Meiotic cells escape prolonged spindle checkpoint activity through kinetochore silencing and slippage.

To prevent chromosome mis-segregation, a surveillance mechanism known as the spindle checkpoint delays the cell cycle if kinetochores are not attached to spindle microtubules, allowing the cell additional time to correct improper attachments. During spindle checkpoint activation, checkpoint proteins bind the unattached kinetochore and send a diffusible signal to inhibit the anaphase promoting complex/cyclosome (APC/C). Previous work has shown that mitotic cells with depolymerized microtubules can escape prolonged spindle checkpoint activation in a process called mitotic slippage. During slippage, spindle checkpoint proteins bind unattached kinetochores, but the cells cannot maintain the checkpoint arrest. We asked if meiotic cells had as robust of a spindle checkpoint response as mitotic cells and whether they also undergo slippage after prolonged spindle checkpoint activity. We performed a direct comparison between mitotic and meiotic budding yeast cells that signal the spindle checkpoint through two different assays. We find that the spindle checkpoint delay is shorter in meiosis I or meiosis II compared to mitosis, overcoming a checkpoint arrest approximately 150 minutes earlier in meiosis than in mitosis. In addition, cells in meiosis I escape spindle checkpoint signaling using two mechanisms, silencing the checkpoint at the kinetochore and through slippage. We propose that meiotic cells undertake developmentally-regulated mechanisms to prevent persistent spindle checkpoint activity to ensure the production of gametes.

The DNA damage checkpoint and the spindle position checkpoint: guardians of meiotic commitment.

Exogenous signals induce cells to enter the specialized cell division process of meiosis, which produces haploid gametes from diploid progenitor cells. Once cells initiate the meiotic divisions, it is imperative that they complete meiosis. Inappropriate exit from meiosis and entrance into mitosis can create polyploid cells and can lead to germline tumors. Saccharomyces cerevisiae cells enter meiosis when starved of nutrients but can return to mitosis if provided nutrient-rich medium before a defined commitment point. Once past the meiotic commitment point in prometaphase I, cells stay committed to meiosis even in the presence of a mitosis-inducing signal. Recent research investigated the maintenance of meiotic commitment in budding yeast and found that two checkpoints that do not normally function in meiosis I, the DNA damage checkpoint and the spindle position checkpoint, have crucial functions in maintaining meiotic commitment. Here, we review these findings and discuss how the mitosis-inducing signal of nutrient-rich medium could activate these two checkpoints in meiosis to prevent inappropriate meiotic exit.

Positive feedback of NDT80 expression ensures irreversible meiotic commitment in budding yeast.

In budding yeast, meiotic commitment is the irreversible continuation of the developmental path of meiosis. After reaching meiotic commitment, cells finish meiosis and gametogenesis, even in the absence of the meiosis-inducing signal. In contrast, if the meiosis-inducing signal is removed and the mitosis-inducing signal is provided prior to reaching meiotic commitment, cells exit meiosis and return to mitosis. Previous work has shown that cells commit to meiosis after prophase I but before entering the meiotic divisions. Since the Ndt80 transcription factor induces expression of middle meiosis genes necessary for the meiotic divisions, we examined the role of the NDT80 transcriptional network in meiotic commitment. Using a microfluidic approach to analyze single cells, we found that cells commit to meiosis in prometaphase I, after the induction of the Ndt80-dependent genes. Our results showed that high-level expression of NDT80 is important for the timing and irreversibility of meiotic commitment. A modest reduction in NDT80 levels delayed meiotic commitment based on meiotic stages, although the timing of each meiotic stage was similar to that of wildtype cells. A further reduction of NDT80 resulted in the surprising finding of inappropriately uncommitted cells: withdrawal of the meiosis-inducing signal and addition of the mitosis-inducing signal to cells at stages beyond metaphase I caused return to mitosis, leading to multi-nucleate cells. Since Ndt80 enhances its own transcription through positive feedback, we tested whether positive feedback ensured the irreversibility of meiotic commitment. Ablating positive feedback in NDT80 expression resulted in a complete loss of meiotic commitment. These findings suggest that irreversibility of meiotic commitment is a consequence of the NDT80 transcriptional positive feedback loop, which provides the high-level of Ndt80 required for the developmental switch of meiotic commitment. These results also illustrate the importance of irreversible meiotic commitment for maintaining genome integrity by preventing formation of multi-nucleate cells.

Chromosome segregation: Mps1 and Dam1 battle to bind a shared interaction site at the kinetochore.

