The aurora B kinase AIR-2 regulates kinetochores during mitosis and is required for separation of homologous Chromosomes during meiosis

BACKGROUND: Mitotic chromosome segregation depends on bi-orientation and capture of sister kinetochores by microtubules emanating from opposite spindle poles and the near synchronous loss of sister chromatid cohesion. During meiosis I, in contrast, sister kinetochores orient to the same pole, and homologous kinetochores are captured by microtubules emanating from opposite spindle poles. Additionally, mechanisms exist that prevent complete loss of cohesion during meiosis I. These features ensure that homologs separate during meiosis I and sister chromatids remain together until meiosis II. The mechanisms responsible for orienting kinetochores in mitosis and for causing asynchronous loss of cohesion during meiosis are not well understood. RESULTS: During mitosis in C. elegans, aurora B kinase, AIR-2, is not required for sister chromatid separation, but it is required for chromosome segregation. Condensin recruitment during metaphase requires AIR-2; however, condensin functions during prometaphase, independent of AIR-2. During metaphase, AIR-2 promotes chromosome congression to the metaphase plate, perhaps by inhibiting attachment of chromatids to both spindle poles. During meiosis in AIR-2-depleted oocytes, congression of bivalents appears normal, but segregation fails. Localization of AIR-2 on meiotic bivalents suggests this kinase promotes separation of homologs by promoting the loss of cohesion distal to the single chiasma. Inactivation of the phosphatase that antagonizes AIR-2 causes premature separation of chromatids during meiosis I, in a separase-dependent reaction. CONCLUSIONS: Aurora B functions to resolve chiasmata during meiosis I and to regulate kinetochore function during mitosis. Condensin mediates chromosome condensation during prophase, and condensin-independent pathways contribute to chromosome condensation during metaphase.     

Slk19p is necessary to prevent separation of sister chromatids in meiosis I

BACKGROUND: A fundamental difference between meiotic and mitotic chromosome segregation is that in meiosis I, sister chromatids remain joined, moving as a unit to one pole of the spindle rather than separating as they do in mitosis. It has long been known that the sustained linkage of sister chromatids through meiotic anaphase I is accomplished by association of the chromatids at the centromere region. The localization of the cohesin Rec8p to the centromeres is essential for maintenance of sister chromatid cohesion through meiosis I, but the molecular basis for the regulation of Rec8p and sister kinetochores in meiosis remains a mystery. RESULTS: We show that the SLK19 gene product from Saccharomyces cerevisiae is essential for proper chromosome segregation during meiosis I. When slk19 mutants were induced to sporulate they completed events characteristic of meiotic prophase I, but at the first meiotic division they segregated their sister chromatids to opposite poles at high frequencies. The vast majority of these cells did not perform a second meiotic division and proceeded to form dyads (asci containing two spores). Slk19p was found to localize to centromere regions of chromosomes during meiotic prophase where it remained until anaphase I. In the absence of Slk19p, Rec8p was not maintained at the centromere region through anaphase I as it is in wild-type cells. Finally, we demonstrate that Slk19p appears to function downstream of the meiosis-specific protein Spo13p in control of sister chromatid behavior during meiosis I. CONCLUSIONS: Our results suggest that Slk19p is essential at the centromere of meiotic chromosomes to prevent the premature separation of sister chromatids at meiosis I.     

