Spindle formation in female meiosis differs from mitosis in many animals,as it takes place independently of centrosomes, and the molecular requirements of this pathway remain to be understood. Here, we report two crucial roles of Incenp, an essential subunit of the chromosomal passenger complex (the Aurora B complex), in centrosome-independent spindle formation in Drosophilafemale meiosis. First, the initial assembly of spindle microtubules is drastically delayed in an incenp mutant. This clearly demonstrates,for the first time, a crucial role for Incenp in chromosome-driven spindle microtubule assembly in living oocytes. Additionally, Incenp is necessary to stabilise the equatorial region of the metaphase I spindle, in contrast to mitosis, where the equivalent function becomes prominent after anaphase onset. Our analysis suggests that Subito, a kinesin-6 protein, cooperates with Incenp for this latter function, but not in microtubule assembly. We propose that the two functions of Incenp are part of the mechanisms that compensate for the lack of centrosomes during meiotic spindle formation.
Cell division is developmentally controlled depending on specific cell requirements. Meiosis is a specialised cell division that produces sperm and oocytes. It differs from mitosis in many aspects, including cell cycle control, chromosome dynamics and spindle morphogenesis. One of the main differences in spindle morphogenesis is the contribution of centrosomes. Unlike mitosis, where centrosomes play a central role in spindle assembly, a bipolar spindle forms without centrosomes during female meiosis in many animals, including humans and Drosophila(McKim and Hawley, 1995; Waters and Salmon, 1997).
The crucial, but unanswered, question is what are the differences in the molecular requirements and regulation of spindle formation between female meiosis and mitosis? Although a bipolar spindle can still form without centrosomes in mitosis if they are artificially eliminated(Khodjakov et al., 2000; Basto et al., 2006), the acentrosomal spindle in female meiosis is much more robust and is likely to possess mechanisms that compensate for the lack of centrosomes.
In the absence of centrosomes, chromosomes play a central role in spindle microtubule assembly. In vitro studies using Xenopus extracts have revealed central roles of the Ran-importin system in chromosome-mediated spindle microtubule assembly (Gruss et al., 2001; Wiese et al.,2001), whereas recent studies in living mouse oocytes suggest the existence of a Ran-independent pathway(Dumont et al., 2007; Schuh and Ellenberg, 2007). In vitro studies found a Ran-independent involvement of the chromosomal passenger complex in spindle microtubule assembly(Sampath et al., 2004; Kelly et al., 2007), but in vivo studies have not so far indicated such a role(Adams et al., 2001; Giet and Glover, 2001; Gassmann et al., 2004; Andrews et al., 2004; Lan et al., 2004; Resnick et al., 2006). Therefore, a crucial issue remains unresolved: whether and how much the chromosomal passenger complex actually contributes to spindle microtubule assembly in living oocytes.
Furthermore, spindle bipolarity needs to be established and maintained without centrosomes in female meiosis. DNA-coated beads in Xenopusextracts can organise a bipolar spindle without centrosomes or kinetochores,indicating that spindle bipolarity is generated by the self-organisation of microtubules into anti-parallel arrays(Heald et al., 1996). Genetic studies in Drosophila revealed that Subito, the MKLP-2 homologue(kinesin-6), localises to the equatorial region of the metaphase I spindle,and is required for its organisation and bipolarity(Giunta et al., 2002; Jang et al., 2005; Jang et al., 2007). The equatorial region in the metaphase I spindle accumulates proteins that normally localise to the central spindle of mitotic anaphase/telophase,including the chromosomal passenger complex(Jang et al., 2005). Therefore, it has been proposed that the equatorial region of the meiotic metaphase I spindle is actually equivalent to the central spindle in mitotic anaphase/telophase, and that this structure (sometimes referred to as the meiotic metaphase central spindle) is crucial to establishing spindle bipolarity in the absence of centrosomes(Jang et al., 2005).
Despite these biochemical and genetic studies, our knowledge of spindle formation in female meiosis is still limited at the molecular level. In this study, we identify an incenp mutant that disrupts the bipolarity of the metaphase I spindle in Drosophila female meiosis. Incenp is an essential subunit of the chromosomal passenger complex containing Aurora B kinase. The chromosomal passenger complex plays multiple roles in mitosis and meiosis (Vagnarelli and Earnshaw,2004; Ruchaud et al.,2007), but no role has been reported in spindle morphogenesis in female meiosis.