The attachment of kinetochores to spindle microtubules is highly regulated to ensure proper chromosome segregation. Three new studies identify an interaction hub at the kinetochore that integrates kinetochore attachment state with spindle checkpoint activity and kinetochore assembly.

Meiotic Cells Escape Prolonged Spindle Checkpoint Activity Through Premature Silencing and Slippage.

To prevent chromosome mis-segregation, a surveillance mechanism known as the spindle checkpoint delays the cell cycle if kinetochores are not attached to spindle microtubules, allowing the cell additional time to correct improper attachments. During spindle checkpoint activation, checkpoint proteins bind the unattached kinetochore and send a diffusible signal to inhibit the anaphase promoting complex/cyclosome (APC/C). Previous work has shown that mitotic cells with depolymerized microtubules can escape prolonged spindle checkpoint activation in a process called mitotic slippage. During slippage, spindle checkpoint proteins bind unattached kinetochores, but the cells cannot maintain the checkpoint arrest. We asked if meiotic cells had as robust of a spindle checkpoint response as mitotic cells and whether they also undergo slippage after prolonged spindle checkpoint activity. We performed a direct comparison between mitotic and meiotic budding yeast cells that signal the spindle checkpoint due to a lack of either kinetochore-microtubule attachments or due to a loss of tension-bearing attachments. We find that the spindle checkpoint is not as robust in meiosis I or meiosis II compared to mitosis, overcoming a checkpoint arrest approximately 150 minutes earlier in meiosis. In addition, cells in meiosis I escape spindle checkpoint signaling using two mechanisms, silencing the checkpoint at the kinetochore and through slippage. We propose that meiotic cells undertake developmentally-regulated mechanisms to prevent persistent spindle checkpoint activity to ensure the production of gametes. AUTHOR SUMMARY: Mitosis and meiosis are the two major types of cell divisions. Mitosis gives rise to genetically identical daughter cells, while meiosis is a reductional division that gives rise to gametes. Cell cycle checkpoints are highly regulated surveillance mechanisms that prevent cell cycle progression when circumstances are unfavorable. The spindle checkpoint promotes faithful chromosome segregation to safeguard against aneuploidy, in which cells have too many or too few chromosomes. The spindle checkpoint is activated at the kinetochore and then diffuses to inhibit cell cycle progression. Although the checkpoint is active in both mitosis and meiosis, most studies involving checkpoint regulation have been performed in mitosis. By activating the spindle checkpoint in both mitosis and meiosis in budding yeast, we show that cells in meiosis elicit a less persistent checkpoint signal compared to cells in mitosis. Further, we show that cells use distinct mechanisms to escape the checkpoint in mitosis and meiosis I. While cells in mitosis and meiosis II undergo anaphase onset while retaining checkpoint proteins at the kinetochore, cells in meiosis I prematurely lose checkpoint protein localization at the kinetochore. If the mechanism to remove the checkpoint components from the kinetochore is disrupted, meiosis I cells can still escape checkpoint activity. Together, these results highlight that cell cycle checkpoints are differentially regulated during meiosis to avoid long delays and to allow gametogenesis.

Distinct Aurora B pools at the inner centromere and kinetochore have different contributions to meiotic and mitotic chromosome segregation.

Proper chromosome segregation depends on establishment of bioriented kinetochore-microtubule attachments, which often requires multiple rounds of release and reattachment. Aurora B and C kinases phosphorylate kinetochore proteins to release tensionless attachments. Multiple pathways recruit Aurora B/C to the centromere and kinetochore. We studied how these pathways contribute to anaphase onset timing and correction of kinetochore-microtubule attachments in budding yeast meiosis and mitosis. We find that the pool localized by the Bub1/Bub3 pathway sets the normal duration of meiosis and mitosis, in differing ways. Our meiosis data suggests that disruption of this pathway leads to PP1 kinetochore localization, which dephosphorylates Cdc20 for premature anaphase onset. For error correction, the Bub1/Bub3 and COMA pathways are individually important in meiosis but compensatory in mitosis. Finally, we find that the haspin and Bub1/3 pathways function together to ensure error correction in mouse oogenesis. Our results suggest that each recruitment pathway localizes spatially distinct kinetochore-localized Aurora B/C pools that function differently between meiosis and mitosis.

Distinct Aurora B pools at the inner centromere and kinetochore have different contributions to meiotic and mitotic chromosome segregation.

