Biology subjects

Wassmann, K.

Publications and source records attributed to Wassmann, K..

9 recordsLinked to original sources

bioRxiv2026

Set mediates chromosome alignment and Cohesin cleavage independently of direct PP2A-B56-binding in oocyte meiosis

Set promotes cohesion removal in mitosis by evicting phosphorylated Histone H1 and counteracting Sgo1. In addition, Set promotes chromosome alignment by counteracting Aurora B activation. The underlying molecular mechanisms through which Set performs these activities remain insufficiently characterized, but roles of Set as a Histone chaperone and PP2A inhibitor have been proposed. Building on our previous observations that Set promotes pericentromeric Cohesin removal in oocyte meiosis II, we generated an oocyte-specific conditional knock-out of Set to address its functions in meiosis. Similar to mitosis, Set depletion caused chromosome alignment and cohesion defects. We found that Set is required for accurate error correction by localizing Aurora B/C, and for efficient cleavage of the meiosis-specific Cohesin subunit Rec8 by Separase. Paired chromosomes and sister chromatids were often incompletely separated, likely a primary cause of missegregation. Set performed both its roles in a Sgo2-dependent manner, but, unexpectedly, independently of interaction with PP2A-B56. In line with a role of Set as a Histone chaperone, accumulation of phosphorylated Histone H1 in Set knock-out oocytes occurs concomitantly with reduction of oocyte-specific H1foo on chromosome arms, indicating that Set is required to create the optimal chromatin environment for efficient Rec8 cleavage by Separase in meiosis.

cell biology↗

bioRxiv2026

Low-input proteomics enables proteome and phosphoproteome-scale molecular phenotyping of separase-deficient oocytes

We performed a comprehensive quantitative proteomic analysis of mouse oocytes using as few as 40 oocytes per condition, comparing wild-type and separase knockout oocytes at metaphase I and metaphase II. To this end, we generated a deep proteomic library spanning oocyte cell cycle stages, enabling the identification of numerous phosphosites without phosphopeptide enrichment. We further combined data-dependent (DDA) and data-independent (DIA) acquisition strategies, analyzed through multiple software pipelines in both library-based and library-free modes. Our results reveal extensive proteome remodeling during the metaphase I to metaphase II transition in wild-type oocytes, consistent with dynamic regulation of meiotic processes. As a proof of concept for our workflow, we asked whether separase knockout oocytes--unable to separate chromosomes in meiosis I--progress into meiosis II. Direct comparison of wild-type and separase knockout oocytes at the metaphase II stage revealed minimal global differences, supporting the idea that both conditions converge toward a comparable metaphase II-like cellular state despite distinct chromosomal configurations. However, at a finer scale, specific alterations were detected among chromosome-associated proteins. Notably, Meikin was enriched in separase-deficient metaphase II oocytes, consistent with defective separase-dependent cleavage and subsequent turnover. More broadly, several proteins involved in chromosome organization displayed behavior similar to Meikin, suggesting that separase activity regulates multiple substrates to orchestrate chromosome segregation during female meiosis.

Cell Biology↗

bioRxiv2025

Elimination of separase inhibition reveals absence of cohesin protection in oocyte metaphase II

The meiotic segregation pattern to generate haploid gametes is mediated by step-wise cohesion removal by separase, first from chromosome arms in meiosis I, and then from the centromere region in meiosis II. In mammalian oocytes, separase is tightly controlled during the hours-long prometaphase and until chromosome segregation in meiosis I, activated for a short time window, and again inhibited until metaphase II arrest is lifted by fertilization. Centromeric cohesin is protected from cleavage by Sgo2-PP2A in meiosis I. It remained enigmatic how tight control of alternating separase activation and inactivation is achieved during the two divisions in oocytes. It was equally unknown when cohesin protection is put in place and removed. Using structure-function assays in knock-out mouse models we established the contributions of cyclin B1 and securin for separase inhibition during both divisions. When eliminating separase inhibition we found that cohesin is not protected early in meiosis I and at metaphase II arrest. Importantly, in meiosis II, the sole event required for cleavage of centromeric cohesin besides separase activation is prior kinetochore individualization in meiosis I.