Here, we show that Incenp has two roles in the acentrosomal spindle formation of Drosophila female meiosis. First, live imaging analysis showed that the initial assembly of spindle microtubules is drastically delayed in the incenp mutant. This is the first and definitive in vivo demonstration of a crucial role for a subunit of the chromosomal passenger complex in the centrosome-independent spindle microtubule assembly. Furthermore, we found that Incenp is required for the stability of the spindle equatorial region in meiotic metaphase I to prevent formation of ectopic poles. This is consistent with the precocious localisation of Incenp to the spindle equatorial region at metaphase. These two functions of Incenp might be part of the mechanisms that compensate for the lack of centrosomes in female meiosis.
MATERIALS AND METHODS
Genetic, molecular and immunological techniques
Standard techniques of fly manipulation were followed(Ashburner et al., 2005). All stocks were grown at 25°C in standard cornmeal media. w1118 was used as wild type. Details of mutations and chromosome aberrations can be found in Lindsley and Zimm(Lindsley and Zimm, 1992) or at FlyBase(http://flybase.org)(Drysdale et al., 2005). For live-imaging, UASp-GFP-α-tubulin and GAL4 under the maternal nanos promotor on the third chromosome were used.
Standard DNA manipulation and immunological techniques were used throughout(Sambrook et al., 1989; Harlow and Lane, 1988). The primary antibodies used in this study include antibodies againstα-tubulin (DM1A; Sigma), γ-tubulin (GLU-88; Sigma), D-TACC(Gergely et al., 2000; Cullen and Ohkura, 2001),Cyclin B (Whitfield et al.,1990), Subito (Jang et al.,2005), Incenp (C. Wu and K.M., unpublished) and Aurora B(Adams et al., 2001). For immunoblots, peroxidase-conjugated antibodies (Jackson Lab) were used as secondary antibodies for western blot and detected by ECL Kit (Pharmacia).
Molecular identification of the gene mutated in QA26
For the molecular identification of the female sterile mutations on the QA26 chromosome, it was first mapped by recombination with chromosomes carrying visible markers. The region was further narrowed by complementation testing using deficiencies in the area. The female sterile mutation in QA26 is located between the proximal breakpoints of Df(2R)Drlrv30and Df(2R)ED1715. A tight linkage of the female sterile mutation with spindle defects in female meiosis was confirmed by cytological analysis of the mutant chromosome over small deficiencies. To test whether the female sterile mutation is in the incenp locus, a lethal incenp3747 mutation(Chang et al., 2006) was used for complementation. For further confirmation, the incenp-coding region was amplified by PCR from the QA26 genomic DNA and sequenced using BigDye (Applied Biosystems).
Immunostaining was carried out, as previously described, for D-TACC/Cyclin B (Tavosanis et al., 1997; Cullen and Ohkura, 2001) or Incenp/Aurora B (Theurkauf and Hawley,1992; Jang et al.,2005) with α-tubulin in non-activated oocytes, and for D-TACC with α-tubulin in activated oocytes(Cullen et al., 2005). Secondary antibodies conjugated with Cy3, Cy5 or Alexa488 (Jackson Lab or Molecular Probes) were used at 1/250-1/1000 dilution. DNA was counterstained with Hoescht, DAPI (0.4 μg/ml; Sigma) or propidium iodide (2 μg/μl,Sigma). The images were taken using a Plan-Apochromat lens (63×, 1.4NA;Zeiss) attached to an Axiovert 200M (Zeiss) with a confocal scan head(LSM510meta; Zeiss). Confocal images were presented as a maximum intensity projection of the z-stacks. All digital images were imported to Photoshop/ImageReady (Adobe), and the brightness and contrast were uniformly adjusted for the whole field without changing features of the images. Statistical significance was calculated using Student's t-test.
Live-imaging of meiosis I spindle was carried out as described(Matthies et al., 1996; Endow and Komma, 1997) except maternally expressed GFP-α-tubulin was used (GAL4-nosUTR combined with UASp-GFP-α-tubulin). Briefly, oocytes were dissected from matured adult females in halocarbon oil (700) and observed using the confocal microscope described above. Typically, a series of z-sections (separated by 1μm) that cover the entire spindle was taken every 20-35 seconds.
