Proc. Nati. Acad. Sci. USA Vol. 84, pp. 8488-8492, December 1987 Cell detection in sea urchin eggs with a monoclonal antibody against Drosophila intermediate filament proteins: Characterization of stages of the division cycle of (cytoskeleton/fertilization/microtubules/) HEIDE SCHATTEN*, MARIKA WALTERt, DANIEL MAZIAt, HARALD BIESSMANNt, NEIDHARD PAWELETZ§, GE2RARD COFFE*, AND GERALD SCHATTEN* *Integrated Resource for Biomedical Research, University of Wisconsin, 1117 West Johnson Street, Madison, WI 53706; tCenter for Developmental Biology, University of California, Irvine, CA 92717; tHopkins Marine Station, Department of Biological Sciences, , Pacific Grove, CA 93950; and 1lnstitute for Cell and Tumor Biology, German Cancer Research Center, D-6900 Heidelberg, Federal Republic of Germany Contributed by , August 27, 1987

ABSTRACT A mouse monoclonal antibody generated Most all of the previous immunocytochemical work on against DrosophUa intermediate filament proteins (designated centrosomes has used an autoimmune serum from a patient Ah6/5/9 and referred to herein as Ah6) is found to cross-react suffering from CREST (calcinosis, Raynaud phenomenon, specifically with centrosomes in sea urchin eggs and with a esophageal dysmotility, sclerodactyly, telangiectasia) scle- 68-kDa antigen in eggs and isolated mitotic apparatus. When roderma (6-10). However, the inability to use this serum for preparations stained with Ah6 are counterstained with a immunoblotting precluded characterization of this centro- human autoimmune serum whose anti-centrosome activity has somal antigen, and its human source presents problems of been established, the immunofluorescence images superimpose routine availability. In the present report, we describe the use exactly. A more severe test of the specificity of the antibody of another probe, a mouse monoclonal antibody designated demands that it display all of the stages of the centrosome cycle Ah6/5/9 and referred to herein as Ah6, which has been in the cell cycle: the flattening and spreading of the compact generated against Drosophila intermediate filament proteins centrosomes followed by their division and the establishment of (11, 12). Ah6 recognizes centrosomes in sea urchin eggs and two compact poles. The test was made by an experimental and cross-reacts with an antigen of 68-kDa. design that uses a period of exposure of the eggs to 2- To test whether the antibody gives a faithful rendering of mercaptoethanol. This treatment allows observation of the the shapes and behavior of sea urchin centrosomes, we stages of the centrosome cycle separation, division, and exploit an experimental design that has provided much ofour bipolarization-while the chromosomes are arrested in meta- information about the centrosome cycle. Fertilized eggs phase. Mitosis is arrested in the presence of 0.1 M 2- approaching first metaphase are exposed to 2-mercaptoeth- mercaptoethanol. Chromosomes remain in a metaphase con- anol (13-17). The chromosomes are arrested in metaphase figuration while the centrosomes divide, producing four poles while the centrosome cycle proceeds. The compact meta- perpendicular to the original spindle axis. Microtubules