Dynamic Live Cell Imaging of Budding Yeast Meiosis.

For over a century, major advances in understanding meiosis have come from the use of microscopy-based methods. Studies using the budding yeast, Saccharomyces cerevisiae, have made important contributions to our understanding of meiosis because of the facility with which budding yeast can be manipulated as a genetic model organism. In contrast, imaging-based approaches with budding yeast have been constrained by the small size of its chromosomes. The advent of advances in fluorescent chromosome tagging techniques has made it possible to use yeast more effectively for imaging-based approaches as well. This protocol describes live cell imaging methods that can be used to monitor chromosome movements throughout meiosis in living yeast cells.

Centromere pairing enables correct segregation of meiotic chromosomes.

Proper chromosome segregation in meiosis I relies on the formation of connections between homologous chromosomes. Crossovers between homologs provide a connection that allows them to attach correctly to the meiosis I spindle. Tension is transmitted across the crossover when the partners attach to microtubules from opposing poles of the spindle. Tension stabilizes microtubule attachments that will pull the partners toward opposite poles at anaphase. Paradoxically, in many organisms, non-crossover partners segregate correctly. The mechanism by which non-crossover partners become bioriented on the meiotic spindle is unknown. Both crossover and non-crossover partners pair their centromeres early in meiosis (prophase). In budding yeast, centromere pairing is correlated with subsequent correct segregation of the partners. The mechanism by which centromere pairing, in prophase, promotes later correct attachment of the partners to the metaphase spindle is unknown. We used live cell imaging to track the biorientation process of non-crossover chromosomes. We find that centromere pairing allows the establishment of connections between the partners that allows their later interdependent attachment to the meiotic spindle using tension-sensing biorientation machinery. Because all chromosome pairs experience centromere pairing, our findings suggest that crossover chromosomes also utilize this mechanism to achieve maximal segregation fidelity.

Cornelia de Lange Syndrome mutations in SMC1A cause cohesion defects in yeast.

Cornelia de Lange Syndrome (CdLS) is a developmental disorder characterized by limb truncations, craniofacial abnormalities, and cognitive delays. CdLS is caused mainly by mutations in genes encoding subunits or regulators of the cohesin complex. Cohesin plays 2 distinct roles in chromosome dynamics as follows: it promotes looping, organization, and compaction of individual chromosomes, and it holds newly replicated sister chromatids together until cell division. CdLS-associated mutations result in altered gene expression likely by affecting chromosome architecture. Whether CdLS mutations cause phenotypes through impact on sister chromatid cohesion is less clear. Here, we show that CdLS-associated mutations introduced into the SMC1A gene of budding yeast had measurable impacts on sister chromatid cohesion, mitotic progression, and DNA damage sensitivity. These data suggest that sister chromatid cohesion-related defects may contribute to phenotypes seen in CdLS affected individuals.

Polyploid yeast are dependent on elevated levels of Mps1 for successful chromosome segregation.

Tumor cell lines with elevated chromosome numbers frequently have correlated elevations of Mps1 expression and these tumors are more dependent on Mps1 activity for their survival than control cell lines. Mps1 is a conserved kinase involved in controlling aspects of chromosome segregation in mitosis and meiosis. The mechanistic explanation for the Mps1-addiction of aneuploid cells is unknown. To address this question, we explored Mps1-dependence in yeast cells with increased sets of chromosomes. These experiments revealed that in yeast, increasing ploidy leads to delays and failures in orienting chromosomes on the mitotic spindle. Yeast cells with elevated numbers of chromosomes proved vulnerable to reductions of Mps1 activity. Cells with reduced Mps1 activity exhibit an extended prometaphase with longer spindles and delays in orienting the chromosomes. One known role of Mps1 is in recruiting Bub1 to the kinetochore in meiosis. We found that the Mps1-addiction of polyploid yeast cells is due in part to its role in Bub1 recruitment. Together, the experiments presented here demonstrate that increased ploidy renders cells more dependent on Mps1 for orienting chromosomes on the spindle. The phenomenon described here may be relevant in understanding why hyper-diploid cancer cells exhibit elevated reliance on Mps1 expression for successful chromosome segregation.

Ycs4 Subunit of Saccharomyces cerevisiae Condensin Binds DNA and Modulates the Enzyme Turnover.