CUL-2 and ZYG-11 promote meiotic anaphase II and the proper placement of the anterior-posterior axis in C. elegans

The faithful segregation of chromosomes during meiosis is vital for sexual reproduction. Currently, little is known about the molecular mechanisms regulating the initiation and completion of meiotic anaphase. We show that inactivation of CUL-2, a member of the cullin family of ubiquitin ligases, delays or abolishes meiotic anaphase II with no effect on anaphase I, indicating differential regulation during the two meiotic stages. In cul-2 mutants, the cohesin REC-8 is removed from chromosomes normally during meiosis II and sister chromatids separate, suggesting that the failure to complete anaphase results from a defect in chromosome movement rather than from a failure to sever chromosome attachments. CUL-2 is required for the degradation of cyclin B1 in meiosis and inactivation of cyclin B1 partially rescued the meiotic delay in cul-2 mutants. In cul-2 mutants, the failure to degrade cyclin B1 precedes the metaphase II arrest. CUL-2 is also required for at least two aspects of embryonic polarity. The extended meiosis II in cul-2 mutants induces polarity reversals that include reversed orientation of polarity proteins, P granules, pronuclei migration and asymmetric cell division. Independently of its role in meiotic progression, CUL-2 is required to limit the initiation/spread of the polarity protein PAR-2 in regions distant from microtubule organizing centers. Finally, we show that inactivation of the leucine-rich repeat protein ZYG-11 produces meiotic and polarity reversal defects similar to those observed in cul-2 mutants, suggesting that the two proteins function in the same pathways.     

SOLO: a meiotic protein required for centromere cohesion,coorientation, and SMC1 localization in Drosophila melanogaster

Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis.     

LAB-1 Targets PP1 and Restricts Aurora B Kinase upon Entrance into Meiosis to Promote Sister Chromatid Cohesion

Successful execution of the meiotic program depends on the timely establishment and removal of sister chromatid cohesion. LAB-1 has been proposed to act in the latter by preventing the premature removal of the meiosis-specific cohesin REC-8 at metaphase I in C. elegans, yet the mechanism and scope of LAB-1 function remained unknown. Here we identify an unexpected earlier role for LAB-1 in promoting the establishment of sister chromatid cohesion in prophase I. LAB-1 and REC-8 are both required for the chromosomal association of the cohesin complex subunit SMC-3. Depletion of lab-1 results in partial loss of sister chromatid cohesion in rec-8 and coh-4 coh-3 mutants and further enhanced chromatid dissociation in worms where all three kleisins are mutated. Moreover, lab-1 depletion results in increased Aurora B kinase (AIR-2) signals in early prophase I nuclei, coupled with a parallel decrease in signals for the PP1 homolog, GSP-2. Finally, LAB-1 directly interacts with GSP-1 and GSP-2. We propose that LAB-1 targets the PP1 homologs to the chromatin at the onset of meiosis I, thereby antagonizing AIR-2 and cooperating with the cohesin complex to promote sister chromatid association and normal progression of the meiotic program.     

Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I

During meiosis, two rounds of chromosome segregation occur after a single round of DNA replication, producing haploid progeny from diploid progenitors. Three innovations in chromosome behaviour during meiosis I accomplish this unique division. First, crossovers between maternal and paternal sister chromatids (detected cytologically as chiasmata) bind replicated maternal and paternal chromosomes together. Second, sister kinetochores attach to microtubules from the same pole (mono-polar orientation), causing maternal and paternal centromere pairs (and not sister chromatids) to be separated. Third, sister chromatid cohesion near centromeres is preserved at anaphase I when cohesion along chromosome arms is destroyed. The finding that destruction of mitotic cohesion is regulated by Polo-like kinases prompted us to investigate the meiotic role of the yeast Polo-like kinase Cdc5. We show here that cells lacking Cdc5 synapse homologues and initiate recombination normally, but fail to efficiently resolve recombination intermediates as crossovers. They also fail to properly localize the Lrs4 (ref. 3) and Mam1 (ref. 4) monopolin proteins, resulting in bipolar orientation of sister kinetochores. Cdc5 is thus required both for the formation of chiasmata and for cosegregation of sister centromeres at meiosis I.     

