Showing papers on "Metaphase published in 2013"
TL;DR: Using polymer simulations, it is found that metaphase Hi-C data are inconsistent with classic hierarchical models and are instead best described by a linearly organized longitudinally compressed array of consecutive chromatin loops.
Abstract: Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two distinct three-dimensional folding states of the human genome. We show that the highly compartmentalized and cell type–specific organization described previously for nonsynchronous cells is restricted to interphase. In metaphase, we identified a homogenous folding state that is locus-independent, common to all chromosomes, and consistent among cell types, suggesting a general principle of metaphase chromosome organization. Using polymer simulations, we found that metaphase Hi-C data are inconsistent with classic hierarchical models and are instead best described by a linearly organized longitudinally compressed array of consecutive chromatin loops.
873 citations
01 Nov 2013
TL;DR: In this article, the authors applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two distinct three-dimensional folding states of the human genome.
Abstract: Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two distinct three-dimensional folding states of the human genome. We show that the highly compartmentalized and cell type–specific organization described previously for nonsynchronous cells is restricted to interphase. In metaphase, we identified a homogenous folding state that is locus-independent, common to all chromosomes, and consistent among cell types, suggesting a general principle of metaphase chromosome organization. Using polymer simulations, we found that metaphase Hi-C data are inconsistent with classic hierarchical models and are instead best described by a linearly organized longitudinally compressed array of consecutive chromatin loops.
753 citations
TL;DR: It is shown that cyclin-dependent kinase (CDK)-mediated, mitosis-specific phosphorylation of SGO1 activates its cohesion-protection function and enables its direct binding to cohesin, and that PP2A-orchestrated, site-selective dephosphorylated of cohes in and its regulators underlies centromeric cohesion protection.
Abstract: Accurate chromosome segregation requires that sister-chromatid cohesion is resolved first at chromosome arms in prophase and then at centromeres in metaphase. In prophase, centromeric cohesion is protected by shugoshin and protein phosphase 2A (SGO1–PP2A). Yu and colleagues show that CDK1-mediated phosphorylation of SGO1 promotes SGO1–PP2A binding to cohesin, and dephosphorylation of the cohesion-promoting component sororin to prevent cohesin removal.
170 citations
TL;DR: This study defines when cells irreversibly commit to enter anaphase and shows that the SAC signal strength correlates with the number of unattached chromosomes.
Abstract: The mitotic spindle assembly checkpoint (SAC) delays anaphase onset until all chromosomes have attached to both spindle poles. Here, we investigated SAC signalling kinetics in response to acute detachment of individual chromosomes using laser microsurgery. Most detached chromosomes delayed anaphase until they had realigned to the metaphase plate. A substantial fraction of cells, however, entered anaphase in the presence of unaligned chromosomes. We identify two mechanisms by which cells can bypass the SAC: first, single unattached chromosomes inhibit the anaphase-promoting complex/cyclosome (APC/C) less efficiently than a full complement of unattached chromosomes; second, because of the relatively slow kinetics of re-imposing APC/C inhibition during metaphase, cells were unresponsive to chromosome detachment up to several minutes before anaphase onset. Our study defines when cells irreversibly commit to enter anaphase and shows that the SAC signal strength correlates with the number of unattached chromosomes. Detailed knowledge about SAC signalling kinetics is important for understanding the emergence of aneuploidy and the response of cancer cells to chemotherapeutics targeting the mitotic spindle.
167 citations
TL;DR: It is shown using in vitro reconstitution assays that PRC1 and kinesin-4, two microtubule-associated proteins required for midzone assembly, can tag microtubULE plus ends, and how biochemically similar microtubules can be differentially marked for selective regulation during the formation of specialized arrays, such as those required for cytokinesis is suggested.
136 citations
TL;DR: The mitotic kinases Aurora B and Cyclin-dependent kinase 1 (Cdk1) destabilize interactions between Sororin and the cohesin subunit precocious dissociation of sisters protein 5 by phosphorylating sororin, leading to release of acetylated cohes in from chromosome arms and loss of cohesion.