Condensins play a key role in higher order chromosome organization. In budding yeast Saccharomyces cerevisiae, a condensin complex consists of five subunits: two conserved structural maintenance of chromosome subunits, Smc2 and Smc4, a kleisin Brn1, and two HEAT repeat subunits, Ycg1, which possesses a DNA binding activity, and Ycs4, which can transiently associate with Smc4 and thereby disrupt its association with the Smc2 head. We characterized here DNA binding activity of the non-SMC subunits using an agnostic, model-independent approach. To this end, we mapped the DNA interface of the complex using sulfo-NHS biotin labeling. Besides the known site on Ycg1, we found a patch of lysines at the C-terminal domain of Ycs4 that were protected from biotinylation in the presence of DNA. Point mutations at the predicted protein-DNA interface reduced both Ycs4 binding to DNA and the DNA stimulated ATPase activity of the reconstituted condensin, whereas overproduction of the mutant Ycs4 was detrimental for yeast viability. Notably, the DNA binding site on Ycs4 partially overlapped with its interface with SMC4, revealing an intricate interplay between DNA binding, engagement of the Smc2-Smc4 heads, and ATP hydrolysis and suggesting a mechanism for ATP-modulated loading and translocation of condensins on DNA.

Mps1 promotes poleward chromosome movements in meiotic prometaphase.

In prophase of meiosis I, homologous chromosomes pair and become connected by cross-overs. Chiasmata, the connections formed by cross-overs, enable the chromosome pair, called a bivalent, to attach as a single unit to the spindle. When the meiotic spindle forms in prometaphase, most bivalents are associated with one spindle pole and then go through a series of oscillations on the spindle, attaching to and detaching from microtubules until the partners of the bivalent become bioriented-attached to microtubules from opposite sides of the spindle. The conserved kinase, Mps1, is essential for the bivalents to be pulled by microtubules across the spindle in prometaphase. Here we show that MPS1 is needed for efficient triggering of the migration of microtubule-attached kinetochores toward the poles and promotes microtubule depolymerization. Our data support the model Mps1 acts at the kinetochore to coordinate the successful attachment of a microtubule and the triggering of microtubule depolymerization to then move the chromosome.

Better safe than sorry-preventing mitotic segregation of meiotic chromosomes.

The distinctive segregation patterns of chromosomes in mitosis and meiosis are dictated in part by the kinetochores, the structures on chromosomes that attach them to the microtubules of the spindle. Inappropriate mitosis-like chromosome segregation in meiosis leads to gametes with incorrect chromosome numbers. New findings by Chen and colleagues (pp. 209-225) in this issue of Genes & Development reveal how cells restructure their kinetochores when they enter meiosis. Their results describe an interconnected set of mechanisms that provides multiple layers of protection from the carryover of mitotic chromosome segregation patterns into meiotic cells.

Shugoshin protects centromere pairing and promotes segregation of nonexchange partner chromosomes in meiosis.

Faithful chromosome segregation during meiosis I depends upon the formation of connections between homologous chromosomes. Crossovers between homologs connect the partners, allowing them to attach to the meiotic spindle as a unit, such that they migrate away from one another at anaphase I. Homologous partners also become connected by pairing of their centromeres in meiotic prophase. This centromere pairing can promote proper segregation at anaphase I of partners that have failed to become joined by a crossover. Centromere pairing is mediated by synaptonemal complex (SC) proteins that persist at the centromere when the SC disassembles. Here, using mouse spermatocyte and yeast model systems, we tested the role of shugoshin in promoting meiotic centromere pairing by protecting centromeric synaptonemal components from disassembly. The results show that shugoshin protects the centromeric SC in meiotic prophase and, in anaphase, promotes the proper segregation of partner chromosomes that are not linked by a crossover.

A ZIP1 separation-of-function allele reveals that centromere pairing drives meiotic segregation of achiasmate chromosomes in budding yeast.