Role of Caenorhabditis elegans protein phosphatase type 1, CeGLC-7 beta,in metaphase to anaphase transition during embryonic development

In Caenorhabditis elegans embryogenesis, phosphorylation events are critical to chromosomal changes. To investigate the dephosphorylation of chromosome behavior, we cloned and characterized the cDNA that encodes C. elegans protein phosphatase type 1 (CeGLC-7 beta), which is composed of 333 amino acids. CeGLC-7 beta possesses a highly conserved amino acid sequence with mammalian and Drosophila protein phosphatase 1. Here, we report on the contribution of CeGLC-7 beta to the dephosphorylation of histone H3 at anaphase. At the embryonic stage, CeGLC-7 beta is associated with the nuclear membrane and chromosomes. The deletion of the Ceglc-7 beta gene and a microinjection of double-stranded RNA produce a disorganized embryogenesis. The Ceglc-7 beta gene mutation causes an abnormal accumulation of phosphorylated histone H3 and delays the mitotic process after anaphase. We propose that CeGLC-7 beta is involved in chromosome dynamics including histone H3 dephosphorylation.     

Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin

It has been proposed but never proven that cohesion between sister chromatids distal to chiasmata is responsible for holding homologous chromosomes together while spindles attempt to pull them toward opposite poles during metaphase of meiosis I. Meanwhile, the mechanism by which disjunction of homologs is triggered at the onset of anaphase I has remained a complete mystery. In yeast, cohesion between sister chromatid arms during meiosis depends on a meiosis-specific cohesin subunit called Rec8, whose mitotic equivalent, Sccl, is cleaved at the metaphase to anaphase transition by an endopeptidase called separin. We show here that cleavage of Rec8 by separin at one of two different sites is necessary for the resolution of chiasmata and the disjunction of homologous chromosomes during meiosis.     

CDC-48/p97 is required for proper meiotic chromosome segregation via controlling AIR-2/Aurora B kinase localization in Caenorhabditis elegans

CDC-48/p97 is a AAA (ATPases associated with diverse cellular activities) chaperone involved in protein conformational changes such as the disassembly of protein complexes. We previously reported that Caenorhabditis elegans CDC-48.1 and CDC-48.2 (CDC-48s) are essential for the progression of meiosis I metaphase. Here, we report that CDC-48s are required for proper chromosome segregation during meiosis in C. elegans. In wild-type worms, at the diakinesis phase, phosphorylation of histone H3, one of the known substrates of aurora B kinase (AIR-2), on meiosis I chromatids correlated with AIR-2 localization at the cohesion sites of homologous chromatids. Conversely, depletion of CDC-48s resulted in a significant expansion of signals for AIR-2 and phosphorylated histone H3 over the entire length of meiotic chromosomes, leading to defective chromosome segregation, while the total amount of AIR-2 in lysates was not changed by the depletion of CDC-48s. The defective segregation of meiotic chromosomes caused by the depletion of CDC-48s was suppressed by the simultaneous depletion of AIR-2 and is similar to that observed following the depletion of protein phosphatase 1 (PP1) phosphatases. However, the amount and localization of PP1 were not changed by the depletion of CDC-48s. These results suggest that CDC-48s control the restricted localization of AIR-2 to the cohesion sites of homologous chromatids in meiosis I.     

Minichromosome analysis of chromosome pairing, disjunction, and sister chromatid cohesion in maize

With the advent of engineered minichromosome technology in plants, an understanding of the properties of small chromosomes is desirable. Twenty-two minichromosomes of related origin but varying in size are described that provide a unique resource to study such behavior. Fourteen minichromosomes from this set could pair with each other in meiotic prophase at frequencies between 25 and 100%, but for the smaller chromosomes, the sister chromatids precociously separated in anaphase I. The other eight minichromosomes did not pair with themselves, and the sister chromatids divided equationally at meiosis I. In plants containing one minichromosome, the sister chromatids also separated at meiosis I. In anaphase II, the minichromosomes progressed to one pole or the other. The maize (Zea mays) Shugoshin protein, which has been hypothesized to protect centromere cohesion in meiosis I, is still present at anaphase I on minichromosomes that divide equationally. Also, there were no differences in the level of phosphorylation of Ser-10 of histone H3, a correlate of cohesion, in the minichromosomes in which sister chromatids separated during anaphase I compared with the normal chromosomes. These analyses suggest that meiotic centromeric cohesion is compromised in minichromosomes depending on their size and cannot be maintained by the mechanisms used by normal-sized chromosomes.     

