Multiple Requirements of PLK1 during Mouse Oocyte Maturation

Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1’s functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.     

MLL5 maintains spindle bipolarity by preventing aberrant cytosolic aggregation of PLK1

Faithful chromosome segregation with bipolar spindle formation is critical for the maintenance of genomic stability. Perturbation of this process often leads to severe mitotic failure, contributing to tumorigenesis. MLL5 has been demonstrated to play vital roles in cell cycle progression and the maintenance of genomic stability. Here, we identify a novel interaction between MLL5 and PLK1 in the cytosol that is crucial for sustaining spindle bipolarity during mitosis. Knockdown of MLL5 caused aberrant PLK1 aggregation that led to acentrosomal microtubule-organizing center (aMTOC) formation and subsequent spindle multipolarity. Further molecular studies revealed that the polo-box domain (PBD) of PLK1 interacted with a binding motif on MLL5 (Thr887-Ser888-Thr889), and this interaction was essential for spindle bipolarity. Overexpression of wild-type MLL5 was able to rescue PLK1 mislocalization and aMTOC formation in MLL5-KD cells, whereas MLL5 mutants incapable of interacting with the PBD failed to do so. We thus propose that MLL5 preserves spindle bipolarity through maintaining cytosolic PLK1 in a nonaggregated form.     

WAVE2 regulates meiotic spindle stability,peripheral positioning and polar body emission in mouse oocytes

During oocyte meiotic maturation, meiotic spindles form in the central cytoplasm and then migrate to the cortex to extrude a small polar body, forming a highly polarized cell through a process involving actin and actin-related molecules. The mechanisms underlying oocyte polarization are still unclear. The Arp2/3 complex regulates oocyte polarization but it is not known whether the WASP family of proteins, a known regulator of the Arp2/3 complex, is involved in this context. In the present study, the role of WASP family member WAVE2 in mouse oocyte asymmetric division was investigated. (1) WAVE2 mRNA and protein were detected during mouse oocyte meiosis. (2) siRNA-mediated and antibody-mediated disruption of WAVE2 resulted in the failure of chromosome congression, spindle formation, spindle positioning and polar body extrusion. (3) WAVE2 regulated actin-driven chromosome migration since chromosomes were arrested in the central cytoplasm by WAVE2 RNAi in the absence of microtubules. (4) Localization of γ-tubulin and MAPK was disrupted after RNAi, confirming the effect of WAVE2 on spindle formation. (5) Actin cap and cortical granule-free domain (CGFD) formation was also disrupted, further confirming the failure of oocyte polarization. Our data suggest that WAVE2 regulates oocyte polarization by regulating meiotic spindle, peripheral positioning, probably via an actin-mediated pathway, and is involved in polar body emission during mouse oocyte meiotic maturation.     

Mps1p regulates meiotic spindle pole body duplication in addition to having novel roles during sporulation

Sporulation in yeast requires that a modified form of chromosome segregation be coupled to the development of a specialized cell type, a process akin to gametogenesis. Mps1p is a dual-specificity protein kinase essential for spindle pole body (SPB) duplication and required for the spindle assembly checkpoint in mitotically dividing cells. Four conditional mutant alleles of MPS1 disrupt sporulation, producing two distinct phenotypic classes. Class I alleles of mps1 prevent SPB duplication at the restrictive temperature without affecting premeiotic DNA synthesis and recombination. Class II MPS1 alleles progress through both meiotic divisions in 30-50% of the population, but the asci are incapable of forming mature spores. Although mutations in many other genes block spore wall formation, the cells produce viable haploid progeny, whereas mps1 class II spores are unable to germinate. We have used fluorescently marked chromosomes to demonstrate that mps1 mutant cells have a dramatically increased frequency of chromosome missegregation, suggesting that loss of viability is due to a defect in spindle function. Overall, our cytological data suggest that MPS1 is required for meiotic SPB duplication, chromosome segregation, and spore wall formation.     