Identification of an incenp mutant defective in the metaphase I spindle in female meiosis
To understand how the acentrosomal spindle is formed in female meiosis, we have cytologically screened collections of female sterile mutants for spindle defects in metaphase I-arrested oocytes. From the collection made by Schupbach and Wieschaus (Schupbach and Wieschaus,1989), we found that a mutant, QA26, showed an abnormal spindle morphology. It was originally classified as showing `no visible sign of development' (Schupbach and Wieschaus,1989) along with γ-tubulin37C (TW1), subito and cks30A (rem), mutants that were later found to have spindle defects at metaphase I(Tavosanis et al., 1997; Giunta et al., 2002; Pearson et al., 2005).
By recombination and deficiency mapping, we localised the mutation to genomic region 43A1-43A4 (see Materials and methods). This region contains eight genes, including incenp, which is known to regulate mitosis. The mutant chromosome did not complement a lethal incenp allele and has a point mutation that alters a conserved residue within the IN box, the domain responsible for interaction of Incenp with Aurora B(Adams et al., 2000). This demonstrates that the mutation is an allele of incenp. A parallel study (Resnick et al., 2006)has already reported QA26 as an incenp allele and described its defects in male meiosis.
In this report, we focused on the study of spindle defects observed in non-activated mutant oocytes, although we also observed abnormal meiotic progression in at least some of the activated mutant oocytes (see Fig. S1 in the supplementary material). As incenp is an essential gene, the phenotype we observed might not represent the full range of Incenp function. Nevertheless, this hypomorphic allele allows us to uncover the role of Incenp in female meiosis.
The incenp mutant forms ectopic spindle poles in female meiosis
Incenp is an essential subunit of the chromosomal passenger complex containing Aurora B kinase. The complex accumulates at centromeres during early mitosis, and then translocates to the spindle equatorial region/central spindle after initiation of anaphase (Adams et al., 2001). In contrast to mitosis, a previous report(Jang et al., 2005) showed that, in female meiosis, Aurora B and Incenp accumulate on the spindle equatorial region in metaphase. We found that the incenpQA26 mutation disrupts the morphology of the metaphase I-arrested spindle in female meiosis.
To determine the role of Incenp in meiotic spindle formation, we examined the morphology of metaphase I-arrested spindles in mature non-activated oocytes by immunostaining. The oocytes were fixed and stained forα-tubulin, the pole protein D-TACC and DNA. In wild type, the metaphase I-arrested spindle is bipolar with tapered poles(Fig. 1A). Bivalent chromosomes are aligned at the spindle equator with the achiasmatic small 4th chromosomes usually located symmetrically closer to the poles. In the incenpmutants, spindle organisation was disrupted in over 50% of oocytes(n=69; Fig. 1D). Although the abnormality varies from oocyte to oocyte, typical defects include the formation of one or more ectopic spindle poles usually around the equator or next to the main poles (Fig. 1B,C). These poles typically have the pole protein D-TACC correctly localised. These results showed that Incenp is required for the proper organisation of the metaphase I-arrested spindle in Drosophilafemale meiosis. Chromosome alignment or location was affected to a lesser extent (32% abnormal compared with 15% in wild type). However, it is difficult to conclude which process is primarily defective, spindle formation or chromosome alignment (or both), as these two processes are inter-dependent during acentrosomal spindle assembly.
Instability of spindle bipolarity during metaphase I arrest in the incenp mutant
To further understand the spindle abnormalities in the incenpmutant, we examined the metaphase I-arrested spindle by live-imaging analysis. We first analysed spindle dynamics in a wild-type background using maternally driven GFP-α-tubulin (see Materials and methods). Oocytes were dissected in halocarbon oil and examined under a confocal microscope. In wild type, a metaphase I-arrested spindle maintained its overall shape and bipolarity over time (Fig. 2A; Movie 1 in the supplementary material), consistent with previous reports using a Ncd-GFP transgene or injection of fluorescently labelled tubulin(Matthies et al., 1996; Endow and Komma, 1997).
