Molecular insights into mechanisms regulating faithful chromosome separation in female meiosis

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1 Cell Cycle ISSN: (Print) (Online) Journal homepage: Molecular insights into mechanisms regulating faithful chromosome separation in female meiosis Shen Yin, Xiao-Fang Sun, Heide Schatten & Qing-Yuan Sun To cite this article: Shen Yin, Xiao-Fang Sun, Heide Schatten & Qing-Yuan Sun (2008) Molecular insights into mechanisms regulating faithful chromosome separation in female meiosis, Cell Cycle, 7:19, , DOI: /cc To link to this article: Copyright 2008 Landes Bioscience Published online: 01 Oct Submit your article to this journal Article views: 122 View related articles Citing articles: 26 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 28 November 2017, At: 03:51

2 [Cell Cycle 7:19, ; 1 October 2008]; 2008 Landes Bioscience Review Molecular insights into mechanisms regulating faithful chromosome separation in female meiosis Shen Yin, 1 Xiao-Fang Sun, 2 Heide Schatten 3 and Qing-Yuan Sun 1, * 1 State Key Laboratory of Reproductive Biology; Institute of Zoology; Chinese Academy of Sciences; Beijing, China; 2 Institute of Gynecology; The Third Affiliated Hospital of Guangzhou Medical College; Guangzhou, China; 3 Department of Veterinary Pathobiology; University of Missouri; Columbia, Missouri USA Abbreviations: APC/C, anaphase promoting complex/cyclosome; M, metaphase; A, anaphase Key words: chromosome separation, cohesin, meiosis, shugoshin, spindle checkpoint The faithful segregation of chromosomes into daughter cells in meiosis is crucial to produce healthy progeny. In gametogenesis, two consecutive rounds of chromosome separation occur with only one round of DNA replication, and the chromosome number is reduced to half to produce haploid gametes. Here, we discuss the molecular mechanisms underlying faithful chromosome separation in meiosis from three aspects: spindle checkpoint, two-step releases of cohesion, and the specific space-time protection of cohesin. Introduction During each cell cycle, accurate segregation of chromosomes must take place to ensure that each daughter cell receives exactly one copy of the genome in order to maintain genetic stability of species. In mitosis, sister chromatids separate during the metaphaseto-anaphase transition so that the two daughter cells receive the same chromosome numbers as the original mother cell. In meiosis, however, two consecutive rounds of chromosome separations take place with only one round of DNA replication, resulting in chromosome number reduction to exactly half to produce haploid gametes. Homologous chromosomes separate from each other during the first meiosis (meiosis I) while sister chromatids segregate during the second meiosis (meiosis II). 1,2 Failures in executing this process accurately results in aneuploidy, which is the cause for genetic disorders and aneuploid embryos In human meiosis, chromosome non-disjunction resulting in aneuploidy is the leading cause of trisomies. 11,12 Meiosis II resembles mitosis, while meiosis I is distinctly different and displays unique characteristics. First, in meiosis I, homologous chromosomes undergo pairing and recombination Single chromatid from each of the homologous chromosomes form synapses and chiasmata that allow and ensure recombination of genetic information. 13,16 Next, the kinetochores of sister chromatids are captured by microtubules that emanate from the same spindle pole (sister kinetochores co-orientation), which ensures that homologous chromosomes separate accurately to opposite poles of the spindle. Finally, a two-step releases of cohesin ensures that the sister chromatids are pulled together to the same pole of the meiosis I spindle; they only separate away from each other in meiosis II. 20,21 To avoid aneuploidy, meiotic cells have developed highly conserved mechanisms to ensure faithful segregation of homologous chromosomes in meiosis I and sister chromatids in meiosis II, respectively. Here, we discuss the underlying molecular mechanisms of faithful chromosome separation in meiosis from three aspects: spindle checkpoint, two-step releases of cohesion, and the specific space-time protection of cohesin. Spindle Checkpoint Kinetochores. In mitosis, sister chromatids are oriented toward opposite spindle poles, thus sister chromatids separate in anaphase to opposite directions. In contrast to mitosis, however, sister