Monopolin Complex Research Articles

Construction of strains to identify novel factors for regulation of centromeric cohesion protection (CCP) and sister kinetochore mono-orientation (SKM)

BackgroundMeiosis-I is a unique type of chromosome segregation where each chromosome aligns and segregates from its homolog. The mechanism of meiosis I homolog separation in different eukaryotes depends on their centromere and kinetochore architecture which in turn relies mainly on two processes, first on a specialized four protein complex known as monopolin and second, the centromeric cohesion protection (CCP). However, in mammals the complex has not been identified. Furthermore, in budding yeast, there could be additional factors in this process which includes some meiosis specific and some non meiosis specific factors.ResultWe constructed two strains. In the first strain we expressed Mam1 and Cdc5 which leads to sister kinetochore monoorientation (SKM) and in the second case we expressed Rec8 and Spo13 which enhanced CCP even in mitosis. The expression of these proteins in mitotically dividing cells caused co-orientation of the chromosomes, which lead to the cell death followed by miss-segregation of chromosomes. Then we utilized these strains to screen the cDNA libraries from yeast and mammals to identify the novel factors which participate in CCP and SKM. Finally, SGY4119 strain expressing Spo13 and Rec8 was transformed with pRS316 gal cDNA library and transformants were screened for lethality on galactose. We screened ~ 105 transformants colonies. Out of these ~ 3000 colonies were able to survive on galactose plate which was narrow down to 6 on the basis of desired phenotype.ConclusionSo far, meiosis specific kinetochore proteins have been identified only in two yeasts. Recently, in mammals a meiosis specific kinetochore protein (MEIKIN) has been identified with similar function. Till now a single protein in mammals and four proteins monopolin complex in budding yeast has been identified to coorient the centromere. Many more novel factors have to be identified yet. That is why we wished to device genetic screen using a functional genomics approach. Since the list of proteins already identified in yeast is not exhaustive as the circumstantial evidence suggests, we wish to use the same yeast strains to identify additional novel yeast proteins that may be involved in the execution of meiosis.

The molecular basis of monopolin recruitment to the kinetochore

The monopolin complex is a multifunctional molecular crosslinker, which in S. pombe binds and organises mitotic kinetochores to prevent aberrant kinetochore-microtubule interactions. In the budding yeast S. cerevisiae, whose kinetochores bind a single microtubule, the monopolin complex crosslinks and mono-orients sister kinetochores in meiosis I, enabling the biorientation and segregation of homologs. Here, we show that both the monopolin complex subunit Csm1 and its binding site on the kinetochore protein Dsn1 are broadly distributed throughout eukaryotes, suggesting a conserved role in kinetochore organisation and function. We find that budding yeast Csm1 binds two conserved motifs in Dsn1, one (termed Box 1) representing the ancestral, widely conserved monopolin binding motif and a second (termed Box 2-3) with a likely role in enforcing specificity of sister kinetochore crosslinking. We find that Box 1 and Box 2-3 bind the same conserved hydrophobic cavity on Csm1, suggesting competition or handoff between these motifs. Using structure-based mutants, we also find that both Box 1 and Box 2-3 are critical for monopolin function in meiosis. We identify two conserved serine residues in Box 2-3 that are phosphorylated in meiosis and whose mutation to aspartate stabilises Csm1-Dsn1 binding, suggesting that regulated phosphorylation of these residues may play a role in sister kinetochore crosslinking specificity. Overall, our results reveal the monopolin complex as a broadly conserved kinetochore organiser in eukaryotes, which budding yeast have co-opted to mediate sister kinetochore crosslinking through the addition of a second, regulatable monopolin binding interface.

Cell scientist to watch – Kevin Corbett

ABSTRACT Kevin Corbett graduated in biology and biochemistry from the University of Virginia. He then went to the University of California, Berkeley, to work on the structure and function of DNA topoisomerases in bacteria and archaea for his PhD with James Berger. In 2005, he moved to the laboratory of Stephen Harrison at Harvard Medical School for his postdoctoral work on kinetochore structure and function, particularly the yeast monopolin complex, which promotes proper chromosome segregation in the first meiotic division. Kevin started his own research group at UC San Diego and the Ludwig Institute for Cancer Research in 2011. He received a Sidney Kimmel Scholar Award in 2012. His current research interests include the molecular mechanisms of homologous chromosome pairing in meiosis I, spindle assembly checkpoint regulation in mitosis and meiosis, and how misregulation of meiotic genes can contribute to carcinogenesis.

