Colchicine permeation is required for inhibition of concanavalin A ...

Proc. Nat. Acad. Sci. USA Vol. 72, No. 11, pp. 4516-4520, November 1975

Cell Biology

Colchicine permeation is required for inhibition of concanavalin A capping in Chinese hamster ovary cells (antimitotic agents/tubulin/membrane mutants)

JANE E. AUBIN, SVEIN A. CARLSEN, AND VICTOR LING Department of Medical Biophysics, University of Toronto, and the Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, M4X 1K9

Communicated by Gerald M. Edelman, September 8,1975

ABSTRACT Several antimitotic, tubulin-binding agents, such as colchicine, colcemid, and podophyllotoxin, inhibit the capping of fluorescent-labeled concanavalin A in Chinese hamster ovary cells. By comparing the effects of these agents on parental cell lines and on several independently selected colchicine-resistant mutants with decreased drug permeability, we have demonstrated that permeation of these drugs is required for inhibition of capping. These data suport the hypothesis that these antimitotic agents interact with an intracellular component, probably microtubules, to prevent the directional movement of concanavalin A receptors on the surface membranes of Chinese hamster ovary cells.

tion of nucleoside transport) has also been reported (13) and is thought to be independent of any action on microtubules. Also, in at least some systems, rather high concentrations of antitubulin drugs are required to elicit a response, concentrations higher than those thought necessary for antimitotic effects. In light of these limitations, we decided to investigate the basic question of whether or not antimitotic agents are required to enter the cell to affect Con A capping. Previously we have isolated and characterized a class of colchicine-resistant mutants in Chinese hamster ovary (CHO) cells that have greatly reduced permeability to colchicine and other compounds (14, 15, 21). The availability of these membrane-altered mutants allows us to pose the fundamental question of whether or not cell permeation is required for colchicine and a number of other agents to affect Con A capping in CHO cells. In this study, we report on the effects of drugs on capping of fluorescent Con A (fl-Con A) in parental and colchicine-resistant (CHR) CHO cells.

Various cell surface receptors, such as those for the mitogenic plant lectins, concanavalin A (Con A) and phytohemagglutinin, exhibit the ability to move in the lipid membrane bilayer. This movement is characterized by two distinct steps involving the redistribution of randomly oriented receptors into clusters or patches and subsequently into one large aggregate or cap. The phenomenon of cap formation has contributed to the current concept of the mammalian cell membrane as being composed of receptors and other surface components that are mobile in the fluid lipid phase

MATERIALS AND METHODS

(1, 2).

Recently it has been shown that the antimitotic agent colchicine, thought to specifically bind microtubule subunits, can alter the redistribution of certain membrane receptors, including those for Con A (3-10). Various drugs that also bind the tubulin subunits of microtubules, for example, colcemid, vinblastine, vincristine, and podophyllotoxin, elicit very similar responses, whereas a nonbinding analogue of colchicine, lumicolchicine, does not (3). These kinds of observations have led Edelman and coworkers (3-5) and others (6-10) to postulate that the nonrandom motion of some cell surface receptors is directed and modulated by a submembranellar cytoplasmic network, composed of microtubules and possibly other fibrous components. While the specificity of various antimitotic drugs to interact with microtubules has not been rigorously challenged, there is as yet little concrete evidence that it is the action of these agents on an intracellular microtubule structure that subsequently results in their effect on capping. As an alternative possibility, these drugs may be acting on the surface membrane modulating the lipid fluidity and thus affecting capping. In support of this hypothesis it has been pointed out that there exist other classes of compounds that inhibit capping, for example, certain local anesthetics and tranquilizers (11) which are known to interact with the cell membrane itself (12). Furthermore, the action of colchicine directly at a membrane level (inhibi-

Cell Culture Conditions. The parental cell line, AUX B1, and colchicine-resistant (CHR) lines, CHR2HA, CHRC4, and CHRC5, were grown routinely at 370 in suspension cultures in a-minimal essential medium (16) supplemented with 10% fetal calf serum (Flow Laboratories) as described (14). Capping of fl-Con A. In preparation for capping, cells were seeded at 5 X 104 cells per cm2 in growth medium and allowed to attach overnight at 370 to glass coverslips (Corning Glassware) contained in 35 mm plastic petri dishes (Falcon). Binding and capping of fl-Con A were performed as follows: medium was removed by aspiration and the cells attached to coverslips were washed twice in phosphate-buffered saline at 40, after which fl-Con A (Miles-Yeda) dissolved in phosphate-buffered saline to a final concentration of 50 ,ug/ml was added, and the cells were incubated at 40 for 30 min. Excess unbound fl-Con A was aspirated off and the cells were washed twice more in cold phosphate-buffered saline. Capping was then allowed to occur by adding warmed phosphate-buffered saline at 370 for an incubation of 45 min at the same temperature. When drugs were tested for their ability to inhibit capping, they were present at both 40 and 370.

