KINESIN-12E regulates metaphase spindle flux and helps control spindle size in Arabidopsis.

The bipolar mitotic spindle is a highly conserved structure among eukaryotes that mediates chromosome alignment and segregation. Spindle assembly and size control are facilitated by force-generating microtubule-dependent motor proteins known as kinesins. In animals, kinesin-12 cooperates with kinesin-5 to produce outward-directed forces necessary for spindle assembly. In plants, the relevant molecular mechanisms for spindle formation are poorly defined. While an Arabidopsis thaliana kinesin-5 ortholog has been identified, the kinesin-12 ortholog in plants remains elusive. In this study, we provide experimental evidence for the function of Arabidopsis KINESIN-12E in spindle assembly. In kinesin-12e mutants, a delay in spindle assembly is accompanied by the reduction of spindle size, demonstrating that KINESIN-12E contributes to mitotic spindle architecture. Kinesin-12E localization is mitosis-stage specific, beginning with its perinuclear accumulation during prophase. Upon nuclear envelope breakdown, KINESIN-12E decorates subpopulations of microtubules in the spindle and becomes progressively enriched in the spindle midzone. Furthermore, during cytokinesis, KINESIN-12E shares its localization at the phragmoplast midzone with several functionally diversified Arabidopsis KINESIN-12 members. Changes in the kinetochore and in prophase and metaphase spindle dynamics occur in the absence of KINESIN-12E, suggest it might play an evolutionarily conserved role during spindle formation similar to its spindle-localized animal kinesin-12 orthologs.

Oocytes can efficiently repair DNA double-strand breaks to restore genetic integrity and protect offspring health.

Female fertility and offspring health are critically dependent on an adequate supply of high-quality oocytes, the majority of which are maintained in the ovaries in a unique state of meiotic prophase arrest. While mechanisms of DNA repair during meiotic recombination are well characterized, the same is not true for prophase-arrested oocytes. Here we show that prophase-arrested oocytes rapidly respond to γ-irradiation-induced DNA double-strand breaks by activating Ataxia Telangiectasia Mutated, phosphorylating histone H2AX, and localizing RAD51 to the sites of DNA damage. Despite mobilizing the DNA repair response, even very low levels of DNA damage result in the apoptosis of prophase-arrested oocytes. However, we show that, when apoptosis is inhibited, severe DNA damage is corrected via homologous recombination repair. The repair is sufficient to support fertility and maintain health and genetic fidelity in offspring. Thus, despite the preferential induction of apoptosis following exogenously induced genotoxic stress, prophase-arrested oocytes are highly capable of functionally efficient DNA repair. These data implicate DNA repair as a key quality control mechanism in the female germ line and a critical determinant of fertility and genetic integrity.

Phosphoregulation of HORMA domain protein HIM-3 promotes asymmetric synaptonemal complex disassembly in meiotic prophase in Caenorhabditis elegans.

In the two cell divisions of meiosis, diploid genomes are reduced into complementary haploid sets through the discrete, two-step removal of chromosome cohesion, a task carried out in most eukaryotes by protecting cohesion at the centromere until the second division. In eukaryotes without defined centromeres, however, alternative strategies have been innovated. The best-understood of these is found in the nematode Caenorhabditis elegans: after the single off-center crossover divides the chromosome into two segments, or arms, several chromosome-associated proteins or post-translational modifications become specifically partitioned to either the shorter or longer arm, where they promote the correct timing of cohesion loss through as-yet unknown mechanisms. Here, we investigate the meiotic axis HORMA-domain protein HIM-3 and show that it becomes phosphorylated at its C-terminus, within the conserved "closure motif" region bound by the related HORMA-domain proteins HTP-1 and HTP-2. Binding of HTP-2 is abrogated by phosphorylation of the closure motif in in vitro assays, strongly suggesting that in vivo phosphorylation of HIM-3 likely modulates the hierarchical structure of the chromosome axis. Phosphorylation of HIM-3 only occurs on synapsed chromosomes, and similarly to other previously-described phosphorylated proteins of the synaptonemal complex, becomes restricted to the short arm after designation of crossover sites. Regulation of HIM-3 phosphorylation status is required for timely disassembly of synaptonemal complex central elements from the long arm, and is also required for proper timing of HTP-1 and HTP-2 dissociation from the short arm. Phosphorylation of HIM-3 thus plays a role in establishing the identity of short and long arms, thereby contributing to the robustness of the two-step chromosome segregation.

Shugoshin protects centromere pairing and promotes segregation of nonexchange partner chromosomes in meiosis.

