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In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in Caenorhabditis elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF, but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated MELT motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1s TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

The α-Aurora kinase is a crucial regulator of spindle microtubule organization during mitosis in plants. Here, we report a post-mitotic role for α-Aurora in reorganizing the phragmoplast microtubule array. In Arabidopsis thaliana, α-Aurora relocated from spindle poles to the phragmoplast midzone, where it interacted with the microtubule cross-linker MAP65-3. In a hypomorphic α-Aurora mutant, MAP65-3 was detected on spindle microtubules, followed by a diffuse association pattern across the phragmoplast midzone. Simultaneously, phragmoplast microtubules remained belatedly in a solid disk array before transitioning to a ring shape. Microtubules at the leading edge of the matured phragmoplast were often disengaged, accompanied by conspicuous retentions of MAP65-3 at the phragmoplast interior edge. Specifically, α-Aurora phosphorylated two residues towards the C-terminus of MAP65-3. Mutation of these residues to alanines resulted in an increased association of MAP65-3 with microtubules within the phragmoplast. Consequently, the expansion of the phragmoplast was notably slower compared to wild-type cells or cells expressing a phospho-mimetic variant of MAP65-3. Moreover, mimicking phosphorylation reinstated disrupted MAP65-3 behaviors in plants with compromised α-Aurora function. Overall, our findings reveal a mechanism in which α-Aurora facilitates cytokinesis progression through phosphorylation-dependent restriction of MAP65-3 associating with microtubules at the phragmoplast midzone.

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during cell division by monitoring kinetochore-microtubule attachment. Plants produce both sequence-conserved and diverged SAC components, and it has been largely unknown how SAC activation leads to the assembly of these proteins at unattached kinetochores to prevent cells from entering anaphase. In Arabidopsis thaliana, the noncanonical BUB3.3 protein was detected at kinetochores throughout mitosis, unlike MAD1 and the plant-specific BUB1/MAD3 family protein BMF3 that associated with unattached chromosomes only. When BUB3.3 was lost by a genetic mutation, mitotic cells often entered anaphase with misaligned chromosomes and presented lagging chromosomes after they were challenged by low doses of the microtubule depolymerizing agent oryzalin, resulting in the formation of micronuclei. Surprisingly, BUB3.3 was not required for the kinetochore localization of other SAC proteins or vice versa. Instead, BUB3.3 specifically bound to BMF3 through two internal repeat motifs that were not required for BMF3 kinetochore localization. This interaction enabled BMF3 to recruit CDC20, a downstream SAC target, to unattached kinetochores. Taken together, our findings demonstrate that plant SAC utilizes unconventional protein interactions for arresting mitosis, with BUB3.3 directing BMF3's role in CDC20 recruitment, rather than the recruitment of BUB1/MAD3 proteins observed in fungi and animals. This distinct mechanism highlights how plants adapted divergent versions of conserved cell cycle machinery to achieve specialized SAC control.

The kinetochore scaffold 1 (KNL1) protein recruits spindle assembly checkpoint (SAC) proteins to ensure accurate chromosome segregation during mitosis. Despite such a conserved function among eukaryotic organisms, its molecular architectures have rapidly evolved so that the functional mode of plant KNL1 is largely unknown. To understand how SAC signaling is regulated at kinetochores, we characterized the function of the KNL1 gene in Arabidopsis thaliana. The KNL1 protein was detected at kinetochores throughout the mitotic cell cycle, and null knl1 mutants were viable and fertile but exhibited severe vegetative and reproductive defects. The mutant cells showed serious impairments of chromosome congression and segregation, that resulted in the formation of micronuclei. In the absence of KNL1, core SAC proteins were no longer detected at the kinetochores, and the SAC was not activated by unattached or misaligned chromosomes. Arabidopsis KNL1 interacted with SAC essential proteins BUB3.3 and BMF3 through specific regions that were not found in known KNL1 proteins of other species, and recruited them independently to kinetochores. Furthermore, we demonstrated that upon ectopic expression, the KNL1 homolog from the dicot tomato was able to functionally substitute KNL1 in A. thaliana, while others from the monocot rice or moss associated with kinetochores but were not functional, as reflected by sequence variations of the kinetochore proteins in different plant lineages. Our results brought insights into understanding the rapid evolution and lineage-specific connection between KNL1 and the SAC signaling molecules.

There is an ongoing need for antifungal agents to treat humans. Identification of new antifungal agents can be based on screening compounds using whole cell assays. Screening compounds that target a particular molecule is possible in budding yeast wherein sophisticated strain engineering allows for controlled expression of endogenous or heterologous genes. We have considered the yeast Mps1 protein kinase as a reasonable target for antifungal agents because mutant or druggable forms of the protein, upon inactivation, cause rapid loss of cell viability. Furthermore, extensive analysis of the Mps1 in budding yeast has offered potential tactics for identifying inhibitors of its enzymatic activity. One such tactic is based on the finding that overexpression of Mps1 leads to cell cycle arrest via activation of the spindle assembly checkpoint. We have endeavored to adapt this assay to be based on the overexpression of Mps1 orthologs from pathogenic yeast in hopes of having a whole-cell assay system to test the activity of these orthologs. Mps1 orthologous genes from seven pathogenic yeast or other pathogenic fungal species were isolated and expressed in budding yeast. Two orthologs clearly produced phenotypes similar to those produced by the overexpression of budding yeast Mps1, indicating that this system for heterologous Mps1 expression has potential as a platform for identifying prospective antifungal agents.

The mitotic spindle is the bipolar, microtubule-based structure that segregates chromosomes at each cell division. Aberrant spindles are frequently observed in cancer cells, but how oncogenic transformation affects spindle mechanics and function, particularly in the mechanical context of solid tumors, remains poorly understood. Here, we constitutively overexpress the oncogene cyclin D1 in human MCF10A cells to probe its effects on spindle architecture and response to compressive force. We find that cyclin D1 overexpression increases the incidence of spindles with extra poles, centrioles, and chromosomes. However, it also protects spindle poles from fracturing under compressive force, a deleterious outcome linked to multipolar cell divisions. Our findings suggest that cyclin D1 overexpression may adapt cells to increased compressive stress, possibly contributing to its prevalence in cancers such as breast cancer by allowing continued proliferation in mechanically challenging environments.