In meiosis I, homologous chromosomes segregate away from each other-the first of two rounds of chromosome segregation that allow the formation of haploid gametes. In prophase I, homologous partners become joined along their length by the synaptonemal complex (SC) and crossovers form between the homologs to generate links called chiasmata. The chiasmata allow the homologs to act as a single unit, called a bivalent, as the chromosomes attach to the microtubules that will ultimately pull them away from each other at anaphase I. Recent studies, in several organisms, have shown that when the SC disassembles at the end of prophase, residual SC proteins remain at the homologous centromeres providing an additional link between the homologs. In budding yeast, this centromere pairing is correlated with improved segregation of the paired partners in anaphase. However, the causal relationship of prophase centromere pairing and subsequent disjunction in anaphase has been difficult to demonstrate as has been the relationship between SC assembly and the assembly of the centromere pairing apparatus. Here, a series of in-frame deletion mutants of the SC component Zip1 were used to address these questions. The identification of a separation-of-function allele that disrupts centromere pairing, but not SC assembly, has made it possible to demonstrate that centromere pairing and SC assembly have mechanistically distinct features and that the centromere pairing function of Zip1 drives disjunction of the paired partners in anaphase I.

Mps1 promotes chromosome meiotic chromosome biorientation through Dam1.

In budding yeast meiosis, homologous chromosomes become linked by chiasmata and then move back and forth on the spindle until they are bioriented, with the kinetochores of the partners attached to microtubules from opposite spindle poles. Certain mutations in the conserved kinase, Mps1, result in catastrophic meiotic segregation errors but mild mitotic defects. We tested whether Dam1, a known substrate of Mps1, was necessary for its critical meiotic role. We found that kinetochore-microtubule attachments are established even when Dam1 is not phosphorylated by Mps1, but that Mps1 phosphorylation of Dam1 sustains those connections. But the meiotic defects when Dam1 is not phosphorylated are not nearly as catastrophic as when Mps1 is inactivated. The results demonstrate that one meiotic role of Mps1 is to stabilize connections that have been established between kinetochores and microtubles by phosphorylating Dam1.

Meiotic Centromere Coupling and Pairing Function by Two Separate Mechanisms in Saccharomyces cerevisiae.

In meiosis I, chromosomes become paired with their homologous partners and then are pulled toward opposite poles of the spindle. In the budding yeast, Saccharomyces cerevisiae, in early meiotic prophase, centromeres are observed to associate in pairs in a homology-independent manner; a process called centromere coupling. Later, as homologous chromosomes align, their centromeres associate in a process called centromere pairing. The synaptonemal complex protein Zip1 is necessary for both types of centromere association. We aimed to test the role of centromere coupling in modulating recombination at centromeres, and to test whether the two types of centromere associations depend upon the same sets of genes. The zip1-S75E mutation, which blocks centromere coupling but no other known functions of Zip1, was used to show that in the absence of centromere coupling, centromere-proximal recombination was unchanged. Further, this mutation did not diminish centromere pairing, demonstrating that these two processes have different genetic requirements. In addition, we tested other synaptonemal complex components, Ecm11 and Zip4, for their contributions to centromere pairing. ECM11 was dispensable for centromere pairing and segregation of achiasmate partner chromosomes; while ZIP4 was not required for centromere pairing during pachytene, but was required for proper segregation of achiasmate chromosomes. These findings help differentiate the two mechanisms that allow centromeres to interact in meiotic prophase, and illustrate that centromere pairing, which was previously shown to be necessary to ensure disjunction of achiasmate chromosomes, is not sufficient for ensuring their disjunction.

Recent advances in cohesin biology.

Sister chromatids are tethered together from the time they are formed in S-phase until they separate at anaphase. A protein complex called cohesin is responsible for holding the sister chromatids together and serves important roles in chromosome condensation, gene regulation, and the repair of DNA damage. Cohesin contains an open central pore and becomes topologically engaged with its DNA substrates. Entrapped DNA can be released either by the opening of a gate in the cohesin ring or by proteolytic cleavage of a component of the ring. This review summarizes recent research that provides important new insights into how DNA enters and exits the cohesin ring and how the rings behave on entrapped DNA molecules to provide functional cohesion.

Ipl1/Aurora-B is necessary for kinetochore restructuring in meiosis I in Saccharomyces cerevisiae.

In mitosis, the centromeres of sister chromosomes are pulled toward opposite poles of the spindle. In meiosis I, the opposite is true: the sister centromeres move together to the same pole, and the homologous chromosomes are pulled apart. This change in segregation patterns demands that between the final mitosis preceding meiosis and the first meiotic division, the kinetochores must be restructured. In budding yeast, unlike mammals, kinetochores are largely stable throughout the mitotic cycle. In contrast, previous work with budding and fission yeast showed that some outer kinetochore proteins are lost in early meiosis. We use quantitative mass spectrometry methods and imaging approaches to explore the kinetochore restructuring process that occurs in meiosis I in budding yeast. The Ndc80 outer kinetochore complex, but not other subcomplexes, is shed upon meiotic entry. This shedding is regulated by the conserved protein kinase Ipl1/Aurora-B and promotes the subsequent assembly of a kinetochore that will confer meiosis-specific segregation patterns on the chromosome.

