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In brief Oocyte quality remains the most important and unsolved issue in reproduction. Our data show that multidrug resistance transporters and oocyte mitochondria are involved in determining oocyte quality in a mouse model. Abstract Multidrug resistance transporter-1 (MDR-1) is a transmembrane ATP-dependent effluxer present in organs that transport a variety of xenobiotics and by-products. Previous findings by our group demonstrated that this transporter is also present in the oocyte mitochondrial membrane and that its mutation led to abnormal mitochondrial homeostasis. Considering the importance of these organelles in the female gamete, we assessed the impact of MDR-1 dysfunction on mouse oocyte quality, with a particular focus on the meiotic spindle organization, aneuploidies, Ca2+ homeostasis, ATP production and mtDNA mutations. Our results demonstrate that young Mdr1a mutant mice produce oocytes characterized by lower quality, with a significant delay in the germinal vesicle to germinal vesicle breakdown transition, an increased percentage of symmetric divisions, chromosome misalignments and a severely altered meiotic spindle shape compared to the wild types. Mutant oocytes exhibit 7000 more SNPs in the exomic DNA and twice the amount of mitochondrial DNA (mtDNA) SNPs compared to the wild-type ones. Ca2+ analysis revealed the inability of MDR-1 mutant oocytes to manage Ca2+ storage content and oscillations in response to several stimuli, and ATP quantification shows that mutant oocytes trend toward lower ATP levels compared to wild types. Finally, 1-year-old mutant ovaries express a lower amount of SIRT1, SIRT3, SIRT5, SIRT6 and SIRT7 compared to wild-type levels. These results together emphasize the importance of MDR-1 in mitochondrial physiology and highlight the influence of MDR-1 on oocyte quality and ovarian aging.

Wnt signalling is an essential player in tissue formation, notably in the regulation of stem cell function. Wnt signalling is best known for its roles in G1/S progression. However, a complex Wnt programme that also mediates mitotic progression and asymmetric cell division (ACD) is emerging. Recent developments in this area have provided mechanistic insights as well as tools to engineer or target Wnt signalling for translational and therapeutic purposes. Here, we discuss the bidirectional relationship between Wnt activity and mitosis. We emphasise how various Wnt-dependent mechanisms control spindle dynamics, chromosome segregation, and ACD. Finally, we illustrate how knowledge about these mechanisms has been successfully employed in tissue engineering for regenerative medicine applications.

In the mitotic spindle, microtubules attach to chromosomes via kinetochores. The microtubule-binding Ndc80 complex is an integral part of kinetochores, and is essential for kinetochores to attach to microtubules and to transmit forces from dynamic microtubule ends to the chromosomes. The Ndc80 complex has a rod-like appearance with globular domains at its ends that are separated by a long coiled coil. Its mechanical properties are considered important for the dynamic interaction between kinetochores and microtubules. Here, we present a novel method that allows us to time-trace the effective stiffness of Ndc80 complexes following shortening microtubule ends against applied force in optical trap experiments. Applying this method to wild type Ndc80 and three variants (CH-domains mutated or Hec1-tail unphosphorylated, phosphorylated, or truncated), we reveal that each variant exhibits strain stiffening, i.e., the effective stiffness increases under tension that is built up by a depolymerizing microtubule. The strain stiffening relation is roughly linear and independent of the state of the microtubule. We introduce structure-based models, which show that the strain stiffening can be traced back to the specific architecture of the Ndc80 complex with a characteristic flexible kink, to thermal fluctuations of the microtubule, and to the bending elasticity of flaring protofilaments, which exert force to move the Ndc80 complexes. Our model accounts for changes in the amount of load-bearing attachments at various force levels and reproduces the roughly linear strain stiffening behavior, highlighting the importance of force-dependent binding affinity.SIGNIFICANCEBy time-tracing the stiffness of microtubule end-tracking Ndc80 complexes in optical trap experiments, we detect strain stiffening, and, thereby, provide new insights into the elastic properties of the Ndc80 complex. The strain stiffening is robust against mutations in the Ndc80 complex. We relate strain stiffening to the structure of the Ndc80 complex by means of a simple polymer model, to thermal fluctuations of the microtubule, and to the flexibility of force-generating flaring protofilaments at the tip of the microtubule. Since Ndc80 complexes play a major role for transmitting force from microtubule ends to the kinetochore, their elastic properties are of great interest for a deeper understanding of chromosome dynamics in the mitotic spindle.

The midbody is a transient structure forming out of the central spindle at late telophase. Both the midbody and central spindle have important functions ensuring completion of cytokinesis and defects in this process may lead to genetic diseases, including cancer. Thus, understanding the mechanisms that control cytokinesis during mitosis can reveal the key components taking part in some of the processes that promote accurate cell division. Our previous study showed that overexpression of FLJ25439 causes cytokinesis defect with midbody arrest and induces tetraploids with prolonged cell growth/cell cycle progression (Pan et al., 2015). Here, we extend our investigation with regard to the expression profile/regulation and cellular localization/function of FLJ25439 during mitosis/cytokinesis. Using a monoclonal antibody 2A4 we found that FLJ25439 expression is cell cycle-dependent and subjected to APC/C complex regulation. Furthermore, it is a novel substrate for the APC/C-Cdc20 complex and its degradation is proteasome-dependent through D-box recognition during mitotic exit. Immunofluorescence microscopy showed it is distributed at the central spindle and midbody, two structures considered important for completion of cell division, in telophase and cytokinesis, respectively, during cell cycle progression. Depletion of FLJ25439 expression revealed defects in chromosome alignment/segregation and delayed mitosis/cytokinesis progression. We thus conclude that FLJ25439 is a hitherto undiscovered factor involved in cytokinesis regulation.

Asymmetric cell division gives rise to two daughter cells that inherit different determinants, thereby acquiring different fates. Polarized trafficking of endosomes containing fate determinants recently emerged as an evolutionarily conserved feature of asymmetric cell division to enhance the robustness of asymmetric cell fate determination in flies, fish and mammals. In particular, polarized sorting of signalling endosomes by an asymmetric central spindle contributes to asymmetric cell division in Drosophila melanogaster. However, how central spindle asymmetry arises remains elusive. Here we identify a moonlighting function of the Elongator complex-an established protein acetylase and tRNA methylase involved in the fidelity of protein translation-as a key factor for central spindle asymmetry. Elongator controls spindle asymmetry by stabilizing microtubules differentially on the anterior side of the central spindle. Accordingly, lowering the activity of Elongator on the anterior side using nanobodies mistargets endosomes to the wrong cell. Molecularly, Elongator regulates microtubule dynamics independently of its acetylation and methylation enzymatic activities. Instead, Elongator directly binds to microtubules and increases their polymerization speed while decreasing their catastrophe frequency. Our data establish a non-canonical role of Elongator at the core of cytoskeleton polarity and asymmetric signalling.