For the scoring of fluorescent caps, coverslips were mounted on glass slides, without prior fixation of the cells, and cells monitored for the appearance of fluorescent label using a Zeiss fluorescent microscope. Duplicate coverslips were prepared for each experimental point, and at least 100 cells were scored per coverslip. Only those cells with fl-Con A discernible as one tight aggregate of label are scored as

Abbreviations: Con A, concanavalin A; fl-Con A, fluorescent-labeled Con A; CHO, Chinese hamster ovary; CHR, colchicine-resistant.

4516

Cell Biology: Aubin et al.

Proc. Nat. Acad. Sci. USA 72 (1975)

4517

FIG. 1. Capping in parental and CHR CHO cells. (a) fl-Con A was bound to cells at 40 and the cells were processed for capping at 370 as described in Materials and Methods. (b) AUX B1 cells allowed to form caps at 37°. (c) CHRC4 cells treated in the same manner as AUX Bi cells in (b). (d) AUX B1 cells processed for capping in the presence of 1O-5 M colchicine.

having a cap. Cells remain firmly attached to coverslips throughout these manipulations. RESULTS Capping of fl-Con A in CHO cells Fig. la shows that if fl-Con A at a concentration of 50 jig/ml is allowed to bind to CHO cells at 40, the label first appears on all cells as a diffuse total surface label or rings. When cells are incubated at 370, randomly oriented, small fluorescent patches are formed which then slowly aggregate to form caps after 20-30 min. By 45 min, the maximum number of cells in the population have capped. Under these conditions, greater than 75% of the cells form caps, which appear as bright fluorescent aggregates located centrally on the cells, as seen in Fig. lb and c, where fl-Con A caps on parental, AUX Bl, and mutant, CHRC4, cells are shown, respectively. These figures indicate the close similarity in the appearance of caps in the parental and mutant cells, pointing out that the membrane alteration resulting in reduced drug permeability in the CHR CHO cells has no effect on the ability of fl-Con A to cap. Very little or no pinocytosis of label occurs in our cells, nor does label disaggregate over the next 2 hr when cells are maintained in phosphate-buffered saline at room temperature. As in other systems, no capping occurs if the cells are maintained at 4°. Binding-and capping can be inhibited by the addition of a-methyl glucoside (0.1 mM) during the 40 incubation with fl-Con A.

Effect of colchicine on Con A capping We next investigated the effect of colchicine on the Con A capping of parental cells and CHR mutant cells. At sufficiently high concentrations of this drug, capping in both parental and mutant cells is inhibited and the fl-Con A appears as a randomly distributed, diffuse pattern (see Fig. Id) different from that of uncapped cells not treated with drug (as seen in Fig. la). This is observed both when colchicine is present during the whole fl-Con A binding and capping procedure or when colchicine is added only during the 370 capping step. This may indicate that, in addition to inhibiting capping, the presence of colchicine also perturbs the patch formation of surface bound fl-Con A in our cells, thus inducing an apparently more diffuse pattern of fl-Con A. It can be seen from Fig. 2 that both the parental and a CHR line are able to form Con A caps at high efficiencies, at greater than 70 and 90%, respectively, of the total cell populations. In all the cell lines that we have examined, Con A cap formation occurs at high efficiencies, although the actual value for any particular line may vary somewhat from experiment to experiment. We have noted as well that in general the CHR mutants display a higher capping efficiency than the parental cells. Whether or not this difference is related to the membrane alteration(s) resulting in drug resistance in CHR cells is not known at present. However, a large difference is observed in the amount of drug required for inhibition between the parental and drug-resistant cells. This

4518


Proc. Nat. Acad. Sci. USA 72 (1975) Table 1. . Effect.of colchicine on the inhibition of capping in AUX B1 and CHR mutant cell lines

V.,~~~~~ 60~~~

20

0

10

106

165

Id

[Colchicine] (M)