Structure of the Saccharomyces cerevisiae Hrr25:Mam1 monopolin subcomplex reveals a novel kinase regulator.

In budding yeast, the monopolin complex mediates sister kinetochore cross-linking and co-orientation in meiosis I. The CK1δ kinase Hrr25 is critical for sister kinetochore co-orientation, but its roles are not well understood. Here, we present the structures of Hrr25 and its complex with the monopolin subunit Mam1. Hrr25 possesses a "central domain" that packs tightly against the kinase C-lobe, adjacent to the binding site for Mam1. Together, the Hrr25 central domain and Mam1 form a novel, contiguous embellishment to the Hrr25 kinase domain that affects Hrr25 conformational dynamics and enzyme kinetics. Mam1 binds a hydrophobic surface on the Hrr25 N-lobe that is conserved in CK1δ-family kinases, suggesting a role for this surface in recruitment and/or regulation of these enzymes throughout eukaryotes. Finally, using purified proteins, we find that Hrr25 phosphorylates the kinetochore receptor for monopolin, Dsn1. Together with our new structural insights into the fully assembled monopolin complex, this finding suggests that tightly localized Hrr25 activity modulates monopolin complex-kinetochore interactions through phosphorylation of both kinetochore and monopolin complex components.

Sister kinetochores are mechanically fused during meiosis I in yeast.

Production of healthy gametes requires a reductional meiosis I division in which replicated sister chromatids comigrate, rather than separate as in mitosis or meiosis II. Fusion of sister kinetochores during meiosis I may underlie sister chromatid comigration in diverse organisms, but direct evidence for such fusion has been lacking. We used laser trapping and quantitative fluorescence microscopy to study native kinetochore particles isolated from yeast. Meiosis I kinetochores formed stronger attachments and carried more microtubule-binding elements than kinetochores isolated from cells in mitosis or meiosis II. The meiosis I-specific monopolin complex was both necessary and sufficient to drive these modifications. Thus, kinetochore fusion directs sister chromatid comigration, a conserved feature of meiosis that is fundamental to Mendelian inheritance.

"Uno, nessuno e centomila": the different faces of the budding yeast kinetochore.

"One, no one and one hundred thousand" is a masterpiece of Italian literature, written by Luigi Pirandello. The central theme is that in each individual there are multiple personalities, since one's perception of one's self differs from the view of others. As a consequence, a unique identity does not exist, but rather one hundred thousand. This concept can be very well applied to the kinetochore, one of the largest macromolecular complexes conserved in eukaryotes. The kinetochore is essential during cell division and fulfills different sophisticated functions, including linking chromosomes to spindle microtubules and delaying anaphase onset in case of incorrect bi-orientation. In order to perform these tasks, the kinetochore shapes its structure by recruiting different subunits, such as the components of the spindle assembly checkpoint (SAC) or the monopolin complex during meiosis. It also modifies its internal organization by rearranging intramolecular connections and acquiring a distinct identity at different time points of cell division. In this review, we describe recent insights into the changes in composition and configuration of the kinetochore in mitosis and meiosis, focusing on the kinetochore of Saccharomyces cerevisiae.