Faithful chromosome segregation during meiosis I depends upon the formation of connections between homologous chromosomes. Crossovers between homologs connect the partners, allowing them to attach to the meiotic spindle as a unit, such that they migrate away from one another at anaphase I. Homologous partners also become connected by pairing of their centromeres in meiotic prophase. This centromere pairing can promote proper segregation at anaphase I of partners that have failed to become joined by a crossover. Centromere pairing is mediated by synaptonemal complex (SC) proteins that persist at the centromere when the SC disassembles. Here, using mouse spermatocyte and yeast model systems, we tested the role of shugoshin in promoting meiotic centromere pairing by protecting centromeric synaptonemal components from disassembly. The results show that shugoshin protects the centromeric SC in meiotic prophase and, in anaphase, promotes the proper segregation of partner chromosomes that are not linked by a crossover.

Kinesin-8 and Dis1/TOG collaborate to limit spindle elongation from prophase to anaphase A for proper chromosome segregation in fission yeast.

High-fidelity chromosome segregation relies on proper microtubule regulation. Kinesin-8 has been shown to destabilise microtubules to reduce metaphase spindle length and chromosome movements in multiple species. XMAP215/chTOG polymerases catalyse microtubule growth for spindle assembly, elongation and kinetochore-microtubule attachment. Understanding of their biochemical activity has advanced, but little work directly addresses the functionality and interplay of these conserved factors. We utilised the synthetic lethality of fission yeast kinesin-8 (Klp5-Klp6) and XMAP215/chTOG (Dis1) to study their individual and overlapping roles. We found that the non-motor kinesin-8 tailbox is essential for mitotic function; mutation compromises plus-end-directed processivity. Klp5-Klp6 induces catastrophes to control microtubule length and, surprisingly, Dis1 collaborates with kinesin-8 to slow spindle elongation. Together, they enforce a maximum spindle length for a viable metaphase-anaphase transition and limit elongation during anaphase A to prevent lagging chromatids. Our work provides mechanistic insight into how kinesin-8 negatively regulates microtubules and how this functionally overlaps with Dis1 and highlights the importance of spindle length control in mitosis.

Nucleolus structural integrity during the first meiotic prophase in rat spermatocytes.

A typical nucleolus structure is shaped by three components. A meshwork of fine fibers forming the fibrillar center (FC) is surrounded by densely packed fibers forming the dense fibrillar component (DFC). Meanwhile, wrapping the FC and DFC is the granular component (GC). During the mitotic prophase, the nucleolus undergoes disassembling of its components. On the contrary, throughout the first meiotic prophase that occurs in the cells of the germ line, small nucleoli are assembled into one nucleolus by the end of the prophase. These nucleoli are transcriptionally active, suggesting that they are fully functional. Electron microscopy analysis has suggested that these nucleoli display their three main components but a typical organization has not been observed. Here, by immunolabeling and electron microscopy, we show that the nucleolus has its three main components. The GC is interlaced with the DFC and is not as well defined as previously thought during leptotene and zygotene stage.

Prophase pathway-dependent removal of cohesin from human chromosomes requires opening of the Smc3-Scc1 gate.

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.

Nuclear envelope-associated dynein drives prophase centrosome separation and enables Eg5-independent bipolar spindle formation.

The microtubule motor protein kinesin-5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti-cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5-independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)-associated dynein that drives centrosome separation in prophase. This NE-dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5-dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin-5 inhibitors.

Releasing prophase arrest in zebrafish oocyte: synergism between maturational steroid and Igf1.

Binding of 17ß-estradiol (E2) to novel G-protein coupled receptor, Gper1, promotes intra-oocyte adenylyl cyclase activity and transactivates epidermal growth factor receptor to ensure prophase-I arrest. Although involvement of either membrane progestin receptor (mPR) or Igf system has been implicated in regulation of meiosis resumption, possibility of concurrent activation and potential synergism between 17α,20ß-dihydroxy-4-pregnen-3-one (DHP)- and Igf-mediated signalling cascades in alleviating E2 inhibition of oocyte maturation (OM) has not been investigated. Here using zebrafish (Danio rerio) defolliculated oocytes, we examined the effect of DHP and Igf1, either alone or in combination, in presence or absence of E2, on OM in vitro. While priming of denuded oocytes with E2 blocked spontaneous maturation, co-treatment with DHP (3 nM) and Igf1 (10 nM), but not alone, reversed E2 inhibition and promoted a robust increase in germinal vesicle breakdown (GVBD). Although stimulation with either Igf1 or DHP promoted Akt phosphorylation, pharmacological inhibition of PI3K/Akt signalling prevented Igf1-induced GVBD but delayed DHP action till 4-5 h of incubation. Moreover, high intra-oocyte cAMP attenuates both DHP and Igf1-mediated OM and co-stimulation with DHP and Igf1 could effectively reverse E2 action on PKA phosphorylation. Interestingly, data from in vivo studies reveal that heightened expression of igf1, igf3 transcripts in intact follicles corresponded well with elevated phosphorylation of Igf1r and Akt, mPRa immunoreactivity, PKA inhibition and accelerated GVBD response just prior to ovulation. This indicates potential synergism between maturational steroid and Igf1 which might have physiological relevance in overcoming E2 inhibition of meiosis resumption in zebrafish oocytes.

