitosis. At this time, the removal of nocodazole induced attachment and spreading of the mitotic cells with a kinetic that wah indistinguishable from the one observed when the cell were immediately placed into fresh medium just after the harvest. These data indicated that during the time course of (maximum 2 hours), the mitotic CHO 15B cells kept their physiological integrity, index ranging from 75 to 82%. A possible loss of the fibronectin receptors from the cell surface just metaphase could account for the rounding up of the mitotic cells. Therefore, we 1431
our experiments and had a mitotic before or during analyzed by flow
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Fig. 1, Spreading of mitotic CHO 15B cells on fibronectin coated dishes after treatment with nocodazole. Mitotic CHO 15B cells were harvested after 3 hours in 35 rig/ml nocodazole as described under Experimental Procedures. They were washed and incubated in fibronectin coated dishes with fresh a MEM (0) or with fresh a MEM supplemented with 35 &ml nocodazole (.). After 150 minutes, nocodazdle was removed (arrow): non adherent cells were-withdrawn, washed and incubated into fresh a MEM. Cells were photographed at different times and the number of spreaded cells was estimated in a constant area. I&& Cell surface expression of integrin receptor (aspi) on mitotic and non mitotic CHO 15B cells. Mitotic (A) and non mitotic (B) CHO 15B cells were fixed with O,l% parafotmaldehyde and incubated with control antibody (dotted line) or anti fibronectin receptor monoclonal antibody PBl (solid line), followed by a second antibody (FITC)-conjugated goat anti mouse IgG as described under Experimental Procedures. For each sample fluorescence intensity of 5000 cells was measured by flow cytometry.
cytometry the number of receptors at the cell surface using the specific monoclonal antibody PBl. This antibody was originally selected for its ability to specifically inhibit the attachment and spreading of the CHO cells onto the fibronectin coat (24). The harvested mitotic cells were kept with nocodazole to maintain their blockade in metaphase and were fixed in 0.1% paraformaldehyde as described under Experimental Procedures. Fig. 2 shows that PBl induced a strong fluorescence signal of the FITC secondary antibody at the cell surface (solid line) as compared to the signal obtained with an irrelevant antibody used as a control (dotted line). With interphasic and mitotic cells those signals were indistinguishable,
indicating that the same fibronectin receptors number
was present on the cell surface during the cell cycle. However, mitotic cells were no longer able to spread on tibronectin coated dishes. In order to quantify the binding parameters of the fibronectin receptor during metaphase, the binding of soluble [ 1251] fibronectin on its specific receptors was assayed. Since the time course of these experiments did not exceed 2 hours, the mitotic cells should keep a high mitotic index at the end of the assay (Fig. 1). Indeed, a typical staining of the cells with Hoechst 33258 at the end of the binding assay revealed that 80% of the cells exhibited a condensed chromatin and the chromosomes were aligned along the equatorial section (not shown). The specific amount of [125I]fibronectin bound to its receptor (i. e., the amount of the radioactive ligand displaced by an excess of cold fibronectin) was assayed using a Scatchard analysis. It revealed a single class of binding sites for interphasic and mitotic cells respectively (Fig. 3). The total number of receptors per cell (1.1kO.l x 105) was approximatly the same. This result 1432
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Fig. Specific binding of [t*sI]FN to mitotic and non mitotic CHO 15B cells. Mitotic (.) and non mitotic (0) CHO 15B cells were incubated with increasing concentrations of [t2sI]FN in the presence or absence of an excess unlabeled fibronectin for 60 min at room temperature as described under Experimental Procedures. Binding results were analyzed by Scatchard analysis: the intercepts corresponded to the number of sites per cell and the slopes of the extrapolated straight lines gave the Kd values. This figure represents a typical experiment, but three distinct experiments gave similar results. For each experiment all the points were performed in triplicate.