Arp2/3 complex regulates asymmetric division and cytokinesis in mouse oocytes

Mammalian oocyte meiotic maturation involves oocyte polarization and a unique asymmetric division, but until now, the underlying mechanisms have been poorly understood. Arp2/3 complex has been shown to regulate actin nucleation and is widely involved in a diverse range of processes such as cell locomotion, phagocytosis and the establishment of cell polarity. Whether Arp2/3 complex participates in oocyte polarization and asymmetric division is unknown. The present study investigated the expression and functions of Arp2/3 complex during mouse oocyte meiotic maturation. Immunofluorescent staining showed that the Arp2/3 complex was restricted to the cortex, with a thickened cap above the meiotic apparatus, and that this localization pattern was depended on actin. Disruption of Arp2/3 complex by a newly-found specific inhibitor CK666, as well as by Arpc2 and Arpc3 RNAi, resulted in a range of effects. These included the failure of asymmetric division, spindle migration, and the formation and completion of oocyte cytokinesis. The formation of the actin cap and cortical granule-free domain (CGFD) was also disrupted, which further confirmed the disruption of spindle migration. Our data suggest that the Arp2/3 complex probably regulates oocyte polarization through its effect on spindle migration, asymmetric division and cytokinesis during mouse oocyte meiotic maturation.     

Asymmetric division in mouse oocytes: with or without Mos

In both vertebrates and invertebrates, meiotic divisions in oocytes are typically asymmetric, resulting in the formation of a large oocyte and small polar bodies. The size difference between the daughter cells is usually a consequence of asymmetric positioning of the spindle before cytokinesis. Spindle movements are often related to interactions between the cell cortex and the spindle asters [1,2]. The spindles of mammalian oocytes are, however, typically devoid of astral microtubules, which normally connect the spindle to the cortex, suggesting that another mechanism is responsible for the unequal divisions in these oocytes. We observed the formation of the first polar body in wild-type oocytes and oocytes derived from c-Mos knockout mice [3]. In wild-type oocytes, the meiotic spindle formed in the centre of the cell and migrated to the cortex just before polar-body extrusion. The spindle did not elongate during anaphase. In mos-/- oocytes, the spindle formed centrally but did not migrate, although an asymmetric division still took place. In these oocytes, the spindle elongated during anaphase and the pole closest to the cortex moved while the other remained in place. Thus, a compensation mechanism exists in mouse oocytes and formation of the first polar body can be achieved in two ways: either after migration of the spindle to the cortex in wild-type oocytes, or after elongation, without migration, of the first meiotic spindle in mos-/- oocytes.     

Mechanisms that prevent catastrophic interactions between paternal chromosomes and the oocyte meiotic spindle

Meiosis produces haploid gametes by accurately reducing chromosome ploidy through one round of DNA replication and two subsequent rounds of chromosome segregation and cell division. The cell divisions of female meiosis are highly asymmetric and give rise to a large egg and two very small polar bodies that do not contribute to development. These asymmetric divisions are driven by meiotic spindles that are small relative to the size of the egg and have one pole juxtaposed against the cell cortex to promote polar body extrusion. An additional unique feature of female meiosis is that fertilization occurs before extrusion of the second polar body in nearly all animal species. Thus sperm-derived chromosomes are present in the egg during female meiosis. Here, we explore the idea that the asymmetry of female meiosis spatially separates the sperm from the meiotic spindle to prevent detrimental interactions between the spindle and the paternal chromosomes.     

