subito
DEVELOPMENTAL BIOLOGY

Antibodies were raised against the poorly conserved C-terminus of the Sub protein that follows the motor domain. These antibodies recognize an ~75-kDa band on a Western blot that is absent in homozygotes of the null alleles sub1 and sub131. To examine the localization of Sub during meiosis, mature Drosophila oocytes (stage 14), which in wild type, are arrested at metaphase I, were stained (Jang, 2005).

Identification of prometaphase and metaphase spindles was based on studies of fixed and living oocytes. There is no congression to a metaphase plate in Drosophila oocytes. Instead, the chromosomes come together and condense into a ball to form the karyosome much earlier in oogenesis. After nuclear envelope breakdown (NEB), the chromosomes in the karyosome initiate spindle formation. The initial phases of spindle assembly are characterized by disorganized arrays of microtubules emanating from the karyosome. Once a bipolar spindle forms, it remains stable until the onset of anaphase when the oocyte is activated by passage through the oviduct. Staining for tubulin in wild-type oocytes revealed that metaphase I spindles have a prominent band of microtubules that run pole-to-pole and do not terminate at the chromosomes. The bright staining in the middle of this region probably represents the antiparallel overlap of microtubules. This region is referred to as the meiotic metaphase central spindle (MMCS) in order to distinguish it from the central spindle or midzone present in anaphase of mitotic cells (Jang, 2005).

Staining of wild-type oocytes with Sub antibodies revealed that Sub protein is found exclusively in the MMCS. Although Sub always colocalizes with tubulin staining, it is also closely associated with the chromosomes. In many spindles, 3D reconstruction revealed that Sub staining appears more concentrated on one side or in some cases in two clusters on either side of the karyosome. What remains to be determined is whether this pattern reflects intrinsic features of the spindle, such as asymmetry within the karyosome, or stochastic properties of central spindle formation. Sub staining was not detected in the null alleles sub1 and sub131 at prometaphase or metaphase, confirming the specificity of the antibody (Jang, 2005).

Because genetic and cytogenetic studies (Giunta, 2002) have suggested that Sub has an important role in spindle assembly, when Sub first appears on the meiotic spindle was investigated. Sub appears from the earliest time points after NEB, even on early prometaphase spindles that are characterized by a disorganized array of microtubules. Sub staining at this stage colocalizes with microtubules in the region that will become the MMCS. Thus, Sub localizes to the central spindle before bipolar spindle formation (Jang, 2005).

Stage 14 oocytes arrest at meiotic metaphase I; therefore, two methods were used to observe anaphase I spindles: (1) mei-218 mutants, in which the metaphase arrest is bypassed (McKim, 1993); (2) embryos were collected after short periods of egg laying, which allows for the isolation of oocytes undergoing the early meiotic divisions. In both of these experiments, Sub remains in the spindle midzone as the chromosomes moved toward the poles (Jang, 2005).

Because sub mutants are viable, it is possible that sub has a meiosis-specific function and is only required for the unique situation of assembling acentrosomal spindles during female meiosis. There is, however, genetic evidence that sub is expressed in mitotically dividing cells (Moore, 1994; Giunta, 2002). Consistent with these genetic observations, Sub staining is observed in mitotically dividing cells of the embryo. Similar to the metaphase oocytes, Sub protein was observed at the middle of the spindle. Sub protein has also been observed at metaphase of larval neuroblast cells. Thus, Sub protein may be a component of most or all metaphase spindles in Drosophila. Although Sub has an important function in pronuclear fusion, some sub mutant embryos commence but never complete the early embryonic divisions (Giunta, 2002). Analysis of these embryos shows evidence of spindle assembly defects and aneuploidy, indicating that sub also has a role in mitotic spindle function (Jang, 2005).

The chromosomal passenger complex is required for meiotic acentrosomal spindle assembly and chromosome biorientation

During meiosis in the females of many species, spindle assembly occurs in the absence of the microtubule-organizing centers called centrosomes. In the absence of centrosomes, the nature of the chromosome-based signal that recruits microtubules to promote spindle assembly as well as how spindle bipolarity is established and the chromosomes orient correctly toward the poles is not known. To address these questions, this study focused on the chromosomal passenger complex (CPC). The CPC localizes in a ring around the meiotic chromosomes that is aligned with the axis of the spindle at all stages. Using new methods that dramatically increase the effectiveness of RNA interference in the germline, it was shown that the CPC interacts with Drosophila oocyte chromosomes and is required for the assembly of spindle microtubules. Furthermore, chromosome biorientation and the localization of the central spindle kinesin-6 protein Subito, which is required for spindle bipolarity, depend on the CPC components Aurora B and Incenp. Based on these data it is proposed that the ring of CPC around the chromosomes regulates multiple aspects of meiotic cell division including spindle assembly, the establishment of bipolarity, the recruitment of important spindle organization factors, and the biorientation of homologous chromosomes (Radford, 2012).

Previous work using Xenopus egg extracts demonstrated that both RanGTP and the CPC are required for chromatin-induced spindle assembly. In contrast, RanGTP appears not to be required for acentrosomal spindle assembly in Drosophila (Cesario, 2011) and mouse oocytes. This study has shown that the CPC is essential for the accumulation of microtubules around the chromosomes in Drosophila oocytes, suggesting that in vivo the CPC is the critical factor for regulating acentrosomal spindle assembly. A model is presented for acentrosomal spindle assembly with implications for how the CPC simultaneously promotes bipolarity and homolog bi-orientation (Radford, 2012).

The results support a model in which the primary step in the establishment of meiotic spindle bipolarity is the accumulation of the CPC in a ring encircling the chromosomes. The enrichment of CPC proteins in a ring around the karyosome may provide the increased local concentration of Aurora B that has been postulated to be necessary to activate the Aurora B kinase for chromosome-based spindle assembly in Xenopus egg extracts. It is proposed that the CPC has two critical functions in Drosophila oocytes: it promotes microtubule accumulation near the chromosomes and also constrains microtubule growth into two poles by establishing the spindle axis. This replaces two functions of the centrosomes: recruitment of microtubules and organizing a bipolar spindle. Previous studies have suggested that the CPC promotes spindle assembly by suppressing the microtubule-depolymerizing activity of a kinesin-13 protein near the chromosomes. In contrast, this study has shown that down-regulating KLP10A, a Drosophila kinesin-13 protein known to regulate spindle length, is not a sufficient explanation for the activity of the CPC. While a role for the CPC in regulating two additional kinesin-13s encoded by the Drosophila genome, KLP59C and KLP59D cannot be ruled out, during acentrosomal spindle assembly, evidence summarized below suggests that the CPC positively regulates spindle assembly factors (Radford, 2012).

For the second function, constraining microtubule assembly towards two poles, a simple model is suggested by the shape of the ring: the ring may act like a tube that restricts microtubules to assemble in only two directions. Additionally, the CPC ring establishes the location for recruitment of other spindle assembly factors that regulate bipolarity, including Subito. A direct physical interaction between Subito and Incenp would be consistent with results showing that the mammalian Subito ortholog MKLP2 physically interacts with Aurora B and Incenp (Gruneberg, 2004). This must depend on Aurora B activity since no Subito localization was observed in plI-aurora-like kinase RNAi oocytes even though Incenp was associated with the chromatin. It is suggested that the CPC interacts with chromosomes in a ring, promotes microtubule accumulation, and recruits proteins like Subito to these microtubules, which results in the establishment or stabilization of antiparallel microtubules, spindle bipolarity, and the formation of two poles (Radford, 2012).

