Report |
Address correspondence to Maurizio Gatti, Dipartimento di Genetica e Biologia Molecolare, Università di Roma "La Sapienza," Piazzale A. Moro 5, 00185 Rome, Italy. Tel.: 39-0649912843. Fax: 39-064456866. E-mail: maurizio.gatti{at}uniroma1.it
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Abstract |
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Key Words: chromosome segregation; chromosome passengers; Aura B; centrosome; microtubule
* Abbreviations used in this paper: fsl, fusolo; MT, microtubule; Pav, Pavarotti; suo, solofuso.
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Introduction |
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A large body of work indicates that chromosomes also play an essential role in the formation of centrosome-containing spindles. When the nucleus of grasshopper spermatocytes is removed by micromanipulation before nuclear envelope breakdown, astral MTs disassemble and the spindle fails to form (Zhang and Nicklas, 1995). Studies performed in echinoderm, Drosophila, and Xenopus early embryos have shown that centrosomes can duplicate and form robust asters in the absence of chromosomes, but these asters fail to evolve into real spindles and do not undergo the ana-telophase morphological transformations that characterize chromosome-containing spindles (Sluder et al., 1986; Picard et al., 1988; Raff and Glover, 1989; Sawin and Mitchison, 1991). Similar results have been recently obtained using PtK homokaryons, where centrosomes lacking associated chromosomes give rise to metaphase-like spindles that fail to turn into normal ana-telophase spindles (Faruki et al., 2002). Interestingly, also in acentrosomal systems, such as mouse meiosis, chromatin-free bipolar spindles do not have the ability to evolve into ana-telophaselike configurations (Brunet et al., 1998). Together, these results have led to the view that chromosomes play an essential role in spindle formation and dynamics both in acentrosomal and centrosome-containing systems (Waters and Salmon, 1997; Karsenti and Vernos, 2001).
Here, we show that Drosophila secondary spermatocytes devoid of chromosomes assemble metaphase-like spindles that evolve into telophase spindles. These chromosome-free cells also assemble regular cytokinetic structures and cleave normally. These results indicate that in Drosophila spermatocytes, spindle formation and dynamics are controlled by chromosome-independent factors.
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Results and discussion |
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To characterize the meiotic phenotype of fsl and suo, we made larval and adult testis preparations that were simultaneously stained for tubulin, centrin, and DNA. The antihuman centrin (HsCen1p) antibody (Paoletti et al., 1996) decorates Drosophila centrioles (Riparbelli et al., 2002), facilitating distinction between first and second meiotic divisions, which display two and one centriole at each pole, respectively. The analysis of fsl1/Df, fsl1/fsl1, fsl2/Df, and fsl2/fsl2 testes showed that these mutant combinations do not substantially differ in terms of severity of the phenotype, displaying a common defect in chromosome segregation. Thus, we focused on fsl1/fsl1 and fsl1/Df for detailed characterization of the meiotic phenotype. In fsl1/fsl1 and fsl1/Df, meiotic prometaphase and metaphase I figures are normal (Fig. 1 c). However, in most ana-telophases, chromosome segregation is disrupted (Fig. 1, d and e; Table I). In approximately half of mutant ana-telophase I cells, all chromosomes segregate to one pole only (Fig. 1 e and Table I), leading to the formation of secondary spermatocytes that are completely devoid of chromosomes (Fig. 2). Chromosome-containing fsl secondary spermatocytes form a regular spindle and exhibit the same aberrant chromosome behavior seen in the first meiotic division (unpublished data; see Fig. 5 a). In fsl secondary spermatocytes without chromosomes, centrosomes nucleate robust astral arrays of MTs that move to the opposite cell poles (Fig. 2 a'). These asters give rise to metaphase-like spindles devoid of chromosomes that differ from their wild-type counterparts only for the absence of kinetochore fibers (Fig. 2, a and a'). It should be noted that in these chromosome-free spindles, there is limited overlapping between the antiparallel MTs emanating from the opposite poles (Fig. 2 a'). However, little or no overlapping of these MTs is also seen in wild-type metaphase spindles (Fig. 2 a; Cenci et al., 1994). Chromosome-free spindles evolve into an anaphase A-like configuration, which again displays little or no MT overlapping at the center of the cell, as occurs in wild-type anaphases (Fig. 2, b and b'; Cenci et al., 1994). These anaphase A-like spindles undergo anaphase B (Fig. 2, c and c'), assemble a morphologically normal central spindle, and elongate to form telophase figures that are indistinguishable from their wild-type counterparts (Fig. 2, de'). It should be noted that in fsl mutants, the frequency of chromosome-free metaphase/early anaphase II figures and the frequency of chromosome-free telophase II figures are comparable (Table I). This indicates that most (if not all) metaphase-like spindles without chromosomes have the ability to form a central spindle and to proceed to telophase.
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In contrast to Drosophila spermatocytes, chromosome-free metaphase-like spindles of PtK homokaryons are unable to evolve into a typical telophase structure. In these peculiar spindles, the antiparallel MTs emanating from the centrosomes give rise to a compact MT bundle that fails to bind the MKLP (CHO1) kinesin, which accumulates at the central spindle midzone in chromosome-containing cells (Faruki et al., 2002). Thus, we asked whether the central spindles of chromosome-free telophases have the ability to bind Pavarotti (Pav), the Drosophila orthologue of MKLP (Adams et al., 1998). This analysis revealed that these telophases normally accumulate Pav in their midzones (Fig. 3), indicating a correct organization of central spindle MTs.