Initial diameter of the polar body contractile ring is minimized by the centralspindlin complex

Polar body formation is an essential step in forming haploid eggs from diploid oocytes. This process involves completion of a highly asymmetric cytokinesis that results in a large egg and two small polar bodies. Unlike mitotic contractile rings, polar body contractile rings assemble over one spindle pole so that the spindle must move through the contractile ring before cytokinesis. During time-lapse imaging of C. elegans meiosis, the contractile ring moved downward along the length of the spindle and completed scission at the midpoint of the spindle, even when spindle length or rate of ring movement was increased. Patches of myosin heavy chain and dynamic furrowing of the plasma membrane over the entire embryo suggested that global cortical contraction forces the meiotic spindle and overlying membrane out through the contractile ring center. Consistent with this model, depletion of myosin phosphatase increased the velocity of ring movement along the length of the spindle. Global dynamic furrowing, which was restricted to anaphase I and II, was dependent on myosin II, the anaphase promoting complex and separase, but did not require cortical contact by the spindle. Large cortical patches of myosin during metaphase I and II indicated that myosin was already in the active form before activation of separase. To identify the signal at the midpoint of the anaphase spindle that induces scission, we depleted two proteins that mark the exact midpoint of the spindle during late anaphase, CYK-4 and ZEN-4. Depletion of either protein resulted in the unexpected phenotype of initial ingression of a polar body ring with twice the diameter of wild type. This phenotype revealed a novel mechanism for minimizing polar body size. Proteins at the spindle midpoint are required for initial ring ingression to occur close to the membrane-proximal spindle pole.     

Chromosomal influence on meiotic spindle assembly: abnormal meiosis I in female Mlh1 mutant mice.

In mouse oocytes, the first meiotic spindle is formed through the action of multiple microtubule organizing centers rather than a pair of centrosomes. Although the chromosomes are thought to play a major role in organizing the meiotic spindle, it remains unclear how a stable bipolar spindle is established. We have studied the formation of the first meiotic spindle in murine oocytes from mice homozygous for a targeted disruption of the DNA mismatch repair gene, Mlh1. In the absence of the MLH1 protein meiotic recombination is dramatically reduced and, as a result, the vast majority of chromosomes are present as unpaired univalents at the first meiotic division. The orientation of these univalent chromosomes at prometaphase suggests that they are unable to establish stable bipolar spindle attachments, presumably due to the inability to differentiate functional kinetochore domains on individual sister chromatids. In the presence of this aberrant chromosome behavior a stable first meiotic spindle is not formed, the spindle poles continue to elongate, and the vast majority of cells never initiate anaphase. These results suggest that, in female meiotic systems in which spindle formation is based on the action of multiple microtubule organizing centers, the chromosomes not only promote microtubule polymerization and organization but their attachment to opposite spindle poles acts to stabilize the forming spindle poles.     

A single internal telomere tract ensures meiotic spindle formation

Contact between telomeres and the fission yeast spindle pole body during meiotic prophase is crucial for subsequent spindle assembly, but the feature of telomeres that confers their ability to promote spindle formation remains mysterious. Here we show that while strains harbouring circular chromosomes devoid of telomere repeat tracts undergo aberrant meiosis with defective spindles, the insertion of a single internal telomere repeat stretch rescues the spindle defects. Moreover, the telomeric overhang‐binding protein Pot1 is dispensable for rescue of spindle formation. Hence, an inherent feature of the double‐strand telomeric region endows telomeres with the capacity to promote spindle formation.     

Symmetry breaking in mouse oocytes requires transient F-actin meshwork destabilization

Female meiotic divisions are extremely asymmetric, giving rise to a large oocyte and small degenerating polar bodies, keeping the maternal stores for further embryo development. This asymmetry is achieved via off-center positioning of the division spindle. Mouse oocytes have developed a formin-2-dependent actin-based spindle positioning mechanism that allows the meiotic spindle to migrate towards the closest cortex. Using spinning disk microscopy and FRAP analysis, we studied the changes in the organization of the cytoplasmic F-actin meshwork during the first meiotic division. It is very dense in prophase I, undergoes a significant density drop upon meiosis resumption and reforms progressively later on. This meshwork remodeling correlates with endogenous formin 2 regulation. High formin 2 levels at meiosis I entry induce meshwork maintenance, leading to equal forces being exerted on the chromosomes, preventing spindle migration. Hence, the meshwork density drop at meiosis resumption is germane to the symmetry-breaking event required for successful asymmetric meiotic divisions.     