chromatids have to be oriented toward the same spindle pole in meiosis I, which ensures monopolar attachment and segregation of homologous chromosomes in meiosis I. The faithful separation of chromosomes requires accurate connections between chromosomes and microtubules, in which kinetochores play most significant roles, both in mitosis and meiosis. Kinetochores serve at least three functions: attaching chromosomes to the spindle, controlling chromosome movement, and maintaining the spindle checkpoint by sensing tension and/or attachment of spindle microtubules to kinetochores In mitosis, sister kinetochores attach to microtubules that emanate from opposite spindle poles prior to the onset of anaphase in a process termed sister kinetochore bi-orientation. In meiosis I, however, sister kinetochores from one homolog must attach to microtubules that extend from the same spindle pole, which is termed sister kinetochore co-orientation, that ensures homologous chromosomes separation in meiosis I, with sister chromatids being pulled to the same pole. Functional genomics has identified monopolin(mam1), a kinetochore protein required for the monopolar attachment and segregation of homologous chromosomes during meiosis I. 26 In monopolin *Correspondence to: Qing-Yuan Sun; State Key Laboratory of Reproductive Biology; Institute of Zoology; Chinese Academy of Sciences; #5 Datun Rd; Chaoyang, Beijing China; Tel./Fax: ; sunqy@ioz.ac.cn; sunqy1@ yahoo.com Submitted: 07/17/08; Accepted: 08/18/08 Previously published online as a Cell Cycle E-publication: Cell Cycle 2997

3 Figure 1. Spindle checkpoint pathway. Checkpoint is turned on when sister chromatids are not correctly attached to the spindle microtubules. All checkpoint proteins, Aurora B, Mps1, Bub1, CENP-E, BubR1, Bub3 Mad1 and Mad2 are sequentially binding to kinetochores. Mad2 directly combines Cdc20, thus inhibits the activity of APC/C. Inactivation of APC/C leads to stabilization of securin, which in turn inhibits separase. Plk1 kinase phosphorylates and removes cohesin along chromosome arms except for centromere. After all chromaids are correctly attached to the spindle, checkpoint is turned off. Mad2 detaches from kinetochores which releases the inhibition on Cdc20, and then Cdc20 activates APC/C, which ubiquitinates securin. Then ubiquitinated securin is degraded by proteasome and separase is activated, resulting in cleavage of centromeric cohesin. Thus, sister chromatids separate from each other in anaphase. mutants, kinetochores of sister chromatids fail to attach to microtubules emanating from the same spindle pole (co-orientation), but attach to opposite poles (bi-orientation) during meiosis I. A protein complex, Csm1/Lrs4, migrates to centromeres and combines with monopolin, which is essential for monopolar attachment of sister kinetochores in meiosis I. 27 When Polo-like kinase Cdc5 is lacking, localization of Lrs4 and monopolin proteins to kinetochores is defective, resulting in bipolar orientation of sister kinetochores in meiosis I. 28 In fission yeast, Aurora kinase Ark1 and Ipl1 is necessary for the faithful mono-orientation of sister chromatids in meiosis I. 29,30 Thus, it is implied that the monopolin complex regulates kinetochore-microtubule attachment by including Aurora B kinase to co-orient sister chromatids during meiosis I. In addition, the kinetochore proteins Pcs1, Mde4, casein kinase1 and heterochromatin are required for mono-polar attachment of sister kinetochores at meiosis I. 31,32 Spindle checkpoint pathway. If sister kinetochores are not correctly attached to spindle microtubules at anaphase onset, chromosome mis-segregation may occur, resulting in aneuploidy. Mitotic cells have developed a high-fidelity surveillance mechanism termed the spindle checkpoint to prevent chromosome missegregation by delaying onset of anaphase until all kinetochores of sister chromatids are correctly attached to the spindle The major components of this surveillance mechanism, originally identified in budding yeast, contain mitotic arrest-deficient (Mad) 1 3 proteins, budding uninhibited by benzimidazole (Bub) 1 3 proteins, and Mps Aside from these proteins, increasing evidence has implies Aurora B as a new important factor in the spindle checkpoint Spindle checkpoint is able to detect spindle damage or a single, unaligned chromosome in the spindle and to arrest the cell cycle at metaphase, which provides more time for all of the chromosomes to move to the spindle equator before chromosomes separate. Although it is not clear by which mechanisms the cells ensure that sister chromatids or homologous chromosomes attach to the microtubules and the spindle checkpoint proteins control anaphase onset, it appears that Aurora B, Mad1-3, Bub1, Bub3 and Mps1 are part of the same metaphase-to-anaphase-transition checkpoint pathway Aurora B may be the first checkpoint protein that binds to the kinetochores. 