was in good agreement with the flow cytometry analysis (Fig. 2). On the other hand, the Kd value estimated for the interphasic cells was 0.07+0.01 PM whereas it was slightly higher for mitotic cells (0.12&0.02 l.tM). The difference observed was small but reproducible. It was not due to an artifactual effect of nocodazole since the experiments performed with interphasic cells in the presence of the drug gave a Kd value of 0.07 PM and a total number of binding sites of 1.4 x 105 receptors per cell (not shown). Some integrins like the platelet integrin CpIIblIIa (aIIbb3) exhibit very different affinities depending on the physical state of their ligands. For example, on resting platelets, the aIIbb3 integrin has a low affinity for soluble fibrinogen but binds insoluble fibrinogen with a high affinity (28). A possible conformational change of CHO fibronectin receptors during mitosis could induce such a sharp loss of affinity for insoluble fibronectin with only a minor effect on the Kd value for the soluble ligand. To rule out this hypothesis, a binding assay of insoluble fibronectin has been designed: the cells were incubated for 30 min. at 20°C with fibronectin coated polystyrene latex beads. Control experiments were carried out with uncoated or BSA coated beads. Typical experiments are shown in Fig. 4: mitotic (Fig. 4B) and interphasic (Fig. 4D) cells did efficiently bind the fibronectin coated beads that formed clusters at the cell surface and induced cell aggregation. Conversely, the beads coated with BSA interacted neither with mitotic nor with interphasic cells (Fig. 4 A and 4C). The same held for uncoated beads (not shown).
DISCUSSION Cell division is a transient event along the cell cycle. At this time, the cells round up and acquire a typical round and smooth shape. This remarkable morphological change may be due to a decrease in the avidity of the integrin receptors for the extracellular matrix. to a disorganization of adhesion plaques, or eventually to the disruption of the actin network. 1433
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Fig. Binding of insoluble fibronectin on mitotic or non mitotic CHO 15B cells. 106 mitotic CHO 15B cells per ml (A-B) or 106 non mitotic CHO 15B cells per ml (C-D) were incubated30 minutes at room temperaturewith 20. lo6 polystyrenelatex beadsper ml. In (A-C) beadswere coated with bovine serumalbumin, in (B-D) beadswere precoatedwith a high concentrationof fibronectin and then blocked with bovine serum albumin. Other details aregiven under ExperimentalProcedures. Note that mitotic CHO 15B cells appearedperceptibly larger than non mitotic CHO 15B cells. Bar is 20 pm.
Herein, we have been using nocodazole on CHO 15B cells to slow down metaphase. We have shown that the blockade of the cells in metaphase is efficient enough to allow the analysis of the function of the fibronectin receptor. On the other hand, our measurements were of a physiological significance since the cells resume their cycle readily upon the removal of the drug. This indicates that during nocodazole treatment, the cells kept their physiological integrity and their viability. We have shown that the fibronectin receptors of CHO 15B cells (the counterpart of the human asp1 integrin) are still present on the surface of mitotic cells with a number similar to the one obsetved on interphasic cells. Furthermore, they keep a high affinity for their ligand either in a soluble or insoluble form. However, a small decrease of the integrin affinity for soluble fibronectin has been detected. It is very unlikely that this small increase in the Kd value or the slight decrease in 1434
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the receptor density (due to the increase of the cell surface) account for the rounding up of the cells, since it occurs quickly at the entry of metaphase. The fact that integrin receptors are functional during mitosis is in good agreement with the observation that the full detachment of the rounded mitotic cells requires a mechanical stress. Thus, it indicates that cell-extracellular matrix interactions still exist during mitosis, but the mechanical strength of the cellular anchorage is considerably reduced. The disruption of the stress fibers and the actin network results in a quite distinct cell morphology than the one observed during mitosis. Mitotic cells appear to be very smooth and the inhibition of the spreading is much faster, since it correlates with the entry of the cells in prometaphase. Therefore, the contol of the microfilaments may not be sufficient alone to account for the rounding up observed during cell division. One possible hypothesis to explain the detachment of the mitotic cells is that the target of the biochemical mechanism that regulates cell adhesion during cell division might be the organization of the adhesion plaques themselves. Such ;I mechanism might or might not involve a chemical modification of the integrins. It could affect the clustering of the receptors, and subsequently, it would lower the mechanical strength of the cell anchorage to the extracellular matrix. Besides the modulation of the integrin affinity (6). our data suggest new ways in controlling cell adhesion.
