AN IMPROVED

PREPARATION TUBULIN

OF HIGHLY

SPECIFIC

ANTIBODIES

G. WICHE and R. D. COLE Depurtment

of Biochemistry.

University

of Culifornicr, Berkeley. CA 94720. USA

SUMMARY Highly specific antibodies to tubulin were obtained by immunization of rabbits with tubulin preparations which had been purified from mouse brain by repeated cycles of in vitro polymerizationldepolymerization and subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis. This purification scheme yielded tubulin preparations absolutely devoid of actin and other minor contaminants commonly observed in such preparations. The specificity of the tubulin antibodies was demonstrated by immunodiffusion, microcomplement fixation and indirect immunofluorescence. Indirect immunofluorescence revealed specific binding of these antibodies to the spindle apparatus of ceils undergoing mitosis and furthermore, to colchicine-sensitive cytoplasmic fibers of tissue culture cells.

Tubulin is considered to be a major protein in eucaryotic cells. Under various physiological conditions tubulin molecules assemble into microtubules, fibrous structures that are associated with such cellular functions as chromosome movement in cell division, cellular motility, development and maintenance of cell shape, and sensory transduction [I]. Much progress has been made recently [2, 31 on the mechanism of in vitro assembly of microtubules, but many details of tubulin distribution in vivo and its possible interactions with organelles remain to be worked out. In studies of the latter type, antibodies specific to tubulin appear to be a powerful analytical tool. Several reports [4-71 have been made on the preparation of such antibodies but the purity of the tubulin injected as antigen for raising antibodies was not clearly established. Purity of the antigen is unusually important in this case, since tubulin is ex2-761802

petted to be weakly immunogenic as a consequence of its ubiquitous occurrence and highly conserved amino acid sequence [8]; even small amounts of immunogenitally potent impurities could give rise to immune responses not specific to tubulin itself. For example, since actin is a common contaminant of tubulin preparations one might confuse actin-containing fibers with tubulin-containing structures if the fibers were visualized with fluorescence antibody techniques using contaminated tubulin preparations. Although in this special case Weber et al. [7] have shown that actin- and tubulin-containing structures can be distinguished by their sensitivity towards colchicine and temperature, it would still seem preferable for other applications of the antibody to use antigen preparations devoid of actin and all other recognized impurities. Fuller et al. [9] have recently reported the preparation of antibodies by Expfl Cell Res 99 (1976)

16

Wiche and Cole

injecting a tubulin preparation that had been purified by in vitro polymerization/depolymerization and column chromatography. Although this purification scheme is extensive the overall procedure did not seem to result in an immune response exclusively directed against tubulin since monospecificity of the antibody preparation was established only after separating the tubulin specific immunoglobulins from others by affinity chromatography. The effect of contaminants (possibly including low levels of actin) in the tubulin preparation might have been exaggerated by the rather large amount of antigen injected. The present report describes an improved method for the production of highly specific tubulin antibodies that avoids column chromatography and affinity chromatography. Preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is incorporated in our procedure, since it seems to allow a fully effective purification of tubulin and has been successful for the preparation of actin specific [lo] and other antibodies [Ill. Our simplified method guarantees the elimination of all known contaminants of tubulin and it proved possible to raise highly specific antibodies with very small injections further minimizing the danger of interference from unknown contaminants that might have accompanied tubulin in repeated cycles of polymerization/depolymerization and that also might have comigrated with tubulin in electrophoresis. The specificity of the antibody preparation was established in immunodiffusion and microcomplement fixation, as well as in immunofluorescent tests based on the decoration of mitotic spindles, as previously reported by Nagayama C!?Z Dales [4] and Fuller et al. [9], and the labeling of cytoplasmic microtubules, as shown recently by Weber et al. [7]. ExptlCell

Res 99 (1976)