45,49 It is implicated that Aurora B play roles in metaphase chromosome alignment, 50,51 microtubule-kinetochore interactions 40 and kinetochore localization of Bub1, Mad2 and Mps1. 52 Then, Mps1 and/or Bub1 bind to kinetochores. As a protein kinase, Bub1 can bind and phosphorylate Bub3, 53 and be sensitive to tension across the centromere Bub1 accumulates on kinetochores in the absence of tension, 44,47,48,54,57 as a platform for other spindle checkpoint proteins. 58 Bub1 is also reported to control centromeric cohesin and loss of Bub1 function causes checkpoint impairment. 44,59-63 The results from depletion of Bub1 and Mps1 suggest that Bub1 and Mps1 bind to kinetochores at the same time and depend on each other, but they bind to kinetochores earlier than other checkpoint proteins ,49,64 CENP-E is a kinase-related microtubule motor protein 64,65 and participates in chromosome movement It also participates in spindle checkpoint by acting as a binding partner of BubR1, 69,70 or forming a link between microtubules and kinetochores. 49,71,72 The next proteins that bind to kinetochors are Bub3 and BubR1 (mammalian homolog of yeast Mad3). 73 As a platform, Bub3 associates with Mad2, Mad3 and Cdc20, which directly inhibit anaphase promoting complex/cyclosome (APC/C). 74,75 BubR1 is required for kinetochore loading of Mad1 and Mad2. 46 The last two checkpoint proteins binding to kinetochores are Mad1 and Mad It appears that Mad1 affects Mad2 binding to kinetochores. 79 As the last one in the checkpoint pathway, Mad2 has been proposed to be a marker for spindle checkpoint, since Mad2 appears to respond directly to microtubule 2998 Cell Cycle 2008; Vol. 7 Issue 19

4 Figure 2. Two-step releases of cohesin and specific space-time protection of centromeric cohesin during meiosis. In meiosis I, sister kinetochores of a homologous chromosome are arranged side-by-side, and they establish attachment to microtubules emanating from the same spindle pole (co-orientation). For synapsis and recombination, sister chromatids from one homologous chromosome are held together by chromosome arm cohesin and centromeric cohesin. Rec8, the target of separase, is the most important cohesin subunit in meiosis. The Plk1 kinase marks Rec8 by phosphorylation at chromatid arms for Rec8 degradation by separase. Shugoshin1 and PP2A are localized at centromeres by Bub1, which results in dephosphorylation of centromeric cohesin. After silencing of spindle checkpoint, at the transition of MI-AI, separase can only cleave phosphorylated arm cohesin along sister chromatids while centromeric cohesin remains unphosporylated by PP2A. Shugoshin1 collaborates with PP2A to protect cohesin by antagonizing phosphorylation from Plk1. In meiosis II, Shugoshin and PP2A disappear from kinetochores, thus centromeric cohesin is cleaved by separase to allow sister chromatid separation as is the case in mitosis. anchoring at the kinetochore plate 80 and directly binds to Cdc20, which is the activator of APC/C. 81 Studies in several model systems have proposed that the downstream target of the spindle checkpoint is Cdc20, an activator of APC/C, which is an 11-subunit complex containing ubiquitin ligase activity. 34 Cdc20 is a member of the cell cycle protein family including Cdc4 in S. cerevisiae, Fzy in Drosophila, Slp1 in S. pombe and p55cdc in human The association of APC/C with either Cdc20 or Cdh1 is essential for its activity. 83 The spindle checkpoint proteins transmit inhibitory signals to APC/C Cdc20 and thus prevents the metaphase-anaphase transition until all chromosomes have established a bipolar attachment to the spindle. 