Quantitative phospho-proteomics to investigate the polo-like kinase 1-dependent phospho-proteome

Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides via methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment via immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by PLK1. Analysis of amino acid sequence motifs among phosphorylation sites down-regulated under PLK1 inhibition in this data set identified two potential novel variants of the PLK1 consensus motif.     

Development of mouse embryos derived from oocytes reconstructed by metaphase I spindle transfer

Abnormal oocyte spindle is frequently associated with the infertility of aged women. Directly manipulating the metaphase I (MI) spindle may be a feasible method to overcome this kind of problem. Here, we report that the MI meiotic spindle can be removed from MI mouse oocytes and will autonomously divide into two daughter cells with the same size, morphology and an equal number of chromosomes after culture for 5 h in maturation medium. The division rate of the MI spindle reached 56% after 10-15 h of culture. After transferring the MI meiotic spindle into synchronous ooplasm by electrofusion, about 61% of the reconstructed oocytes continued to complete the first meiosis and extruded a normal first polar body. The matured reconstructed oocytes can also be fertilised. Approximately 50% of the 2-cell embryos developed to the morula stage after in vitro culture.     

Centrobin/NIP2 Is a Microtubule Stabilizer Whose Activity Is Enhanced by PLK1 Phosphorylation during Mitosis

Centrobin/NIP2 is a centrosomal protein that is required for centrosome duplication. It is also critical for microtubule organization in both interphase and mitotic cells. In the present study, we observed that centrobin is phosphorylated in a cell cycle stage-specific manner, reaching its maximum at M phase. PLK1 is a kinase that is responsible for M phase-specific phosphorylation of centrobin. The microtubule forming activity of centrobin was enhanced by PLK1 phosphorylation. Furthermore, mitotic spindles were not assembled properly with the phospho-resistant mutant of centrobin. Based on these results, we propose that centrobin functions as a microtubule stabilizing factor and PLK1 enhances centrobin activity for proper spindle formation during mitosis.     

Small Molecule Inhibitor of Formin Homology 2 Domains (SMIFH2) Reveals the Roles of the Formin Family of Proteins in Spindle Assembly and Asymmetric Division in Mouse Oocytes

Dynamic actin reorganization is the main driving force for spindle migration and asymmetric cell division in mammalian oocytes. It has been reported that various actin nucleators including Formin-2 are involved in the polarization of the spindle and in asymmetric cell division. In mammals, the formin family is comprised of 15 proteins. However, their individual roles in spindle migration and/or asymmetric division have not been elucidated yet. In this study, we employed a newly developed inhibitor for formin family proteins, small molecule inhibitor of formin homology 2 domains (SMIFH2), to assess the functions of the formin family in mouse oocyte maturation. Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels. In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation. Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells. Collectively, these results suggested that besides Formin-2, the other proteins of the formin, including mDia family play a role in asymmetric division and meiotic spindle formation in mammalian oocytes.     

Distribution and association of mTOR with its cofactors,raptor and rictor,in cumulus cells and oocytes during meiotic maturation in mice

Mammalian target of rapamycin (mTOR), a Ser/Thr protein kinase, is the catalytic component of two distinct signaling complexes, mTOR‐raptor complex (mTORC1) and mTOR‐rictor complex (mTORC2). Recently, studies have demonstrated mitosis‐specific roles for mTORC1, but the functions and expression dynamics of mTOR complexes during meiotic maturation remain unclear. In the present study, to evaluate the roles of respective mTOR complexes in maternal meiosis and compare them with those in mitosis, we sought to elucidate the spatiotemporal immunolocalization of mTOR, the kinase‐active Ser2448‐ and Ser2481‐phosphorylated mTOR, and raptor and rictor during cumulus‐cell mitosis and oocyte meiotic maturation in mice. mTOR principally accumulated around the chromosomes and on the spindle. Phosphorylated mTOR (Ser2448 and Ser2481) exhibited elevated fluorescence intensities in the cytoplasm and punctate localization adjacent to the chromosomes, on the spindle poles, and on the midbody during mitotic and meiotic maturation, suggesting functional homology of mTOR between the two cell division systems, despite their mechanistically distinctive spindles. Raptor colocalized with mTOR during both types of cell division, indicating that mTORC1 is predominantly associated with these events. Mitotic rictor uniformly distributed through the cytoplasm, and meiotic rictor localized around the spindle poles of metaphase‐I oocytes, suggesting functional divergence of mTORC2 between mitosis and female meiosis. Based on the general function of mTORC2 in the organization of the actin cytoskeleton, we propose that mTORC1 controls spindle function during mitosis and meiosis, while mTORC2 contributes to actin‐dependent asymmetric division during meiotic maturation in mice. Mol. Reprod. Dev. 80: 334–348, 2013. © 2013 Wiley Periodicals, Inc.     