MATERIALS

AND METHODS

Preparation of tuhulin Tubulin was purified from the brains of about 80 young mice (23-27 g) by repeated cvcles of polvmerigation and depolym&iz&ion according td tie method of Shelanski et al. [3]. Final pellets of polymerized tubulin were dissolved by boiling in IO’% glycerol-5 % mercaetoethanol-3 % SDS fsodium dddecyl sulfate)-62.j mM Tris/HCI, pH 6.8 and applied to 10% polyacrylamide slab gels prepared according to Ferro-Luzzi Ames [ 121.The discontinuous buffer system of Laemmli [I31 was used in running the gels and the gels were stained with Coomassie brilliant blue and destained according to Fairbanks et al. [l4]. Tubulin was recovered from the gel by excision of the stained band and electrophoretic elution. The protein eluted from about 15aels was oooled, concentrated by lyophilization and &ed as gntigen after dialysis against IO mM phosphate- 150 mM NaCl, pH 7.0 (phosphate-buffered saline, PBS) to which was added 0.2% SDS. Hinhlv purified hog brain tubulin was generously supplied hy *Larry Honig (Univ. of Calif., Berkeley).

Preparation of antibodies Approx. 500 pg pure mouse tubulin was injected into each rabbit (New Zealand White or Dutch belted) intradermally in complete Freund’s adjuvant. Five and seven weeks later additional injections were made intravenously using each time the same amount of antigen (500 pg). Blood was collected one week after the final injection and serum was obtained by centrifugation at 8 000 g. The immunoglobulins were purified by repeated ammonium sulfate precipitation (3 X) and subsequent chromatography on DEAE Sephadex [15]. This preparation was then dialyzed against PBS and stored frozen at concentrations of 10-15 mg protein per ml. Immunoglobulins from blood of normal rabbits were purified and stored under identical conditions. Prior to use the immunoglobulin preparations were diluted with PBS to the concentrations indicated in the text. Protein was determined according to Lowry et al.

[W.

Indirect immunojhorescence Mouse BALB/c 3T3 fibroblast and rat kangaroo kidney cells (Ptk,) were grown on coverslips in Dulbecco’s modified Eagle’s medium supplemented with IO and 20% fetal calf serum, respectively. For the visualization of cvtoolasmic microtubules bv indirect immunofluorescence the cells were processed essentially as described by Weber et al. [7]. In brief, cells were washed quickly in PBS fixed in 3.5 % formaldehyde in PBS for 5 min at room temperature, washed again, immersed in methanol first at -20°C for 4 min and then in acetone at -20°C for 2 min and air dried. In attempts to visualize the spindle apparatus of Ptk, cells we followed essentially a procedure described by Lazarides & Weber [IO] which differs from the above in an extended period of formaldehyde fixation (20 min at room temperature) and omis-

Tuhulin untihodir.v

I?

depolymerization according to the method of Shelanski et al. [3] was identified as tubulin by the following criteria: On SDS polyacrylamide cells it comigrates with highly purified hog brain tubulin at a mol. wt of 55 000 (fig. 1a, 6) and with a colchitine-binding protein isolated from neuroblastoma cells [17] by DEAE chromatography [ 181. The electrophoretic analysis of a heavily overloaded slab gel of the same mouse tubulin preparation reveals minor bands predominantly in the high molecular weight range (fig. 1~). In addition there was a faint protein band that migrated slightly in front of tubulin (fig. 0 lc) and this band could be identified as actin by comigration on SDS polyacryla b C amide gels with highly purified mouse musFig. 1. SDS-polyacrylamide gel electrophoresis of cle actin (data not shown). tubulin. Electrophoresis was performed on 10% polyTo ensure the highest possible purity of acrylamide slabs in 0.1% SDS as described in Materials and Methods. (a) 2.5 pg mouse brain tubulin the tubulin preparations used as antigens purified by three cycles of in vitro polymerization/ depolymerization; (b) hog brain tubulin; (c) 13 kg for antibody production in rabbits we premouse tubulin purified as in (a). HMW, high molecu- pared tubulin which was free even of the lar weight protein. minor contaminants shown in fig. 1c. For this purpose tubulin from mouse brain sion of the methanol treatment. Immersion in acetone prepurified by three cycles of polymerizawas done in this case at -10°C for 10 min. Incuba- tion and depolymerization was run on tion with antitubulin or normal immunoglobulins were performed at 37°C for 1 h. After several rinses in PBS preparative slab gels under denaturing conthe coverslips were then incubated with fluorescein- ditions in SDS. The protein bands were labeled goat anti-rabbit immunoglobulin. Tine fluoresthen stained and the tubulin material was cent probe. obtained from Antibodies Incorporated, Davis, CA, was diluted l/IO in PBS and supple- eluted after cutting out the bands from the mented with 10% normal goat serum prior to use. The coverslips were finally washed in PBS, mounted slab gel with a razor blade; particular cauin 90% glycerol and viewed in a Leitz Orthoplan mi- tion was exercised to achieve complete croscope with ultraviolet optics using epifluorescence and a 40x objective. Photographs were taken using separation of the tubulin from the actin Tri-X Pan film (Kodak). band. Colchicine sensitivity of cytoplasmic microtubules A total of 4.5 mg denatured protein was assayed as follows: Trypsinized 3T3 cells were suspended in medium, containing the drug at 15 wg/ eluted from the tubulin bands of several ml, then spread over glass coverslips and subsequently incubated at 37°C for 24 h. Thereafter the at- preparative slab gels was used as antigen tached cells were processed for indirect immuno- in the immunization of three rabbits, two fluorescence. of which gave a positive immune response. The immunoglobulin preparations obtained RESULTS were shown to be specific for tubulin by The major protein purified from mouse both immunodiffusion and microcomplebrain by three cycles of polymerization and ment fixation. As shown in fig. 2 the antiErprl CellRrs