Centromere pairing--tethering partner chromosomes in meiosis I.

In meiosis, homologous chromosomes face the obstacle of finding, holding onto and segregating away from their partner chromosome. There is increasing evidence, in a diverse range of organisms, that centromere-centromere interactions that occur in late prophase are an important mechanism in ensuring segregation fidelity. Centromere pairing appears to initiate when homologous chromosomes synapse in meiotic prophase. Structural proteins of the synaptonemal complex have been shown to help mediate centromere pairing, but how the structure that maintains centromere pairing differs from the structure of the synaptonemal complex along the chromosomal arms remains unknown. When the synaptonemal complex proteins disassemble from the chromosome arms in late prophase, some of these synaptonemal complex components persist at the centromeres. In yeast and Drosophila these centromere-pairing behaviors promote the proper segregation of chromosome partners that have failed to become linked by chiasmata. Recent studies of mouse spermatocytes have described centromere pairing behaviors that are similar in several respects to what has been described in the fly and yeast systems. In humans, chromosomes that fail to experience crossovers in meiosis are error-prone and are a major source of aneuploidy. The finding that centromere pairing is a conserved phenomenon raises the possibility that it may play a role in promoting the segregation fidelity of non-exchange chromosome pairs in humans.

Dual mechanisms prevent premature chromosome segregation during meiosis.

In meiosis I, homologous chromosomes pair and then attach to the spindle so that the homologs can be pulled apart at anaphase I. The segregation of homologs before pairing would be catastrophic. We describe two mechanisms that prevent this. First, in early meiosis, Ipl1, the budding yeast homolog of the mammalian Aurora B kinase, triggers shedding of a kinetochore protein, preventing microtubule attachment. Second, Ipl1 localizes to the spindle pole bodies (SPBs), where it blocks spindle assembly. These processes are reversed upon expression of Ndt80. Previous studies have shown that Ndt80 is expressed when homologs have successfully partnered, and this triggers a rise in the levels of cyclin-dependent kinase (CDK). We found that CDK phosphorylates Ipl1, delocalizing it from SPBs, triggering spindle assembly. At the same time, kinetochores reassemble. Thus, dual mechanisms controlled by Ipl1 and Ntd80 coordinate chromosome and spindle behaviors to prevent the attachment of unpartnered chromosomes to the meiotic spindle.

Attaching to spindles before they form: do early incorrect chromosome-microtubule attachments promote meiotic segregation fidelity?

The proper partitioning of the genome during meiosis depends on the correct segregation of chromosomes. Errors in this process result in the production of aneuploid gametes, a major cause of birth defects and infertility in humans. In order to segregate properly in meiosis, homologous chromosome partners must attach to microtubules that emanate from opposites poles of the spindle. However, a recent study in yeast has shown that, remarkably, the initial attachments between microtubules and the chromosomes are usually incorrect, which would lead to catastrophic segregation errors, but they are nearly always corrected through the detachment and reattachment of the microtubules. Here we review the reasons for the initial incorrect attachments, which stem from the timing of their formation early in the spindle assembly process, and the fact that the microtubule organizers, called spindle pole bodies in yeast, are not equal. One spindle pole body is older and better able to produce microtubules that attach to the chromosomes. We draw parallels to recent findings in animal cells and suggest that these early microtubule attachments, while often incorrect, may serve an important role in spindle assembly, which, in the long-term, promotes high-fidelity chromosome segregation.

Mps1 and Ipl1/Aurora B act sequentially to correctly orient chromosomes on the meiotic spindle of budding yeast.

The conserved kinases Mps1 and Ipl1/Aurora B are critical for enabling chromosomes to attach to microtubules so that partner chromosomes will be segregated correctly from each other, but the precise roles of these kinases have been unclear. We imaged live yeast cells to elucidate the stages of chromosome-microtubule interactions and their regulation by Ipl1 and Mps1 through meiosis I. Ipl1 was found to release kinetochore-microtubule (kMT) associations after meiotic entry, liberating chromosomes to begin homologous pairing. Surprisingly, most chromosome pairs began their spindle interactions with incorrect kMT attachments. Ipl1 released these improper connections, whereas Mps1 triggered the formation of new force-generating microtubule attachments. This microtubule release and reattachment cycle could prevent catastrophic chromosome segregation errors in meiosis.

