Mutations in the Drosophila mei-S332 gene cause premature separation of the sister chromatids in late anaphase of meiosis I. Therefore, the mei-S332 protein was postulated to hold the centromere regions of sister chromatids together until anaphase II. The mei-S332 gene encodes a novel 44 kDa protein. Mutations in mei-S332 that differentially affect function in males or females map to distinct domains of the protein. A fusion of mei-S332 to the green fluorescent protein (GFP) is fully functional and localizes specifically to the centromere region of meiotic chromosomes. When sister chromatids separate at anaphase II, Mei-S332-GFP disappears from the chromosomes, suggesting that the destruction or release of this protein is required for sister-chromatid separation (Kerrebrock, 1995).

The Drosophila MEI-S332 protein has been shown to be required for the maintenance of sister-chromatid cohesion in male and female meiosis. The protein localizes to the centromeres during male meiosis when the sister chromatids are attached, and it is no longer detectable after they separate. Drosophila melanogaster male meiosis is atypical in several respects, making it important to define Mei-S332 behavior during female meiosis, which better typifies meiosis in eukaryotes. Mei-S332 localizes to the centromeres of prometaphase I chromosomes in oocytes, remaining there until it is delocalized at anaphase II. By using oocytes sufficient material was obtained to investigate the fate of Mei-S332 after the metaphase II-anaphase II transition. The levels of Mei-S332 protein are unchanged after the completion of meiosis, even when translation is blocked, suggesting that the protein dissociates from the centromeres but is not degraded at the onset of anaphase II. Unexpectedly, Mei-S332 is present during embryogenesis, localizes onto the centromeres of mitotic chromosomes, and is delocalized from anaphase chromosomes. Thus, Mei-S332 associates with the centromeres of both meiotic and mitotic chromosomes and dissociates from them at anaphase (Moore, 1998).

Attachment, or cohesion, between sister chromatids is essential for their proper segregation in mitosis and meiosis. Sister chromatids are tightly apposed at their centromeric regions, but it is not known whether this is due to cohesion at the functional centromere or at flanking centric heterochromatin. The Drosophila Mei-S332 protein maintains sister-chromatid cohesion at the centromeric region. By analyzing Mei-S332's localization requirements at the centromere on a set of minichromosome derivatives, the role of heterochromatin and the relationship between cohesion and kinetochore formation in a complex centromere of a higher eukaryote was tested. The frequency of Mei-S332 localization is decreased on minichromosomes with compromised inheritance, despite the consistent presence of two kinetochore proteins. Furthermore, Mei-S332 localization is not coincident with kinetochore outer-plate proteins, suggesting that it is located near the DNA. It is concluded that Mei-S332 localization is driven by the functional centromeric chromatin, and binding of Mei-S332 is regulated independently of kinetochore formation. These results suggest that in higher eukaryotes cohesion is controlled by the functional centromere, and that, in contrast to yeast, the requirements for cohesion are separable from those for kinetochore assembly (Lopez, 2000).

Given the role of Polo in promoting release of sister chromatid cohesion in mitosis and meiosis, the relationship between Polo and Mei-S332 was tested by investigating genetic interactions between polo and mei-S332 mutants in Drosophila. Although homozygous strong polo mutations are lethal (polo⁹ and polo¹⁰) and weak mutations are sterile (polo¹), tests were performed for dominant enhancement or suppression of the mei-S332 meiosis II nondisjunction phenotype in the presence of one mutant copy of the polo gene. The mei-S332⁸ allele was used that results in intermediate levels of chromosome missegregation in males; this mutant form of the protein still localizes to centromeres (Kerrebrock, 1992; Tang, 1998; Clarke, 2005).

