In syncytial embryos, Drosophila INCENP associates with condensing chromatin during prophase, before becoming focussed to the centromeric regions of metaphase chromosomes. Upon entry into anaphase, the protein leaves the chromosomes to form a ring of spots between the segregating chromatids. Each spot seems to be at the converging focus of bundles of microtubules. As telophase progresses, the INCENP ring decreases in diameter until it becomes a single midbody-like structure between the central spindle microtubule bundles. A similar distribution of INCENP was also observed in cellularized embryos and Dmel2 cultured cells (Adams, 2001b).
The distribution of Drosophila Aurora B resembles that of Drosophila INCENP throughout mitosis. However, interphase nuclei show no detectable staining for Aurora B (they do for INCENP), and as nuclei enter prophase, Aurora B appears first at the centromere: no staining was observed along the chromosome arms. In an embryo being traversed by a wave of mitosis, nuclei just entering mitosis (i.e., adjacent to interphase nuclei) accumulate DmAurora B only at the centromere. Aurora B remains at the centromere until the metaphase to anaphase transition, when it transfers to the central spindle and subsequently to the midbody. This distribution is also observed in cellularised embryos and in cultured cells. It is concluded that INCENP and DmAurora B are chromosomal passenger proteins whose distribution in mitosis resembles their vertebrate counterparts (Adams, 2001b).
A genetic screen for new Incenp alleles and interactors
To obtain new alleles or interactors of Incenp, a mutagenesis screen was carried out using a previously identified allele of Incenp (incenpP(EP)2340). incenpp(EP)2340 is a P-element insertion into the third exon of the Drosophila Incenp gene. The presence and location of the P-element was confirmed by plasmid rescue and sequencing of the neighbouring genomic region. incenpP(EP)2340 is a homozygous lethal mutation and was described originally as a female sterile dominant. The chromosome carrying the insertion was recombined over a wild-type chromosome to get rid of possible second-site mutations. The P-element insertion is lethal over a chromosomal deletion uncovering the Incenp gene. incenpP(EP)2340 heterozygous individuals produce a smaller transcript of around 600 bp, which is not present in the wild-type control. This is predicted to potentially produce a 91 aa N-terminal peptide, however, no truncated peptide was detected in protein extracts from homozygous embryos by Western blot (Chang, 2006).
Male flies carrying the incenpP(EP)2340 insertion show a dominant meiotic phenotype that results in low fertility. The main defects observed include misshapen meiotic spindles, tetrapolar or multipolar spindles and defects in onion stage spermatids consistent with problems both in chromosome disjunction and cytokinesis. Heterozygous individuals also show defects in larval mitosis consistent with those shown by S2 cells depleted of Drosophila Incenp by double-stranded RNA interference (dsRNAi) (Adams, 2001c). Additionally, heterozygous adults show mild external defects consistent with abnormalities during imaginal mitosis, including slightly roughened eyes, nicked wings and defects in the abdominal terguites. The Incenp locus is not haplo-insufficient: individuals heterozygous for a deficiency uncovering the Incenp gene do not show any of the phenotypes described above (Chang, 2006).
The precise excision of the P-element, using an exogenous source of transposase, reverted the lethality and the dominant phenotypes, demonstrating that the insertion is responsible for both effects. Precise excision was confirmed by sequencing the genomic region of the excised chromosome. Twenty-one imprecise excisions obtained were lethal and also showed dominant meiotic phenotypes. Genomic DNA was isolated from six of these lines and a PCR strategy was devised to characterize them at the molecular level. All the lines analysed (Delta8, Delta9, Delta14, Delta17, Delta18 and Delta24) were internal deletions in the P-element, and are predicted to produce the same truncated peptide as the original insertion (Chang, 2006).
To date, no mutations in any of the genes coding for passenger complex proteins have been described in Drosophila. Therefore, to isolate new recessive alleles of Incenp and also possible new interactors, a second-site non-complementor screen was performed using ethyl methanesulfonate (EMS) as mutagen. EMS was chosen because it is more likely to induce point mutations that can disrupt specific protein-protein interactions. The Incenp gene is located on the second chromosome (43A04), so to simplify the screen, mutations only in the second chromosome were isolated. Mutations were isolated and analyzed that when in trans to incenpP(EP)2340 were either lethal, male sterile or showed an enhancement of the dominant visible phenotype (Chang, 2006).