ACKNOWLEDGMENTS We thank Dr R.L. Juliano (Chapel Hill University N.C., USA) for providing us with the PBI antibody and Mrs M.-C. Jacob (Grenoble Blood Center) for her excellent technical assistance and helpful discussion during flow cytometry experiments. This work was supported in part by a grant from the C.N.R.S (ATIPE 900041), a Contrat de Recherche Externe I.N.S.E.R.M. No900102 and by a grant from the Association pour la Recherche contre le Cancer (A.R.C.). P.P. is supported by a fellowship from the Ministere pour la Recherche et la Technologie.
REFERENCES Yamashiro, S., Yamakita, Y., Ishikawa, R., and Matsumura, F. (1990) Nature 344, 675-678. Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172. Ridley, A.J., and Hall, A. (1992) Cell 70, 389-399. Aktories, K., and Wegner, A. (1989) J. Cell Biol. 109, 13851387. Paterson, H.F., Self, A.J., Garrett, M.D., Just, I., Aktories. K., and Hall, A. (1990) J. Cell. Biol. 111, 1001-1007. 6. Hynes, R.O. (1992) Cell 69, 1 l-25. 7. Shattil, S.J., Hoxie, J.A., Cunningham, M., and Brass, L.F. (1985) J. Biol. Chem. 260, 1110711114. 8. Gulino, D., Ryckewaert, J.J., Andrieux, A., Rabiet, M.J., and Marguerie, G. (1990) J. Biol. Chem. 265,9575-9581. 9. Kouns, W.C., Wall, CD., White, M.M., Fox, C.F., and Jennings, L.K. (1990) J. Biol. Chem. 265,20594-20601. ..-_-. __ _ 10. O’Toole, T.E., Loftus, J.C., Du, X., Glass, A.A., Ruggeri, Z.M., Shattil, S.J., Plow, E.F., and Ginsberg, M.H. (1990) Cell Regul. 1,883-893. 11. Andrieux, A., Rabiet, M.J., Chapel, A., Concord, E., and Marguerie, G. (1991) J. Biol. Chem. 266, 14202-14207. 12. Keizer, G.D., Visser, W., Vliem, M., and Figdor, C.G. (1988) J. Immunol. 140, 1393-1400. 13. Van Kooyk, Y., van de Wiel-van Kemenade, P., Weder, P., Kuijpers, T.W., and Figdor, C.G. (1989) Nature 342, 811-813.
1. 2. 3. 4. 5.
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14. Parise, L.V., Helgerson, S.L., Steiner, B., Nannizzi, L., and Phillips, D.R. (1987) J. Biol. Chem. 62, 12597-12602. 15. Sims, P.J., Ginsberg, M.H., Plow, E.F., and Shattil, S.J. (1991) J. Biol. Chem. 266,7345-7352. 16. O’Toole, T.E., Mandelman, D., Forsyth, J., Shattil, S.J., Plow, E.F., and Ginsberg, M.H. (1991) Science 254,845-847. 17. Hibbs, M.L., Xu, H., Stacker, S.A., Springer, T.A. (1991) Science 251, 1611-1613. 18. Shimizu, Y., Van Seventer, G.A., Horgan, K.J., and Shaw, S. (1990) Nature 345,250-253. 19. Neugebauer, K.M., and Reichardt, L.F. (1991) Nature 68-7 1. 20. Adams, J.C., and Watt, F.M. (1990) Cell 63,425-435. 21. Marie, C., Tranqui, L., Soyez, S., and Block, M.R. (1991) Exp. Cell Res. 192, 173-181. 22. Arroyo, A.G., Sanchez-Mateos, P., Campanero, M.R., Martin-Padura, I., Dejana, E., and Sanchez-Madrid, F. (1992) J. Cell Biol. 117,659-670. 23. Brady, R.C., Schibler, M.J., and Cabral, F. (1986) Methods Enzymol. 134,217-225. 24. Brown, P.J., and Juliano, R.L. (1985) Science 228, 1448-1450. 25. Engvall, E., and Ruoslahti, E. (1977) Int. J. Cancer 20, l-5. 26. Fraker, P.J., and Speck, J.C. (1978) Biochem. Biophys. Res. Commun. 80,849-857. 27. Akiyama, S.K., and Yamada, K.M. (1985) J. Biol. Chem. 260,4492-4500. 28. Packham, M.A., Evans, G., Glynn, M.F., and Mustard, J.F. (1969) J. Lab. Clin. Med. 73. 686697.