Male flies homozygous for the mei-S332⁸ mutation and heterozygous either for a deficiency that removes the polo gene, Df(3L)rdgC-co2, or for the strong polo alleles, polo⁹ or polo¹⁰, were scored for segregation of the X and Y chromosomes and compared to sibling controls mutant only for mei-S332⁸. All three polo mutants dominantly suppress the mei-S332⁸ nondisjunction phenotype to degrees proportional to the severity of the polo defect. The polo deficiency suppresses to the greatest extent, decreasing total nondisjunction by 72%. The polo⁹ allele suppresses with a 52% decrease and polo¹⁰ with a 46% decrease, correlating with allele strength. In addition, the weak polo¹ allele also dominantly reduces mei-S332⁸ nondisjunction, although to a lesser extent. Thus, the effect of the polo deficiency and polo mutants from three different genetic backgrounds is most likely due to a reduction of Polo activity. The striking ability of decreased polo activity to suppress sister chromatid missegregation in mei-S332 mutants indicates that Polo antagonizes the function of Mei-S332 in promoting sister chromatid cohesion, raising the possibility that Polo inactivates or delocalizes Mei-S332 (Clarke, 2005).

Mei-S332 localizes to meiotic centromeres from prometaphase I until the metaphase II/anaphase II transition, correlating with its role in maintaining sister chromatid cohesion until segregation in meiosis II (Moore, 1998). Whether heterozygous or homozygous polo mutants affect the centromere localization or delocalization of Mei-S332 in male meiosis was tested. polo⁹ and polo¹⁰ were examined as heterozygotes, but homozygous mutants die in the third instar larval stage so spermatocytes could not be recovered. It was found, however, that polo⁹/polo¹ transheterozygous animals survive to adulthood but are sterile. Mei-S332 localization was examined in these transheterozygotes (Clarke, 2005).

A striking effect of polo mutations on Mei-S332 centromere localization was observed: Mei-S332 remains localized to chromosomes after the metaphase II/anaphase II transition in both heterozygous polo⁹/+ or polo¹⁰/+ and transheterozygous polo⁹/polo¹ mutant spermatocytes and fail to delocalize at the proper time. The loss of polo activity primarily affects centromere dissociation of Mei-S332 but not association, since foci of Mei-S332 bound to centromeres are clearly visible in the polo⁹/polo¹ cells. Although Mei-S332 is somewhat diffusely spread on chromosomes in the polo¹⁰/+ telophase II cells, some concentrated foci at centromeres are present. Mei-S332 persists at centromeres in some but not all polo⁹/+ anaphase II cells. Some lagging chromosomes were observed in the polo⁹/+ heterozygotes, and obvious chromosome segregation defects occur in the polo⁹/polo¹ transheterozygotes (Clarke, 2005).

The finding that Polo function is needed for delocalization of Mei-S332 from centromeres in meiosis could provide an explanation for the suppression of the mei-S332⁸ nondisjunction phenotype. Reduced Polo function may suppress premature sister separation in mei-S332 mutants either by retention of Mei-S332 longer at centromeres or by increased Mei-S332 activitys (Clarke, 2005).

Mei-S332 has a striking localization pattern during the mitotic cell cycle; the protein localizes to centromeres during prometaphase and dissociates at the metaphase/anaphase transition. To determine whether Polo kinase regulates Mei-S332 in mitosis, the localization of Mei-S332 was examined in polo mutant larval brain neuroblasts. This cell type was examined because cells in the larval brain are actively dividing, and the polo⁹ and polo¹⁰ mutant phenotype is manifested at this stage of development. These alleles provided the additional advantage that some cells arrest in metaphase with centromeres separated while others proceed into anaphase (Clarke, 2005).

In both polo⁹ and polo¹⁰ heterozygous and homozygous mutant neuroblasts, Mei-S332 localized normally to centromeres during metaphase. In the heterozygotes, Mei-S332 delocalized from centromeres in anaphase as in wild-type. In homozygous polo mutants arrested at metaphase with separated centromeres, Mei-S332 fails to delocalize from centromeres, even when the small fourth chromosomes completely separates and migrates to the poles. These results suggest that Polo kinase is required for the release of Mei-S332 from centromeres in mitosis as in meiosis II (Clarke, 2005).

