Search

Cell aging is the result of deteriorating competence in maintaining cellular homeostasis and quality control. Certain cell types are able to rejuvenate through asymmetric cell division by excluding aging factors, including damaged cellular compartments and extra chromosomal rDNA circles, from entering the daughter cell. Recent findings from the budding yeast S. cerevisiae have shown that gametogenesis represents another type of cellular rejuvenation. Gametes, whether produced by an old or a young mother cell, are granted a renewed replicative lifespan through the formation of a fifth nuclear compartment that sequesters the harmful senescence factors accumulated by the mother. Here, we describe the importance and mechanism of cellular remodeling at the nuclear envelope mediated by ESCRT-III and the LEM-domain proteins, with a focus on nuclear pore biogenesis and chromatin interaction during gamete rejuvenation.

Centrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single-pass SUN-domain protein anchored at the inner nuclear membrane and concentrated at the nuclear side of the half-bridge. Using the unique feature in yeast meiosis that centrosomes are linked for hours before their separation, we have revealed that Mps3 is cleaved at its nucleus-localized N-terminal domain, the process of which is regulated by its phosphorylation at serine 70. Cleavage of Mps3 takes place at the yeast centrosome and requires proteasome activity. We show that noncleavable Mps3 (Mps3-nc) inhibits centrosome separation during yeast meiosis. In addition, overexpression of mps3-nc in vegetative yeast cells also inhibits centrosome separation and is lethal. Our findings provide a genetic mechanism for the regulation of SUN-domain protein-mediated activities, including centrosome separation, by irreversible protein cleavage at the nuclear periphery., Author summary The nucleus, where the eukaryotic chromosomes are stored, is enclosed by a double-membrane structure called the nuclear envelope. Located at the inner nuclear membrane, a class of highly conserved proteins called SUN-domain proteins regulates a range of nuclear activities at the nuclear periphery, including tethering the centrosomes to the nuclear membranes. Defective SUN-domain proteins have been linked to certain types of muscular dystrophy and premature aging in humans. In this study, we use budding yeast as a model and show that its sole SUN-domain protein, Mps3, a key component of the yeast centrosome, is phosphorylated and then subject to proteolytic cleavage in order to properly split the centrosomes before spindle formation and chromosome segregation. Understanding how SUN-domain proteins are posttranslationally modified can shed light on the regulation of centrosome separation; it also provides insight into the homeostasis of SUN-domain proteins that are associated with many human diseases of the nuclear envelope.

Condensin undergoes a sequestration, release, and reloading cycle at the rDNA array in budding yeast meiosis. It regulates rDNA stability by suppressing double-strand break (DSB) formation and promoting DSB processing., During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity.

A prerequisite for determination of chromosome dynamics in live cells is development of a method for staining or marking the chromosome of interest. We describe here a unique chromosome-tracking system that differentially marks two large chromosome segments from homologs in the budding yeast Saccharomyces cerevisiae. Using yeast genetics and the special features at the repetitive ribosomal RNA (rRNA) gene cluster, we incorporated arrays of the tet operator and the lac operator into each repeat of the two rDNA homologs by homologous recombination. Expression of tet repressor-fused green fluorescent protein and lac repressor-fused red fluorescent protein in engineered cells led to the differential labeling of rDNA homologs. Using live-cell three-dimensional fluorescence microscopy, we showed that homologs undergo contraction and expansion cycles in an actin-dependent manner during meiosis and that chromosome mobility appears to be correlated with nuclear positioning. Our observations further revealed that, in contrast to mitosis, in meiosis the yeast nucleolus, the site of rRNA processing, was disassembled upon anaphase onset, suggesting a differential regulation of the rDNA array during meiotic chromosome segregation. Because rRNA genes are highly conserved, a similar chromosome-engineering approach may be adaptable in other eukaryotes for functional assays of chromosome organization in live cells.

