Proc. Nail. Acad. Sci. USA Vol. 91, pp. 11358-11362, November 1994 Cell Biology Microtubule dynamics in vitro are regulated by the tubulin isotype composition DULAL PANDA*, HERBERT P. MILLER*, ASOK BANERJEEt, RICHARD F. LUDUENAt, AND LESLIE WILSON*t *Department of Biological Sciences, University of California, Santa Barbara, CA 93106; and tDepartment of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78284 Communicated by John A. Carbon, July 22, 1994 (receivedfor review June 6, 1994)
ABSTRACT The growing and shortening dynamics of called isotypes. For example, mammalian brain tubulin con- individual bovine brain microtubules at their plus ends at sists of at least five a- and five j-tubulin isotypes (17, 18). steady state in vitro, assembled from isotypically pure a4u, However, despite considerable effort, the functional signif- abim, or acsv tubulin dimers, were determined by differential icance of the multiple isotypes in cells remains obscure (for interference contrast video microscopy. Microtubules assem- review, see ref. 19). Recent evidence indicates that some bled from the purified afm isotype were considerably more tubulin isotypes may have distinct functions. For example, dynamic than microtubules made from the apu or a4iv when Hoyle and Raff (20) expressed the Drosophila (3 isotypes or from unfractionated phosphocellulose-purified tu- isotype in Drosophila male germ cells in place of the testis- bulin. Furthermore, increasing the proportion of the aoil specific (32 isotype, axoneme formation and meiotic spindle isotype in a mixture of the alI and atom isotypes suppressed formation did not occur, while other microtubule-dependent microtubule dynamics, demonstrating that microtubule dy- processes occurred normally. Similarly, Falconer et al. (21) namics can be influenced by the tubulin isotype composition. found that the a(ii isotype was incorporated preferentially The data support the hypothesis that cells might determine the into a colchicine-stable subset of microtubules, whereas the dynamic properties and functions ofits microtubules in part by a(3IuI isotype was preferentially incorporated into a colchi- altering the relative amounts of the different tubulin isotypes. cine-labile microtubule subset in cultured neuronal cells. These results indicated that the two isotypes could segregate Microtubules (1) are required for structural organization and into different microtubule populations. for many kinds of movements within the eukaryotic cell Recently, Banarjee et al. (22-25) purified the major (3-tu- cytoplasm. They are especially prominent in the central bulin isotypes from bovine brain by immunoaffinity chroma- nervous system where they appear to be essential for the tography with specific antibodies to the isotypes. By analyz- organization and function of axonal and dendritic processes ing the polymerization properties of the different isotypes in of neurons. Although it is unclear how microtubule functions suspension in vitro, Banerjee et al. (22-25) and Lu and in cells are controlled, a large body of evidence indicates that Luduefia (26) found that the polymerization properties ofthe the dynamic properties ofthe microtubules play an important isotypes differed from one another and that the pattern of the role (for reviews, see refs. 2 and 3). For example, microtubule differences varied with the polymerization conditions. No- dynamics increase dramatically when cells progress from tably, the polymerization properties of the a(3m isotype were interphase to mitosis (4-6), and the rapid dynamics ofspindle significantly different from those of the a(3u and a(3Iv iso- microtubules appear to be important for establishing spindle types. microtubule organization, for facilitating the linkage of chro- While the general polymerization properties of the pure mosomes to the spindles, and for chromosome movement (3-tubulin isotypes have been analyzed in