Proc. Nat. Acad. Sci. USA Vol. 73, No. 4, pp. 1227-1231, April 1976
Cell Biology
Reversible in vitro polymerization of tubulin from a cultured cell line (rat glial cell clone C6) (control of microtubule assembly/tissue culture)
GERHARD WICHE AND R. DAVID COLE Department of Biochemistry, University of California, Berkeley, Calif. 94720
Communicated by Bruce N. Ames, December 22,1975
Tubulin from cultures of the rat glial cell ABSTRACT clone Ce could be polymerized in vitro into intact microtubules. The polymerization was reversible and spontaneous, i.e., no addition of heterologous nucleation centers was necessary. Two cycles of polymerization/depolymerization yielded tubulin preparations of 95% purity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron microscopy was used to show that the microtubules assembled in vitro by two cycles of polymerization/depolymerization were morphologically intact and temperature sensitive. In contrast, tubuin from neuroblastoma cells, clone Neuro-2A, could not be polymerized in a reversible fashion. The discovery of a cell line from which tubulin can be reversibly polymerized in vitro establishes a model system for studies of cell-cycle- and cell-type-dependent regulatory mechanisms controlling the assembly of microtubules.
The ordered assembly of tubulin into microtubules seems to play a significant role in several different cellular functions, including formation of the mitotic spindle, maintenance and generation of the cell shape, neuronal outgrowth, flagellar development, and possibly anchorage of cell surface receptors (1, 2). An understanding of these diverse cellular processes must include knowledge of the regulatory mechanisms controlling the assembly of microtubules and, in particular, the way in which those mechanisms differ among the various processes. While the cell-cycle-dependent regulation of microtubules in the mitotic apparatus is probably common to all cells, most of the other regulatory mechanisms would differ from one cell type to another. In considering experiments that might elucidate the mechanisms for control of microtubule assembly it seems advantageous to study mammalian tissue culture cells rather than to use tissues. Whole tissues cannot be used for synchronization of cell cycles and they are scarcely suitable for pulsed label experiments. Obviously the multiplicity of cell types in tissues would complicate immensely attempts to compare cell types in the ways in which they control microtubule assembly. In contrast, homogeneous cell lines maintained in culture would be well suited to all these kinds of experiments. In spite of these obvious advantages of cell culture systems for molecular biological experiments related to microtubules, most of the published reports on their in vitro assembly have concerned tubulin isolated from tissues. Recently, brain tissue was used to develop procedures (3-5) that allow the reversible in vitro formation of microtubules that resemble the structures formed in vwo in morphology and cold sensitivity (6). Furthermore, the in vitro formation process is similar to the in vivo one in being colchicine-sensitive (6). These same polymerization procedures have been applied to Abbreviation: EGTA, ethylene glycol bis(fl-aminoethyl ether)N,N'-tetraacetic acid. 1227
tissues of bovine anterior pituitary (7) and to pig platelets (8), although reversibility of the microtubule formation was not established in the latter case. Unfortunately, however, the procedures that were successful in the case of brain tubulin have appeared to be unsuccessful in cell culture systems, because attempts (9, 10) to obtain tubulin preparations capable of spontaneous microtubule formation from cell culture systems failed in all cases studied. Indeed, in the present work we encountered similar failures with neuroblastoma tubulin preparations. That such failures are not general for cell culture systems after all can be seen in the work presented here, since a spontaneous microtubule assembly system has been obtained from rat glial C6 cells. The establishment of a successful microtubule formation system from a homogeneous cell line in the experiments reported here has several attractive features. The availability of this system as a model not only provides the potential for studying cell-cycle-dependent control mechanisms, but it should make it possible also to study the role of microtubule assembly in the induction of distinct morphological changes such as the outgrowth of cell extensions induced by various treatments. Moreover, the differences among cell types in the competence of their tubulin preparations offers an entry into the study of cell-specific factors that regulate the assembly of microtubules. MATERIALS AND METHODS Cells. The cloned rat glial cell strain, C6 (11), was originally obtained from the American Type Culture Collection in the 37th passage and maintained in our laboratory for approximately another 30 passages. Cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (growth medium). Stock cultures were maintained in plastic T flasks and cells used for experiments were grown in roller bottles containing 150 ml of growth medium. Cells were collected from the bottles at the time they had reached confluence (approximately 4.7 X 108 cells per bottle) by removing the growth medium, rinsing with phosphate-buffered saline (10 mM sodium phosphate-150 mM NaCl, pH 7.0), and subsequently dislodging the cells from the glass surface with the aid of a rubber policeman. Cells were then pelleted by low-speed centrifugation. The pellet was resuspended, first in phosphate-buffered saline and then in assembly buffer (see below), and finally pelleted. The neuroblastoma clone Neuro-2A (12) was obtained from the American Type Culture Collection in the 172nd passage. Cells were grown in spinner flasks in Minimal Essential Medium (Joklik-Modified; no. F13, Grand Island Biological Co.) supplemented with 5% fetal calf serum, or in roller bottles, or in immobile glass bottles with the same
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Proc. Nat. Acad. Sci. USA 73 (1976)
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FIG. 1. Electrophoresis of fractions from glial C6 tubulin preparations. Tubulin was isolated from soluble cell extracts of glial C6 cells by two cycles of in vitro polymerization/depolymerization. Aliquots of pellet 1 (first cycle) and pellet 3 (second cycle) were run on sodium dodecyl sulfate-polyacrylamide slab gels. (A) Muscle actin (3 jig), (B) pellet 1 (25 jig), (C) mouse brain tubulin (3 jig), (D) pellet 3 (10 ,ug). Migration was from top (-) to bottom (+).