Meiotic Scc2 recruits cohesin to the chromosome for two important functions: activation of gene expression and mediation of sister-chromatid cohesion. This study reveals that cohesin positively regulates transcription in a position-dependent manner. Therefore cohesin can also act as a transcriptional activator in budding yeast., To tether sister chromatids, a protein-loading complex, including Scc2, recruits cohesin to the chromosome at discrete loci. Cohesin facilitates the formation of a higher-order chromosome structure that could also influence gene expression. How cohesin directly regulates transcription remains to be further elucidated. We report that in budding yeast Scc2 is required for sister-chromatid cohesion during meiosis for two reasons. First, Scc2 is required for activating the expression of REC8, which encodes a meiosis-specific cohesin subunit; second, Scc2 is necessary for recruiting meiotic cohesin to the chromosome to generate sister-chromatid cohesion. Using a heterologous reporter assay, we have found that Scc2 increases the activity of its target promoters by recruiting cohesin to establish an upstream cohesin-associated region in a position-dependent manner. Rec8-associated meiotic cohesin is required for the full activation of the REC8 promoter, revealing that cohesin has a positive feedback on transcriptional regulation. Finally, we provide evidence that chromosomal binding of cohesin is sufficient for target-gene activation during meiosis. Our data support a noncanonical role for cohesin as a transcriptional activator during cell differentiation.

Sister-chromatid cohesion mediated by cohesin ensures proper chromosome segregation during cell division. Cohesin is also required for postreplicative DNA double-strand break repair and gene expression. The molecular mechanisms of these diverse cohesin functions remain to be elucidated. Here we report that the cohesin subunits Scc3 and Smc1 are both required for the production of the meiosis-specific subunit Rec8 in the budding yeast Saccharomyces cerevisiae. Using a genetic approach, we depleted Scc3 and Smc1 independently in cells that were undergoing meiosis. Both Scc3- and Smc1-depleted cells were inducible for meiosis, but the REC8 promoter was only marginally activated, leading to reduced levels of REC8 transcription and protein production. In contrast, the expression of MCD1, the mitotic counterpart of REC8, was not subject to Scc3 regulation in vegetative cells. We provide genetic evidence to show that sister-chromatid cohesion is not necessary for activation of REC8 gene expression. Cohesin appears to positively regulate the expression of a variety of genes during yeast meiosis. Our results suggest that the cohesin complex plays a dual role in gene regulation and sister-chromatid cohesion during meiotic differentiation in 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 (coorientation). The monopolin complex, consisting of Lrs4, Csm1, and the meiosis-specific component Mam1, brings about meiosis I coorientation. 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.

Homologue segregation during the first meiotic division requires the proper spatial regulation of sister chromatid cohesion and its dissolution along chromosome arms, but its protection at centromeric regions. This protection requires the conserved MEI-S332/Sgo1 proteins that localize to centromeric regions and also recruit the PP2A phosphatase by binding its regulatory subunit, Rts1. Centromeric Rts1/PP2A then locally prevents cohesion dissolution possibly by dephosphorylating the protein complex cohesin. We show that Aurora B kinase in Saccharomyces cerevisiae (Ipl1) is also essential for the protection of meiotic centromeric cohesion. Coupled with a previous study in Drosophila melanogaster, this meiotic function of Aurora B kinase appears to be conserved among eukaryotes. Furthermore, we show that Sgo1 recruits Ipl1 to centromeric regions. In the absence of Ipl1, Rts1 can initially bind to centromeric regions but disappears from these regions after anaphase I onset. We suggest that centromeric Ipl1 ensures the continued centromeric presence of active Rts1/PP2A, which in turn locally protects cohesin and cohesion.

Condensin is an evolutionarily conserved protein complex that helps mediate chromosome condensation and segregation in mitotic cells. Here, we show that condensin has two activities that contribute to meiotic chromosome condensation in Saccharomyces cerevisiae. One activity, common to mitosis, helps mediate axial length compaction. A second activity promotes chromosome individualization with the help of Red1 and Hop1, two meiotic specific components of axial elements. Like Red1 and Hop1, condensin is also required for efficient homologue pairing and proper processing of double strand breaks. Consistent with these functional links condensin is necessary for proper chromosomal localization of Red1 and Hop1 and the subsequent assembly of the synaptonemal complex. Finally, condensin has a Red1/Hop1-independent role in the resolution of recombination-dependent linkages between homologues in meiosis I. The existence of distinct meiotic activities of condensin (axial compaction, individualization, and resolution of recombination-dependent links) provides an important framework to understand condensin's role in both meiotic and mitotic chromosome structure and function.