9'4 11'976)

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Wiche and Cole

4

b

C

Fig. 2. Ouchterlony test. lmmunodiffusion was performed on a 1%’ agar plate in isotris buffer [l9] at 4°C for 48 h. The plate was then immersed in phosphate buffered saline at room temperature for 48 h, stained in 0.25 % Coomassie brilliant blue-50% methanol-7.5% acidic acid for I h and destained in 7.S% methanol-7.5% acetic acid. (a) 4 wg mouse brain tubulin, partially purified by two cycles of in vitro polymerization/depolymerization; (b) rabbit antiserum partially purified by ammonium sulfate precipitation; (c) 3 kg hog brain tubulin.

ment is in the pg range. Whereas this is higher than the optima of most proteins [ 191 it is comparable to that of histones [20] which are similar to tubulin in being ubiquitous proteins with highly conserved amino acid sequences [21]. The antiserum also appeared to be monospecific when whole cell extract (mouse brain) was tested as antigen in place of partially purified tubulin. When the extract was used over a concentration range of 1 pg/ml to 10 mglml a single peak of complement fixation was observed. To further establish the specificity of the antibody, attempts were made to stain the spindle apparatus of cells in mitosis by indirect immunofluorescence, a characteristic of tubulin antibodies as demonstrated by Nagayama & Dales [4] and Fuller et al. [9]. Rat kangaroo Ptk, cells grown on coverslips were processed for indirect

serum reacts with undenatured mouse or hog brain tubulin to give a single precipitin line; the undenatured tubulin was prepared by two cycles of polymerization/depolymerization without electrophoretic purification. Control experiments substituting normal rabbit serum or an unrelated antiserum for the tubulin antiserum did not result in precipitin lines. Neither was a precipitate observed when bovine serum albumin was substituted for tubulin at the same concentration in this assay. The antiserum also reacted with the highly purified SDS-denatured antigen (originally used to immunize the rabbits) to give a sharp precipitin band. In this case a second faint curved band was also seen but control exI I I I periments showed this to be due to the 01 4’ 1.5 3 6 12 24 precipitation of serum components by SDS Fig. 3. Abscissa: pg protein; ordinarec % complement itself. fixed. Complement fixation by partially purified mouse The results of a microcomplement fixaRabbit tubulin antiserum diluted I :75 was tion test made with a 1 : 75 dilution of the tubulin. reacted with tubulin partially purified from mouse antiserum and partially purified mouse brain (two cycles of polymerization/depolymerizain the presence of guinea pig complement and tubulin is shown in fig. 3. The optimum con- tion) sheep erythrocytes under the conditions described by centration of tubulin for fixation of comple- Champion et al. [ 191.