FIG. 2. Dose response curves for the inhibition of capping in AUX B1 (@) and CHRC5 (A) CHO cells. Cells were treated with various concentrations of colchicine during incubation at 4° and 370, as described in Materials and Methods.

is evident from Fig. 2, where it can be seen that capping in AUX Bi is inhibited by 10-5 M colchicine, while in CHRC5 cells, a concentration of 10-3 M is required. Since we have

previously demonstrated that the mechanism of colchicine resistance in the CHR mutants is the result of reduced membrane permeability rather than altered drug binding or increased drug degradation (14), these results are consistent with the hypothesis that colchicine must get inside the cell in order to disrupt Con A capping. Thus it follows that in the resistant mutant high extracellular concentrations of drug would be required for the same amount of drug to get inside the cell in the time allotted. To test the above hypothesis, we determined the effects of colchicine on two other independently selected colchicineresistant mutants with different degrees of drug resistance (14). Since it is known that the relative rate of colchicine uptake in these mutants is correlated with their relative resistance to colchicine, we reasoned that if colchicine permeability is important for inhibition of Con A capping, there should be also some relationship between the concentration of colchicine required for capping inhibition and the degree of colchicine resistance in these cells. That such a relationship exists is shown in Table 1, where it can be seen that with the substantially less resistant CHR2HA cells, less colchicine is required to inhibit capping, while another highly resistant line, CHRC4, selected independently from CHRC5, requires a much higher dose. We have previously observed that nonionic detergents at nontoxic doses increase the rate of colchicine uptake into CHO cells (14; Carlsen, unpublished observation). Thus, to further test that permeation of colchicine is required for the inhibition of capping, we examined the effect of a nonionic detergent, Triton X-100, on the dose response of colchicine inhibition in AUX B1 cells. For this experiment, we chose a concentration of Triton X-100 (1.0 ,g/ml) that stimulates the rate of colchicine uptake by 2-fold in AUX B1 cells (Carlsen, unpublished data) but which by itself has no effect on capping. As can be seen in Fig. 3, the presence of the detergent when compared with the control sample without detergent decreases by approximately 2-fold the amount of drug required to reduce cap formation by 50%. This again lends support to the concept that the drug must get inside the cell and not merely interact at the membrane. Effect of other antimitotic agents If one is to implicate directly microtubules in the directional movement of Con A receptors, then other microtubule dis-

Cell line

Drug resistance

AUXB1 CHRS42HA CHRC4 CHRC5

1 16 250 300

Concentration (M) of colchicine required to inhibit capping to 50%

5x10-6 5 x 10-5 -1 -3 10-3

Concentration of drug required for inhibition of capping to 50% of the value obtained in the absence of drug was determined from dose-response curves, as shown for AUX B1 and CHRC5 in Fig. 1. Values for drug resistance were from Ling and Thompson (14).

ruptive drugs should also inhibit fl-Con A capping in CHO cells. Thus we determined whether or not inhibition of Con A capping is also demonstrated by two other microtubuleactive agents, podophyllotoxin (17) and colcemid, a close analogue of colchicine, and by lumicolchicine (18), a photodegradation product of colchicine that does not bind tubulin. In addition, we were able to determine whether or not permeation of these compounds is required also for inhibition of Con A capping in the same manner as that described for colchicine in Fig. 2. This was possible since colchicine-resistant cells are cross-resistant to various drugs, e.g., actinomycin D, puromycin, colcemid, and podophyllotoxin (14, 21), and the resistance to these drugs also results from reduced permeability since it has been demonstrated previously that the mutant cells were less permeable to labeled puromycin and colcemid (15; Carlsen and Ling, unpublished observation). The results of these experiments are summarized in Table 2. It is seen that both colcemid and podophyllotoxin are able to inhibit Con A capping in AUX B1 cells at very low concentrations: 7 X 10-9 M and 3 X 10-8 M, respectively, while lumicolchicine has no effect up to 10-4 M. Moreover, it is observed from Table 2 that the drug-resistant mutant required substantially higher concentrations of colcemid and podophyllotoxin to inhibit capping than the parental cell line, implying that the permeation of these antimicrotubule drugs also is required to inhibit capping of Con A. These results are completely compatible with the concept of microtubule involvement in Con A capping in CHO cells.

