The cohesin stabilizer Sororin drives G2-M transition and spindle assembly in mammalian oocytes.
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ABSTRACT:
SUBMITTER: Zhou C
PROVIDER: S-EPMC8462903 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mammalian oocytes segregate chromosomes with a microtubule spindle that lacks centrosomes, but the mechanisms by which acentrosomal spindles are organized and function are largely unclear. In this study, we identify a conserved subcellular structure in mammalian oocytes that forms by phase separation. This structure, which we term the liquid-like meiotic spindle domain (LISD), permeates the spindle poles and forms dynamic protrusions that extend well beyond the spindle. The LISD selectively concentrates multiple microtubule regulatory factors and allows them to diffuse rapidly within the spindle volume. Disruption of the LISD via different means disperses these factors and leads to severe spindle assembly defects. Our data suggest a model whereby the LISD promotes meiotic spindle assembly by serving as a reservoir that sequesters and mobilizes microtubule regulatory factors in proximity to spindle microtubules.
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Project description:Patterned cell divisions require a precisely oriented spindle that segregates chromosomes and determines the cytokinetic plane. In this study, we investigated how the meiotic spindle orients through an obligatory rotation during meiotic division in mouse oocytes. We show that spindle rotation occurs at the completion of chromosome segregation, whereby the separated chromosome clusters each define a cortical actomyosin domain that produces cytoplasmic streaming, resulting in hydrodynamic forces on the spindle. These forces are initially balanced but become unbalanced to drive spindle rotation. This force imbalance is associated with spontaneous symmetry breaking in the distribution of the Arp2/3 complex and myosin-II on the cortex, brought about by feedback loops comprising Ran guanosine triphosphatase signaling, Arp2/3 complex activity, and myosin-II contractility. The torque produced by the unbalanced hydrodynamic forces, coupled with a pivot point at the spindle midzone cortical contract, constitutes a unique mechanical system for meiotic spindle rotation.
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Project description:Since the dissolution of sister chromatid cohesion by separase and cyclin B destruction is irreversible, it is essential to delay both until all chromosomes have bioriented on the mitotic spindle. Kinetochores that are not correctly attached to the spindle generate the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex/cyclosome (APC/C) and blocks anaphase onset. This process is known as the spindle assembly checkpoint (SAC). The SAC is especially important in meiosis I, where bivalents consisting of homologous chromosomes held together by chiasmata biorient. Since the first meiotic division is unaffected by rare achiasmatic chromosomes or misaligned bivalents, it is thought that several tensionless kinetochores are required to produce sufficient MCC for APC/C inhibition. Consistent with this, univalents lacking chiasmata elicit a SAC-mediated arrest in Mlh1(-/-) oocytes. In contrast, chromatids generated by TEV protease-induced cohesin cleavage in Rec8(TEV/TEV) oocytes merely delay APC/C activation. Since the arrest of Mlh1(-/-)Rec8(TEV/TEV) oocytes is alleviated by TEV protease, even when targeted to kinetochores, we conclude that their SAC depends on cohesin as well as dedicated kinetochore proteins. This has important implications for aging oocytes, where cohesin deterioration will induce sister kinetochore biorientation and compromise MCC production, leading to chromosome missegregation and aneuploid fetuses.
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Project description:In both mitosis and meiosis, metaphase to anaphase transition requires the activity of a ubiquitin ligase known as anaphase promoting complex/cyclosome (APC/C). The activation of APC/C in metaphase is under the control of the checkpoint mechanism, called the spindle assembly checkpoint (SAC), which monitors the correct attachment of all kinetochores to the spindle. It has been shown previously in somatic cells that exposure to a small molecule inhibitor, prodrug tosyl-l-arginine methyl ester (proTAME), resulted in cell cycle arrest in metaphase, with low APC/C activity. Interestingly, some reports have also suggested that the activity of SAC is required for this arrest. We focused on the characterization of proTAME inhibition of cell cycle progression in mammalian oocytes and embryos. Our results show that mammalian oocytes and early cleavage embryos show dose-dependent metaphase arrest after exposure to proTAME. However, in comparison to the somatic cells, we show here that the proTAME-induced arrest in these cells does not require SAC activity. Our results revealed important differences between mammalian oocytes and early embryos and somatic cells in their requirements of SAC for APC/C inhibition. In comparison to the somatic cells, oocytes and embryos show much higher frequency of aneuploidy. Our results are therefore important for understanding chromosome segregation control mechanisms, which might contribute to the premature termination of development or severe developmental and mental disorders of newborns.
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Project description:Oocyte meiosis is a transcription quiescence process and the cell-cycle progression is coordinated by multiple post-translational modifications, including SUMOylation. SENP3 an important deSUMOylation protease has been intensively studied in ribosome biogenesis and oxidative stress. However, the roles of SENP3 in cell-cycle regulation remain enigmatic, particularly for oocyte meiotic maturation. Here, we found that SENP3 co-localized with spindles during oocyte meiosis and silencing of SENP3 severely compromised the M phase entry (germinal vesicle breakdown, GVBD) and first polar body extrusion (PBI). The failure in polar body extrusion was due to the dysfunction of γ-tubulin that caused defective spindle morphogenesis. SENP3 depletion led to mislocalization and a substantial loss of Aurora A (an essential protein for MTOCs localization and spindle dynamics) while irregularly dispersed distribution of Bora (a binding partner and activator of Aurora A) in cytoplasm instead of concentrating at spindles. The SUMO-2/3 but not SUMO-1 conjugates were globally decreased by SENP3 RNAi. Additionally, the spindle assembly checkpoint remained functional upon SENP3 RNAi. Our findings renew the picture of SENP3 function by exploring its role in meiosis resumption, spindle assembly and following polar body emission during mouse oocyte meiotic maturation, which is potentially due to its proteolytic activity that facilitate SUMO-2/3 maturation.