Inverted meiosis and its place in the evolution of sexual reproduction pathways

Inverted meiosis is observed in plants (Cyperaceae and Juncaceae) and insects (Coccoidea, Aphididae) with holocentric chromosomes, the centromeres of which occupy from 70 to 90% of the metaphase chromosome length. In the first meiotic division (meiosis I), chiasmata are formed and rodlike bivalents orient equationally, and in anaphase I, sister chromatids segregate to the poles; the diploid chromosome number is maintained. Non-sister chromatids of homologous chromosomes remain in contact during interkinesis and prophase II and segregate in anaphase II, forming haploid chromosome sets. The segregation of sister chromatids in meiosis I was demonstrated by example of three plant species that were heterozygous for chromosomal rearrangements. In these species, sister chromatids, marked with rearrangement, segregated in anaphase I. Using fluorescent antibodies, it was demonstrated that meiotic recombination enzymes Spo11 and Rad5l, typical of canonical meiosis, functioned at the meiotic prophase I of pollen mother cells of Luzula elegance and Rhynchospora pubera. Moreover, antibodies to synaptonemal complexes proteins ASY1 and ZYP1 were visualized as filamentous structures, pointing to probable formation of synaptonemal complexes. In L. elegance, chiasmata are formed by means of chromatin threads containing satellite DNA. According to the hypothesis of the author of this review, equational division of sister chromatids at meiosis I in the organisms with inverted meiosis can be explained by the absence of specific meiotic proteins (shugoshins). These proteins are able to protect cohesins of holocentric centromeres from hydrolysis by separases at meiosis I, as occurs in the organisms with monocentric chromosomes and canonical meiosis. The basic type of inverted meiosis was described in Coccoidea and Aphididae males. In their females, the variants of parthenogenesis were also observed. Until now, the methods of molecular cytogenetics were not applied for the analysis of inverted meiosis in Coccoidea and Aphididae. Evolutionary, inverted meiosis is thought to have appeared secondarily as an adaptation of the molecular mechanisms of canonical meiosis to chromosome holocentrism.     

Rec8 cleavage by separase is required for meiotic nuclear divisions in fission yeast

Sister chromatid cohesion in meiosis is established by cohesin complexes, including the Rec8 subunit. During meiosis I, sister chromatid cohesion is destroyed along the chromosome arms to release connections of recombined homologous chromosomes (homologues), whereas centromeric cohesion persists until it is finally destroyed at anaphase II. In fission yeast, as in mammals, distinct cohesin complexes are used depending on the chromosomal region; Rec8 forms a complex with Rec11 (equivalent to SA3) mainly along chromosome arms, while Psc3 (equivalent to SA1 and SA2) forms a complex mainly in the vicinity of the centromeres. Here we show that separase activation and resultant Rec8 cleavage are required for meiotic chromosome segregation in fission yeast. A non-cleavable form of Rec8 blocks disjunction of homologues at meiosis I. However, displacing non-cleavable Rec8 restrictively from the chromosome arm by genetically depleting Rec11 alleviated the blockage of homologue segregation, but not of sister segregation. We propose that the segregation of homologues at meiosis I and of sisters at meiosis II requires the cleavage of Rec8 along chromosome arms and at the centromeres, respectively.     