Proper chromosome segregation depends on the establishment of bioriented kinetochore-microtubule attachments, which often requires multiple rounds of release and reattachment. Aurora B and C kinases phosphorylate kinetochore proteins to release tensionless attachments. Multiple pathways recruit Aurora B/C to the centromere and kinetochore. We studied how these pathways contribute to anaphase onset timing and correction of kinetochore-microtubule attachments in budding yeast meiosis and mitosis. We find that the pool localized by the Bub1/Bub3 pathway sets the normal duration of meiosis and mitosis, in differing ways. Our meiosis data suggests a model that disruption of this pathway leads to PP1 kinetochore localization, which dephosphorylates Cdc20 for premature anaphase onset. For error correction, the Bub1/Bub3 and COMA pathways are individually important in meiosis but compensatory in mitosis. Finally, we find that the haspin and Bub1/3 pathways function together to ensure error correction in mouse oogenesis. Our results suggest that each recruitment pathway localizes spatially distinct kinetochore-localized Aurora B/C pools that function differently between meiosis and mitosis.

High-throughput genetic screening of meiotic commitment using fluorescence microscopy in Saccharomyces cerevisiae.

Simple genetic screens in budding yeast have identified many conserved meiotic regulators. However, the identification of genes involved in specific steps of meiosis may require a more complex genetic screen that allows visualization of meiosis. Here, we describe a high-throughput protocol using fluorescence microscopy to systematically screen an overexpression library to identify genes involved in meiotic commitment. We also explain how this protocol can be adapted for identifying proteins that function at different stages of meiosis. For complete details on the use and execution of this protocol, please refer to Gavade et al. (2022).

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. Cerevisiae.

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

Identification of 14-3-3 proteins, Polo kinase, and RNA-binding protein Pes4 as key regulators of meiotic commitment in budding yeast.

The initiation of the cell division process of meiosis requires exogenous signals that activate internal gene regulatory networks. Meiotic commitment ensures the irreversible continuation of meiosis, even upon withdrawal of the meiosis-inducing signals. A loss of meiotic commitment can cause highly abnormal polyploid cells and can ultimately lead to germ cell tumors. Despite the importance of meiotic commitment, only a few genes involved in commitment are known. In this study, we have discovered six new regulators of meiotic commitment in budding yeast: the Bcy1 protein involved in nutrient sensing, the meiosis-specific kinase Ime2, Polo kinase Cdc5, RNA-binding protein Pes4, and the 14-3-3 proteins Bmh1 and Bmh2. Decreased levels of these proteins cause a failure to establish or maintain meiotic commitment. Importantly, we found that Bmh1 and Bmh2 are involved in multiple processes throughout meiosis and in meiotic commitment. First, cells depleted of both Bmh1 and Bmh2 trigger the pachytene checkpoint, likely due to a role in DNA double-strand break repair. Second, Bmh1 interacts directly with the middle meiosis transcription factor Ndt80, and both Bmh1 and Bmh2 maintain Ndt80 levels. Third, Bmh1 and Bmh2 bind to Cdc5 and enhance its kinase activity. Finally, Bmh1 binds to Pes4, which regulates the timing of the translation of several mRNAs in meiosis II and is required to maintain meiotic commitment. Our results demonstrate that meiotic commitment is actively maintained throughout meiosis, with the 14-3-3 proteins and Polo kinase serving as key regulators of this developmental program.

The mitotic exit network regulates the spatiotemporal activity of Cdc42 to maintain cell size.

During G1 in budding yeast, the Cdc42 GTPase establishes a polar front, along which actin is recruited to direct secretion for bud formation. Cdc42 localizes at the bud cortex and then redistributes between mother and daughter in anaphase. The molecular mechanisms that terminate Cdc42 bud-localized activity during mitosis are poorly understood. We demonstrate that the activity of the Cdc14 phosphatase, released through the mitotic exit network, is required for Cdc42 redistribution between mother and bud. Induced Cdc14 nucleolar release results in premature Cdc42 redistribution between mother and bud. Inhibition of Cdc14 causes persistence of Cdc42 bud localization, which perturbs normal cell size and spindle positioning. Bem3, a Cdc42 GAP, binds Cdc14 and is dephosphorylated at late anaphase in a Cdc14-dependent manner. We propose that Cdc14 dephosphorylates and activates Bem3 to allow Cdc42 inactivation and redistribution. Our results uncover a mechanism through which Cdc14 regulates the spatiotemporal activity of Cdc42 to maintain normal cell size at cytokinesis.

