Spindle migration

  • 文章类型: Journal Article
    Sirtuin5(Sirt5),Sirtuin家族的一员,参与各种细胞内生物过程。然而,Sirt5在卵母细胞成熟中的功能尚未明确阐明。在这项研究中,我们观察到Sirt5在小鼠卵母细胞减数分裂过程中持续表达,在衰老的卵母细胞中表达显着下降。Sirt5抑制导致第一极体挤压失败并诱导细胞周期停滞,表明卵母细胞成熟不成功。此外,Sirt5抑制与异常大的极体的挤压有关,提示不对称卵母细胞分裂被破坏。机械上,Sirt5的抑制导致卵母细胞中纺锤体组装异常和染色体排列紊乱。此外,Sirt5抑制导致纺锤体位于卵母细胞的中央而不迁移到皮质区域,从而防止肌动蛋白帽的形成。进一步的研究表明,Sirt5抑制显着降低了磷酸化的cofilin和profilin1的表达,同时增加了细胞质F-肌动蛋白水平。这些发现表明,卵母细胞成熟过程中的Sirt5抑制会对纺锤体组装和染色体排列产生不利影响,并破坏肌动蛋白动力学,从而损害纺锤体的迁移并导致对称卵母细胞分裂和成熟的失败。
    Sirtuin 5 (Sirt5), a member of the Sirtuin family, is involved in various intracellular biological processes. However, the function of Sirt5 in oocyte maturation has not been clearly elucidated. In this study, we observed that Sirt5 was persistently expressed during the meiotic division of mouse oocytes, with a notable decline in expression in aging oocytes. Sirt5 inhibition led to the failure of the first polar body extrusion and induced cell cycle arrest, indicative of unsuccessful oocyte maturation. Furthermore, Sirt5 inhibition was associated with the extrusion of abnormally large polar bodies, suggesting disrupted asymmetric oocyte division. Mechanistically, the inhibition of Sirt5 resulted in aberrant spindle assembly and disordered chromosome alignment in oocytes. Moreover, Sirt5 inhibition caused the spindle to be centrally located in the oocyte without migrating to the cortical region, consequently preventing the formation of the actin cap. Further investigation revealed that Sirt5 inhibition notably diminished the expression of phosphorylated cofilin and profilin1, while increasing cytoplasmic F-actin levels. These findings suggest that Sirt5 inhibition during oocyte maturation adversely affects spindle assembly and chromosome alignment and disrupts actin dynamics impairing spindle migration and contributing to the failure of symmetric oocyte division and maturation.
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  • 文章类型: Journal Article
    PLD1与体细胞中的细胞骨架重组和囊泡运输有关;然而,其在卵母细胞减数分裂中的功能尚不清楚。在这里,我们发现PLD1在小鼠卵母细胞减数分裂中稳定表达,与主轴直接相互作用,RAB11A+囊泡和大型自噬/自噬液泡。PLD1的遗传或化学抑制干扰了MTOC聚类,纺锤体组装及其皮质迁移,也减少了PtdIns(4,5)P2,磷酸化CFL1(p-CFL1[Ser3])和ACTR2,以及它们在MTOC上的局部分布,纺锤体和囊泡。此外,在PLD1抑制的卵母细胞中,囊泡大小显着减少,而F-肌动蛋白密度在细胞质中急剧增加,自噬空泡的不对称分布被打破,整个自噬过程大大增强,如自噬体的特征性变化所示,自溶酶体的形成和ATG5,BECN1,LC3-II的水平,SQSTM1和UB。外源性给予PtdIns(4,5)P2或过表达CFL1过度磷酸化突变体(CFL1S3E)可以显着改善PLD1耗竭卵母细胞的极性MTOC聚焦和纺锤体结构,而ACTR2的过表达不仅可以挽救MTOC聚集,和主轴组件,但也不对称定位。有趣的是,自噬激活导致纺锤体结构和定位类似缺陷;相反,它的抑制作用减轻了PLD1耗竭卵母细胞的改变,这在很大程度上归因于PtdIns(4,5)P2,ACTR2和p-CFL1(Ser3)的恢复水平。一起,PLD1促进卵母细胞减数分裂中的纺锤体组装和迁移,通过以调节自噬通量的方式维持ACTR2,PtdIns(4,5)P2和p-CFL1(Ser3)的合理水平。这项研究首次介绍了自噬活性和功能在卵母细胞减数分裂发育中的独特观点。缩写:ACTR2/ARP2:肌动蛋白相关蛋白2;ACTR3/ARP3:肌动蛋白相关蛋白3;ATG5:自噬相关5;Baf-A1:bafilomycinA1;BFA:brefeldinA;GAPDH:甘油醛-3-磷酸脱氢酶;GOLGA2/GM130:golginmeginamycinA2;GtulebRAS癌基因家族成员;RPS6KB1/S6K1:核糖体蛋白S6激酶B1;SQSTM1/p62:螯合体1;TEM:透射电子显微镜;TUBA/α-微管蛋白:微管蛋白α;TUBG/γ-微管蛋白:微管蛋白γ;UB:泛素;WASL/N-WASP:WASP样肌动蛋白成核促进因子。
    PLD1 has been implicated in cytoskeletal reorganization and vesicle trafficking in somatic cells; however, its function remains unclear in oocyte meiosis. Herein, we found PLD1 stably expresses in mouse oocytes meiosis, with direct interaction with spindle, RAB11A+ vesicles and macroautophagic/autophagic vacuoles. The genetic or chemical inhibition of PLD1 disturbed MTOC clustering, spindle assembly and its cortical migration, also decreased PtdIns(4,5)P2, phosphorylated CFL1 (p-CFL1 [Ser3]) and ACTR2, and their local distribution on MTOC, spindle and vesicles. Furthermore in PLD1-suppressed oocytes, vesicle size was significantly reduced while F-actin density was dramatically increased in the cytoplasm, the asymmetric distribution of autophagic vacuoles was broken and the whole autophagic process was substantially enhanced, as illustrated with characteristic changes in autophagosomes, autolysosome