suspension in vitro, (2-7). the studies thus far have not provided insight about the Little is known about how microtubule dynamics are growing and shortening events that occur at the ends of the regulated in cells. In vitro and in cells, microtubule ends individual microtubules-events closely linked to the func- switch between states of growing and shortening, a process tions of individual microtubules within cells. Thus, in the known as "dynamic instability" (8-13), apparently due to the present study we used video microscopy to analyze the gain and loss ofa stabilizing GTP- or GDP-P1-liganded tubulin dynamic instability parameters of individual microtubules cap at the microtubule ends. Also, in vitro and in cells, net made from different (3-tubulin isotypes. Our results indicate growing of microtubules can occur at one microtubule end that at steady state, microtubules made from the brain- and net shortening can occur at the opposite end, a process specific a(uIl isotype were considerably more dynamic than termed "treadmilling" or "flux" (9, 14-16). Both dynamic microtubules made from unfractionated tubulin or from the instability and treadmilling are logical targets for control. purified a(3II or a(3Iv isotypes. In addition, inclusion of the Biochemical studies in vitro have indicated that microtubule a(3u isotype along with the a,8111 isotype modulated the dynamics in cells could be regulated at several levels. One dynamics of the microtubules. The results support the hy- possibility is that control of microtubule dynamics involves potheses that the (3-tubulin isotype composition may modu- regulation ofthe gain and loss ofthe stabilizing cap. A second late microtubule polymerization dynamics in cells and that possibility is that control of microtubule dynamics occurs the a(i3I isotype might perform a unique function in neurons. through interactions of microtubule-associated proteins (MAPs) with microtubule surfaces and ends. Another possible mechanism for control of microtubule MATERIALS AND METHODS polymerization dynamics could involve the isotypic compo- Purification of Isotopicafly Purified Tubulin Dimers and sition ofthe tubulin itself. Tubulin is composed oftwo 50-kDa Axonemes. Purified a3Iu, a(3m, and a(3Iv isotypes were pre- polypeptide subunits, a and (3, which exist in multiple forms pared by immunoaffinity chromatography as described (24,
The publication costs of this article were defrayed in part by page charge Abbreviations: MAP, microtubule-associated protein; PC-tubulin, payment. This article must therefore be hereby marked "advertisement" phosphocellulose-purified tubulin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. $To whom reprint requests should be addressed. 11358 Downloaded by guest on September 27, 2021 Cell Biology: Panda et al. Proc. Natl. Acad. Sci. USA 91 (1994) 11359 26). The purified isotypes were not detectably contaminated 12 with other f3-tubulin isotypes as determined by SDS/ A polyacrylamide gel electrophoresis and immunoblot analysis (24, 27). [Like 8 tubulin, a tubulin consists of various 8 isotypes. However, the a-tubulin isotype composition of the isotypically pure 3-tubulin dimers used in this work is not AAAA . AA ..A'.A..' . known. Subjection of the a subunits to isoelectric focusing 4 indicates that there are relatively minor differences in the isotypic compositions ofthe a isotypes bound to the different ,8 isotypes (24, 28)]. /3-tubulin isotypes were stored frozen in 0 0.1 M Pipes/1 mM EGTA/1 mM Mg2+ (PEM) containing 4 M 12 glycerol and 1 mM GTP at -700C. Immediately prior to use, the isotypes were transferred to PEM (no glycerol) by E passage through a Bio-Gel P-2 (Bio-Rad) column previously equilibrated with PEM plus 0.1 mM GTP. Protein concen- 0) CD trations were determined by the method of Bradford (29) B 4 [I| A A -i ~~~~~~~~~~~~~~w using bovine serum albumin as the standard. Video Microscopy. Differential interference contrast video microscopy was carried out as described (12). Solutions of 0 individual &3-tubulin isotypes (1.6 mg of protein per ml) were 12 incubated with sea urchin flagellar axonemal "seeds" at 30°C in PEM containing 1 mM GTP. The seed concentration was .a- optimized at three to six seeds per field in all ix.