Wnt and CDK-1 regulate cortical release of WRM-1/ß-catenin to control cell division orientation in early Caenorhabditis elegans embryos.

In early Caenorhabditis elegans embryos, the Wingless/int (Wnt)- and Src-signaling pathways function in parallel to induce both the division orientation of the endomesoderm (EMS) blastomere and the endoderm fate of the posterior EMS daughter cell, called E. Here, we show that, in addition to its role in endoderm specification, the ß-catenin-related protein Worm armadillo 1 (WRM-1) also plays a role in controlling EMS division orientation. WRM-1 localizes to the cortex of cells in both embryos and larvae and is released from the cortex in a Wnt-responsive manner. We show that WRM-1 cortical release is disrupted in a hypomorphic cyclin-dependent protein kinase 1 (cdk-1) mutant and that WRM-1 lacking potential CDK-1 phosphoacceptor sites is retained at the cortex. In both cases, cortical WRM-1 interferes with EMS spindle rotation without affecting endoderm specification. Finally, we show that removal of WRM-1 from the cortex can restore WT division orientation, even when both Wnt- and Src-signaling pathways are compromised. Our findings are consistent with a model in which Wnt signaling and CDK-1 modify WRM-1 in a temporal and spatial manner to unmask an intrinsic polarity cue required for proper orientation of the EMS cell division axis.

Full-length synaptonemal complex grows continuously during meiotic prophase in budding yeast.

the synaptonemal complex (SC) links two meiotic prophase chromosomal events: homolog pairing and crossover recombination. SC formation involves the multimeric assembly of coiled-coil proteins (Zip1 in budding yeast) at the interface of aligned homologous chromosomes. However, SC assembly is indifferent to homology and thus is normally regulated such that it occurs only subsequent to homology recognition. Assembled SC structurally interfaces with and influences the level and distribution of interhomolog crossover recombination events. Despite its involvement in dynamic chromosome behaviors such as homolog pairing and recombination, the extent to which SC, once installed, acts as an irreversible tether or maintains the capacity to remodel is not clear. Experiments presented here reveal insight into the dynamics of the full-length SC in budding yeast meiotic cells. We demonstrate that Zip1 continually incorporates into previously assembled synaptonemal complex during meiotic prophase. Moreover, post-synapsis Zip1 incorporation is sufficient to rescue the sporulation defect triggered by SCs built with a mutant version of Zip1, Zip1-4LA. Post-synapsis Zip1 incorporation occurs initially with a non-uniform spatial distribution, predominantly associated with Zip3, a component of the synapsis initiation complex that is presumed to mark a subset of crossover sites. A non-uniform dynamic architecture of the SC is observed independently of (i) synapsis initiation components, (ii) the Pch2 and Pph3 proteins that have been linked to Zip1 regulation, and (iii) the presence of a homolog. Finally, the rate of SC assembly and SC central region size increase in proportion to Zip1 copy number; this and other observations suggest that Zip1 does not exit the SC structure to the same extent that it enters. Our observations suggest that, after full-length assembly, SC central region exhibits little global turnover but maintains differential assembly dynamics at sites whose distribution is patterned by a recombination landscape.

Telomere distribution pattern and synapsis initiation during spermatogenesis in zebrafish.

BACKGROUND: Telomeres are located at ends of eukaryotic chromosomes and can affect proper chromosomal positioning. During spermatogenesis, the appropriate dynamics and behavior of chromosomes is crucial to generate haploid cells through meiosis. Here, we describe telomere distribution patterns during spermatogenesis in zebrafish, especially during meiotic prophase I, using fluorescence in situ hybridization. This was combined with synaptonemal complex protein 3 immunostaining, which allows the staging of spermatocytes. RESULTS: During spermatogonial proliferation and the preleptotene stage, telomeres were dispersed throughout the nucleus. During the leptotene stage, telomeres temporarily moved to one pole of the nucleus at which γ-tubulin was located, forming the telomere bouquet. The cluster lasted until the onset of zygotene where it coincided with terminal synapsis initiation. They then spread around the periphery of the nucleus during the zygotene to pachytene stages. During postmeiotic stages, telomeres in spermatids and sperm were again dispersed throughout the nuclei. Application of this procedure in meiotic mutants confirmed that meiotic telomere clustering is independent of axial element formation of the synaptonemal complex. CONCLUSIONS: These data clearly showed the clustering and distributions of telomeres throughout spermatogenesis in zebrafish. This procedure could be used to screen for mutants that have primary defects in telomere clustering.

