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Summary: Cell cycle progression is regulated by the orderly balance between kinase and phosphatase activities. PP2A phosphatase holoenzymes containing the B55 family of regulatory B subunits function as major CDK1-counteracting phosphatases during mitotic exit in mammals. However, the identification of the specific mitotic roles of these PP2A-B55 complexes has been hindered by the existence of multiple B55 isoforms. Here, through the generation of loss-of-function genetic mouse models for the two ubiquitous B55 isoforms (B55α and B55δ), we report that PP2A-B55α and PP2A-B55δ complexes display overlapping roles in controlling the dynamics of proper chromosome individualization and clustering during mitosis. In the absence of PP2A-B55 activity, mitotic cells display increased chromosome individualization in the presence of enhanced phosphorylation and perichromosomal loading of Ki-67. These data provide experimental evidence for a regulatory mechanism by which the balance between kinase and PP2A-B55 phosphatase activity controls the Ki-67-mediated spatial organization of the mass of chromosomes during mitosis.

The cell surface protease-activated receptor 1 (PAR1) is overexpressed in glioblastoma multiforme (GBM). We studied the function and structure of intracellular microtubule (MT) and PAR1 in a tubulin-mediated process. We found that exposure to thrombin increased the percentage of proliferative, S, and M phases cells, affected morphology, and increased process elongation. PAR1 antagonist inversely affects these measures, increases tubulin end-binding protein 3 (EB3) mRNA expression in C6 cells, and reduces EB3 comet length, track length, and duration in neuroblastoma cells. In addition, immunofluorescence staining suggests that PAR1 is in close association with the MT α-tubulin and with coagulation cascade proteins during cell division stages. Our findings support PAR1 involvement in MT dynamics.

Summary: Background: Poly(ADP-ribose) polymerase (PARP) inhibitors have emerged as promising chemotherapeutic drugs primarily against BRCA1/2-associated tumours, known as synthetic lethality. However, recent clinical trials reported patients’ survival benefits from PARP inhibitor treatments, irrelevant to homologous recombination deficiency. Therefore, revealing the therapeutic mechanism of PARP inhibitors beyond DNA damage repair is urgently needed, which can facilitate precision medicine. Methods: A CRISPR-based knock-in technology was used to establish stable BRCA1 mutant cancer cells. The effects of PARP inhibitors on BRCA1 mutant cancer cells were evaluated by biochemical and cell biological experiments. Finally, we validated its in vivo effects in xenograft and patient-derived xenograft (PDX) tumour mice. Findings: In this study, we uncovered that the majority of clinical BRCA1 mutations in breast cancers were in and near the middle of the gene, rather than in essential regions for DNA damage repair. Representative mutations such as R1085I and E1222Q caused transient extra spindle poles during mitosis in cancer cells. PAR, which is synthesized by PARP2 but not PARP1 at mitotic centrosomes, clustered these transient extra poles, independent of DNA damage response. Common PARP inhibitors could effectively suppress PARP2-synthesized PAR and induce cell senescence by abrogating the correction of mitotic extra-pole error. Interpretation: Our findings uncover an alternative mechanism by which PARP inhibitors efficiently suppress tumours, thereby pointing to a potential new therapeutic strategy for centrosome error-related tumours. Funding: Funded by National Natural Science Foundation of China (NSFC) (T2225006, 82272948, 82103106), Beijing Municipal Natural Science Foundation (Key program Z220011), and the National Clinical Key Specialty Construction Program, P. R. China (2023).

Resumen: Introducción y objetivo: El fibroxantoma atípico (FXA) y el sarcoma pleomórfico dérmico (SPD) son neoplasias de origen mesenquimal poco frecuentes. Debido a la baja incidencia del SPD y a una nomenclatura históricamente confusa existe poca información acerca de la verdadera agresividad de este tumor. Realizamos el presente estudio con el objetivo de identificar qué características clínicas y/o histológicas del SPD son predictoras de riesgo de recidiva. Material y método: Se diseñó un estudio bicéntrico observacional retrospectivo de 31 casos de SPD diagnosticados y tratados en el Hospital Clínico Universitario de Valencia y el Instituto Valenciano de Oncología, entre los años 2005 y 2020. Se realizó un análisis descriptivo de las características clínicas e histológicas, un análisis inferencial univariado y un análisis multivariado mediante la regresión de Cox. Resultados: En el análisis univariado, la recidiva tumoral (p

Background and objective: Atypical fibroxanthoma and pleomorphic dermal sarcoma (PDS) are rare mesenchymal tumors. Due to the low incidence of PDS and a historically confusing nomenclature, little is known about the true aggressiveness of this tumor. The aim of this study was to investigate clinical and histologic risk factors for recurrence in PDS. Material and methods: Retrospective, observational, bicentric study of 31 PDSs diagnosed and treated at Hospital Clínico Universitario de Valencia and Instituto Valenciano de Oncología in Valencia, Spain, between 2005 and 2020. We described the clinical and histologic features of these tumors and performed univariate analysis and multivariate Cox regression analysis. Results: In the univariate analysis, tumor recurrence (P

Mutations in CSA and CSB proteins cause Cockayne syndrome, a rare genetic neurodevelopment disorder. Alongside their demonstrated roles in DNA repair and transcription, these two proteins have recently been discovered to regulate cytokinesis, the final stage of the cell division. This last finding allowed, for the first time, to highlight an extranuclear localization of CS proteins, beyond the one already known at mitochondria. In this study, we demonstrated an additional role for CSA protein being recruited at centrosomes in a strictly determined step of mitosis, which ranges from pro-metaphase until metaphase exit. Centrosomal CSA exerts its function in specifically targeting the pool of centrosomal Cyclin B1 for ubiquitination and proteasomal degradation. Interestingly, a lack of CSA recruitment at centrosomes does not affect Cyclin B1 centrosomal localization but, instead, it causes its lasting centrosomal permanence, thus inducing Caspase 3 activation and apoptosis. The discovery of this unveiled before CSA recruitment at centrosomes opens a new and promising scenario for the understanding of some of the complex and different clinical aspects of Cockayne Syndrome.