Abstract: Sister chromatid cohesion depends on Sororin, a protein that stabilizes acetylated cohesin complexes on DNA by antagonizing the cohesin release factor Wings-apart like protein (Wapl). Cohesion is essential for chromosome biorientation but has to be dissolved to enable sister chromatid separation. To achieve this, the majority of cohesin is removed from chromosome arms in prophase and prometaphase in a manner that depends on Wapl and phosphorylation of cohesin’s subunit stromal antigen 2 (SA2), whereas centromeric cohesin is cleaved in metaphase by the protease separase. Here we show that the mitotic kinases Aurora B and Cyclin-dependent kinase 1 (Cdk1) destabilize interactions between Sororin and the cohesin subunit precocious dissociation of sisters protein 5 (Pds5) by phosphorylating Sororin, leading to release of acetylated cohesin from chromosome arms and loss of cohesion. At centromeres, the cohesin protector shugoshin (Sgo1)-protein phosphatase 2A (PP2A) antagonizes Aurora B and Cdk1 partly by dephosphorylating Sororin and thus maintains cohesion until metaphase. We propose that the stepwise loss of cohesion between chromosome arms and centromeres is caused by local regulation of Wapl activity, which is controlled by the phosphorylation state of Sororin.
134 citations
TL;DR: Stability of k–MT attachments increases decisively in a coordinated fashion among all chromosomes as cells transit from prometaphase to metaphase, and Cyclin A creates a cellular environment that promotes microtubule detachment from kinetochores in prometAPHase to ensure efficient error correction and faithful chromosome segregation.
Abstract: The most conspicuous event in the cell cycle is the alignment of chromosomes in metaphase. Chromosome alignment fosters faithful segregation through the formation of bi-oriented attachments of kinetochores to spindle microtubules. Notably, numerous kinetochore-microtubule (k-MT) attachment errors are present in early mitosis (prometaphase), and the persistence of those errors is the leading cause of chromosome mis-segregation in aneuploid human tumour cells that continually mis-segregate whole chromosomes and display chromosomal instability. How robust error correction is achieved in prometaphase to ensure error-free mitosis remains unknown. Here we show that k-MT attachments in prometaphase cells are considerably less stable than in metaphase cells. The switch to more stable k-MT attachments in metaphase requires the proteasome-dependent destruction of cyclin A in prometaphase. Persistent cyclin A expression prevents k-MT stabilization even in cells with aligned chromosomes. By contrast, k-MTs are prematurely stabilized in cyclin-A-deficient cells. Consequently, cells lacking cyclin A display higher rates of chromosome mis-segregation. Thus, the stability of k-MT attachments increases decisively in a coordinated fashion among all chromosomes as cells transit from prometaphase to metaphase. Cyclin A creates a cellular environment that promotes microtubule detachment from kinetochores in prometaphase to ensure efficient error correction and faithful chromosome segregation.
132 citations
TL;DR: This work has identified REEP4, a previously uncharacterized endoplasmic reticulum (ER) protein that functions redundantly to clear the ER from metaphase chromatin, thereby ensuring correct progression through mitosis and proper nuclear envelope architecture.
120 citations
TL;DR: It is shown that displacement of human cohesin from early mitotic chromosomes requires dissociation of Smc3 from Scc1 but no opening of the other two gates, and proposed that the use of differently regulated gates for loading and release facilitates unidirectionality of DNA's entry into and exit from the cohesIn ring.
Abstract: Faithful transmission of chromosomes during eukaryotic cell division requires sister chromatids to be paired from their generation in S phase until their separation in M phase. Cohesion is mediated by the cohesin complex, whose Smc1, Smc3 and Scc1 subunits form a tripartite ring that entraps both DNA double strands. Whereas centromeric cohesin is removed in late metaphase by Scc1 cleavage, metazoan cohesin at chromosome arms is displaced already in prophase by proteolysis-independent signalling. Which of the three gates is triggered by the prophase pathway to open has remained enigmatic. Here, we show that displacement of human cohesin from early mitotic chromosomes requires dissociation of Smc3 from Scc1 but no opening of the other two gates. In contrast, loading of human cohesin onto chromatin in telophase occurs through the Smc1–Smc3 hinge. We propose that the use of differently regulated gates for loading and release facilitates unidirectionality of DNA's entry into and exit from the cohesin ring.
118 citations
TL;DR: The kinesin-13 kif2a as a driver of developmental spindle scaling whose microtubule-destabilizing activity is inhibited in stage 3 spindles by the transport receptor Importin α, and activated in stage 8 when importin α partitions to a membrane pool.