86 At the transition of metaphase to anaphase, APC/C Cdc20 activity triggers securin (separase inhibitor) degradation, thereby allowing separase to become active and dissolve the cohesin between sister chromatids. This cascade is highly conserved from yeast to human All these proteins work together and form a complicated checkpoint system. Once any spindle checkpoint protein is out of order, then the checkpoint system is turned off and faithful chromosome segregation is affected, resulting in aneuploidy. Spindle checkpoint proteins in mammalian meiosis I. Although the checkpoint pathway in mitosis has been widely studied, only a few spindle checkpoint proteins have been reported in meiosis. Mad1, 90 Mad2, and Bub1, 95 function during meiosis as checkpoint proteins, which ensures high fidelity meiotic chromosome segregation. 49,96 Mad1 is present in mouse oocytes and its localization is not exactly the same as in mitosis. 90,97 Mad1 functions by sensing attachment of chromosomes to microtubules rather than tension between microtubules and chromosomes, since Mad1 localization is not altered when tension is changed. 90,98 Adding anti-mad1 antibody to Xenopus egg extracts de-activates the checkpoint and prevents Mad2 from binding to unattached kinetochores. 79 Mad2 becomes activated and dissociated from Mad1 at kinetochores and is replenished by the pool of Mad1- free Mad2. 78 Thus, Mad1 participates in spindle checkpoint in meiosis by recruiting Mad2 binding to kinetochores. Mad2 is one of the best-studied spindle checkpoint proteins in mammalian meiosis. Similar Mad2 localization was observed in the pre-metaphse of meiosis I (Pre-MI) stage and differences in Mad2 localization were observed after reaching MI and past MI stages during meiosis in rat, 93 mouse 91 and pig. 99 The localization of Mad2 is similar to that of Mad1, but is clearly different from other checkpoint proteins, which still bind to kinetochores of metaphase stage cells. However, one common characteristic is that Mad2 binds to unattached kinetochores and is released from kinetochores when microtubules attach to kinetochores. 49 The function of Mad2 has been studied by Mad2 depletion, 100 antibody inhibition, overexpression, microtubule disruption and stabilization. Blocking Mad2 function induces premature anaphase in which chromosomes start to separate before they are correctly aligned at the metaphase plate in rat, 93 mouse 91,101 and pig oocytes. 99 On the other hand, overexpression of Mad2 results in metaphase arrest by activation of the spindle checkpoint in mammalian oocytes, 93,101 which indicates that excess of Mad2 prevents silencing of the spindle checkpoint. It appears that Mad2 senses the defect of microtubule attachment rather than tension. 54, We also reported the same results in oocyte meiosis, in which Mad2 can sense unattached kinetochores, but can not sense unaligned chromosomes. 93 It has been implicated that dynein, one spindle motor protein, also participates in the spindle checkpoint mechanism. 106,107 In mammalian oocytes, we reported that cytoplasmic dynein participates in meiotic checkpoint inactivation by transporting cytoplasmic Mad1 and Mad2 proteins from kinetochores to spindle poles. 98 Evidences for Bub1 as spindle checkpoint protein in meiosis came from mouse oocyte. 95,108 We reported that Bub1 could prevent precocious anaphase and chromosome misalignment, 95 which is consistent to the dual role for Bub1 in the spindle checkpoint and chromosome congression. 109 For the latter role, Bub1 may function through controlling Shugoshin (see below) localization according to results obtained from mitosis. 61,62 CENP-E function is essential for meiosis I in pig, 110 mouse, rat and frog, 111,112 since it is a binding partner of BubR1. The localization of MAP kinase kinase (MEK) and MAP kinase is similar to some spindle checkpoint proteins in oocytes. Immunodepletion of MAP kinase prevents checkpoint activation, which is rescued Cell Cycle 2999

5 by adding external MAP kinase. 119 Knockdown of MEK causes misalignment of chromosomes and error in chromosome separation. 118 Thus, MEK/MAP kinase may also participate in the spindle checkpoint pathway regulation in meiosis All these observations support the notion that spindle checkpoint is required for faithful segregation of chromosomes in oocyte meiosis I. Therefore, premature silencing of spindle checkpoint may be associated with an increase in aneuploidy of oocytes. Two-Step Releases of Cohesion When all homologous chromosomes are correctly attached to the spindle, the cohesion between chromosomes is cut off to initiate anaphase. It is known that cohesion of sister chromatids in eukaryotes is largely achieved by the cohesin complex, which is important for high fidelity chromosome segregation during anaphase. 