Background: Although the tumor-suppressive effects of ginsenosides in cell cycle have been well established, their pharmacological properties in mitosis have not been clarified yet. The chromosomal instability resulting from dysregulated mitotic processes is usually increased in cancer. In this study, we aimed to investigate the anticancer effects of ginsenoside Rg1 on mitotic progression in cancer. Materials and methods: Cancer cells were treated with ginsenoside Rg1 and their morphology and intensity of different protein were analyzed using immunofluorescence microscopy. The level of proteins in chromosomes was compared through chromosomal fractionation and Western blot analyses. The location and intensity of proteins in the chromosome were confirmed through immunostaining of mitotic chromosome after spreading. The colony formation assays were conducted using various cancer cell lines. Results: Ginsenoside Rg1 reduced cancer cell proliferation in some cancers through inducing mitotic arrest. Mechanistically, it inhibits the phosphorylation of histone H3 Thr3 (H3T3ph) mediated by Haspin kinase and concomitant recruitment of chromosomal passenger complex (CPC) to the centromere. Depletion of Aurora B at the centromere led to abnormal centromere integrity and spindle dynamics, thereby causing mitotic defects, such as increase in the width of the metaphase plate and spindle instability, resulting in delayed mitotic progression and cancer cell proliferation. Conclusion: Ginsenoside Rg1 reduces the level of Aurora B at the centromere via perturbing Haspin kinase activity and concurrent H3T3ph. Therefore, ginsenoside Rg1 suppresses cancer cell proliferation through impeding mitotic processes, such as chromosome alignment and spindle dynamics, upon depletion of Aurora B from the centromere.

In mammalian cells, the Golgi apparatus undergoes fragmentation for its correct partition into two daughter cells during mitosis. Several Golgi structural proteins have been demonstrated to regulate Golgi disassembly/reassembly and spindle formation. However, it is largely unknown whether Golgi proteins mediate other major events in mitosis. Here, we report that Golgin45, a Golgi tethering protein, participates in recruiting PLK1 to the kinetochores. Upon entry into mitosis, Golgin45 binds PLK1 and a nuclear import protein, importin β2. Enriched RanGTP at kinetochores in prometaphase and metaphase sequesters importin β2 from Golgin45 and liberates Golgin45-PLK1 complex, which then gets further delivered to the kinetochores by Golgin45-KNL1 interaction. R375A mutation in Golgin45 that specifically disrupts Golgin45-importin β2 interaction impairs PLK1 localization to the kinetochores, leading to mitotic arrest. Our findings reveal a novel role of a golgin tether protein in mediating Ran-dependent PLK1 enrichment on the kinetochores for proper progression of mitosis., Competing Interests: Declaration of competing interest We have no conflicts of interest to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Proteins localized in the inner nuclear membrane (INM) engage in various fundamental cellular processes via their interactions with outer nuclear membrane (ONM) proteins and nuclear lamina. LAP2-emerin-MAN1 domain (LEMD) family proteins, predominantly positioned in the INM, participate in the maintenance of INM functions, including the reconstruction of the nuclear envelope during mitosis, mechanotransduction, and gene transcriptional modulation. Malfunction of LEMD proteins leads to severe tissue-restricted diseases, which may manifest as fatal deformities and defects. In this review, we summarize the significant roles of LEMD proteins in cellular processes, explains the mechanisms of LEMD protein-related diseases, and puts forward questions in less-explored areas like details in tissue-restricted phenotypes. It intends to sort out previous works about LEMD proteins and pave way for future researchers who might discover deeper mechanisms of and better treatment strategies for LEMD protein-related diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cell division is a highly regulated process that secures the generation of healthy progeny in all organisms, from yeast to human. Dysregulation of this process can lead to uncontrolled cell proliferation and genomic instability, both which are hallmarks of cancer. Cell cycle progression is dictated by a complex network of kinases and phosphatases. These enzymes act on their substrates in a highly specific temporal manner ensuring that the process of cell division is unidirectional and irreversible. Key events of the cell cycle, such as duplication of genetic material and its redistribution to daughter cells, occur in S-phase and mitosis, respectively. Deciphering the dynamics of phosphorylation/dephosphorylation events during these cell cycle phases is important. Here, we showcase a quantitative proteomic and phosphoproteomic mass spectrometry dataset that profiles both early and late phosphorylation events and associated proteome alterations that occur during S-phase and mitotic arrest in the model organism S. cerevisiae. This dataset is of broad interest as the molecular mechanisms governing cell cycle progression are conserved throughout evolution. The data has been deposited in ProteomeXchange with the dataset identifier PXD037291.

To understand general features in evolution of kinetochore organization, investigating a wide range of mitotic mechanisms in various non-model eukaryotes is necessary. A binucleate flagellate Giardia intestinalis is a representative of highly divergent eukaryotic lineage of Metamonads. FIB/SEM tomography was used to investigate ultrastructural details of its mitotic architecture, including kinetochores. Giardia undergoes semi-open mitosis, with the nuclear envelope remaining intact except for polar fenestrae, allowing microtubules to enter the nucleoplasm. At the onset of mitosis, the nuclear envelope bends inward, forming a concave depression at the spindle poles. Spindle microtubules emanate from a cytoplasmic fuzzy microtubule organizing center near the flagellar basal bodies. Kinetochoral microtubules enter the nucleoplasm and bind to kinetochores. A small bipartite kinetochore composed of a dense inner disk, approximately 46 nm in diameter, and a two-armed outer fork, is attached to just one microtubule. To our knowledge, this is the first in situ evidence of a one-microtubule attachment to a kinetochore, which could represent a basic eukaryotic situation.

Background: Silybum marianum (SM) seeds contain a family of flavonolignans. Many studies have revealed the role of SM in regulating cancer cell proliferation and apoptosis, but research has rarely focused on the correlation between cancer cell regulation and SM seed (SMS). Breast cancer is the most commonly diagnosed cancer in women worldwide. In addition to clinical treatments, natural products reportedly act as auxiliary agents or treatments for breast cancer. Purpose: This study investigated the effect of SMS water extract (SMSWE) on breast cancer and explored the potential molecular mechanism of its effect. Methods: We used SMSWE to treat breast cancer cells and animals orthotopically injected with breast cancer cells. Results: The results indicated that SMSWE decreased breast cancer cell viability by disrupting mitosis. The mechanism was modulated by the downregulation of proteins involved in spindle assembly, including polo-like kinase 1 (PLK1) and the EXTAH complex (Eg5, XMAP215, Tpx2, Aurora A, and HURP). In mice orthotopically injected with breast cancer cells and treated with SMSWE, tumor growth significantly decreased and PLK1 expression decreased compared with controls. Conclusions: These current evidences provide that SMSWE treatment can decelerate breast cancer cell growth, and the possible mechanism involves mitosis disruption through decreased PLK1 expression.