growth medium as used for the glial C6 cells. Morphological differentiation of Neuro-2A cells grown in immobile glass bottles was induced by keeping the cultures in growth medium devoid of serum for 72 hr prior to harvest. Neuro-2A cells were collected from roller and immobile bottles as described for the glial C6 cells. Cells grown in suspension were pelleted and resuspended several times in phosphate-buffered saline and finally in assembly buffer. Tubulin Preparations. Tubulin was prepared from cells essentially by the method of Shelanski et al. (5) with the single modification that the cells were disrupted by sonication [Biosonik II, (Bronwill-Scientific, Rochester, N.Y.); three 30 sec exposures] after having been suspended in the assembly buffer, 0.1 M sodium morpholinoethane sulfonate-1 mM GTP-1 mM ethylene glycol bis(,B-aminoethyl ether)-N,N'tetraacetic acid (EGTA)-0.5 mM MgCl2, at a concentration of 1 g of cells (wet weight) per ml. All centrifugations were performed at 100,000 X g for 1 hr at either 40 or 25°. Highly purified mouse brain tubulin was prepared by three cycles of in vitro polymerization/depolymerization. Purified muscle actin was a generous gift of J. Spudich, University of California at San Francisco Medical School. Protein was determined according to Lowry et al. (13). Gel Electrophoresis. Samples were dissolved by boiling in 10% glycerol (vol/vol)-5% mercaptoethanol-3% sodium dodecyl sulfate-62.5 mM Tris-HCl, pH 6.8, and applied to discontinuous 10% polyacrylamide slab gels prepared according to Ferro-Luzzi Ames (14). The discontinuous buffer system of Laemmli (15) was used in running the gels and the gels were stained with Coomassie brilliant blue and destained according to Fairbanks et al. (16). Tube gels were run and stained in a similar fashion.
HMW
FIG. 2. Electrophoresis of glial C6 tubulin: densitometry scan. Aliquots of pellet 3 from a tubulin preparation were dissolved and run on a cylindrical sodium dodecyl sulfate-polyacrylamide gel. The gel was stained with Coomassie blue and destained as described in Materials and Methods. Scanning was performed at 570 nm. HMW is a high-molecular-weight component.
RESULTS We attempted to isolate tubulin from cultured glial C6 cells by repeated cycles of in vitro polymerization/depolymerization. The experimental procedure we used was that described by Shelanski et al. (5), originally designed for the isolation of tubulin from mammalian brain. Soluble cell extracts prepared from cell homogenates by high-speed centrifugation were incubated in 4 M glycerol at 370 to induce the in vitro formation of microtubules. Microtubules and other aggregates formed were then pelleted (pellet 1). The pelleted material was rehomogenized and incubated in ice to allow the depolymerization of microtubules. The material that did not depolymerize was then centrifuged to yield a pellet (pellet 2) and disassembled tubulin in the supernatant was polymerized in vitro for a second time. Reformed microtubules were finally collected by high-speed centrifugation (pellet 3). The microtubules present in this pellet had undergone two cycles of polymerization/depolymerization. Microtubule formation was routinely monitored by electrophoretic analysis of the material in pellets 1 and 3 on sodium dodecyl sulfate polyacrylamide slab gels, using highly purified mouse brain tubulin as marker. The results of an analysis of pellets 1 and 3 obtained from a culture of glial C6 cells grown to confluence in roller bottles are shown in Fig. 1. It is apparent that tubulin constituted the main component of pellet 1. However, several other proteins were also contained in this pellet in various amounts. One of them, present in relatively high amounts, probably was actin, as was indicated by the coincidence of its electrophoretic mobility with that of pure muscle actin. Resuspension of the material constituting pellet 1 and induction of a second cycle of depolymerization/polymerization of microtubules dramatically improved the purity of the tubulin preparations. As is obvious from the analysis of pellet 3, shown in Fig. 1, most of the contaminants present in pellet 1 do not copurify with tubulin during the second cycle of
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FIG. 3. Cold sensitivity of glial C6 microtubules formed in vitro. Microtubules prepared by two cycles of polymerization/depolymerization (pellet 3) were resuspended at room temperature (2-4 mg of protein per ml) in 50 mM ammonium acetate, pH 6.4-1 mM EGTA-0.5 mM MgCl2-1 mM GTP. After incubation at the indicated temperatures, samples were deposited upon carbon-coated collodion specimen films by spraying them from a dual nebulizer (18) with 5% uranyl acetate. Left panel: incubation at 370, 5 min. Right panel: incubation at 0o-4o, 20 min. The white spheres are particles of tomato bushy stunt virus, added as size marker (diameter 31 nm).