A total of 5000 individual mutagenised chromosomes were generated and analyzed. This screen yielded 16 homozygous lethal mutations corresponding to 13 complementation groups. Most of the mutants were isolated on the basis of lethality in trans with incenpP(EP)2340, except for five (EC282, EC2388, EC2394, EC2519, EC3959) that showed defects in the eyes, bristles or wings. Two of the mutants had a dominant female sterile phenotype (EC3322, EC4330). When analyzed carefully on a larger scale, some of the trans-heterozygote combinations turned out to be semi-lethal or viable. The same results were obtained when trans-heterozygote combinations with imprecise excisions of P(EP)2340 (Delta9, Delta24) were analyzed. However, when in trans with a precise excision of P(EP)2340 (Delta11) the EC mutations show no phenotype (Chang, 2006).
Whenever the lethal phase allowed the analysis, the phenotype in male meiosis of the trans-heterozygous combinations [EMS mutant/P(EP)2340] were examined. Some combinations showed defects in primary spermatocytes as well as in the postmeiotic onion-stage spermatids. This is consistent with disruption of a function required for both mitosis and meiosis. In other cases, i.e., EC666/P(EP)2340, the defects were found exclusively in the meiotic divisions, which raises the possibility of identifying meiotic specific functions of the chromosomal passenger proteins (Chang, 2006).
Deficiency and, in some cases, meiotic-recombination mapping were used to further characterize the new mutations. For the deficiency mapping a second chromosome deficiency kit (a set of overlapping deletions of chromosome 2) was used. Four of the mutants, corresponding to two complementation groups (EC1650/EC2388 and EC2364/EC2374) are semi-lethal over a chromosomal deficiency for the Drosophila Aurora B kinase gene [Df(2L)1469]. In addition, two of these mutations (EC1650/EC2388) do not complement a chromosomal deficiency for the Drosophila borealin gene - Df(2L)2892 (Chang, 2006).
These experiments showed that the chromosomes typically contained more than a single EMS hit. Further analysis of the mutants requires the separation of the different mutations by recombination. As yet, the molecular identity of only one of the new mutants has been determined (Chang, 2006).
One of the newly generated mutants (EC3747) did not complement Df(2R)pk78k (42E03-43C03), a chromosomal deficiency uncovering the Incenp gene. EC3747 was therefore selected for further characterization as a putative new allele of Incenp. Genetic recombination with a multiply marked chromosome was used to minimize the presence of other spurious EMS mutations on the chromosome containing the EC3747 mutation. To further refine the mapping of EC3747 six strains were used carrying chromosomal deficiencies with breakpoints in the Incenp gene region. EC3747 was mapped to region 43A04, the same location as the Incenp gene. In addition, EC3747 failed to complement any of the imprecise excisions generated by the mobilization of the P-element. Therefore, all of the available genetic evidence suggests that EC3747 is a new allele of Incenp (Chang, 2006).
Molecular characterization confirmed that EC3747 was indeed a new allele of Incenp. To identify the mutation in EC3747, genomic DNA was extracted from single EC3747-homozygous embryos, PCR was used to amplify the entire coding region of the Incenp gene, and then the PCR products were sequenced directly. Genomic DNA from four single embryos was prepared as template to perform four separate PCR reactions. Comparison of the sequences revealed a point mutation in exon 4 of the Incenp gene. This single base change created a stop codon in position 457 of the open reading frame. The predicted molecular weight of the putative truncated gene product is 61 kDa (Chang, 2006).
Although the sequence analysis of EC3747 predicted that the product of this mutated gene would be a truncated version of Drosophila Incenp, EC3747 homozygous embryos were analyzed by immunoblotting no product of the predicted size was detected . The expected full-length protein with molecular weight of 110 kDa was detected in wild type, but was absent from embryos homozygous for Df(2R)pk78k, EC3747 and incenpP(EP)2340. This suggests that the truncated mutant protein is unstable. The antibody used for immunoblots, Rb801, was raised against the N-terminal 348 amino acids of Drosophila Incenp (Adams, 2001c), which is wholly contained within the 457 aa polypeptide encoded by EC3747 (Chang, 2006).
The new recessive null allele of Incenp shows a genetic interaction with several of the mutants generated in the screen: incenpEC3747 is semi-lethal over EC666 and EC2519, and male sterile over EC549, EC666, and EC1608. This confirms the genetic interaction between these as-yet-unidentified genes and Incenp, and excludes the notion that the interaction is specific to the dominant P-element insertion (Chang, 2006).