The microtubule (MT) cytoskeleton provides the architecture that governs intracellular organization and the regulated motion of macromolecules through the crowded cytoplasm. The key to establishing a functioning cytoskeletal architecture is regulating when and where new MTs are nucleated. Within the spindle, the vast majority of MTs are generated through a pathway known as branching MT nucleation, which exponentially amplifies MT number in a polar manner. Whereas other MT nucleation pathways generally require a complex organelle such as the centrosome or Golgi apparatus to localize nucleation factors, the branching site is based solely on a simple, preformed MT, making it an ideal system to study MT nucleation. In this review, we address recent developments in characterizing branching factors, the branching reaction, and its regulation, as well as branching MT nucleation in systems beyond the spindle and within human disease.

Cilia and mitotic spindles are microtubule (MT)-based, macromolecular machines that consecutively assemble and disassemble during interphase and M phase of the cell cycle, respectively, and play fundamental roles in how eukaryotic cells swim through a fluid, sense their environment, and divide to reproduce themselves. The formation and function of these structures depend on several types of cytoskeletal motors, notably MT-based kinesins and dyneins, supplemented by actin-based myosins, which may function independently or collaboratively during specific steps in the pathway of mitosis or ciliogenesis. System-specific differences in these pathways occur because, instead of conforming to a simple one motor–one function rule, ciliary and mitotic motors can be deployed differently by different cell types. This reflects the well-known influence of natural selection on basic molecular processes, creating diversity at subcellular scales. Here we review our current understanding of motor function and cooperation during the assembly–disassembly, maintenance, and functions of cilia and mitotic spindles.

Early cellular patterning is a critical step of embryonic development that determines the proper progression of morphogenesis in all metazoans. It relies on a series of rapid reductive divisions occurring simultaneously with the specification of the fate of different subsets of cells. Multiple species developmental strategies emerged in the form of a unique cleavage pattern with stereotyped division geometries. Cleavage geometries have long been associated to the emergence of canonical developmental features such as cell cycle asynchrony, zygotic genome activation and fate specification. Yet, the direct causal role of division positioning on blastomere cell behavior remain partially understood. Oriented and/or asymmetric divisions define blastomere cell sizes, contacts and positions, with potential immediate impact on cellular decisions, lineage specification and morphogenesis. Division positions also instruct daughter cells polarity, mechanics and geometries, thereby influencing subsequent division events, in an emergent interplay that may pattern early embryos independently of firm deterministic genetic programs. We here review the recent literature which helped to delineate mechanisms and functions of division positioning in early embryos.

SummaryMicrotubule-severing proteins (MTSPs) play important roles in mitosis and interphase. However, to the best of our knowledge, no previous studies have evaluated the role of MTSPs in female meiosis in mammals. It was found that FIGNL1, a member of MTSPs, was predominantly expressed in mouse oocytes and distributed at the spindle poles during meiosis in the present study. FIGNL1 was co-localized and interacted with γ-tubulin, an important component of the microtubule tissue centre (MTOC). Fignl1 knockdown by specific small interfering RNA caused spindle defects characterized by an abnormal length:width ratio and decreased microtubule density, which consequently led to aberrant chromosome arrangement, oocyte maturation and fertilization obstacles. In conclusion, the present results suggested that FIGNL1 may be an essential factor in oocyte maturation by influencing the meiosis process via the formation of spindles.

The kinetochore is a large proteinaceous structure assembled on the centromeres of chromosomes. The complex machinery links chromosomes to the mitotic spindle and is essential for accurate chromosome segregation during cell division. The kinetochore is composed of two submodules: the inner and outer kinetochore. The inner kinetochore is assembled on centromeric chromatin and persists with centromeres throughout the cell cycle. The outer kinetochore attaches microtubules to the inner kinetochore, and assembles only during mitosis. The review focuses on recent advances in our understanding of the mechanisms governing the proper assembly of the outer kinetochore during mitosis and highlights open questions for future investigation.

Cytokinesis is a mechanism that separates dividing cells via constriction of a supramolecular structure, the contractile ring. In animal cells, three modes of symmetry-breaking of cytokinesis result in unilateral cytokinesis, asymmetric cell division, and oriented cell division. Each mode of cytokinesis plays a significant role in tissue patterning and morphogenesis by the mechanisms that control the orientation and position of the contractile ring relative to the body axis. Despite its significance, the mechanisms involved in the symmetry-breaking of cytokinesis remain unclear in many cell types. Classical embryologists have identified that the geometric relationship between the mitotic spindle and cell cortex induces cytokinesis asymmetry; however, emerging evidence suggests that a concerted flow of compressional cell-cortex materials (cortical flow) is a spindle-independent driving force in spatial cytokinesis control. This review provides an overview of both classical and emerging mechanisms of cytokinesis asymmetry and their roles in animal development.

Sirtuins play an important role in female mammalian reproductive function, participating in folliculogenesis and oocyte maturation. Studies exploring the consequences of inhibition/deletion of a specific sirtuin (SIRT) have demonstrated a deleterious effect on follicular growth, oocyte maturation, fertilization rates and embryo development, suggesting that sirtuins must have a relevant role in these processes. However, the exact mechanisms behind sirtuin function are still unclear. Most of the knowledge currently available derives from mouse studies and the literature is scarce in other species. So far, there is insufficient information about the subcellular localization of sirtuins during bovine meiosis, which would contribute to understanding the role and participation of sirtuins in the process of oocyte maturation, due to the close relation between location and function. Using in vitro maturation (IVM) of bovine oocytes we comprehensively documented and illustrated the subcellular localization pattern and distribution of SIRT1, 2 and 3 during meiotic progression. Moreover, we also detailed and quantified the colocalization of those sirtuins with the meiotic spindle, from the germinal-vesicle (GV)-stage until the Metaphase-II (MII)-stage. Our study demonstrated an increase in the expression of SIRT1, 2 and 3 during in vitro oocyte maturation and, for the first time, colocalization of SIRT1, 2 and 3 with both metaphase-I and metaphase-II spindles. These findings suggest that all three sirtuins may have a role in meiotic spindle assembly and microtubule dynamics in the bovine model. In addition, we have demonstrated the nuclear presence of SIRT1 and SIRT2 in the GV-stage. The apparent perinucleolar location of SIRT2 suggests that SIRT2 may shuttle into the nucleus at the GV-stage to regulate heterochromatin. This study reinforces the value of sirtuins during in vitro bovine meiotic progression and indicates potential molecular targets to improve maturation rates and embryo development.