Subito and the CPC appear to have a mutual dependency. It has been shown previously that the meiotic central spindle localization of the CPC depended on Subito (Jang, 2005). To explain these results, it is suggested that the CPC is first recruited to the chromosomes, and then moves to the central spindle microtubules. In the absence of Subito and the central spindle microtubules, the interaction of Incenp with the chromosomes persists and the CPC does not move to the microtubules. While interacting with the chromosomes the CPC can apparently promote spindle assembly, but not bi-orientation (Radford, 2012).

What controls the localization of the CPC ring and how it gets targeted to the region between bi-oriented centromeres remains to be uncovered. In the absence of Aurora B, the localization pattern of Incenp within the karyosome is disorganized, suggesting that the kinase activity of the CPC may play a role in shaping the ring, but underlying features of the chromosomes may also be important. It is intriguing that the passenger proteins are not detected in the centromere regions as they are in mitotic and centrosomal meiotic cells. The results are consistent with data from C. elegans oocytes, showing that the CPC interacts with non-centromeric chromatin at metaphase of meiosis I. In C. elegans, the CPC forms a ring at the center of each bivalent that colocalizes with cohesion proteins distal to chiasmata. The C. elegans CPC ring is a complex structure which, like in Drosophila, contains motor proteins (Klp-19) and is required for segregation of homologs at meiosis I. The importance of non-centromeric CPC in a variety of organisms suggests that the unique demands of acentrosomal meiosis have resulted in a meiosis-specific CPC/central spindle localization pattern with a conserved role in spindle assembly and chromosome segregation. Finding out the identity or structural features of the chromosome locations to which the CPC ring localizes will be critical to understanding how the chromosomes organize acentrosomal spindles (Radford, 2012).

Centromeres are paired in Drosophila oocytes prior to NEB. Based on examination of oocytes depleted of the CPC and spindle assembly motors Subito and NCD, the following pathway leading to homolog bi-orientation is proposed. First, the CPC binds in a ring to the chromosomes and recruits spindle assembly factors such as Subito. This stage is defined by the observation that the CPC can bind chromosomes independent of microtubules and, in its absence, the microtubules and Subito fail to accumulate around the chromosomes. Second, microtubules with attachments to the chromosomes provide a poleward force on the centromeres. This stage is defined by the observation that, in the absence of the CPC, and consequently the absence of microtubules, the homologous centromeres fail to separate. Third, the homologs bi-orient through interactions with the central spindle microtubules. This stage is defined by the observation that, in sub mutants, the central spindle is absent but microtubules with attachments to the chromosomes still form and the homologous centromeres separate but fail to bi-orient (Radford, 2012).

The nature of the microtubule attachments to the chromosomes that lead to centromere separation is not known. Some previous studies have suggested that chromosome alignment depends on lateral interactions during acentrosomal meiosis. However, an alternative model incorporates an important role for kinetochore microtubules). Kinetochore microtubules in oocytes have been inferred by Hughes (2011) and could be the cold-resistant karyosome-associated microtubules observed in previous studies. Whether the microtubules connect to the chromosomes though traditional end-on kinetochore attachments or lateral attachments, it is proposed that these microtubules are bundled with central spindle microtubules to achieve bi-orientation. Interactions between central spindle microtubules and the microtubules with attachments to the chromosomes could be mediated by the kinesin-5 KLP61F or the kinesin-14 NCD. Indeed, this study has shown that NCD is required for homolog bi-orientation. The frayed spindles that are typical of ncd mutants could be explained by the loss of bundling between chromosome and central spindle microtubules (Radford, 2012).

A possible mechanism for how the CPC ring may facilitate bi-orientation at meiosis is suggested by two recent studies in mammalian mitotic and meiotic cells (Kitajima, 2011; Magidson, 2011). In both systems, prometaphase chromosomes move towards the outside edges of the developing spindle and then congress via lateral interactions to a ring around the central part of the spindle. This 'prometaphase belt' facilitates and enhances the rate of bi-orientation by bringing kinetochores into the vicinity of a high density of microtubules, which leads to stable kinetochore-microtubule attachments. It is proposed that the ring of CPC protein promotes a prometaphase belt-like organization to enhance the interaction of centromeres with a high density of microtubules in Drosophila oocytes (Radford, 2012).

Chromosome-based spindle assembly is a well described phenomenon, but the responsible chromatin-based factors in intact oocytes have not been previously identified. The current data suggests that the CPC interacts with noncentromeric chromatin and not only promotes the accumulation of microtubules around the chromosomes, but also regulates multiple aspects of spindle function, including the establishment of bipolarity and bi-orientation of homologs. Indeed, the localization to a central spindle ring and not centromeres may be critical for these functions. At this location, the CPC could regulate several different types of target protein that organize microtubules. One type is represented by Subito, which is required for spindle bipolarity, perhaps through the stabilization of antiparallel microtubules in the central spindle. Another type of target protein may function to promote microtubule attachment to the chromosomes. Indeed, these results provide the starting point for investigating what controls the localization of the CPC and what are its critical targets during acentrosomal meiosis (Radford, 2012).

Effects of Mutation or Deletion

sub null mutant females develop monopolar and tripolar spindles in a majority of meiosis I figures and are sterile because of a requirement during early embryogenesis. In addition, sub hypomorphic mutants exhibit a high frequency of homologous chromosome nondisjunction at meiosis I (Giunta, 2002). Considered along with the Sub staining pattern, these results suggest that the MMCS may have an important role for in bipolar spindle formation and chromosome segregation. For this study, the effects on central spindle formation were reexamined with a new sub mutant data set. Similar to the previous report, 14 out of 17 sub1/sub131 mutant oocytes had abnormal spindle organization compared with only 4 out of 36 in wild-type oocytes. Among the 14 abnormal sub1/sub131 spindles, 3 were monopolar, 9 were tripolar, and 2 had other problems such as fraying of the microtubules. Thus, sub mutant spindles have a defect in maintaining or establishing bipolarity, although in a minority of cases, relatively normal bipolar spindles were observed. Notwithstanding these defects, the ability to taper microtubules into poles is not usually affected in sub mutants (Jang, 2005).

Given the localization of Sub, the MMCS in sub mutants were examined. Strikingly, all sub mutant spindles, even those that were bipolar, lacked metaphase central spindle tubulin staining. Although the amount of central spindle microtubules was variable in wild type, the prominent bundles of central spindle microtubules often observed in wild-type oocytes are never observed in sub mutant oocytes. Instead, there are often small gaps of microtubules staining in the middle of the spindle or only limited evidence of antiparallel microtubule overlap. Additional evidence that the central spindle fails to form in sub mutants is that midzone proteins such as Incenp and AurB did not localize to this region in sub mutants. These results suggest that the MMCS is normally organized or maintained by Sub (Jang, 2005).