1436
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Pages 1429-1236
30. 1992
RECEPTORS ARE FUNCTIONAL
ON MITOTIC CHINESE HAMSTER
OVARY CELLS Pascal POMIES and Marc R. BLOCK Laboratoire d’Etude des Systemes Adhesifs Cellulaires, A.T.I.P.E. de I’URA 1178 du CNRS, Universite Joseph Fourier, BP 53X, F38041 GRENOBLE CEDEX-FRANCE Received
November
3,
1992
In this paper, evidence is provided indicating that the blockade of presynchronized CHO 15B cells in prometaphase by nocodazole is fully reversible and efficient enough to allow us to analyze the function of the integrin receptors. Flow cytometry analysis using a specific antibody raised against the fibronectin receptor, and binding studies of the radiolabeled fibronectin on the cell membrane, indicated a stable number of receptors at the cell surface during mitosis. Furthermore, in the mean time, only a slight increase in the Kd value of the fibronectin-receptor interaction was detected. A binding assay designed to test the affinity of the receptor for its extracellular ligand in an insoluble form was used. No difference was observed between mitotic and interphasic cells. Taken together, these results indicate that the rounding up of the cells observed during mitosis is not due to a loss of the receptor affinity for its extracellular ligand. c 1992ACadEmlC PrPss,Inc.
One of the most striking change during mitosis is the rounding up of the cultured cells accompanied by the disassembly of the microfilament bundles. It has been proposed that these morphological changes may be due to the disruption of the stress fibers: the dissociation from the microfilaments
of the phosphorylated caldesmon would result in a subsequent release of the
inhibition of gelsolin activity (1,2). Since the organization of stress fibers is under the control of the small GTP binding protein rho (3), one can imagine a regulation of the cell spreading along the cell cycle by this small G protein. Yet, the disruption of the actin network by the Clostridial toxins C2 or C3 or by the microinjection of recombinant p21 rho does not result in a fast disorganization of adhesion plaques (4, 5). Dramatic morphological changes are observed after 30 to 60 minutes (5), but the cells remain firmly attached to the substratum with a typical irregular shape quite different from what is observed during mitosis. Therefore, the disruption of the actin bundles may not be sufficient alone to account for the rapid detachment of the cells from the extracellular matrix during metaphase. Cell adhesion and spreading mostly occur through membrane receptors of the integrin family . Therefore an alternative explanation would be that the rounding of the cells during mitosis could be due to a loss of integrin affinity for their respective ligand after the phosphorylation of the cytoplasmic domain of these receptors (6). Such a modulation of activity has been well documented for the anbP3 platelet integrin and cxLp2 (LFA-1) integrin. It is due to a conformational change that can be detected immunologically
(7-13) or physically (14, 15). Whereas allh cytoplasmic domain
1429
0006-?9 I x/92 84.~10 C’opwight 0 1992 by Amdernic Prrss. I/w. All rights of reproduction in any ,fim~~ re.wrwd
Vol.
189,
No.