DISCUSSION The effect of antimitotic agents on Con A capping has been used to support the hypothesis that a cytoplasmic network of microtubules modulates the movement of some cell surface receptors (3-10). Very little definitive evidence has been presented that would prove the notion that it is the action of these drugs on the microtubules themselves which is causing the effect on capping. In this report, we have presented several lines of evidence that demonstrate that microtubule-disrupting drugs must get inside the cell to be effective in the inhibition of Con A capping in CHO cells and support the concept that these drugs interfere with capping as a result of their specific interaction with intracellular microtubules. First, we have shown that the concentration of colchicine required to inhibit Con A capping correlates with the degree of reduced drug permeability in a number of independently selected mutant lines (Table 1). Second; when permeation of

Proc. Nat. Acad. Sci. USA 72 (1975)


4519

Table 2. Effect of colcemid and podophyllotoxin on the inhibition of capping in AUX Bi and CHRC4 Cohcentration (M) of drug required to inhibit capping to 50% Drug

AUX B1

CHRC4

Colcemid Podophyllotoxin Lumicolchicine

7 x 10-9 3 X 10" No effect up to 104

8 x 10-5 10-6 No effect up to 10-4

The concentration of drug required for 50% inhibition of capping was determined as described in Table 1.

colchicine is stimulated by 2-fold by a concentration of Triton X-100 that by itself has no effect on capping, effective inhibitory doses of colchicine shift to lower concentrations by approximately 2-fold (Fig. 3). Third, cell permeation is required also for two other tubulin-binding agents, colcemid and podophyllotoxin, to inhibit Con A capping. A nonbinding colchicine analogue, lumicolchicine, had no effect

(Table 2). There is one additional observation which leads further support to these lines of evidence. As mentioned above, colchicine and colcemid have been reported to exert some influence on at least one other membrane function, that of nucleoside transport, in a manner that does not involve their binding to microtubule components. This function is not altered in the colchicine-resistant mutants; that is, when colchicine inhibition of nucleoside transport is studied, inhibitory doses of colchicine are the same for mutant and parental cell lines (Carlsen, unpublished data). This latter observation suggests that at least some membrane sites must be as accessible in the mutant cells as in parental cells and argues against some alteration in the mutant cells which prevents colchicine from approaching the cell surface per se. Thus, assuming that the plasma membranes of both cell lines are equally accessible to colchicine, one would not expect the observed large concentration difference required to inhibit the capping in these lines (Table 1) if the cell surface were in fact the colchicine target for this effect. This provides some indirect evidence that inhibition of Con A capping in CHO cells by colchicine is not the result of an interaction of the drug with the cell surface membrane. We believe that all the above data provide some definitive evidence that interaction with an intracellular target is required for inhibition of Con A capping in CHO cells by the antimitotic agents and are consistent with the hypothesis that directional receptor motion in mammalian cells is modulated by a cytoplasmic component, probably the microtubules. It is of interest to compare the characteristics of Con A capping in CHO cells to those in a number of other cell types. As documented above, Con A forms a central cap which is inhibited by colchicine. We have observed also that other cells, e.g., hamster embryo fibroblasts and mouse L cells, behave similarly (Aubin, unpublished data). However, in contrast to these cells, lymphocytes form polar Con A caps and their formation is stimulated by colchicine and other tubulin-binding agents (3-5, 9, 10). Whether or not the position of the Con A cap relates to its response to antimitotic agents is not known, although studies with polymorphonuclear leukocytes seem to imply that such a relationship exists (7). For example, motile polymorphonuclear leukocytes

KC)-

[Colchicine]

(M )

FIG. 3. Potentiation of the inhibition of cap'ping by colchicine in the presence of nonionic detergent. AUX B1 cells were incubated with 1.0 gg/ml of Triton X-100 (0) or without detergent (0), in various concentrations of colchicine, and the number of capped cells was scored as in Fig. 2.