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We have shown that chromosome-free spermatocytes assemble regular cytokinetic structures and cleave normally, indicating that chromosomes are not the source of signals that stimulate cytokinesis. These findings are consistent with the micromanipulation experiments on grasshopper spermatocytes, showing that elimination of chromosomes from metaphase cells does not prevent them to proceed through anaphase and telophase and undergo cytokinesis (Zhang and Nicklas, 1996). They also agree with the classic Rappaport's experiments on echinoderm eggs (Rappaport, 1986), and with more recent experiments on vertebrate cells, showing that ectopic cytokinesis can occur between adjacent asters of different chromosome-containing spindles placed in the same cytoplasm (Eckley et al., 1997; Rieder et al., 1997; Savoian et al., 1999). Previously, we have shown that in Drosophila spermatocytes the signals for cytokinesis are not generated by the asters. asterless spermatocytes, which are devoid of asters due to a primary defect in centrosome assembly, form poorly focused anastral spindles. However, these spindles eventually organize morphologically normal central spindles that are fully able to support cytokinesis (Bonaccorsi et al., 1998). Thus, the results on asterless, fsl, and suo indicate that neither the asters nor the chromosomes are required for signaling cytokinesis in Drosophila spermatocytes. This suggests that in this system, the central spindle is both necessary and sufficient to stimulate cytokinesis.
Our results indicate that Drosophila secondary spermatocytes can form a morphologically normal spindle in the absence of chromosomes, and thus, in the absence of a high concentration of Ran-GTP in the center of the cell. The assembly of a metaphase-like bipolar spindle in the absence of chromosomes has been observed in several systems (see Introduction), including mouse oocytes (Brunet et al., 1998) and PtK homokaryons (Faruki et al., 2002). However, all these metaphase-like MT arrays are unstable and fail to proceed through ana-telophase. Thus, it has been suggested that in most centrosome-containing animal cells, chromosomes are not required for initial spindle morphogenesis, but for the stabilization of the structure and its evolution toward an ana-telophase configuration (Faruki et al., 2002).
In contrast with these systems, the metaphase-like chromosome-free spindles of Drosophila spermatocytes are sufficiently stable to undergo anaphase and telophase. We would like to point out that Drosophila spermatocytes behave differently from those of grasshopper, where enucleation of late prophase spermatocytes inhibits spindle assembly (Zhang and Nicklas, 1995). Yet, elimination of chromosomes from grasshopper metaphase spermatocytes does not affect the ability of the spindle to proceed through ana-telophase (Zhang and Nicklas, 1996). However, the latter finding may reflect incomplete elimination of a critical chromosome-associated factor (e.g., Ran-GTP) from the micromanipulated cell. Regardless the interpretation of these grasshopper experiments, it is clear that in Drosophila spermatocytes, chromosome-independent factors control spindle formation and dynamics. However, both the nature of these factors and the mechanisms underlying progression of chromosome-free spindles from a metaphase-like to a telophase-like structure remain to be determined.
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Materials and methods |
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To map suo and fsl, we used the second and third chromosome deficiency kits (provided by the Bloomington Stock Center, Indiana University, Bloomington, IN; http://flystocks.bio.indiana.edu/), respectively. Each of these kits includes a set of selected deficiencies that uncover about two thirds of the chromosome. fsl/TM6C and suo/CyO females were crossed to males from each pertinent deficiency stock, and the mutant/Df males from each cross were tested for fertility. Sterile mutant/Df males were then examined cytologically to determine their meiotic phenotype.
Cytological procedures
The double-staining techniques for actin/tubulin, myosin II/tubulin, and anillin/tubulin (the anti-myosin and -anillin antibodies were provided by C. Field, Harvard Medical School, Boston, MA) were described previously (Giansanti et al., 1999). For double immunostaining of centrin/tubulin and Aurora B/tubulin, testes were fixed according to protocol 3 of Giansanti et al. (1999). Testis preparations were incubated for 1 h in 1% BSA in PBS. They were then incubated overnight with both a monoclonal anti-tubulin antibody (Amersham Biosciences) diluted 1:100 in PBS and either a rabbit anti-HsCen1p antibody (provided by M. Bornens, Institut Curie, Paris, France; Paoletti et al., 1996) or a rabbit anti-Aurora B antibody (provided by D. Glover, University of Cambridge, Cambridge, UK; Giet and Glover, 2001) diluted 1:500 and 1:200 in PBS, respectively. Primary antibodies were detected using a FITC-conjugated antimouse (diluted 1:20; Jackson ImmunoResearch Laboratories) and a CY3-conjugated antirabbit (diluted 1:100; Jackson ImmunoResearch Laboratories) secondary antibodies. After two washes (5 min each) in PBS, slides were mounted in Vectashield® plus DAPI (Vector Laboratories) to stain DNA. Live spermatid preparations were obtained as described by Cenci et al. (1994), and were analyzed by phase contrast optics.
Immunostained and live preparations were examined using a microscope (Axioplan; Carl Zeiss MicroImaging, Inc.) equipped with an HBO 50-W mercury lamp for epifluorescence and with a cooled charge-coupled device (CCD; Photometrics), as described by Bonaccorsi et al. (1998). Grayscale images were collected separately using the IPLab Spectrum software (Scanalytics). They were then converted into Photoshop® 5.5 and used as such, or merged in pseudocolors.
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Acknowledgments |
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Submitted: 7 November 2002
Revised: 31 January 2003
Accepted: 6 February 2003
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References |
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