Abstract: Early development of many animals is characterized by rapid cleavages that dramatically decrease cell size, but how the mitotic spindle adapts to changing cell dimensions is not understood. To identify mechanisms that scale the spindle during Xenopus laevis embryogenesis, we established an in vitro system using cytoplasmic extracts prepared from embryos that recapitulates in vivo spindle size differences between stage 3 (4 cells, 37 µm) and stage 8 (∼4000 cells, 18 µm). We identified the kinesin-13 kif2a as a driver of developmental spindle scaling whose microtubule-destabilizing activity is inhibited in stage 3 spindles by the transport receptor importin α, and activated in stage 8 when importin α partitions to a membrane pool. Altering spindle size in developing embryos impaired spindle orientation during metaphase, but chromosome segregation remained robust. Thus, spindle size in Xenopus development is coupled to cell size through a ratiometric mechanism controlling microtubule destabilization.DOI:http://dx.doi.org/10.7554/eLife.00290.001.
113 citations
TL;DR: Upon CDK1 inactivation in anaphase, the rise in dephosphorylated NuMA at the cell cortex leads to cortical dynein enrichment, and thus to robust spindle elongation, finding a mechanism whereby the status of NuMA phosphorylation coordinates mitotic progression with proper spindle function.
Abstract: Spindle positioning and spindle elongation are critical for proper cell division. In human cells, an evolutionary conserved ternary complex (NuMA/LGN/Gαi) anchors dynein at the cortex during metaphase, thus ensuring correct spindle positioning. Whether this complex contributes to anaphase spindle elongation is not known. More generally, the mechanisms coupling mitotic progression with spindle behaviour remain elusive. Here, we uncover that levels of cortical dynein markedly increase during anaphase in a NuMA-dependent manner. We demonstrate that during metaphase, CDK1-mediated phosphorylation at T2055 negatively regulates NuMA cortical localization and that this phosphorylation is counteracted by PPP2CA phosphatase activity. We establish that this tug of war is essential for proper levels of cortical dynein and thus spindle positioning during metaphase. Moreover, we find that upon CDK1 inactivation in anaphase, the rise in dephosphorylated NuMA at the cell cortex leads to cortical dynein enrichment, and thus to robust spindle elongation. Our findings uncover a mechanism whereby the status of NuMA phosphorylation coordinates mitotic progression with proper spindle function.
TL;DR: Results suggest that MPS1 inhibitors may exert robust anticancer activity, either as standalone therapeutic interventions or combined with microtubule-targeting chemicals.
Abstract: Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A synergistic interaction between paclitaxel and MPS1 inhibitors could also be demonstrated in vivo, as the combination of these agents efficiently reduced the growth of tumor xenografts and exerted superior antineoplastic effects compared with either compound employed alone. Altogether, these results suggest that MPS1 inhibitors may exert robust anticancer activity, either as standalone therapeutic interventions or combined with microtubule-targeting chemicals.
TL;DR: A comprehensive, cross-species electron tomography analysis of kinetochore–microtubule interfaces has provided insight into shared structural features and their likely functional consequences.
Abstract: Interfaces between spindle microtubules and kinetochores were examined in diverse species by electron tomography and image analysis. Overall structures were conserved in a mammal, an alga, a nematode, and two kinds of yeasts; all lacked dense outer plates, and most kinetochore microtubule ends flared into curved protofilaments that were connected to chromatin by slender fibrils. Analyses of curvature on >8,500 protofilaments showed that all classes of spindle microtubules displayed some flaring protofilaments, including those growing in the anaphase interzone. Curved protofilaments on anaphase kinetochore microtubules were no more flared than their metaphase counterparts, but they were longer. Flaring protofilaments in budding yeasts were linked by fibrils to densities that resembled nucleosomes; these are probably the yeast kinetochores. Analogous densities in fission yeast were larger and less well-defined, but both yeasts showed ring- or partial ring-shaped structures girding their kinetochore microtubules. Flaring protofilaments linked to chromatin are well placed to exert force on chromosomes, assuring stable attachment and reliable anaphase segregation.
TL;DR: It is found that Arabidopsis KINETOCHORE NULL2 (KNL2) colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase.