The targeting protein for Xklp2 (TPX2) is a spindle assembly factor, that can stimulate microtubule formation and promote spindle completion during mitosis. However, the role of TPX2 in mammalian oocyte meiotic maturation is still not fully understood. This study was conducted to address the dynamic distribution and potential roles of TPX2 in microtubule nucleation during meiotic maturation in porcine oocytes by microinjecting specific siRNAs. Western blotting results revealed that the expression of TPX2 displayed a lower level from 0 to 22 h of culture, while its expression exhibited a higher level after 28 h of culture. Immunofluorescence staining demonstrated that TPX2 was distributed along the microtubules and enriched in the poles after meiotic spindle formation at the 28 and 44 h of culture. From immunoprecipitation, TPX2 can interact with the microtubule-associated proteins aurora kinase A (AURKA) and transforming acidic coiled-coil 3 (TACC3). Meanwhile, the dynamic changes in the expression and localization of AURKA and TACC3 were highly consistent with TPX2 during meiotic maturation. After knocking down TPX2 by siRNA injection, the proportion of oocytes with aberrant spindles and scattered cytoplasmic actin filaments was significantly increased. In addition, TPX2 depletion markedly downregulated the expression of AURKA and TACC3. Thus, these results suggested that TPX2 is essential for meiotic spindle formation in the porcine oocyte, and that this regulation is related to its effect on AURKA and TACC3 expression.

We have presented an in vitro trackable model system, atavistic induced from conservation in our genome, which strongly is applicable to tumorigenesis start and evolution. The inducing factor was death signals to proliferating normal human cells (primary cell strains), which respon-ded by a special type of tetraploidization, chromosomes with 4-chromatids (diplochromosomes, earlier described in cancer cells). The response included cell cycle stress, which prolonged S-period with result of mitotic slippage process, forming the special 4n cells by re-replication of diploid cells, which showed cell division capability to unexpected, genome reduced diploid cells which remarkably, showed fitness gain. This unique response through cell cycle stress and mitotic slippage process was further discovered to be linked to a rather special characteristic of the, 4n nucleus. The nucleus turned, self-inflicted, 90° perpendicular to the cell's cytoskeleton axis, importantly, before the special 4n-division system produced genome reduce diploid cells, we call "first cells", because of fitness gain. These 2n cells also showed the nuclear dependent 90° turn, which in both cases was associated with cells gaining cell shape changes, herein illustrated from normal fibroblastic cells changing to roundness cells, indistinguishable from todays' diagnostic cancer cell morphology. This 3-D ball-like cell shape, in metastasis, sque-ezing in and out between (?) endothelial cells in the lining of blood veins during disbursement, would be advantageous.

The spindle is a dynamic intracellular structure self-organized from microtubules and microtubule-associated proteins. The spindle’s bipolar morphology is essential for the faithful segregation of chromosomes during cell division, and it is robustly maintained by multifaceted mechanisms. However, abnormally shaped spindles, such as multipolar spindles, can stochastically arise in a cell population and cause chromosome segregation errors. The physical basis of how microtubules fail in bipolarization and occasionally favor nonbipolar assembly is poorly understood. Here, using live fluorescence imaging and quantitative shape analysis in Xenopus egg extracts, we find that spindles of varied shape morphologies emerge through nonrandom, bistable self-organization paths, one leading to a bipolar and the other leading to a multipolar phenotype. The bistability defines the spindle’s unique morphological growth dynamics linked to each shape phenotype and can be promoted by a locally distorted microtubule flow that arises within premature structures. We also find that bipolar and multipolar spindles are stable at the steady-state in bulk but can infrequently switch between the two phenotypes. Our microneedle-based physical manipulation further demonstrates that a transient force perturbation applied near the assembled pole can trigger the phenotypic switching, revealing the mechanical plasticity of the spindle. Together with molecular perturbation of kinesin-5 and augmin, our data propose the physical and molecular bases underlying the emergence of spindle-shape variation, which influences chromosome segregation fidelity during cell division.

Oriented cell divisions balance self-renewal and differentiation in stratified epithelia such as the skin epidermis. During peak epidermal stratification, the distribution of division angles among basal keratinocyte progenitors is bimodal, with planar and perpendicular divisions driving symmetric and asymmetric daughter cell fates, respectively. An apically-polarized, evolutionarily-conserved spindle orientation complex that includes the scaffolding protein LGN/Pins/Gpsm2 plays a central role in promoting perpendicular divisions and stratification, but little is known about the molecular regulation of planar divisions. Here, we demonstrate that the LGN paralog, AGS3/Gpsm1, is a novel negative regulator of LGN, and inhibits perpendicular divisions. Static and ex vivo live imaging reveal that AGS3 overexpression displaces LGN from the apical cortex and increases planar orientations, while AGS3 loss prolongs cortical LGN localization and leads to a perpendicular orientation bias. Genetic epistasis experiments in double mutants confirm that AGS3 operates through LGN. Finally, clonal lineage tracing shows that LGN and AGS3 promote asymmetric and symmetric fates, respectively, while also influencing differentiation through delamination. Collectively, these studies shed new light into how spindle orientation influences epidermal stratification.

Chromosome segregation requires load-bearing interactions across kinetochore fibers and antiparallel microtubule bundles, which constitute the spindle midzone. Mechanical properties of kinetochore fibers have been characterized during metaphase, when the mitotic spindle achieves steady state. However, it has been difficult to probe the mechanics of the spindle midzone that elongates during anaphase. Here, we combine superresolution expansion and electron microscopies, lattice light-sheet imaging, and laser microsurgery to examine how midzone organization sets its mechanics. We find that individual midzone bundles extend out to multiple positions across chromosomes and form multiple apparent microtubule-based connections with each other. Across the spindle’s short axis, these microtubule bundles exhibit restricted, submicrometer-amplitude motions, which are weakly correlated on

Genetic information is replicated and transmitted from a parent cell to two identical daughter cells through mitotic cell division. To accomplish this dynamic process with high accuracy and precision, various motor proteins work in a concerted manner. Especially in the metaphase, mitotic chromosomes are delivered by the motor protein of centromere-associated protein E (CENP-E) to the cell equatorial plane (metaphase plate) along mitotic spindles. However, the critical functional failure of CENP-E can activate the spindle assembly checkpoint through the misalignment of chromosomes at the metaphase plate. In this symposium review, the reversibly photoswitchable CENP-E inhibitor PCEI-HU (5) is reported. Compound 5 exhibited almost quantitative trans-cis photoisomerization of the arylazopyrazole photoswitch by illuminating light at 365 nm and 510 nm. Depending on the photoisomerization, CENP-E activity was regulated not only in vitro but also in cells. We successfully established a novel technique using 5 to dynamically photocontrol the CENP-E-dependent chromosome movement and mitotic progression in a living cell.