depolymerization/polymerization. Tubulin represented approximately 95% of the material in pellet 3, as was determined from the densitometry gel scan shown in Fig. 2. This scan also revealed the presence of a high-molecular-weight component copurifying with tubulin through both cycles of polymerization/depolymerization. The separation of the two subunits of tubulin was obvious visually but some of the resolution is lost in the densitometric scanning so that Fig. 2 shows only a shoulder in the main tubulin peak. The amount of protein in pellet 3 varied according to the preparation from 0.4 to 0.7 mg of protein per g of packed cells (2.5-4% of the total protein of the soluble cell extract). To further authenticate the in vitro polymerized material as tubulin we submitted samples of pellet 3 to amino-acid analysis (Beckman analyzer) after hydrolysis in vacuo at 1200 in 6 M HCI for 7 hr. The analysis was in excellent agreement with published data (e.g., ref. 17). Final proof for the reversibility of the in vtnro polymerization/depolymerization of tubulin from cultured C6 cells was established by electron microscopy. The protein material constituting pellet 3 was resuspended at 370 in ammonium acetate buffer and negatively stained with uranyl acetate. As shown in the left panel of Fig. 3, a large number of intact microtubules with a typical diameter of 260 A could be observed. However, no microtubules could be observed in the electron microscope after keeping the resuspended pellet 3 material at 00-4' for 20 min prior to staining with uranyl acetate. A typical picture of such a sample is shown in the right panel of Fig. 3.
In addition to the C6 cell line maintained in our laboratory we tested four other Cr, cell lines, obtained from different laboratories. Tubulin could be isolated by reversible in vitro polymerization from all of them with the exception of a C6 line which had been cloned in the laboratory of Dr. J. deVellis at the University of California, Los Angeles. The relative amount of presumed actin copurifying with tubulin through two cycles of polymerization was slightly higher, however, in these cases than with the cell clone maintained in our own laboratory. Attempts to isolate tubulin by repeated cycles of in vitro polymerization/depolymerization from the neuroblastoma cell clone Neuro-2A were unsuccessful. Fig. 4 shows the analysis of pellets 1, 2, and 3 obtained from a culture of Neuro-2A cells grown in suspension. Pellet 1 contains three major components; the one present in the highest relative amount comigrates with brain tubulin. However, pellet 3 did not contain tubulin in any appreciable amount. The tubulin of pellet 1 apparently did not disassemble, since pellet 2 displayed a protein composition very similar to that of pellet 1. Pellet 1 presumably contained nonordered tubulin aggregates rather than intact microtubules, since such microtubules ought to have depolymerized at low temperature. However, it is also possible that the conditions for reversible in vitro polymerization, even though having been satisfactory in the case of cultured glial C6 cells, were not satisfactory in the case of Neuro-2A cells, and therefore did not allow a second cycle of polymerization. The only protein which seemed to get purified fairly extensively by repeated in
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FIG. 4. Electrophoresis of fractions from neuroblastoma-2A tubulin preparations. Soluble cell extracts of Neuro-2A cells grown in suspensions were fractionated as described in Materials and Methods. Aliquots of fractions corresponding to microtubules isolated by one (pellet 1) or two cycles (pellet 3) of in vitro polymerization/depolymerization as well as an intermediate fraction (pellet 2) were redissolved and run on sodium dodecyl sulfate-polyacrylamide slab gels. (A) Mouse brain tubulin, (B) pellet 1, (C) pellet 2, (D) pellet 3. Migration was from top (-) to bottom (+). vitro polymerization/depolymerization pose
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Although the failure of the Neuro-2A system may be due changes in components regulating assembly, technical factors might have accounted for the failure. Some modifications of the conditions maintained for polymerizing tubulin in vitro and culturing Neuro-2A cells were investigated. These included: (i) homogenization of the cells in a glassTeflon homogenizer instead of disruption by sonication, (ii) inclusion of 1 mM EDTA in all buffers, (iii) sonication of the resuspended material of pellet 1, (iv) preincubation of the soluble cell extract with pure submaxillary nerve growth factor (0.08 mg/ml) and its inclusion in all buffers used, (v) growth of cells in roller bottles or immobile glass bottles instead of spinner flasks, (vi) induction of morphological differentiation of Neuro-2A cells prior to harvest by removal of serum from the growth medium. None of these modifications had any effect on the results shown in Fig. 4. to
DISCUSSION