formation and levels of ATG5, BECN1, LC3-II, SQSTM1 and UB. Exogenous administration of PtdIns(4,5)P2 or overexpression of CFL1 hyperphosphorylation mutant (CFL1S3E) could significantly improve polar MTOC focusing and spindle structure in PLD1-depleted oocytes, whereas overexpression of ACTR2 could rescue not only MTOC clustering, and spindle assembly but also its asymmetric positioning. Interestingly, autophagy activation induced similar defects in spindle structure and positioning; instead, its inhibition alleviated the alterations in PLD1-depleted oocytes, and this was highly attributed to the restored levels of PtdIns(4,5)P2, ACTR2 and p-CFL1 (Ser3). Together, PLD1 promotes spindle assembly and migration in oocyte meiosis, by maintaining rational levels of ACTR2, PtdIns(4,5)P2 and p-CFL1 (Ser3) in a manner of modulating autophagy flux. This study for the first time introduces a unique perspective on autophagic activity and function in oocyte meiotic development.Abbreviations: ACTR2/ARP2: actin related protein 2; ACTR3/ARP3: actin related protein 3; ATG5: autophagy related 5; Baf-A1: bafilomycin A1; BFA: brefeldin A; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GV: germinal vesicle; GVBD: germinal vesicle breakdown; IVM: in vitro maturation; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MI: metaphase of meiosis I; MII: metaphase of meiosis II; MO: morpholino; MTOC: microtubule-organizing center; MTOR: mechanistic target of rapamycin kinase; PB1: first polar body; PLA: proximity ligation assay; PLD1: phospholipase D1; PtdIns(4,5)P2/PIP2: phosphatidylinositol 4,5-bisphosphate; RAB11A: RAB11A, member RAS oncogene family; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TUBA/α-tubulin: tubulin alpha; TUBG/γ-tubulin: tubulin gamma; UB: ubiquitin; WASL/N-WASP: WASP like actin nucleation promoting factor.
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  • 文章类型: Journal Article
    小鼠卵母细胞中的第一次不对称减数分裂细胞分裂是由纺锤体周围的形式蛋白2(FMN2)成核的肌动蛋白聚合驱动的。在这项研究中,我们研究了FMN2是如何被招募到纺锤体外周ER的,以及它的活性是如何在小鼠减数分裂I(MI)卵母细胞中被调节的。我们证明这个过程是由RanGTP酶调节的,染色质信号的保守介质,和ER相关蛋白VAPA。FMN2在先前显示为FMN2定位至纺锤体外周所需的结构域(SLD)内含有核定位序列(NLS)。FMN2NLS与导入蛋白α1/β复合物结合,RanGTP破坏这种相互作用是FMN2在染色质和MI纺锤体附近区域积累所必需的。然后通过SLD与ER-膜蛋白VAPA的结合,将不含导入蛋白的FMN2募集到纺锤体周围的ER表面。我们进一步表明,FMN2通过SLD与使肌动蛋白丝成核的C末端形式蛋白同源2(FH2)结构域之间的分子内相互作用而被自动抑制。与SLD结合的VAPA减轻了FMN2的自抑制,导致局部肌动蛋白聚合。这种对formin介导的肌动蛋白组装的双重控制允许肌动蛋白聚合启动减数分裂纺锤体向皮质的运动,哺乳动物雌配子成熟的重要步骤。
    The first asymmetric meiotic cell divisions in mouse oocytes are driven by formin 2 (FMN2)-nucleated actin polymerization around the spindle. In this study, we investigated how FMN2 is recruited to the spindle peripheral ER and how its activity is regulated in mouse meiosis I (MI) oocytes. We show that this process is regulated by the Ran GTPase, a conserved mediator of chromatin signal, and the ER-associated protein VAPA. FMN2 contains a nuclear localization sequence (NLS) within a domain (SLD) previously shown to be required for FMN2 localization to the spindle periphery. FMN2 NLS is bound to the importin α1/β complex, and the disruption of this interaction by RanGTP is required for FMN2 accumulation in the area proximal to the chromatin and the MI spindle. The importin-free FMN2 is then recruited to the surface of ER around the spindle through the binding of the SLD with the ER-membrane protein VAPA. We further show that FMN2 is autoinhibited through an intramolecular interaction between the SLD with the C-terminal formin homology 2 (FH2) domain that nucleates actin filaments. VAPA binding to SLD relieves the autoinhibition of FMN2, leading to localized actin polymerization. This dual control of formin-mediated actin assembly allows actin polymerization to initiate the movement of the meiotic spindle toward the cortex, an essential step in the maturation of the mammalian female gamete.