0 0 0i 0Il 4 Transition frequencies, 0 200 400 -400 200 0 200 400 -400 200 events per min S -GorA 3.1 3.2 3.7 Rate. dimers per s GorA-- S 1.6 1.2 0.9 A-* GorS 2.5 2.1 1.5 of FIG. 2. Frequency distribution histograms growing and short- GorS -> A 1.1 1.8 2.3 ening rates at microtubule plus ends made from unfractionated PC-tubulin (A) or purified apli (B), a,(3ii (C), or a4iv (D) tubulin. Mixtures of a(31 and aftI tubulin isotypes at the indicated ratios Positive rates indicate growing and negative rates indicate shorten- were polymerized to steady state at the ends ofaxonemal seeds. The ing; rates between +23 and -23 dimer per s were below the total protein concentration in all experiments was 1.6 mg/ml. Values resolution of the microscope and are reported as zero. are the mean + SEM. Downloaded by guest on September 27, 2021 Cell Biology: Panda et al. Proc. Natl. Acad. Sci. USA 91 (1994) 11361 80 100 from three purified 8-tubulin isotypes. The purified a(,, a8iii, and a,83v isotypes used in this work account, respec- tively, for 58%, 25%, and 13% ofthe total f3 tubulin in bovine V) ID 60 80 0m brain tubulin preparations (22). We found that pure apj3 -o microtubules were substantially more dynamic than micro- En o tubules made from the a,,ii or afiv isotypes or from unfrac- 60 tionated PC-tubulin. In addition, including the a,3iI isotype at0 40 along with ap311 during polymerization modulated the dy- 40 c namics of the microtubules. >,200 .0 Methodological Considerations. The dynamic instability Q behaviors reported in the present study for microtubules 2) made from the isotypically purified 3-tubulin dimers were co 20 similar qualitatively to the behaviors observed for microtu- bules made from unfractionated PC-tubulin (e.g., refs. 10- 12). However, the magnitudes of the parameters obtained in 100 80 50 20 the present work were smaller than those reported in other % apIll studies and, in general, the magnitudes of the parameters FIG. 3. Effect of different percentages of the aP,, and a,38II have varied substantially from one study to another. For isotypes on the percentage of total time the microtubules spent in the example, Walker et al. (10) observed microtubules in vitro to attenuated state (cross-hatched bars) and dynamicity (solid bars). be in a pause (attenuated) state very infrequently, whereas in Experiments were performed by mixing the al3ii and a8III isotypes the present work microtubules made from PC-tubulin spent at different ratios keeping total protein concentration constant at 1.6 -42% of the total time in the attenuated slate. Such quanti- mg/ml. The percentage of time in the attenuated state is a measure tative differences in polymerization dynamics are most likely of the time the microtubules spent neither growing nor shortening due to the use of different experimental systems, to the detectably. Dynamicity is the mean tubulin dimer exchange rate at different conditions used, and to the use of different methods microtubule ends calculated from all detectable growing and short- ening events including time spent in the attenuated state. Error bars of analysis. Important in this work is that microtubule are the SEM. dynamics were analyzed at steady state; most dynamic instability measurements have not been made at steady state ratios while keeping the total protein concentration constant (9-11). At steady state, microtubules appear to exhibit (1.6 mg/ml). smaller length excursions than microtubules undergoing net The relatively rapid growing and shortening dynamics of growing or shortening (16, 30). In addition, the different