To address whether Sub localization is dependent on bipolar spindle formation, Sub localization was examined in mutants with disrupted spindle organization, including ncd, tacc, alpha-tub67C, and gamma-tub37C. Like sub, ncd encodes a kinesin required for bipolar spindle formation. Double mutants with the hypomorph sub1794 have a meiotic phenotype similar to that of the single mutants (Giunta, 2002). tacc has an important role in meiotic bipolar spindle formation with some similar phenotypes to sub mutants (Cullen, 2001). gamma-Tub37C is one of two Drosophila gamma-tubulin isoforms and has previously been shown to have a role in female meiotic spindle formation. Mutants of the female-specific isoform alpha-Tub67C are sterile, although defects in spindle formation have not previously been shown. Despite severe defects in bipolar spindle formation, these mutants exhibit Sub staining in association with the central spindle. In addition, meiotic spindle defects have been documented in alpha-tub67C mutant females. These results suggest that the MMCS and Sub staining are not dependent on bipolar spindle formation. Instead, Sub most likely localizes and functions before the formation of a bipolar spindle. Sub may simply localize to any region of the spindle containing antiparallel microtubules (Jang, 2005).

One candidate for regulating Sub localization is Polo kinase because, the Sub ortholog MKLP2 is phosphorylated by Polo in human cells. Observing the effects of polo null mutants is problematic because the homozygotes are lethal. It was, however, possible to examine females with a viable but female sterile allele heterozygous to a null allele, and it was found that Sub staining was normal. Indeed, these mutants did not have gross defects in meiotic spindle formation, except for a possible reduction in kinetochore microtubules. The absence of an effect on Sub staining in polo mutants is consistent with the results that in HeLa-S3 cells, MKLP2 is a target for Polo phosphorylation (Neef, 2003) but this is not required for localization (Jang, 2005).

Because MKLP2 is required for the localization of Polo kinase to the midzone in HeLa-S3 cells (Neef, 2003), Polo localization was examined during Drosophila meiosis. In addition, Drosophila Polo has been shown to have a localization pattern similar in mitotic cells to proteins such as AurB and Incenp, which localize to the MMCS in Drosophila oocytes and depend on Sub activity. Polo antibody staining in wild-type metaphase Drosophila oocytes appeared weakly in the MMCS and was not visible in all images. In contrast, it was stronger in foci that colocalized with the DNA. These Polo foci are probably the kinetochores, consistent with previous studies of larval neuroblasts. In these mitotically dividing cells, Polo is localized to kinetochores during metaphase, and midzone staining is strong only during anaphase. In sub mutant oocytes, the foci of Polo staining are still observed, which is consistent with the absence of Sub protein at kinetochores. However, the level of Polo staining was variable and may be dependent on spindle structure, because in some sub mutant oocytes, particularly those with the most disorganized spindles, Polo staining was weak (Jang, 2005).

These results suggest that the MMCS has an important function in organizing the meiotic spindle. This led to an examination of what other proteins are located in this region of the meiotic spindle and could contribute to microtubule assembly. Several proteins have been localized to the spindle midzone at anaphase of mitotic cells including the passenger proteins AurB/Ial and Incenp. In mitotic cells, the localization pattern of the passenger proteins depends on the mitotic stage. They localize to the centromeres during metaphase and then move to the spindle midzone at anaphase. The metaphase I localization pattern of AurB and Incenp was examined by antibody staining to determine if the MMCS also contains these proteins and whether they show an early (mitotic metaphase) or late (mitotic anaphase) staining pattern. RacGap50C, another mitotic midzone component that forms a complex with the Sub paralog Pav was examined (Jang, 2005).

For Incenp, AurB, and RacGap50C, staining similar or identical to Sub was found. For example, Incenp perfectly colocalizes with Sub in the MMCS, including early prometaphase staining before a bipolar spindle has formed. To determine the relationship of these proteins to the centromeres, staining was examined with an antibody to MEI-S332. Sub and MEI-S332 always occupied distinct regions around the karyosome in both disorganized prometaphase and bipolar metaphase spindles. By extension, because the midzone proteins and Sub always colocalize, Incenp and AurB do not localize to the centromere regions during meiotic metaphase I. Indeed, the centromeres and MMCS appear to be properly organized and oriented before a bipolar spindle forms (Jang, 2005).

The localization of these proteins to the MMCS is dependent on sub activity. In sub mutants, AurB accumulates in a region surrounding the karyosome but not in the region where the MMCS would have been. Almost identical localization defects were observed with RacGap50C and Incenp. This pattern appears to be more extensive than just centromere staining, at least when compared with the MEI-S332 staining and Polo staining described above. Instead, these proteins concentrate in the region where the microtubules are in close proximity to the karyosome. This could occur if, in the absence of a central spindle in sub mutants, these proteins concentrate near the plus-ends of microtubules. A similar effect of midzone disruption on AurB and MKLP1 staining has been observed in HeLa cells (Kurasawa, 2004). It was suggested that localization toward the plus ends was an intermediate stage in development of the midzone. It is possible these proteins have an affinity for the plus ends of microtubules or other motors, such as the Drosophila MKLP1 ortholog Pavarotti, that actively transport them there. Unfortunately, it was not possible to test the role of the passenger proteins in acentrosomal spindle formation because Incenp appears to be required during early stages of oogenesis (Jang, 2005).

Although Sub has an important role in spindle formation, most sub mutant spindles retain the ability for form poles. Therefore, Sub-independent activities must be functioning to organize spindle poles. TACC and MSPS may have a role in this function because these proteins localize to meiotic spindle poles. Previous genetic and cytological studies have shown that tacc and msps have an important role in meiotic bipolar spindle formation (Cullen, 2001), and the mutants have been reported to have phenotypes similar to sub mutants. To investigate the relationship between TACC and Sub, TACC localization was examined in sub mutants, and the phenotype of double mutant combinations was examined. TACC localizes to the acentrosomal spindle poles in wild-type oocytes. In sub mutants, however, the spindle pole staining was weaker and in some cases accumulated near the chromosomes. Thus, it is possible that the phenotype of Sub mutants may be related to defects in TACC localization (Jang, 2005).

To test if Sub and TACC function in distinct spindle forming activities or have similar functions during spindle assembly, sub; tacc double mutants were constructed. The sub1; taccstella592 double mutant has severe spindle formation defects, more dramatic than either single mutant. Unlike the single mutants, there were often multiple bundles of microtubules, some associating with chromosomes. Although microtubules were still associating with the chromosomes, most bundles of microtubules are randomly organized. Similar defects were also observed in sub1794/sub1; taccstella592 but not sub1794/sub1794; taccstella592 females, demonstrating this phenotype is dependent on severe loss of sub function. The double mutants appear to retain the ability to assemble kinetochore microtubules. This phenotype could be explained by a combination of tacc and sub mutant defects: a failure to stabilize the spindle poles (tacc) and a failure to organize the microtubules around the chromosomes (sub). These results suggest that sub and tacc contribute to different pathways that function to organize the microtubules of acentrosomal spindles. This is consistent with the observation that Sub and TACC associate with distinct structures or populations of microtubules (Jang, 2005).