Vinca alkaloids, a class of tubulin-binding agent, are widely used in treating cancer, yet the emerging resistance compromises their efficacy. Hepatoma up-regulated protein (HURP), a microtubule-associated protein displaying heightened expression across various cancer types, reduces cancer cells' sensitivity to vinca-alkaloid drugs upon overexpression. However, the molecular basis behind this drug resistance remains unknown. Here we discover a tubulin-binding domain within HURP, and establish its role in regulating microtubule growth. Cryo-EM analysis reveals interactions between HURP's tubulin-binding domain and the vinca domain on β-tubulin -- the site targeted by vinca alkaloid drugs. Importantly, HURP competes directly with vinorelbine, a vinca alkaloid-based chemotherapeutic agent, countering microtubule growth defects caused by vinorelbine both in vitro and in vivo. Our findings elucidate a mechanism driving drug resistance in HURP-overexpressing cancer cells and emphasize HURP tubulin-binding domain's role in mitotic spindle assembly. This underscores its potential as a therapeutic target to improve cancer treatment. Vinca alkaloids, tubulin-binding agents that impair microtubule growth, are widely used in cancer treatment. Here, the authors show that resistance to these drugs is due to Hepatoma up-regulated protein (HURP) competitively binding to the same site on tubulin, countering the drug's effects. [ABSTRACT FROM AUTHOR]

BACKGROUND Bisphenol A (BPA) is an endocrine disrupting chemical released from plastic materials, including food packaging and dental sealants, persisting in the environment and ubiquitously contaminating ecosystems and human populations. BPA can elicit an array of damaging health effects and, alarmingly, 'BPA-free' alternatives mirror these harmful effects. Bisphenol exposure can negatively impact female fertility, damaging both the ovary and oocytes therein. Such damage can diminish reproductive capacity, pregnancy success, and offspring health. Despite global government regulations in place to indicate 'safe' BPA exposure levels, these policies have not considered the effects of bisphenols on oocyte health. OBJECTIVE AND RATIONALE This scoping review was conducted to evaluate evidence on the effects of BPA and BPA alternatives on standardized parameters of oocyte health. In doing so, this review addresses a critical gap in the literature providing a comprehensive, up-to-date synthesis of the effects of bisphenols on oocyte health. SEARCH METHODS This scoping review was conducted in accordance with PRISMA guidelines. Four databases, Medline, Embase, Scopus, and Web of Science, were searched twice (23 February 2022 and 1 August 2023) to capture studies assessing mammalian oocyte health post-bisphenol exposure. Search terms regarding oocytes, ovarian follicles, and bisphenols were utilized to identify relevant studies. Manuscripts written in English and reporting the effect of any bisphenol on mammalian oocyte health from all years were included. Parameters for toxicological studies were evaluated, including the number of bisphenol concentrations/doses tested, dosing regimen, biological replicates and/or animal numbers, and statistical information (for human studies). Standardized parameters of oocyte health including follicle counts, oocyte yield, oocyte meiotic capacity, morphology of oocyte and cumulus cells, and oocyte meiotic spindle integrity were extracted across the studies. OUTCOMES After screening 3147 studies, 107 studies of either humans or mammalian animal models or humans were included. Of the in vitro exposure studies, 96.3% (26/27) and 94.1% (16/17) found at least one adverse effect on oocyte health using BPA or BPA alternatives (including BHPF, BPAF, BPB, BPF, and BPS), respectively. These included increased meiotic cell cycle arrest, altered morphology, and abnormal meiotic spindle/chromosomal alignment. In vivo , 85.7% (30/35) of studies on BPA and 92.3% (12/13) on BPA alternatives documented adverse effects on follicle development, morphology, or spindle/chromosome alignment. Importantly, these effects were recorded using levels below those deemed 'safe' for human exposure. Over half (11/21) of all human observational studies showed associations between higher urinary BPA levels and reduced antral follicle counts or oocyte yield in IVF patients. Recommendations are presented based on the identified shortcomings of the current evidence, incorporating elements of FDA requirements for future research in the field. WIDER IMPLICATIONS These data highlight the detrimental impacts of low-level BPA and BPA alternative exposure, contributing to poor oocyte quality and reduced fertility. These outcomes are valuable in promoting the revision of current policies and guidelines pertaining to BPA exposure internationally. This study serves as a valuable resource to scientists, providing key recommendations on study design, reporting elements, and endpoint measures to strengthen future studies. Ultimately, this review highlights oocyte health as a fundamentally important endpoint in reproductive toxicological studies, indicating an important direction for future research into endocrine disrupting chemicals to improve fertility outcomes. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) ranks among the most aggressive types of cancer globally. Currently, clinical tumor prognostic biomarkers still lack accuracy. Mitotic spindle positioning (MISP) protein connects microtubules to the actin cytoskeleton and adhesive plaques, playing a critical role in spindle positioning, orientation, and the process of cell division. MISP can regulate the malignant biological functions of pancreatic cancer and intrahepatic cholangiocarcinoma and it acts as biomarker for prognosis, but its role in CRC remains unclear. Methods: This study has collected 37 CRC tissue samples and 37 corresponding adjacent nontumor tissue samples, and 57 additional CRC tissues samples. Clinical data were obtained from the patients with CRC. MISP mRNA and protein expression levels were analyzed in normal control and CRC tissues using the GEPIA and Human Protein Atlas website. MISP protein levels in the collected tissues were analyzed using immunohistochemistry. Results: MISP mRNA and protein expression levels were significantly increased in CRC tissues compared to adjacent nontumor tissues. Higher MISP protein levels were associated with distant metastasis, recurrence, and lower survival rates. Kaplan–Meier analysis showed that high expression levels of MISP protein were associated with recurrence and death in CRC patients. In addition, a high expression level of MISP protein, lymph node metastasis, and distance metastasis were risk factors for recurrence and a poor prognosis in patients with CRC. Conclusion: Elevated MISP protein correlated with tumor metastasis, recurrence, and lower survival rates in patients with CRC, and thus, MISP has the potential to become a prognostic marker for CRC. [ABSTRACT FROM AUTHOR]

Red Melon (RM) and Red Cover (RC) discus (Symphysodon spp.) are ornamental fish varieties that were selectively bred from the wild parental lineages of the brown discus S. aquafaciatus over many generations, resulting in distinct cutaneous patterns from juveniles to adults. To better understand the underlying mechanisms, skin samples were collected from juveniles aged 60 days and adults aged 1 year from RM and RC for investigations. Microscopic observation detected xanthophores and erythrophores in all samples, except RC juveniles with no erythrophores. Melanophores were presented only in RC. The comparative analysis revealed that genes involved in pteridine synthesis (gch1 and zgc:153031), one‐carbon metabolism (aldh1l2 and zgc153031), and lipid metabolism (apoda and klf1) were differentially expressed in RM juveniles, which may be associated with the development of erythrophores and xanthophores. The temporal inhibition of melanophore differentiation and development was observed in RM juveniles, coupled with elevated expression of notum2 and sost, two antagonist genes in Wnt‐signaling, suggesting their roles in melanophore development. Distinct pigment pattern between RM and RC since the juvenile stage may be driven by the differential expression of multiple axial developmental genes, including GATA, ankyrin, and mitotic spindle orientation proteins. This is the first report to describe the differential growth of cutaneous pigments and the molecular processes involved in red discus. The results provided valuable insights into pigment pattern differences in an interesting ornamental fish model. [ABSTRACT FROM AUTHOR]