Cyclin B staining confirmed that some cells with separated centromeres showing Mei-S332 staining were no longer in metaphase, since it is present in metaphase cells but is degraded at the metaphase/anaphase transition and thus absent in anaphase cells. In contrast to wild-type cells, a significant number of polo mutant neuroblasts showing two rows of Mei-S332 staining had low levels of Cyclin B (35% in polo⁹ mutants and 40% in polo¹⁰ mutants). These cells may be in anaphase, or it is possible that some cells showing no Cyclin B staining had also reached interphase. The presence of localized Mei-S332 in cells lacking Cyclin B (localization never observed in wild-type cells), suggests that Polo kinase activity is needed for Mei-S332 delocalization from centromeres (Clarke, 2005).

To investigate whether Mei-S332 remains localized to centromeres in interphase in polo mutants, the presence of phosphorylated histone H3 was used to mark cells in mitosis and, conversely, its absence was used to identify cells in interphase. Mei-S332 is not normally detected on chromosomes in interphase cells. In contrast, in polo mutant neuroblasts with Mei-S332 staining, 33% of polo⁹ cells and 28% of polo¹⁰ cells had no phospho-histone H3 staining. These results indicate that Mei-S332 is unable to be released from centromeres in polo mutants (Clarke, 2005).

Faithful segregation of sister chromatids during cell division requires properly regulated cohesion between the sister centromeres. The sister chromatids are attached along their lengths, but particularly tightly in the centromeric regions. Therefore specific cohesion proteins may be needed at the centromere. Drosophila Mei-S332 protein localizes to mitotic metaphase centromeres. Both overexpression and mutation of Mei-S332 increase the number of apoptotic cells. In mei-S332 mutants the ratio of metaphase to anaphase figures is lower than wild type, but it is higher if Mei-S332 is overexpressed. In chromosomal squashes centromeric attachments appear weaker in mei-S332 mutants than wild type and tighter when Mei-S332 is overexpressed. These results are consistent with Mei-S332 contributing to centromeric sister-chromatid cohesion in a dose-dependent manner. Mei-S332 is the first member identified of a predicted class of centromeric proteins that maintain centromeric cohesion (LeBlanc, 1999).

The Drosophila mei-S332 gene acts to maintain sister-chromatid cohesion before anaphase II of meiosis in both males and females. By isolating and analyzing seven new alleles and a deficiency uncovering the mei-S332 gene it has been demonstrated that the onset of the requirement for mei-S332 is not until late anaphase I. All of the alleles result primarily in equational (meiosis II) nondisjunction with low amounts of reductional (meiosis I) nondisjunction. Cytological analysis revealed that sister chromatids frequently separate in late anaphase I in these mutants. Since the sister chromatids remain associated until late in the first division, chromosomes segregate normally during meiosis I, and the genetic consequences of premature sister-chromatid dissociation are seen as nondisjunction in meiosis II. The late onset of mei-S332 action demonstrated by the mutations was not a consequence of residual gene function because two strong, and possibly null, alleles give predominantly equational nondisjunction both as homozygotes and in trans to a deficiency. mei-S332 is not required until after metaphase I, when the kinetochore differentiates from a single hemispherical kinetochore jointly organized by the sister chromatids into two distinct sister kinetochores. Therefore, it is proposed that the mei-S332 product acts to hold the doubled kinetochore together until anaphase II. All of the alleles are fully viable when in trans to a deficiency, thus mei-S332 is not essential for mitosis. Four of the alleles show an unexpected sex specificity (Kerebrock, 2002).