Since the formation of microtubules seems to play an imporrole in rather diverse cell functions the postulation of different cell-cycle- and even cell-type-dependent regulatory mechanisms controlling microtubule assembly appears reasonable. In order to study such mechanisms a well-defined biological system is needed. Brain tissue, which has been used for the vast majority of studies concerning the isolation and characterization of tubulin, is not suitable for these studies, especially since it represents a heterogeneous mixture of several cell types. Cell culture, on the other hand, tant
seems to be the system of choice. Our finding, that microtubules can be polymerized in vitro from the cultured rat glial cell line C6 in a reversible manner, therefore seems to establish a model system that would allow one to study the mechanism of cell-cycle-dependent microtubule assembly. By comparison of different cell lines, cell-specific regulatory mechanisms might be found. Since all eukaryotic cells obviously contain microtubules in vivo one might expect to be able to isolate a competent microtubule assembly system as well from one cell type as any other. However, our results with neuroblastoma cells and those obtained by others with other cell lines (9, 10) show that this expectation is not realized. This does not mean that crude tubulin cannot be isolated from these cell lines in the aggregate that forms upon incubation of the cell extract at 370, but it does mean that a competent assembly system requires much more. Competence of the assembly system cannot be defined merely by the first step of aggregation of crude tubulin. A successful system must allow repeated disaggregation at low temperature, and repolymerization upon warming. Moreover, the assembly must be spontaneous, that is, it ought not to require the addition of fragments of microtubules from heterologous systems as nucleation centers. It is in the sense of this full definition of competence that the present work has established a competent microtubule assembly system from glial cells. Among other possibilities we think that reduced resistance of Neuro-2A tubulin or of other essential components such as tau factor (19) towards inactivation during the process of cell homogenization and fractionation could be responsible for the observed lack of reversible in vitro polymerization of tubulin from this cell line. This would then become a question of different stabilities of the tubulin subunit itself or more likely the stability of factors of the microtubule assembly machinery in the different cell lines. Another possibility is that the regulatory mechanisms controlling microtubule assembly are different in cultured neuroblastoma and in glial cells. Preliminary experiments in our laboratory using a number of different cell lines seem to indicate that the competence of an in vitro assembly system might be dependent on certain growth properties of the cells from which it was isolated. The discovery of a cultured cell line from which large quantities of intact tubulin can be isolated by a relatively simple in vitro polymerization/depolymerization procedure should be of advantage also for mere preparative purposes, e.g., it should be possible now to isolate in vivo labeled radioactive tubulin exhibiting very high specific activities. This task could hitherto only be achieved to a limited extent by the rather tedious method of injecting radioactive precursors into chicken embryo brain and subsequent isolation of the labeled tubulin. We thank Dr. Norman Wessells and Dr. Jean deVellis for their generous gifts of cell lines, and Dr. James Spudich for donating the pure actin. To Ms. Angela Longo we are grateful for a preparation of nerve growth factor. We are especially indebted to Mr. Larry Honig for the electron microscopy of our samples. This work was supported by Grant GB 38658 from the National Science Foundation and Contract NOI-CB-43866 from the National Cancer Institute. 1. Olmsted, J. B. & Borisy, G. G. (1973) Annu. Rev. Biochem. 42, 507-540. 2. Yahara, I. & Edelman, G. M. (1975) Proc. Nat. Acad. Sci. USA 72, 1579-1583.
Cell Biology: Wiche and Cole 3. Weisenberg, R. C. (1972) Science 177, 1104-1105. 4. Borisy, G. G., Olmsted, J. B. & Klugman, R. A. (1972) Proc. Nat. Acad. Sci. USA 69,2890-2894. 5. Shelanski, M. L., Gaskin, F. & Cantor, C. R. (1973) Proc. Nat. Acad. Sci. USA 70,765-768. 6. Borisy, G. G., Olmsted, J. B., Marcum, J. M. & Allen, C. (1974) Fed. Proc. 33, 167-174. 7. Sheterline, P. & Schofield, J. G. (1975) FEBS Lett. 56, 297302. 8. Castle, A. G. & Crawford, N. (1975) FEBS Lett. 51, 195-200. 9. Bryan, J. (1975) Am. Zool. 15,649-660. 10. Rebhun, L. I., Jemiolo, D., Ivy, N., Mellon, M. & Nath, J. (1975) Ann. N.Y. Acad. Sci. 253, 362-377. 11. Benda, P., Lightbody, J., Sato, G., Levine, L. & Sweet, W.
Proc. Nat. Acad. Sci. USA 73 (1976)
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(1968) Science 161,370-371. 12. Klebe, R. J. & Ruddle, F. H. (1969) J. Cell Biol. 43, 69A. 13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 14. Ferro-Luzzi Ames, G. (1974) J. Biol. Chem. 249,634-644. 15. Laemmli, U. K. (1970) Nature 227,680-85. 16. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Bibchemistry 10, 2606-2617. 17. Eipper, B. A. (1974) J. Biol. Chem. 249, 1407-1416. 18. Kirschner, M. W., Honig, L. S. & Williams, R. C. (1975) J. Mol. Biol., 99,263-276. 19. Weingarten, M. D., Lockwood, A. H., Hwo Shu-Ying & Kirschner, M. W. (1975) Proc. Nat. Acad. Scd. USA 72, 1858-1862.