USP11 is overexpressed in colorectal cancer (CRC) and breast cancer tissues compared to normal tissues, suggesting a role in promoting cell proliferation and inhibiting cell death. In this study, we observed that depleting USP11 inhibits cell proliferation and delays cell cycle progression. This depletion leads to increased p53 protein levels due to an extended half-life, resulting in elevated p21 mRNA levels in a p53-dependent manner. The rise in p53 protein upon USP11 depletion is linked to a reduced half-life of MDM2, a known E3 ligase for p53, via enhanced polyubiquitination of MDM2. These findings indicate that USP11 might act as a deubiquitinase for MDM2, regulating the MDM2-p53-p21 axis. Additionally, USP11 depletion promotes the induction of senescent cells in a manner dependent on its deubiquitinase activity. Our findings provide insights into the physiological significance of high USP11 expression in primary tumors and its reduction in senescent cells, highlighting its potential as a therapeutic target., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Epigenetics, particularly deacetylation, plays a critical role in tumorigenesis as many carcinogens are under tight control by post-translational modification. HDAC6, an important and special histone deacetylase (HDAC) family member, has been indicated to increase carcinogenesis through various functions. Recent studies demonstrated the effects of HDAC6 inhibitors in mitotic arrest, however, detailed mechanisms still remain unknown. Herein, we review and summarize HDAC6-associated proteins that have been implicated in important roles in mitosis. We also discuss the development of medicinal agents targeting HDAC6., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Objective: Mitotically active cellular fibroma (MACF) of the ovary, characterized by relatively high mitotic activity without severe atypia, was first described in the WHO classification in 2014. However, due to its rarity, the clinicopathological characteristics of ovarian MACF have not been established. This study was performed to describe the clinical, radiological, and pathological features of MACF by analyzing 11 cases of ovarian MACF., Materials and Methods: Between 2015 and 2022, 11 patients with ovarian MACFs underwent surgical treatment at our institution. Clinicopathologic data of the patients were retrospectively reviewed from their medical records., Results: Median patient age was 53.7 years (range 21-77 years), and median tumor diameter was 7.8 cm (range 4.3-14.0 cm). Preoperative CA125 was elevated in 4 cases. Four of the eleven patients had abdominal pain, and two presented with vulvar pain or a palpable abdominal mass, respectively. Preoperative radiological impressions included fibroma, fibrothecoma, stromal tumor, and cystadenocarcinoma. A laparoscopic approach was adopted in 7 cases (64%). Intraoperative frozen section was performed in 5 patients, and all demonstrated the presence of a benign, fibromatous stromal tumor. Three patients underwent fertility-sparing surgery, including laparoscopic ovarian cystectomy and unilateral salpingo-oophorectomy. Median follow-up was 37.7 months (range 2-84 months), and no patient experienced disease relapse or died of their disease., Conclusion: This study shows that ovarian MACF has a benign clinical course. Fertility-sparing surgery provides a safe therapeutic option for MACF, which can be managed safely by laparoscopy. Imaging findings and final pathological diagnosis were not well matched. Intraoperative frozen section is important for determining surgical extent in mitotically active cellular fibroma of the ovary., Competing Interests: Conflicts of interest The authors declare no conflicts of interest related to this manuscript., (Copyright © 2024. Published by Elsevier B.V.)

Chaperonin Containing Tailless complex polypeptide 1 (CCT) is a molecular chaperone composed of eight distinct subunits that can exist as individual monomers or as components of a double oligomeric ring, which is essential for the folding of actin and tubulin and other substrates. Here we assess the role of CCT subunits in the context of cell cycle progression by individual subunit depletions upon siRNA treatment in mammalian cells. The depletion of individual CCT subunits leads to variation in the distribution of cell cycle phases and changes in mitotic index. Mitotic defects, such as unaligned chromosomes occur when CCTδ is depleted, concurrent with a reduction in spindle pole-localised p150 Glued , a component of the dynactin complex and a binding partner of monomeric CCTδ. In CCTδ-depleted cells, changes in the elution profile of p150 Glued are observed consistent with altered conformations and or assembly states with the dynactin complex. Addition of monomeric CCTδ, in the form of GFP-CCTδ, restores correct p150 Glued localisation to the spindle poles and rescues the mitotic segregation defects that occur when CCTδ is depleted. This study demonstrates a requirement for CCTδ in its monomeric form for correct chromosome segregation via a mechanism that promotes the correct localisation of p150 Glued , thus revealing further complexities to the interplay between CCT, tubulin folding and microtubule dynamics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The Eyes Absent family (EYA1-4) are a group of dual function proteins that act as both tyrosine phosphatases and transcriptional co-activators. EYA proteins play a vital role in development, but are also aberrantly overexpressed in cancers, where they often confer an oncogenic effect. Precisely how the EYAs impact cell biology is of growing interest, fuelled by the therapeutic potential of an expanding repertoire of EYA inhibitors. Recent functional studies suggest that the EYAs are important players in the regulation of genome maintenance pathways including DNA repair, mitosis, and DNA replication. While the characterized molecular mechanisms have predominantly been ascribed to EYA phosphatase activities, EYA co-transcriptional activity has also been found to impact the expression of genes that support these pathways. This indicates functional convergence of EYA phosphatase and co-transcriptional activities, highlighting the emerging importance of the EYA protein family at the intersection of genome maintenance mechanisms. In this review, we discuss recent progress in defining EYA protein substrates and transcriptional effects, specifically in the context of genome maintenance. We then outline future directions relevant to the field and discuss the clinical utility of EYA inhibitors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Piwi proteins and Piwi interacting RNAs, piRNAs, presented in germline cells play a role in transposon silencing during germline development. In contrast, the role of somatic Piwi proteins and piRNAs still remains obscure. Here, we characterize the expression pattern and distribution of piRNAs in human renal cells in terms of their potential role in kidney development. Further, we show that all PIWI genes are expressed at the RNA level, however, only PIWIL1 gene is detected at the protein level by western blotting in healthy and cancerous renal cells. So far, the expression of human Piwil1 protein has only been shown in testes and cancer cells, but not in healthy somatic cell lines. Since we observe only Piwil1 protein, the regulation of other PIWI genes is probably more intricated, and depends on environmental conditions. Next, we demonstrate that downregulation of Piwil1 protein results in a decrease in the rate of cell proliferation, while no change in the level of apoptotic cells is observed. Confocal microscopy analysis reveals that Piwil1 protein is located in both cellular compartments, cytoplasm and nucleus in renal cells. Interestingly, in nucleus region Piwil1 is observed close to the spindle during all phases of mitosis in all tested cell lines. It strongly indicates that Piwil1 protein plays an essential role in proliferation of somatic cells. Moreover, involvement of Piwil1 in cell division could, at least partly, explain invasion and metastasis of many types of cancer cells with upregulation of PIWIL1 gene expression. It also makes Piwil1 protein as a potential target in the anticancer therapy., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2024. Published by Elsevier GmbH.)