3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
controls the binding affinity of the extracellular segment of the platelet integrin (16), it is the p2 cytoplasmic domain that seems to play this role for LFA- 1 (17). Beside these well-studied models, modulations of the affinities of constitutively active integrins have been reported. For instance, the affinity of VLA (pl) integrins on T cells are rapidly and dramatically augmented after antigen stimulation, without any change of the level of expression (18). Similarly, inhibition of a5Pl and a6Pl activity during cell differentiation has been reported (19, 20). On CHO and HUVEC cells in culture, it has been demonstrated in an irl vim assay that the binding activities of the fibronectin and the vitronectin-fibrinogen receptors became dependent on cytosolic proteins and micromolar concentrations of intracellular calcium. These data suggest that calcium or calcium-dependent protein may play a key role in the control of integrin activity (21, 22). To address the question of a possible change in the integrin activity during mitosis, the affinity
of the fibronectin
receptor has been studied on CHO cells (clone 15B) blocked in
prometaphase. Here, we report that the fibronectin receptor normally present at the cell surface of CHO cells (the counterpart of the human a5Pl) is in a high affinity conformational state during mitosis. Our results indicate that the interaction between the integrin and the extracellular matrix is not the target of the biochemical mechanism underlying the rounding up of the cells during mitosis.
EXPERIMENTAL
PROCEDURES
Cell culture and synchronization. CHO 15B cells were grown on plates in the minimum essential medium with alpha modification (a MEM) without nucleoside and supplemented with 7.5% fetal calf serum (v:v) at 37’C in a humidified 5% CO2 / 95% air atmosphere. Cells were harvested with a Hank’s Balanced Salt Solution (HBSS) supplemented with 1mM EDTA and 0.05% trypsin (w:v). Mitotic cells were obtained according to (23). CHO 15B cells (3.105 per ml) were incubated in aMEM containing 5 mM thymidine. After 16 hours, cells were washed with HBSS and the incubation was carried out for another 4 hours in fresh cxMEM. Then 35 rig/ml of nocodazole (Sigma) dissolved in DMSO was added. The incubation was continued for 3 hours. At the end of this period approximately 70% of the cells were blocked in prometaphase and mitotic cells were halvested by gently tapping the dishes without any proteases. A mitotic index of 95% was obtained on the harvested cells, as disclosed by the staining of the chromosomes with a 1 PM Hoechst 33258 solution in HBSS, for 45 minutes at room temperature and in the dark. Cell adhesion experiments. Experiments were performed in bacterial plastic dishes coated with 0.25 mg/ml fibronectin in 2M urea and PBS for 2 hours at 37°C. Using this material, no block with bovine serum albumin was required. Mitotic cells were pelleted at 4”C, resuspended in a MEM with 7.5% (v:v) fetal calf serum and incubated at 37’C on fibronectin coated petri dishes with or without 35 @ml nocodazole. After 150 minutes the cells in suspension were harvested, washed once and resuspended in fresh a MEM. At different times cells were observed under phase contrast and photographed using a Diaphot inverted microscope (Nikon) and counted. The number of spreaded cells in a constant area (0,03 mm?) was estimated. All assays were run in triplicate. Flow cytometry. Mitotic and non mitotic CHO 15B cells were harvested, pelleted at 4’C. They were resuspended and fixed in HBSS supplemented with 0.1% paraformaldehyde for 15 min at 20°C as described in (10). Nocodazole (35 rig/ml) was present or not for mitotic or control cells respectively. The fixed cells were pelleted, resuspended in HBSS and incubated with the antifibronectin receptor monoclonal antibody PBl (24), or with a control antibody for 90 min at 4°C. After two washes, cells were incubated for 30 min with fluorescein isothiocyanate (FITC)conjugated goat anti-mouse IgG (Bio-Rad Laboratories). Then, 5000 cells were analyzed on a FACScan (Becton Dickinson) equiped with Argon laser (x=488 nm). Fibronectin purification and radiolabeling. Fibronectin was purified by affinity chromatography from bovine plasma according to (25). Radiolabeling of fibronectin with carrier free [125]Iodine (Amersham) was performed using 1,3,4,6-tetrachloro-3a,6a diphenyl glycouril (Sigma) according to (26). Elimination of free iodine was achieved by desalting onto a lOm1 Biogel P6-DG column (Bio-Rad Laboratories). 1430