Timely and accurate centrosome separation is critical for bipolar spindle organization and faithful chromosome segregation during cell division. Kinesin-5 Eg5 is essential for centrosome separation and spindle organization in somatic cells; however, the detailed functions and mechanisms of Eg5 in spermatocytes remain unclear. In this study, we show that Eg5 proteins are located at spindle microtubules and centrosomes in spermatocytes both in vivo and in vitro. We reveal that the spermatocytes are arrested at metaphase I in seminiferous tubules after Eg5 inhibition. Eg5 ablation results in cell cycle arrest, the formation of monopolar spindle, and chromosome misalignment in cultured GC-2 spd cells. Importantly, we find that the long-term inhibition of Eg5 results in an increased number of centrosomes and chromosomal instability in spermatocytes. Our findings indicate that Eg5 mediates centrosome separation to control spindle assembly and chromosome alignment in spermatocytes, which finally contribute to chromosome stability and faithful cell division of the spermatocytes.

Oocyte vitrification, while beneficial for research and species conservation applications, has limited success due to cryoinjury to the meiotic spindle. This study aimed to improve meiotic spindle recovery in vitrified bovine oocytes by investigating the effects of treatment with either a microtubule stabilizing agent, or a microtubule recovery agent. In the first two experiments, either paclitaxel or epothilone B were used to treat bovine oocytes before vitrification. Both compounds have microtubule stabilizing properties and are known antimitotic compounds used to disrupt microtubule dynamics in rapidly proliferating cancer cells. Paclitaxel treatment at 2.0 μM significantly increased the proportion of oocytes with normal microtubule distribution and chromosome arrangement after warming. Treatment with 1.0 μM had no effect and 0.5 μM had a negative effect on meiotic spindle recovery. Epothilone B treatment at all concentrations significantly increased the proportion of oocytes with meiotic spindle disruption and abnormally dispersed chromosomes. In the second set of experiments, Rho-associated coiled-coil kinase inhibition and glutathione accumulation were investigated as recovery treatments after vitrification. Oocytes were incubated with either Y-27632 or combinations of cysteine and cysteamine for 4 h after warming. Treatment with 5 μM and 10 μM of Y-27632 to inhibit rho-associated coiled-coil kinase activity significantly increased the proportion of vitrified oocytes with normal microtubule distribution and chromosome arrangement. When oocytes were incubated with 20 μM of Y-27632 there was no effect on spindle recovery. Incubation with 100 μM of cysteamine also had no effect on spindle recovery while 0.6 mM of cysteine and both 0.6 mM of cysteine and 100 μM of cysteamine significantly increased oocytes with normal microtubule distribution and chromosome arrangement.

Stratification of the epidermis is essential for the barrier function of the skin. However, the molecular mechanisms governing epidermal stratification are not fully understood. Herein, we demonstrate that enkurin domain-containing protein 1 (ENKD1) contributes to epidermal stratification by modulating the cell-division orientation of basal keratinocytes. The epidermis of Enkd1 knockout mice is thinner than that of wild-type mice due to reduced generation of suprabasal cells from basal keratinocytes through asymmetric division. Depletion of ENKD1 impairs proper orientation of the mitotic spindle and delays mitotic progression in cultured cells. Mechanistic investigation further reveals that ENKD1 is a novel microtubule-binding protein that promotes the stability of astral microtubules. Introduction of the microtubule-binding domain of ENKD1 can largely rescue the spindle orientation defects in ENKD1-depleted cells. These findings establish ENKD1 as a critical regulator of astral microtubule stability and spindle orientation that stimulates epidermal stratification in mammalian cells.

The mitotic spindle is a highly dynamic bipolar structure that emerges from the self-organization of microtubules, molecular motors and other proteins. Sustained motor-driven poleward flows of dynamic microtubules play a key role in the bipolar organization of spindles. However, it is not understood how the local activity of motor proteins generates these large-scale coherent poleward flows. Here we show that a gelation transition enables long-range microtubule transport causing the spindles to self-organize into two oppositely polarized microtubule gels. Laser ablation experiments reveal that local active stresses generated at the spindle midplane propagate through the structure, thereby driving global coherent microtubule flows. Simulations show that microtubule gels undergoing rapid turnover can exhibit long stress relaxation times, in agreement with the long-range flows observed in experiments. Finally, our model predicts that in the presence of branching microtubule nucleation, either disrupting such flows or decreasing the network connectivity can lead to a microtubule polarity reversal in spindles. We experimentally confirm this inversion of polarity by abolishing the microtubule transport in spindles. Overall, we uncover a connection between spindle rheology and architecture in spindle self-organization.

The Chromosomal Passenger Complex (CPC) regulates a plethora of processes during multiple stages of nuclear and cytoplasmic division. Early during mitosis, the CPC is recruited to centromeres and kinetochores, and ensures that the duplicated chromosomes become properly connected to microtubules from opposite poles of the mitotic spindle. Progression into anaphase is accompanied by a striking relocation of the CPC from centromeres to the antiparallel microtubule overlaps of the anaphase spindle and to the equatorial cortex. This translocation requires direct interactions of the CPC with the kinesin-6 family member MKLP2/KIF20A, and the inactivation of cyclin B-cyclin-dependent kinase-1 (CDK1). Here, we review recent progress in the regulation of this relocation event. Furthermore, we discuss why the CPC must be relocated during early anaphase in light of recent advances in the functions of the CPC post metaphase.

Objective To determine if a cytoplasmic diameter of ≥130 μm can help identify human giant oocytes (GOs) in clinical practice and confirm the presence of genetic abnormalities in GOs by assessing the spindle length and centromere numbers. Design Case–control study Setting Private in vitro fertilization clinic Patient(s) The subjects were females aged 20–49 years who underwent oocyte retrieval following ovarian stimulation from January 2014 to December 2020. Intervention(s) None Main Outcome Measure(s) Oocyte diameter was measured; immunofluorescent staining was performed to assess the spindle diameter and centromere numbers. Results Among the oocytes examined, 0.22% (561/254,337) had a diameter of ≥130 μm. The mean diameter range in the normal-sized metaphase II (MII) oocytes (MII group) was 103.0–119.0 μm and 132.3–175.9 μm in the GO group. Spindle size could be measured in 6 GOs with one spindle (GO1), 10 GOs with 2 spindles (GO2) and 16 MII groups. The equatorial plane and pole-to-pole distance in the GO1 were significantly longer than GO2 and MII groups. A median of 86 centromeres in GOs with one spindle and 42 in each spindle for GOs with 2 spindles was observed among 11 GO1s and 5 GO2s. Conclusions This study is the first to define GOs as oocytes with a diameter of ≥130 μm and is a large-scale study surveying the incidence of GO. It is also the first study to analyze and elucidate the relationship between spindle numbers within the cytoplasm of GOs and spindle size and centromeres.

