116 Brief Communication
Zygotic development without functional mitotic centrosomes Timothy L. Megraw, Ling-Rong Kao and Thomas C. Kaufman The centrosome is the dominant microtubuleorganizing center in animal cells. At the onset of mitosis, each cell normally has two centrosomes that lie on opposite sides of the nucleus. Centrosomes nucleate the growth of microtubules and orchestrate the efficient assembly of the mitotic spindle. Recent studies in vivo and in vitro have shown that the spindle can form even in the absence of centrosomes and demonstrate that individual cells can divide without this organelle [1–8]. However, since centrosomes are involved in multiple processes in vivo, including polarized cell divisions, which are an essential developmental mechanism for producing differentiated cell types [9, 10], it remains to be shown whether or not a complete organism can develop without centrosomes. Here we show that in Drosophila a centrosomin (cnn) null mutant, which fails to assemble fully functional mitotic centrosomes and has few or no detectable astral microtubules, can develop into an adult fly. These results challenge long-held assumptions that the centrosome and the astral microtubules emanating from it are essential for development and are required specifically for spindle orientation during asymmetric cell divisions.
mutant embryos [4, 5, 11], and asterless mutant cells [7]. In cultured cells, laser ablation of centrosomes halts neither the assembly of spindles nor the process of cell division [6]. In Drosophila, ganglion mother cells appear to assemble bipolar spindles in the presence of centrosomes without using them [7]. There is also a stable Drosophila cell line that lacks centrioles, yet it assembles spindles and divides perpetually in culture [8]. Furthermore, normal female meiosis in Drosophila, Xenopus, and the mouse occurs without centrosomes. These findings demonstrate that particular cells can divide in the absence of centrosomes, but they do not address the issue of complete animal development. Centrosomin (Cnn) is a core component of centrosomes [12]. In most cell types, Cnn associates with the centrosome at mitosis and is dispersed at interphase. Cnn is found in the centrosomes of embryos, imaginal disks, the central nervous system, testis, ovaries, and all dividing cells examined with the exception of the oocyte at meiosis. Immunoelectron microscopy has shown that Cnn is a component of the pericentriolar matrix of the centrosome (our unpublished data).
Figure 1
Address: Department of Biology and Howard Hughes Medical Institute, Indiana University, Bloomington, Indiana 47405, USA. Correspondence: Timothy L. Megraw E-mail:
[email protected] Received: 3 November 2000 Revised: 27 November 2000 Accepted: 27 November 2000 Published: 23 January 2001 Current Biology 2001, 11:116–120 0960-9822/01/$ – see front matter 2001 Elsevier Science Ltd. All rights reserved.
Results and discussion All of the components necessary and sufficient for producing a functional centrosome in animal cells have not been elucidated, but these requirements are likely to vary with cell type. In cell-free extracts of Xenopus oocytes, spindles can be assembled upon DNA beads that do not contain centromeres [1]; however, spindle assembly in this system is more efficient when a centrosome is added [2]. Moreover, mitotic spindles assemble in the absence of functional centrosomes in unfertilized Sciarid embryos [3], cnn
cnn mutants develop into adult flies. (a) Schematic representation of centrosomin with the position of the nonsense mutation in cnnhk21 indicated with an asterisk. The predicted leucine zipper domains are shown in red, and the predicted coiled-coil domains are in green. (b) Images of (1) wild-type and (2) cnnhk21 mutant flies. A closeup of wings from (3) wild-type and (4) cnnhk21 flies shows the wing dots seen on cnn mutant wings [arrows in (4)]. The cnn mutant fly carries a white mutation.
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Figure 2
Mitotic divisions in the absence of Cnn lack normal centrosomes and astral microtubules. (a) Mitotic figures from (1) wild-type and (2) cnnhk21 third-instar larval brains stained for centrosomin (red), ␣-tubulin (green), and DNA (blue). Centrioles are present in [(4) and inset] cnnhk21 imaginal disks and appear normal compared to the (3) wild type. (b) Western blot of time course RNAi-treated S2 cells probed with anti-Cnn and anti–␣-tubulin antibodies (loading control). Lanes were loaded with protein extracts of S2 cells treated with mock (control) or cnn dsRNA for the number of days indicated before samples were removed from culture for protein analysis. Protein molecular weights
in kDa are indicated on the right of the blot. (c,d) Mitotic figures from S2 cells stained as in (a) from S2 cells treated with (c) mock and (d) cnn RNAi. Panels 1–6 in (c) and (d) are sequential stages of the cell cycle, as follows: (1), early prophase; (2), late prophase; (3), metaphase; (4), anaphase; (5), telophase; and (6), cytokinesis. In (d2), the spindle appears to be in the midst of assembly from within the chromosomes [compare to (c2)]. In (c6) the centrosome is dividing in each daughter cell. Following centrosome division, Cnn is dispersed from the centrosome (not shown).