Two disjunction defective meiotic mutants, ord and mei-S332, each of which disrupts meiosis in both male and female Drosophila melanogaster, were analyzed cytologically and genetically in the male germ-line. It was observed that sister-chromatids are frequently associated abnormally during prophase I and metaphase I in ord. Sister chromatid associations in mei-S332 are generally normal during prophase I and metaphare I. By telophase I, sister chromatids have frequently precociously separated in both mutants. During the first division sister chromatids disjoin from one another frequently in ord and rarely in mei-S332. It is argued that the simplest interpretation of the observations is that each mutant is defective in sister chromatid cohesiveness and that the defect in ord manifests itself earlier than does the defect in mei-S332. In addition, based on these mutant effects, several conclusions regarding normal meiotic processes are drawn. (1) The phenotype of these mutants support the proposition that the second meiotic metaphase (mitotic-type) position of chromosomes and their equational orientation is a consequence of the equilibrium, at the metaphase plate, of pulling forces acting at the kinetochores and directed towards the poles. (2) Chromosomes which lag during the second meiotic division tend to be lost. (3) Sister chromatid cohesiveness, or some function necessary for sister chromatid cohesivenss, is required for the normal reductional orientation of sister kinetochores during the first meiotic division. (4) The kinetochores of a half-bivalent are double at the time of chromosome orientation during the first meiotic division. Finally, functions which are required throughout meiosis in both sexes must be considered in the pathways of meiotic control (Goldstein, 1980).

The Drosophila mei-S332 and ord gene products are essential for proper sister-chromatid cohesion during meiosis in both males and females. Flies were constructed that contain null mutations for both genes. Double-mutant flies are viable and fertile. Therefore, the lack of an essential role for either gene in mitotic cohesion cannot be explained by compensatory activity of the two proteins during mitotic divisions. Analysis of sex chromosome segregation in the double mutant indicates that ord is epistatic to mei-S332. ord is not required for Mei-S332 protein to localize to meiotic centromeres. Although overexpression of either protein in a wild-type background does not interfere with normal meiotic chromosome segregation, extra ORD⁺ protein in mei-S332 mutant males enhances nondisjunction at meiosis II. These results suggest that a balance between the activity of mei-S332 and ord is required for proper regulation of meiotic cohesion and demonstrate that additional proteins must be functioning to ensure mitotic sister-chromatid cohesion (Bickel, 1998).

Approximately one-third of the Drosophila kinome has been ascribed some cell-cycle function. However, little is known about which of its 117 protein phosphatases (PPs) or subunits have counteracting roles. This study investigated mitotic roles of PPs through systematic RNAi. It was found that G2-M progression requires Puckered, the JNK MAP-kinase inhibitory phosphatase and PP2C in addition to string (Cdc25). Strong mitotic arrest and chromosome congression failure occurs after Pp1-87B downregulation. Chromosome alignment and segregation defects also occurs after knockdown of PP1-Flapwing, not previously thought to have a mitotic role. Reduction of several nonreceptor tyrosine phosphatases produced spindle and chromosome behavior defects, and for corkscrew, premature chromatid separation. RNAi of the dual-specificity phosphatase, Myotubularin, or the related Sbf 'antiphosphatase' resulted in aberrant mitotic chromosome behavior. Finally, for PP2A, knockdown of the catalytic or A subunits led to bipolar monoastral spindles, knockdown of the Twins B subunit led to bridged and lagging chromosomes, and knockdown of the B' Widerborst subunit led to scattering of all mitotic chromosomes. Widerborst is associated with MEI-S332 (Shugoshin) and is required for its kinetochore localization. This study has identified cell-cycle roles for 22 of 117 Drosophila PPs. Involvement of several PPs in G2 suggests multiple points for its regulation. Major mitotic roles are played by PP1 with tyrosine PPs and Myotubularin-related PPs having significant roles in regulating chromosome behavior. Finally, depending upon its regulatory subunits, PP2A regulates spindle bipolarity, kinetochore function, and progression into anaphase. Discovery of several novel cell-cycle PPs identifies a need for further studies of protein dephosphorylation (Chen, 2007).