Drosophila male meiosis as a model system for the study of cytokinesis in animal cells

Drosophila male meiosis offers unique opportunities for mutational dissection of cytokinesis. This system allows easy and unambiguos identification of mutants defective in cytokinesis through the examination of spermatid morphology. Moreover, cytokinesis defects and protein immunostaining can be analyzed with exquisite cytological resolution because of the large size of meiotic spindles. In the past few years several mutations have been isolated that disrupt meiotic cytokinesis in Drosophila males. These mutations specify genes required for the assembly, proper constriction or disassembly of the contractile ring. Molecular characterization of these genes has identified essential components of the cytokinetic machinery, highlighting the role of the central spindle during cytokinesis. This structure appears to be both necessary and sufficient for signaling cytokinesis. In addition, many data indicate that the central spindle microtubules cooperatively interact with elements of the actomyosin contractile ring, so that impairment of either of these structures prevents the formation of the other.     

The kinesinlike protein Subito contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes

In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila melanogaster oocyte chromosomes have a major role in nucleating microtubules, which precedes the bundling and assembly of these microtubules into a bipolar spindle. Here we present evidence that a region similar to the anaphase central spindle functions to organize acentrosomal spindles. Subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. Subito encodes a kinesinlike protein and associates with the meiotic central spindle, consistent with its classification in the Kinesin 6/MKLP1 family. This class of proteins is known to be required for cytokinesis, but our results suggest a new function in spindle formation. The meiotic central spindle appears during prometaphase and includes passenger complex proteins such as AurB and Incenp. Unlike mitotic cells, the passenger proteins do not associate with centromeres before anaphase. In the absence of Subito, central spindle formation is defective and AurB and Incenp fail to properly localize. We propose that Subito is required for establishing and/or maintaining the central spindle in Drosophila oocytes, and this substitutes for the role of centrosomes in organizing the bipolar spindle.     

Meiosis in a temperature-sensitive DNA-synthesis mutant and in an apomictic yeast strain (Saccharomyces cerevisiae).

It is shown that in the temperature-sensitive yeast mutant (Saccharomyces cerevisiae) spo 11 at the restrictive temperature of 34 degrees C. (1) premeiotic DNA synthesis is nearly completely blocked; (2) the nucleus enters meiotic prophase indicated by the formation of axial cores and polysynaptonemal complexes; (3) the kinetic apparatus functions normally at meiosis I and II; (4) early spore formation occurs in nearly all cells but it is variable and all spores eventually degenerate. It is concluded that chromosome replication is not a prerequisite for the functions listed above. The apomictic yeast strain 4117 produces 2 diploid spores. It is shown that a diploid which produces 2-spored asci, synthesized from 4117, no. 5, and an adenine requiring strain (1) has a normal meiotic prophase with abundant synaptonemal complexes; (2) has only one meiotic spindle; (3) has spores which form red clones more frequently than normal or u.v.-treated vegetative cells form ade/ade red sectors through mitotic recombination. It is concluded that this apomictic yeast has maintained meiotic prophase, but that one of the two meiotic divisions is suppressed.