Analysis of the lethal phase of homozygous incenpEC3747 individuals showed that they died late in embryonic development. The rare first larval instar escapers show reduced mobility and abnormal wandering behavior consistent with defects in the nervous system. To investigate this further, late-stage embryos were stained for the marker Mab22C10 that stains neurons and their processes throughout the nervous system. Homozygous mutants were distinguished from heterozygous embryos using the CyOKrüppel-GFP balancer marker. Focusing on the peripheral nervous system (PNS), the heterozygous animals were observed to have a wild-type neural pattern. However, homozygous mutant embryos exhibited a range of defects, from severe cases of neuronal loss to perturbation of the neural clusters and missing subsets of neurons (Chang, 2006).
To determine the time in which the first defects in mitosis arose in these individuals, embryos were fixed at different stages and stained with anti-Drosophila Incenp and anti-tubulin antibodies. During the first 13 rapid synchronous cycles no difference was observed between wild-type and incenpEC3747 embryos. Since Incenp has been proposed to be required for completion of cytokinesis, special attention was paid to the process of blastodermal cellularization. This process, which occurs in mitotic cycle 14 when membranes grow inward between the blastoderm nuclei, in some ways resembles the process of cytokinesis. incenpEC3747 embryos showed no defect in cellularization and Incenp could still be observed to be localized properly. This reflects perdurance of the wild-type maternal product. No significant defects were observed in early-stage embryos, although from mitotic cycle 15, a very low percentage of cells were observed with a reduced amount of Drosophila Incenp and chromosome segregation defects (Chang, 2006).
By embryonic stage 13, Drosophila Incenp was no longer detectable by immunostaining in incenpEC3747 embryos. Consistent with this, phosphorylation of histone H3 on Ser10 (a known Aurora B kinase substrate) was also no longer detected. This phenotype confirms that Incenp is required for Aurora B kinase to function as a histone H3 Ser10 kinase. At this stage, cells of the central nervous system (CNS) in incenpEC3747 embryos showed enlarged nuclei compared with wild-type as observed by DAPI staining for DNA. Staining with anti-gamma-tubulin antibody showed that these enlarged cells contain bigger than normal centrosomes or multiple centrosomes. This phenotype, detectable in mutant embryos following the complete disappearance of the Drosophila Incenp, can be explained as the result of failure of cytokinesis in the previous division and is consistent with a lack of Aurora B kinase function (Adams, 2001c; Oegema, 2001) in the early development of the CNS (Chang, 2006).
Asymmetric cell division is key to the development of the Drosophila nervous system. Each dividing neuroblast produces one large daughter cell that remains a multipotent neuroblast and continues to divide, and a smaller daughter cell that becomes a ganglion mother cell that divides once more asymmetrically to produce neurons or glia cells. This cell-fate decision hinges on the segregation of Prospero, a homeodomain transcription factor that is segregated largely, if not exclusively, into the ganglion mother cell. This is accomplished by sequestering Prospero into a basal cortical crescent in the dividing neuroblast from prophase onwards (Chang, 2006).
In wild-type embryos, the expected asymmetric distribution of Prospero at the basal cell surface of dividing cells was observed. However, in early prophase, Prospero transiently associates with the condensing chromatin on entry into mitosis. The distribution of Prospero was abnormal in neuroblasts lacking detectable Incenp. Abnormalities observed in neuroblasts of incenpEC3747 embryos included defects in the shape and orientation of the basal Prospero crescent. Mitotic neuroblasts were observed with Prospero distributed all around the cell cortex, and not restricted to a basal crescent. These results reveal that Drosophila Incenp and, therefore, presumably the chromosomal passenger complex, is required for the correct localization of Prospero during asymmetric cell division in the developing Drosophila nervous system (Chang, 2006).
The chromosomal passenger complex protein INCENP is required in mitosis for chromosome condensation, spindle attachment and function, and cytokinesis. INCENP has an essential function in the specialized behavior of centromeres in meiosis. Mutations affecting Drosophila incenp profoundly affect chromosome segregation in both meiosis I and II, due, at least in part, to premature sister chromatid separation in meiosis I. INCENP binds to the cohesion protector protein MEI-S332, which is also an excellent in vitro substrate for Aurora B kinase. A MEI-S332 mutant that is only poorly phosphorylated by Aurora B is defective in localization to centromeres. These results implicate the chromosomal passenger complex in directly regulating MEI-S332 localization and, therefore, the control of sister chromatid cohesion in meiosis (Resnick, 2006).