Loss of Heterozygosity (LOH) due to mitotic recombination is frequently associated with the development of various cancers (e.g. retinoblastoma). LOH is also an important source of genetic diversity, especially in organisms where meiosis is infrequent. Irc20 is a putative helicase, and E3 ubiquitin ligase involved in DNA double-strand break repair pathway. We analyzed genome-wide LOH events, gross chromosomal changes, small insertion-deletions and single nucleotide mutations in eleven S. cerevisiae mutation accumulation lines of irc20∆, which underwent 50 mitotic bottlenecks. LOH enhancement in irc20∆ was small (1.6 fold), but statistically significant as compared to the wild type. Short (≤ 1 kb) and long (> 10 kb) LOH tracts were significantly enhanced in irc20∆. Both interstitial and terminal LOH events were also significantly enhanced in irc20∆ compared to the wild type. LOH events in irc20∆ were more telomere proximal and away from centromeres compared to the wild type. Gross chromosomal changes, single nucleotide mutations and in-dels were comparable between irc20∆ and wild type. Locus based and genome-wide analysis of meiotic recombination showed that meiotic crossover frequencies are not altered in irc20∆. These results suggest Irc20 primarily regulates mitotic recombination and does not affect meiotic crossovers. Our results suggest that the IRC20 gene is important for regulating LOH frequency and distribution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Over the past few decades, unbiased approaches such as genetic screening and protein affinity purification have unveiled numerous proteins involved in DNA double-strand break (DSB) repair and maintaining genome stability. However, despite our knowledge of these protein factors, the underlying molecular mechanisms governing key cellular events during DSB repair remain elusive. Recent evidence has shed light on the role of non-protein factors, such as RNA, in several pivotal steps of DSB repair. In this review, we provide a comprehensive summary of these recent findings, highlighting the significance of ribosomal RNA (rRNA) as a critical mediator of DNA damage response, meiosis, and mitosis. Moreover, we discuss potential mechanisms through which rRNA may influence genome integrity., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Summary: Functional tags are ubiquitous in cell biology, and for studies of one chromosomal locus, the centromere, tags have been remarkably useful. The centromere directs chromosome inheritance at cell division. The location of the centromere is defined by a histone H3 variant, CENP-A. The regulation of the chromatin assembly pathway essential for centromere inheritance and function includes posttranslational modification (PTM) of key components, including CENP-A itself. Others have recently called into question the use of functional tags, with the claim that at least two widely used tags obscured the essentiality of one particular PTM, CENP-AK124 ubiquitination (ub). Here, we employ three independent gene replacement strategies that eliminate large, lysine-containing tags to interrogate these claims. Using these approaches, we find no evidence to support an essential function of CENP-AK124ub. Our general methodology will be useful to validate discoveries permitted by powerful functional tagging schemes at the centromere and other cellular locations.

Maintenance of proper mitotic spindle structure is necessary for error-free chromosome segregation and cell division. Spindle assembly is controlled by force-generating kinesin motors that contribute to its geometry and bipolarity, and balancing motor-dependent forces between opposing kinesins is critical to the integrity of this process. Non-claret dysjunctional (Ncd), a Drosophila kinesin-14 member, crosslinks and slides microtubule minus-ends to focus spindle poles and sustain bipolarity. However, mechanisms that regulate Ncd activity during mitosis are underappreciated. Here, we identify Mushroom body defect (Mud), the fly ortholog of human NuMA, as a direct Ncd binding partner. We demonstrate this interaction involves a short coiled-coil domain within Mud (MudCC) binding the N-terminal, non-motor microtubule-binding domain of Ncd (NcdnMBD). We further show that the C-terminal ATPase motor domain of Ncd (NcdCTm) directly interacts with NcdnMBD as well. Mud binding competes against this self-association and also increases NcdnMBD microtubule binding in vitro. Our results describe an interaction between two spindle-associated proteins and suggest a potentially new mode of minus-end motor protein regulation at mitotic spindle poles.

Summary: Stable transmission of genetic material during cell division requires accurate chromosome segregation. PLK1 dynamics at kinetochores control establishment of correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the regulatory mechanism responsible for PLK1 activity in prometaphase has not yet been affirmatively identified. Here we identify Apolo1, which tunes PLK1 activity for accurate kinetochore-microtubule attachments. Apolo1 localizes to kinetochores during early mitosis, and suppression of Apolo1 results in misaligned chromosomes. Using the fluorescence resonance energy transfer (FRET)-based PLK1 activity reporter, we found that Apolo1 sustains PLK1 kinase activity at kinetochores for accurate attachment during prometaphase. Apolo1 is a cognate substrate of PLK1, and the phosphorylation enables PP1γ to inactivate PLK1 by dephosphorylation. Mechanistically, Apolo1 constitutes a bridge between kinase and phosphatase, which governs PLK1 activity in prometaphase. These findings define a previously uncharacterized feedback loop by which Apolo1 provides fine-tuning for PLK1 to guide chromosome segregation in mitosis.

Extraventricular neurocytoma is a rare tumor, and only a small number of case series of extraventricular neurocytoma have been reported. Because of the rarity of extraventricular neurocytoma, especially disseminated cases, it is difficult to diagnose these cases before surgery. The patient was a 57-year-old man complaining of headaches. Head magnetic resonance imaging demonstrated multiple disseminated lesions. Over two months, the patient was examined for various infectious diseases at the outpatient clinic. However, no abnormalities were detected. He was therefore referred to our neurosurgery department for a histological examination. MRI performed at this time showed the progression of the tumor. The right temporal tumor was partially surgically removed. We diagnosed the tumor as extraventricular neurocytoma. The preoperative clinical and radiological findings suggested a more malignant tumor. The atypical tumor cells and high Ki-67 index were malignant findings. However, neither mitosis nor necrosis was found, and tumor shrinkage was observed after surgery. One year after the surgery, this patient was alive. Brain MRI at this time showed mild tumor regrowth without mass effect. Thus, this may have been an intermediate malignancy.