Microtubules are dynamic filaments that assemble spindles for eukaryotic cell division. As the concentration profiles of soluble tubulin and regulatory proteins are non-uniform during spindle assembly, we asked if diffusiophoresis - motion of particles under solute gradients - can act as a motorless transport mechanism for microtubules. We identify the migration of stable microtubules along cytoplasmic and higher concentration gradients of soluble tubulin, MgCl2, Mg-ATP, Mg-GTP, and RanGTP at speeds O(100) nm/s, validating the diffusiophoresis hypothesis. Using two buffers (BRB80 and CSF-XB), microtubule behavior under MgCl2 gradients is compared with negatively charged particles and analyzed with a multi-ion diffusiophoresis and diffusioosmosis model. Microtubule diffusiophoresis under gradients of tubulin and RanGTP is also compared with the charged particles and analyzed with a non-electrolyte diffusiophoresis model. Further, we find that tubulin and RanGTP display concentration dependent cross-diffusion that influences microtubule diffusiophoresis. Finally, using Xenopus laevis egg extract, we show that diffusiophoretic transport occurs in an active cytoplasmic environment. The authors show microtubule motion along concentration gradients of proteins and salts similar to those found in the cytoplasm. Based on their observations they propose diffusiophoresis as a potential motorless transport mechanism for cytoplasmic particles along intracellular solute gradients. [ABSTRACT FROM AUTHOR]

Acrylamide (AAM), a compound extensively utilized in various industrial applications, has been reported to induce toxic effects across multiple tissues in living organisms. Despite its widespread use, the impact of AAM on ovarian function and the mechanisms underlying these effects remain poorly understood. Here, we established an AAM‐exposed mouse toxicological model using 21 days of intragastric AAM administration. AAM exposure decreased ovarian coefficient and impaired follicle development. Further investigations revealed AAM would trigger apoptosis and disturb tricarboxylic acid cycle in ovarian tissue, thus affecting mitochondrial electron transport function. Moreover, AAM exposure decreased oocyte and embryo development potential, mechanically associated with pericentrin and phosphorylated Aurora A cluster failure, leading to meiotic spindle assembly defects. Collectively, these results suggest that AAM exposure may lead to apoptosis, glucose metabolic disorders, and mitochondrial dysfunction in ovary tissue, ultimately compromising oocyte quality. [ABSTRACT FROM AUTHOR]

Catharanthine, a component of the anticancer drug vinblastine along with vindoline, disrupts the cell cycle by interfering with mitotic spindle formation. Apart from their antioxidant properties, vinca alkaloids like catharanthine inhibit phosphodiesterase activity and elevate intracellular cAMP levels. The aim of this study was to investigate how catharantine affects apoptosis and autophagy. This study conducted experiments on HepG2 liver carcinoma cells with varying doses of catharanthine to evaluate cell death rates and viability and determine the IC50 concentration via MTT assays. The apoptotic and autophagic effects of catharanthine were assessed using flow cytometry with annexin V and PI staining, while the expression of autophagy-related genes was analyzed through quantitative PCR. Additionally, molecular docking and molecular dynamics simulations were employed to further investigate catharanthine's impact on autophagy mechanisms. The study showed that catharanthine reduced oxidative stress and triggered apoptosis in HepG2 cells in a dose-dependent manner. Catharanthine also upregulated the expression of autophagy-related genes like LC3, Beclin1, and ULK1. Notably, catharanthine increased sirtuin-1 levels, a known autophagy inducer, while decreasing Akt expression compared to untreated cells. Molecular docking results indicated rapamycin had a stronger binding affinity with FRB (−10.7 KJ/mol−1) than catharanthine (−7.3 KJ/mol−1). Additionally, molecular dynamics simulations revealed that catharanthine interacted effectively with the FRB domain of mTOR, displaying stability and a strong binding affinity, although not as potent as rapamycin. In summary, besides its cytotoxic and pro-apoptotic effects, catharanthine activates autophagy signaling pathways and induces autophagic necrosis by inhibiting mTOR. [ABSTRACT FROM AUTHOR]

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Centrosomes are the major microtubule organizing centers of animal cells. Supernumerary centrosomes are a common feature of human tumors and associated with karyotype abnormalities and aggressive disease, but whether they are cause or consequence of cancer remains controversial. Here, we analyzed the consequences of centrosome amplification by generating transgenic mice in which centrosome numbers can be increased by overexpression of the structural centrosome protein STIL. We show that STIL overexpression induces centrosome amplification and aneuploidy, leading to senescence, apoptosis, and impaired proliferation in mouse embryonic fibroblasts, and microcephaly with increased perinatal lethality and shortened lifespan in mice. Importantly, both overall tumor formation in mice with constitutive, global STIL overexpression and chemical skin carcinogenesis in animals with inducible, skin-specific STIL overexpression were reduced, an effect that was not rescued by concomitant interference with p53 function. These results suggest that supernumerary centrosomes impair proliferation in vitro as well as in vivo, resulting in reduced lifespan and delayed spontaneous as well as carcinogen-induced tumor formation. Author summary: Already at the beginning of the last century, Theodor Boveri suggested that supernumerary centrosomes might be a cause of cancer via the generation of abnormal mitotic spindles and subsequent chromosome missegregation. In the meantime, centrosome amplification has been observed in most tumor types and is viewed as a "hallmark" of cancer cells. However, studies on tumor formation in mice by overexpression of PLK4, the principal kinase regulating centrosome duplication, led to ambiguous results, as some but not all models resulted in increased tumorigenesis. In this study, we generated transgenic mice, which overexpress the structural centrosome protein STIL. Similar to PLK4, overexpression of STIL caused centrosome amplification and aneuploidy in mouse cells. Our analyses revealed that powerful mechanisms lead to the elimination of cells with extra centrosomes and/or aneuploidy by impaired proliferation, senescence and apoptosis, thereby delaying both spontaneous tumor formation and chemical skin carcinogenesis, and explaining the reduced life span of STIL-transgenic mice. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is an etiologically complex disease characterized by acute exacerbations and stable phases. We aimed to identify biological functions modulated in specific COPD conditions, using whole blood samples collected in the AERIS clinical study (NCT01360398). Considered conditions were exacerbation onset, severity of airway obstruction, and presence of respiratory pathogens in sputum samples. With an integrative multi-network gene community detection (MNGCD) approach, we analyzed expression profiles to identify communities of correlated genes. The approach combined different layers of gene interactions for each explored condition/subset of samples: gene expression similarity, protein-protein interactions, transcription factors, and microRNAs validated regulons. Heme metabolism, interferon-alpha, and interferon-gamma pathways were modulated in patients at both exacerbation and stable-state visits, but with the involvement of distinct sets of genes. An important gene community was enriched with G2M checkpoint, E2F targets, and mitotic spindle pathways during exacerbation. Targets of TAL1 regulator and hsa−let−7b − 5p microRNA were modulated with increasing severity of airway obstruction. Bacterial infections with Moraxella catarrhalis and, particularly, Haemophilus influenzae triggered a specific cellular and inflammatory response in acute exacerbations, indicating an active reaction of the host to infections. In conclusion, COPD is a complex multifactorial disease that requires in-depth investigations of its causes and features during its evolution and whole blood transcriptome profiling can contribute to capturing some relevant regulatory mechanisms associated with this disease. In this work, we explored multi-network modeling that integrated diverse layers of regulatory gene networks and enhanced our comprehension of the biological functions implicated in the COPD pathogenesis. [ABSTRACT FROM AUTHOR]