P2A is a heterotrimeric serine/threonine phosphatase composed of invariant catalytic (C) and structural (A) subunits together with one member of a family of B regulatory subunits thought to direct the AC core to different substrates. The Drosophila gene for the catalytic subunit of type 2A protein serine/threonine phosphatase (PP2A) is known as microtubule star (mts) because mutant embryos show multiple individual centrosomes with disorganized astral arrays of microtubules. In agreement with this mutant phenotype, it was found that S2 cells depleted for Mts (PP2A-C) displayed aberrant elongated arrays of microtubules with a high proportion (5- to 10-fold increase over the control) of bipolar monoastral spindles or monopolar spindles emanating from a single centrosomal mass. This phenotype is also consistent with the observations in Xenopus egg extracts where mitotic microtubules grow longer and bipolar spindles can not be assembled after inhibition of PP2A by low concentrations of okadaic acid (OA). It is speculated that the monopolar spindle phenotype after mts dsRNA treatment is a consequence of the spindle collapse rather than a failure in centrosome duplication or separation because most of the RNAi-treated cells showed well-separated centrosomes during prophase. In support of this view, spindle bipolarity can be rescued by restoration of microtubule dynamics in OA-treated Xenopus egg extracts (Chen, 2007).

In Drosophila, as in many other eukaryotes, mitosis-specific phosphorylation of histone H3 requires Aurora B activity, but the identity of the opposing phosphatase remains unclear. Because P-H3 (Ser 10) levels were used for monitoring the mitotic index in this analysis, it is possible that a high mitotic index observed after RNAi for PPs may also reflect a defect in dephosphorylating P-H3 in the absence of PPs upon mitotic exit. The phosphorylation state of this histone was therefore studied after RNAi for PPs that displayed a significant increase in the mitotic index in the screen. The immunostaining of control cells showed that P-H3 signals began to decrease at early telophase and then disappeared completely at late telophase. After RNAi knockdown of Mts (PP2A-C) or Pp1-87B, however, the majority of mitotic cells were arrested at prometaphase, but late telophase figures could occassionally be found showing an abnormal accumulation of P-H3 on decondensed chromosomes. To better assess the effect of depletion of these two PPs on P-H3 dephosphorylation, the spindle-assembly checkpoint was inactivated by simultaneously knocking down BubR1. It was found that this rescued the prometaphase arrest of cells simultaneously depleted for Mts or Pp1-87B; this allowed a study of telophase cells. P-H3 was present in the majority of such telophase cells compared to control cells, indicating that both PPs are required for P-H3 dephosphorylation. These results are in accordance with previous studies showing that reduction of PP1 activity can partially suppress defects in the mitotic histone H3 phosphorylation in yeast and C. elegans (Chen, 2007).

Downregulation of Pp2A-29B, the structural A subunit, revealed almost identical aberrant phenotypes to those observed after mts (PP2A-C) RNAi. Consistently, knockdown of Pp2A-29B (PP2A-A) led to a reduction of the protein level of Mts (PP2A-C) (Chen, 2007).

The Drosophila genome contains 4 B-type PP2A regulatory subunits, twins/tws/aar (B sub-type), widerborst/wdb (B' sub-type), Pp2A-B' (B' sub-type), and Pp2A-B" (B" sub-type), but mitotic defects have so far only been reported for mutants of tws. Consistent with the phenotype of tws mutants, it was observed that RNAi for this gene led to an increased proportion of anaphase figures showing lagging chromosomes and chromosome bridges (Chen, 2007).