Direct Evidence for Sister Kinetochore Fusion in Meiosis I

For decades, biologists have wondered how replicated sister chromatids, which always separate from one another during normal mitotic cell division, instead co-migrate together during phase I of meiosis, the specialized division that produces eggs and sperm. Genetic, cytological, and biochemical studies using budding yeast have suggested that sister kinetochores, which link the sister chromatids to microtubules and drive their movement, may be mechanically fused by a meiosis I-specific kinetochore-binding factor called monopolin. But thus far the evidence for monopolin-dependent sister kinetochore fusion has been indirect, and the possibility that monopolin could instead promote co-migration by binding to and inhibiting one of the two sister kinetochores has not been excluded. To test these ideas directly, we are isolating native meiotic and mitotic kinetochore particles, reconstituting their function in vitro, and applying advanced tools for manipulating and tracking individual molecules. Using laser trapping, we find that kinetochore particles from meiosis I form substantially stronger attachments to dynamic microtubule tips than those from mitosis or from meiosis II. The high strength of meiosis I particles is lost if DNA replication is blocked during pre-meiotic S-phase, demonstrating that sisters are required. High strength is also lost if monopolin is disrupted prior to kinetochore particle isolation and, conversely, the strength of mitotic kinetochores can be increased by artificially inducing monopolin expression. Quantitative fluorescence microscopy also confirms that meiosis I particles have more copies of the core microtubule-binding component, Nuf2. Together these data suggest that sister kinetochores are mechanically fused by the monopolin complex during meiosis I, in such a way that the microtubule-binding elements from the two sister kinetochores can cooperate together to form a single attachment site.

Monopolin recruits condensin to organize centromere DNA and repetitive DNA sequences

The establishment and maintenance of higher-order structure at centromeres is essential for accurate chromosome segregation. The monopolin complex is thought to cross-link multiple kinetochore complexes to prevent merotelic attachments that result in chromosome missegregation. This model is based on structural analysis and the requirement that monopolin execute mitotic and meiotic chromosome segregation in Schizosaccharomyces pombe, which has more than one kinetochore-microtubule attachment/centromere, and co-orient sister chromatids in meiosis I in Saccharomyces cerevisiae. Recent data from S. pombe suggest an alternative possibility: that the recruitment of condensin is the primary function of monopolin. Here we test these models using the yeast Candida albicans. C. albicans cells lacking monopolin exhibit defects in chromosome segregation, increased distance between centromeres, and decreased stability of several types of repeat DNA. Of note, changing kinetochore-microtubule copy number from one to more than one kinetochore-microtubule/centromere does not alter the requirement for monopolin. Furthermore, monopolin recruits condensin to C. albicans centromeres, and overexpression of condensin suppresses chromosome segregation defects in strains lacking monopolin. We propose that the key function of monopolin is to recruit condensin in order to promote the assembly of higher-order structure at centromere and repetitive DNA.

Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I.

Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore.

Molecular Architecture of the Yeast Monopolin Complex

The Saccharomyces cerevisiae monopolin complex directs proper chromosome segregation in meiosis I by mediating co-orientation of sister kinetochores on the meiosis I spindle. The monopolin subunits Csm1 and Lrs4 form a V-shaped complex that may directly crosslink sister kinetochores. We report here biochemical characterization of the monopolin complex subunits Mam1 and Hrr25 and of the complete four-protein monopolin complex. By purifying monopolin subcomplexes with different subunit combinations, we have determined the stoichiometry and overall architecture of the full monopolin complex. We have determined the crystal structure of Csm1 bound to a Mam1 fragment, showing how Mam1 wraps around the Csm1 dimer and alters the stoichiometry of kinetochore-protein binding by Csm1. We further show that the kinase activity of Hrr25 is altered by Mam1 binding, and we identify Hrr25 phosphorylation sites on Mam1 that may affect monopolin complex stability and/or kinetochore binding in meiosis.

The Lrs4-Csm1 monopolin complex associates with kinetochores during anaphase and is required for accurate chromosome segregation

Lrs4 and Csm1, components of the monopolin complex, localize to the rDNA where they regulate rDNA maintenance and segregation. During meiosis, the complex also associates with kinetochores to bring about sister kinetochore co-orientation, an essential aspect of meiosis I chromosome segregation. We show here that the Lrs4-Csm1 complex associates with kinetochores during mitosis. This kinetochore localization is observed during anaphase and depends on the on the Mitotic Exit Network, a signaling cascade essential for the completion of mitosis. Furthermore, we find that Lrs4 and Csm1 are important for chromosome segregation fidelity. Our results reveal a previously unanticipated function for Lrs4-Csm1 in mitotic chromosome segregation.