The female meiotic spindle lacks a centrosome or microtubule-organizing center in many organisms. During cell division, these spindles are organized by the chromosomes and microtubule-associated proteins. Previous studies in Drosophila have implicated at least one kinesin motor protein, NCD, in tapering the microtubules into a bipolar spindle. A second Drosophila kinesin-like protein, Sub, has been identified that is required for meiotic spindle function. At meiosis I in males and females, sub mutations affect only the segregation of homologous chromosomes. In female meiosis, sub mutations have a similar phenotype to ncd; even though chromosomes are joined by chiasmata they fail to segregate at meiosis I. Cytological analyses have revealed that sub is required for bipolar spindle formation. In sub mutations, spindles were observed that were unipolar, multipolar, or frayed with no defined poles. On the basis of these phenotypes and the observation that sub mutations genetically interact with ncd, it is proposed that Sub is one member of a group of microtubule-associated proteins required for bipolar spindle assembly in the absence of the centrosomes. sub is also required for the early embryonic divisions but is otherwise dispensable for most mitotic divisions (Giunta, 2002).

The sub genetic and cytological mutant phenotypes are similar to those previously described for mutants in ncd. Most important, both mutants cause nondisjunction of homologous chromosomes at the first meiotic division but have no effect on the second meiotic division. On the basis of a live analysis, it has been proposed that Ncd is required in the acentrosomal spindle to taper the microtubules into a pole with its minus-end-directed motor moving outward from the chromosomes, bundling together microtubules in the process. It has also been proposed that at least one additional motor is involved in the process because poles can still form in the absence of Ncd. Thus, one possible function of Sub is to bundle microtubules to form that portion of the poles that is not handled by Ncd. This model predicts that a sub; ncd double mutant would have a more severe defect in spindle pole formation than would either single mutant. Double mutant analysis showed this is not the case; the double mutant is able to make spindle poles with a similar array of defects as the single mutants. Therefore, it is concluded that both ncd and sub are involved in the same process of spindle formation (Giunta, 2002).

The subDub mutation changes a highly conserved amino acid in the motor domain. The original study of the dominant subDub mutation did not distinguish between an antimorph or neomorph (Moore, 1994). The subDub meiotic phenotypes are almost identical to the null alleles, arguing that it is an antimorph. The kinesin motor nod also has a dominant antimorphic allele (nodDTW) that is associated with a single amino acid change in a highly conserved region of the ATP-binding domain. Similar to subDub, nodDTW dominantly affects chiasmate and achiasmate chromosomes. Both the subDub and nodDTW proteins could have altered microtubule-binding activities that lead to interference with other proteins on the meiotic spindle. Both dominant mutations also cause lethality due to mitotic defects, and this phenotype is lessened by the presence of wild-type gene activity, suggesting that both sub and nod gene products interact with the mitotic spindle. The observation that ncd1 and subnull alleles are synthetically lethal also argues that sub has a function in mitotic cells (Giunta, 2002).

The Drosophila female meiotic spindle must organize in the absence of centrosomes and segregate homologs at the reductional division. Genes likes sub that are not required for the typical mitotic division or meiosis II may be required for these unique properties of the meiotic spindle. The simplest hypothesis is that SUB is a kinesin that interacts with spindle microtubules. This is supported by the specific genetic interactions with the ncdD and nod mutations. Interestingly, the Sub homologs MKLP1 and Pav have been shown to localize at centrosomes in mitotic metaphase. In addition, MKLP1 is known to bundle microtubules and be a plus-end-directed motor. Indeed, most of the members in the MKLP1 group have this property, although one, RB6K, has been reported to associate with the Golgi. Although a more recent report demonstrates that RB6K has an important role in cytokinesis, an indirect role for Sub in spindle formation cannot be ruled out (Giunta, 2002).

Spindle assembly in the absence of centrosomes can be divided into four stages. Stage 1: the nucleation or capture of microtubules by the chromosomes. Stage 2: the microtubules are bundled together by proteins that can form bridges between parallel and/or antiparallel microtubules. Stage 3: extension of the spindle by antiparallel microtubule sliding or a 'polar ejection force' exerted by motors associated with the chromosomes. Stage 4: the minus-ends of the microtubules are focused to produce defined spindle poles. Although these stages probably overlap and share genetic requirements, the function of Sub appears to be most important for the last stage of bipolar spindle formation. In sub mutants, microtubule arrays of wild-type length are able to form, but they fail to be focused into only two poles. It has been proposed that an inherent product of the microtubule bundling process is the formation of a single axis and therefore at least a crude bipolar spindle. A relationship between maintaining the integrity of the poles and constructing a spindle with only two poles would explain why in sub mutants the spindle poles are often frayed and/or they are tripolar or monopolar (Giunta, 2002).

One aspect of the sub mutation phenotype could be the inability to generate poleward forces. In sub mutants the position of the chromosomes within the karyosome is abnormal. Thus, Sub could facilitate interactions between the chromosomes and microtubules that are part of the process that organizes meiotic spindles. As was argued from an analysis of alphaTubulin67C mutants, defects in sub could result in a disruption of poleward forces, leading to a failure in centromere positioning. This would provide a link between the chromosomes and spindle pole organization, which is plausible considering that the chromosomes have a role in organizing the spindle (Giunta, 2002).

In addition to female spindle formation, sub is required for at least two other cell divisions: male meiosis and the early embryonic cleavage divisions. There are significant differences between the Drosophila male and female meiotic divisions; for example, in male meiosis crossing over does not occur and centrosomes are present. In both male and female meiosis, however, there is a reductional division involving the segregation of homologs. Thus, the importance of SUB may not be for spindle pole formation, but to organize a spindle where bivalents must be oriented and segregated. It is noteworthy that the sub alleles are the only Drosophila mutants that are defective at the first meiotic division of both males and females without affecting the segregation of sister chromatids (Giunta, 2002).

The null alleles of sub are female sterile due to a failure in the early embryonic cell divisions. The early defects in sub mutation embryos have some similarities to mutants in other genes with a variety of roles in spindle function such as Klp3A, alpha-Tubulin67C, polo, and wispy. In all these cases, it was suggested that the embryonic arrest was due to a defect in pronuclear migration, although the variety of defects observed in sub and the other mutants make it difficult to define a precise function for these proteins. In addition, pronuclear fusion may be a sensitive point for a wide variety of defects in microtubule-based processes, leading to the arrest prior to pronuclear fusion in many different mutants. It will be interesting to determine the nature of the sub function that is required for reductional meiotic divisions and an early event in embryogenesis (Giunta, 2002).

A Drosophila mutation, Double or nothing (Dub), causes meiotic nondisjunction in a conditional, dominant manner. Previously isolated mutations in Drosophila specifically affect meiosis either in females or males, with the exception of the mei-S332 and ord genes, which are required for proper sister-chromatid cohesion. Dub is unusual in that it causes aberrant chromosome segregation almost exclusively in meiosis I in both sexes. In Dub mutant females both nonexchange and exchange chromosomes undergo nondisjunction, but the effect of Dub on nonexchange chromosomes is more pronounced. Dub reduces recombination levels slightly. Multiple nondisjoined chromosomes frequently cosegregate to the same pole. Dub results in nondisjunction of all chromosomes in meiosis I of males, although the levels are lower than in females. When homozygous, Dub is a conditional lethal allele and exhibits phenotypes consistent with cell death (Moore, 1994).