Abstract: The centromeric histone H3 variant cenH3 is an essential centromeric protein required for assembly, maintenance, and proper function of kinetochores during mitosis and meiosis. We identified a KINETOCHORE NULL2 (KNL2) homolog in Arabidopsis thaliana and uncovered features of its role in cenH3 loading at centromeres. We show that Arabidopsis KNL2 colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase. KNL2 is regulated by the proteasome degradation pathway. The KNL2 promoter is mainly active in meristematic tissues, similar to the cenH3 promoter. A knockout mutant for KNL2 shows a reduced level of cenH3 expression and reduced amount of cenH3 protein at chromocenters of meristematic nuclei, anaphase bridges during mitosis, micronuclei in pollen tetrads, and 30% seed abortion. Moreover, knl2 mutant plants display reduced expression of suppressor of variegation 3-9 homologs2, 4 ,a nd9 and reduced DNA methylation, suggesting an impact of KNL2 on the epigenetic environment for centromere maintenance.
TL;DR: The budding yeast Wee1 kinase Swe1 restrains the metaphase-to-anaphase transition by preventing the Cdk1-dependent phosphorylation and activation of APCCdc20.
Abstract: Cdk1 drives both mitotic entry and the metaphase-to-anaphase transition. Past work has shown that Wee1 inhibition of Cdk1 blocks mitotic entry. Here we show that the budding yeast Wee1 kinase, Swe1, also restrains the metaphase-to-anaphase transition by preventing Cdk1 phosphorylation and activation of the mitotic form of the anaphase-promoting complex/cyclosome (APC(Cdc20)). Deletion of SWE1 or its opposing phosphatase MIH1 (the budding yeast cdc25(+)) altered the timing of anaphase onset, and activation of the Swe1-dependent morphogenesis checkpoint or overexpression of Swe1 blocked cells in metaphase with reduced APC activity in vivo and in vitro. The morphogenesis checkpoint also depended on Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A). cdc55Δ checkpoint defects were rescued by mutating 12 Cdk1 phosphorylation sites on the APC, demonstrating that the APC is a target of this checkpoint. These data suggest a model in which stepwise activation of Cdk1 and inhibition of PP2A(Cdc55) triggers anaphase onset.
TL;DR: The slow increase in CDK1 activity in meiosis I acts as a timing mechanism to allow stable kinetochore–microtubule attachments only after bipolar spindle formation, thus preventing attachment errors.
Abstract: Chromosome segregation during cell division depends on stable attachment of kinetochores to spindle microtubules. Mitotic spindle formation and kinetochore–microtubule (K-MT) capture typically occur within minutes of nuclear envelope breakdown. In contrast, during meiosis I in mouse oocytes, formation of the acentrosomal bipolar spindle takes 3–4 h, and stabilization of K-MT attachments is delayed an additional 3–4 h. The mechanism responsible for this delay, which likely prevents stabilization of erroneous attachments during spindle formation, is unknown. Here we show that during meiosis I, attachments are regulated by CDK1 activity, which gradually increases through prometaphase and metaphase I. Partial reduction of CDK1 activity delayed formation of stable attachments, whereas a premature increase in CDK1 activity led to precocious formation of stable attachments and eventually lagging chromosomes at anaphase I. These results indicate that the slow increase in CDK1 activity in meiosis I acts as a timing mechanism to allow stable K-MT attachments only after bipolar spindle formation, thus preventing attachment errors.
TL;DR: It is revealed here that movements of neighboring sister-kinetochore pairs in metaphase are correlated in a distance-dependent manner, suggesting that periodic movements of bioriented chromosomes limit the correlated motion of nonsisters.
TL;DR: A computational model of kinetochore dynamics suggests that differences in the distribution of polar ejection forces at the periphery and in the middle of PtK1 cell spindles underlie the observed position-dependence of metaphase chromosome behavior.
Abstract: Duplicated mitotic chromosomes aligned at the metaphase plate maintain dynamic attachments to spindle microtubules via their kinetochores, and multiple motor and nonmotor proteins cooperate to regulate their behavior. Depending on the system, sister chromatids may display either of two distinct behaviors, namely (1) the presence or (2) the absence of oscillations about the metaphase plate. Significantly, in PtK1 cells, in which chromosome behavior appears to be dependent on the position along the metaphase plate, both types of behavior are observed within the same spindle, but how and why these distinct behaviors are manifested is unclear. Here, we developed a new quantitative model to describe metaphase chromosome dynamics via kinetochore–microtubule interactions mediated by nonmotor viscoelastic linkages. Our model reproduces all the key features of metaphase sister kinetochore dynamics in PtK1 cells and suggests that differences in the distribution of polar ejection forces at the periphery and in the middle of PtK1 cell spindles underlie the observed dichotomy of chromosome behavior.