Meiotic cohesin REC8 marks the axial elements of rat synaptonemal complexes before cohesins SMC1beta and SMC3

In meiotic prophase, the sister chromatids of each chromosome develop a common axial element (AE) that is integrated into the synaptonemal complex (SC). We analyzed the incorporation of sister chromatid cohesion proteins (cohesins) and other AE components into AEs. Meiotic cohesin REC8 appeared shortly before premeiotic S phase in the nucleus and formed AE-like structures (REC8-AEs) from premeiotic S phase on. Subsequently, meiotic cohesin SMC1beta, cohesin SMC3, and AE proteins SCP2 and SCP3 formed dots along REC8-AEs, which extended and fused until they lined REC8-AEs along their length. In metaphase I, SMC1beta, SMC3, SCP2, and SCP3 disappeared from the chromosome arms and accumulated around the centromeres, where they stayed until anaphase II. In striking contrast, REC8 persisted along the chromosome arms until anaphase I and near the centromeres until anaphase II. We propose that REC8 provides a basis for AE formation and that the first steps in AE assembly do not require SMC1beta, SMC3, SCP2, and SCP3. Furthermore, SMC1beta, SMC3, SCP2, and SCP3 cannot provide arm cohesion during metaphase I. We propose that REC8 then provides cohesion. RAD51 and/or DMC1 coimmunoprecipitates with REC8, suggesting that REC8 may also provide a basis for assembly of recombination complexes.     

Holocentric plant meiosis: first sisters,then homologues

Meiosis is a crucial process of sexual reproduction by forming haploid gametes from diploid precursor cells. It involves 2 subsequent divisions (meiosis I and meiosis II) after one initial round of DNA replication. Homologous monocentric chromosomes are separated during the first and sister chromatids during the second meiotic division. The faithful segregation of monocentric chromosomes is realized by mono-orientation of fused sister kinetochores at metaphase I and by bi-orientation of sister kinetochores at metaphase II. Conventionally this depends on a 2-step loss of cohesion, along chromosome arms during meiosis I and at sister centromeres during meiosis II.     

Spo13 maintains centromeric cohesion and kinetochore coorientation during meiosis I

BACKGROUND: The meiotic cell cycle, the cell division cycle that leads to the generation of gametes, is unique in that a single DNA replication phase is followed by two chromosome segregation phases. During meiosis I, homologous chromosomes are segregated, and during meiosis II, as in mitosis, sister chromatids are partitioned. For homolog segregation to occur during meiosis I, physical linkages called chiasmata need to form between homologs, sister chromatid cohesion has to be lost in a stepwise manner, and sister kinetochores must attach to microtubules emanating from the same spindle pole (coorientation). RESULTS: Here we show that the meiosis-specific factor Spo13 functions in two key aspects of meiotic chromosome segregation. In cells lacking SPO13, cohesin, which is the protein complex that holds sister chromatids together, is not protected from removal around kinetochores during meiosis I but is instead lost along the entire length of the chromosomes. We furthermore find that Spo13 promotes sister kinetochore coorientation by maintaining the monopolin complex at kinetochores. In the absence of SPO13, Mam1 and Lrs4 disassociate from kinetochores prematurely during pro-metaphase I and metaphase I, resulting in a partial defect in sister kinetochore coorientation in spo13 Delta cells. CONCLUSIONS: Our results indicate that Spo13 has the ability to regulate both the stepwise loss of sister chromatid cohesion and kinetochore coorientation, two essential features of meiotic chromosome segregation.     

Involvement of chromatid cohesiveness at the centromere and chromosome arms in meiotic chromosome segregation: A cytological approach