Background The neurofibromatosis type 2 (NF2) gene mutation is the leading genetic event in meningiomas, usually accompanied by malignant features. Dysfunction of the spindle assembly checkpoint (SAC) induces tumorigenesis. However, the crosstalk between NF2 and SAC in meningiomas remains unclear. Methods Cell proliferation, invasion, apoptosis, and cell cycle of meningiomas were determined by cell counting kit-8 assay, transwell assay, and flow cytometry, respectively. The expression of SAC in meningioma cells was detected by quantitative real-time polymerase chain reaction and Western blot. The interaction between anaphase promoting complex/cyclosome (APC/C) and cell division cycle 20 (Cdc20) protein in meningioma cells was further explored by co-immunoprecipitation. Results We found that the expression of NF2/merlin was low or absent in malignant meningiomas. Overexpression of NF2 suppressed the proliferation and invasion of meningioma cells, prolonged the G2/M phase, and elevated the expression of SAC proteins at posttranscription. Furthermore, the interaction between APC/C and Cdc20 was inhibited by NF2. Conclusions Our findings suggested that NF2 might restore SAC function by impairing the binding of APC/C and Cdc20, thereby limiting the mitotic rate and inhibiting proliferation of meningiomas.

Funder: Boehringer Ingelheim Fonds, Funder: Bonn University, Funder: Max Planck Society, Funder: Carnegie Mellon University, The key to ensuring proper chromosome segregation during mitosis is the kinetochore (KT), a tightly regulated multiprotein complex that links the centromeric chromatin to the spindle microtubules and as such leads the segregation process. Understanding its architecture, function, and regulation is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in structural detail so far. In this study, we construct a nanometer-precise in situ map of the human-like regional KT of Schizosaccharomyces pombe using multi-color single-molecule localization microscopy. We measure each protein of interest (POI) in conjunction with two references, cnp1CENP-A at the centromere and sad1 at the spindle pole. This allows us to determine cell cycle and mitotic plane, and to visualize individual centromere regions separately. We determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI and, consequently, build an in situ KT model with unprecedented precision, providing new insights into the architecture.

Backgrounds & Aims Increased permeability is implicated in the pathogenesis of intestinal disease. In vitro and in vivo studies have linked down-regulation of the scaffolding protein ZO-1, encoded by the TJP1 gene, to increased tight junction permeability. This has not, however, been tested in vivo. Here, we assessed the contributions of ZO-1 to in vivo epithelial barrier function and mucosal homeostasis. Methods Public Gene Expression Omnibus data sets and biopsy specimens from patients with inflammatory bowel disease (IBD) and healthy control individuals were analyzed. Tjp1f/f;vil-CreTg mice with intestinal epithelial–specific ZO-1 knockout (ZO-1KO.IEC) mice and Tjp1f/f mice littermates without Cre expression were studied using chemical and immune-mediated models of disease as well as colonic stem cell cultures. Results ZO-1 transcript and protein expression were reduced in biopsy specimens from patients with IBD. Despite mildly increased intestinal permeability, ZO-1KO.IEC mice were healthy and did not develop spontaneous disease. ZO-1KO.IEC mice were, however, hypersensitive to mucosal insults and displayed defective repair. Furthermore, ZO-1–deficient colonic epithelia failed to up-regulate proliferation in response to damage in vivo or Wnt signaling in vitro. ZO-1 was associated with centrioles in interphase cells and mitotic spindle poles during division. In the absence of ZO-1, mitotic spindles failed to correctly orient, resulting in mitotic catastrophe and abortive proliferation. ZO-1 is, therefore, critical for up-regulation of epithelial proliferation and successful completion of mitosis. Conclusions ZO-1 makes critical, tight junction–independent contributions to Wnt signaling and mitotic spindle orientation. As a result, ZO-1 is essential for mucosal repair. We speculate that ZO-1 down-regulation may be one cause of ineffective mucosal healing in patients with IBD.

The effects of ionizing radiation on centrosomes have been well documented and reviewed by Saladino et al. (2012) and are only briefly addressed here. These results showed that exposure of tumor cells to ionizing radiation causes centrosome overduplication and the formation of multipolar mitotic spindles, resulting in nuclear fragmentation and subsequent cell death (Sato et al. 2000). By using a variety of cell lines derived from different types of human solid tumors, it was shown that exposure to 10 Gy γ-radiation resulted in a substantial increase in cells containing an abnormally high number of aberrant centrosomes that formed multipolar spindles, resulting in imbalanced chromosome separation followed by mitotic cell death and formation of multi- or micronucleated cells.

The ability to proliferate is a common feature of most T-cell populations. However, proliferation follows different cell-cycle dynamics and is coupled to different functional outcomes according to T-cell subsets. Whether the mitotic machineries supporting these qualitatively distinct proliferative responses are identical remains unknown. Here, we show that disruption of the microtubule-associated protein LIS1 leads to proliferative defects associated with a blockade of T-cell development after β-selection and of peripheral CD4+ T cell expansion after antigen priming. In contrast, cell divisions in CD8+ T cells occurred independently of LIS1 following T-cell antigen receptor stimulation, although LIS1 was required for proliferation elicited by pharmacological activation. In thymocytes and CD4+ T cells, LIS1-deficiency did not affect signaling events leading to activation but led to an interruption of proliferation after the initial round of division and to p53-induced cell death. Proliferative defects resulted from a mitotic failure, characterized by the presence of extra-centrosomes and the formation of multipolar spindles, causing abnormal chromosomes congression during metaphase and separation during telophase. LIS1 was required to stabilize dynein/dynactin complexes, which promote chromosome attachment to mitotic spindles and ensure centrosome integrity. Together, these results suggest that proliferative responses are supported by distinct mitotic machineries across T-cell subsets.