Cell Biology
Reversible in vitro polymerization of tubulin from a cultured cell line (rat glial cell clone C6) (control of microtubule assembly/tissue culture)
GERHARD WICHE AND R. DAVID COLE Department of Biochemistry, University of California, Berkeley, Calif. 94720
Communicated by Bruce N. Ames, December 22,1975
Tubulin from cultures of the rat glial cell ABSTRACT clone Ce could be polymerized in vitro into intact microtubules. The polymerization was reversible and spontaneous, i.e., no addition of heterologous nucleation centers was necessary. Two cycles of polymerization/depolymerization yielded tubulin preparations of 95% purity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron microscopy was used to show that the microtubules assembled in vitro by two cycles of polymerization/depolymerization were morphologically intact and temperature sensitive. In contrast, tubuin from neuroblastoma cells, clone Neuro-2A, could not be polymerized in a reversible fashion. The discovery of a cell line from which tubulin can be reversibly polymerized in vitro establishes a model system for studies of cell-cycle- and cell-type-dependent regulatory mechanisms controlling the assembly of microtubules.
The ordered assembly of tubulin into microtubules seems to play a significant role in several different cellular functions, including formation of the mitotic spindle, maintenance and generation of the cell shape, neuronal outgrowth, flagellar development, and possibly anchorage of cell surface receptors (1, 2). An understanding of these diverse cellular processes must include knowledge of the regulatory mechanisms controlling the assembly of microtubules and, in particular, the way in which those mechanisms differ among the various processes. While the cell-cycle-dependent regulation of microtubules in the mitotic apparatus is probably common to all cells, most of the other regulatory mechanisms would differ from one cell type to another. In considering experiments that might elucidate the mechanisms for control of microtubule assembly it seems advantageous to study mammalian tissue culture cells rather than to use tissues. Whole tissues cannot be used for synchronization of cell cycles and they are scarcely suitable for pulsed label experiments. Obviously the multiplicity of cell types in tissues would complicate immensely attempts to compare cell types in the ways in which they control microtubule assembly. In contrast, homogeneous cell lines maintained in culture would be well suited to all these kinds of experiments. In spite of these obvious advantages of cell culture systems for molecular biological experiments related to microtubules, most of the published reports on their in vitro assembly have concerned tubulin isolated from tissues. Recently, brain tissue was used to develop procedures (3-5) that allow the reversible in vitro formation of microtubules that resemble the structures formed in vwo in morphology and cold sensitivity (6). Furthermore, the in vitro formation process is similar to the in vivo one in being colchicine-sensitive (6). These same polymerization procedures have been applied to Abbreviation: EGTA, ethylene glycol bis(fl-aminoethyl ether)N,N'-tetraacetic acid. 1227
tissues of bovine anterior pituitary (7) and to pig platelets (8), although reversibility of the microtubule formation was not established in the latter case. Unfortunately, however, the procedures that were successful in the case of brain tubulin have appeared to be unsuccessful in cell culture systems, because attempts (9, 10) to obtain tubulin preparations capable of spontaneous microtubule formation from cell culture systems failed in all cases studied. Indeed, in the present work we encountered similar failures with neuroblastoma tubulin preparations. That such failures are not general for cell culture systems after all can be seen in the work presented here, since a spontaneous microtubule assembly system has been obtained from rat glial C6 cells. The establishment of a successful microtubule formation system from a homogeneous cell line in the experiments reported here has several attractive features. The availability of this system as a model not only provides the potential for studying cell-cycle-dependent control mechanisms, but it should make it possible also to study the role of microtubule assembly in the induction of distinct morphological changes such as the outgrowth of cell extensions induced by various treatments. Moreover, the differences among cell types in the competence of their tubulin preparations offers an entry into the study of cell-specific factors that regulate the assembly of microtubules. MATERIALS AND METHODS Cells. The cloned rat glial cell strain, C6 (11), was originally obtained from the American Type Culture Collection in the 37th passage and maintained in our laboratory for approximately another 30 passages. Cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (growth medium). Stock cultures were maintained in plastic T flasks and cells used for experiments were grown in roller bottles containing 150 ml of growth medium. Cells were collected from the bottles at the time they had reached confluence (approximately 4.7 X 108 cells per bottle) by removing the growth medium, rinsing with phosphate-buffered saline (10 mM sodium phosphate-150 mM NaCl, pH 7.0), and subsequently dislodging the cells from the glass surface with the aid of a rubber policeman. Cells were then pelleted by low-speed centrifugation. The pellet was resuspended, first in phosphate-buffered saline and then in assembly buffer (see below), and finally pelleted. The neuroblastoma clone Neuro-2A (12) was obtained from the American Type Culture Collection in the 172nd passage. Cells were grown in spinner flasks in Minimal Essential Medium (Joklik-Modified; no. F13, Grand Island Biological Co.) supplemented with 5% fetal calf serum, or in roller bottles, or in immobile glass bottles with the same
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Proc. Nat. Acad. Sci. USA 73 (1976)
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FIG. 1. Electrophoresis of fractions from glial C6 tubulin preparations. Tubulin was isolated from soluble cell extracts of glial C6 cells by two cycles of in vitro polymerization/depolymerization. Aliquots of pellet 1 (first cycle) and pellet 3 (second cycle) were run on sodium dodecyl sulfate-polyacrylamide slab gels. (A) Muscle actin (3 jig), (B) pellet 1 (25 jig), (C) mouse brain tubulin (3 jig), (D) pellet 3 (10 ,ug). Migration was from top (-) to bottom (+).