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Soluble fibronectin binding assays.CHO 15B cells were pelleted, washed two times with HBSS and 25 m M Hepes, 5% BSA (w:v), 150 m M NaCI. They were resuspended in this later medium supplemented with 1 m M MgCl2 and used in our binding assays. Fibronectin binding on mitotic or non mitotic CHO 15B cells in suspension was carried out as described in (27), with 35 rig/ml nocodazole for mitotic cells. Insoluble fibronectin binding assays.Polystyrene latex beads LB-30 (Sigma) with an average diameter of 3.0 pm were used for these binding assays.The beads were precoated with 0.1 mg/ml of fibronectin for 2 hours and subsequently blocked with bovine serum albumin at the concentration of 50 mg/ml for 18 hours. For the controls, beads incubated only with bovine serum albumin (50 mg/ml) or with HBSS for 20 hours were used. All the incubations were carried out at room temperature. Immediately before use, the beads were submitted to a sonication for 1s in order to disperse the bead aggregates. Binding assays with mitotic and non mitotic CHO 15B cells were made at room temperature for 30 minutes. After two washes in HBSS lo6 cells/ml were incubated with 20.106 beads/ml in 25 m M Hepes pH 7.2, 5% BSA (w:v), 150 m M NaCl, 1 m M MgC12. With mitotic cells, 35 @ml nocodazole was also added in the binding buffer. At the end of the incubation time, a drop of the cell suspension was withdrawn and observed under phase contrast using a FxA microscope equiped with a plan 20X/ph (na=0.5) objective lens.
RESULTS To be significant, any experiment performed with nocodazole must fulfill the following conditions: i) the cells must stay in prometaphase during the whole time course of the experiment; ii) the cells should maintain a physiological state, i.e., their viability must be preserved, and the cell cycle must immediately resume upon the removal of the drug. Long telm exposure of the cells to nocodazole results in an efficient blockade of the cells in mitosis. However, it impairs the reversibility of this process and shar@y reduces the viability of the cells. The procedure described in (23) relies on the presynchronization of the cells in S phase by a thymidine block. Then. a short exposure to nocodazole at low concentration is sufficient to allow the cells to reach prometaphase. Under these experimental conditions, the cells exit from metaphase within 10 min. after the removal of nocodazole. Using this procedure we have obtained a population of round CHO 15B cells with a mitotic index of 95% determined according to the staining of the DNA with Hoechst 33258. The spreading of these cells onto a fibronectin coat was analyzed and correlated to the mitotic index. Fig. 1 shows that upon the removal of nocodazole, more that 90% of the cells have exited from mitosis and were spread on fibronectin within less than 100 min. at 37’C. This indicates the excellent viability of our biological material at the begining of the experiment. Conversely, when the concentration of 35 rig/ml of nocodazole was maintained in a MEM, 72 to 78% of the cells remained unattached after 2.5 hours and were still mitotic as disclosed by the Hoechst staining. The other cells were attached and spread on the fibronectin coated petri dishes and the observation ot their DNA content revealed that they were no longer in mitosis. At this time, the removal of nocodazole induced attachment and spreading of the mitotic cells with a kinetic that wah indistinguishable from the one observed when the cell were immediately placed into fresh medium just after the harvest. These data indicated that during the time course of (maximum 2 hours), the mitotic CHO 15B cells kept their physiological integrity, index ranging from 75 to 82%. A possible loss of the fibronectin receptors from the cell surface just metaphase could account for the rounding up of the mitotic cells. Therefore, we 1431
our experiments and had a mitotic before or during analyzed by flow
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Fig. 1, Spreading of mitotic CHO 15B cells on fibronectin coated dishes after treatment with nocodazole. Mitotic CHO 15B cells were harvested after 3 hours in 35 rig/ml nocodazole as described under Experimental Procedures. They were washed and incubated in fibronectin coated dishes with fresh a MEM (0) or with fresh a MEM supplemented with 35 &ml nocodazole (.). After 150 minutes, nocodazdle was removed (arrow): non adherent cells were-withdrawn, washed and incubated into fresh a MEM. Cells were photographed at different times and the number of spreaded cells was estimated in a constant area. I&& Cell surface expression of integrin receptor (aspi) on mitotic and non mitotic CHO 15B cells. Mitotic (A) and non mitotic (B) CHO 15B cells were fixed with O,l% parafotmaldehyde and incubated with control antibody (dotted line) or anti fibronectin receptor monoclonal antibody PBl (solid line), followed by a second antibody (FITC)-conjugated goat anti mouse IgG as described under Experimental Procedures. For each sample fluorescence intensity of 5000 cells was measured by flow cytometry.