Kinetochores are large protein complexes that bind to centromeres. By interacting with microtubules and their associated motor proteins, kinetochores both generate and regulate chromosome movement. Kinetochores also function in the spindle checkpoint; a surveillance mechanism that ensures that metaphase is complete before anaphase begins. Although the ultrastructure of plant kinetochores has been known for many years, only recently have specific kinetochore proteins been identified. The recent data indicate that plant kinetochores contain homologs of many of the proteins implicated in animal and fungal kinetochore function, and that the plant kinetochore is a redundant structure with distinct biochemical subdomains.

Centrosomes are microtubule-organizing centers that nucleate spindle microtubules during cell division. In budding yeast, the centrosome, often referred to as the spindle pole body, shares structural components with the centriole, the central core of the animal centrosome. The parental centrosome is duplicated when DNA replication takes place. Like sister chromatids tethered together by cohesin, duplicated centrosomes are linked and then separate to form the bipolar spindle necessary for chromosome segregation. Recent studies have shown that cohesin is also localized to the animal centrosome and is perhaps directly involved in engaging paired centrioles. Here we discuss the potential role of cohesin in mediating spindle-pole-body cohesion in the context of yeast meiosis. We propose that the coordination of chromosome segregation with centrosome cohesion and duplication is mediated by the antagonistic interaction between the Aurora kinase and the Polo kinase and that the role of cohesin in centrosome regulation appears to be indirect in budding yeast.

The attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The spindle checkpoint can sense the lack of tension on chromosomes as well as detached kinetochores to prevent anaphase onset. In budding yeast Saccharomyces cerevisiae, tension checkpoint proteins Aurora/Ipl1 kinase and centromere-localized Sgo1 are required to sense the absence of tension but are dispensable for the checkpoint response to detached kinetochores. We have found that the loss of function of a motor protein complex Cik1/Kar3 in budding yeast leads to syntelic attachments. Inactivation of either the spindle or tension checkpoint enables premature anaphase entry in cells with dysfunctional Cik1/Kar3, resulting in co-segregation of sister chromatids. Moreover, the abolished Kar3-kinetochore interaction in cik1 mutants suggests that the Cik1/Kar3 complex mediates chromosome movement along microtubules, which could facilitate bipolar attachment. Therefore, we can induce syntelic attachments in budding yeast by inactivating the Cik1/Kar3 complex, and this approach will be very useful to study the checkpoint response to syntelic attachments., Author Summary Chromosome bipolar attachment occurs when sister chromatids are attached by microtubules emanating from opposite spindle poles and is essential for faithful sister-chromatid segregation. Chromosomes are under tension once bipolar attachment is established. The absence of tension is sensed by the tension checkpoint that prevents chromosome segregation. The attachment of sister chromatids by microtubules from the same spindle pole generates syntelic attachment, which fails to generate tension on chromosomes. However, a reliable method to induce syntelic attachment is not available. Our findings indicate that the inactivation of the motor complex, Cik1/Kar3, results in chromosomes with syntelic attachment in budding yeast. In the absence of the tension checkpoint, yeast cells with dysfunctional Cik1/Kar3 enter anaphase, resulting in co-segregation of sister chromatids. Therefore, with this method we can experimentally induce syntelic attachment in yeast and investigate how cells respond to this incorrect attachment.

A meiosis-conditional pds5 allele in yeast provides a more detailed understanding of homologue pairing and synaptonemal complex formation., During meiosis, homologues become juxtaposed and synapsed along their entire length. Mutations in the cohesin complex disrupt not only sister chromatid cohesion but also homologue pairing and synaptonemal complex formation. In this study, we report that Pds5, a cohesin-associated protein known to regulate sister chromatid cohesion, is required for homologue pairing and synapsis in budding yeast. Pds5 colocalizes with cohesin along the length of meiotic chromosomes. In the absence of Pds5, the meiotic cohesin subunit Rec8 remains bound to chromosomes with only minor defects in sister chromatid cohesion, but sister chromatids synapse instead of homologues. Double-strand breaks (DSBs) are formed but are not repaired efficiently. In addition, meiotic chromosomes undergo hypercondensation. When the mitotic cohesin subunit Mcd1 is substituted for Rec8 in Pds5-depleted cells, chromosomes still hypercondense, but synapsis of sister chromatids is abolished. These data suggest that Pds5 modulates the Rec8 activity to facilitate chromosome morphological changes required for homologue synapsis, DSB repair, and meiotic chromosome segregation.

