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.

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.

Homeostatic Control of Meiotic Prophase Checkpoint Function by Pch2 and Hop1.

Checkpoint cascades link cell cycle progression with essential chromosomal processes. During meiotic prophase, recombination and chromosome synapsis are monitored by what are considered distinct checkpoints. In budding yeast, cells that lack the AAA+ ATPase Pch2 show an impaired cell cycle arrest in response to synapsis defects. However, unperturbed pch2Δ cells are delayed in meiotic prophase, suggesting paradoxical roles for Pch2 in cell cycle progression. Here, we provide insight into the checkpoint roles of Pch2 and its connection to Hop1, a HORMA domain-containing client protein. Contrary to current understanding, we find that Pch2 (together with Hop1) is crucial for checkpoint function in response to both recombination and synapsis defects, thus revealing a shared meiotic checkpoint cascade. Meiotic checkpoint responses are transduced by DNA break-dependent phosphorylation of Hop1. Based on our data and on the described effect of Pch2 on HORMA topology, we propose that Pch2 promotes checkpoint proficiency by catalyzing the availability of signaling-competent Hop1. Conversely, we demonstrate that Pch2 can act as a checkpoint silencer, also in the face of persistent DNA repair defects. We establish a framework in which Pch2 and Hop1 form a homeostatic module that governs general meiotic checkpoint function. We show that this module can-depending on the cellular context-fuel or extinguish meiotic checkpoint function, which explains the contradictory roles of Pch2 in cell cycle control. Within the meiotic prophase checkpoint, the Pch2-Hop1 module thus operates analogous to the Pch2/TRIP13-Mad2 module in the spindle assembly checkpoint that monitors chromosome segregation.

Biochemical and functional characterization of a meiosis-specific Pch2/ORC AAA+ assembly.

Pch2 is a meiosis-specific AAA+ protein that controls several important chromosomal processes. We previously demonstrated that Orc1, a subunit of the ORC, functionally interacts with budding yeast Pch2. The ORC (Orc1-6) AAA+ complex loads the AAA+ MCM helicase to origins of replication, but whether and how ORC collaborates with Pch2 remains unclear. Here, we show that a Pch2 hexamer directly associates with ORC during the meiotic G2/prophase. Biochemical analysis suggests that Pch2 uses its non-enzymatic NH2-terminal domain and AAA+ core and likely engages the interface of ORC that also binds to Cdc6, a factor crucial for ORC-MCM binding. Canonical ORC function requires association with origins, but we show here that despite causing efficient removal of Orc1 from origins, nuclear depletion of Orc2 and Orc5 does not trigger Pch2/Orc1-like meiotic phenotypes. This suggests that the function for Orc1/Pch2 in meiosis can be executed without efficient association of ORC with origins of replication. In conclusion, we uncover distinct functionalities for Orc1/ORC that drive the establishment of a non-canonical, meiosis-specific AAA+ assembly with Pch2.

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.

Extranuclear Structural Components that Mediate Dynamic Chromosome Movements in Yeast Meiosis.

Telomere-led rapid chromosome movements or rapid prophase movements direct fundamental meiotic processes required for successful haploidization of the genome. Critical components of the machinery that generates rapid prophase movements are unknown, and the mechanism underlying rapid prophase movements remains poorly understood. We identified S. cerevisiae Mps2 as the outer nuclear membrane protein that connects the LINC complex with the cytoskeleton. We also demonstrate that the motor Myo2 works together with Mps2 to couple the telomeres to the actin cytoskeleton. Further, we show that Csm4 interacts with Mps2 and is required for perinuclear localization of Myo2, implicating Csm4 as a regulator of the Mps2-Myo2 interaction. We propose a model in which the newly identified functions of Mps2 and Myo2 cooperate with Csm4 to drive chromosome movements in meiotic prophase by coupling telomeres to the actin cytoskeleton.

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.

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.

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.

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells.

The mitotic spindle is a dynamic bipolar structure that mediates chromosome segregation in mitosis. In most organisms, spindle formation requires the action of kinesin-5 motor proteins that generate outward force on antiparallel microtubules to establish spindle bipolarity. Previous work has shown that Eg5 and TPX2, a spindle microtubule-associated protein that suppresses Eg5 motor activity, are enriched on parallel microtubules near spindle poles. This distribution is inconsistent with the requirement for Eg5-dependent force production during mitosis. To investigate this, we used CRISPR/Cas9 gene editing to tag Eg5 and TPX2 with EGFP and quantify protein distribution throughout mitosis. The results show that at metaphase both Eg5-EGFP and TPX2-EGFP are enriched toward spindle poles, but only TPX2-EGFP is enriched relative to microtubules. Eg5-EGFP and TPX2-EGFP show distinct localization patterns in anaphase, with Eg5-EGFP relocalizing to the midzone earlier than TPX2-EGFP. Analysis of spindles oriented at 90° to the coverslip confirmed that Eg5-EGFP was present on bridge microtubules in metaphase and anaphase; in contrast, TPX2 was not enriched, or enriched at later times, on these microtubules. Overall, TPX2 was present at 3.6X the level of Eg5 on the spindle and Eg5 was locally enriched at the prophase centrosome (~7×) compared to the whole cell. Our results show that using cells with fluorescent tags at the endogenous locus can provide novel insight into protein distribution during mitosis.