TL;DR: It is shown that the spindle midzone proteins kinesin-5 cut7p and MT bundler ase1p contribute to outward-pushing forces and that theSpindle kinetochore proteins klesin-8 klp5/6p and dam1p contributed to inward-pulling forces, and that removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects.
TL;DR: Data suggest that DNA damage inhibits or delays the G2/M transition, but once GVBD occurs, DNA-damaged oocytes can complete chromosome separation and polar body extrusion even under a higher SAC activity, causing the formation of numerous micronuclei in early embryos.
Abstract: Both endogenous and exogenous factors can induce DNA double-strand breaks (DSBs) in oocytes, which is a potential risk for human-assisted reproductive technology as well as animal nuclear transfer. Here we used bleomycin (BLM) and laser micro-beam dissection (LMD) to induce DNA DSBs in germinal vesicle (GV) stage oocytes and compared the germinal vesicle breakdown (GVBD) rates and first polar body extrusion (PBE) rates between DNA DSB oocytes and untreated oocytes. Employing live cell imaging and immunofluorescence labeling, we observed the dynamics of DNA fragments during oocyte maturation. We also determined the cyclin B1 expression pattern in oocytes to analyze spindle assembly checkpoint (SAC) activity in DNA DSB oocytes. We used parthenogenetic activation to determine if the DNA DSB oocytes could be activated. As a result, we found that the BLM- or LMD-induced DSB oocytes showed lower GVBD rates and took a longer time to undergo GVBD compared with untreated oocytes. PBE was also delayed in DSB oocytes, but once GVBD had occurred, PBE was not affected, even in oocytes with severe DSBs. Compared with control oocytes, the DSB oocytes showed higher SAC activity, as indicated by less Ccnb1-GFP degradation during metaphase I to anaphase I transition. Parthenogenetic activation could activate the metaphase to interphase transition in the DNA DSB mature oocytes, but many oocytes contained multiple pronuclei or numerous micronuclei. These data suggest that DNA damage inhibits or delays the G2/M transition, but once GVBD occurs, DNA-damaged oocytes can complete chromosome separation and polar body extrusion even under a higher SAC activity, causing the formation of numerous micronuclei in early embryos.
TL;DR: Ser99 on Plk1 is identified as a novel mitosis-specific phosphorylation site, which operates independently of Plk 1–Thr210 phosphorylated, which controls proper progression from metaphase to anaphase.
Abstract: Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1-Thr210 phosphorylation. Plk1-Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1-Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1-Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1-Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1-Ser99 phosphorylation downstream of the PI3K-Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase.
TL;DR: P phenotypes caused by overexpression of RNF213 wild type and R4810K variant in the cell cycle are characterized to investigate the mechanism of proliferation inhibition and induced mitotic abnormalities and increased risk of genomic instability.
TL;DR: Morphometric spindle parameters were very comparable between oocytes matured in vivo and in vitro and were unable to predict chromosome alignment, while a flattened shape of both poles was positively associated with chromosome displacement from the metaphase plate.
Abstract: Study question Are morphometric and morphological parameters of the metaphase II (MII) spindle of human oocytes matured in vivo or in vitro predictive of chromosome alignment on the metaphase plate? Summary answer Morphometric spindle parameters were very comparable between oocytes matured in vivo and in vitro and were unable to predict chromosome alignment, while a flattened shape of both poles was positively associated with chromosome displacement from the metaphase plate. What is known already The relationship between MII spindle morphometry and chromosome alignment has only been sporadically investigated in human oocytes. The possible implications of spindle pole morphology are totally unrecognized. Study design, size, duration Morphometric and morphological analysis of the MII spindle of donated supernumerary human oocytes (N = 93) aimed at investigating possible associations between novel microtubule parameters and chromosome arrangement. Participants/materials, setting, methods MII oocytes from three sources were analysed: (i) stimulated cycles matured in vivo (ivo-MII), (ii) leftover cumulus-free germinal vesicle oocytes from stimulated cycles matured in vitro (lgv-MII) and (iii) immature cumulus-cell oocyte complexes (COCs) recovered from in vitro maturation (IVM) cycles and matured in vitro (ivm-MII). Oocytes were fixed and stained for tubulin, chromatin and actin. Optical sections were collected at 0.3 µm intervals by high-performance confocal microscopy and three-dimensionally reconstructed for assignment of specific spindle and chromosomal properties. Spindle pole morphology was classified as either focused or flattened depending on whether microtubule ends were more or less convergent, respectively. Optical density measurements were generated