In metazoans, the B' regulatory subunits of PP2A have evolved into two related subclasses with conserved central regions and diverged amino and carboxy termini. The protein encoded by widerborst (wdb) is more closely related to the human α and ɛ subunits (79%-80% identity) than to the β, γ, or δ subunits (69%-75% identity). Whereas RNAi for tws led to lagging chromosomes, wdb RNAi led to dramatic scattering of chromosomes throughout the spindle. Whether this dramatic effect of wdb RNAi on chromosome segregation reflected any particular subcellular localization of this regulatory subunit was examined. To this end, a GFP-tagged Wdb was expressed in S2 cells. During interphase and prophase, Wdb::GFP partially colocalized with the centromeric marker CID (CENP-A). After spindle formation, Wdb::GFP was found adjacent and external to the centromeres. Although less pronounced, this distribution remained during chromosome segregation at anaphase. Because MEI-S332 (Drosophila Shugoshin) is a dynamic centromeric marker, its distribution was examined in wdb RNAi cells. In control cells, MEI-S332 localized in a band between each pair of the centromeres at metaphase. After downregulation of wdb, however, greatly reduced MEI-S332 staining was found on the metaphase chromosome. In contrast, depletion of MEI-S332 by RNAi did not affect the normal localization of the Wdb B' PP2A subunit. Thus, it is concluded that the Wdb B' subunit is required for correct localization of MEI-S332 but not vice versa. Whether the two proteins existed in the same complex was examined. To address this, a Protein A (PrA)-tagged form of MEI-S332 was expressed in S2 cells to purify potential protein complexes and identify its components by mass spectrometry. The catalytic C (Mts), the structural A (PP2A-29B), and the regulatory B' (Wdb) and B (Tws) subunits of PP2A were identified associated with MEI-S332. Three recent studies also identified PP2A complexed to the B' subunit bound to Shugoshin (Sgo) in human and yeast cells, where they are thought to protect centromeric cohesin subunits from phosphorylation that would promote premature sister-chromatid separation. As with the archetypal family member, Drosophila MEI-S332, the Shugoshins function primarily to protect sister chromatids from separation in the first meiotic division but are also present in mitotic divisions. Consistent with these observations in Drosophila S2 cells, it has been found that depletion of PP2A in human cells led to premature dissociation of Shugoshin 1 (Sgo1) from the kinetochore and loss of mitotic centromere cohesion. The finding of Shugoshin complexed to PP2A/B' in yeast and human, and now in Drosophila, points to a highly evolutionally conserved role for this particular PP2A heterotrimer in regulating sister-chromatid cohesion. Interestingly, Tws B regulatory subunit was also recovered associated with MEI-S332. How this subunit of PP2A might function with MEI-S332 should be the subject of future investigations (Chen, 2007).

Only a moderatedly elevated mitotic index (by approximately 10%) was observed after downregulation of the second Drosophila B' regulatory subunit (Pp2A-B'/B56-1). However, when this second B' subunit was simultaneously knocked down with Wdb, this led to similar phenotypes seen in Mts (PP2A-C) or Pp2A-29B (PP2A-A)-depleted cells. Western-blot analysis showed that the Mts (PP2A-C) level decreased after simultaneous knockdown of both B' subunits, suggesting that this phenotype could be partially due to the loss of PP2A catalytic subunit, although the possibility that the two B' subunits share partially redundant mitotic functions cannot be excluded (Chen, 2007).

Cell-cycle kinases represent a large family of enzymes governing the cell division cycle. It is therefore not surprising that a considerable number of counteracting cell-cycle phosphatases (19% of the genes for tested) were identified in the current study. In addition to finding all the well-known PPs required for cell-cycle progression in Drosophila (Mts, Tws, String, Pp4-19C, and Pp1-87B), the Drosophila counterparts of some eight PPs implicated in cell-cycle functions were identified from studies on other organisms together with six PPs for which novel cell-cycle roles were ascribe. These results were validated by confirming the observed phenotypes with a second nonoverlapping dsRNA. In two cases (flw and csw), their mitotic roles were confirmed through the analysis of phenotypes in mutant larval neuroblasts. The RNAi phenotypes of catalytic subunits were evaluated by observing similar phenotypes after downregulation of the corresponding regulatory subunits (e.g., Pp4-19C and PPP4R2r, Mts/PP2A-C and Pp2A-29B/PP2A-A, and simultaneous RNAi of the two PP2A-B' regulatory subunits). Although a recent large-scale RNAi screen based solely on flow cytometry in Drosophila S2 cells identified many regulators of the cell cycle, cell size, and cell death, this study showed a very low degree of overlap with the cureent analysis (only six), reflecting the need for more sensitive small-scale screens that can examine the functional requirements of assayed proteins in greater detail. These results have provided novel insights into the cell-cycle functions of the Drosophila PPs, and it is likely that, in many cases, these functions have been conserved in other metazoans including humans. This study should guide future work aimed at elucidating the significance and mechanisms of the balanced activities of PKs and PPs in regulating the cell division cycle. The challenge ahead will be to match up the functions of the PPs that were identified with their corresponding counteracting PKs and to identify their common key substrates (Chen, 2007).

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