124 In meiosis I, homologous chromosomes are connected by chiasmata. Then the kinetochores of sister chromatids are captured by the spindle fiber complex emanating from the same pole of spindle. When the homologous chromosomes are aligned correctly on the equator of spindle, anaphase I is initiated and sister chromatids are pulled to the same pole of the spindle. Importantly, only cohesins along the chromosome arms are removed and centromeric cohesins are maintained in meiosis I. Retention of centromeric cohesin is necessary for faithful segregation of sister chromatids in meiosis II. Thereby, the two-step releases of arm and centromeric cohesin accounts for segregation of homologous chromosomes in meiosis I and sister chromatids in meiosis II. The structure of cohesin. Cohesin is a multiprotein complex, conserved from yeast to humans, that mediates linkage of sister chromatids. It combines sister chromatids in the S stage and disappears from sister chromatids during the transition from metaphase to anaphase. 124 In mitosis of Saccharomyces cerevisiae, it includes at least four subunits, Smc1, Smc3 (Structural maintenance of chromosome, SMC), Scc1/Mcd1 and Scc3 (Sister chromaitd cohesion, SCC) Up to date, the paralogs of four subunits have been identified in Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Mus musculus, Homo sapiens. In human, three paralogs of Scc3 are found, namely SA1, SA2 and SA3, and SA3 is a meiosis-specific cohesin. 127 Two paralogs of Smc1 are found, namely Smc1α and Smc1β, the latter is meiosis-specific. 128 With the help of Scc3, Scc1 combines the Smc1-Smc3 heterodimer through its N and C motif, to form a large protein ring, which holds the sister chromatids together. 129,130 The ring-shaped structure holding sister chromatids tightly together contributes to the recombination of DNA and faithful chromosome segregation. 131 When the cell undergoes the transition from metaphase to anaphase, Scc1 cleavage by separase causes the protein ring to open and cohesins dissociate from centromeres, which initiates the segregation of sister chromatids. 129 Therefore, Scc1 is an important target controlling the transition from metaphase to anaphase. Except for the proteins described, one new cohesin protein, Pds5, has been identified in yeast and human, which is in physical contact with the cohesin complex. But their connection is weak, and thus Pds5 may be a cohesin-raleated protein. 132,133 In meiosis, Scc1/Mcd1 is replaced by Rec8, the specific meiotic protein, while the other three subunits, Scc3, Smc1 and Smc3 are consistent with those of mitosis. 134 Rec8 is the most important meiosis-specific cohesin subunit, which is degraded by separase and necessary for the segregation of chromosomes The paralog proteins have been identified in homo sapiens, Mus musculus, Caenorhabditis elegans, Drosophila melanogaster, Xenous laevis Arabidopsis thaliana. 1,127, These proteins are composed of a conserved protein family, the Rad21/Rec8 family. According to amino acid sequence analysis, Scc1 and Rec8 paralogs in higher eukaryotes contain highly conserved N and C-terminal motif. 140,141 Localization and degradation of cohesion. The combination of cohesin with chromosomes initiated at the S stage largely depends on the loading proteins, Scc2 and Scc Cohesin localizes strongly to the centromeres and randomly associates with multiple sites along the chromosome arms depending on the transcription convergent regions, 143 about every 5 10 kb. 144,145 The different localizations on chromosome arms and centromeres are the perquisite for the two-step release, which is necessary for segregation of homologous choromosomes in meiosis I and sister chromatids in meiosis II. In mitosis, during the transition from metaphase to anaphase, cohesin cleavage initiates the segregation of chromosomes. The specific endopeptidase, separase, is responsible for timely cohesin degradation The conserved His and Cys amino acids in the C terminal motif of separase are necessary for the enzyme activity, since mutation in either one of them causes the inactivation of separase. 149,150 Two different pathways remove mammalian cohesin from chromosome arms in prophase and from centromeres in anaphase. 151 The first is prophase pathway. In prophase and pre-metaphase, the chromosome arm cohesin is first degraded, independent of separase. 