| S-EPMC4616058 | biostudies-literature
Project description:The transition from meiosis to mitosis, classically defined by fertilization, is a fundamental process in development. However, its mechanism remains largely unexplored. In this paper, we report a surprising gradual transition from meiosis to mitosis over the first eight divisions of the mouse embryo. The first cleavages still largely share the mechanism of spindle formation with meiosis, during which the spindle is self-assembled from randomly distributed microtubule-organizing centers (MTOCs) without centrioles, because of the concerted activity of dynein and kinesin-5. During preimplantation development, the number of cellular MTOCs progressively decreased, the spindle pole gradually became more focused, and spindle length progressively scaled down with cell size. The typical mitotic spindle with centrin-, odf2-, kinesin-12-, and CP110-positive centrosomes was established only in the blastocyst. Overall, the transition from meiosis to mitosis progresses gradually throughout the preimplantation stage in the mouse embryo, thus providing a unique system to study the mechanism of centrosome biogenesis in vivo.
| S-EPMC3413348 | biostudies-literature
Project description:Mammalian oocytes assemble a bipolar acentriolar microtubule spindle to segregate chromosomes during asymmetric division. There is increasing evidence that actin in the spindle interior not only participates in spindle migration and positioning but also protects oocytes from chromosome segregation errors leading to aneuploidy. Here we show that actin is an integral component of the meiotic machinery that closely interacts with microtubules during all major events of human oocyte maturation from the time point of spindle assembly till polar body extrusion and metaphase arrest. With the aid of drugs selectively affecting cytoskeleton dynamics and transiently disturbing the integrity of the two cytoskeleton systems, we identify interdependent structural rearrangements indicative of a close communication between actin and microtubules as fundamental feature of human oocytes. Our data support a model of actin-microtubule interplay that is essential for bipolar spindle assembly and correct partitioning of the nuclear genome in human oocyte meiosis.
| S-EPMC6789129 | biostudies-literature
Project description:BackgroundAnastral spindles assemble by a mechanism that involves microtubule nucleation and growth from chromatin. It is still uncertain whether γ-tubulin, a microtubule nucleator essential for mitotic spindle assembly and maintenance, plays a role. Not only is the requirement for γ-tubulin to form anastral Drosophila oocyte meiosis I spindles controversial, but its presence in oocyte meiosis I spindles has not been demonstrated and is uncertain.ResultsWe show, for the first time, using a bright GFP fusion protein and live imaging, that the Drosophila maternally-expressed γTub37C is present at low levels in oocyte meiosis I spindles. Despite this, we find that formation of bipolar meiosis I spindles does not require functional γTub37C, extending previous findings by others. Fluorescence photobleaching assays show rapid recovery of γTub37C in the meiosis I spindle, similar to the cytoplasm, indicating weak binding by γTub37C to spindles, and fits of a new, potentially more accurate model for fluorescence recovery yield kinetic parameters consistent with transient, diffusional binding.ConclusionsThe FRAP results, together with its mutant effects late in meiosis I, indicate that γTub37C may perform a role subsequent to metaphase I, rather than nucleating microtubules for meiosis I spindle formation. Weak binding to the meiosis I spindle could stabilize pre-existing microtubules or position γ-tubulin for function during meiosis II spindle assembly, which follows rapidly upon oocyte activation and completion of the meiosis I division.
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Project description:Establishing the bipolar spindle in mammalian oocytes after their prolonged arrest is crucial for meiotic fidelity and subsequent development. In contrast to somatic cells, the first meiotic spindle assembles in the absence of centriole-containing centrosomes. Ran-GTP can promote microtubule nucleation near chromatin, but additional unidentified factors are postulated for the activity of multiple acentriolar microtubule organizing centers in the oocyte. We now demonstrate that partially overlapping, nonredundant functions of Aurora A and Plk4 kinases contribute to initiate acentriolar meiosis I spindle formation. Loss of microtubule nucleation after simultaneous chemical inhibition of both kinases can be significantly rescued by drug-resistant Aurora A alone. Drug-resistant Plk4 can enhance Aurora A-mediated rescue, and, accordingly, Plk4 can phosphorylate and potentiate the activity of Aurora A in vitro. Both kinases function distinctly from Ran, which amplifies microtubule growth. We conclude that Aurora A and Plk4 are rate-limiting factors contributing to microtubule growth as the acentriolar oocyte resumes meiosis.
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Project description:During the maturation of Xenopus oocytes, Cdc6 expression is necessary to establish replication competence to support early embryonic DNA replication. However, Cdc6 is expressed before the completion of MI, at a time when its function as a replication factor is not required, suggesting additional roles for Cdc6 in meiosis. Confocal immunofluorescence microscopy revealed that Cdc6 protein was distributed around the spindle precursor at the time of germinal vesicle breakdown (GVBD), and localized to the margin of the nascent spindle early in prometaphase. Cdc6 subsequently localized to spindle poles in late prometaphase, where it remained until metaphase arrest. Microinjection of antisense oligonucleotides specific for Cdc6 mRNA disrupted spindle assembly, resulting in defects including delayed spindle assembly, misoriented and unattached anaphase spindles, monasters, multiple spindles, microtubule aggregates associated with condensed chromosomes, or the absence of recognizable spindle-like structures, depending on the level of residual Cdc6 expression. Furthermore, Cdc6 co-localized with γ-tubulin in centrosomes during interphase in all somatic cells analyzed, and associated with spindle poles in mitotic COS cells. Our data suggest a role for Cdc6 in spindle formation in addition to its role as a DNA replication factor.
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