growth medium as used for the glial C6 cells. Morphological differentiation of Neuro-2A cells grown in immobile glass bottles was induced by keeping the cultures in growth medium devoid of serum for 72 hr prior to harvest. Neuro-2A cells were collected from roller and immobile bottles as described for the glial C6 cells. Cells grown in suspension were pelleted and resuspended several times in phosphate-buffered saline and finally in assembly buffer. Tubulin Preparations. Tubulin was prepared from cells essentially by the method of Shelanski et al. (5) with the single modification that the cells were disrupted by sonication [Biosonik II, (Bronwill-Scientific, Rochester, N.Y.); three 30 sec exposures] after having been suspended in the assembly buffer, 0.1 M sodium morpholinoethane sulfonate-1 mM GTP-1 mM ethylene glycol bis(,B-aminoethyl ether)-N,N'tetraacetic acid (EGTA)-0.5 mM MgCl2, at a concentration of 1 g of cells (wet weight) per ml. All centrifugations were performed at 100,000 X g for 1 hr at either 40 or 25°. Highly purified mouse brain tubulin was prepared by three cycles of in vitro polymerization/depolymerization. Purified muscle actin was a generous gift of J. Spudich, University of California at San Francisco Medical School. Protein was determined according to Lowry et al. (13). Gel Electrophoresis. Samples were dissolved by boiling in 10% glycerol (vol/vol)-5% mercaptoethanol-3% sodium dodecyl sulfate-62.5 mM Tris-HCl, pH 6.8, and applied to discontinuous 10% polyacrylamide slab gels prepared according to Ferro-Luzzi Ames (14). The discontinuous buffer system of Laemmli (15) was used in running the gels and the gels were stained with Coomassie brilliant blue and destained according to Fairbanks et al. (16). Tube gels were run and stained in a similar fashion.
HMW
FIG. 2. Electrophoresis of glial C6 tubulin: densitometry scan. Aliquots of pellet 3 from a tubulin preparation were dissolved and run on a cylindrical sodium dodecyl sulfate-polyacrylamide gel. The gel was stained with Coomassie blue and destained as described in Materials and Methods. Scanning was performed at 570 nm. HMW is a high-molecular-weight component.
RESULTS We attempted to isolate tubulin from cultured glial C6 cells by repeated cycles of in vitro polymerization/depolymerization. The experimental procedure we used was that described by Shelanski et al. (5), originally designed for the isolation of tubulin from mammalian brain. Soluble cell extracts prepared from cell homogenates by high-speed centrifugation were incubated in 4 M glycerol at 370 to induce the in vitro formation of microtubules. Microtubules and other aggregates formed were then pelleted (pellet 1). The pelleted material was rehomogenized and incubated in ice to allow the depolymerization of microtubules. The material that did not depolymerize was then centrifuged to yield a pellet (pellet 2) and disassembled tubulin in the supernatant was polymerized in vitro for a second time. Reformed microtubules were finally collected by high-speed centrifugation (pellet 3). The microtubules present in this pellet had undergone two cycles of polymerization/depolymerization. Microtubule formation was routinely monitored by electrophoretic analysis of the material in pellets 1 and 3 on sodium dodecyl sulfate polyacrylamide slab gels, using highly purified mouse brain tubulin as marker. The results of an analysis of pellets 1 and 3 obtained from a culture of glial C6 cells grown to confluence in roller bottles are shown in Fig. 1. It is apparent that tubulin constituted the main component of pellet 1. However, several other proteins were also contained in this pellet in various amounts. One of them, present in relatively high amounts, probably was actin, as was indicated by the coincidence of its electrophoretic mobility with that of pure muscle actin. Resuspension of the material constituting pellet 1 and induction of a second cycle of depolymerization/polymerization of microtubules dramatically improved the purity of the tubulin preparations. As is obvious from the analysis of pellet 3, shown in Fig. 1, most of the contaminants present in pellet 1 do not copurify with tubulin during the second cycle of
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FIG. 3. Cold sensitivity of glial C6 microtubules formed in vitro. Microtubules prepared by two cycles of polymerization/depolymerization (pellet 3) were resuspended at room temperature (2-4 mg of protein per ml) in 50 mM ammonium acetate, pH 6.4-1 mM EGTA-0.5 mM MgCl2-1 mM GTP. After incubation at the indicated temperatures, samples were deposited upon carbon-coated collodion specimen films by spraying them from a dual nebulizer (18) with 5% uranyl acetate. Left panel: incubation at 370, 5 min. Right panel: incubation at 0o-4o, 20 min. The white spheres are particles of tomato bushy stunt virus, added as size marker (diameter 31 nm).
