Biology subjects

Lacefield, S.

Publications and source records attributed to Lacefield, S..

7 recordsLinked to original sources

bioRxiv2025

Disrupted MOS signaling alters meiotic cell cycle regulation and the egg transcriptome

Mammalian female meiosis is tightly regulated to produce a developmentally competent egg. Oocytes enter meiosis in the fetal ovary and then arrest at prophase I until sexual maturation. Upon hormonal stimulation, a subset of oocytes resumes meiosis at which time, new transcription is halted. Oocytes then complete meiosis I, enter metaphase II, and arrest until fertilization, a process essential for egg competency and fertility. The MOS kinase is a key regulator of the metaphase II arrest, activating the MAPK signaling cascade. Loss of MOS in female mice disrupts the maintenance of the metaphase II arrest, leading some eggs to extrude two polar bodies and some to divide beyond anaphase II. To investigate the consequences of the Mos deletion, we performed live imaging and found that mos-/- eggs exhibit transient chromosome separation events in meiosis I, suggesting a role for MOS in coordinating the timing of meiotic divisions. Further analysis showed that new transcription is required for mos-/- eggs to undergo additional divisions but not for second polar body extrusion. Surprisingly, single-egg sequencing revealed extensive differences in gene expression between wildtype and mos-/-eggs, including those with only one polar body. Many of the differentially expressed genes were involved in cell cycle regulation, including Aurka, Bub3, and Cdk7. Other upregulated pathways included metabolism of RNA, transcription, and neddylation. Furthermore, the gene expression profile of mos-/- eggs was markedly different from that of wildtype eggs chemically activated to undergo embryo-like divisions. Our findings demonstrate that MOS plays a crucial role in meiotic cell cycle regulation and helps ensure that the egg maintains the proper transcriptome necessary for developmental competence.

cell biology↗

bioRxiv2024

The B-type cyclin Clb4 prevents meiosis I sister centromere separation in budding yeast

In meiosis, one round of DNA replication followed by two rounds of chromosome segregation halves the ploidy of the original cell. Accurate chromosome segregation in meiosis I depends on recombination between homologous chromosomes. Sister centromeres attach to the same spindle pole in this division and only segregate in meiosis II. We used budding yeast to select for mutations that produced viable spores in the absence of recombination. The most frequent mutations inactivated CLB4, which encodes one of four B-type cyclins. In two wild yeast isolates, Y55 and SK1, but not the W303 laboratory strain, deleting CLB4 causes premature sister centromere separation and segregation in meiosis I and frequent termination of meiosis after a single division, demonstrating a novel role for Clb4 in meiotic chromosome dynamics and meiotic progression. This role depends on the genetic background since meiosis in W303 is largely independent of CLB4.

genetics↗

bioRxiv2024

Aneuploidy of Specific Chromosomes is Beneficial to Cells Lacking Spindle Checkpoint Protein Bub3

Aneuploidy typically poses challenges for cell survival and growth. However, recent studies have identified exceptions where aneuploidy is beneficial for cells with mutations in certain regulatory genes. Our research reveals that cells lacking the spindle checkpoint gene BUB3 exhibit aneuploidy of select chromosomes. While the spindle checkpoint is not essential in budding yeast, the loss of BUB3 and BUB1 increases the probability of chromosome missegregation compared to wildtype cells. Contrary to the prevailing assumption that the aneuploid cells would be outcompeted due to growth defects, our findings demonstrate that bub3{Delta} cells consistently maintained aneuploidy of specific chromosomes over many generations. We investigated whether the persistence of these additional chromosomes in bub3{Delta} cells resulted from the beneficial elevated expression of certain genes, or mere tolerance. We identified several genes involved in chromosome segregation and cell cycle regulation that confer an advantage to Bub3-depleted cells. Overall, our results suggest that the upregulation of specific genes through aneuploidy may provide a survival and growth advantage to strains with poor chromosome segregation fidelity. AUTHOR SUMMARYAccurate chromosome segregation is crucial for the proper development of all living organisms. Errors in chromosome segregation can lead to aneuploidy, characterized by an abnormal number of chromosomes, which generally impairs cell survival and growth. However, under certain stress conditions, such as in various cancers, cells with specific mutations and extra copies of advantageous chromosomes exhibit improved survival and proliferation. In our study, we discovered that cells lacking the spindle checkpoint protein Bub3 became aneuploid, retaining specific chromosomes. This finding was unexpected because although bub3{Delta} cells have a higher rate of chromosome mis-segregation, they were not thought to maintain an aneuploid karyotype. We investigated whether the increased copy number of specific genes on these acquired chromosomes offered a benefit to Bub3-deficient cells. Our results revealed that several genes involved in chromosome segregation and cell cycle regulation prevented the gain of chromosomes upon Bub3-depletion, suggesting that these genes confer a survival advantage. Overall, our study demonstrates that cells lacking Bub3 selectively retain specific chromosomes to increase the copy number of genes that promote proper chromosome segregation.

cell biology↗

bioRxiv2023

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.

cell biology↗

bioRxiv2023

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 SUMMARYMitosis 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.

cell biology↗

bioRxiv2021

The 14-3-3 Proteins Bmh1 and Bmh2 Are Key Regulators of Meiotic Commitment

Induction 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. Budding yeast cells have a unique property in that cells enter meiosis when starved, but if given nutrient-rich medium prior to the commitment point, they exit meiosis and enter mitosis. After the meiotic commitment point in prometaphase I, cells remain in meiosis even with addition of nutrients. Despite the importance of meiotic commitment in ensuring the production of gametes, only a few genes involved in the commitment process are known. We performed a genome-scale screen in budding yeast and discovered new regulators of meiotic commitment including Bcy1, which is involved in nutrient sensing, the meiosis-specific kinase Ime2, Polo kinase Cdc5, and the 14-3-3 proteins Bmh1 and Bmh2. Importantly, we found that Bmh1 and Bmh2 are involved in multiple processes throughout meiosis including the maintenance of the middle meiosis transcription factor Ndt80, activation of Cdc5, and interaction with an RNA-binding protein Pes4, which is important for regulating the timing of translation of several mRNAs in meiosis II. This study identifies a meiotic commitment regulatory network with the 14-3-3 proteins functioning as central regulators.

cell biology↗

bioRxiv2019

Bub3 and Bub1 maintain the balance of kinetochore-localized Aurora B Kinase and Protein Phosphatase I to Regulate Chromosome Segregation and Anaphase Onset in Meiosis

Accurate chromosome segregation depends on proper attachment of kinetochores to spindle microtubules prior to 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 mis-segregation 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.\n\nSummaryCairo et al show that in S. cerevisiae meiosis, spindle checkpoint proteins Bub1 and Bub3 have an essential role in preventing chromosome mis-segregation and setting the normal duration of anaphase I and anaphase II onset by regulating the kinetochore-localization of Ipl1 and PP1.

cell biology↗