cell biology↗

bioRxiv2023

Prolonged metaphase II arrest weakens Aurora B/C- dependent error correction in mouse oocytes

Chromosome segregation during meiosis is highly error-prone in mammalian oocytes. The mechanisms controlling chromosome attachments and the Spindle Assembly Checkpoint (SAC) have been extensively studied in meiosis I, but our knowledge of these mechanisms during meiosis II is rather limited. Although mammalian oocytes arrest in metaphase II for an extended period awaiting fertilization, some misattached chromosomes may persist. This suggests that the mechanism correcting misattachments is not fully functional during the arrest. In this study, we investigated whether low inter-kinetochore tension, which characterizes incorrect attachments, can be detected by Aurora B/C-dependent error correction in meiosis II. We found that low tension in early metaphase II does indeed mediate microtubule detachment by Aurora B/C and consequently, anaphase II delay through SAC activation. Surprisingly, we also found that during prolonged metaphase II arrest, Aurora B/C activity is no longer sufficient to detach low-tension attachments, concomitantly with high accumulation of PP2A at kinetochores. As a result, the SAC is not activated and sister chromatids segregate in anaphase II without delay even in the presence of low tension. Hence, during the prolonged metaphase II arrest to await fertilization, oocytes become unable to discriminate between correct and incorrect attachments and may allow errors to persist.

cell biology↗

bioRxiv2023

Qualitative rather than quantitative phosphoregulation shapes the end of meiosis I in budding yeast

Exit from mitosis is brought about by dramatic changes in the phosphoproteome landscape. A drop in Cyclin-dependent kinase (Cdk), the master regulatory kinase, and activation of counteracting phosphatases such as Cdc14 in budding yeast, results in ordered substrate dephosphorylation, allowing entry into a new cell cycle and replication licensing. In meiosis however, two cell divisions have to be executed without intermediate DNA replication, implying that global phosphorylation and dephosphorylation have to be adapted to the challenges of meiosis. Using a global time-resolved phosphoproteomics approach in budding yeast, we compared the phosphoproteome landscape between mitotic exit and the transition from meiosis I to meiosis II. We found that unlike exit from mitosis, Cdk phosphomotifs remain mostly stably phosphorylated at the end of meiosis I, whereas a majority of Cdk-unrelated motifs are reset by dephosphorylation. However, inducing an artificial drop of Cdk at metaphase of meiosis I leads to ordered substrate dephosphorylation, comparable to mitosis, indicating that phosphoregulation of substrates at the end of meiosis I is thus mainly qualitatively rather than quantitatively ordered.

cell biology↗

bioRxiv2022

Cyclin B3 implements timely vertebrate oocyte arrest for fertilization

To ensure successful offspring ploidy, vertebrate oocytes must halt the cell cycle in meiosis II until sperm entry. Emi2 is essential to keep oocytes arrested until fertilization. Yet, how this arrest is implemented exclusively in meiosis II and not prematurely in meiosis I remained enigmatic. Using mouse and frog oocytes, we show here that cyclin B3, an understudied B- type cyclin, is essential to keep Emi2 levels low in meiosis I. Direct phosphorylation of Emi2 at an evolutionarily highly conserved site by Cdk1/cyclin B3 targets Emi2 for degradation. In contrast, Cdk1/cyclin B1 is inefficient in Emi2 phosphorylation providing a molecular explanation for the requirement of different B-type cyclins for oocyte maturation. Cyclin B3 degradation at exit from meiosis I enables Emi2 accumulation and thus, timely arrest in meiosis II. Our findings illuminate the evolutionarily conserved mechanisms controlling oocyte arrest for fertilization at the correct cell cycle stage, essential for embryo viability.