CSN5/JAB1 interacts with the centromeric components CENP-T and CENP-W and regulates their proteasome-mediated degradation.

The CENP-T·CENP-W complex is a recently identified inner centromere component that plays crucial roles in the formation of a functional kinetochore involved in cell division during mitosis. Using yeast two-hybrid screening, we identified an interaction between CENP-T and CSN5, the fifth component of the COP9 signalosome and a key modulator of the cell cycle and cancer. Co-immunoprecipitation revealed that CSN5 directly interacts with both CENP-T and CENP-W. Ectopically expressed CSN5 promoted the ubiquitin- and proteasome-dependent degradation of CENP-T·CENP-W. The formation of a CENP-T·CENP-W complex greatly enhanced the stabilities of the respective proteins, possibly by blocking CSN5-mediated degradation. Furthermore, dysregulation of CSN5 induced severe defects in the recruitment of CENP-T·CENP-W to the kinetochore during the prophase stage of mitosis. Thus, our results indicate that CSN5 regulates the stability of the inner kinetochore components CENP-T and CENP-W, providing the first direct link between CSN5 and the mitotic apparatus, highlighting the role of CSN5 as a multifunctional cell cycle regulator.

Bivalent associations in Mus domesticus 2n = 40 spermatocytes. Are they random?

The establishment of associations between bivalents from Mus domesticus 2n = 40 spermatocytes is a common phenomenon that shows up during the first prophase of meiotic nuclei. In each nucleus, a seemingly random display of variable size clusters of bivalents in association is observed. These associations originate a particular nuclear architecture and determine the probability of encounters between chromosome domains. Hence, the type of randomness in associations between bivalents has nontrivial consequences. We explore different models for randomness and the associated bivalent probability distributions and find that a simple model based on randomly coloring a subset of vertices of a 6-regular graph provides best agreement with microspreads observations. The notion of randomness is thereby explained in conjunction with the underlying local geometry of the nuclear envelope.

Aurora-A phosphorylates Augmin complex component Hice1 protein at an N-terminal serine/threonine cluster to modulate its microtubule binding activity during spindle assembly.

Proper assembly of mitotic spindles requires Hice1, a spindle-associated protein. Hice1 possesses direct microtubule binding activity at its N-terminal region and contributes to intraspindle microtubule nucleation as a subunit of the Augmin complex. However, whether microtubule binding activity of Hice1 is modulated by mitotic regulators remains unexplored. Here, we found that Aurora-A kinase, a major mitotic kinase, specifically binds to and phosphorylates Hice1. We identified four serine/threonine clusters on Hice1 that can be phosphorylated by Aurora-A in vitro. Of the four clusters, the Ser/Thr-17-21 cluster was the most critical for bipolar spindle assembly, whereas other phospho-deficient point mutants had a minimal effect on spindle assembly. Immunostaining with a phospho-Ser-19/20 phospho-specific antibody revealed that phosphorylated Hice1 primarily localizes to spindle poles during prophase to metaphase but gradually diminishes after anaphase. Consistently, the phospho-mimic 17-21E mutant reduced microtubule binding activity in vitro and diminished localization to spindles in vivo. Furthermore, expression of the 17-21E mutant led to decreased association of Fam29a, an Augmin component, with spindles. On the other hand, expression of the phospho-deficient 17-21A mutant permitted intraspindle nucleation but delayed the separation of early mitotic spindle poles and the timely mitotic progression. Taken together, these results suggest that Aurora-A modulates the microtubule binding activity of Hice1 in a spatiotemporal manner for proper bipolar spindle assembly.

Interaction of Sororin protein with polo-like kinase 1 mediates resolution of chromosomal arm cohesion.