Carbon dots (CDs) possess considerable potentials in fields like biomarker and cell imaging due to its good fluorescence properties. Nevertheless, the molecular mechanism concerning influences of CDs on plant growth still remains unknown. In this study, the subcellular localization of CDs in Arabidopsis and the molecular mechanism of CDs toxicity to plants were investigated. Results demonstrate that CDs tend to accumulate in meristematic nucleus of root tips. CDs can inhibit growth of meristem zone of primary root (PR) of Arabidopsis seedlings significantly. The transcription level of auxin biosynthesis related genes decreases and the abundance of auxin efflux carriers PIN1 and PIN2 declines after 40 mg/L CDs treatment, thus lowering the auxin level in root tips. Moreover, CDs weaken activity of cell division in meristem zone by disturbing expressions of DNA damage repair genes and cell cycle regulation genes, thus enabling to inhibit growth of the meristem zone. To sum up, CDs inhibit growth of Arabidopsis seedlings through above pathways. These results provide useful information to elaborate potential toxicity mechanism of CDs on terrestrial plants.

Summary: Mitochondria are highly dynamic organelles subjected to fission and fusion events. During mitosis, mitochondrial fission ensures equal distribution of mitochondria to daughter cells. If and how this process can actively drive mitotic progression remains largely unknown. Here, we discover a pathway linking mitochondrial fission to mitotic progression in mammalian cells. The mitochondrial fission factor (MFF), the main mitochondrial receptor for the Dynamin-related protein 1 (DRP1), is directly phosphorylated by Protein Kinase D (PKD) specifically during mitosis. PKD-dependent MFF phosphorylation is required and sufficient for mitochondrial fission in mitotic but not in interphasic cells. Phosphorylation of MFF is crucial for chromosome segregation and promotes cell survival by inhibiting adaptation of the mitotic checkpoint. Thus, PKD/MFF-dependent mitochondrial fission is critical for the maintenance of genome integrity during cell division.

We are investigating plants from the prairie ecological zone of Canada to identify natural products that inhibit mitosis in cancer cells. Investigation of plant parts from the Canadian plant species Hymenoxys richardsonii (Asteraceae) revealed that leaf extracts (PP-360A) had anti-mitotic activity on human cancer cell lines. Cells treated with leaf extracts acquired a rounded morphology, similar to that of cells in mitosis. We demonstrated that the rounded cells contained mitotic spindles and phospho-histone H3 using the techniques of immunofluorescence microscopy. By biology-guided fractionation of H. richardsonii leaves, we isolated a sesquiterpene lactone named hymenoratin, which had not been previously assigned a biological activity. Cells treated with hymenoratin have phospho-histone H3 positive chromosomes, a mitotic spindle, and enter a prolonged mitotic arrest in which the spindles become distorted. By Western blot analysis, hymenoratin treated cells acquire high levels of cyclin B and dephosphorylated Cdk1. There is a growing body of evidence that select members of the sesquiterpene lactone chemical family have anti-mitotic activity.

Summary: Most solid tumors are aneuploid, and p53 has been implicated as the guardian of the euploid genome. Previous experiments using human cell lines showed that aneuploidy induction leads to p53 accumulation and p21-mediated G1 cell cycle arrest. We find that adherent 2-dimensional (2D) cultures of human immortalized or cancer cell lines activate p53 upon aneuploidy induction, whereas suspension cultures of a human lymphoid cell line undergo a p53-independent cell cycle arrest. Surprisingly, 3D human and mouse organotypic cultures from neural, intestinal, or mammary epithelial tissues do not activate p53 or arrest in G1 following aneuploidy induction. p53-deficient colon organoids have increased aneuploidy and frequent lagging chromosomes and multipolar spindles during mitosis. These data suggest that p53 may not act as a universal surveillance factor restricting the proliferation of aneuploid cells but instead helps directly or indirectly ensure faithful chromosome transmission likely by preventing polyploidization and influencing spindle mechanics.