form polar caps that are not inhibited by colchicine, while the same cells rendered nonmotile by cytochalasin B or by low serum form central caps that are inhibited by colchicine (7). These results also suggest that the position of the Con A cap may be a consequence of the relative motility of the cells. However, the mechanism determining the position of the Con A caps may be relatively complex, and this may involve microtubular structure as well. This has recently been demonstrated by Rutishauser et al. (20) in experiments with lymphocytes immobilized on nylon fibers. They observed that the position of Con A caps on the immobilized lymphocytes did not depend on lateral movements, but rather caps formed opposite the site of attachment of cells undergoing shape changes, and that the position of the caps became random after treatment with colchicine. The capping of a number of other surface receptors has also been investigated in various laboratories, most notably the capping of surface receptors to specific IgG has been compared to that of Con A. It is becoming obvious from the growing body of data on the effects of various agents on the capping ability of these receptors that while the mobility of Con A and IgG receptors are related, they are probably not identical, at least in response to various chemical stimuli. This is true of the responses elicited by the colchicine-like compounds where, for example, it has been observed that the normal capping of surface IgG in mouse fibroblasts is inhibited by colcemid (19). In contrast to this, normal capping of antisera to surface components in lymphocytes and motile polymorphonuclear leukocytes is unaffected by the addition of colchicine or other tubulin binding agents (3-10). However, in these latter systems, IgG capping is inhibited by the binding of Con A, but this inhibition of the IgG capping is released by treatment with colchicine-like compounds. While it seems clear that there may be differences in the process of capping by different surface receptors and in the capping of a particular receptor in different cells, yet in each case, antitubulin agents perturb these systems. The underlying basis for the different responses is presently not understood but may be related to the pleotropic nature of mammalian cell membranes. An interesting possibility is the selection of colchicine-resistant mutants in a number of other systems to determine whether or not perturbation of the capping response (either positively or negatively) also requires colchicine permeation. The availability of well characterized drug-resistant permeability mutants used in this study has clearly helped us to define the question of whether

4520

Proc. Nat. Aced. Sci. USA 72 (1975)


or not permeation is required for antimitotic agents to inhibit the phenomenon of Con A capping in CHO cells. Success in obtaining mutants altered in their intracellular colchicinebinding proteins or in the process of capping itself will greatly facilitate further delineation of this complex capping process and understanding of the intricacies of the cell membrane. We thank Drs. Charles Waldren and Brian J. Storrie for helpful discussions concerning Con A capping in CHO cells. This work has been supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada, and by a contract from the National Institutes of Health of the United States. J.E.A. and S.A.C. are recipients of Medical Research Council of Canada Studentships. 1. Singer, S. J. & Nicholson, G. L. (1972) Science 175,720-731. 2. Taylor, R. B., Duffus, W. P. H., Raff, M. C. & de Petris, S. (1971) Nature New Biol. 223,225-229. 3. Edelman, G. M., Yahara, I. & Wang, J. L. (1973) Proc. Nat. Acad. Sci. USA 70, 1442-1446. 4. Yahara, I. & Edelman, G. M. (1975) Exp. Cell Res. 91, 125142. 5. Yahara, I. & Edelman, G. M. (1975) Proc. Nat. Acad. Sci. USA 72, 1579-1583.

6. Ukena, T. E., Borysenko, J. F., Karnovsky, M. J. & Berlin, R. D. (1974) J. Cell Biol. 61, 70-82. 7. Ryan, G. B., Borysenko, J. F. & Karnovsky, M. J. (1974) J. Cell Biol. 62,351-65. 8. Unanue, E. R. & Karnovsky, M. T. (1974) J. Exp. Med. 140, 1207-1220. 9. de Petris, S. (1974) Nature 250,54-55. 10. de Petris, S. (1975) J. Cell Biol. 65, 123-146. 11. Ryan, G. B., Unanue, E. R. & Karnovsky, M. J. (1974) Nature

250,56-57. 12. Seeman, P. (1972) Pharmacol. Rev. 24,583-65. 13. Mizel, S. B. & Wilson, L. (1972) Biochemistry 11, 2573-2578. 14. Ling, V. & Thompson, L. H. (1974) J. Cell. Physiol. 83,103116. 15. See, Y. P., Carlsen, S. A., Till, J. E. & Ling, V. (1974) Biochim. Biophys. Acta 373,242-252. 16. Stanners, C. P., Eliceiri, G. & Green, H. (1971) Nature New Biol. 230,52-54. 17. Wilson, L. & Friedkin, M. (1967) Biochemistry 6,3126-3135. 18. Wilson, L. & Friedkin, M. (1966) Biochemistry 5,2463-2468. 19. Edidin, M. & Weiss, A. (1972) Proc. Nat. Acad. Sci. USA 69, 2456-2459. 20. Rutishauser, U.; Yahara, I. & Edelman, G. M. (1974) Proc. Nat. Acad. Sci. USA 71,1149-1153. 21. Bech-Hansen, N. T., Till, J. E. & Ling, V. (1975) J. Cell. Physlol., in press.