During cell division, the microtubule cytoskeleton undergoes dramatic cell cycle-driven reorganizations of its architecture. Coordinated by changes in the phosphorylation patterns of a multitude of microtubule associated proteins, the mitotic spindle first self-assembles to capture the chromosomes and then reorganizes in anaphase as the chromosomes are segregated. A key protein for this reorganization is PRC1 which is differentially phosphorylated by the mitotic kinases CDK1 and PLK1. How the phosphorylation state of PRC1 orchestrates spindle reorganization is not understood mechanistically. Here, we reconstitute in vitro the transition between metaphase and anaphase-like microtubule architectures triggered by the changes in PRC1 phosphorylation. We find that whereas PLK1 regulates its own recruitment by PRC1, CDK1 controls the affinity of PRC1 for antiparallel microtubule binding. Dephosphorylation of CDK1-phosphorylated PRC1 is required and sufficient to trigger the reorganization of a minimal anaphase midzone in the presence of the midzone length controlling kinesin KIF4A. These results demonstrate how phosphorylation-controlled affinity changes regulate the architecture of active microtubule networks, providing new insight into the mechanistic underpinnings of the cell cycle-driven reorganization of the central spindle during mitosis. Lim et al. reconstitute in vitro the transition between metaphase and anaphase-like spindle midzone architectures and dissect the role of PRC1 phosphorylation by PLK and CDK1 in reorganizing the central spindle during mitosis. [ABSTRACT FROM AUTHOR]

Kinesin-5 motors play an essential role during mitotic spindle assembly in many organisms 1,2,3,4,5,6,7,8,9,10,11 : they crosslink antiparallel spindle microtubules, step toward plus ends, and slide the microtubules apart. 12,13,14,15,16,17 This activity separates the spindle poles and chromosomes. Kinesin-5s are not only plus-end-directed but can walk or be carried toward MT minus ends, 18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 where they show enhanced localization. 3,5,7,27,29,32 The kinesin-5 C-terminal tail interacts with and regulates the motor, affecting structure, motility, and sliding force of purified kinesin-5 35,36,37 along with motility and spindle assembly in cells. 27,38,39 The tail contains phosphorylation sites, particularly in the conserved BimC box. 6,7,40,41,42,43,44 Nine mitotic tail phosphorylation sites were identified in the kinesin-5 motor of the fission yeast Schizosaccharomyces pombe , 45,46,47,48 suggesting that multi-site phosphorylation may regulate kinesin-5s. Here, we show that mutating all nine sites to either alanine or glutamate causes temperature-sensitive lethality due to a failure of bipolar spindle assembly. We characterize kinesin-5 localization and sliding force in the spindle based on Cut7-dependent microtubule minus-end protrusions in cells lacking kinesin-14 motors. 39,49,50,51,52 Imaging and computational modeling show that Cut7p simultaneously moves toward the minus ends of protrusion MTs and the plus ends of spindle midzone MTs. Phosphorylation mutants show dramatic decreases in protrusions and sliding force. Comparison to a model of force to create protrusions suggests that tail truncation and phosphorylation mutants decrease Cut7p sliding force similarly to tail-truncated human Eg5. 36 Our results show that C-terminal tail phosphorylation is required for kinesin-5/Cut7 sliding force and bipolar spindle assembly in fission yeast. [Display omitted] • Phosphorylation site mutations in the kinesin-5 tail affect motor function • Phosphorylation site mutants show impaired mitotic spindle assembly and elongation • Kinesin-5 sliding force is reduced in phosphorylation mutants • Computational modeling reproduces kinesin-5 accumulation in protrusions Jones et al. show that phosphorylation of the kinesin-5 C-terminal tail is required for proper spindle assembly in fission yeast. Mutations in multiple tail phosphorylation sites impair both spindle function and motor sliding force, similarly to full tail truncations of fission yeast and (by comparison to a model of force) human kinesin-5s. [ABSTRACT FROM AUTHOR]

Taxanes are one of the most widely used classes of breast cancer (BC) therapeutics. Despite the long history of clinical usage, the molecular mechanisms of their action and cancer resistance are still not fully understood. Here we aimed to identify gene expression and molecular pathway activation biomarkers of BC sensitivity to taxane drugs paclitaxel and docetaxel. We used to our knowledge the biggest collection of clinically annotated publicly available literature BC gene expression data (12 datasets, n = 1250) and the experimental clinical BC cohort (n = 12). Seven literature datasets were used for biomarker discovery (n = 914), and the remaining five literature plus one experimental datasets (n = 336) – for the validation. We totally found 34 genes and 29 molecular pathways which could strongly discriminate good and poor responders to taxane treatments. The biomarker genes and pathways were associated with molecular processes related to formation of mitotic spindle and centromeres, and with the spindle assembly mitotic checkpoint. Furthermore, we created gene expression and pathway activation signatures predicting BC response to taxanes. These signatures were tested on the validation BC cohort and demonstrated strong biomarker potential reflected by mean AUC values of 0.76 and 0.77, respectively, which outperforms previously reported analogs. Taken together, these findings can deepen our understanding of mechanism of action of taxanes and potentially improve personalization of treatment in BC. [ABSTRACT FROM AUTHOR]

Candida albicans is themost prevalent fungal pathogen associated with candidemia. Similar to other fungi, the complex life cycle of C. albicans has been challenging to study with high-resolution microscopy due to its small size. Here, we employed ultrastructure expansion microscopy (UExM) to directly visualise subcellular structures at high resolution in the yeast and during its transition to hyphal growth. N-hydroxysuccinimide (NHS)-ester pan-labelling in combination with immunofluorescence via snapshots of various mitotic stages provided a comprehensive map of nucleolar and mitochondrial segregation dynamics and enabled the resolution of the inner and outer plaque of spindle pole bodies (SPBs). Analyses of microtubules (MTs) and SPBs suggest that C. albicans displays a side-by-side SPB arrangement with a short mitotic spindle and longer astral MTs (aMTs) at the pre-anaphase stage. Modifications to the established U-ExM protocol enabled the expansion of six other human fungal pathogens, revealing that the side-by-side SPB configuration is a plausibly conserved feature shared by many fungal species. We highlight the power of U-ExM to investigate subcellular organisation at high resolution and low cost in poorly studied and medically relevant microbial pathogens. [ABSTRACT FROM AUTHOR]

Microtubule organising centres (MTOCs) are sites of localised microtubule nucleation in eukaryotic cells. Regulation of microtubule dynamics often involves KATANIN (KTN): a microtubule severing enzyme that cuts microtubules to generate new negative ends, leading to catastrophic depolymerisation. In Arabidopsis thaliana, KTN is required for the organisation of microtubules in the cell cortex, preprophase band, mitotic spindle and phragmoplast. However, as angiosperms lack MTOCs, the role of KTN in MTOC formation has yet to be studied in plants. Two unique MTOCs - the polar organisers - form on opposing sides of the preprophase nucleus in liverworts. Here, we show that KTN-mediated microtubule depolymerisation regulates the number and organisation of polar organisers formed in Marchantia polymorpha. Mpktn mutants that lacked KTN function had supernumerary disorganised polar organisers compared with wild type. This was in addition to defects in the microtubule organisation in the cell cortex, preprophase band, mitotic spindle and phragmoplast. These data are consistent with the hypothesis that KTN-mediated microtubule dynamics are required for the de novo formation of MTOCs, a previously unreported function in plants. [ABSTRACT FROM AUTHOR]