Unlike in budding yeast, sister chromatid cohesion in vertebrate cells is resolved in two steps: cohesin complexes are removed from sister chromatid arms during prophase via phosphorylation, whereas centromeric cohesins are removed at anaphase by Separase. Phosphorylation of cohesin subunit SA2 by polo-like kinase 1 (Plk1) is required for the removal of cohesins at prophase, but how Plk1 is recruited to phosphorylate SA2 during prophase is currently not known. Here we report that Sororin, a cohesin-interacting protein essential for sister chromatid cohesion, plays a novel role in the resolution of sister chromatid arms by direct interaction with Plk1. We identified an evolutionarily conserved motif (ST(159)P) on Sororin, which was phosphorylated by Cdk1/cyclin B and bound to the polo box domain of Plk1. Mutating Thr(159) into alanine prevented the interaction of Plk1 and Sororin and inhibited the resolution of chromosomal arm cohesion. We propose that Sororin is phosphorylated by Cdk1/cyclin B at prophase and acts as a docking protein to bring Plk1 into proximity with SA2, resulting in the phosphorylation of SA2 and the removal of cohesin complexes from chromosomal arms.

Drosophila Klp67A binds prophase kinetochores to subsequently regulate congression and spindle length.

The kinesin-8 proteins are a family of microtubule-depolymerising motor molecules, which, despite their highly conserved roles in chromosome alignment and spindle dynamics, remain poorly characterised. Here, we report that the Drosophila kinesin-8 protein, Klp67A, exists in two spatially and functionally separable metaphase pools: at kinetochores and along the spindle. Fixed and live-cell analyses of different Klp67A recombinant variants indicate that this kinesin-8 first collects at kinetochores during prophase and, by metaphase, localises to the kinetochore outerplate. Although the catalytic motor activity of Klp67A is required for efficient kinetochore recruitment at all times, microtubules are entirely dispensable for this process. The tail of Klp67A does not play a role in kinetochore accumulation, but is both necessary and sufficient for spindle association. Using functional assays, we reveal that chromosome position and spindle length are determined by the microtubule-depolymerising motor activity of Klp67A exclusively when located at kinetochores, but not along the spindle. These data reveal that, unlike other metazoan kinesin-8 proteins, Klp67A binds the nascent prophase and mature metaphase kinetochore. From this location, Klp67A uses its motor activity to ensure chromosome alignment and proper spindle length.

DNA methylation, histone modifications and behaviour of AKAP95 during mouse oocyte growth and upon nuclear transfer of foreign chromatin into fully grown prophase oocytes.

The poor efficiency of mammalian cloning is due to inappropriate/incomplete epigenetic reprogramming of the donor chromatin. As the success in reprogramming of the donor nucleus may require activity of similar mechanisms which reprogram the chromatin in the course of gametogenesis, we decided to follow the status of some epigenetic markers in the late phase of oogenesis in mice, i.e. in prophase oocytes during their growth and after completion of the growth phase. Our analysis reveals an increase in the level of global DNA methylation starting in oocytes with diameters around 60 microm which was further elevated until completion of oocyte growth. A similar increase was observed in respect to the acetylation of histone H4. On the other hand, the methylation of histone H4 Arg3 was constantly high until the end of oocyte growth, although it differed between fully grown oocytes depending on the type of spatial chromatin organization. We have also studied the AKAP95 protein which was abundant at earlier stages but decreased in fully grown oocytes according to changes in their chromatin organization. The nuclear transfer of different types of donor nuclei with hypomethylated DNA into fully grown prophase oocytes did not increase the global level of methylation of transferred foreign chromatin, regardless if the recipient oocyte was devoid of its own nucleus or its nucleus was left intact. This suggests a major problem in the ability of recipient oocytes to modify donor DNA methylation.

Microtubule dynamics in mitosis in Aspergillus nidulans.

Mitosis in Aspergillus nidulans is very rapid, requiring less than 5 min at 37 °C in germlings (Bergen and Morris, 1983). In this time the cytoplasmic microtubules (MTs) must disassemble, the mitotic spindle assemble, function and disassemble, and cytoplasmic MTs reassemble. It follows that cytoplasmic MTs must be extremely dynamic in this period and we were interested, in particular, in examining the processes of MT disassembly in prophase and reassembly in anaphase and telophase. We observed a diploid strain that expressed GFP-α-tubulin. We used a spinning disk confocal microscope that allowed rapid image capture, which proved necessary because microtubule dynamics were extremely rapid. We found, for the first time, that microtubule severing occurs in prophase in a filamentous fungus and that catastrophe rather than nucleation limits astral microtubule growth.

Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C.

Nuclear events of mitosis are initiated when the protein kinase cyclin-B1-Cdk1 is translocated into the nucleus during prophase. Recent work has unveiled many of the mechanisms that govern the localization of cyclin-B1-Cdk1 and its regulator Cdc25C. Phosphorylation-dependent changes in the rate of nuclear import and export of these proteins help to control the onset of mitosis both in normal cells and in cells delayed before mitosis by DNA damage.