The number of mitotic cells is an important indicator of grading invasive breast cancer. It is very challenging for pathologists to identify and count mitotic cells in pathological sections with naked eyes under the microscope. Therefore, many computational models for the automatic identification of mitotic cells based on machine learning, especially deep learning, have been proposed. However, converging to the local optimal solution is one of the main problems in model training. In this paper, we proposed a novel multilevel iterative training strategy to address the problem. To evaluate the proposed training strategy, we constructed the mitotic cell classification model with ResNet50 and trained the model with different training strategies. The results showed that the models trained with the proposed training strategy performed better than those trained with the conventional strategy in the independent test set, illustrating the effectiveness of the new training strategy. Furthermore, after training with our proposed strategy, the ResNet50 model with Adam optimizer has achieved 89.26% F1 score on the public MITOSI14 dataset, which is higher than that of the state-of-the-art methods reported in the literature., Competing Interests: Declaration of competing interest We hereby declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research presented in the manuscript. No author has any financial or non-financial interests, affiliations, or involvements that may influence the interpretation of the findings or the presentation of the work in this manuscript. Furthermore, there are no sources of financial support, sponsorship, or grants that have influenced the research or outcomes reported in the manuscript. We affirm that our manuscript has been prepared in accordance with the ethical guidelines, and we have adhered to the highest standards of scientific integrity and transparency in conducting and reporting our research., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Recognition of mitotic figures in histologic tumor specimens is highly relevant to patient outcome assessment. This task is challenging for algorithms and human experts alike, with deterioration of algorithmic performance under shifts in image representations. Considerable covariate shifts occur when assessment is performed on different tumor types, images are acquired using different digitization devices, or specimens are produced in different laboratories. This observation motivated the inception of the 2022 challenge on MItosis Domain Generalization (MIDOG 2022). The challenge provided annotated histologic tumor images from six different domains and evaluated the algorithmic approaches for mitotic figure detection provided by nine challenge participants on ten independent domains. Ground truth for mitotic figure detection was established in two ways: a three-expert majority vote and an independent, immunohistochemistry-assisted set of labels. This work represents an overview of the challenge tasks, the algorithmic strategies employed by the participants, and potential factors contributing to their success. With an F 1 score of 0.764 for the top-performing team, we summarize that domain generalization across various tumor domains is possible with today's deep learning-based recognition pipelines. However, we also found that domain characteristics not present in the training set (feline as new species, spindle cell shape as new morphology and a new scanner) led to small but significant decreases in performance. When assessed against the immunohistochemistry-assisted reference standard, all methods resulted in reduced recall scores, with only minor changes in the order of participants in the ranking., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Counting of mitotic figures is a fundamental step in grading and prognostication of several cancers. However, manual mitosis counting is tedious and time-consuming. In addition, variation in the appearance of mitotic figures causes a high degree of discordance among pathologists. With advances in deep learning models, several automatic mitosis detection algorithms have been proposed but they are sensitive to domain shift often seen in histology images. We propose a robust and efficient two-stage mitosis detection framework, which comprises mitosis candidate segmentation (Detecting Fast) and candidate refinement (Detecting Slow) stages. The proposed candidate segmentation model, termed EUNet, is fast and accurate due to its architectural design. EUNet can precisely segment candidates at a lower resolution to considerably speed up candidate detection. Candidates are then refined using a deeper classifier network, EfficientNet-B7, in the second stage. We make sure both stages are robust against domain shift by incorporating domain generalization methods. We demonstrate state-of-the-art performance and generalizability of the proposed model on the three largest publicly available mitosis datasets, winning the two mitosis domain generalization challenge contests (MIDOG21 and MIDOG22). Finally, we showcase the utility of the proposed algorithm by processing the TCGA breast cancer cohort (1,124 whole-slide images) to generate and release a repository of more than 620K potential mitotic figures (not exhaustively validated)., Competing Interests: Declaration of competing interest Mostafa Jahanifar, Simon Graham, Shan E Ahmad Raza, Fayyaz Minhas and Nasir Rajpoot report financial support provided by UK Research and Innovation (UKRI). Nasir Rajpoot and Fayyaz Minhas report financial support from GlaxoSmithKline, outside of the submitted work. Adam Shephard reports financial support from Cancer Research UK (CRUK), outside of the submitted work. Nasir Rajpoot and Simon Graham are co-founders of Histofy Ltd., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The chromosome passenger complex (CPC) is a kinase complex formed by Aurora B, borealin, survivin and inner centromere protein (INCENP). The CPC is active during mitosis and contributes to proper chromosome segregation via the phosphorylation of various substrates. Overexpression of each CPC component has been reported in most cancers. However, its significance remains unclear, as only survivin is known to confer chemoresistance. This study showed that the overexpression of borealin, a CPC component, stabilized survivin protein depending on its interaction with survivin. Unexpectedly, the accumulation of survivin by borealin overexpression did not affect the well-characterized functions of survivin, such as chemoresistance and cell proliferation. Interestingly, the overexpression of borealin promoted lactate production but not the overexpression of the deletion mutant that lacks the ability to bind to survivin. Consistent with these findings, the expression levels of glycolysis-related genes were enhanced in borealin-overexpressing cancer cells. Meanwhile, the overexpression of survivin alone did not promote lactate production. Overall, the accumulation of the borealin-survivin complex promoted glycolysis in squamous cell carcinoma cells. This mechanism may contribute to cancer progression via excessive lactate production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Skp2, the substrate recognition component of the SCF Skp2 ubiquitin ligase, has been implicated in the targeted destruction of a number of key cell cycle regulators and the promotion of S-phase. One of its critical targets is the Cyclin dependent kinase (Cdk) inhibitor p27, and indeed the overexpression of Skp2 in a number of cancers is directly correlated with the premature degradation of p27. Skp2 was first identified as a protein that interacts with Cyclin A in transformed cells, but its role in this complex has remained unclear. In this paper, we demonstrate that Skp2 interacts with Cyclin A in Drosophila and is required to maintain Cyclin A levels and permit mitotic entry. Failure of mitotic entry in Skp2 mutant cells results in polyploidy. If these cells enter mitosis again they are unable to properly segregate their chromosomes, leading to checkpoint dependent cell cycle arrest or apoptosis. Thus, Skp2 is required for mitosis and for maintaining diploidy and genome stability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy, with a median survival of less than 12 months and a 5-year survival of less than 10 %. Here, we have established an image-based screening pipeline for quantifying single PDAC spheroid dynamics in genetically and phenotypically diverse PDAC cell models. Wild-type KRas PDAC cells formed tight/compact spheroids - compaction of these structures was completely blocked by cytoplasmic dynein and focal adhesion kinase (FAK) inhibitors. In contrast, PDAC cells containing mutant KRas formed loosely aggregated spheroids that grew significantly slower following inhibition of polo-like kinase 1 (PLK1) or focal adhesion kinase (FAK). Independent of genetic background, multicellular PDAC-mesenchymal stromal cell (MSC) spheroids self-organized into structures with an MSC-dominant core. The inclusion of MSCs into wild-type KRas PDAC spheroids modestly affected their compaction; however, MSCs significantly increased the compaction and growth of mutant KRas PDAC spheroids. Notably, exogenous collagen 1 potentiated PANC1 spheroid compaction while ITGA1 knockdown in PANC1 cells blocked MSC-induced PANC1 spheroid compaction. In agreement with a role for collagen-based integrin adhesion complexes in stromal cell-induced PDAC phenotypes, we also discovered that MSC-induced PANC1 spheroid growth was completely blocked by the ITGB1 immunoneutralizing antibody mAb13. Finally, multiplexed single-cell immunohistochemical analysis of a 25 patient PDAC tissue microarray revealed a relationship between decreased variance in Spearman r correlation for ITGA1 and PLK1 expression within the tumor cell compartment of PDAC in patients with advanced disease stage, and elevated expression of both ITGA1 and PLK1 in PDAC was found to be associated with decreased patient survival. Taken together, this work uncovers new therapeutic vulnerabilities in PDAC that are relevant to the progression of this stromal cell-rich malignancy and which may reveal strategies for improving patient outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The Aurora-A kinase (AurkA) and its major regulator TPX2 (Targeting Protein for Xklp2) are key mitotic players frequently co-overexpressed in human cancers, and the link between deregulation of the AurkA/TPX2 complex and tumourigenesis is actively investigated. Chromosomal instability, one of the hallmarks of cancer related to the development of intra-tumour heterogeneity, metastasis and chemo-resistance, has been frequently associated with TPX2-overexpressing tumours. In this study we aimed to investigate the actual contribution to chromosomal instability of deregulating the AurkA/TPX2 complex, by overexpressing it in nontransformed hTERT RPE-1 cells. Our results show that overexpression of both AurkA and TPX2 results in increased AurkA activation and severe mitotic defects, compared to AurkA overexpression alone. We also show that AurkA/TPX2 co-overexpression yields increased aneuploidy in daughter cells and the generation of micronucleated cells. Interestingly, the p53/p21 axis response is impaired in AurkA/TPX2 overexpressing cells subjected to different stimuli; consistently, cells acquire increased ability to proliferate after independent induction of mitotic errors, i.e. following nocodazole treatment. Based on our observation that increased levels of the AurkA/TPX2 complex affect chromosome segregation fidelity and interfere with the activation of a pivotal surveillance mechanism in response to altered cell division, we propose that co-overexpression of AurkA and TPX2 per se represents a condition promoting the generation of a genetically unstable context in nontransformed human cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Summary: CENP-A incorporation is critical for centromere specification and is mediated by the chaperone HJURP. The CENP-A-targeting domain (CATD) of CENP-A specifically binds to HJURP, and this binding is conserved. However, the binding interface of CENP-A-HJURP is yet to be understood. Here, we identify the critical residues for chicken CENP-A or HJURP. The A59Q mutation in the α1-helix of chicken CENP-A causes CENP-A mis-incorporation and subsequent cell death, whereas the corresponding mutation in human CENP-A does not. We also find that W53 of HJURP, which is a contact site of A59 in CENP-A, is also essential in chicken cells. Our comprehensive analyses reveal that the affinities of HJURP to CATD differ between chickens and humans. However, the introduction of two arginine residues to the chicken HJURP αA-helix suppresses CENP-A mis-incorporation in chicken cells expressing CENP-AA59Q. Our data explain the mechanisms and evolution of CENP-A essentiality by the CENP-A-HJURP interaction.