PIWI proteins, traditionally associated with germline development, have recently gained attention for their expression in various cancers, including colorectal cancer. However, the molecular mechanisms underlying their reactivation and impact on cancer initiation and progression remain elusive. Here, we found that PIWIL1 is expressed at relatively high levels in CRC-derived samples and cell lines, where it undergoes a dynamic relocalization to the centrosome during mitosis. Knockdown of PIWIL1 induces G2/M arrest associated with disruption of the mitotic spindle and aberrant metaphase events, highlighting its role in cell cycle progression. We also found that the expression of PIWIL1 is lost during the differentiation of Caco-2 cells into enterocytes and that PIWIL1 is expressed in cells at the base of the intestinal crypts in normal human colon tissue, where intestinal stem cells are known to reside. Thus, it is possible that the presence of PIWIL1 in cancer cells reflects a physiological role of this protein in stem cell maintenance, which would argue in favor of the proposed stem cell origin of CRC. Supporting this view, dedifferentiation of human fibroblasts into induced pluripotent stem cells (iPSCs) involves the reactivation of PIWIL2 expression, another member of the PIWI protein family. Overall, our findings suggest a role of PIWIL1 in mediating cell cycle dynamics, both in colorectal cancer cells and possibly also in intestinal stem cells. In a broader aspect, we provide evidence supporting an involvement of PIWI proteins in somatic stem cell maintenance, thus expanding the known non-gonadal functions of this protein family. [ABSTRACT FROM AUTHOR]

Mitotic Arrest Deficient 1 (gene name MAD1L1), an essential component of the mitotic spindle assembly checkpoint, is frequently overexpressed in colon cancer, which correlates with poor disease-free survival. MAD1 upregulation induces two phenotypes associated with tumor promotion in tissue culture cells–low rates of chromosomal instability (CIN) and destabilization of the tumor suppressor p53. Using CRISPR/Cas9 gene editing, we generated a novel mouse model by inserting a doxycycline (dox)-inducible promoter and HA tag into the endogenous mouse Mad1l1 gene, enabling inducible expression of HA-MAD1 following exposure to dox in the presence of the reverse tet transactivator (rtTA). A modest 2-fold overexpression of MAD1 in murine colon resulted in decreased p53 expression and increased mitotic defects consistent with CIN. After exposure to the colon-specific inflammatory agent dextran sulfate sodium (DSS), 31% of mice developed colon lesions, including a mucinous adenocarcinoma, while none formed in control animals. Lesion incidence was particularly high in male mice, 57% of which developed at least one hyperplastic polyp, adenoma or adenocarcinoma in the colon. Notably, mice expressing HA-MAD1 also developed lesions in tissues in which DSS is not expected to induce inflammation. These findings demonstrate that MAD1 upregulation is sufficient to promote colon tumorigenesis in the context of inflammation in immune-competent mice. Author summary: Worldwide, colorectal cancer is the third most commonly diagnosed cancer and the second-highest cause of cancer-related deaths. A better understanding of the molecular causes of colorectal cancer could provide novel therapeutic targets for this disease. Mitotic Arrest Deficient-1 (MAD1) is commonly overexpressed in colorectal cancers, and elevated expression of MAD1 is associated with worse prognosis. Here we generated a new mutant mouse with inducible, modest overexpression of MAD1. First identified for its role in mitosis, MAD1 is required to ensure proper chromosome segregation. Elevated expression of MAD1 causes chromosome mis-segregation in the colons of these mice. During interphase, overexpressed MAD1 destabilizes the well-known tumor suppressor protein, p53. Consistent with this, mouse colons modestly overexpressing MAD1 show decreased expression of p53. Since inflammation is a risk factor for colorectal cancer, we induced inflammation in the colon using dextran sulfate sodium (DSS) and found that MAD1 overexpression significantly increased the incidence of colon tumors. Like inflammation-mediated colon tumors in humans, in the mice these tumors were more common in males than females. This work shows that MAD1 overexpression can promote colon cancer and suggests that MAD1 may be a novel drug target for this disease. [ABSTRACT FROM AUTHOR]

Hematologic abnormalities are the most common symptoms of Shwachman-Diamond syndrome (SDS). The causative gene for SDS is the Shwachman-Bodian-Diamond syndrome (SBDS) gene; however, the function of SBDS and pathogenesis of each condition in SDS are largely unknown. SBDS is known to be localized at mitotic spindles and stabilizes microtubules. Previously, we demonstrated that SBDS is ubiquitinated and subsequently degraded in the mitotic phase, thereby accelerating mitotic progression. In this study, we examined mitosis in a myeloid cell model of SDS (SDS cells). 4',6-Diamidino-2-phenylindole (DAPI)-stained chromosome observation and cell cycle analysis of nocodazolesynchronized cells revealed that the SDS cells have abnormally rapid mitosis. In addition, many lagging chromosomes and micronuclei were detected. Moreover, the phosphorylation of threonine tyrosine kinase, the crucial kinase of the spindle assembly checkpoint (SAC), was suppressed. Chromosomal instability caused by SAC dysfunction may cause a variety of clinical conditions, including hematologic tumors in patients with SDS. [ABSTRACT FROM AUTHOR]

Amino acid transporters play a vital role in cellular homeostasis by maintaining protein synthesis. L-type amino acid transporter 1 (LAT1/SLC7A5/CD98lc) is a major transporter of large neutral amino acids in cancer cells because of its predominant expression. Although amino acid restriction with various amino acid analog treatments is known to induce mitotic defects, the involvement of amino acid transporters in cell division remains unclear. In this study, we identified that LAT1 is responsible for mitotic progression in a transport activity-independent manner. LAT1 knockdown activates the spindle assembly checkpoint, leading to a delay in metaphase. LAT1 maintains proper spindle orientation with confinement of the lateral cortex localization of the NuMA protein, which mediates the pulling force against the mitotic spindle toward the lateral cortex. Unexpectedly, JPH203, an inhibitor of LAT1 amino acid transport activity, does not affect mitotic progression. Moreover, the transport activity-deficient LAT1 mutant maintains the proper spindle orientation and mitotic progression. LAT1 forms a heterodimer with CD98 (SLC3A2/CD98hc) both in interphase and mitosis. Although CD98 knockdown decreases the plasma membrane localization of LAT1, it does not affect mitotic progression. LAT1 is localized to the Golgi and ER not only at the plasma membrane in interphase, and promotes Golgi unlinking during the mitotic entry, leading to centrosome maturation. These results suggest that LAT1 supports mitotic progression in an amino acid transport activity-independent manner and that Golgi-localized LAT1 is important for mitotic progression through the acceleration of Golgi unlinking and centrosome maturation. These findings reveal a novel LAT1 function in mitosis. [ABSTRACT FROM AUTHOR]