to estimate microtubule abundance in chromosome to pole domains proximal and distal to the oolemma. Main results and the role of chance In ivo-MII oocytes, the sizes (mean ± SD) of major and minor axes were 11.8 ± 2.6 and 8.9 ± 1.7 μm, respectively, while maximum projection was 88.8 ± 29.5 μm(2). Very comparable values of these parameters were found in lgv-MII and ivm-MII oocytes. Double-focused spindles were rarely found (3.1%), unlike those with a double-flattened conformation (47.7%). Spindles with both focused and flattened poles amounted to almost half of the sample set (49.2%), but in this subgroup it was very infrequent (4.6%) to observe the flattened pole oriented towards the oolemma. Overall, differences in the relative proportions of pole morphology categories in ivo-MII, lgv-MII and ivm-MII oocytes were not statistically significant. For both the distal and proximal spindle hemidomains, optical intensity profiles were also comparable between ivo-MII, lgv-MII and ivm-MII oocytes. None of the morphometric parameters (major and minor axes, their ratio, maximum projection, distances of the metaphase plate from the poles) was associated with chromosome alignment on the metaphase plate or arrangement inside and outside the spindle. Importantly, a double-flattened outline of pole morphology was positively associated with the displacement of one or more chromosomes from the metaphase plate. Moreover, when a flattened pole was oriented towards the oolemma, a higher rate of chromosome displacement was observed. Limitations, reasons for caution The findings of the study will require confirmation by further in-depth analysis and extension of the database, especially regarding the relationship between microtubule abundance and chromosome arrangement. Furthermore, considering the high number of comparisons, the observed statistical differences will require future 'ad hoc' analysis. Wider implications of the findings Collectively, this work provides a robust database for future research on the human oocyte cytoskeleton, and contributes to a better definition of oocyte quality in assisted reproduction technology. Also, these data support the notion that IVM does not affect spindle morphometry and morphology. Study funding/competing interest(s) Part of this work was supported by a grant awarded by the Italian Ministry of Labour, Health and Social Policies. The authors have no conflicts of interest to declare. Trial registration number Not applicable.
TL;DR: Overexpression and RNAi studies demonstrate that SENP1 and SENP2 are important modulators of SUMO function in mitosis.
Abstract: Sumoylation of centromere, kinetochore, and other mitotic chromosome-associated proteins is essential for chromosome segregation. The mechanisms regulating spatial and temporal sumoylation of proteins in mitosis, however, are not well understood. Here we show that the small ubiquitin-related modifier (SUMO)–specific isopeptidases SENP1 and SENP2 are targeted to kinetochores in mitosis. SENP2 targeting occurs through a mechanism dependent on the Nup107-160 subcomplex of the nuclear pore complex and is modulated through interactions with karyopherin α. Overexpression of SENP2, but not other SUMO-specific isopeptidases, causes a defect in chromosome congression that depends on its precise kinetochore targeting. By altering SENP1 kinetochore associations, however, this effect on chromosome congression could be phenocopied. In contrast, RNA interference–mediated knockdown of SENP1 delays sister chromatid separation at metaphase, whereas SENP2 knockdown produces no detectable phenotypes. Our findings indicate that chromosome segregation depends on precise spatial and temporal control of sumoylation in mitosis and that SENP1 and SENP2 are important mediators of this control.
TL;DR: This work identifies a plant Bub1-related kinase 1 (BRK1) in rice and suggests that this protein corrects the merotelic attachment of paired sister kinetochores during metaphase I, which affects the synchronicity of homolog separation at the onset of anaphase I.
Abstract: Bub1 (for budding uninhibited by benzimidazole 1), one of the main spindle checkpoint kinases, acts as a kinetochore scaffold for assembling other checkpoint proteins. Here, we identify a plant Bub1-related kinase 1 (BRK1) in rice (Oryza sativa). The brk1 mutants are sterile due to the precocious separation of sister chromatids at the onset of anaphase I. The centromeric recruitment of SHUGOSHIN1 and phosphorylation of histone H2A at Thr-134 (H2A-pT134) depend on BRK1. Although the homologs can faithfully separate from each other at the end of meiosis I, the uncorrected merotelic attachment of paired sister kinetochores at the early stage of metaphase I in brk1 reduces the tension across homologous kinetochores, causes the metaphase I spindle to be aberrantly shaped, and subsequently affects the synchronicity of homolog separation at the onset of anaphase I. In addition, the phosphorylation of inner centromeric histone H3 at Ser-10 (H3-pS10) during diakinesis depends on BRK1. Therefore, we speculate that BRK1 may be required for normal localization of Aurora kinase before the onset of metaphase I, which is responsible for correcting the merotelic attachment.