cell biology↗

bioRxiv2021

Aurora B/C-dependent phosphorylation promotesRec8 cleavage in mammalian oocytes

To generate haploid gametes, cohesin is removed in a step-wise manner from chromosome arms in meiosis I and the centromere region in meiosis II, to segregate chromosomes and sister chromatids, respectively. Meiotic cohesin removal requires cleavage of the meiosis-specific kleisin subunit Rec8 by the protease Separase[1, 2]. In yeast, Rec8 is kept in a non-phosphorylated state by the action of PP2A-B56, which is localised to the centromere region, thereby preventing cohesin removal from this region in meiosis I[3-5]. However, it is unknown whether Rec8 has to be equally phosphorylated for cleavage, and whether centromeric cohesin protection is indeed brought about by dephosphorylation of Rec8 preventing cleavage, in mammalian meiosis. The identity of one or several potential Rec8-specific kinase(s) is also unknown. This is due to technical challenges, as Rec8 is poorly conserved preventing a direct translation of the knowledge gained from model systems such as yeast and C. elegans to mammals, and additionally, there is no turn-over of Rec8 after cohesion establishment, preventing phosphomutant analysis of functional Rec8. To address how Rec8 cleavage is brought about in mammals, we adapted a biosensor for Separase to study Rec8 cleavage in single mouse oocytes by live imaging, and identified phosphorylation sites promoting cleavage. We found that Rec8 cleavage by Separase depends on Aurora B/C kinase activity, and identified a residue promoting cleavage and being phosphorylated in an Aurora B/C kinase-dependent manner. Accordingly, inhibition of Aurora B/C kinase during meiotic maturation impairs endogenous Rec8 phosphorylation and chromosome segregation.

cell biology↗

bioRxiv2020

Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II

Partitioning of the genome in meiosis occurs through two highly specialized cell divisions, named meiosis I and II. Step-wise cohesin removal is required for chromosome segregation in meiosis I, and sister chromatid segregation in meiosis II. In meiosis I, mono-oriented sister kinetochores appear as fused together when examined by high resolution confocal microscopy, whereas they are clearly separated in meiosis II, when attachments are bipolar. It has been proposed that bipolar tension applied by the spindle is responsible for the physical separation of sister kinetochores, removal of cohesin protection and chromatid separation in meiosis II. We show here that this is not the case, and initial separation of sister kinetochores occurs already in anaphase I, when attachments are still monopolar, and independently of pericentromeric Sgo2 removal. This kinetochore individualization occurs also independently of spindle forces applied on sister kinetochores, but importantly, depends on cleavage activity of Separase. Crucially, without kinetochore individualization by Separase in meiosis I, oocytes separate bivalents into chromosomes and not sister chromatids in meiosis II, showing that whether centromeric cohesin is removed or not is determined by the kinetochore structure prior to meiosis II.

cell biology↗

bioRxiv2018

Cyclin B3 promotes APC/C activation and anaphase I onset in oocyte meiosis

As obligate kinase partners, cyclins control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation of genomes. Meiosis, the cell division that generates gametes for sexual reproduction, poses unique challenges because two rounds of chromosome segregation must be executed without intervening DNA replication. Mammalian cells express numerous, temporally regulated cyclins, but how these proteins collaborate to control meiosis remains poorly understood. Here, we delineate an essential function for mouse cyclin B3 in the first meiotic division of oocytes. Females genetically ablated for cyclin B3 are viable, indicating the protein is dispensable for mitotic divisions, but are sterile. Mutant oocytes appear normal until metaphase I but then display a highly penetrant failure to transition to anaphase I. They arrest with hallmarks of defective APC/C activation, including no separase activity and high MPF, cyclin B1, and securin levels. Partial APC/C activation occurs, however, as exogenously expressed APC/C substrates can be degraded and arrest can be suppressed by inhibiting MPF kinase. Cyclin B3 is itself targeted for degradation by the APC/C. Cyclin B3 forms active kinase complexes with CDK1, and meiotic progression requires cyclin B3-associated kinase activity. Collectively, our findings indicate that cyclin B3 is essential for oocyte meiosis because it fine-tunes APC/C activity as a kinase-activating CDK partner. Cyclin B3 homologs from frog, zebrafish, and fruitfly rescue meiotic progression in cyclin B3-deficient mouse oocytes, indicating conservation of the biochemical properties and possibly cellular functions of this germline-critical cyclin.

cell biology↗