Funder: Gates Cambridge Scholarship, Funder: University of Cambridge; Id: http://dx.doi.org/10.13039/501100000735, The spindle assembly checkpoint (SAC) is a cellular surveillance mechanism that functions to ensure accurate chromosome segregation during mitosis. Macromolecular complexes known as kinetochores, act as the interface of sister chromatid attachment to spindle microtubules. In response to unattached kinetochores, the SAC activates its effector, the mitotic checkpoint complex (MCC), which delays mitotic exit until all sister chromatid pairs have achieved successful attachment to the bipolar mitotic spindle. Formation of the MCC (composed of Mad2, BubR1, Bub3 and Cdc20) is regulated by an Mps1 kinase-dependent phosphorylation signaling cascade which assembles and repositions components of the MCC onto a catalytic scaffold. This scaffold functions to catalyze the conversion of the HORMA-domain protein Mad2 from an "inactive" open-state (O-Mad2) into an "active" closed-Mad2 (C-Mad2), and simultaneous Cdc20 binding. Here, our current understanding of the molecular mechanisms underlying the kinetic barrier to C-Mad2:Cdc20 formation will be reviewed. Recent progress in elucidating the precise molecular choreography orchestrated by the catalytic scaffold to rapidly assemble the MCC will be examined, and unresolved questions will be highlighted. Ultimately, understanding how the SAC rapidly activates the checkpoint not only provides insights into how cells maintain genomic integrity during mitosis, but also provides a paradigm for how cells can utilize molecular switches, including other HORMA domain-containing proteins, to make rapid changes to a cell's physiological state.

Mitosis is the cellular process that ensures accurate segregation of the cell's genetic material into two daughter cells. Mitosis is often deregulated in cancer; thus drugs that target mitosis-specific proteins represent attractive targets for anticancer therapy. Numerous inhibitors have been developed against kinesin-5 Eg5, a kinesin essential for bipolar spindle assembly. Unfortunately, Eg5 inhibitors (K5Is) have been largely ineffective in the clinic, possibly due to the activity of a second kinesin, KIF15, that can suppress the cytotoxic effect of K5Is by driving spindle assembly through an Eg5-independent pathway. We hypothesized that pairing of K5Is with small molecule inhibitors of KIF15 will be more cytotoxic than either inhibitor alone. Here we present the results of a high-throughput screen from which we identified two inhibitors that inhibit the motor activity of KIF15 both in vitro and in cells. These inhibitors selectively inhibit KIF15 over other molecular motors and differentially affect the ability of KIF15 to bind microtubules. Finally, we find that chemical inhibition of KIF15 reduces the ability of cells to acquire resistance to K5Is, highlighting the centrality of KIF15 to K5I resistance and the value of these inhibitors as tools with which to study KIF15 in a physiological context.

Meiotic spindle assembly ensures proper chromosome segregation in oocytes. However, the mechanisms behind spindle assembly in human oocytes remain largely unknown. We used three-dimensional high-resolution imaging of more than 2000 human oocytes to identify a structure that we named the human oocyte microtubule organizing center (huoMTOC). The proteins TACC3, CCP110, CKAP5, and DISC1 were found to be essential components of the huoMTOC. The huoMTOC arises beneath the oocyte cortex and migrates adjacent to the nuclear envelope before nuclear envelope breakdown (NEBD). After NEBD, the huoMTOC fragments and relocates on the kinetochores to initiate microtubule nucleation and spindle assembly. Disrupting the huoMTOC led to spindle assembly defects and oocyte maturation arrest. These results reveal a physiological mechanism of huoMTOC-regulated spindle assembly in human oocytes.

The purpose of meiosis is to generate developmentally competent, haploid gametes with the correct number of chromosomes. For reasons not completely understood, female meiosis is more prone to chromosome segregation errors than meiosis in males, leading to an abnormal number of chromosomes, or aneuploidy, in gametes. Meiotic spindles are the cellular machinery essential for the proper segregation of chromosomes. One unique feature of spindle structures in female meiosis is spindles poles that lack centrioles. The process of building a meiotic spindle without centrioles is complex and requires precise coordination of different structural components, assembly factors, motor proteins, and signaling molecules at specific times and locations to regulate each step. In this review, we discuss the basics of spindle formation during oocyte meiotic maturation focusing on mouse and human studies. Finally, we review different factors that could alter the process of spindle formation and its stability. We conclude with a discussion of how different assisted reproductive technologies could affect spindles and the consequences these perturbations may have for subsequent embryo development.

Chromosomal instability (CIN) is a driving force for cancer development. The most common causes of CIN include the dysregulation of the spindle assembly checkpoint (SAC), which is a surveillance mechanism that prevents premature chromosome separation during mitosis by targeting anaphase-promoting complex/cyclosome (APC/C). DAB2IP is frequently silenced in advanced prostate cancer (PCa) and is associated with aggressive phenotypes of PCa. Our previous study showed that DAB2IP activates PLK1 and functions in mitotic regulation. Here, we report the novel mitotic phosphorylation of DAB2IP by Cdks, which mediates DAB2IP’s interaction with PLK1 and the activation of the PLK1-Mps1 pathway. DAB2IP interacts with Cdc20 in a phosphorylation-independent manner. However, the phosphorylation of DAB2IP inhibits the ubiquitylation of Cdc20 in response to SAC, and blocks the premature release of the APC/C-MCC. The PLK1-Mps1 pathway plays an important role in mitotic checkpoint complex (MCC) assembly. It is likely that DAB2IP acts as a scaffold to aid PLK1-Mps1 in targeting Cdc20. Depletion or loss of the Cdks-mediated phosphorylation of DAB2IP destabilizes the MCC, impairs the SAC, and increases chromosome missegregation and subsequent CIN, thus contributing to tumorigenesis. Collectively, these results demonstrate the mechanism of DAB2IP in SAC regulation and provide a rationale for targeting the SAC to cause lethal CIN against DAB2IP-deficient aggressive PCa, which exhibits a weak SAC.

Human embryonic stem cells (hESCs) are self-renewing and pluripotent cells that originate from the inner cell mass of the blastocyst. Mitosis is fundamental to organism survival and reproduction and is responsible for the equal distribution of duplicated chromosomes into daughter cells. Mitotic dysfunction is associated with a wide variety of human diseases, not least cancer. hESCs have a unique cell cycle distribution, but it is unclear exactly how the mitotic activity of hESCs is related to their proliferation and differentiation. Here, we established a cell line of hESCs stably expressing GFP-α-tubulin and mCherry-H2B by lentiviral infection to analyze and visualize mitosis in detail. During metaphase, the mitotic spindle was smaller and wider and contained a greater proportion of astral microtubules than normal cells. In addition, spindle microtubules were more stable, and chromosome alignment was faster in hESCs than in somatic cells. We also found that the spindle assembly checkpoint was functional in hESCs. These findings thus reveal a specialized mitotic behavior of hESCs.