TL;DR: Synchrotron X-ray fluorescence microscopy of non-synchronized NIH 3T3 fibroblasts revealed an intriguing redistribution dynamics that defines the inheritance of trace metals during mitosis, suggesting a prominent physiological role that lies beyond the requirement of Zn as a cofactor in metalloproteins or messenger in signaling pathways.
Abstract: Synchrotron X-ray fluorescence microscopy of non-synchronized NIH 3T3 fibroblasts revealed an intriguing redistribution dynamics that defines the inheritance of trace metals during mitosis. At metaphase, the highest density areas of Zn and Cu are localized in two distinct regions adjacent to the metaphase plate. As the sister chromatids are pulled towards the spindle poles during anaphase, Zn and Cu gradually move to the center and partition into the daughter cells to yield a pair of twin pools during cytokinesis. Colocalization analyses demonstrated high spatial correlations between Zn, Cu, and S throughout all mitotic stages, while Fe showed consistently different topographies characterized by high-density spots distributed across the entire cell. Whereas the total amount of Cu remained similar compared to interphase cells, mitotic Zn levels increased almost 3-fold, suggesting a prominent physiological role that lies beyond the requirement of Zn as a cofactor in metalloproteins or messenger in signaling pathways.
TL;DR: How the standard somatic cell cycle is modulated to meet the specific requirements of different developmental stages is discussed, focusing on cell cycle regulation in mature vertebrate eggs arrested at metaphase II (MII‐arrest), the first mitotic cell cycle, and early embryonic divisions.
Abstract: Ex ovo omnia—all animals come from eggs—this statement made in 1651 by the English physician William Harvey marks a seminal break with the doctrine that all essential characteristics of offspring are contributed by their fathers, while mothers contribute only a material substrate. More than 360 years later, we now have a comprehensive understanding of how haploid gametes are generated during meiosis to allow the formation of diploid offspring when sperm and egg cells fuse. In most species, immature oocytes are arrested in prophase I and this arrest is maintained for few days (fruit flies) or for decades (humans). After completion of the first meiotic division, most vertebrate eggs arrest again at metaphase of meiosis II. Upon fertilization, this second meiotic arrest point is released and embryos enter highly specialized early embryonic divisions. In this review, we discuss how the standard somatic cell cycle is modulated to meet the specific requirements of different developmental stages. Specifically, we focus on cell cycle regulation in mature vertebrate eggs arrested at metaphase II (MII-arrest), the first mitotic cell cycle, and early embryonic divisions.
TL;DR: It is concluded that NEDL2 is a novel substrate of APC/C-Cdh1 as cells exit mitosis and functions as a regulator of the metaphase to anaphase transition.
TL;DR: In contrast to the situation in mammalian oocytes, the metaphase-to-anaphase transition in frog oocytes is not regulated by a spindle assembly checkpoint.
Abstract: The spindle assembly checkpoint (SAC) functions as a surveillance mechanism to detect chromosome misalignment and to delay anaphase until the errors are corrected. The SAC is thought to control mitosis and meiosis, including meiosis in mammalian eggs. However, it remains unknown if meiosis in the eggs of nonmammalian vertebrate species is also regulated by SAC. Using a novel karyotyping technique, we demonstrate that complete disruption of spindle microtubules in Xenopus laevis oocytes did not affect the bivalent-to-dyad transition at the time oocytes are undergoing anaphase I. These oocytes also acquired the ability to respond to parthenogenetic activation, which indicates proper metaphase II arrest. Similarly, oocytes exhibiting monopolar spindles, via inhibition of aurora B or Eg5 kinesin, underwent monopolar anaphase on time and without additional intervention. Therefore, the metaphase-to-anaphase transition in frog oocytes is not regulated by SAC.
TL;DR: A role for Mad1p in regulating nuclear import is described that employs its ability to sense a disruption of kinetochore-microtubule interactions during mitosis and, by altering NPC transport activity, regulates the nuclear environment of the spindle.