The dominant Dub mutation is the first mutation isolated in Drosophila that affects the three known pathways of homolog segregation in meiosis 1. Both nonexchange and exchange chromosomes in females undergo nondisjunction in Dub mutant females, and segregation of homologs is aberrant in mutant males. The segregation of all four chromosomes is disrupted in Dub mutant females and males. Four results demonstrate that Dub causes nondisjunction of nonexchange chromosomes in females: (1) the achiasmate chromosome 4 undergoes nondisjunction at high frequencies in females; (2) diplo-X ova from Dub females show an increased percentage of nonexchange tetrads compared to normal, mono-X ova, indicating that achiasmate chromosomes are more likely to nondisjoin in the Dub mutant; (3) the segregation of compound-X chromosomes from a Y chromosome is affected by the Dub mutation, a segregation previously shown to be mediated by the distributive system; and (4) nondisjunction frequencies for the X chromosome increase dramatically when the X chromosome is made non-exchange by making it heterozygous with a balancer chromosome. The fact that both the segregation of chromosome 4 and the disjunction of a compound X from a Y chromosome are altered indicates that both the homologous and heterologous systems of achiasmate segregation are disrupted by the Dub mutation (Moore, 1994).

Although Dub predominantly affects nonexchange chromosomes, it also results in nondisjunction of exchange chromosomes. Dub reduces recombination frequencies only slightly, so the frequency of X chromosome nondisjunction (16%-18%) in the female is too high to be the consequence of failure of only nonexchange chromosomes to segregate. In addition, in diplo-X exceptional ova, 49% of the tetrads had one or more exchange (Moore, 1994).

Dub mutant males also exhibit nondisjunction. The frequencies of nondisjunction in the male are considerably less than in the female. The interpretation of this difference depends on whether the Dub mutation is antimorphic or neomorphic. If the mutation is antimorphic, the requirement of the gene product in male meiosis may be lower than in female meiosis, or redundant functions may exist in the male. If the allele is neomorphic, it may not interfere with meiosis in the male to as great an extent as in the female. Dub differs from mutations in the ord and mei-S332 genes, which also cause nondisjunction in both sexes, in that Dub causes nondisjunction in meiosis I almost exclusively. In ord mutants, nondisjunction occurs in both meiosis I and I1 in a ratio suggesting that the four sister chromatids of the bivalent separate prematurely and then segregate randomly through two divisions. Indeed, precocious sister-chromatid separation is observed as early as prometaphase I in ord mutants. In contrast, mei-S332 mutations result primarily in meiosis I1 nondisjunction. Although the sister chromatids also prematurely disjoin in mei-S332 mutants, the sister chromatids do not separate until late in anaphase I. Thus the ord and mei-S332 genes control the behavior of sister chromatids, whereas the Dub mutation causes aberrant segregation of the homologs (Moore, 1994).

The Dub mutation is conditional lethal when homozygous. The homozygous larvae and pupae exhibit phenotypes indicative of extensive cell death such as small or missing imaginal discs, melanotic tumors, rough eyes, etched tergites, and missing bristles. This suggests that when homozygous the Dub mutation affects mitotic chromosome segregation. Gynandremorphs were observed in the progeny of Dub mutant females, consistent with abnormal mitotic chromosome segregation. However, abnormal mitotic figures were not found in neuroblast squashes from homozygous Dub larvae at a frequency that could account for the observed cell death. One possibility is that Dub affects mitosis in tissues other than the brain. This is consistent with the observation that while the imaginal discs are small or missing in homozygous Dub larvae, the brain appears normal in size. An alternative possibility is that the homozygous mutation affects other cell processes in such a manner that results in cell death (Moore, 1994).

Kinesin 6 family member Subito participates in mitotic spindle assembly and interacts with mitotic regulators

Drosophila Subito is a kinesin 6 family member and ortholog of mitotic kinesin-like protein (MKLP2) in mammalian cells. Based on the previously established requirement for Subito in meiotic spindle formation and for MKLP2 in cytokinesis, the function of Subito in mitosis was investigated. During metaphase, Subito localizes to microtubules at the center of the mitotic spindle, probably interpolar microtubules that originate at the poles and overlap in antiparallel orientation. Consistent with this localization pattern, subito mutants improperly assembled microtubules at metaphase, causing activation of the spindle assembly checkpoint and lagging chromosomes at anaphase. These results are the first demonstration of a kinesin 6 family member with a function in mitotic spindle assembly, possibly involving the interpolar microtubules. However, the role of Subito during mitotic anaphase resembles other kinesin 6 family members. Subito localizes to the spindle midzone at anaphase and is required for the localization of Polo, Incenp and Aurora B. Genetic evidence suggested that the effects of subito mutants are attenuated as a result of redundant mechanisms for spindle assembly and cytokinesis. For example, subito double mutants with ncd, polo, Aurora B or Incenp mutations are synthetic lethal with severe defects in microtubule organization (Cesario, 2006).

Subito is one of the two Drosophila kinesin 6 family members and probably the ortholog of MKLP2. In support of this classification, there are striking similarities between Subito and MKLP2. Both are required for localization of the passenger proteins to the midzone during anaphase. In addition, both Subito and MKLP2 interact with Polo kinase (or Plk1 in human) and are required for its localization to the midzone during anaphase. Plk1 phosphorylates MKLP2 at Ser528 and this phosphorylation promotes Plk1 binding to MKLP2. Plk1 phosphorylation negatively regulates MKLP2 microtubule bundling activity in vitro but is not required for the localization of MKLP2 to the midzone (Cesario, 2006).

Despite belonging to the same family, the two kinesin 6 family members probably have unique functions. The distinct phenotypes of sub and pav mutants indicate they have non-overlapping functions. Similarly, and despite having similar localization patterns, MKLP2 and MKLP1 have nonredundant functions in cytokinesis. MKLP2, but not MKLP1, has been shown to physically interact with Aurora B and Incenp. However, it has also been suggested that the MKLP2-dependent localization of Aurora B to the midzone is required for it to phosphorylate MKLP1. The importance of this phosphorylation on MKLP2 localization is unclear and the results are consistent with this indirect relationship between Subito and Pavarotti (Cesario, 2006).

It is possible that all members of the kinesin 6 group interact with antiparallel microtubules. Immunolocalization data is consistent with this because Subito is found on interpolar microtubules, which are characterized by an overlap of antiparallel microtubules in the midzone at mitotic anaphase in embryos, brains and testis. However, the localization of Subito to metaphase interpolar microtubules in the vicinity of the centromeres was a surprising finding. Although it is likely that Subito also associates with antiparallel microtubules at metaphase, the possibility that Subito interacts with the plus ends of the microtubules that interact with the kinetochores cannot be ruled. Surprisingly, a specific localization pattern of other kinesin 6 family members to metaphase microtubules has not been observed. This is not due to the absence of the appropriate substrate, since metaphase interpolar microtubules are present in most spindles. Either Subito is regulated differently than MKLP2, with an associated additional function in spindle assembly, or the localization pattern of MKLP2 at metaphase has not been informative with respect to its function (Cesario, 2006).