This analysis of Drosophila incenp mutants reveals for the first time a crucial role for INCENP in regulating centromeric cohesion during the reductional division of meiosis. INCENP influences the localization and/or function of MEI-S332: precocious sister chromatid separation is observed at the centromeres in the mutants, the distribution of MEI-S332 is abnormal when INCENP levels are decreased, INCENP can bind MEI-S332 in vitro, the protein is phosphorylated in vitro by Aurora B, and MEI-S332 localization to centromeres in mitosis is perturbed when its preferred Aurora B phosphorylation site is mutated (Resnick, 2006).
The QA26 incenp mutation perturbs chromosome condensation and causes precocious separation of the sister chromatids in spermatocytes. Quantitative genetic nondisjunction tests showed that chromosome segregation fails in both meiosis I and II, and that these nondisjunction events are consistent with premature separation of sister chromatids and random segregation in both meiotic divisions. This genetic analysis is likely to underestimate the true rates of nondisjunction because many of the defects caused by loss of passenger function (e.g., defective spindle organization or cytokinesis) would not yield functional gametes, thereby preventing the scoring all of the nondisjunction events. Although the aberrant condensation in prophase and prometaphase I made direct visualization of the onset of loss of cohesion difficult, completely separated sister chromatids could unambiguously be seen in mutant anaphase I cells, confirming one mechanism that contributes to the genetic nondisjunction phenotype (Resnick, 2006).
In C. elegans meiosis, the chromosome passenger complex is necessary for chiasma resolution. If chromosomal passengers were to participate both in regulation of centromeric cohesion as well as processing of chiasmata in C. elegans, essential roles in the latter might obscure roles in the former. In Drosophila male meiosis, there is no synapsis of homologs or recombination. Rather, segregation of homologous chromosomes is regulated via specific pairing sites. The analysis of passenger function was therefore simplified in Drosophila males, where chiasmata do not form (Resnick, 2006).
The MEI-S332-related yeast Shugoshin proteins are critical for the maintenance of the meiotic-specific cohesin subunit Rec8 at centromeres during anaphase Interestingly, no Rec8 homolog has yet been found in Drosophila. The only Drosophila meiotic kleisin, C(2)M, is a component of the synaptonemal complex and has been shown to have an earlier role in female and male meiosis. Thus, what MEI-S332 protects at centromeres in meiosis remains unclear. In mitosis, ablation of MEI-S332 does not lead to premature loss of the mitotic cohesin Rad21 (Resnick, 2006).
In both incenp mutants, impaired INCENP function results in a failure of MEI-S332 localization to centromeres in meiosis. This presumably leads to defects in the protection of cohesion at sister centromeres and contributes to the observed increase in meiotic nondisjunction. The failure to localize MEI-S332 in the incenp mutants is not a general secondary effect of prophase I condensation defects or of premature sister chromatid separation prior to the onset of anaphase I: ord mutants, which display both of those phenotypes, localize MEI-S332 normally. Although the data support a role for MEI-S332 in the increased nondisjunction in incenp mutants, mei-S332 mutants predominantly lead to meiosis II nondisjunction, whereas the incenp alleles show defects in both meiotic divisions. Thus, INCENP must be required for additional functions beyond its role in MEI-S332 regulation described in this study (Resnick, 2006).
One mechanism by which INCENP could promote MEI-S332 function is through its role in establishing or maintaining the specialized chromatin structure around centromeres. The chromosomal passenger complex is involved in regulation of chromatin remodeling complexes like ISWI, and it interacts with histone and nonhistone proteins from the pericentric heterochromatin. Recent studies show a direct link between Aurora B activity and regulation of HP1 localization in mitosis, suggesting a possible role in the regulation of heterochromatin structure. Since heterochromatin is important for cohesin binding to centromeres, it is possible that modifications of both MEI-S332 and the underlying heterochromatin are important for stabilizing centromeric cohesion during meiosis I (Resnick, 2006).
Alternatively, INCENP could act as a platform for the regulation of MEI-S332 at centromeres. The direct binding between INCENP and MEI-S332 could target MEI-S332 to heterochromatin, or it could help to direct its regulation by protein kinases. MEI-S332 binds better in vitro to a mixture of INCENP and Aurora B than to INCENP alone, suggesting that the interaction is strengthened by phosphorylation of either INCENP or MEI-S332. In addition to its role in binding and activating Aurora B, INCENP that has been phosphorylated by CDK1 can bind to Plk1, the human homolog of POLO kinase (Resnick, 2006).