A

E.rpfl Cdl Res 99 (1976)

I

Fig. 4. Visualization of the spindle apparatus of F’tk, cells. Ceils were processed for indirect immunofluorescence and treated with tubulin antibody preparation

(6 mg/ml) as described in Materials and Methods. (tl) View of cell in metaphase by fluorescence microscopy: (b) view of the same cell by phase contrast.

immunofluorescence as described in Materials and Methods. Treatment with tubulin specific antibodies gave the result shown in fig. 4~. The spindle apparatus clearly stains much brighter than the remainder of the cell. Fig. 4b shows the same cell in phase contrast. Several coverslips were examined and every mitotic cell detected with phase contrast optics showed specific staining of the spindle region when examined for fluorescence. No staining of the spindle above background was observed in control experiments using equivalent concentrations of two different preparations of immunoglobulins purified from normal rabbits. Another characteristic demonstrated [7] for tubulin antibodies is their

ability to stain cytoplasmic microtubules of tissue culture cells by indirect immunofluorescence. Therefore mouse 3T3 were processed for indirect immunofluorescence and indirectly stained essentially as described by Weber et al. [7]. As shown in fig. 5a a network of fine fibers was observed throughout the cytoplasm and across the nuclei of most cells. This staining pattern of 3T3 cells is similar to the one observed by Weber et al. [7]. In addition we observed such staining patterns with rat kangaroo Ptk, cells. The drug colchicine disrupts cytoplasmic microtubular structures [l], and its effect on the fibers observed by indirect immunofluorescence was used to authenticate

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Wiche and Cole

Fig. 5. Cytoplasmic microtubules visualized by indirect immunofluorescence in mouse 3T3 cells. Cells were processed for indirect immunofluorescence and then treated with tubulin antibody preparation (I mg/

the fibers as microtubules. Fig. 56 shows 3T3 cells which were treated with colchicine as described in Materials and Methods and subquently processed for immunofluorescence using tubulin specific antibodies. It is obvious that colchicine caused the disappearance of most of the cytoplasmic fibers that had reacted with tubulin antibodies similar to the results of Weber et al. [7]. By using phase contrast optics it also became apparent that most cells lose their distinct cell shape when treated with colchicine. Weaker staining of some cytoplasmic fibers was also observed in control experiments with immunoglobulins purified from normal rabbit serum. However, most of these fibers did not seem to be colchicine sensitive (to be reported elsewhere). Exptl Cell Res 99 (1976)

ml) essentially as described by Weber et al. [7]. (a) Cells stained with tubulin antibody; (b) as ((I) but treated with colchicine. Bar 25 pm.

DISCUSSION Special care seems necessary in preparing specific antibodies to ubiquitous proteins like tubulin, that appear to have rather strictly conserved amino acid sequences. Since one can expect these proteins to be only weakly immunogenic even small amounts of immunogenically potent impurities could give rise to an immune response which is not specific to the weak immunogen. We have therefore attempted to reduce to a minimum possible contamination of the tubulin antigen preparation. Mouse tubulin was first partially purified by three cycles of in vitro polymerization and then run on preparative SDS polyacrylamide slab gels to separate tubulin quantitatively from high molecular weight

Tubulin

proteins and actin-like material, which are the only recognized contaminants as judged by SDS polyacrylamide gel electrophoresis. That the specificity of the antibody preparation obtained by use of this highly purified tubulin preparation as antigen, was indeed directed towards tubulin only (monospecificity) was established by the following criteria: (i) in Ouchterlony tests only a single immunoprecipitin band was observed using either the electrophoretitally purified mouse brain tubulin originally used as antigen in the immunization of the rabbits or less pure tubulin preparations from mouse or hog brain; (ii) microcomplement fixation showed only one optimum either with tubulin purified by repeated polymerization/depolymerization or, even more important, with crude cell extract from mouse brain. Additional evidence for the specificity of the antibody preparation and proof of its utility in cytological studies comes from indirect immunofluorescence data. We observed intense staining of the spindle apparatus of cells in mitosis and specific decoration of colchicine-sensitive cytoplasmic fibers of tissue culture cells. The latter observation needs to be emphasized since to the best of our knowledge there has been only one report on the visualization of cytoplasmic microtubules by indirect immunofluorescence [7]. However, the tubulin antibody preparation used in that study was described as not being monospecific and so the staining of cytoplasmic fibers might have been caused by other components in addition to tubulin; indeed such non-specific staining of other cellular components was pointed out by the authors. Our observations of similar cytoplasmic fibers by using a more specific antibody preparation, reduce the possibility of non-specific staining and therefore