depolymerization/polymerization. Tubulin represented approximately 95% of the material in pellet 3, as was determined from the densitometry gel scan shown in Fig. 2. This scan also revealed the presence of a high-molecular-weight component copurifying with tubulin through both cycles of polymerization/depolymerization. The separation of the two subunits of tubulin was obvious visually but some of the resolution is lost in the densitometric scanning so that Fig. 2 shows only a shoulder in the main tubulin peak. The amount of protein in pellet 3 varied according to the preparation from 0.4 to 0.7 mg of protein per g of packed cells (2.5-4% of the total protein of the soluble cell extract). To further authenticate the in vitro polymerized material as tubulin we submitted samples of pellet 3 to amino-acid analysis (Beckman analyzer) after hydrolysis in vacuo at 1200 in 6 M HCI for 7 hr. The analysis was in excellent agreement with published data (e.g., ref. 17). Final proof for the reversibility of the in vtnro polymerization/depolymerization of tubulin from cultured C6 cells was established by electron microscopy. The protein material constituting pellet 3 was resuspended at 370 in ammonium acetate buffer and negatively stained with uranyl acetate. As shown in the left panel of Fig. 3, a large number of intact microtubules with a typical diameter of 260 A could be observed. However, no microtubules could be observed in the electron microscope after keeping the resuspended pellet 3 material at 00-4' for 20 min prior to staining with uranyl acetate. A typical picture of such a sample is shown in the right panel of Fig. 3.
In addition to the C6 cell line maintained in our laboratory we tested four other Cr, cell lines, obtained from different laboratories. Tubulin could be isolated by reversible in vitro polymerization from all of them with the exception of a C6 line which had been cloned in the laboratory of Dr. J. deVellis at the University of California, Los Angeles. The relative amount of presumed actin copurifying with tubulin through two cycles of polymerization was slightly higher, however, in these cases than with the cell clone maintained in our own laboratory. Attempts to isolate tubulin by repeated cycles of in vitro polymerization/depolymerization from the neuroblastoma cell clone Neuro-2A were unsuccessful. Fig. 4 shows the analysis of pellets 1, 2, and 3 obtained from a culture of Neuro-2A cells grown in suspension. Pellet 1 contains three major components; the one present in the highest relative amount comigrates with brain tubulin. However, pellet 3 did not contain tubulin in any appreciable amount. The tubulin of pellet 1 apparently did not disassemble, since pellet 2 displayed a protein composition very similar to that of pellet 1. Pellet 1 presumably contained nonordered tubulin aggregates rather than intact microtubules, since such microtubules ought to have depolymerized at low temperature. However, it is also possible that the conditions for reversible in vitro polymerization, even though having been satisfactory in the case of cultured glial C6 cells, were not satisfactory in the case of Neuro-2A cells, and therefore did not allow a second cycle of polymerization. The only protein which seemed to get purified fairly extensively by repeated in
1230
Cell Biology: Wiche and Cole
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Proc. Nat. Acad. Sci. USA 73 (1976)
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FIG. 4. Electrophoresis of fractions from neuroblastoma-2A tubulin preparations. Soluble cell extracts of Neuro-2A cells grown in suspensions were fractionated as described in Materials and Methods. Aliquots of fractions corresponding to microtubules isolated by one (pellet 1) or two cycles (pellet 3) of in vitro polymerization/depolymerization as well as an intermediate fraction (pellet 2) were redissolved and run on sodium dodecyl sulfate-polyacrylamide slab gels. (A) Mouse brain tubulin, (B) pellet 1, (C) pellet 2, (D) pellet 3. Migration was from top (-) to bottom (+). vitro polymerization/depolymerization pose
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Although the failure of the Neuro-2A system may be due changes in components regulating assembly, technical factors might have accounted for the failure. Some modifications of the conditions maintained for polymerizing tubulin in vitro and culturing Neuro-2A cells were investigated. These included: (i) homogenization of the cells in a glassTeflon homogenizer instead of disruption by sonication, (ii) inclusion of 1 mM EDTA in all buffers, (iii) sonication of the resuspended material of pellet 1, (iv) preincubation of the soluble cell extract with pure submaxillary nerve growth factor (0.08 mg/ml) and its inclusion in all buffers used, (v) growth of cells in roller bottles or immobile glass bottles instead of spinner flasks, (vi) induction of morphological differentiation of Neuro-2A cells prior to harvest by removal of serum from the growth medium. None of these modifications had any effect on the results shown in Fig. 4. to
DISCUSSION