Since Subito is required to localize Polo, Aurora B and Incenp to the spindle midzone at anaphase, it is surprising that sub mutants are viable. Loss of MKLP2 causes cytokinesis defects. Drosophila mutants with strong defects in cytokinesis fall into the categories of male sterile, embryonic lethal (e.g. pav mutants) or pupal lethal. In fact, Incenp and polo mutants have embryonic lethal phenotypes that may be caused by a failure of cytokinesis. Unlike the loss of Incenp, Aurora B or Polo, sub mutants do not have any of these phenotypes and appear to complete cytokinesis most of the time in larval brains. In addition, because sub mutant males are fertile, and most mutants with strong defects in cytokinesis during spermatogenesis are male sterile, Subito does not appear to be essential for cytokinesis in the testis. A cytokinesis phenotype was also not evident in cultured Drosophila cells depleted of Subito by RNAi. These same studies did identify cytokinesis defects when Polo, Aurora B and Incenp were depleted. Thus, it seems likely that in some cell types, such as larval brains, the presence of Subito and the localization of the passenger proteins are not required for cytokinesis to occur (Cesario, 2006).

A close examination of sub mutants, however, revealed that anaphase did not proceed normally. In addition to the failure to accumulate Polo, Aurora B and Incenp in the midzone, the absence of Subito resulted in disorganized midzone microtubules at anaphase and a small increase in the frequency of polyploid cells. When the dosage of Incenp was reduced in sub mutants, the frequency of polyploidy was markedly increased. Therefore, Subito appears to have a similar function to MKLP2 in promoting cytokinesis, although there may be functional redundancy. Since the ability to complete cytokinesis in sub mutants depends on Incenp and Aurora B dosage, it is possible that unlocalized Incenp or Aurora B may promote cytokinesis. However, the observation that Incenp and Aurora B have a limited ability to spread along anaphase microtubules in the absence of Subito suggests an alternative; enough passenger protein activity may be present to promote cytokinesis. This model can account for the sensitivity of sub mutants to Incenp or Aurora B dosage because high levels of these proteins may be needed to promote cytokinesis if not concentrated in the midzone. It is also possible that anaphase may last longer and/or the microtubule organization improves with time in sub mutants. This would account for the relatively normal Fascetto localization and high success completing cytokinesis in sub mutants (Cesario, 2006).

Several lines of evidence suggest that Subito has a role in mitotic spindle assembly: (1) Subito initially localizes to interpolar microtubules at metaphase; (2) abnormally formed metaphase spindles were found in sub mutants more frequently than in the wild type; (3) sub mutant brains have an elevated mitotic index. Although the magnitude of the increase in sub mutants was lower than reported in some other mutants with spindle assembly defects, these mutants are lethal. Consistent with the conclusion that sub mutants have a defect in spindle assembly, the elevated mitotic index was dependent on BubR1, suggesting that the spindle assembly checkpoint is activated in the absence of Subito. (4) sub mutations exhibit synthetic lethality in combination with polo, Incenp and Aurora B mutations, and the cytological phenotype includes defects in spindle assembly and increased mitotic index. (5) RNAi of sub in Drosophila S2 cells results in frequent mitotic spindle abnormalities. These observations all point to a role for Subito in spindle assembly (Cesario, 2006).

The defects associated with sub mutants are less severe in mitotic cells than during female meiosis, possibly because of redundant spindle assembly pathways in mitosis. The double mutant studies suggest that the defects in spindle assembly or chromosome alignment in sub mutants are compensated for in two ways. First, the activation of the spindle assembly checkpoint allows defects in microtubule organization to be corrected. Second, the presence of redundant spindle assembly pathways allows microtubules to be assembled in the absence of sub. Double mutant studies support both of these mechanisms (Cesario, 2006).

The phenotype of the sub;polo16-1/+ double mutant is consistent with a redundant role for Subito in spindle assembly. Compared with the single mutants, the double mutants exhibit grossly abnormal metaphase and anaphase spindles. Similar to the results with sub, a role for Polo in spindle assembly has been shown through the analysis of polo hypomorphs that have an elevated mitotic index in larval brains, indicating that the spindle assembly checkpoint is activated. During metaphase, Polo localizes to the centromeres where it has a role in spindle formation but during anaphase it localizes to the spindle midzone where it has a role in cytokinesis. The very high mitotic index in the double mutants, however, suggests a more severe defect in spindle assembly than either single mutant. It is suggested that the abnormal spindle phenotype in sub/sub;polo/+ mutants arise from a combination of defects in two partially redundant spindle assembly pathways: improper assembly of kinetochore microtubules in polo/+ mutants and a reduction in assembling interpolar microtubules in sub mutants. Although polo mutants are recessive lethal, there is other evidence for dominant phenotypes, such as an elevated mitotic index in polo16-1/+ brains (Cesario, 2006).

The combination of these two spindle assembly defects in polo/+;sub/sub mutants might result in the severe spindle assembly phenotype and lethality in the double mutant. Similar conclusions apply for the interactions between sub and Incenp or Aurora B. Like Polo, the passenger proteins have an important role in spindle assembly. Indeed, the effects of all three mutants are strikingly similar, suggesting that Subito, Polo and the passenger proteins have important interactions during metaphase and anaphase. Interestingly, there is evidence of a direct interaction between Plk and Incenp in mammalian cells (Cesario, 2006).

Like its kinesin 6 homolog MKLP1, Subito is probably a plus-end-directed motor that crosslinks and slides interpolar antiparallel microtubules. The results suggest that this activity is important from metaphase through anaphase. Interestingly, the metaphase and anaphase interpolar microtubules have functional differences. Metaphase interpolar microtubules are observed in the absence of Subito whereas their anaphase counterparts depend on Subito. Another important difference is that Polo and the passenger proteins localize only to anaphase interpolar microtubules in the midzone. It has been suggested that the precocious appearance of anaphase-like interpolar microtubules is an important feature of acentrosomal meiotic spindle assembly in Drosophila oocytes. The passenger proteins Aurora B and Incenp localize to the interpolar microtubules at metaphase of meiosis I, rather than the centromeres, which is typical during mitotic metaphase. Therefore, the regulation of the passenger protein localization pattern is modified in oocytes to bypass the centromere localization that is characteristic of mitotic metaphase, resulting in precocious localization to interpolar microtubules (Cesario, 2006).

Despite these differences, the same biochemical activities of Subito could be used to organize both centrosomal mitotic and female acentrosomal meiotic spindles. In mitotic cells, kinetochores can initiate microtubule fiber formation, but these fibers are not directed toward either spindle pole. Failure to organize these fibers could result in disorganized and frayed spindles, as has been observed in sub mutants. A function for Subito and interpolar microtubules could be to properly orient undirected kinetochore fibers. Interpolar microtubules could interact with and direct the organization of kinetochore microtubules via motors that bundle parallel microtubules. This mechanism has been proposed for organizing a bipolar spindle in the acentrosomal meiosis of Drosophila oocytes. With motor-driven sliding of antiparallel microtubules, this is an example of a centrosome-independent model for the spindle assembly pathway. This is consistent with previous conclusions that centrosome-independent mechanisms for spindle assembly are active in mitotic cells. Indeed, since bipolar spindles can form in the absence of centrosomes in neuroblasts and ganglion mother cells, it appears that centrosome-independent mechanisms for spindle assembly are active in the mitotic cells analyzed (Cesario, 2006).