Mutations in cnn prevent the assembly of centrosomal proteins such as CP60, CP190, and ␥-tubulin into centrosomes [4, 5]. Without Cnn, spindles are formed aberrantly with few or no astral microtubules emanating from their poles [4, 5]. It is therefore surprising that cnn null (cnnhk21) mutant animals survive to adulthood. Development is not slower for mutant animals than for their wild-type siblings, and the numbers of mutant animals produced from a cross of heterozygotes indicate no loss of viability. Figure 1a is a schematic representation of Cnn protein structure with the position of the cnnhk21 nonsense mutation indicated. Figure 1b shows a cnn null mutant fly. The phenotypes associated with the cnn null mutant are male sterility [13], maternal-effect lethality [4, 5], and the presence of black dots on the wings (Figure 1b4). Although cnn is required
for male meiosis and for cellularization of blastoderm embryos, homozygous mutants develop into apparently normal adults before these phenotypes are manifest. Adults walk, eat, court, and mate and show no obvious abnormalities aside from the phenotypes described above. This result implies that cell divisions can occur in the absence of a complete centrosome and a normal array of astral microtubules and that the necessary asymmetric cell divisions are occurring as well. Upon closer inspection, we observed that the cell divisions in cnn mutant larval brains and imaginal disks display several unique characteristics. Figure 2a2 shows a mitotic figure from a cnnhk21 mutant larval brain. No centrosomin protein is detected at the spindle poles, which also appear
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Figure 3
␥-tubulin is present at interphase centrosomes but fails to accumulate at mitotic spindle poles in cnn mutant cells. Cells were stained for ␣-tubulin (green), ␥-tubulin (red), and phosphohistone H3 (blue). The phosphohistone signal is a chromosome marker for cells in mitosis. (a) In wild-type neuroblasts, ␥-tubulin is localized at the centrosome during interphase and mitosis. (b) In cnnhk21 mutant neuroblasts,
␥-tubulin is not detected at the spindle poles at mitosis but is localized at the interphase centrosomes. (c) In control S2 cells, ␥-tubulin is localized at the centrosome during interphase and mitosis. (d) RNAitreated S2 cells. In the absence of Cnn, ␥-tubulin accumulates at interphase centrosomes but not at mitotic spindle poles.
anastral. In addition, cnn mutant spindles appear meiotic in character, with the smaller, fourth chromosomes migrating to the spindle poles precociously (Figure 2a2; also see Figures 3b and 4c). Despite the absence of mitotic centrosomes in cnn mutant imaginal cells, centrioles with normal morphology can be found (Figure 2a4). We have not determined where the centrioles are located in mutant cells at mitosis.
ment, Cnn is no longer detected at the mitotic spindle poles, and the spindle microtubules appear to assemble outward from the chromosomes (compare Figure 2c2,d2). Spindles are assembled similarly in cnn mutant neuroblasts (Figure 3b). At prophase, astral microtubules are prominent in wild-type cells but are apparent in neither the cnn mutant (compare the panels labeled “late prophase” in Figure 3a,b) nor in the RNAi-treated S2 cells (compare panels labeled “prophase” in Figure 3c,d). Mitosis in the cnn RNAi-treated S2 cells does not obviously appear to segregate the fourth chromosomes precociously as described above for cnn mutant cells.
Using double-stranded RNA–mediated interference (RNAi) [14], we looked at the consequences of Cnn loss in cultured Drosophila S2 cells. This method reduces Cnn protein levels within 3–4 days (Figure 2b). Cnn normally accumulates at centrosomes in S2 cells at mitosis and disperses at cytokinesis (Figure 2c). Upon RNAi treat-
In the cnn mutant, ␥-tubulin is absent from the spindle poles. In wild-type cells, ␥-tubulin is a component of
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Figure 4
Here, cytoplasmic dynein is required to anchor the spindle pole [17]. In the absence of polarized mitotic spindles during mitosis, the normal cyst, containing 15 nurse cells and one oocyte, fails to form an oocyte [18]. Likewise, in the developing nervous system, asymmetric divisions are essential for establishing different neuronal-cell fates [10]. Asymmetric cell divisions require a mechanism to orient mitotic spindles. This mechanism is thought to involve astral microtubules and centrosomes [19]. During Drosophila neurogenesis, the mitotic spindle poles orient relative to polarizing factors at the cell cortex. Such factors include Prospero, Numb, Miranda, and others [10]. We looked at the distribution of Prospero in cnn mutant larval neuroblasts and found it to be asymmetrically distributed at the cell cortex as in the wild type (Figure 4). However, we found that the spindle is oriented improperly approximately 22% of the time (n ⫽ 36) in the cnn mutant, with Prospero aligned adjacent to the metaphase plate rather than to one spindle pole (Figure 4c). We observed no disoriented spindles in wild-type neuroblasts (n ⫽ 28). This degree of error in the cnn mutant neuroblasts apparently has no serious consequences on the development of the nervous system.