are phase centrosome divides and condenses into two compact still present in the mitotic apparatus, as indicated by im- poles. munofluorescence and transmission electron microscopy. When 2-mercaptoethanol is removed, the chromosomes reori- MATERIALS AND METHODS ent to the poles of a tetrapolar (sometimes tripolar) mitotic apparatus. During the following cycle, the blastomeres form a Sea urchin fertilization and 2-mercaptoethanol application monopolar mitotic apparatus. The observations of the centro- was performed as described (14-16). Anti-centrosomal and some cycle with the Ah6 antibody display very clearly all the anti-tubulin immunofluorescence microscopy on Strongylo- stages that have been seen or deduced from work with other centrotus purpuratus cells followed the methods as described probes. The 68-kDa antigen that reacts with the Ah6 mono- (10). In experiments comparing the detection of centrosomes clonal antibody to Drosophila intermediate filament proteins with the autoimmune human serum (no. 5051; ref. 6) and the must be a constant component of sea urchin centrosomes mouse monoclonal antibody to Drosophila intermediate fil- because it is present at all stages of the centrosome cycle. ament proteins (Ah6/5/9; refs. 11 and 12), cells were first labeled with the human serum 5051 and then counterstained Historical doubts and misconceptions about centrosomes with the mouse monoclonal antibody Ah6. Immunoblotting have now been overcome by evidence gained from the use of was performed by the methods of Walter and Biessmann (12) anti-centrosome antibodies. Centrosomes are not necessarily with homogenates from unfertilized sea urchin eggs or mitotic corpuscular structures, and they can be found in many shapes apparatus isolated by the methods of Salmon and Segall (18). in many kinds of cells including cells of higher plants. A Transmission electron microscopy was performed as de- strong case can now be made for the universal occurrence of scribed by Paweletz et al. (16). centrosomes. Boveri's proposal of a centrosome cycle (1), involving a sequence of changes of shape of centrosomes RESULTS through the cell cycle, has been reinforced (reviewed in ref. 2). Recent years have seen the revival of Boveri's strong To demonstrate that the monoclonal antibody detects cen- conception of the centrosome as a major permanent organ of trosomal material, double immunofluorescence experiments the cell. Going further, some workers (2-5) propose that the were performed on mitotic sea urchin eggs by using the centrosome is a bearer of information for cell morphology. mouse monoclonal antibody and the autoimmune human serum to centrosomal material (serum 5051; ref. 6). This The publication costs of this article were defrayed in part by page charge serum was shown by Schatten et al. (10) to bind to the payment. This article must therefore be hereby marked "advertisement" centrosomes in mitotic sea urchin eggs. In Fig. 1, the human in accordance with 18 U.S.C. §1734 solely to indicate this fact. serum 5051 (Left) and the mouse monoclonal antibody Ah6 8488 Downloaded by guest on September 24, 2021 Cell Biology: Schatten et al. Proc. Natl. Acad. Sci. USA 84 (1987) 8489