14-3-3 proteins are conserved, dimeric, acidic proteins that regulate multiple cellular pathways. Loss of either 14-3-3e or 14-3-3γ leads to centrosome amplification. However, we find that while the knockout of 14-3-3e leads to multi-polar mitoses, the knockout of 14-3-3γ results in centrosome clustering and pseudo-bipolar mitoses. 14-3-3γ knockouts demonstrate compromised desmosome function and a decrease in keratin levels, leading to decreased cell stiffness and an increase in centrosome clustering. Restoration of desmosome function increased multi-polar mitoses, whereas knockdown of either plakoglobin or keratin 5 led to decreased cell stiffness and increased pseudo-bipolar mitoses. These results suggest that the ability of the desmosome to anchor keratin filaments maintains cell stiffness, thus inhibiting centrosome clustering, and that phenotypes observed upon 14-3-3 loss reflect the dysregulation of multiple pathways.

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link between kinetochore core MIS13 complex and spindle microtubule plus-ends to achieve accurate chromosome alignment in mitosis. However, it remains elusive how SKAP regulates kinetochore attachment from lateral association to end-on attachment during metaphase alignment. Here, we identify a novel interaction between Aurora B and SKAP that orchestrates accurate interaction between the kinetochore and dynamic spindle microtubules. Interestingly, SKAP spontaneously phase-separates in vitro via weak, multivalent interactions into droplets with fast internal dynamics. SKAP and Aurora B form heterogeneous coacervates in vitro, which recapitulate the dynamics and behavior of SKAP comets in vivo. Importantly, SKAP interaction with Aurora B via phase separation is essential for accurate chromosome segregation and alignment. Based on those findings, we reason that SKAP–Aurora B interaction via phase separation constitutes a dynamic pool of Aurora B activity during the lateral to end-on conversion of kinetochore–microtubule attachments to achieve faithful cell division.

Microtubules and the mitotic spindle have become an important target for cancer treatment due to their critical role in cell division. In this work, a novel series of benzofuran and indole derivatives were designed and synthesized, to be evaluated as tubulin polymerization inhibitors. 2-Acetylbenzofuran derivatives 1a,b and 3-acetylindole 1c were condensed with Wittig reagents 2a-d and Wittig-Horner reagents 3a-e to afford the respective 2-ethylidene derivatives 5a-j and 7a-e. Also, iminomethylene triphenylphosphine (2e) reacted with 1a,b to afford benzofuran-2-ylethylidene aniline derivatives 6a,b. In addition, compounds 1a,b reacted with trialkylphosphites 4a-c to give 1:1 adduct for which the Oxaphospholo[4,3-b]benzofuran-7-yl)diazene derivatives 8a-f, were assigned. The possible reactions mechanisms were discussed and structural reasoning for the new compounds were based upon spectroscopic data. Their antiproliferative activities against two cell lines namely, HepG2 and MCF7 cells were then evaluated. It was found that the benzofuran compounds 5b, 6a, and 8c exhibited the strongest antiproliferative activities against both cell lines compared to doxorubicin. By studying the mechanism of action, compound 6a showed good inhibition of tubulin polymerization which leads to mitotic spindle formation disruption, cell cycle arrest in the G2/M phase, and apoptosis of HepG2 cells. A conducted docking study confirmed the in vitro results indicating that compound 6a fitted properly at the colchicine binding site of tubulin. Based on these findings, compound 6a can be considered as a promising anticancer candidate that can be subjected for further development as a tubulin polymerization inhibitor for treating liver and breast cell carcinoma.

Mitotic spindle is a self-assembling macromolecular machine responsible for the faithful segregation of chromosomes during cell division. Assembly of the spindle is believed to be governed by the ‘Search & Capture’ (S&C) principle in which dynamic microtubules explore space in search of kinetochores while the latter capture microtubules and thus connect chromosomes to the spindle. Due to the stochastic nature of the encounters between kinetochores and microtubules, the time required for incorporating all chromosomes into the spindle is profoundly affected by geometric constraints, such as the size and shape of kinetochores as well as their distribution in space at the onset of spindle assembly. In recent years, several molecular mechanisms that control these parameters have been discovered. It is now clear that stochastic S&C takes place in structured space, where components are optimally distributed and oriented to minimize steric hindrances. Nucleation of numerous non-centrosomal microtubules near kinetochores accelerates capture, while changes in the kinetochore architecture at various stages of spindle assembly promote proper connection of sister kinetochores to the opposite spindle poles. Here we discuss how the concerted action of multiple facilitating mechanisms ensure that the spindle assembles rapidly yet with a minimal number of errors.

This study was conducted to elucidate the effects of introducing conjugated linoleic acid (CLA) on meiotic spindle organization of heat stressed (HS) matured oocytes and the resulting blastocysts DNA methylation as well as the expression of the genes involved in DNA methylation (DNMT3a, DNMT3b and DNMT1). Immature bovine oocytes were cultured at 41 °C for the first 12 h and 38.5 °C for the second 12 h of maturation time in the presence of 0 and 50 μM of CLA (HS and HS + CLA groups, respectively). A group of oocytes cultured in medium with no CLA supplementation at normal temperature (38.5 °C for 24 h) was considered as negative control (C). Percentage of normal spindle, and cleavage and blastocyst rates were significantly decreased in the HS group compared to the C group (P 0.05). The global DNA methylation and expression level of DNMT3a gene were increased in HS group compared to the C groups (P 0.05), while the expression level of DNMT3b was decreased. The CLA supplementation improved the percentage of normal microtubules shape in MII oocytes as well as the developmental competence in the HS + CLA group compared to the HS group (P 0.05). However, global DNA methylation and expression level of DNMT3a/b were not ameliorated by CLA supplementation (P 0.05). Based on the obtained results, CLA proved to be capable of improving the oocyte developmental competence as well as decreased the aberrant spindle organization of heat-stressed oocytes and it would not cause epigenetic alteration in the obtained blastocysts.

Generation of mechanical oscillations is ubiquitous to a wide variety of intracellular processes ranging from activity of muscle fibres to oscillations of the mitotic spindle. The activity of motors plays a vital role in maintaining the integrity of the mitotic spindle structure and in generating spontaneous oscillations. While the structural features and properties of the individual motors are well characterized, their implications on the functional behaviour of motor-filament complexes is more involved. We show that force-induced allosteric deformations in dynein, which results in catchbonding behaviour, provide a generic mechanism to generate spontaneous oscillations in motor-cytoskeletal filament complexes. The resultant phase diagram of such motor-filament systems - characterized by force-induced allosteric deformations - exhibits bistability and sustained limit cycle oscillations in biologically relevant regimes, such as for catchbonded dynein. The results reported here elucidate the central role of this mechanism in fashioning a distinctive stability behaviour and oscillations in motor-filament complexes, such as mitotic spindles.