Mitotic asymmetry in cnn mutant neuroblasts is not always correctly established. (a) Wild-type and (b,c) cnnhk21 mutant larval neuroblasts were stained with antibodies to Prospero. This allowed for the examination of spindle polarity. Cells were stained for ␣-tubulin (green), Prospero (red), and phosphohistone H3 (blue) (row labeled “DNA”). In (c) the spindle is misaligned by 90⬚. Note that in (c) the fourth chromosomes can be seen closer to the spindle poles than the other chromosomes in this anaphase cell.
interphase and mitotic centrosomes (Figure 3a,c). In cnn mutant cells, and cells phenocopied by RNAi, ␥-tubulin is still observed at interphase centrosomes, but as cells enter mitosis this localization is lost, and there is no detectable staining at the spindle poles (Figure 3b,d). This result is demonstrated in Figure 3, where ␥-tubulin is seen at the centrosome at all stages in wild-type animals and control RNAi cells. Cells entering mitosis are distinguished by the appearance of the phosphohistone H3 signal. In the cnn mutant and RNAi-treated S2 cells, the focused signal for ␥-tubulin becomes undetectable and then reappears as the cells exit mitosis. Thus, Cnn is required at the mitotic spindle pole to recruit or retain ␥-tubulin. The centrosome appears to be important in animal development for multiple processes, including polarized mitotic divisions that establish asymmetric fates of daughter cell types [10, 15]. Polarized mitoses are essential from the first division in some animals, such as the nematode C. elegans [16]. In the Drosophila ovary, the development of the egg chamber depends on polarized mitotic divisions.
Our results show that it is possible for animal development to occur without completely functional mitotic centrosomes. Furthermore, we detect few or no astral microtubules at the spindle poles in cnn mutant cells. cnn mutant cells still have centrioles. We do not know whether they are located at the spindle poles at mitosis, though their transmission at cell division suggests this may be the case. The absence of ␥-tubulin at the mitotic centrosome in cells that lack Cnn provides some explanation for why the spindle poles appear anastral since ␥-tubulin ring complexes (␥TuRCs) are the nucleation sites for microtubules at mitotic centrosomes [20, 21]. Furthermore, these results show that the modes of association for ␥-tubulin at interphase and at mitotic centrosomes are different, which suggests that there may be alternate roles for ␥-tubulin at these stages. The possibility of two functionally distinct pools of ␥-tubulin has been indicated by other studies [22, 23]. In support of a role in the interphase centriole, ␥-tubulin has been found to be required for basal-body duplication in Paramecium [24]. Although mutations in ␥-tubulin genes are lethal [25–27], the data presented here show that the protein is not required at mitotic centrosomes. Additionally, we found that the asymmetry of neuroblast divisions is mostly normal, with spindle polarity being aberrant about one-fifth of the time. These results suggest that there is a mechanism to anchor spindle poles to the cortex, although less efficiently, when astral microtubules are absent or dramatically reduced. Finally, these findings may not be unique to Drosophila since we have recently found apparent homologs of cnn in the
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silkworm, zebrafish, rat, and human genomes (our unpublished data).
15.
Materials and methods
16.
Drosophila stocks An Oregon-R stock was used for the wild type. The cnnhk21 mutant was provided by Trudi Schupbach and is described in Megraw et al [4]. Mutant analysis utilized animals of the genotype cnnhk21/Df(2R)cnn, obtained by crossing stocks balanced over CyO marked with a Kruppelgreen fluorescent protein transgene [28]. Mutant larvae were selected by the absence of GFP fluorescence.
17.
18. 19. 20.
Cytology Brains were dissected from third-instar larvae. Tissues were fixed and stained as described [29]. RNAi on S2 cells was performed as described [14] except that the culture medium was a supplemented M3 medium “BPYE” with 10% fetal bovine serum [30]. Double-stranded RNA for cnn corresponded to bases 970–1908 of the cDNA (Genbank accession number U35621). The mock RNAi used a 400 bp double-stranded RNA made from the pACT2 plasmid vector. S2 cells were fixed in methanol at ⫺20⬚C for 15 min and were stained as described [4]. A monoclonal antibody to Prospero was a gift from Chris Doe. Images were collected on a Leica TCS confocal microscope.
21. 22.
23. 24.
Acknowledgements We especially thank Bill Sullivan for his encouragement and enthusiastic support of our efforts. We are grateful to Sandhya Kilaru for a critical reading of the manuscript. We thank C. Doe for the anti-Prospero antibody. This work was supported by the National Science Foundation and the Howard Hughes Medical Institute. T. C. K. is an investigator of the Howard Hughes Medical Institute.
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