Sul Kc

- 80 I_*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~t~~~~~~~~~~~~~~~~~~~~~~~~~~~~~6

FIG. 1. Ah6 reacts with centrosomes and a 68-kDa protein in sea urchin eggs. The mouse monoclonal antibody to Drosophila intermediate filament proteins (Ah6) detects the identical material stained with a characterized human autoimmune serum to centrosomes, serum 5051 (A and B), and it reacts with a 68-kDa protein (C). (A) Sea urchin egg at first metaphase triple-stained with fluorescein-labeled human serum 5051 (Left), rhodamine-labeled mouse monoclonal antibody Ah6 (Center), and the Hoechst DNA dye 33258 (Right). (B) An egg at first telophase stained as in A. (Bars = 10 Am.) (C) Immunoblot: Ah6 specifically reacts with a68-kDa protein in isolated mitotic spindles from sea urchin zygotes (lane Sul, arrowhead) and in unfertilized egg homogenates (data not shown). Ah6 also recognizes five major proteins in Drosophila Kc cells at 110, 80, 68, 46, and 40 kDa (lane Kc).

(Center) both show identical staining patterns ofcentrosomes shows that microtubules can be found in the spindles and at metaphase and telophase in mitotic sea urchin zygotes. At asters and at the center of the centrosomes, consistent with metaphase (Fig. 1A) the centrosomes are highly compacted the anti-tubulin images. spherical bodies, whereas by telophase (Fig. 1B) they have Recovery from 2-mercaptoethanol was studied at various flattened and spread into broad ovoid disks. Immunoblots timepoints after resuspension in normal sea water (Fig. 4). At show that an antigen corresponding to 68-kDa was found in 15 min post recovery of cells that had been incubated in unfertilized sea urchin eggs (data not shown) and in isolated 2-mercaptoethanol for 15 min (Fig. 4A), the chromosomes mitotic apparatus (Fig. 1C). havejust begun their anaphase movement. The centrosomes Stages of the centrosome cycle in control cells are shown separate perpendicular to the spindle axis and are apparent as in Fig. 2. The sequence of stages of the centrosome cycle two distinct foci at each mitotic pole (Fig. 4A Left). Micro- conforms to the plan investigated in theory by Mazia (figure tubules comprise a barrel-shaped spindle with asters extend- 2 in ref. 2). The centrosome, brought into the egg by the ing from large ovoid clear zones, more typical of telophase sperm, remains associated with the male pronucleus and asters than of those seen at anaphase. As the chromosomes organizes the radially arrayed microtubules of the sperm continue to move towards the poles, the centrosomes con- aster (Fig. 2A). By prophase the centrosomes have separated tinue their separation (Fig. 4B). into two arcs positioned on opposing poles of the condensing Recovery from 2-mercaptoethanol results first in a tetra- chromatin; the bipolar mitotic apparatus begins to emerge polar division and then in the next cycle in the formation of (Fig. 2B). At metaphase, the centrosomes are maximally a monopolar division (reviewed in ref. 2). Upon recovery, the condensed as compact spheres found at the astral centers and four compact centrosomes ofthe tetrapolar mitotic apparatus spindle poles (Fig. 2C). By telophase, the centrosomes have can be clearly distinguished (Fig. 5 Center). The chromo- flattened and spread into broad plates (Fig. 2D). somes are now reoriented towards the poles (Fig. 5 Right). When cells are transferred into 0.1 M 2-mercaptoethanol at The mitotic apparatus (Fig. SA Left) has four asters and prometaphase, the centrosome cycle continues on schedule, complex spindles, and often these cells divide into four but the chromosome cycle arrests at metaphase (14). In Fig. blastomeres. During the next cycle, the classic monopolar 3, centrosome separation progresses in the presence of division occurs. In Fig. SB a representative half-spindle and 2-mercaptoethanol even though the centrosomes stain as aster (Left) and chromosomes (Right) aligned on the equator compact spheres as they move apart (Fig. 3A). Microtubules of the half-spindle are shown. The centrosomes are con- remain assembled in the presence of 2-mercaptoethanol, densed as a pair of compact particles (arrows in Center). demonstrating that the arrest of mitosis is probably not due to any direct depolymerization ofmicrotubules. In addition to DISCUSSION microtubules extending around the centrosomes, there is the The molecular composition of centrosomes has been ad- appearance of some tubulin staining at the interior of the dressed only recently. The discovery by Calarco-Gillam et al. centrosome (Fig. 3A Center, arrow). In Fig. 3A, the anti- (6) ofa human serum reacting with centrosomes has led to the tubulin immunofluorescence microscopy shows the config- immunocytochemical detection of this structure in a variety uration of a multipolar mitotic apparatus as the centrosomes of cells including sea urchin eggs (10). Autoimmune sera move apart. Transmission electron microscopy (Fig. 3B) against centrosomes have been found in rabbits (19, 20), and Downloaded by guest on September 24, 2021 8490 Cell Biology: Schatten et al. Proc. Natl. Acad. Sci. USA 84 (1987)

FIG. 2. Centrosomes in sea urchin eggs during fertilization and mitosis. (A) The sperm aster. The centrosome (Center) embedded at the center of the microtubules comprising the sperm aster (Left) is a compact particle adjacent to the male pronucleus (Right). (B) Early Prophase. The centrosome (Center) has separated during interphase and aggregates into two arc-shaped structures condensed along opposing poles of the nucleus (Right). Microtubules of the mitotic apparatus begin to emerge (Left). (C) Metaphase. The centrosomes (Center) are maximally condensed at the poles of the mitotic apparatus (Left). (D) Telophase. The centrosomes (Center) expand and flatten into ovoid plates with axes predictive of the axes for the next mitotic apparatus. (A is double-stained for microtubules and DNA with a centrosome image of a cell at the same stage, and B-D are triple-stained for microtubules, centrosomes, and DNA; bars = 10 Am.)