Since it was first identified in Drosophila as a centromeric protein, MEI-S332/Shugoshin (Sgo1) has been an enigma. It is neither a kinase nor a phosphatase, but Sgo1's many functions are often attributable to protein-protein interactions that require Sgo1's presence to localize and/or function properly in a timely manner. Though much has been reported about Sgo1's role in ensuring proper genome segregation through its regulatory role in maintaining cohesion through protection of centromeric cohesin, several additional functions have been identified that are less well characterized. In higher eukaryotes Sgo2 and a splice variant of Sgo1 make up a small family of shugoshin proteins, whereas in budding yeast a single Sgo1 exists, and available data suggest that the many functions carried out by Sgo2 and the splice variant are handled exclusively by the Sgo1 homolog in yeast. In fact, the function of the mammalian splice variant is the factor that has revealed a new possible role for shugoshin in centrosome regulation, which may be related mechanistically to its task in protection of centromeric cohesin. New evidence in budding yeast supports this line of reasoning, which may represent an exciting new advance in exposing the myriad faces of the guardian spirit.

During meiosis, segregation of homologous chromosomes necessitates the coordination of sister chromatid cohesion, chromosome condensation, and recombination. Cohesion and condensation require the SMC complexes, cohesin and condensin, respectively. Here we use budding yeast Saccharomyces cerevisiae to show that condensin and Cdc5, a Polo-like kinase, facilitate the removal of cohesin from chromosomes prior to the onset of anaphase I when homologs segregate. This cohesin removal is critical for homolog segregation because it helps dissolve the recombination-dependent links between homologs that form during prophase I. Condensin enhances the association of Cdc5 with chromosomes and its phosphorylation of cohesin, which in turn likely stimulates cohesin removal. Condensin/Cdc5-dependent removal of cohesin underscores the potential importance of crosstalk between chromosome structural components in chromosome morphogenesis and provides a mechanism to couple chromosome morphogenesis with other meiotic events.

In eukaryotic cells, cohesin holds sister chromatids together until they separate into daughter cells during mitosis. We have used chromatin immunoprecipitation coupled with microarray analysis (ChIP chip) to produce a genome-wide description of cohesin binding to meiotic and mitotic chromosomes of Saccharomyces cerevisiae. A computer program, PeakFinder, enables flexible, automated identification and annotation of cohesin binding peaks in ChIP chip data. Cohesin sites are highly conserved in meiosis and mitosis, suggesting that chromosomes share a common underlying structure during different developmental programs. These sites occur with a semiperiodic spacing of 11 kb that correlates with AT content. The number of sites correlates with chromosome size; however, binding to neighboring sites does not appear to be cooperative. We observed a very strong correlation between cohesin sites and regions between convergent transcription units. The apparent incompatibility between transcription and cohesin binding exists in both meiosis and mitosis. Further experiments reveal that transcript elongation into a cohesin-binding site removes cohesin. A negative correlation between cohesin sites and meiotic recombination sites suggests meiotic exchange is sensitive to the chromosome structure provided by cohesin. The genome-wide view of mitotic and meiotic cohesin binding provides an important framework for the exploration of cohesins and cohesion in other genomes.

Genes for three maize homologs (CenpcA, CenpcB, and CenpcC) of the conserved kinetochore assembly protein known as centromere protein C (CENPC) have been identified. The C-terminal portion of maize CENPC shares similarity with mammalian CENPC and its yeast homolog Mif2p over a 23-amino acid region known as region I. Immunolocalization experiments combined with three-dimensional light microscopy demonstrated that CENPC is a component of the kinetochore throughout interphase, mitosis, and meiosis. It is shown that sister kinetochore separation occurs in two discrete phases during meiosis. A partial separation of sister kinetochores occurs in prometaphase I, and a complete separation occurs in prometaphase II. CENPC is absent on structures known as neocentromeres that, in maize, demonstrate poleward movement but lack other important features of centromeres/kinetochores. CENPC and a previously identified centromeric DNA sequence interact closely but do not strictly colocalize on meiotic chromosomes. These and other data indicate that CENPC occupies an inner domain of the maize kinetochore.