Another possibility is that Subito functions as part of the centrosomal assembly pathway. For example, an array of interpolar microtubules could help channel centrosome microtubules towards the kinetochores. This activity could reduce the element of chance associated with making contacts between centrosome microtubules and kinetochores. It has also been proposed that centrosomal microtubules may capture the minus ends of kinetochore microtubules. An involvement of Subito in this process would be surprising, however, because the ability to bundle microtubules in parallel has not been described for a kinesin 6 family member. Nonetheless, if Subito was involved in the interactions of centrosomal and kinetochore microtubules, subsequent plus-end-directed movement would explain why Subito localization overlaps with centromeres. Whether or not these models are correct, the redundant nature of spindle assembly and function may explain why a role for kinesin 6 motor proteins in spindle assembly has not been described previously (Cesario, 2006).

Misregulation of the kinesin-like protein Subito induces meiotic spindle formation in the absence of chromosomes and centrosomes

Bipolar spindles assemble in the absence of centrosomes in the oocytes of many species. In Drosophila oocytes, the chromosomes have been proposed to initiate spindle assembly by nucleating or capturing microtubules, although the mechanism is not understood. An important contributor to this process is Subito, a kinesin-6 protein that is required for bundling interpolar microtubules located within the central spindle at metaphase I. This study characterized the domains of Subito that regulate its activity and its specificity for antiparallel microtubules. This analysis has revealed that the C-terminal domain may interact independently with microtubules while the motor domain is required for maintaining the interaction with the antiparallel microtubules. Surprisingly, deletion of the N-terminal domain resulted in a Subito protein capable of promoting the assembly of bipolar spindles that do not include centrosomes or chromosomes. Bipolar acentrosomal spindle formation during meiosis in oocytes may be driven by the bundling of antiparallel microtubules. Furthermore, these experiments have revealed evidence of a nuclear- or chromosome-based signal that acts at a distance to activate Subito. Instead of the chromosomes directly capturing microtubules, signals released upon nuclear envelope breakdown may activate proteins like Subito, which in turn bundles together microtubules (Jang, 2007).

Subito is a kinesin motor protein that contributes to acentrosomal meiotic spindle formation, possibly by stabilizing the overlap of antiparallel microtubules located in the central spindle during meiotic metaphase. The object of this study was to investigate the characteristics of Subito that facilitate spindle formation. The results have stimulated a model for acentrosomal spindle assembly that emphasizes the bundling of antiparallel microtubules without direct contacts with the chromosomes (Jang, 2007).

When a fragment containing only the C-terminal domain of Subito was expressed, the protein localized to the spindle microtubules. It is also likely that that there is competition between the C-terminal domain and full-length Subito for binding sites. This is based on the observation that when the C-terminal domain was expressed in a wild-type background, there was less wild-type protein localized to the central spindle at metaphase I and an increased incidence of chromosome segregation errors. Similarly, the C-terminal domain of MKLP2 has been shown to have microtubule binding activity in vitro and in vivo. These results indicate that Subito, and other MKLP2 paralogs, have two microtubule-binding domains, one each in the C-terminal and motor domains. This feature of the MKLP2 proteins may enable them to form cross-bridges between antiparallel microtubules. Specificity may also reside in the protein-protein interactions involving Subito. Similar to MKLP2, the C-terminal domain of Subito may interact with the Passenger proteins and CDC14. Indeed, the passenger proteins and Subito or MKLP2 may be obligate partners during meiosis and mitosis (Jang, 2007).

Two mutations were characterized that affect conserved amino acids in the motor domain and a third that affects a motor domain sequence specific to kinesin-6 proteins. All three mutants exhibit dominant nondisjunction and weak spindle staining, suggesting they have similar defects in motor activity. The subDub mutation changes an E to K at position 385. Although this mutation is outside of the microtubule-binding region, it is the last residue in a group of seven amino acids that are invariant in all kinesin-like proteins. This mutant has a dominant nondisjunction phenotype and the protein fails to accumulate in the central spindle. The failure to localize to microtubules is surprising because similar mutants (E --> A) in minus end-directed motors such as Kar3 or NCD bind strongly to microtubules but lack a microtubule-stimulated ATPase activity. Thus, the SubitoDub protein is predicted to bind microtubules but have an inactive motor. A similar array of phenotypes was observed when a mutation was generated that changes the three invariant amino acids GKT in the ATP-binding domain to AAA (subATP). This change has been made in other kinesins and similar changes of the GKT sequence have been made in Pavarotti (EKT) and the kinesin-5 homolog Eg5 (GKN, GKI) or kinesin heavy chain (GKN). Most of these mutants exhibit 'rigor' binding phenotypes associated with excessive binding of microtubules in vivo. In contrast, the SubitoATP protein failed to localize strongly to meiotic spindles (Jang, 2007).

Despite the weak localization of the motor domain mutants, several observations indicate these motor domain mutant proteins interact with the spindle microtubules: (1) these mutants have dominant effects on meiotic chromosome segregation and spindle organization; (2) these mutants cause reductions in the localization of wild-type protein to the spindle. Indeed, interfering with the localization of wild-type Subito protein could be the cause of the dominant nondisjunction phenotype. (3) At least one of the motor defective proteins (SubitoATP) localizes strongly to metaphase microtubules in mitotic cells, although not to the central spindle like wild-type protein. These observations suggest that in the presence of motor domain defective proteins, wild-type Subito engages in interactions that lead to its removal from the spindle (Jang, 2007).

Motor defective Subito protein may be able to initially associate with the microtubules but then be rapidly displaced toward the poles. This would explain the observation that motor domain mutant proteins fail to localize on the spindle despite containing an intact C-terminal domain that can independently interact with the spindle. Such a 'polar wind' has been implicated in previous studies of Drosophila oocytes. It is also possible that the motor-inactive Subito proteins could be dislodged from the spindle by another mechanism. Whatever the mechanism by which the motor domain mutant proteins fail to remain on the spindle, these results suggest that the motor domain is required to retain Subito on the spindle in oocytes (Jang, 2007).

Two factors were identified that regulate Subito, by characterizing stage 14 oocytes expressing a Subito mutant lacking the N-terminal domain (SubitoδNT). The first regulator of Subito is shown by the observation that the subδNT mutant forms a large number of ectopic spindles, indicating there is a mechanism to limit where Subito interacts with microtubules. The second regulator of Subito is shown by the observation that the unregulated microtubule bundling activity in subδNT mutants is dependent on NEB. This is a different result from overexpressing the kinesin-6 member Pavarotti, which has effects on oogenesis prior to NEB. Possibly, NEB releases a diffusible factor into the cytoplasm that activates Subito microtubule binding and bundling (Jang, 2007).