137,152,153 Kinase phosphorylation plays important roles in this process. 154 The second is the anaphase pathway. The remaining centromeric cohesin is cleaved by separase at the transition from metaphase to anaphase. 144,151,155 The cleavage of arm cohesin and centromeric cohesin is triggered in two different ways, which may be the evolutionary predecessor of the two-step releases of cohesin in meiosis. In meiosis I, due to crossovers between homologous chromosome arms, cohesion along the arms must first be dissociated to allow the segregation of the homologous chromosomes. The arm Rec8 first dissociates from homologous chromosomes and sister chromatids, which introduces segregation of homologous chromosomes and the two arms of sister chromatids. 136, This is the first step release of cohesin from chromosomes. Notably, the removal of arm cohesin in meiosis I is a separase-dependent mechanism, 159 while in mitosis it is a separase-independent mechanism so-called prophase pathway. However, the centromeric Rec8 between sister chromatids persists until meiosis II, so that sister chromatids are held together and pulled towards the same pole of the spindle. 134,160 The second step release of cohesin occurs in meiosis II. As in mitosis, the cleavage of centromeric Rec8 by separase initiates the segregation of sister chromatids in mammalian meiosis II. 157 The two-step releases of arm cohesin and centromeric cohesin is the important molecular mechanism regulating two subsequent meiotic divisions. Activation of separase for cohesin release. It has been shown that separase is responsible for cleavage of cohesin subunit Rec8, the resolution of chiasmata and transition from metaphase to anaphase in meiosis. 159,161,162 For most of the cell cycle, separase combines with its inhibitory factor, securin, and loses its ability to dissociate cohesin. 148, Spindle checkpoint and APC/C control securin dissociation from 3000 Cell Cycle 2008; Vol. 7 Issue 19

6 separase. Receiving the inhibitory signals from checkpoint, stabilized securin with separase prevents cleavage of cohesin and metaphaseanaphase transition. 86, After all chromosomes have established correct attachments to the spindle fibers, the spindle checkpoint is inactivated and the APC/C Cdc20 ubiquitinates securin, and degrades securin by proteasomes, which in turn allows separase to become active and thus cohesion is eliminated. 162,172 In meiosis, mechanisms regulating separase are similar to those in mitosis. APC/C and securin are also key regulators in meiosis. Accumulation and degradation of securin is observed in both meiosis I and meiosis II, 173,174 which is the key for the timely activation of separase. At the onset of anaphase, APC/C-dependent cleavage of securin causes loss of its inhibition to separase, which in turn cleaves Rec8 and causes opening of the cohesin ring along chromosome arm. 162 Since in Xenopus oocytes APC/C is supposedly not required for meiosis I, the arm cohesin is removed by a separase-independent mechanism called prophase pathway. 175,176 Nevertheless, it is given serious credence by other results. 159,177 In mouse oocytes, conditional knockout of separase prevents removal of cohesin s Rec8 from chromosome arm and resolution of chiasma. mrna encoding wild-type but not catalytically inactive separase restores chiasma resolution. Thus, proteolytic activity of separase is essential for cohesin s removal from chromosome arms and chiasma resolution. 159 In conclusion, removal of cohesin along chromosome arms triggers segregation of homologs during meiosis I. However, cohesin localized around centromeres is not degraded during meiosis I, allowing sister chromatids to be moved to the same direction. 134,157,160,178 During second meiosis, the centromeric cohesin is removed, thus allowing the sister chromatids to separate. What are the mechanisms responsible for preventing centromeric cohesin from degradation in meiosis I? The following part will address this question. Specific Space-time Protection of Centromeric Cohesins During the First Meiosis Mei-S332/shugoshin. It is not clear how meiotic cells protect centromeric cohesins from separase cleavage in meiosis I but not in meiosis II. It had been suggested that there might be specific spacetime protecting mechanisms of centromeric cohesin in meiosis. Several factors had been proposed as candidates, such as Spo13, Bub1 and heterochromatin. In Saccharomyces cerevisiae, mutant Spo13 introduces defects of centromeric cohesin protection, and overexpression of Spo13 will block degradation of Rec It has been proposed that Spo13 protects meiotic cohesin at centromeres in meiosis I. 180 However, Spo13 is not a centromere-specific protein and no homologue has been found in other species, which leaves the possibility that it might indirectly play protective functions. Studies on Bub1 provided promising results. 