antibodies

21

add further strength to conclusions reached in the previous report. In conclusion, the tubulin antibody preparation described in this paper seems to be highly specific and therefore apt for cytological studies. Since the method of preparing the antibody is relatively simple and has advantages over previous ones, it should hold great promise for further studies. For example, we are applying such preparations to a study of the microtubules located in the spindle apparatus and their interaction with chromosomes. We thank Dr Allan Wilson for helpful advice and discussions, Dr Vincent Sarich for help in the preparation of antibody, Dr Ellen Prager for providing advice and materials for microcomplement fixation, and Misz Victoria Lundblad for excellent assistance in carrying out the microcomplement fixation. We are indebted to Dr Leon Wofsy for the use of his fluorescence microscope. Mr Larry Honig’s generous gift of purified hog brain tubulin is also gratefully acknowledged. The rat kangaroo cell line Ptk, was provided by Contract E73-200l-NOI within the special Virus Cancer Program, NIH, PHS, through the courtesy of Dr Jack Weaver. This work was supported by the Max Kade Postdoctoral Research Exchange Grant (to G. W.) and by grants GM 20338from the NIH and GB 38658from the NSF.

REFERENCES I. Olmsted, J B & Borisv, G G. Ann rev biochem 42 (1973) 507. . 2. WeisenberE. R C. Science 177(1972) 1104. 3. She1anski.lh L, Gaskin, F & Cantor, C R, Proc natl acad sci US 70 (1973) 765. 4. Nagayama, A & Dales, S, Proc natl acad sci US 66 (1970) 464. 5. Fulton, C, Kane, R E & Stephens, R E, J cell biol 50 (1971) 762. 6. Kowit, J D & Fulton, C, J biol them 249 (1974) 3638. 7. Weber, K, Pollack, R & Bibring, T, Proc natl acad sci US 72 (1975) 459. 8. Luduena, R F & Woodward, D 0, Proc natl acad sci US 70 (1973) 3594. 9. Fuller, G M, Brinkley, B R & Boughter. J M, Science 187 (1975) 948. 10. Lazarides, E & Weber, K, Proc natl acad sci US 71 (1974) 2268. II. Stumph, W E, Elgin, S C R & Hood, L, J immunol 113 (1974) 1752. 12. Fe&L&i Ames, G, J biol them 249 (1974) 634. 13. Laemmli, U K, Nature 227 (1970) 680. 14. Fairbanks, G, Steck, T L & Wallach, D F H, Biochemistry 10 (1971) 2606. Exptl Cell Res 99 (1976)

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15. Campbell, D H, Garvey, J S, Cremer. N E & Sussdorf, D H, Methods in immunology. Isolation and purification of rabbit antibodies, pp. 114-l 15. Benjamin, New York (1963). 16. Lowrv, 0 H, Rosebrough, N J, Farr. A L & Randall, R J, J biol chemj93 (1951) 265. 17. Wiche, G, Zomzely-Neurath, C & Blume, A J, Proc natl acad sci.US 71 (1974) 1446. 18. Weisenberg, R C, Borisy, G G & Taylor, E W, Biochemistry 7 (1968) 4466.

Exptl Cell Res 99 (1976)

19. Champion, A B, Prager, E M, Wachter, D & Wilson, A C, Biochemical and immunological taxonomy of animals (ed C A Wright) pp. 397-416. Academic Press, London (1974). 20. Bustin, M, Nature new biol 245 (1973) 207. 21. DeLange, R J & Smith, E L, Acct them res 5 (1972) 368. Received June 4, 1975 Accepted September 23, 1975