Aside from being numerous, the most striking aspect of the ectopic spindles of subδNT mutants is that they were not built around chromosomes. It is suggested that, through a still-unknown mechanism, the N-terminal domain regulates Subito activity to ensure that microtubules are bundled only in the direct vicinity of the chromosomes. The N-terminal domain could regulate Subito activity in a spatial manner. For example, this domain could promote interactions with a membranous sheath that has been proposed to surround the developing spindle. Interestingly, the initial studies of the Subito homolog MKLP2 demonstrated an interaction with Rab6, a Golgi-associated Rab protein, although through the C-terminal domain of MKLP2. Another possible mechanism is that the N-terminal domain may respond to a diffusible substance from the karyosome (Jang, 2007).

Rather than regulate when or where the motor is active, the N-terminal domain could affect the biochemical activity of the motor. For example, unregulated plus end-directed motor activity could lead to lengthening of the spindle through the sliding of antiparallel microtubules, causing the karyosome to be pulled apart and leaving the chromosomes scattered in the oocyte cytoplasm. The scattered chromosomes could go through repeated cycles of stimulating microtubule assembly followed by detachment from the spindle to generate the ectopic spindles observed in subδNT oocytes. More studies, including understanding the details of karyosome structure and the biochemical properties of Subito and the SubitoδNT mutant, are needed to distinguish these possibilities (Jang, 2007).

It has been suggested that the antiparallel overlaps of microtubules in the central spindle play an important role early in spindle assembly. The current results are also consistent with previous studies suggesting that interpolar microtubules are more sensitive to destabilizing agents like temperature and colchicine than kinetochore microtubules. Subito is critical for the central spindle since it is required for the interpolar microtubules. Like other members of the kinesin-6 family, Subito probably cross-links antiparallel microtubules. Subito has two microtubule binding domains, which may cooperate to facilitate interactions with antiparallel microtubules. In addition, motor activity may play a role in the localization of Subito. However, previous studies have also suggested that spindle assembly in Drosophila oocytes involves the recruitment of microtubules by the chromosomes and subsequent bundling of parallel microtubules by motor proteins such as NCD (Jang, 2007).

Spindles appear in subδNT mutant oocytes, however, that do not have direct contacts to the chromosomes. Therefore, conditions exist in the Drosophila oocyte cytoplasm in which recruitment and assembly of microtubules into a spindle may occur without direct contacts with the chromosomes. The concept of a cytoplasmic state permissive to spindle assembly has been proposed in Xenopus oocytes to explain how the injection of DNA can stimulate spindle assembly but only in cytoplasm from M phase eggs. Normally, however, chromosomes are needed to stimulate the process, leading to the idea that there is an 'organizational field' around the chromosomes. This two-component model of acentrosomal spindle assembly is consistent with current observations. Alternatively, it cannot be ruled out that one signal in high concentration near the chromosomes is responsible for generating both the permissive cytoplasmic state for spindle assembly and the organizational field around the chromosomes. However, the results from the subδNT mutant, which interacts with microtubules but is not restricted to the chromosomes, suggest activation is separable from restriction around the chromosomes (Jang, 2007).

Spindle assembly in Drosophila oocytes begins immediately following NEB, suggesting NEB somehow triggers the process. There is evidence that Subito activity is also regulated by NEB. Subito, even the unregulated form, does not bundle microtubules until after NEB. Nonetheless, spindle assembly is constrained such that microtubules assemble only around the chromosomes. On the basis of the phenotype of the subδNT mutant, Subito may also be regulated by proximity to the chromosomes. Since spindle assembly may be initiated by overlapping microtubules rather than direct contacts with the chromosomes, tightly regulating a protein like Subito that can bundle microtubules could be particularly important. In contrast, however, Subito is not essential for spindle assembly. There are probably several proteins or redundant mechanisms for recruiting microtubules to the spindle (Jang, 2007).

A key part of this model is that the chromosomes may not be essential for the polymerization of microtubules but may regulate the number and size of the spindles. A similar situation may occur during acentrosomal spindle formation in mammalian meiosis. Mouse oocyte microtubules can polymerize and be organized into bipolar spindles without the presence of chromosomes. Furthermore, several bipolar spindles of varying sizes tended to form, indicating that chromosomes may be needed to control spindle formation and growth. In mouse or Drosophila oocytes, therefore, it may be necessary to regulate the interaction of microtubules with motor proteins to occur in the vicinity of chromosomes. There are also other effects of the chromosomes. It is possible that the presence of chromosomes may promote localization of spindle pole proteins like TACC (Jang, 2007).

Interestingly, microtubules do not attach to kinetochores in mouse oocytes throughout most of prometaphase I. Instead, kinetochores are not competent to anchor and stabilize microtubule ends until the end of prometaphase I, ~8 hr after NEB. Thus, in both Drosophila and mammal oocytes, spindle assembly may be initiated by the interaction of nonkinetochore microtubules with motor proteins. Interpolar microtubules, which depend on Subito, play a critical role in organizing these bundles into the bipolar spindle. Kinetochore microtubules have a secondary role in spindle formation, being assimilated into the bipolar structure by interacting with the interpolar microtubules (Jang, 2007).

Some of the current observations can be explained if a signal spreads out from the nucleus or the chromosomes themselves into the cytoplasm of stage 14 oocytes after NEB. One candidate for a signal gradient is the active form (GTP bound) of Ran, which emanates from the chromosomes and has been proposed to promote microtubule assembly around chromosomes. Of relevance to these studies is the observation that the addition of RCC1 or an activated form of Ran (RanG19V) stimulated microtubule assembly in the absence of chromatin. Drosophila RCC1, a Ran cofactor, is found in the oocyte nucleus before and after NEB and Ran has also been suggested to have a role in meiotic spindle assembly in mouse oocytes although there is evidence for RanGTP-independent pathways as well. Whether Ran signaling is involved in meiotic spindle assembly of Drosophila oocytes is currently being investigated, and whether it is responsible for cytoplasmic state permissive to spindle assembly or the 'organizational field' around the chromosomes or both (Jang, 2007).

Evidence for a gene involved in multiple and diverse rearrangements in the Drosophila genus

In Drosophila, chromosomes have been extensively reorganized during evolution, with most rearrangements affecting the gene order in chromosomal elements but not their gene content. The level of reorganization and the evidence for breakpoint reuse vary both between and within elements. The subito gene stands out as a gene involved in multiple rearrangements both because of its active single-gene transposition and because it is the nearest gene to diverse rearrangements breakpoints. Indeed, subito has undergone three single-gene transpositions and it is the nearest gene to the breakpoints of other single-gene transpositions and of two chromosomal inversions. Given that subito is involved in meiosis and therefore active in the female germ line, the high number of nearby fixed breakages might be related among others to the presumed high accessibility of the subito region to the machinery associated with double-strand breaks repair. A second important contributor would be the reduced and simple regulatory region of subito, which would imply that a fraction of the rearrangements originating from subito nearby breakages would have not affected either its pattern or timing of expression and would have, thus, not resulted in reduced fitness (Puerma, 2014).


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subito: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation

date revised: 25 June 2013

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