The Monopolin Complex Crosslinks Kinetochore Components to Regulate Chromosome-Microtubule Attachments

The monopolin complex regulates different types of kinetochore-microtubule attachments in fungi, ensuring sister chromatid co-orientation in Saccharomyces cerevisiae meiosis I and inhibiting merotelic attachment in Schizosaccharomyces pombe mitosis. In addition, the monopolin complex maintains the integrity and silencing of ribosomal DNA (rDNA) repeats in the nucleolus. We show here that the S. cerevisiae Csm1/Lrs4 monopolin subcomplex has a distinctive V-shaped structure, with two pairs of protein-protein interaction domains positioned approximately 10 nm apart. Csm1 presents a conserved hydrophobic surface patch that binds two kinetochore proteins: Dsn1, a subunit of the outer-kinetochore MIND/Mis12 complex, and Mif2/CENP-C. Csm1 point-mutations that disrupt kinetochore-subunit binding also disrupt sister chromatid co-orientation in S. cerevisiae meiosis I. We further show that the same Csm1 point-mutations affect rDNA silencing, probably by disrupting binding to the rDNA-associated protein Tof2. We propose that Csm1/Lrs4 functions as a molecular clamp, crosslinking kinetochore components to enforce sister chromatid co-orientation in S. cerevisiae meiosis I and to suppress merotelic attachment in S. pombe mitosis, and crosslinking rDNA repeats to aid rDNA silencing.

Condensins Promote Coorientation of Sister Chromatids During Meiosis I in Budding Yeast

The condensin complex is a key determinant of higher-ordered chromosome structure. We show here that the complex is also important for the correct alignment of chromosomes on the meiosis I spindle. Unlike during mitosis and meiosis II, when sister chromatids attach to microtubules emanating from opposite spindle poles (biorientation), accurate meiosis I chromosome segregation requires that sister chromatids attach to microtubules emanating from the same spindle pole (co-orientation). The monopolin complex, consisting of Lrs4, Csm1, and the meiosis-specific component Mam1, brings about meiosis I co-orientation. We find that in the absence of functional condensin complexes, a fraction of sister kinetochores biorient on the meiosis I spindle and association of the monopolin complex subunit Mam1 with kinetochores is decreased. Our studies uncover a new locus-specific effect of the condensin complex.

Chromosome Segregation: Monopolin Goes Spindle

At anaphase onset the mitotic spindle undergoes dramatic changes in order to segregate sister chromatids. Surprisingly, the monopolin complex, best known for its role at kinetochores in meiosis, is now shown to localize to, and stabilize, the mitotic anaphase spindle.

Phosphorylation State Defines Discrete Roles for Monopolin in Chromosome Attachment and Spindle Elongation

It is unknown how oscillations in Cdk1 activity drive the dramatic changes in chromosome and spindle dynamics that occur at the metaphase/anaphase transition. We show that the Schizosaccharomyces pombe monopolin complex has distinct functions in metaphase and anaphase that are determined by the phosphorylation state of its Mde4 subunit. When Cdk1 activity is high in metaphase, Mde4 is hyperphosphorylated on Cdk1 phosphorylation sites and localizes to kinetochores. A nonphosphorylatable mutant of Mde4 does not localize to kinetochores, appears prematurely on the metaphase spindle, and interferes with spindle dynamics and chromosome segregation, illustrating the importance of Cdk1 phosphorylation in regulating metaphase monopolin activity. When Cdk1 activity drops in anaphase, dephosphorylation of Mde4 triggers monopolin localization to the mitotic spindle, where it promotes spindle elongation and integrity, coupling the late mitotic loss of Cdk1 activity to anaphase spindle dynamics. Together, these findings illustrate how the sequential phosphorylation and dephosphorylation of monopolin helps ensure the orderly execution of discrete steps in mitosis.