181 When Bub1 gene is deleted in Schizosaccharomyces pombe, the cohesin Rec8 is never retained at centromeres at anaphase I and sister chromatids often move to opposite spindle poles during meiosis I. 63 But Bub1 is better known as a spindle checkpoint protein rather than a protector of Rec8. Similarly, pericentromeric heterochromatin is important in recruiting Rec8 in fission yeast, 156 but heterochromatin persists in both meiosis cycles and is not meiosis I-specific. The first break came from Mei-S332, which has been identified in Drosophila melanogaster. 182 The Mei-S332 gene encodes a 44 kda protein, and mutations in Mei-S332 cause premature separation of sister chromatids in late anaphase of meiosis I. 183 It contains two separable functional domains. The carboxy-terminal basic region is required for chromosomal localization and the amino-terminal coiled-coil domain may facilitate protein-protein interactions between MEI-S332 and male meiotic proteins. 184 Mei-S332 disappears from the chromosomes when sister chromatids separate at anaphase II. Therefore, the Mei-S332 protein was proposed to hold the centromere regions of sister chromatids together until anaphase II. 183 Mei-S332 localization is driven by the functional centromeric chromatin, and binding of Mei-S332 is regulated independent of kinetochore formation. 185 As a centromeric protein, Mei-S332 is the most likely candidate which is necessary for the protection of centromeric cohesin in meiosis I. However, the counterparts of Mei-S332 so far have not been identified in other species for over ten years, which obscures its significance. In 2004, the protector of centromeric cohesin Rec8, Shugoshin was first identified in yeast. 186,187 Shugoshin1 localizes at pericentromeric regions but dissociates at the onset of anaphase II. In Shugoshin1-deleted cells, centromeric Rec8 was lost during anaphase I. These data implied that Shugoshuin1 protects centromeric cohesion at anaphase I by safeguarding cohesin Rec8 from separase cleavage. 186,187 As of now, both Shugoshin 1 and 2 have been identified in Schizosaccharomyces pombe, A.thaliana, mouse and human, but only Shugoshin1 in Saccharomyces cerevisiae, C. elegans and N. crassa Notably, all Shugoshins share structural similarity to Mei-S332, for their similarity of the sequence architecture, including conserved N-terminal coiled-coil and C-terminal motif. 186,188,190 Therefore, these proteins define an orthologous family conserved in eukaryotic cells. The localization of Shuhoshin was controlled by Bub1 and Aurora B. 61,62,191,192 Shugoshin is degraded by the anaphasepromoting complex, allowing separation of sister centromeres in anaphase. 193 In meiosis I, the amount of meiotic cohesin subunit Rec8 retained at centromeres is reduced in Sgo1 mutant, but not in Sgo2 mutant cells, and Shugoshin1 appears to regulate cleavage of Rec8 by separase. 188 Depletion of Sgo1 in budding yeast not only causes frequent non-disjunction of homologous chromosomes at meiosis I but also random segregation of sister centromeres at meiosis II. 189 In maize, Shugoshin homolog ZMSgo1 is required for maintenance of centromeric cohesin during meiosis but it does not have mitotic functions. 194 Evidence of Shogoshin as a protector of centromeric cohesin was also reported in mammalian meiosis. In mouse oocytes, both Shogoshin proteins are centromeric proteins. Sister chromatids initiate precocious separation in Sgo2-RNAi oocytes while modest defect was observed in Sgo1-RNAi oocytes. 195 Thus, it is accepted that Shugoshin prevents precocious dissociation of cohesin from centromeres in meiosis I. The mechanism is conserved from yeast to mammals. These findings on Shugoshin protection of centromeric cohesin provide significant insights into the mechanism of faithful segregation of homologous chromosomes and sister chromatids in meiosis. In addition, Shugoshin may also be acting as a tension sensor for the spindle checkpoint. In fission yeast, centromeric Shugosin1 is required to sense lack of tension on mitotic chromosomes. 196 Vertebrate Shugoshin interacts with microtubules in vitro and it regulates kinetochore microtubule stability in vivo, consistent with direct microtubule interactions. 193 RNAi assays of Shugoshin1 Cell Cycle 3001