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  • 文章类型: Journal Article
    在女性减数分裂I(MI)期间,纺锤体定位必须严格调节,以确保第一次不对称分裂的保真度和忠实的染色体分离。尽管已经广泛研究了F-肌动蛋白在调节这些关键过程中的作用,对于微管(MT)是否参与调节这些过程知之甚少。使用小鼠卵母细胞作为模型系统,我们描述了不直接促进主轴装配的MT组织中心的子集,称为mcMTOC。使用激光烧蚀,STED超分辨率显微镜,和化学操纵,我们表明mcMTOC需要调节MI期间的纺锤体定位和忠实的染色体分离。我们讨论了mcMTOC成核的MT如何平衡F-肌动蛋白在主轴上施加的力,以集中锚定主轴并调节其及时迁移。我们的发现为不对称细胞分裂提供了一个模型,补充当前基于F-肌动蛋白的模型,并暗示mcMTOC是调节主轴定位的主要参与者。
    During female meiosis I (MI), spindle positioning must be tightly regulated to ensure the fidelity of the first asymmetric division and faithful chromosome segregation. Although the role of F-actin in regulating these critical processes has been studied extensively, little is known about whether microtubules (MTs) participate in regulating these processes. Using mouse oocytes as a model system, we characterize a subset of MT organizing centers that do not contribute directly to spindle assembly, termed mcMTOCs. Using laser ablation, STED super-resolution microscopy, and chemical manipulation, we show that mcMTOCs are required to regulate spindle positioning and faithful chromosome segregation during MI. We discuss how forces exerted by F-actin on the spindle are balanced by mcMTOC-nucleated MTs to anchor the spindle centrally and to regulate its timely migration. Our findings provide a model for asymmetric cell division, complementing the current F-actin-based models, and implicate mcMTOCs as a major player in regulating spindle positioning.
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  • 文章类型: Journal Article
    In female meiosis, oocyte meiotic maturation is a form of asymmetric cell division, producing the first polar body and a large oocyte, in which the asymmetry of oocyte meiotic division depends on spindle migration and positioning, and cortical polarization. In this study, we conclude that WDR62 (WD40-repeat protein 62) plays an important role in asymmetric meiotic division during mouse oocyte maturation. Our initial study demonstrated that WDR62 mainly co-localized with chromosomes during mouse oocyte meiotic maturation. Interference of Wdr62 by siRNA microinjection did not affect germinal vesicle breakdown (GVBD) but compromised the first polar body extrusion (PBE) with the large polar bodies generated, which is coupled with a higher incidence of spindle abnormality and chromosome misalignment. Further analysis concluded that loss of WDR62 blocked asymmetric spindle positioning and actin cap formation, which should be responsible for large polar body extrusion. Moreover, WDR62 decline intervened with the Arp2/3 complex, an upstream regulator for the cortical actin. Besides for p-MAPK, a critical regulator for the asymmetric division of oocyte, WDR62-depleted oocytes showed perturbation only in localization pattern but not expression level. In summary, our study defines WDR62 as an essential cytoskeletal regulator of spindle migration and asymmetric division during mouse oocyte meiotic maturation.
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  • 文章类型: Journal Article
    The inhibitor Y-27632 is a specific selective inhibitor of Rho-associated protein kinases (ROCKs), which are downstream effectors of Rho guanosine triphosphatease (GTPases) and regulate Rho-associated cellular functions, including actin cytoskeletal organization. Little is known regarding the effects of Y-27632 on mammalian oocyte maturation. In the present study, we investigated the effects of Y-27632 on porcine oocyte meiosis and possible regulatory mechanisms of ROCK during porcine oocyte maturation. We found that ROCK accumulated not only at spindles, but also at the cortex in porcine oocytes. Y-27632 treatment reduced ROCK expression, and inhibited porcine oocyte meiotic maturation, which might be because of the impairment of actin expression and actin-related spindle positioning. Y-27632 treatment also disrupted the formation of actin cap and cortical granule-free domain, which further confirmed a spindle positioning failure. Thus, Y-27632 has significant effects on the meiotic competence of mammalian oocytes by reducing ROCK expression, and the regulation is related to its effects on actin-mediated spindle positioning.
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  • 文章类型: Journal Article
    During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.
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