Dbf4-Dependent Cdc7 Kinase Links DNA Replication to the Segregation of Homologous Chromosomes in Meiosis I

Meiosis differs from mitosis in that DNA replication is followed by the segregation of homologous chromosomes but not sister chromatids. This depends on the formation of interhomolog connections through crossover recombination and on the attachment of sister kinetochores to microtubules emanating from the same spindle pole. We show that in yeast, the Dbf4-dependent Cdc7 kinase (DDK) provides a link between premeiotic S phase, recombination, and monopolar attachment. Independently from its established role in initiating DNA replication, DDK promotes double-strand break formation, the first step of recombination, and the recruitment of the monopolin complex to kinetochores, which is essential for monopolar attachment. DDK regulates monopolin localization together with the polo-kinase Cdc5 bound to Spo13, probably through phosphorylation of the monopolin subunit Lrs4. Thus, activation of DDK both initiates DNA replication and commits meiotic cells to reductional chromosome segregation in the first division of meiosis.

Kinetochore Orientation during Meiosis Is Controlled by Aurora B and the Monopolin Complex

Kinetochores of sister chromatids attach to microtubules emanating from the same pole (coorientation) during meiosis I and microtubules emanating from opposite poles (biorientation) during meiosis II. We find that the Aurora B kinase Ipl1 regulates kinetochore-microtubule attachment during both meiotic divisions and that a complex known as the monopolin complex ensures that the protein kinase coorients sister chromatids during meiosis I. Furthermore, the defining of conditions sufficient to induce sister kinetochore coorientation during mitosis provides insight into monopolin complex function. The monopolin complex joins sister kinetochores independently of cohesins, the proteins that hold sister chromatids together. We propose that this function of the monopolin complex helps Aurora B coorient sister chromatids during meiosis I.

Monopolar Attachment of Sister Kinetochores at Meiosis I Requires Casein Kinase 1

In meiosis, a single round of DNA replication is followed by two consecutive rounds of chromosome segregation, called meiosis I and II. Disjunction of maternal from paternal centromeres during meiosis I depends on the attachment of sister kinetochores to microtubules emanating from the same pole. In budding yeast, monopolar attachment requires recruitment to kinetochores of the monopolin complex. How monopolin promotes monopolar attachment was unclear, as its subunits are poorly conserved and lack similarities to proteins with known functions. We show here that the monopolin subunit Mam1 binds tightly to Hrr25, a highly conserved casein kinase 1 delta/epsilon (CK1delta/epsilon), and recruits it to meiosis I centromeres. Hrr25 kinase activity and Mam1 binding are both essential for monopolar attachment. Since CK1delta/epsilon activity is important for accurate chromosome segregation during meiosis I also in fission yeast, phosphorylation of kinetochore proteins by CK1delta/epsilon might be an evolutionary conserved process required for monopolar attachment.

Spo13 Facilitates Monopolin Recruitment to Kinetochores and Regulates Maintenance of Centromeric Cohesion during Yeast Meiosis

Cells undergoing meiosis perform two consecutive divisions after a single round of DNA replication. During the first meiotic division, homologous chromosomes segregate to opposite poles. This is achieved by (1) the pairing of maternal and paternal chromosomes via recombination producing chiasmata, (2) coorientation of homologous chromosomes such that sister chromatids attach to the same spindle pole, and (3) resolution of chiasmata by proteolytic cleavage by separase of the meiotic-specific cohesin Rec8 along chromosome arms. Crucially, cohesin at centromeres is retained to allow sister centromeres to biorient at the second division. Little is known about how these meiosis I-specific events are regulated. Here, we show that Spo13, a centromere-associated protein produced exclusively during meiosis I, is required to prevent sister kinetochore biorientation by facilitating the recruitment of the monopolin complex to kinetochores. Spo13 is also required for the reaccumulation of securin, the persistence of centromeric cohesin during meiosis II, and the maintenance of a metaphase I arrest induced by downregulation of the APC/C activator CDC20. Spo13 is a key regulator of several meiosis I events. The presence of Spo13 at centromere-surrounding regions is consistent with the notion that it plays a direct role in both monopolin recruitment to centromeres during meiosis I and maintenance of centromeric cohesion between the meiotic divisions. Spo13 may also limit separase activity after the first division by ensuring securin reaccumulation and, in doing so, preventing precocious removal from chromatin of centromeric cohesin.

Spo13 Maintains Centromeric Cohesion and Kinetochore Coorientation during Meiosis I

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). 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. 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.