Summary: The Drosophila midgut is an excellent system for characterizing cell cycle regulation in the context of tissue homeostasis. Two major progenitor cell types populate the midgut: mitotic intestinal stem cells and their post-mitotic daughters, enteroblasts. Although regulatory networks that control stem cell proliferation are well characterized, how enteroblast mitotic-cell-cycle exit is coordinated with endocycle entry and enterocyte specification remains poorly defined. Myt1 is a conserved Cdk1 inhibitory kinase that regulates mitotic timing during animal development. Here, we use myt1-null mutants and cell-specific RNA interference to investigate Myt1 function in stem cells and enteroblast progenitors. Myt1 depletion alters cell cycle kinetics and promotes ectopic stem cell and enteroblast mitoses at the expense of enteroblast-enterocyte differentiation. These aberrant enteroblast mitoses rely upon cyclin A, implicating Myt1 inhibition of cyclin A/Cdk1 as a mechanism for the coupling mitotic exit with differentiation in enteroblasts.

Aim: The purpose of this study was to use a simple, cost-effective technique for studying mitosis in various grades of oral squamous cell carcinoma (OSCC) using 1% Crystal-violet stain and to correlate mitotic frequency index (MFI) obtained by it with Ki67 labeling index (Ki67LI) so as to validate its usefulness as a selective stain for evaluating proliferation. Materials and Methods: The invasive front grading score (IFG Score) was recorded in 40 patients of OSCC. Mitotic figures were assessed in hematoxylin and eosin (H and E) stained section as well as in 1% crystal-violet stained section using MFI (MFI = Mitosis/total number of cell counted X100). Comparison between MFI obtained by 1% crystal violet stain and H and E stain was done. Ki67LI was assessed using Ki67 immunohistochemical (IHC) marker. Correlation between Ki67LI and MFI obtained by using 1% crystal-violet stain was performed. Results: There was statistically significant increase in MFI obtained by using 1% crystal-violet stain compared to routine H and E stain. Statistically significant positive correlation was observed between Ki67LI and mitosis in well and moderately differentiated OSCC. Positive correlation was also observed in poorly differentiated OSCC, but it was not statistically significant. Both mean MFI and mean Ki67LI significantly increased from grade I to grade II to grade III OSCC. Conclusion: 1% crystal violet stain provides a definite advantage over the H and E stained sections. Thus crystal violet stain is easy and cost effective to evaluate proliferation when compared with expensive proliferative IHC marker.

Although aneuploidy is found in the majority of tumors, the degree of aneuploidy varies widely. It is unclear how cancer cells become aneuploid or how highly aneuploid tumors are different from those of more normal ploidy. We developed a simple computational method that measures the degree of aneuploidy or structural rearrangements of large chromosome regions of 522 human breast tumors from The Cancer Genome Atlas (TCGA). Highly aneuploid tumors overexpress activators of mitotic transcription and the genes encoding proteins that segregate chromosomes. Overexpression of three mitotic transcriptional regulators, E2F1, MYBL2, and FOXM1, is sufficient to increase the rate of lagging anaphase chromosomes in a non-transformed vertebrate tissue, demonstrating that this event can initiate aneuploidy. Highly aneuploid human breast tumors are also enriched in TP53 mutations. TP53 mutations co-associate with the overexpression of mitotic transcriptional activators, suggesting that these events work together to provide fitness to breast tumors.

Summary: Stem cells of the Drosophila midgut (ISCs) are the only mitotically dividing cells of the epithelium and, therefore, presumably the only epithelial cells that require functional kinetochores for microtubule spindle attachment during mitosis. The histone variant CENP-A marks centromeric chromatin as the site of kinetochore formation and spindle attachment during mitotic chromosome segregation. Here, we show that centromeric proteins distribute asymmetrically during ISC division. Whereas newly synthesized CENP-A is enriched in differentiating progeny, CENP-C is undetectable in these cells. Remarkably, CENP-A persists in ISCs for weeks without being replaced, consistent with it being an epigenetic mark responsible for maintaining stem cell properties. Furthermore, CENP-A and its loading factor CAL1 were found to be essential for post-mitotic, differentiating cells; removal of any of these factors interferes with endoreduplication. Taken together, we propose two additional roles of CENP-A: to maintain stem cell-unique properties and to regulate post-mitotic cells.