For live-imaging observation of mutant spindles, we introduced GFP-α-tubulin transgenes into the incenp mutant by successive genetic crosses. Consistent with our immunostaining results, among the 47 metaphase I-arrested spindles we observed, about 40% (19) showed an abnormal morphology, typically exhibiting ectopic or split poles, at the beginning of the observation. Half of these abnormal spindles (9) became bipolar during our observation that typically lasted for 20-40 minutes, whereas some of the others (6) changed their morphology but stayed abnormal. Conversely, most of the initially bipolar spindles (20) remained bipolar during the observation;however, others (8) lost their bipolarity through the appearance of ectopic poles in most cases or, less often, splitting of poles. Ectopic poles usually grew from the spindle equatorial region, and spindle bipolarity was restored by disassembling the ectopic pole or merging it with one of the main poles(Fig. 2B; see Movie 2 in the supplementary material, which shows the clearest example). In total, more than a third of all the observed spindles (17) showed at least one inter-conversion of their morphology between bipolar and abnormal during our observation. Therefore, Incenp is required for the stability of spindle bipolarity during metaphase I arrest.
To understand the requirement for Incenp during spindle formation in female meiosis, we followed spindle formation from beginning of nuclear envelope breakdown. Prophase oocytes expressing GFP-α-tubulin were selected for study and their progression was followed over time. In wild type(Fig. 3A; see Movie 3 in the supplementary material), GFP-α-tubulin was excluded from the prophase nucleus, until just before nuclear envelope breakdown, when it entered the nucleus. After nuclear envelope breakdown, the GFP-α-tubulin diffused into the cytoplasm. After a short gap, microtubules assembled around the cluster of meiotic chromosomes (called the karyosome), which can be recognised as a dark spherical shape (that excludes the GFP signal). Multiple transitory poles were formed during very early stages of spindle assembly, but one axis quickly became dominant. Once one axis was established, it was maintained without forming other poles. Then the poles were focused and the spindle elongated before arresting in metaphase. These observations were in agreement with previous reports using Ncd-GFP or injection of fluorescently labelled tubulin (Matthies et al.,1996; Endow and Komma,1997).
Similarly to wild type, in the incenp mutant, spindle microtubules were assembled around the chromosomes after nuclear envelope breakdown, and multiple transitory poles appeared at the beginning of spindle formation. However, unlike wild type, even after one spindle axis became dominant, other poles continued to be formed. These ectopic poles eventually fused with the original poles during spindle formation. In the time sequence shown in Fig. 3B (see Movie 4 in the supplementary material), soon after microtubules were assembled around the chromosomes, multiple poles were temporarily formed. Eventually two dominant poles were established. Then a third pole (arrow in Fig. 3B) appeared near the spindle equatorial region and merged with one of the main poles to re-establish bipolarity. In total, six out of 14 incenp oocytes observed by us showed abnormalities during the formation of the meiotic spindle, whereas all the 10 wild-type oocytes observed behaved normally. In summary, time-lapse observation of meiotic spindle formation revealed that the incenp mutant exhibits instability of spindle bipolarity,particularly near the spindle equatorial region, before and after the metaphase I arrest.
The spindle equatorial region is partially defective in the incenp mutant
The instability of the spindle equatorial region in the incenpmutant might be caused by a failure to recruit other proteins to this region and/or by defective organisation of this region. First, we examined the localisation of Incenp and Aurora B in incenp mutant oocytes. We found that the mutant Incenp protein localised to the spindle equatorial region (Fig. 4B) as does the wild-type Incenp protein (Fig. 4A). Aurora B was also accumulated in the equatorial region,although we are uncertain about the level of the accumulation relative to wild type, owing to a high background (Fig. 4B). Next, we examined the effect of the incenp mutation on Cyclin B which also localises to the spindle equatorial region in wild-type female meiosis (Pearson et al.,2005). Immunostaining indicated that Cyclin B was still localised to the spindle equatorial region in the incenp mutant(Fig. 4C).
To quantify the integrity of the spindle equatorial region in the incenp mutant, we compared the relative microtubule density of this spindle region in wild-type and mutant oocytes expressing GFP-α-tubulin. We measured the intensity of GFP-α-tubulin along the spindle axis(Fig. 4E). In wild type, the spindle equatorial region gave an average of 40% higher GFP-tubulin signal than the pole regions, probably representing the overlapping anti-parallel microtubule array in the equatorial region(Fig. 4D,F). In the incenp mutant, by contrast, the GFP-tubulin signal at the spindle equatorial region was significantly reduced (P<0.01) to a level comparable with that of the pole regions(Fig. 4D,F). This result showed that the spindle equatorial region is structurally, as well as functionally,defective in the incenp mutant.