Binding to phosphorylated INCENP is required to target Plk1 to centromeres in mitosis. Thus, INCENP could potentially coordinate the functions of POLO and Aurora B, both of which have been implicated in the regulation of cohesin (and also in the regulation of MEI-S332 in the case of POLO). These kinases have been shown to cooperate in the release of arm cohesion in chromosomes assembled in Xenopus extract. In contrast to Aurora B, however, POLO promotes the dissociation of MEI-S332 from centromeres during mitosis and meiosis. In polo mutants, MEI-S332 persists on the centromere, and mutation of two POLO box domains disrupts POLO binding and phosphorylation of MEI-S332 in vitro, as well as MEI-S332 dissociation from the centromeres (Resnick, 2006).
Together, these observations suggest that INCENP may act to integrate the various pathways controlling MEI-S332 function in meiosis I. Early in meiosis I, INCENP/Aurora B complexes may stabilize centromeric MEI-S332 through direct binding or modification of the underlying chromatin as described above. Similar to what happens in mitosis, CDK1 could phosphorylate INCENP at the POLO binding site, and phosphorylation-dependent binding of POLO to INCENP could target the kinase to the centromere. This binding might also render the kinase unavailable to phosphorylate MEI-S332. During the metaphase-anaphase I transition, INCENP remains on the centromeres and might therefore prevent MEI-S332 from being phosphorylated by POLO. At the onset of anaphase II, however, as INCENP transitions off the centromere, POLO may be free to phosphorylate MEI-S332, thereby releasing it from centromeres, allowing the release of sister chromatid cohesion (Resnick, 2006).
INCENP is emerging as a key regulator of kinase signaling pathways in mitosis. The present study reveals that this versatile protein may have a similar role in meiosis and may use its interactions with Aurora B and POLO to coordinate the specialized behavior of sister chromatids in meiosis I (Resnick, 2006).
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 was have 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).
Production of haploid gametes relies on the specially regulated meiotic cell cycle. Analyses of the role of essential mitotic regulators in meiosis have been hampered by a shortage of appropriate alleles in metazoans. This study has characterized female-sterile alleles of the condensin complex component dcap-g (Cap-g) and used them to define roles for condensin in Drosophila female meiosis. In mitosis, the condensin complex is required for sister-chromatid resolution and contributes to chromosome condensation. In meiosis, a role for dcap-g is demonstrate in disassembly of the synaptonemal complex and for proper retention of the chromosomes in a metaphase I-arrested state. The chromosomal passenger complex also is known to have mitotic roles in chromosome condensation and is required in some systems for localization of the condensin complex. The QA26 allele of passenger component incenp was used to investigate the role of the passenger complex in oocyte meiosis. Strikingly, in incenpQA26 mutants maintenance of the synaptonemal complex is disrupted. In contrast to the dcap-g mutants, the incenp mutation leads to a failure of paired homologous chromosomes to biorient, such that bivalents frequently orient toward only one pole in prometaphase and metaphase I. incenp interacts genetically with ord, suggesting an important functional relationship between them in meiotic chromosome dynamics. The dcap-g and incenp mutations cause maternal effect lethality, with embryos from mutant mothers arrested in the initial mitotic divisions (Resnick, 2009).
The condensin and chromosomal passenger complexes both have important roles in chromosome condensation in mitosis, and the passenger complex has been shown in many systems to be required for localization or phosphorylation of condensin proteins. This study observed distinct meiotic consequences of mutations in dcap-g and incenp. Strikingly, SC disassembly was premature in incenp mutants but delayed in dcap-g mutants, and prometaphase I and metaphase I chromosome configurations were disrupted in both mutants, but in clearly distinguishable ways (Resnick, 2009).