Summary: The spindle assembly checkpoint (SAC) prevents premature segregation of chromosomes during mitosis. This process requires structural remodeling of MAD2 from O-MAD2 to C-MAD2 conformation. After the checkpoint is satisfied, C-MAD2 is reverted to O-MAD2 to allow anaphase-promoting complex/cyclosome (APC/C) to trigger anaphase. Recently, the AAA+-ATPase TRIP13 was shown to act in concert with p31comet to catalyze C- to O-MAD2. Paradoxically, although C-MAD2 is present in TRIP13-deficient cells, the SAC cannot be activated. Using a degron-mediated system to uncouple TRIP13 from O- and C-MAD2 equilibrium, we demonstrated that the loss of TRIP13 did not immediately abolish the SAC, but the resulting C-MAD2-only environment was insufficient to enable the SAC. These results favor a model in which MAD2-CDC20 interaction is coupled directly to the conversion of O- to C-MAD2 instead of one that involves unliganded C-MAD2. TRIP13 replenishes the O-MAD2 pool for activation by unattached kinetochores.

Aurora B kinase has emerged as a key regulator of mitosis and deregulation of Aurora B activity is closely related to the development and progression of human cancers. In the present study, we found that Aurora B is overexpressed in human esophageal squamous cell carcinoma (ESCC), high levels of Aurora B protein were associated with a worse overall survival rate in ESCC patients. Depleting of Aurora B blunted the malignant phenotypes in ESCC cells. Importantly, we demonstrated that a natural compound, deguelin, has a profound anti-tumor effect on ESCC via inhibiting Aurora B activity. Deguelin potently inhibited in vitro Aurora B kinase activity. The in silico docking study further indicated that deguelin was docked into the ATP-binding pocket of Aurora B. Inhibition of Aurora B activity attenuated growth of ESCC cells, resulted in G2/M cell cycle arrest, polyploidy cells formation, and apoptosis induction. Knocking down of Aurora B decreased the sensitivity of ESCC cells to deguelin. The in vivo results showed that deguelin blocked the phosphorylation of histone H3 and inhibited the growth of ESCC tumor xenografts. Overall, we identified deguelin as an effective Aurora B inhibitor, which deserves further studies in other animal models and ESCC treatment.

Chromosomal instability is a hallmark of cancer and correlates with the presence of extra centrosomes, which originate from centriole overduplication. Overduplicated centrioles lead to the formation of centriole rosettes, which mature into supernumerary centrosomes in the subsequent cell cycle. While extra centrosomes promote chromosome missegregation by clustering into pseudo-bipolar spindles, the contribution of centriole rosettes to chromosome missegregation is unknown. We used multi-modal imaging of cells with conditional centriole overduplication to show that mitotic rosettes in bipolar spindles frequently harbor unequal centriole numbers, leading to biased chromosome capture that favors binding to the prominent pole. This results in chromosome missegregation and aneuploidy. Rosette mitoses lead to viable offspring and significantly contribute to progeny production. We further show that centrosome abnormalities in primary human malignancies frequently consist of centriole rosettes. As asymmetric centriole rosettes generate mitotic errors that can be propagated, rosette mitoses are sufficient to cause chromosome missegregation in cancer.

The second most commonly mutated gene in primary microcephaly (MCPH) patients is wd40-repeat protein 62 (wdr62), but the relative contribution of WDR62 function to the growth of major brain lineages is unknown. Here, we use Drosophila models to dissect lineage-specific WDR62 function(s). Interestingly, although neural stem cell (neuroblast)-specific depletion of WDR62 significantly decreased neuroblast number, brain size was unchanged. In contrast, glial lineage-specific WDR62 depletion significantly decreased brain volume. Moreover, loss of function in glia not only decreased the glial population but also non-autonomously caused neuroblast loss. We further demonstrated that WDR62 controls brain growth through lineage-specific interactions with master mitotic signaling kinase, AURKA. Depletion of AURKA in neuroblasts drives brain overgrowth, which was suppressed by WDR62 co-depletion. In contrast, glial-specific depletion of AURKA significantly decreased brain volume, which was further decreased by WDR62 co-depletion. Thus, dissecting relative contributions of MCPH factors to individual neural lineages will be critical for understanding complex diseases such as microcephaly.

Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. While we have previously established that the timely activation of CyclinB1-Cdk1 in late G2 triggers mitotic entry, the upstream regulatory mechanisms remain unclear. Here, we report that Polo-like kinase 1 (Plk1) is required for entry into mitosis during an unperturbed cell cycle and is rapidly activated shortly before CyclinB1-Cdk1. We determine that Plk1 associates with the Cdc25C1 phosphatase and induces its phosphorylation before mitotic entry. Plk1-dependent Cdc25C1 phosphosites are sufficient to promote mitotic entry, even when Plk1 activity is inhibited. Furthermore, we find that activation of Plk1 during G2 relies on CyclinA2-Cdk activity levels. Our findings thus elucidate a critical role for Plk1 in CyclinB1-Cdk1 activation and mitotic entry and outline how CyclinA2-Cdk, an S-promoting factor, poises cells for commitment to mitosis.

The recent establishment of mammalian haploid embryonic stem cells (ESCs) provides new possibilities for genetic screening and for understanding genome evolution and function. However, the dynamics of mitosis in haploid ESCs is still unclear. Here, we report that the duration of mitosis in haploid ESCs, especially the metaphase, is significantly longer than that in diploid ESCs. Delaying mitosis by chemicals increased self-diploidization of haploid ESCs, while shortening mitosis stabilized haploid ESCs. Taken together, our study suggests that the delayed mitosis of haploid ESCs is associated with self-diploidization.

The mitotic checkpoint ensures proper segregation of chromosomes by delaying anaphase until all kinetochores are bound to microtubules. This inhibitory signal is composed of a complex containing Mad2, which inhibits anaphase progression. The complex can be disassembled by p31comet and TRIP13; however, TRIP13 knockdown has been shown to cause only a mild mitotic delay. Overexpression of checkpoint genes, as well as TRIP13, is correlated with chromosomal instability (CIN) in cancer, but the initial effects of Mad2 overexpression are prolonged mitosis and decreased proliferation. Here, we show that TRIP13 overexpression significantly reduced, and TRIP13 reduction significantly exacerbated, the mitotic delay associated with Mad2 overexpression, but not that induced by microtubule depolymerization. The combination of Mad2 overexpression and TRIP13 loss reduced the ability of checkpoint complexes to disassemble and significantly inhibited the proliferation of cells in culture and tumor xenografts. These results identify an unexpected dependency on TRIP13 in cells overexpressing Mad2.