cytometry the number of receptors at the cell surface using the specific monoclonal antibody PBl. This antibody was originally selected for its ability to specifically inhibit the attachment and spreading of the CHO cells onto the fibronectin coat (24). The harvested mitotic cells were kept with nocodazole to maintain their blockade in metaphase and were fixed in 0.1% paraformaldehyde as described under Experimental Procedures. Fig. 2 shows that PBl induced a strong fluorescence signal of the FITC secondary antibody at the cell surface (solid line) as compared to the signal obtained with an irrelevant antibody used as a control (dotted line). With interphasic and mitotic cells those signals were indistinguishable,
indicating that the same fibronectin receptors number
was present on the cell surface during the cell cycle. However, mitotic cells were no longer able to spread on tibronectin coated dishes. In order to quantify the binding parameters of the fibronectin receptor during metaphase, the binding of soluble [ 1251] fibronectin on its specific receptors was assayed. Since the time course of these experiments did not exceed 2 hours, the mitotic cells should keep a high mitotic index at the end of the assay (Fig. 1). Indeed, a typical staining of the cells with Hoechst 33258 at the end of the binding assay revealed that 80% of the cells exhibited a condensed chromatin and the chromosomes were aligned along the equatorial section (not shown). The specific amount of [125I]fibronectin bound to its receptor (i. e., the amount of the radioactive ligand displaced by an excess of cold fibronectin) was assayed using a Scatchard analysis. It revealed a single class of binding sites for interphasic and mitotic cells respectively (Fig. 3). The total number of receptors per cell (1.1kO.l x 105) was approximatly the same. This result 1432
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Fig. Specific binding of [t*sI]FN to mitotic and non mitotic CHO 15B cells. Mitotic (.) and non mitotic (0) CHO 15B cells were incubated with increasing concentrations of [t2sI]FN in the presence or absence of an excess unlabeled fibronectin for 60 min at room temperature as described under Experimental Procedures. Binding results were analyzed by Scatchard analysis: the intercepts corresponded to the number of sites per cell and the slopes of the extrapolated straight lines gave the Kd values. This figure represents a typical experiment, but three distinct experiments gave similar results. For each experiment all the points were performed in triplicate.
was in good agreement with the flow cytometry analysis (Fig. 2). On the other hand, the Kd value estimated for the interphasic cells was 0.07+0.01 PM whereas it was slightly higher for mitotic cells (0.12&0.02 l.tM). The difference observed was small but reproducible. It was not due to an artifactual effect of nocodazole since the experiments performed with interphasic cells in the presence of the drug gave a Kd value of 0.07 PM and a total number of binding sites of 1.4 x 105 receptors per cell (not shown). Some integrins like the platelet integrin CpIIblIIa (aIIbb3) exhibit very different affinities depending on the physical state of their ligands. For example, on resting platelets, the aIIbb3 integrin has a low affinity for soluble fibrinogen but binds insoluble fibrinogen with a high affinity (28). A possible conformational change of CHO fibronectin receptors during mitosis could induce such a sharp loss of affinity for insoluble fibronectin with only a minor effect on the Kd value for the soluble ligand. To rule out this hypothesis, a binding assay of insoluble fibronectin has been designed: the cells were incubated for 30 min. at 20°C with fibronectin coated polystyrene latex beads. Control experiments were carried out with uncoated or BSA coated beads. Typical experiments are shown in Fig. 4: mitotic (Fig. 4B) and interphasic (Fig. 4D) cells did efficiently bind the fibronectin coated beads that formed clusters at the cell surface and induced cell aggregation. Conversely, the beads coated with BSA interacted neither with mitotic nor with interphasic cells (Fig. 4 A and 4C). The same held for uncoated beads (not shown).