microtubules typical of ai3lii microtubules were not appre- buffer conditions and temperatures used in the various stud- ciably affected when the a/38i isotype was included along with ies must contribute to the quantitative differences reported. the amI1 isotype during polymerization at a ratio of 20% apil For example, our studies were carried out at 30TC rather than and 80% aplin. For example, the growing and shortening rates at 37TC, which results in reduced dynamics (D.P. and L.W., (Tables 1 and 2), the percent time the microtubules spent in unpublished data). the attenuated state and the overall dynamicity of the micro- Importantly, values for the growing and shortening rates or tubules (Fig. 3) were not significantly different for the mi- for the fraction of total time in a particular phase are highly crotubules made with pure aiii or 20% apii and 80% aliiI. dependent upon the method of analysis. Because we com- However, when the ratio of a,1ii to ai3iii was increased to 50% pared the three /3-tubulin isotypes to each other and to a4II and 50% afki1, the dynamics parameters of the resulting PC-tubulin using identical conditions and the same method of microtubules became very similar to those of isotypically analysis, the quantitative differences we observed are valid pure a,81i microtubules. For example, the mean growing rates indicators of differences in dynamic behaviors. were 49 dimers per s with microtubules made from 100% al1ii, Microtubules Made from Isotypically Pure agm Tubulin 50 dimers per s with microtubules made from a 50:50 mixture Dimers Have Unique Polymerization Dynamics. Microtubules of the al311 and aj3III isotypes, and 77 dimers per s with made from the purified a(3iii isotype were significantly more microtubules made from 100% a(3li, (Tables 1 and 2). Simi- dynamic at their plus ends than microtubules made from the larly, both the percentage of time that the microtubules spent al3ii or aBiv isotypes or PC-tubulin (Fig. 1 and Table 1). For in the attenuated state and the overall dynamicity (Fig. 3) example, the growing and shortening rates and the overall were not significantly different for the microtubules made dynamicity of the a,3iti microtubules were more than double from the 50:50 mixture of aPi1 and a,3iii and microtubules those of microtubules made from the a,1ii dimers, aPiv made from the pure also isotype. dimers, or PC-tubulin (Table 1). Interestingly, when the ratio of a,8ii to a,3iii was increased Previous evidence has also indicated that the aiii isotype to 80%o aji3 and 20% aI,3m, the dynamics of the resulting has unique polymerization properties (23, 24, 26). For ex- microtubules became distinctly slower than the dynamics of ample, in the presence of 4 M glycerol and 6 mM MgCl2, the microtubules made from the pure a8jii isotype. For exam- assembly of the purified a3iii isotype occurred after a pro- ple, microtubules made from 100% a/3II spent 47% of the total nounced delay in nucleation while nucleation with the other time in the attenuated state, whereas microtubules made from 1-tubulin isotypes occurred quickly (26). Once nucleated, a mixture of 80%o a(3i1 and 20% a(3i11 spent 62% ofthe total time polymerization ofthe aX3iii isotype proceeded at a slower rate in the attenuated state (Fig. 3). Similarly, the dynamicity ofthe and occurred to a lesser extent than did polymerization ofthe microtubules made from 100% al3ii was 28 dimers per s, aj8II or ativ isotypes. Different rates of polymerization but a whereas the dynamicity of the microtubules made from a similar conclusion regarding the unique properties of aPiii mixture of 80% aPii and 20% aPiii was reduced to 18 dimers microtubules were obtained by Banerjee et al. (24) who per s. Thus, the dynamics of the microtubules polymerized analyzed polymerization of the P-tubulin isotypes in the from mixtures of the two isotypes do not reflect the simple presence of MAPs. averages of