Kinetochores and chromatid cores of meiotic chromosomes of the grasshopper species Arcyptera fusca and Eyprepocnemis plorans were differentially silver stained to analyse the possible involvement of both structures in chromatid cohesiveness and meiotic chromosome segregation. Special attention was paid to the behaviour of these structures in the univalent sex chromosome, and in B univalents with different orientations during the first meiotic division. It was observed that while sister chromatid of univalents are associated at metaphase I, chromatid cores are individualised independently of their orientation. We think that cohesive proteins on the inner surface of sister chromatids, and not the chromatid cores, are involved in the chromatid cohesiveness that maintains associated sister chromatids of bivalents and univalents until anaphase I. At anaphase I sister chromatids of amphitelically oriented B univalents or spontaneous autosomal univalents separate but do not reach the poles because they remain connected at the centromere by a long strand which can be visualized by silver staining, that joins stretched sister kinetochores. This strand is normally observed between sister kinetochores of half-bivalents at metaphase II and early anaphase II. We suggest that certain centromere proteins that form the silver-stainable strand assure chromosome integrity until metaphase II. These cohesive centromere proteins would be released or modified during anaphase II to allow normal chromatid segregation. Failure of this process during the first meiotic division could lead to the lagging of amphitelically oriented univalents. Based on our results we propose a model of meiotic chromosome segregation. During mitosis the cohesive proteins located at the centromere and chromosome arms are released during the same cellular division. During meiosis those proteins must be sequentially inactivated, i.e. those situated on the inner surface of the chromatids must be eliminated during the first meiotic division while those located at the centromere must be released during the second meiotic division.by D.P. Bazett-Jones     

Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.     

Phosphorylation of histone H2A is associated with centromere function and maintenance in meiosis

Histone phosphorylation is dynamically regulated during cell division, for example phosphorylation of histone H3 (H3)-Ser10, H3-Thr11 and H3-Ser28. Here we analyzed maize (Zea mays L) for Thr133-phosphorylated histone H2A, which is important for spindle checkpoint control and localization of the centromere cohesion protector Shugoshin in mammals and yeast. Immunostaining results indicate that phosphorylated H2A-Thr133 signals bridged those of the centromeric H3 histone variant CENH3 by using a plant displaying yellow fluorescent protein-CENH3 signals and H2A-Thr133 is phosphorylated in different cell types. During mitosis, H2A-Thr133 phosphorylation becomes strong in metaphase and is specific to centromere regions but drops during later anaphase and telophase. Immunostaining for several maize dicentric chromosomes revealed that the inactive centromeres have lost phosphorylation of H2A-Thr133. During meiosis in maize meiocytes, H2A phosphorylation becomes strong in the early pachytene stage and increases to a maximum at metaphase I. In the maize meiotic mutant afd1 (absence of first division), sister chromatids show equational separation at metaphase I, but there are no changes in H2A-Thr-133 phosphorylation during meiosis compared with the wild type. In sgo1 mutants, sister chromatids segregate randomly during meiosis II, and phosphorylation of H2A-Thr-133 is observed on the centromere regions during meiosis II. The availability of such mutants in maize that lack sister cohesion and Shugoshin indicate that the signals for phosphorylation are not dependent on cohesion but on centromere activity.     

Meiotic cohesion requires accumulation of ORD on chromosomes before condensation

Cohesion between sister chromatids is a prerequisite for accurate chromosome segregation during mitosis and meiosis. To allow chromosome condensation during prophase, the connections that hold sister chromatids together must be maintained but still permit extensive chromatin compaction. In Drosophila, null mutations in the orientation disruptor (ord) gene lead to meiotic nondisjunction in males and females because cohesion is absent by the time that sister kinetochores make stable microtubule attachments. We provide evidence that ORD is concentrated within the extrachromosomal domains of the nuclei of Drosophila primary spermatocytes during early G2, but accumulates on the meiotic chromosomes by mid to late G2. Moreover, using fluorescence in situ hybridization to monitor cohesion directly, we show that cohesion defects first become detectable in ord(null) spermatocytes shortly after the time when wild-type ORD associates with the chromosomes. After condensation, ORD remains bound at the centromeres of wild-type spermatocytes and persists there until centromeric cohesion is released during anaphase II. Our results suggest that association of ORD with meiotic chromosomes during mid to late G2 is required to maintain sister-chromatid cohesion during prophase condensation and that retention of ORD at the centromeres after condensation ensures the maintenance of centromeric cohesion until anaphase II.