Establishing correct kinetochore-microtubule attachments in mitosis and meiosis.

Faithful chromosome segregation in mitosis and meiosis requires that chromosomes properly attach to spindle microtubules. Initial kinetochore-microtubule attachments are often incorrect and rely on error correction mechanisms to release improper attachments, allowing the formation of new attachments. Aurora B kinase and, in mammalian germ cells, Aurora C kinase function as the enzymatic component of the Chromosomal Passenger Complex (CPC), which localizes to the inner centromere/kinetochore and phosphorylates kinetochore proteins for microtubule release during error correction. In this review, we discuss recent findings of the molecular pathways that regulate the chromosomal localization of Aurora B and C kinases in human cell lines, mice, fission yeast, and budding yeast. We also discuss differences in the importance of localization pathways between mitosis and meiosis.

CDK Regulation of Meiosis: Lessons from S. cerevisiae and S. pombe.

Meiotic progression requires precise orchestration, such that one round of DNA replication is followed by two meiotic divisions. The order and timing of meiotic events is controlled through the modulation of the phosphorylation state of proteins. Key components of this phospho-regulatory system include cyclin-dependent kinase (CDK) and its cyclin regulatory subunits. Over the past two decades, studies in budding and fission yeast have greatly informed our understanding of the role of CDK in meiotic regulation. In this review, we provide an overview of how CDK controls meiotic events in both budding and fission yeast. We discuss mechanisms of CDK regulation through post-translational modifications and changes in the levels of cyclins. Finally, we highlight the similarities and differences in CDK regulation between the two yeast species. Since CDK and many meiotic regulators are highly conserved, the findings in budding and fission yeasts have revealed conserved mechanisms of meiotic regulation among eukaryotes.

Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation.

Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore-microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1-Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome-spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1-Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

Autophagy prevents runaway meiotic divisions.

In budding yeast, macroautophagy/autophagy is required for cells to enter into the meiotic divisions. Our recent publication showed that autophagy is also required for meiotic exit. Inhibition of autophagy as cells enter into the meiotic divisions results in additional rounds of spindle formation, spindle elongation, and aberrant chromosome segregation leading to cell death. Under these conditions, the meiosis II-specific cyclin Clb3 is absent, and two substrates of the anaphase-promoting complex/cyclosome (APC/C) persist into the additional divisions instead of being degraded after meiosis II. We found that the translational repressor Rim4 is a substrate of autophagy, which could explain these observations through its known role in repressing synthesis of Clb3 and the meiosis-specific co-activator of the APC/C, Ama1. Combined, these results provide new mechanistic insight into the control of meiotic exit through timed autophagic degradation of a master regulator of gene expression.

Autophagy of an Amyloid-like Translational Repressor Regulates Meiotic Exit.

We explored the potential for autophagy to regulate budding yeast meiosis. Following pre-meiotic DNA replication, we blocked autophagy by chemical inhibition of Atg1 kinase or engineered degradation of Atg14 and observed homologous chromosome segregation followed by sister chromatid separation; cells then underwent additional rounds of spindle formation and disassembly without DNA re-replication, leading to aberrant chromosome segregation. Analysis of cell-cycle regulators revealed that autophagy inhibition prevents meiosis II-specific expression of Clb3 and leads to the aberrant persistence of Clb1 and Cdc5, two substrates of a meiotic ubiquitin ligase activated by Ama1. Lastly, we found that during meiosis II, autophagy degrades Rim4, an amyloid-like translational repressor whose timed clearance regulates protein production from its mRNA targets, which include CLB3 and AMA1. Strikingly, engineered Clb3 or Ama1 production restored meiotic termination in the absence of autophagy. Thus, autophagy destroys a master regulator of meiotic gene expression to enable irreversible meiotic exit.

Signal transduction: external signals influence spore-number control.

In budding yeast, the supply of external nutrients controls both sporulation and spore number. A new study has investigated how an external signal can be propagated internally to digitize an output of the number of spores formed.

Detaching the tether: Remodeling mitochondrial localization during meiosis.

During meiosis, many organelles including mitochondria undergo dramatic remodeling to be inherited in gametes. In this issue, new work from Sawyer et al. (2019. J. Cell. Biol. https://doi.org/10.1083/jcb.201807097) demonstrates that the developmentally programmed destruction of a tether releases mitochondria from the cell cortex during meiosis in budding yeast.