recently Gosti-Testu et al. (21) have demonstrated that a intermediate filament rabbit serum Drosophila proteins, while there is not that stains centrosomes reacts with a family of yet evidence that intermediate filaments are found in sea proteins from enriched human lymphocyte centrosomes urchin eggs, let alone centrosomes. It is also including one conceivable that characterized by a doublet of 60-65 kDa in the monoclonal antibody Ah6 recognizes an epitope on an immunoblots. Constituents of centrosomes in mammalian otherwise unrelated polypeptide. Until this issue is resolved cultured cells include cyclic AMP-dependent protein kinase by the 68-kDa centrosomal 11 (22), characterizing protein, the Ah6 phosphoproteins during mitosis (23), and a 36-kDa antibody can nevertheless be used as a tool to study sea epitope shared in common with lactate dehydrogenase (24). urchin centrosomes. Turksen et al. (25) describe a 50-kDa antigen in basal bodies. 2-Mercaptoethanol treatment is now a classic method for In addition, some centrospheric constituents include a 50- division of kDa permitting centrosomes, while arresting chromo- protein (26) and components of a calcium-transport some separation (14, 16, 17, 29). By the use of these system (27, 28). immunocytochemical methods, the sequence of changes in It is intriguing that this antibody was produced against centrosome configuration during incubation in 2-mercapto- Downloaded by guest on September 24, 2021 Cell Biology: Schatten et al. Proc. Natl. Acad. Sci. USA 84 (1987) 8491

FIG. 3. Centrosomes, microtubules, and chromosomes in the presence of2-mercaptoethanol, as shown by anti-tubulin immunofluorescence microscopy (A) and transmission electron microscopy (B). Cells treated with 0.1 M 2-mercaptoethanol at prometaphase continue to undergo the flattening and splitting of centrosomes during the subsequent 30 min (Left). Microtubules (Center) remain present in the presence of 2-mercaptoethanol and appear to undergo changes in configurations as the centrosomes alter their shapes; a few microtubules remain near the (arrows in A and B). (Bars = 10 ,um in A and 100 nm in B.) ethanol and during recovery can be described. The centro- and decondensation are not observed. Microtubule arrays somes separate in its presence yet remain condensed. This not only remain assembled in 2-mercaptoethanol, they un- observation will permit future inquiries into the normal dergo dynamic shape changes as centrosomal configurations events when duplication and division are coupled with cycles alter. In 2-mercaptoethanol, the chromosomes remain arrest- of condensation, flattening, separation, and expansion, as ed at metaphase. During recovery, chromosome separation is opposed to the events in 2-mercaptoethanol when flattening now asynchronous with the centrosome cycle.

FIG. 4. Centrosomes, microtubules, and chromosomes during recovery from 2-mercaptoethanol: 15 min (A) and 20 min (B). Cells permitted to recover for 15 min from a 15-min treatment with 2-mercaptoethanol display a flattening and spreading of centrosomal material (Left). Microtubules are organized into mitotic apparatus with large elliptical clear zones at the mitotic poles and with wide barrel-shaped spindles (Center). The chromosomes separate upon recovery (Right). (A and B are triple-stained for centrosomes, microtubules, and chromosomes; bars = 10 /Lm.) Downloaded by guest on September 24, 2021 8492 Cell Biology: Schatten et al. Proc. Natl. Acad. Sci. USA 84 (1987)