Since the formation of microtubules seems to play an imporrole in rather diverse cell functions the postulation of different cell-cycle- and even cell-type-dependent regulatory mechanisms controlling microtubule assembly appears reasonable. In order to study such mechanisms a well-defined biological system is needed. Brain tissue, which has been used for the vast majority of studies concerning the isolation and characterization of tubulin, is not suitable for these studies, especially since it represents a heterogeneous mixture of several cell types. Cell culture, on the other hand, tant
seems to be the system of choice. Our finding, that microtubules can be polymerized in vitro from the cultured rat glial cell line C6 in a reversible manner, therefore seems to establish a model system that would allow one to study the mechanism of cell-cycle-dependent microtubule assembly. By comparison of different cell lines, cell-specific regulatory mechanisms might be found. Since all eukaryotic cells obviously contain microtubules in vivo one might expect to be able to isolate a competent microtubule assembly system as well from one cell type as any other. However, our results with neuroblastoma cells and those obtained by others with other cell lines (9, 10) show that this expectation is not realized. This does not mean that crude tubulin cannot be isolated from these cell lines in the aggregate that forms upon incubation of the cell extract at 370, but it does mean that a competent assembly system requires much more. Competence of the assembly system cannot be defined merely by the first step of aggregation of crude tubulin. A successful system must allow repeated disaggregation at low temperature, and repolymerization upon warming. Moreover, the assembly must be spontaneous, that is, it ought not to require the addition of fragments of microtubules from heterologous systems as nucleation centers. It is in the sense of this full definition of competence that the present work has established a competent microtubule assembly system from glial cells. Among other possibilities we think that reduced resistance of Neuro-2A tubulin or of other essential components such as tau factor (19) towards inactivation during the process of cell homogenization and fractionation could be responsible for the observed lack of reversible in vitro polymerization of tubulin from this cell line. This would then become a question of different stabilities of the tubulin subunit itself or more likely the stability of factors of the microtubule assembly machinery in the different cell lines. Another possibility is that the regulatory mechanisms controlling microtubule assembly are different in cultured neuroblastoma and in glial cells. Preliminary experiments in our laboratory using a number of different cell lines seem to indicate that the competence of an in vitro assembly system might be dependent on certain growth properties of the cells from which it was isolated. The discovery of a cultured cell line from which large quantities of intact tubulin can be isolated by a relatively simple in vitro polymerization/depolymerization procedure should be of advantage also for mere preparative purposes, e.g., it should be possible now to isolate in vivo labeled radioactive tubulin exhibiting very high specific activities. This task could hitherto only be achieved to a limited extent by the rather tedious method of injecting radioactive precursors into chicken embryo brain and subsequent isolation of the labeled tubulin. We thank Dr. Norman Wessells and Dr. Jean deVellis for their generous gifts of cell lines, and Dr. James Spudich for donating the pure actin. To Ms. Angela Longo we are grateful for a preparation of nerve growth factor. We are especially indebted to Mr. Larry Honig for the electron microscopy of our samples. This work was supported by Grant GB 38658 from the National Science Foundation and Contract NOI-CB-43866 from the National Cancer Institute. 1. Olmsted, J. B. & Borisy, G. G. (1973) Annu. Rev. Biochem. 42, 507-540. 2. Yahara, I. & Edelman, G. M. (1975) Proc. Nat. Acad. Sci. USA 72, 1579-1583.
Cell Biology: Wiche and Cole 3. Weisenberg, R. C. (1972) Science 177, 1104-1105. 4. Borisy, G. G., Olmsted, J. B. & Klugman, R. A. (1972) Proc. Nat. Acad. Sci. USA 69,2890-2894. 5. Shelanski, M. L., Gaskin, F. & Cantor, C. R. (1973) Proc. Nat. Acad. Sci. USA 70,765-768. 6. Borisy, G. G., Olmsted, J. B., Marcum, J. M. & Allen, C. (1974) Fed. Proc. 33, 167-174. 7. Sheterline, P. & Schofield, J. G. (1975) FEBS Lett. 56, 297302. 8. Castle, A. G. & Crawford, N. (1975) FEBS Lett. 51, 195-200. 9. Bryan, J. (1975) Am. Zool. 15,649-660. 10. Rebhun, L. I., Jemiolo, D., Ivy, N., Mellon, M. & Nath, J. (1975) Ann. N.Y. Acad. Sci. 253, 362-377. 11. Benda, P., Lightbody, J., Sato, G., Levine, L. & Sweet, W.
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(1968) Science 161,370-371. 12. Klebe, R. J. & Ruddle, F. H. (1969) J. Cell Biol. 43, 69A. 13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 14. Ferro-Luzzi Ames, G. (1974) J. Biol. Chem. 249,634-644. 15. Laemmli, U. K. (1970) Nature 227,680-85. 16. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Bibchemistry 10, 2606-2617. 17. Eipper, B. A. (1974) J. Biol. Chem. 249, 1407-1416. 18. Kirschner, M. W., Honig, L. S. & Williams, R. C. (1975) J. Mol. Biol., 99,263-276. 19. Weingarten, M. D., Lockwood, A. H., Hwo Shu-Ying & Kirschner, M. W. (1975) Proc. Nat. Acad. Scd. USA 72, 1858-1862.