That both the condensin and passenger complexes affect SC disassembly is intriguing because little is known about regulation of this process. BubR1 has recently been shown to be required for SC maintenance, although the mechanism has not yet been established. A suggestion that condensin might be required for SC disassembly arose from the observation that mutation of nhk-1 disrupts both condensin loading and C(3)G unloading from the chromosomes, within the same developmental window. In the germarium, multiple cells within each cyst initiate meiosis and form SC but ultimately the SC disassembles in all the cells except the oocyte. Interestingly, the dcap-g mutations affect SC disassembly solely in the oocyte, not in the nurse cells at the earlier developmental stage. This corresponds to when condensin assembles on the oocyte chromosomes and raises the possibility of a distinct mechanism of SC disassembly in the nurse cells. Condensin seems not to be required for SC assembly or disassembly in C. elegans, whereas it is required for proper SC assembly in S. cerevisiae. Understanding the differences among these systems and the manner in which condensin is required for SC disassembly in Drosophila remain important questions for future study (Resnick, 2009).
A requirement for the passenger complex in maintenance of the SC is striking in combination with the result that incenp and ord interact genetically. ord is required, as well, for SC maintenance. incenp could regulate ord, and the SC phenotype seen in the incenp mutant could be due to defects in ORD localization or activity. In addition, SC disassembly in C. elegans has been suggested to play a role in positioning AIR-2 (Aurora B) for its role in releasing cohesion at the onset of anaphase I, suggesting that these two complexes might dynamically regulate each other's localization (Resnick, 2009).
The defects in prometaphase I and metaphase I configurations lead to several important conclusions about the functions of the passenger and condensin complexes. First, mutations in both complexes resulted in clear abnormalities, strongly supporting roles for both in a stable metaphase I chromosome configuration. Second, the defects from the dcap-g and incenp mutations were different from each other, suggesting that their roles are distinct and that the phenotypes observed in the incenp mutants are not mediated by defects in condensin localization or activity. Third, ord dominantly enhances the incenpQA26 mutation. Taken together with the observations that ord10 does not display dominant behavior alone or in a mei-S332 background, this enhancement of incenp may reveal an important functional relationship between the two proteins (Resnick, 2009).
In the dcap-g mutants bivalents biorient, and sister chromatids are not prematurely separated, but in metaphase I the homologous chromosomes frequently prematurely separate and begin poleward migration. One explanation for this phenotype is that the chiasmata, which normally provide the physical linkage between the homologs in the bivalent, are not present. This could be due either to an absence of recombination, a failure to form chiasmata, or the premature loss of chiasmata. The latter two effects could result from loss of arm cohesion. Given the female sterility of the dcap-g alleles, it is not possible to recover viable progeny to measure recombination frequencies. Thus currently it is difficult to distinguish between these possibilities. In addition to the distinct functions of condensin in the dynamics of the synaptonemal complex in different organisms, the results highlight differences in the role of condensin in homolog attachments. In Drosophila males, in which recombination does not occur, the condensin II complex is required for homolog separation in anaphase I (Hartl, 2008). In budding yeast condensin is needed to resolve recombination linkages between homologs by promoting the release of cohesin. In C. elegans, the dpy28 gene that encodes a Cap-D2 protein (Tsai, 2008) blocks recombination and affects interference, although it is not yet clear whether it has this function as part of a condensin complex (Resnick, 2009).
The incenpQA26 Df(2L)Exel7049/incenpQA26 mutants show a high frequency of failure of bivalents to biorient. This result indicates that in metazoan meiosis, as in budding and fission yeast, the chromosome passenger complex and Aurora B are likely to have the ability to destabilize improper microtubule kinetochore attachments. Homolog monoorientation is observed in meiosis I in yeast mutant for Aurora B. The biorientation failure explains the multiple chromosome masses observed in prometaphase I, although it is surprising that the monooriented bivalents nevertheless are in a normal compacted chromosome mass by metaphase I. It most likely results from the shortening of the spindle that occurs in the transition from prometaphase I to metaphase I. While this work was under review Colombie (2008) reported that incenpQA26 mutant oocytes have spindle defects. They found that the assembly time for the spindle was extended in prometaphase I and that the central spindle region was less stable in metaphase I. This effect on the metaphase I spindle stability correlates with the observation that INCENP protein localizes to the spindle midregion. The aberrant bivalent orientation observed raises the possibility that the chromosome defects in incenpQA26 are responsible for the delayed bipolar spindle assembly and metaphase I spindle instability, given that the chromosomes organize this acentriolar spindle (Resnick, 2009).