Our analysis demonstrated that Incenp is required for the stability and organisation of the spindle equatorial region in prometaphase and metaphase in female meiosis. This is consistent with a previous report showing that Incenp precociously localises to the spindle equatorial region in prometaphase and metaphase in female meiosis (Jang et al.,2005), which is in contrast to mitosis or male meiosis when it is localised to centromeres prior to anaphase(Adams et al., 2001; Resnick et al., 2006).
The incenp mutation delays spindle microtubule assembly in female meiosis
In addition to defects in spindle bipolarity, we also noticed that the initiation of spindle microtubule assembly was considerably delayed in the incenp mutant. For quantification, we measured the time between nuclear envelope breakdown and the first appearance of microtubules around the chromosomes. This process took an average of 354 seconds (∼6 minutes) in wild-type oocytes, whereas it was delayed threefold to 1128 seconds (∼19 minutes) in the incenp mutant(Fig. 5A). The difference is statistically significant (P<0.001).
To confirm that this delay was not due to reduced levels of tubulin, we examined the amounts of α- and γ-tubulin in ovaries. Immunoblots showed that the levels of the tubulins were not significantly affected by the incenp mutation (Fig. 5C). However, the length of the metaphase I spindle in the incenp mutant was not significantly different from that in the wild-type oocytes (Fig. 5B). Therefore, the spindle length appears to be determined by mechanisms that do not crucially depend on Incenp activity.
In conclusion, these results showed a crucial role of Incenp in the initial assembly of microtubules around chromosomes. Although this function was previously indicated by a study using Xenopus extract(Sampath et al., 2004), this is the first in vivo evidence to demonstrate the involvement of Incenp, or any subunits of the chromosomal passenger complex, in the assembly of spindle microtubules.
A subito-null mutation induces instability of the central spindle but does not delay microtubule assembly
Subito, a kinesin-6 protein, has previously been shown to localise to the equatorial region of the meiotic metaphase I spindle and is required for the localisation of the chromosomal passenger complex and all known proteins recruited to the equatorial region (Jang et al., 2005). Consistent with this, immunostaining indicated that the incenp and subito mutants show similar phenotypes.
To further explore the relationship between Incenp and Subito, we first examined whether Subito localisation is affected by the incenpmutation. Metaphase I-arrested spindles were immunostained for α-tubulin and Subito. We found that the Subito protein still localised to the spindle equatorial region in the incenp mutant(Fig. 6A), contrasting with Incenp delocalisation in a subito mutant(Jang et al., 2005). This shows that the defects observed in the incenp mutant are not due to Subito delocalisation.
Next, we examined spindle organisation in a subito-null mutant by live-imaging analysis. We introduced GFP-α-tubulin and GAL4 transgenes into the subito mutant, and dissected oocytes were observed under a confocal microscope. Live-imaging of metaphase I-arrested oocytes revealed spindle instability. During our observations, an ectopic pole formed around the equatorial region in about half of the spindles (11/18) that were initially bipolar. In most cases, the bipolarity was restored as this ectopic pole merged with one of the main poles(Fig. 6B; see Movie 5 in the supplementary material). Additionally, among the spindles that exhibited ectopic poles at the beginning of our observation, about a half (9/16) became bipolar before the end.
Consistent with a requirement of Subito for Incenp localisation, the instability of spindle bipolarity is shared by both incenp and subito mutants. In addition, both mutants exhibited low microtubule density at the spindle equatorial region (this study)(Jang et al., 2005). However,there are some notable differences between the subito and incenp mutants. First, in the subito mutant, splitting of the poles was not observed as it was in the incenp mutant. Second,the subito mutant showed much more frequent and dynamic inter-conversions of spindle morphology between bipolar and tripolar. These differences might be due to the stronger nature of the subitomutation and/or to other spindle equatorial region proteins affected by the subito mutation.
To assess whether Subito functions in the assembly of spindle microtubules,we also measured the time from nuclear envelope breakdown to the first appearance of spindle microtubules in the subito mutant. Unlike the incenp mutant, which takes three times longer than the wild type to begin spindle microtubule assembly, the subito mutant did not show a significant delay (354 seconds in wild type versus 408 seconds in subito; P=0.45). These results clearly showed that this function of Incenp is independent of Subito.