Telomeres, the ends of eukaryotic linear chromosomes, are composed of repeated DNA sequences and specialized proteins, with the conserved telomeric Cdc13/CTC1-Stn1-Ten1 (CST) complex providing chromosome stability via telomere end protection and the regulation of telomerase accessibility. In this study, SIZ1, coding for a SUMO E3 ligase, and TOP2 (a SUMO target for Siz1 and Siz2) were isolated as extragenic suppressors of Saccharomyces cerevisiae CST temperature-sensitive mutants. ten1-sz, stn1-sz and cdc13-sz mutants were isolated next due to being sensitive to intracellular Siz1 dosage. In parallel, strong negative genetic interactions between mutants of CST and septins were identified, with septins being noticeably sumoylated through the action of Siz1. The temperature-sensitive arrest in these new mutants of CST was dependent on the G2/M Mad2-mediated and Bub2-mediated spindle checkpoints as well as on the G2/M Mec1-mediated DNA damage checkpoint. Our data suggest the existence of yet unknown functions of the telomeric Cdc13-Stn1-Ten1 complex associated with mitotic spindle positioning and/or assembly that could be further elucidated by studying these new ten1-sz, stn1-sz and cdc13-sz mutants. [ABSTRACT FROM AUTHOR]

Cell division is a highly regulated process essential for the accurate segregation of chromosomes. Central to this process is the assembly of a bipolar mitotic spindle, a highly dynamic microtubule (MT)‐based structure responsible for chromosome movement. The nucleation and dynamics of MTs are intricately regulated by MT‐binding proteins. Over the recent years, various MT‐binding proteins have been reported to undergo liquid–liquid phase separation, forming either single‐ or multi‐component condensates on MTs. Herein, we provide a comprehensive summary of the phase separation characteristics of these proteins. We underscore their critical roles in MT nucleation, spindle assembly and kinetochore‐MT attachment during the cell division process. Furthermore, we discuss the current challenges and various remaining unsolved problems, highlights the ongoing research efforts aimed at a deeper understanding of the role of the phase separation process during spindle assembly and orientation. Our review aims to contribute to the collective knowledge in this area and stimulate further investigations that will enhance our comprehension of the intricate mechanisms governing cell division. [ABSTRACT FROM AUTHOR]

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, also called American trypanosomiasis. This neglected tropical disease affects millions of individuals across the Americas. To complete its life cycle, T. cruzi parasitizes both vertebrate hosts and its vector, commonly known as the 'kissing bug'. The parasite's survival and proliferation strategies are driven by the diverse environments it encounters. Despite being described by Carlos Chagas in 1909, significant knowledge gaps persist regarding the parasite's various life forms and adaptive capabilities in response to environmental cues. In this study, we employed Ultrastructure Expansion Microscopy to explore the intricate journey of T. cruzi within the host cell. Upon entry into the host cell, trypomastigotes undergo folding, resulting in intermediate forms characterized by a rounded cell body, anterior positioning of basal bodies, and a shortened flagellum. The repositioning of basal bodies and the kinetoplast and the shortening of the flagella mark the culmination of intracellular amastigogenesis. Furthermore, we analyzed intracellular trypomastigogenesis, identifying discrete intermediate forms, including leaf-shaped stages and epimastigote-like forms, which suggests a complex differentiation process. Notably, we did not observe any dividing intracellular epimastigotes, indicating that these may be non-replicative forms within the host cell. Our detailed examination of amastigote cell division revealed semi-closed nuclear mitosis, with mitotic spindle formation independent of basal bodies. This study provides new insights into the morphological and cytoskeletal changes during the intracellular stages of T. cruzi, providing a model for understanding the dynamics of intracellular amastigogenesis and trypomastigogenesis. [ABSTRACT FROM AUTHOR]

The ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) and its regulatory protein Cdc20 play important roles in the control of different stages of mitosis. APC/C associated with Cdc20 is active and promotes metaphase-anaphase transition by targeting for degradation inhibitors of anaphase initiation. Earlier in mitosis, premature action of APC/C is prevented by the mitotic checkpoint (or spindle assembly checkpoint) system, which ensures that anaphase is not initiated until all chromosomes are properly attached to the mitotic spindle. The active mitotic checkpoint system promotes the assembly of a Mitotic Checkpoint Complex (MCC), which binds to APC/C and inhibits its activity. The interaction of MCC with APC/C is strongly enhanced by Cdc20 bound to APC/C. While the association of Cdc20 with APC/C was known to be essential for both these stages of mitosis, it was not known how Cdc20 remains bound in spite of ongoing processes, phosphorylation and ubiquitylation, that stimulate its release from APC/C. We find that MCC strongly inhibits the release of Cdc20 from APC/C by the action of mitotic protein kinase Cdk1-cyclin B. This is not due to protection from phosphorylation of specific sites in Cdc20 that affect its interaction with APC/C. Rather, MCC stabilizes the binding to APC/C of partially phosphorylated forms of Cdc20. MCC also inhibits the autoubiquitylation of APC/C-bound Cdc20 and its ubiquitylation-promoted release from APC/C. We propose that these actions of MCC to maintain Cdc20 bound to APC/C in mitosis are essential for the control of mitosis during active mitotic checkpoint and in subsequent anaphase initiation. [ABSTRACT FROM AUTHOR]

Microtubules, polymers of αβ-tubulin heterodimers, are essential for various cellular processes. The incorporation of different tubulin isotypes, each encoded by distinct genes, is proposed to contribute to the functional diversity observed in microtubules. However, the functional roles of each tubulin isotype are not completely understood. In this study, we investigated the role of the β4B-tubulin isotype (Tubb4b) in spermatogenesis, utilizing a Tubb4b knockout mouse model. We showed that β4B-tubulin is expressed in the germ cells throughout spermatogenesis. β4B-tubulin was localized to cytoplasmic microtubules, mitotic spindles, manchette, and axonemes of sperm flagella. We found that the absence of β4B-tubulin resulted in male infertility and failure to produce sperm cells. Our findings demonstrate that a lack of β4B-tubulin leads to defects in the initial stages of spermatogenesis. Specifically, β4B-tubulin is needed for the expansion of differentiating spermatogonia, which is essential for the subsequent progression of spermatogenesis. [ABSTRACT FROM AUTHOR]

[Objective] The genes related to pollen abortion of blueberry were screened to provide reference for its cloning and related research and breeding. (Method) Illumina NovaSeq 6000 was used to sequence the transcriptomes of pollen from three rabbiteyed blueberry cultivars, Brightwell (BT)', 'Powderblue (PB)' and 'Premier (PM)', which were derived from the same female parent and had significant differences in pollen viability. The genes related to pollen abortion of blueberry were screened based on the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Protein family (Pfam). (Result) The pollen viability of PM was significantly lower than that of 'BT' and 'PB', and 2717 and 3208 differentially expressed genes were detected in the comparison combinations of BT' vs 'PM' and 'PB' and 'PM', respectively. There were 1124, 1020 and 1791 differentially expressed genes in the GO, KEGG and Pfam databases of the 2717 differentially expressed genes in the comparison combination of BT' vs 'PM', while the 3208 differentially expressed genes of PB' vs 'PM' were annotated 1350,1144 and 2197 differentially expressed genes in GO, KEGG and Pfam databases, respectively. Further screening of GO and Pfam annotation results of differentially expressed genes revealed that 27 differentially expressed genes were related to pollen abortion, of which 22 genes were up-expressed and 5 genes were down-expressed. (Conclusion] The annotation results of GO and Pfam differentially expressed genes related to pollen abortion show that pollen development, inflorescence development, sporopollenin biosynthetic process, formation of pollen exine, cell differentiation of anther wall tapetum, positive regulation of cytokinesis, regulation of mitotic spindle organization, brassinosteroid mediated signal process and other pathways are closely related to pollen abortion in blueberry. It can be used as a reference for further exploring the mechanism of pollen abortion and mining the key genes of pollen abortion in blueberry. [ABSTRACT FROM AUTHOR]

miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including β-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis. [ABSTRACT FROM AUTHOR]