FIG. 5. Centrosomes, microtubules, and chromosomes during recovery from 2-mercaptoethanol: the tetrapolar (A) and monopolar (B) divisions. (A) Four centrosomes are apparent (Center), and the microtubules are organized into tetrapolar mitotic apparatus (Left). (B) A pair of centrosomes (Center, arrows) at the pole of the half-spindle (Left) during monopolar division. (A is double-stained for centrosomes and DNA, with a complimentary cell stained for microtubules at the same stage, and B is double-stained for microtubules and DNA; bars = 10 aim.) In summary, a monoclonal antibody to invertebrate inter- 10. Schatten, H., Schatten, G., Mazia, D., Balczon, R. & Simerly, mediate filament proteins detects sea urchin centrosomes and C. (1986) Proc. Natl. Acad. Sci. USA 83, 105-109. cross-reacts with a 68-kDa protein. The animation of the 11. Falkner, F.-G., Saumweber, H. & Biessmann, H. (1981) J. centrosomes and microtubules in the presence of 2-mercap- Cell Biol. 91, 175-183. is all 12. Walter, M. F. & Biessmann, H. (1984) J. Cell Biol. 99, toethanol depicted, the while the chromosomes are 1468-1477. stationary at their metaphase configuration. 13. Mazia, D. & Zimmerman, A. M. (1958) Exp. Cell Res. 15, 138-153. It is a pleasure to acknowledge the generous donations of human 14. Mazia, D., Harris, P. J. & Bibring, T. (1960) J. Biophys. autoimmune serum 5051 from Dr. Patricia Calarco and Dr. Marc Biochem. Cytol. 7, 1-20. Kirschner (University of California, San Francisco Medical Center). 15. Mazia, D., Paweletz, N., Sluder, G. & Finze, E.-M. (1981) The support of this research by grants from the National Institutes Proc. Natl. Acad. Sci. USA 78, 377-381. of Health (to G.S. and H.S.), the National Science Foundation (to 16. Paweletz, N., Mazia, D. & Finze, E.-M. (1984) Exp. Cell Res. D.M. and to H.B. and M.W.) and the German Cancer Research 152, 47-65. Center (to N.P.) is gratefully acknowledged. G.C. is supported by a 17. Sluder, G. & Begg, D. A. (1983) J. Cell Biol. 97, 877-886. Centre National de la Recherche Scientifique-National Science 18. Salmon, E. D. & Segall, R. R. (1980) J. Cell Biol. 86, 355-365. Foundation Postdoctoral Fellowship. The Integrated Microscopy 19. Connolly, J. A. & Kalnins, V. I. (1978) J. Cell Biol. 79, Resource at Madison is a National Institutes of Health Biomedical 526-532. Research Technology Resource (RR-570). 20. Maunoury, R. (1978) C.R. Hebd. Seances Acad. Sci. Ser. C 286, 503-506. 1. Boveri, T. (1900) Zellen-Studien: Ueber die Natur der Centro- 21. Gosti-Testu, F., Marty, M.-C., Berges, J., Maunoury, R. & somen (Fischer, Jena, G.D.R.), Vol. 4. Bornens, M. (1987) EMBO J. 5, 2545-2550. 2. Mazia, D. (1987) Int. Rev. Cytol. 100, 49-92. 22. Nigg, E. A., Schafer, G., Hilz, H. & Eppenberger, H. M. 3. Albrecht-Buehler, G. (1985) in Muscle and Cell Motility, ed. (1985) Cell 41, 1039-1051. Shay, J. (Plenum, New York), Vol. 6, pp. 1-21. 23. Vandre, D. P., Davis, F. M., Rao, P. N. & Borisy, G. (1984) 4. N. K. R. & Proc. Natl. Acad. Sci. USA 81, 4439-4443. Gershon, D., Porter, McNiven, M. A. (1986) 24. Gosti, F., Marty, M.-C., Courvalin, J. C., Maunoury, R. & Biophys. J. 49, 65-66. Bornens, M. (1987) Proc. Natl. Acad. Sci. USA 84, 1000-1004. 5. Mazia, D. (1984) Exp. Cell Res. 153, 1-15. 25. Turksen, K., Aubin, J. E. & Kalnins, V. I. (1982) 6. Calarco-Gillam, P. D., Siebert, M. C., Hubble, R., Mitchison, (London) 298, 763-765. T. & Kirschner, M. (1983) Cell 35, 621-629. 26. Kuriyama, R. & Borisy, G. G. (1985) J. Cell Biol. 101, 524- 7. Clayton, L., Black, C. M. & Lloyd, C. W. (1985) J. Cell Biol. 530. 101, 319-324. 27. Petzelt, C. & Hafner, M. (1986) Proc. Natl. Acad. Sci. USA 8. Maro, B., Howlett, S. K. & Webb, M. (1985) J. Cell Biol. 101, 83, 1719-1722. 1665-1672. 28. Silver, R. (1986) Proc. Natl. Acad. Sci. USA 83, 4302-4306. 9. Wick, S. M. (1985) Cell Biol. Int. Rep. 9, 357-371. 29. Sluder, G. & Rieder, C. L. (1985) J. Cell Biol. 100, 887-890. Downloaded by guest on September 24, 2021