From a screen of female sterile mutants for spindle defects in female meiosis, we identified a mutant of Incenp, an essential subunit of the chromosomal passenger complex containing Aurora B kinase. Live-imaging analysis of the mutant revealed roles of Incenp in two crucial steps of the acentrosomal spindle formation in female meiosis. The first is to assemble spindle microtubules around chromosomes, and the second is to stabilise the spindle equatorial region to maintain spindle bipolarity. These two functions are separable and differentially regulated in terms of their requirement for Subito, a kinesin-like protein.
The function of Incenp in spindle microtubule assembly in female meiosis
In the absence of centrosomes, which are the major sites of microtubule nucleation in mitosis, chromosomes appear to play a crucial role in the assembly of spindle microtubules. In recent years, the molecular basis of this activity of chromosomes has been under intense investigation. Beads coated with phage DNA can assemble microtubules in Xenopus extract without centrosomes or kinetochores (Heald et al.,1996). Mainly using the Xenopus in vitro system, a great deal of evidence has been accumulated to support the hypothesis that Ran activated by a chromosome-associated factor, Rcc1, plays a central role in the assembly of spindle microtubules in the absence of centrosomes(Carazo-Salas et al., 1999; Kalab et al., 2002; Goodman and Zheng, 2006). Despite this compelling evidence obtained in in vitro studies, the extent of Ran involvement in the process is more ambiguous in vivo. Recent studies in mouse oocytes suggest the existence of a Ran-independent spindle assembly pathway in female meiosis (Dumont et al.,2007; Schuh and Ellenberg,2007).
Candidates responsible for this Ran-independent spindle assembly pathway include the chromosomal passenger complex, evidence for which again comes from an in vitro study using the Xenopus system(Sampath et al., 2004). In this system, depletion of Incenp or other subunits of the chromosomal passenger complex prevents spindle microtubule assembly(Sampath et al., 2004), and Aurora B can be activated by chromosomes independently from Ran(Kelly et al., 2007). Consistent with this, Aurora B can phosphorylate and inhibit the microtubule destabilising proteins Kinesin-13 and Op18(Andrews et al., 2004; Lan et al., 2004; Ohi et al., 2004; Zhang et al., 2007; Gadea and Ruderman, 2006). In contrast to this in vitro evidence, inhibition of the chromosomal passenger complex (or inhibition of Kinesin-13 phosphorylation) produces only a limited defect in microtubule assembly in mitosis in vivo(Adams et al., 2001; Giet and Glover, 2001; Gassmann et al., 2004; Andrews et al., 2004; Lan et al., 2004). We found that our incenp mutant takes three times longer to initiate spindle microtubule assembly after nuclear envelope breakdown in oocytes. Our results thus provide the first and definitive in vivo demonstration that a subunit of the chromosomal passenger complex is required for efficient assembly of spindle microtubules in female meiosis.
The role of Incenp in the equatorial region of the meiotic metaphase spindle
Evidence from the Xenopus in vitro system indicated that bipolar spindles can be formed without centrosomes or kinetochores, suggesting that microtubules can self-organise into a bipolar spindle(Heald et al., 1996). A likely candidate for the basis of spindle bipolarity is anti-parallel bundling of spindle microtubules at the spindle equatorial region.
The incenp mutant reduces microtubule density in the spindle equatorial region relative to the polar regions, which suggests that the overlap and/or bundling of anti-parallel microtubules is compromised. A bipolar spindle can assemble, but tends to lose its bipolarity by forming ectopic poles around the spindle equatorial region. Eventually, the bipolarity is restored by the merging of poles. The origin of the ectopic poles is unclear, as single microtubules cannot be resolved in our live-imaging analysis in oocytes. The microtubules forming the ectopic poles may originally be derived from spindle microtubules, may grow from chromosomes, or may be spontaneously nucleated in the cytoplasm. In wild type, these microtubules are likely to be quickly bundled and aligned with existing spindle microtubules. However, in the incenp mutant, they can temporarily retain an independent orientation from existing spindle microtubules, possibly owing to compromised anti-parallel bundling in the spindle equatorial region.
Incenp and Aurora B kinase localise to the equatorial region of the meiotic metaphase I spindle (Jang et al.,2005). Consistent with this, we have shown that Incenp function is essential for the organisation of the spindle equatorial region in meiotic metaphase I, and for the establishment and maintenance of spindle bipolarity. Our results reinforce the hypothesis that anti-parallel microtubule bundling in the spindle equatorial region plays a central role in establishing bipolarity of meiotic metaphase I spindles in order to compensate for the lack of centrosomal activity.