The preprophase band of microtubules controls the robustness of division orientation in plants.

Controlling cell division plane orientation is essential for morphogenesis in multicellular organisms. In plant cells, the future cortical division plane is marked before mitotic entry by the preprophase band (PPB). Here, we characterized an Arabidopsis trm (TON1 Recruiting Motif) mutant that impairs PPB formation but does not affect interphase microtubules. Unexpectedly, PPB disruption neither abolished the capacity of root cells to define a cortical division zone nor induced aberrant cell division patterns but rather caused a loss of precision in cell division orientation. Our results advocate for a reassessment of PPB function and division plane determination in plants and show that a main output of this microtubule array is to limit spindle rotations in order to increase the robustness of cell division.

The Evolution of Cell Division: From Streptophyte Algae to Land Plants.

The mechanism of cell division has undergone significant alterations during the evolution from aquatic streptophyte algae to land plants. Two new structures evolved, the cytokinetic phragmoplast and the preprophase band (PPB) of microtubules, whereas the ancestral mechanism of cleavage and the centrosomes disappeared. We map cell biological data onto the recently emerged phylogenetic tree of streptophytes. The tree suggests that, after the establishment of the phragmoplast mechanism, several groups independently lost their centrosomes. Surprisingly, the phragmoplast shows reductions in the Zygnematophyceae (the sister to land plants), many of which returned to cleavage. The PPB by contrast evolved stepwise and, most likely, originated in the algae. The phragmoplast/PPB mechanism established in this way served as a basis for the 3D development of land plants.

Sister chromatid resolution is an intrinsic part of chromosome organization in prophase.

The formation of mitotic chromosomes requires both compaction of chromatin and the resolution of replicated sister chromatids. Compaction occurs during mitotic prophase and prometaphase, and in prophase relies on the activity of condensin II complexes. Exactly when and how sister chromatid resolution occurs has been largely unknown, as has its molecular requirements. Here, we established a method to visualize sister resolution by sequential replication labelling with two distinct nucleotide derivatives. Quantitative three-dimensional imaging then allowed us to measure the resolution of sister chromatids throughout mitosis by calculating their non-overlapping volume within the whole chromosome. Unexpectedly, we found that sister chromatid resolution starts already at the beginning of prophase, proceeds concomitantly with chromatin compaction and is largely completed by the end of prophase. Sister chromatid resolution was abolished by inhibition of topoisomerase IIα and by depleting or preventing mitotic activation of condensin II, whereas blocking cohesin dissociation from chromosomes had little effect. Mitotic sister chromatid resolution is thus an intrinsic part of mitotic chromosome formation in prophase that relies largely on DNA decatenation and shares the molecular requirement for condensin II with prophase compaction.

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.

Double-strand break repair on sex chromosomes: challenges during male meiotic prophase.

During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with many DNA damage response proteins and form a unique chromatin structure known as the XY body. Interestingly, associated with the prolonged DSB repair, transcription is repressed in the XY body but not in autosomes, a phenomenon known as meiotic sex chromosome inactivation (MSCI), which is critical for male meiosis. Here using mice as model organisms, we briefly summarize recent progress on DSB repair in meiotic prophase and focus on the mechanism and function of DNA damage response in the XY body.

Microtubule organization and microtubule-associated proteins in plant cells.

Plants have unique microtubule (MT) arrays, cortical MTs, preprophase band, mitotic spindle, and phragmoplast, in the processes of evolution. These MT arrays control the directions of cell division and expansion especially in plants and are essential for plant morphogenesis and developments. Organizations and functions of these MT arrays are accomplished by diverse MT-associated proteins (MAPs). This review introduces 10 of conserved MAPs in eukaryote such as γ-TuC, augmin, katanin, kinesin, EB1, CLASP, MOR1/MAP215, MAP65, TPX2, formin, and several plant-specific MAPs such as CSI1, SPR2, MAP70, WVD2/WDL, RIP/MIDD, SPR1, MAP18/PCaP, EDE1, and MAP190. Most of the studies cited in this review have been analyzed in the particular model plant, Arabidopsis thaliana. The significant knowledge of A. thaliana is the important established base to understand MT organizations and functions in plants.

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.

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.

The role of chromatin modifications in progression through mouse meiotic prophase.

Meiosis is a key event in gametogenesis that generates new combinations of genetic information and is required to reduce the chromosome content of the gametes. Meiotic chromosomes undergo a number of specialised events during prophase to allow meiotic recombination, homologous chromosome synapsis and reductional chromosome segregation to occur. In mammalian cells, DNA physically associates with histones to form chromatin, which can be modified by methylation, phosphorylation, ubiquitination and acetylation to help regulate higher order chromatin structure, gene expression, and chromosome organisation. Recent studies have identified some of the enzymes responsible for generating chromatin modifications in meiotic mammalian cells, and shown that these chromatin modifying enzymes are required for key meiosis-specific events that occur during meiotic prophase. This review will discuss the role of chromatin modifications in meiotic recombination, homologous chromosome synapsis and regulation of meiotic gene expression in mammals.

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.