It is important to note that incenpQA26 exhibits somewhat distinct effects in male and female meiosis. In male meiosis in these mutants chromosome condensation is defective and premature loss of sister-chromatid cohesion occurs, but the bivalents biorient. In contrast, in female meiosis in the mutants sister-chromatid cohesion is not lost, but bivalent orientation and spindle formation are defective. These phenotypes may result from important regulatory differences in male and female meiosis, indeed the differing mechanisms of meiotic spindle formation necessitate variations in chromosome-spindle interactions. Alternatively, the distinct phenotypes could arise due to the hypomorphic incenpQA26 allele differentially affecting passenger complex activity in the two tissues (Resnick, 2009).
The identification of female-sterile alleles of dcap-g and incenp has led to the demonstration of critical roles for these proteins, and presumably the protein complexes in which they participate, in several critical aspects of chromosome dynamics during oocyte meiosis. These mutants point to distinct roles for the condensin and chromosome passenger complexes in control of the synaptonemal complex, and they illustrate the crucial role of each of these complexes in formation of a stable metaphase I configuration (Resnick, 2009).
Animal mitotic spindle assembly relies on centrosome-dependent and centrosome-independent mechanisms, but their relative contributions remain unknown. This study investigated the molecular basis of the centrosome-independent spindle assembly pathway by performing a whole-genome RNAi screen in Drosophila S2 cells lacking functional centrosomes. This screen identified 197 genes involved in acentrosomal spindle assembly, eight of which had no previously described mitotic phenotypes and produced defective and/or short spindles. All 197 genes also produced RNAi phenotypes when centrosomes were present, indicating that none were entirely selective for the acentrosomal pathway. However, a subset of genes produced a selective defect in pole focusing when centrosomes were absent, suggesting that centrosomes compensate for this shape defect. Another subset of genes was specifically associated with the formation of multipolar spindles only when centrosomes were present. It was further shown that the chromosomal passenger complex orchestrates multiple centrosome-independent processes required for mitotic spindle assembly/maintenance. On the other hand, despite the formation of a chromosome-enriched RanGTP gradient, S2 cells depleted of RCC1, the guanine-nucleotide exchange factor for Ran on chromosomes, established functional bipolar spindles. Finally, it was shown that cells without functional centrosomes have a delay in chromosome congression and anaphase onset, which can be explained by the lack of polar ejection forces. Overall, these findings establish the constitutive nature of a centrosome-independent spindle assembly program and how this program is adapted to the presence/absence of centrosomes in animal somatic cells (Moutinho-Pereira, 2013).
This study has identified eight genes involved in spindle assembly in S2 cells. Mitotic spindle organization in the presence/absence of centrosomes is driven by a common set of genes. However, a specific cohort of genes was identified that differentially affect the formation of a bipolar spindle depending upon whether the centrosomes are present or not. In particular, knockdown of γ-TuRC, 26S proteasome, and the chaperone complex t-complex polypeptide-1 (TCP-1) subunits (involved in folding various proteins, including actin and tubulin) all produced a much more obvious pole-focusing defect specifically when centrosomes are absent. Finally, it was also found that spindle assembly in S2 cells is not affected by >95% depletion of the RanGTP effector RCC1 (Moutinho-Pereira, 2013).
These results suggest that either Drosophila S2 cell spindles are very robust to a decrease in RanGTP or that, alternatively, Ran-independent pathways compensate for the loss of RanGTP. The recently discovered Aurora B phosphorylation gradient along the spindle (Tan, 2011) may provide the necessary spatiotemporal cues for centrosome-independent MT stabilization and bipolar spindle formation. Indeed, previous findings have implicated the human CPC in spindle assembly and in SAC response. The present data link both processes and support that the CPC [see Incenp, an essential subunit of the CPC (the Aurora B complex)] has an impact on mitotic spindle assembly/maintenance, in part, by regulating the duration of mitosis, regardless of the presence/ absence of functional centrosomes (Moutinho-Pereira, 2013).
Collectively, this study's RNAi screening results in Drosophila S2 cells suggest that a centrosome-independent spindle pathway operates constitutively during spindle assembly and does not require a distinct backup genetic mechanism. These data are fully consistent with recent transcriptome profiling studies of acentrosomal cells in Drosophila brains and wing discs. However, certain cell systems demonstrate a greater dependence on centrosomes for spindle assembly and furrow positioning during early embryonic divisions or centrosomes might enhance cell division fidelity in mammalian somatic cells. Nevertheless, the conclusions support the view that centrosomes are not main drivers of spindle assembly during mitosis (Moutinho-Pereira, 2013).
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date revised: 10 March 2013
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