Cytoskeleton‐associated protein 2‐like (CKAP2L) is a paralogue of cytoskeleton‐associated protein 2 (CKAP2). We characterized the expression pattern, subcellular localization, and microtubule‐stabilizing properties of human CKAP2L. The levels of both CKAP2L transcript and protein were cell cycle phase‐dependent, peaking during the G2/M phase and relatively high in certain human tissues, including testis, intestine, and spleen. CKAP2L protein was detectable in all human cancer cell lines we tested. CKAP2L localized to the mitotic spindle apparatus during mitosis, as reported previously. During interphase, however, CKAP2L localized mainly to the nucleus. Ectopic overexpression of CKAP2L resulted in 'microtubule bundling', and, consequently, an elevated CKAP2L level led to prolonged mitosis. These findings support the mitotic role of CKAP2L during the human cell cycle. [ABSTRACT FROM AUTHOR]

Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. We demonstrate in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, we present evidence that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. We postulate that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.

Neuroepithelial transforming gene 1 (NET1) is a RhoA subfamily-specific guanine/nucleotide-exchange factor that exhibits critical roles in diverse biological processes. However, the functions in mouse preimplantation embryonic development have not yet determined. In the present study we demonstrated that NET1 is a key factor in the outcome of early mouse embryonic development. Immunofluorescence detection showed that NET1 is principally localized to the nucleus during mouse pre-implantation embryonic development. Silencing Net1 at the zygote stage using a specific siRNA impaired the developmental competence of early mouse embryos, and Net1 -knockdown (Net1 -KD) induced mitotic spindle-assembly defects and chromosomal alignment abnormalities at the first embryonic cleavage. In addition, reduced NET1 exacerbated reactive oxygen species production and DNA lesions in two-cell stage embryos, further augmenting cellular apoptosis in the preimplantation blastocyst. In summary, our data display key roles for NET1 in mitotic spindle assembly, oxidative stress, and DNA damage during early mouse embryonic development. [ABSTRACT FROM AUTHOR]

Oocyte aging is a key constraint on oocyte quality, leading to fertilization failure and abnormal embryonic development. In addition, it is likely to generate unfavorable assisted reproductive technology (ART) outcomes. SCM-198, a synthetic form of leonurine, was found to rescue the rate of oocyte fragmentation caused by postovulatory aging. Therefore, the aim of this study was to conduct a more in-depth investigation of SCM-198 by exploring its relationship with aged oocytes after ovulation or maternal aging and clarifying whether it affects cell quality. The results indicate that, compared to the postovulatory aged group, the 50 µM SCM-198 group significantly improved sperm-egg binding and increased fertilization of aged oocytes, restoring the spindle apparatus/chromosome structure, cortical granule distribution, and ovastacin and Juno protein distribution. The 50 µM SCM-198 group showed significantly normal mitochondrial distribution, low levels of reactive oxygen species (ROS), and a small quantity of early oocyte apoptosis compared to the postovulatory aged group. Above all, in vivo supplementation with SCM-198 effectively eliminated excess ROS and reduced the spindle/chromosome structural defects in aged mouse oocytes. In summary, these findings indicate that SCM-198 inhibits excessive oxidative stress in oocytes and alters oocyte quality both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

The genetic basis of vertebrate emergence during metazoan evolution has remained largely unknown. Understanding vertebrate‐specific genes, such as the tight junction protein Occludin (Ocln), may help answer this question. Here, it is shown that mammary glands lacking Ocln exhibit retarded epithelial branching, owing to reduced cell proliferation and surface expansion. Interestingly, Ocln regulates mitotic spindle orientation and function, and its loss leads to a range of defects, including prolonged prophase and failed nuclear and/or cytoplasmic division. Mechanistically, Ocln binds to the RabGTPase‐11 adaptor FIP5 and recruits recycling endosomes to the centrosome to participate in spindle assembly and function. FIP5 loss recapitulates Ocln null, leading to prolonged prophase, reduced cell proliferation, and retarded epithelial branching. These results identify a novel role in OCLN‐mediated endosomal trafficking and potentially highlight its involvement in mediating membranous vesicle trafficking and function, which is evolutionarily conserved and essential. [ABSTRACT FROM AUTHOR]

Mitosis exhibits astonishing evolutionary plasticity, with dividing eukaryotic cells differing in the organization of the mitotic spindle and the extent of nuclear envelope breakdown. A new study suggests that a multinucleated lifestyle may favor the evolution of closed nuclear division. [ABSTRACT FROM AUTHOR]

The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a 'timely two-ness' that allows cell division to occur in absence of a SAC-dependent mitotic delay. [ABSTRACT FROM AUTHOR]

Cervical cancer is a malignant disease that seriously affects women's health, and the cause of cervical cancer is complex. Microtubule-associated protein 9 (MAP9) localizes to the mitotic spindle and it plays an important role in spindle assembly and centrosome integrity maintenance. The expression and function of MAP9 is unclear in cervical cancer. This study aims to explore the relationship between DNA methylation of MAP9 and cervical cancer, and the function of MAP9 in cervical cancer. qRT-PCR was used to detect the transcriptional expression of MAP9 in cervical cancer tissues and cell lines. The methylation status of MAP9 promoter was determined by bisulfite sequencing PCR (BSP). The DNA methyltransferase inhibitor, 5-Aza-CdR, was used to inhibit the activity of methyltransferase. Then, the cell proliferation was performed by CCK-8 assay after the transfection of MAP9 plasmids. Furthermore, the cell cycle and apoptosis were performed by flow cytometry. It was found that MAP9 was down-regulated both in cervical cancer tissues and cell lines. The promoter and the first exon region of MAP9 was hypermethylated and its mRNA expression could be restored after the treatment of 5-Aza-CdR. Ectopic expression of MAP9 could inhibit cell proliferation and cell cycle, without effecting the cell apoptosis. The low expression of MAP9 in cervical cancer dues to its hypermethylation and acts a potential tumor suppressor gene. MAP9 might be a novel biomarker in cervical cancer. [ABSTRACT FROM AUTHOR]

Accurate chromosome segregation during cell division relies on coordinated actions of microtubule (MT)-based motor proteins in the mitotic spindle. Kinesin-14 motors play vital roles in spindle assembly and maintenance by crosslinking antiparallel MTs at the spindle midzone and anchoring spindle MTs' minus ends at the poles. In this study, we investigate the force generation and motility of the Kinesin-14 motors HSET and KlpA. Our findings reveal that both motors are non-processive, producing single load-dependent power strokes per MT encounter, with estimated load-free power strokes of ~30 and ~35 nm, respectively. Each homodimeric motor generates forces of ~0.5 pN, but when assembled in teams, they cooperate to generate forces of 1 pN or more. Notably, the cooperative activity among multiple motors leads to increased MT-sliding velocities. These results quantitatively elucidate the structure-function relationship of Kinesin-14 motors and underscore the significance of cooperative behavior in their cellular functions. This study reveals that Kinesin14 motors, HSET and KlpA, generate single load-dependent power strokes, producing forces of ~0.5 pN each. When working in teams, these motors achieve forces of 1 pN or more, enhancing MT-sliding velocities and highlighting their cooperative behaviour in cell division. [ABSTRACT FROM AUTHOR]