the isotypes that are present in solution. Our data support the hypothesis that the ai,8i1 isotype may play a unique role in neurons. Only the neurons in brain and DISCUSSION in dorsal root ganglia and Sertoli cells have been shown to We have examined the dynamic instability parameters at express the alIII isotype (see ref. 19). The amino acid steady state at the plus ends of individual microtubules made sequences ofthe f3-tubulin isotypes are substantially different Downloaded by guest on September 27, 2021 11362 Cell Biology: Panda et al. Proc. NatL Acad. Sci. USA 91 (1994) in their C termini (17-19) and the C terminus appears to play 2. McIntosh, R. (1994) in Microtubules, eds, Lloyd, C. & Hyams, an important role in microtubule assembly (31-33). For J. (Wiley, New York), pp. 413-434. example, the ability of MAPs to stabilize microtubule dy- 3. Wordeman, L. & Mitchison, T. J. (1994) in Microtubules, eds. namics appears to occur by MAP binding to the C terminus Lloyd, C. & Hyams, J. (Wiley, New York), pp. 287-301. of tubulin 4. Saxton, W. M. Stemple, D. L., Leslie, R. J., Salmon, E. D., (3, 31, 32). All the post-translational modifications Zavortink, M. & McIntosh, J. R. (1984) J. Cell Biol. 99, known to occur on (3 tubulin also occur in the C-terminal 2175-2186. region, and the api3 isotype is uniquely phosphorylated at its 5. Belmont, L. D., Hyman, A. A., Sawin, K. E. & Mitchison, C terminus (34). Perhaps the C-terminal domain of the apm T. J. (1990) Cell 62, 579-589. isotype interacts more strongly than other /3-tubulin isotypes 6. Hush, J. M., Wadsworth, P., Callaham, D. A. & Hepler, P. K. with a neuron-specific MAP, thereby selectively stabilizing (1994) J. Cell Sci. 107, 775-784. 7. Jordan, M. A., Thrower, D. & Wilson, L. (1992) J. Cell Sci. neuronal microtubules when the MAP is present and making 102, 401-416. the microtubules especially dynamic when the MAP is either 8. Michison, T. & Kirschner, M. W. (1984) Nature (London) 312, absent or modified. 232-237. The Tubulin Isotpe Composition Can Specify the Polymer- 9. Hotani, H. & Horio, T. (1988) Cell Motil. Cytoskeleton 10, ization Dynamics of a Microtubule. Mixing the a/gX and ahm 229-236. isotypes resulted in the formation of microtubules with 10. Walker, R. A., O'Brien, E. T., Pryer, N. K., Sobdeiro, M., significantly different assembly dynamics depending upon Voter W. A., Erickson, H. P. & Salmon, E. D. (1988) J. Cell the ratio of the Biol. 107, 1437-1448. two isotypes present during polymerization. 11. Gildersleeve, R. F., Cross, A. R., Cullen, R. E., Fagen, A. P. Despite the fact that microtubules made from the pure ahm & Williams, R. C. (1992) J. Biol. Chem. 267, 7995-8006. isotype were significantly more dynamic than microtubules 12. Toso, R. J., Jordan, M. A., Farrell, K. W., Matsumoto, B. & made from the pure ap3 isotype, microtubules formed from Wilson, L. (1993) Biochemistry 32, 1285-1293. amixture of20%' ahm and 80% a/31 dimers were less dynamic 13. Sheldon, E. & Wadsworth, P. (1993) J. Cell Biol. 4, 935-945. than microtubules made from isotypically pure af43 dimers. 14. Margolis, R. L. & Wilson, L. (1978) Cell 13, 1-8. These data support the idea that presence ofa specific isotype 15. Margolis, R. L. & Wilson, L. (1981) Nature (London) 293, in 705-711. solution or its incorporation into a microtubule may confer 16. Farrell, K. W., Jordan, M. A., Miller, H. P. & Wilson, L. unique polymerization properties to that microtubule. Mat- (1987) J. Cell Biol. 104, 1035-1046. thews et al. (35) observed that mutation and alteration of the 17. Villasante, A., Wang, D., Dobner, P., Dolph, P., Lewis, S. A. normal isotype ratio in Drosophila produced severe effects & Cowan, N. G. (1986) Mol. Cell. Biol. 6, 2409-2419. both on mitosis and meiosis and speculated that the isotype 18. Sullivan, K. F. & Cleveland, D. W. (1986) Proc. Natl. Acad. composition might control microtubule-dependent cell func- Sci. USA 83, 4327-4331. tion. Hoyle and Raff (20) expressed the Drosophila /33 19. Luduefia, R. F. (1993) Mol. Biol. Cell 4, 445-457. isotype in 20. Hoyle, H. D. & Raff, E. C. (1990) J. Cell Biol. 111, 1009-1026. the male germ cell line ofDrosophila in place ofthe 21. Falconer, M. M., Echeverri, C. J. & Brown, D. L. (1992) Cell P2 isotype and observed that altering the isotype ratios Motil. Cytoskeleton 21, 313-325. allowed certain microtubule-dependent functions to occur 22. Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., normally, but because all such males were sterile, other Cleveland, D. W. & Luduefia, R. F. (1988) J. Biol. Chem. 263, microtubule functions must have been defective. Replace- 5625-5630. ment of the ,32 isotype with the /3 isotype in the experiment 23. Banerjee, A., Roach, M. C., Trcka, P. & Luduena, R. F. (1990) of Hoyle and Raff (20) could have resulted in the formation J. Biol. Chem. 265, 1794-1799. of 24. Banedjee, A., Roach, M. C., Trcka, P. & Luduefia, R. F. (1992) microtubules with significantly altered dynamics that may J. Biol. Chem. 267, 5625-5630. have adversely affected the functions of a specific microtu- 25. Banerjee, A. & Luduefia, R. F. (1992) J. Biol. Chem. 267, bule subset required for spermiogenesis. 13335-13339. In summary, the functional significance oftubulin isotypes 26. Lu, Q. & Luduenia, R. F. (1994) J. Biol. Chem. 269, 2041-2047. remains unclear. Because some isotypes have different tissue 27. Baneijee, A. & Luduefia, R. F. (1991) J. Biol. Chem. 266, distributions and because the sequence differences among 1689-1691. the isotypes are very strongly conserved in evolution suggest 28. Luduefia, R. F., Roach, M. C., Lee, M. K., Banerjee, A., that isotypes may have a functional significance as first Trcka, P. P., Frankfurter, A., Bulinski, J. C. & Asal, D. J. suggested by Fulton and Simpson (36). Due to their amino (1989) J. Cell Biol. 109, 338a (abstr.). acid sequence the 29. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. differences, (3-tubulin isotypes may adopt 30. Bayley, P. M., Sharma, K. K. & Martin, S. R. (1994) in different conformations leading to differences in their tubu- Microtubules, eds. Lloyd, C. & Hyams, J. (Wiley, New York), lin-tubulin interaction sites in the microtubule and to differ- pp. 111-137. ences in the dynamics. Incorporation of a specific tubulin 31. Serrano, L. de la Torre, J., Maccioni, R. B. & Avila, J. (1984) isotype into a microtubule may modulate the dynamic be- Proc. Natd. Acad. Sci. USA 81, 5989-5993. havior or function of a microtubule directly, or it may do so 32. Littauer, U. Z., Giveon, D., Thierauf, M., Ginzburg, I. & less directly by providing specific recognition sites for MAPs Ponstingl, H. (1986) Proc. Natl. Acad. Sci. USA 83,7162-7166. or other regulators of microtubule dynamics. One could 33. Cross, D., Dominguez, J., Maccioni, R. B. & Avila, J. (1991) imagine that cells might regulate the dynamic properties of Biochemistry 30, 4362-4366. their microtubules by altering the relative amounts of the 34. Alexander, J. E., Hunt, D. F., Lee, M. K., Shabanowitz, J., Michel, H., Berlin, S. C., Macdonald, T. L., Sundberg, R. J., different tubulin isotypes that they express. Rebhun, L. I. & Frankfurter, A. (1991) Proc. Natd. Acad. Sci. USA 88, 4685-4689. This work was supported by National Institutes of Health Grant 35. Matthews, K. A., Rees, D. & Kaufman, T. C. (1993) Devel- NS13560 (to L.W.) and by National Institutes of Health Grants opment (Cambridge, U.K.) 117, 977-991. CA26376 and GM23476 and by Grant AQ-0726 from the Robert A. 36. Fulton, C. & Simpson, P. A. (1976) in Cell Motility: Cold Welch Foundation (to R.F.L.). Spring Harbor Conferences on Cell Proliferation, eds. Gold- man, R., Pollard, T. & Rosenbaum, J. (Cold Spring Harbor 1. Dustin, P. (1984) Microtubules (Springer, Berlin), 2nd Ed. Lab. Press, Plainview, NY), pp. 987-1005. Downloaded by guest on September 27, 2021