DISCUSSION Cell division is a transient event along the cell cycle. At this time, the cells round up and acquire a typical round and smooth shape. This remarkable morphological change may be due to a decrease in the avidity of the integrin receptors for the extracellular matrix. to a disorganization of adhesion plaques, or eventually to the disruption of the actin network. 1433
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Fig. Binding of insoluble fibronectin on mitotic or non mitotic CHO 15B cells. 106 mitotic CHO 15B cells per ml (A-B) or 106 non mitotic CHO 15B cells per ml (C-D) were incubated30 minutes at room temperaturewith 20. lo6 polystyrenelatex beadsper ml. In (A-C) beadswere coated with bovine serumalbumin, in (B-D) beadswere precoatedwith a high concentrationof fibronectin and then blocked with bovine serum albumin. Other details aregiven under ExperimentalProcedures. Note that mitotic CHO 15B cells appearedperceptibly larger than non mitotic CHO 15B cells. Bar is 20 pm.
Herein, we have been using nocodazole on CHO 15B cells to slow down metaphase. We have shown that the blockade of the cells in metaphase is efficient enough to allow the analysis of the function of the fibronectin receptor. On the other hand, our measurements were of a physiological significance since the cells resume their cycle readily upon the removal of the drug. This indicates that during nocodazole treatment, the cells kept their physiological integrity and their viability. We have shown that the fibronectin receptors of CHO 15B cells (the counterpart of the human asp1 integrin) are still present on the surface of mitotic cells with a number similar to the one obsetved on interphasic cells. Furthermore, they keep a high affinity for their ligand either in a soluble or insoluble form. However, a small decrease of the integrin affinity for soluble fibronectin has been detected. It is very unlikely that this small increase in the Kd value or the slight decrease in 1434
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the receptor density (due to the increase of the cell surface) account for the rounding up of the cells, since it occurs quickly at the entry of metaphase. The fact that integrin receptors are functional during mitosis is in good agreement with the observation that the full detachment of the rounded mitotic cells requires a mechanical stress. Thus, it indicates that cell-extracellular matrix interactions still exist during mitosis, but the mechanical strength of the cellular anchorage is considerably reduced. The disruption of the stress fibers and the actin network results in a quite distinct cell morphology than the one observed during mitosis. Mitotic cells appear to be very smooth and the inhibition of the spreading is much faster, since it correlates with the entry of the cells in prometaphase. Therefore, the contol of the microfilaments may not be sufficient alone to account for the rounding up observed during cell division. One possible hypothesis to explain the detachment of the mitotic cells is that the target of the biochemical mechanism that regulates cell adhesion during cell division might be the organization of the adhesion plaques themselves. Such ;I mechanism might or might not involve a chemical modification of the integrins. It could affect the clustering of the receptors, and subsequently, it would lower the mechanical strength of the cell anchorage to the extracellular matrix. Besides the modulation of the integrin affinity (6). our data suggest new ways in controlling cell adhesion.
ACKNOWLEDGMENTS We thank Dr R.L. Juliano (Chapel Hill University N.C., USA) for providing us with the PBI antibody and Mrs M.-C. Jacob (Grenoble Blood Center) for her excellent technical assistance and helpful discussion during flow cytometry experiments. This work was supported in part by a grant from the C.N.R.S (ATIPE 900041), a Contrat de Recherche Externe I.N.S.E.R.M. No900102 and by a grant from the Association pour la Recherche contre le Cancer (A.R.C.). P.P. is supported by a fellowship from the Ministere pour la Recherche et la Technologie.
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