Introduction: We have developed a risk-scoring model using gene expression levels related to mitotic spindle assembly (MSA) to predict the prognosis of liver cancer. Methods and results: Initially, we identified 470 genes related to MSA from public databases. Subsequently, through analysis of sequencing data from liver cancer patient samples in online databases, we identified 7 genes suitable for constructing the risk-scoring model. We validated the predictive accuracy and clinical utility of the model. Through drug sensitivity analysis, we identified SAC3D1 as a gene sensitive to the most common anti-tumor drugs among these 7 genes. We propose SAC3D1 as a significant target for future clinical treatment. Furthermore, we conducted in vivo and in vitro experiments to validate the relevance of SAC3D1 to MSA and found its significant impact on the PI3K/Akt signaling pathway and spindle function. Conclusion: Our research introduces a novel risk-scoring model that accurately predicts liver cancer prognosis. Additionally, our findings suggest SAC3D1 as a promising therapeutic target for hepatocellular carcinoma, potentially revealing new mechanisms underlying liver cancer development. [ABSTRACT FROM AUTHOR]

Fertilization occurs before the completion of oocyte meiosis in the majority of animal species and sperm contents move long distances within the zygotes of mouse and C. elegans. If incorporated into the meiotic spindle, paternal chromosomes could be expelled into a polar body resulting in lethal monosomy. Through live imaging of fertilization in C. elegans, we found that the microtubule disassembling enzymes, katanin and kinesin-13 limit long-range movement of sperm contents and that maternal ataxin-2 maintains paternal DNA and paternal mitochondria as a cohesive unit that moves together. Depletion of katanin or double depletion of kinesin-13 and ataxin-2 resulted in the capture of the sperm contents by the meiotic spindle. Thus limiting movement of sperm contents and maintaining cohesion of sperm contents within the zygote both contribute to preventing premature interaction between maternal and paternal genomes. [ABSTRACT FROM AUTHOR]

Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors. [ABSTRACT FROM AUTHOR]

Precise segregation of chromosomes during mitosis requires assembly of a bipolar mitotic spindle followed by correct attachment of microtubules to the kinetochores. This highly spatiotemporally organized process is controlled by various mitotic kinases and molecular motors. We have recently shown that Casein Kinase 1 (CK1) promotes timely progression through mitosis by phosphorylating FAM110A leading to its enrichment at spindle poles. However, the mechanism by which FAM110A exerts its function in mitosis is unknown. Using structure prediction and a set of deletion mutants, we mapped here the interaction of the N-and C-terminal domains of FAM110A with actin and tubulin, respectively. Next, we found that the FAM110A-Δ40-61 mutant deficient in actin binding failed to rescue defects in chromosomal alignment caused by depletion of endogenous FAM110A. Depletion of FAM110A impaired assembly of F-actin in the proximity of spindle poles and was rescued by expression of the wild-type FAM110A, but not the FAM110A-Δ40-61 mutant. Purified FAM110A promoted binding of F-actin to microtubules as well as bundling of actin filaments in vitro. Finally, we found that the inhibition of CK1 impaired spindle actin formation and delayed progression through mitosis. We propose that CK1 and FAM110A promote timely progression through mitosis by mediating the interaction between spindle microtubules and filamentous actin to ensure proper mitotic spindle formation. [ABSTRACT FROM AUTHOR]

Background: Prostate cancer (PrCa) is the most frequently diagnosed cancer in men. Variants in known moderate- to high-penetrance genes explain less than 5% of the cases arising at early-onset (< 56 years) and/or with familial aggregation of the disease. Considering that BubR1 is an essential component of the mitotic spindle assembly checkpoint, we hypothesized that monoallelic BUB1B variants could be sufficient to fuel chromosomal instability (CIN), potentially triggering (prostate) carcinogenesis. Methods: To unveil BUB1B as a new PrCa predisposing gene, we performed targeted next-generation sequencing in germline DNA from 462 early-onset/familial PrCa patients and 1,416 cancer patients fulfilling criteria for genetic testing for other hereditary cancer syndromes. To explore the pan-cancer role of BUB1B, we used in silico BubR1 molecular modeling, in vitro gene-editing, and ex vivo patients' tumors and peripheral blood lymphocytes. Results: Rare BUB1B variants were found in ~ 1.9% of the early-onset/familial PrCa cases and in ~ 0.6% of other cancer patients fulfilling criteria for hereditary disease. We further show that BUB1B variants lead to decreased BubR1 expression and/or stability, which promotes increased premature chromatid separation and, consequently, triggers CIN, driving resistance to Taxol-based therapies. Conclusions: Our study shows that different BUB1B variants may uncover a trigger for CIN-driven carcinogenesis, supporting the role of BUB1B as a (pan)-cancer predisposing gene with potential impact on genetic counseling and treatment decision-making. [ABSTRACT FROM AUTHOR]

Centrioles are the core constituent of centrosomes, microtubule-organizing centers involved in directing mitotic spindle assembly and chromosome segregation in animal cells. In sexually reproducing species, centrioles degenerate during oogenesis and female meiosis is usually acentrosomal. Centrioles are retained during male meiosis and, in most species, are reintroduced with the sperm during fertilization, restoring centriole numbers in embryos. In contrast, the presence, origin, and function of centrioles in parthenogenetic species is unknown. We found that centrioles are maternally inherited in two species of asexual parthenogenetic nematodes and identified two different strategies for maternal inheritance evolved in the two species. In Rhabditophanes diutinus, centrioles organize the poles of the meiotic spindle and are inherited by both the polar body and embryo. In Disploscapter pachys, the two pairs of centrioles remain close together and are inherited by the embryo only. Our results suggest that maternally-inherited centrioles organize the embryonic spindle poles and act as a symmetry-breaking cue to induce embryo polarization. Thus, in these parthenogenetic nematodes, centrioles are maternally-inherited and functionally replace their sperm-inherited counterparts in sexually reproducing species. Centrioles are broken down during oogenesis and are provided by the sperm during sexual reproduction, though how centriole numbers are regulated during asexual reproduction is unclear. Here they study two asexually reproducing nematodes and identify distinct mechanisms for maternal centrosome inheritance. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, PfNDC80 and PfNuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites. Using Ultra-Expansion Microscopy of the malaria parasite, P. falciparum, Li et al. show that disruption of kinetochore components breaks a nexus between the mitotic spindle and the nascent apical organelles. [ABSTRACT FROM AUTHOR]

Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis and has a role in localizing CENP-E, but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild-type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time-lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Long-term inhibition of CNEP-E via GSK923295 recapitulates CTCF knockdown abnormal mitotic spindles with polar chromosomes and increased nuclear sizes. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild-type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting that population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus likely through its known role in recruiting CENP-E. [ABSTRACT FROM AUTHOR]