Synaptonemal complex components persist at centromeres and are required for homologous centromere pairing in mouse spermatocytes.

Recent studies in simple model organisms have shown that centromere pairing is important for ensuring high-fidelity meiotic chromosome segregation. However, this process and the mechanisms regulating it in higher eukaryotes are unknown. Here we present the first detailed study of meiotic centromere pairing in mouse spermatogenesis and link it with key events of the G2/metaphase I transition. In mouse we observed no evidence of the persistent coupling of centromeres that has been observed in several model organisms. We do however find that telomeres associate in non-homologous pairs or small groups in B type spermatogonia and pre-leptotene spermatocytes, and this association is disrupted by deletion of the synaptonemal complex component SYCP3. Intriguingly, we found that, in mid prophase, chromosome synapsis is not initiated at centromeres, and centromeric regions are the last to pair in the zygotene-pachytene transition. In late prophase, we first identified the proteins that reside at paired centromeres. We found that components of the central and lateral element and transverse filaments of the synaptonemal complex are retained at paired centromeres after disassembly of the synaptonemal complex along diplotene chromosome arms. The absence of SYCP1 prevents centromere pairing in knockout mouse spermatocytes. The localization dynamics of SYCP1 and SYCP3 suggest that they play different roles in promoting homologous centromere pairing. SYCP1 remains only at paired centromeres coincident with the time at which some kinetochore proteins begin loading at centromeres, consistent with a role in assembly of meiosis-specific kinetochores. After removal of SYCP1 from centromeres, SYCP3 then accumulates at paired centromeres where it may promote bi-orientation of homologous centromeres. We propose that, in addition to their roles as synaptonemal complex components, SYCP1 and SYCP3 act at the centromeres to promote the establishment and/or maintenance of centromere pairing and, by doing so, improve the segregation fidelity of mammalian meiotic chromosomes.

Slk19p of Saccharomyces cerevisiae regulates anaphase spindle dynamics through two independent mechanisms.

Slk19p is a member of the Cdc-14 early anaphase release (FEAR) pathway, a signaling network that is responsible for activation of the cell-cycle regulator Cdc14p in Saccharomyces cerevisiae. Disruption of the FEAR pathway results in defects in anaphase, including alterations in the assembly and behavior of the anaphase spindle. Many phenotypes of slk19Δ mutants are consistent with a loss of FEAR signaling, but other phenotypes suggest that Slk19p may have FEAR-independent roles in modulating the behavior of microtubules in anaphase. Here, a series of SLK19 in-frame deletion mutations were used to test whether Slk19p has distinct roles in anaphase that can be ascribed to specific regions of the protein. Separation-of-function alleles were identified that are defective for either FEAR signaling or aspects of anaphase spindle function. The data suggest that in early anaphase one region of Slk19p is essential for FEAR signaling, while later in anaphase another region is critical for maintaining the coordination between spindle elongation and the growth of interpolar microtubules.

Meiotic cohesin promotes pairing of nonhomologous centromeres in early meiotic prophase.

A period of pairing between nonhomologous centromeres occurs early in meiosis in a diverse collection of organisms. This early, homology-independent, centromere pairing, referred to as centromere coupling in budding yeast, gives way to an alignment of homologous centromeres as homologues synapse later in meiotic prophase. The regulation of centromere coupling and its underlying mechanism have not been elucidated. In budding yeast, the protein Zip1p is a major component of the central element of the synaptonemal complex in pachytene of meiosis, and earlier, is essential for centromere coupling. The experiments reported here demonstrate that centromere coupling is mechanistically distinct from synaptonemal complex assembly. Zip2p, Zip3p, and Red1p are all required for the assembly of Zip1 into the synaptonemal complex but are dispensable for centromere coupling. However, the meiotic cohesin Rec8p is required for centromere coupling. Loading of meiotic cohesins to centromeres and cohesin-associated regions is required for the association of Zip1 with these sites, and the association of Zip1 with the centromeres then promotes coupling. These findings reveal a mechanism that promotes associations between centromeres before the assembly of the synaptonemal complex, and they demonstrate that chromosomes are preloaded with Zip1p in a manner that may promote synapsis.