Proc. Nati. Acad. Sci. USA Vol. 84, pp. 8488-8492, December 1987 Cell Biology Centrosome detection in sea urchin eggs with a monoclonal antibody against Drosophila intermediate filament proteins: Characterization of stages of the division cycle of centrosomes (cytoskeleton/fertilization/microtubules/mitosis) HEIDE SCHATTEN*, MARIKA WALTERt, DANIEL MAZIAt, HARALD BIESSMANNt, NEIDHARD PAWELETZ§, GE2RARD COFFE*, AND GERALD SCHATTEN* *Integrated Microscopy 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, Stanford University, Pacific Grove, CA 93950; and 1lnstitute for Cell and Tumor Biology, German Cancer Research Center, D-6900 Heidelberg, Federal Republic of Germany Contributed by Daniel Mazia, 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 embryos 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