7 diminished Plk1 kinetochore binding and Shugoshin1 is phosphorylated by Plk1 in vitro. Thus, Shogoshin1 may play a role as tension sensor by regulating Plk1 kinetochore affinity. 197 A novel requirement for Shugoshin1 is to bias sister kinetochores toward bi-orientation in meiosis I. 198 Shugoshin2 is important for chromosome bi-orientation and it controls docking of the Passenger proteins on chromosomes in early mitotic cells. 199 In Schizosaccharomyces pombe, Shugoshin2 interacts with Bir1/Survivin and promotes Aurora kinase complex localization to the pericentromeric region, to correct erroneous attachment of kinetochores, thereby enabling tension-generating attachment. 200 Thus, Shugoshin2 might also be a component of the tension-sensing machinery. 201 The understanding of Shugoshin function gives new insights on spindle checkpoint and chromosome separation. Thus, except for protecting centrometic cohesin, Shugoshin plays a key role in ensuring faithful chromosome segregation in another way, i.e., sensing the absence of tension from sister kinetochores, which could explain Bub1 s dual roles in meiosis. 53,95 Shugoshin/PP2A: dephosphorylation antagonizes phosphorylation. Although it has been recognized that Shugoshin1 closely combines with Rec8 and protects it from separase in anaphase I, the underlying mechanisms are still elusive. The important role of protein phosphatase 2A (PP2A) and dephosphorylation of cohesin was found independently by two labs in ,203 It was demonstrated that Shgoshin1 recruits PP2A to centromeres and inactivation of PP2A causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at meiosis II in both budding and fission yeast. 203 It was also shown that PP2A colocalizes with Shugoshin at centromeres and is required for the protection of centermeric cohesin in mitosis and in fission yeast meiosis. 202 Thus, phosphorylation of cohesin mediated by Plk1 kinase is required for degradation of cohesion by separase, while dephosphorylation of cohesin by PP2A is refractory to separase. It seems that the phosphorylation of cohesin is the trigger for separase to cleave cohesin. Shugoshin1 localizes to centromeres at anaphase I but not anaphase II, so PP2A only dephosphrylates cohesin at anaphase I and cohesin resists degradation by separase in anaphase I. Shugoshin1 and PP2A disappear from centromeres before anaphase II, not to antagonize phosphorylation of Plk1 on Rec8, which is then degraded by separase. Thus, sister chromatids can not segregate at anaphase I but only separate at anaphase II. PP2A and Shugoshin1 may complex and interact with each other, since PP2A is also required for centromeric localization of Shugoshin1 during mitosis of human cells. 204 Interestingly, mouse Shugoshin2 is solely responsible for the centromeric localization of PP2A and the protection of cohesin Rec PP2A comprises a group of protein family that is highly abundant and ubiquitously expressed serine/threonine phosphatases in eukaryotes. It is a large and complicated complex with multiple functions, composed of three distinct subunits (A, B and C subunits). The A subunit serves as a scaffold to accommodate the other two subunits as a structural subunit. The C subunit is the catalytic unit, which could combine with the A subunit and form the dimeric core enzyme. 205 In many cases, it is the B regulatory subunit that associates with the core catalytic subunits of PP2A to define the specific functions of PP2A. Multiple subunits allow generation of over 60 different heterotrimeric PP2A holoenzymes. PP2A accounts for the majority of the Ser/Thr phosphatase activity and has been implicated in the regulation of many signaling pathways, such as G 1 /S transition, mitotic entry and exit, cytokinesis, checkpoint, apoptosis. Recruiting PP2A to centromeres to antagonize phosphorylation by Polo kinase is the key protection of centromeric cohesin in anaphase I. Therefore, it seems that the balance of kinase and phosphatase activities is the key to the conserved mechanism that protects centromeric cohesin from separase-mediated cleavage during meiosis I. 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