Independent regulation of the two Incenp functions
Our study uncovered two functions of Incenp for acentrosomal spindle formation in oocytes. Both functions are likely to be mediated by Aurora B,although we cannot exclude the possibility that Incenp has roles independent of Aurora B.
Subito is a kinesin-like protein that plays a crucial role in the assembly of the spindle equatorial region. It is required for the localisation of other proteins to this region, including the chromosomal passenger complex(Jang et al., 2005). Consistent with this, our immunostaining and live-imaging showed that the subito mutant and the incenp mutant produce similar defects in spindle bipolarity. The stronger phenotype in the subito mutant is likely to be due to other proteins affected by the subito mutation,or to the hypomorphic nature of the incenp mutation.
Crucially, we found that the assembly of spindle microtubules is not delayed in the subito mutant, whereas it is greatly delayed in the incenp mutant. This indicates that the early function of Incenp in spindle microtubule assembly is independent of Subito. This function may be mediated through phosphorylation and inhibition of microtubule depolymerising proteins by Aurora B. In conclusion, the two functions of Incenp in spindle microtubule assembly and stabilisation of spindle bipolarity are differentially regulated in female meiosis.
The chromosomal passenger complex in centrosome dependent and independent spindle formation
In the light of our findings in female meiosis, the issue is whether the chromosomal passenger complex plays similar roles in centrosome-dependent spindle formation in mitosis or male meiosis. It has been proposed that Aurora B activity is involved in the regulation of microtubule dynamics at kinetochores upon improper microtubule attachment(Lampson et al., 2004; Andrews et al., 2004; Lan et al., 2004; Ohi et al., 2004). However,there is little evidence to support the possibility that the chromosomal passenger complex is required for general microtubule assembly in mitosis. Although the centrosome is the major microtubule nucleation site in mitotic cells, the activity of chromosomes to stabilise microtubules is thought to be important for the efficient capture of kinetochores by spindle microtubules(Wollman et al., 2005). Furthermore, when centrosomes are eliminated in mitosis, spindle microtubules are still assembled around chromosomes(Khodjakov et al., 2000; Basto et al., 2006). Ran-GTP is proposed to be responsible for these activities(Caudron et al., 2005), but involvement of Aurora B should be considered.
Does the chromosomal passenger complex play a role in spindle morphogenesis prior to anaphase in centrosome-dependent spindle formation? In Drosophila, before anaphase, the chromosomal passenger complex localises to centromeres in male meiosis but to the spindle equatorial region in female meiosis. The same hypomorphic mutation disrupts chromosome alignment in male meiosis but spindle bipolarity in female meiosis, without strong effects on the other functions (Resnick et al., 2006) (this study).
Although the pre-anaphase function of the chromosomal passenger complex at the spindle equatorial region has not attracted much attention in the past,there is some evidence to support a role for the complex in the spindle equatorial region prior to anaphase in mitosis. In some vertebrate cell lines,Incenp was observed to localise to the spindle equatorial region during late metaphase (Earnshaw et al., 1991). RNAi of the chromosomal passenger complex in Drosophila S2 cells induces defects in spindle bipolarity(Goshima et al., 2007). RNAi of Borealin in mammalian cells disrupts spindle bipolarity as ectopic poles split off from the bipolar spindle after establishment of metaphase but prior to anaphase in mitosis (Gassmann et al.,2004). Therefore, it is likely that the chromosomal passenger complex functions in the spindle equatorial region prior to anaphase both in mitosis/male meiosis and female meiosis. However, in female meiosis, this function becomes crucial, owing to the absence of centrosomes.
In summary, our study in female meiosis has revealed roles of Incenp in two vital steps of acentrosomal spindle formation: the assembly of spindle microtubules and the formation of a robust spindle equatorial region. So far,most studies of the chromosomal passenger complex have focused on centromeric functions in prometaphase and the central spindle/cytokinesis function in telophase in mitosis. Further studies will be required to establish to what extent the chromosomal passenger complex contributes to bipolar spindle assembly in mitosis and how different the regulation of the complex is between mitosis and acentrosomal meiosis.
We thank Will Whitfield for kindly providing Cyclin B antibody, Terry Orr-Weaver for sharing information and Sandrin Ruchaud for critical reading of the manuscript. We also thank Bloomington stock centre for providing fly stocks. This work is supported by The Wellcome Trust (051091, 081849) and NIH(R01GM067142).