Accurate chromosome segregation requires a complete restructuring of cellular organization. Microtubules remodel to assemble a mitotic spindle and the actin cytoskeleton rearranges to form a stiff actomyosin cortex. These cytoplasmic events must be spatially and temporally coordinated with mitotic chromosome condensation and nuclear envelope permeabilization, in order to ensure mitotic timing and fidelity. Here, we discuss the main cytoskeletal and nuclear events that occur during mitotic entry in proliferating animal cells, focusing on their coordinated contribution for early mitotic spindle assembly. We will also explore recent progress in understanding their regulatory biochemical and mechanical pathways.

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

The shortening of microtubules attached to kinetochores is the driving force of chromosome movement during cell division. Specific kinesins are believed to shorten microtubules but are dispensable for viability in yeast, implying the existence of additional factors responsible for microtubule shortening. Here, we demonstrate that Dis1, a TOG/XMAP215 ortholog in fission yeast, promotes microtubule shortening to carry attached chromosomes. Although TOG/XMAP215 orthologs are generally accepted as microtubule polymerases, Dis1 promoted microtubule catastrophe in vitro and in vivo. Notably, microtubule catastrophe was promoted when the tip was attached to kinetochores, as they steadily anchored Dis1 at the kinetochore-microtubule interface. Engineered Dis1 oligomers artificially tethered at a chromosome arm region induced the shortening of microtubules in contact, frequently pulling the chromosome arm towards the vicinity of spindle poles in meiocytes. Thus, unlike Alp14 and other TOG/XMAP215 orthologs, Dis1 plays an unconventional role in promoting microtubule catastrophe, thereby driving chromosome movement.

Microcephaly often results from mitotic defects in neuronal progenitors, frequently by decreasing proliferation rates or shifting cell fates. During neurogenesis, oriented cell division-the molecular control of mitotic spindle positioning to control the axis of division-represents an important mechanism to balance expansion of the progenitor pool with generating cellular diversity. While mostly studied in the context of cortical development, more recently, spindle orientation has emerged as a key player in the formation of other brain regions such as the cerebellum. Here we describe methods to perform automated dual-color fluorescent immunohistochemistry on murine cerebellar sections using the mitotic markers phospho-Histone H3 and Survivin, and detail analytical and statistical approaches to display and compare division orientation datasets.

During cell division, the spindle generates force to move chromosomes. In mammals, microtubule bundles called kinetochore-fibers (k-fibers) attach to and segregate chromosomes. To do so, k-fibers must be robustly anchored to the dynamic spindle. We previously developed microneedle manipulation to mechanically challenge k-fiber anchorage, and observed spatially distinct response features revealing the presence of heterogeneous anchorage (Suresh et al. 2020). How anchorage is precisely spatially regulated, and what forces are necessary and sufficient to recapitulate the k-fiber’s response to force remain unclear. Here, we develop a coarse-grained k-fiber model and combine with manipulation experiments to infer underlying anchorage using shape analysis. By systematically testing different anchorage schemes, we find that forces solely at k-fiber ends are sufficient to recapitulate unmanipulated k-fiber shapes, but not manipulated ones for which lateral anchorage over a 3 μm length scale near chromosomes is also essential. Such anchorage robustly preserves k-fiber orientation near chromosomes while allowing pivoting around poles. Anchorage over a shorter length scale cannot robustly restrict pivoting near chromosomes, while anchorage throughout the spindle obstructs pivoting at poles. Together, this work reveals how spatially regulated anchorage gives rise to spatially distinct mechanics in the mammalian spindle, which we propose are key for function.

How cells adjust their growth to the spatial and mechanical constraints of their surrounding environment is central to many aspects of biology. Here, we examined how extracellular matrix (ECM) rigidity affects cell division. We found that cells divide more rapidly when cultured on rigid substrates. While we observed no effect of ECM rigidity on rounding or postmitotic spreading duration, we found that changes in matrix stiffness impact mitosis progression. We noticed that ECM elasticity up-regulates the expression of the linker of nucleoskeleton and cytoskeleton (LINC) complex component SUN2, which in turn promotes metaphase-to-anaphase transition by acting on mitotic spindle formation, whereas when cells adhere to soft ECM, low levels of SUN2 expression perturb astral microtubule organization and delay the onset of anaphase.

The spindle position checkpoint (SPOC) is a mitotic surveillance mechanism in Saccharomyces cerevisiae that prevents cells from completing mitosis in response to spindle misalignment, thereby contributing to genomic integrity. The kinase Kin4, one of the most downstream SPOC components, is essential to stop the mitotic exit network (MEN), a signalling pathway that promotes the exit from mitosis and cell division. Previous work, however, suggested that a Kin4-independent pathway contributes to SPOC, yet the underlying mechanisms remain elusive. Here, we established the glycogen-synthase-kinase-3 (GSK-3) homologue Mck1, as a novel component that works independently of Kin4 to engage SPOC. Our data indicate that both Kin4 and Mck1 work in parallel to counteract MEN activation by the Cdc14 early anaphase release (FEAR) network. We show that Mck1's function in SPOC is mediated by the pre-replication complex protein and mitotic cyclin-dependent kinase (M-Cdk) inhibitor, Cdc6, which is degraded in a Mck1-dependent manner prior to mitosis. Moderate overproduction of Cdc6 phenocopies MCK1 deletion and causes SPOC deficiency via its N-terminal, M-Cdk inhibitory domain. Our data uncover an unprecedented role of GSK-3 kinases in coordinating spindle orientation with cell cycle progression.

During mitosis, spindle assembly relies on centrosomal and acentrosomal microtubule nucleation pathways that all require the γ-Tubulin Ring Complex (γ-TuRC) and its adaptor protein NEDD1. The activity of these different pathways needs to be coordinated to ensure bipolar spindle assembly ( Cavazza et al., 2016) but the underlying mechanism is still unclear. Previous studies have identified three sites in NEDD1 (S377, S405 and S411) that when phosphorylated drive MT nucleation at the centrosomes, around the chromosomes and on pre-existing MTs respectively ( Lüders et al., 2006; Pinyol et al., 2013; Sdelci et al., 2012). Here we aimed at getting additional insights into the mechanism that coordinates the different MT nucleation pathways in dividing cells using a collection of HeLa stable inducible cell lines expressing NEDD1 phospho-variants at these three sites and Xenopus egg extracts. Our results provide further support for the essential role of phosphorylation at the three residues. Moreover, we directly demonstrate that S411 phosphorylation is essential for MT branching using TIRF microscopy in Xenopus egg extracts and we show that it plays a crucial role in ensuring the balance between centrosome and chromosome-dependent MT nucleation required for bipolar spindle assembly in mitotic cells.