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Introduction: Medullary carcinoma (MTC) is a rare neuroendocrine thyroid neoplasm. The international medullary thyroid carcinoma grading scheme (IMTCGS), which has prognostic significance, has been introduced recently. The present study graded MTC cases using the IMTCGS and evaluated it in our study cohort. Methods: All MTC thyroidectomy cases over 6 years were evaluated. Low-grade (LG) and high-grade (HG) were compared. Survival analysis included overall survival (OS), loco-regional free survival and distant metastasis free survival (DMFS). Results: Of 32 cases, 31.25% were HG and 68.75% LG. The mean age was 44.0 years and M:F ratio 1:1.146. HG patients were older and had tumour cells with high-grade nuclear features and prominent nucleoli and showed distant metastasis. Necrosis was found more in patients with high grade nuclear features. There was discordance between the high Ki67 (60%) and increased mitotic activity (20%). Univariate survival analysis revealed poor DMFRS and OS in the cohorts with high grade, Ki67 > 5% and coagulative necrosis. The multivariate cox regression analysis showed IMTCGS significantly associated with overall survival (HR 28.30, p = 0.009) and DMFS (HR 15.70, p = 0.02). Discussion and conclusion: This is the first Indian study evaluating IMTCGS, a very simple and convenient grading system that can be readily used in any tertiary health care centre. IHC for Ki 67 should mandatorily be done irrespective of the low mitotic activity on the HPE and necrosis should be diligently searched in cases with high-grade nuclear morphology. HG MTC cohorts were associated with poor OS as well as DMFRS. [ABSTRACT FROM AUTHOR]

The efficacy of current antimitotic cancer drugs is limited by toxicity in highly proliferative healthy tissues. A cancer-specific dependency on the microtubule motor protein KIF18A therefore makes it an attractive therapeutic target. Not all cancers require KIF18A, however, and the determinants underlying this distinction remain unclear. Here, we show that KIF18A inhibition drives a modest and widespread increase in spindle assembly checkpoint (SAC) signaling from kinetochores which can result in lethal mitotic delays. Whether cells arrest in mitosis depends on the robustness of the metaphase-to-anaphase transition, and cells predisposed with weak basal anaphase-promoting complex/cyclosome (APC/C) activity and/or persistent SAC signaling through metaphase are uniquely sensitive to KIF18A inhibition. KIF18A-dependent cancer cells exhibit hallmarks of this SAC:APC/C imbalance, including a long metaphase-to-anaphase transition, and slow mitosis overall. Together, our data reveal vulnerabilities in the cell division apparatus of cancer cells that can be exploited for therapeutic benefit. Synopsis: The kinesin KIF18A has emerged as an important new drug target, but why ~25% of cancers are addicted to this protein remains unclear. This work provides a mechanistic model implicating altered APC/C activity and spindle assembly checkpoint (SAC) signaling in the cancer cell-specific dependence on KIF18A. KIF18A inhibition weakens kinetochore-microtubule interactions, leading to modest but widespread SAC activation in both KIF18A-dependent and KIF18A-agnostic cell lines. Genome-wide CRISPR-Cas9 screens demonstrate that reducing the barrier to mitotic exit or stabilizing kinetochore-microtubule interactions relieves KIF18A inhibitor toxicity. Whole genome doubling magnifies total SAC output after KIF18A inhibition, increasing the probability of lethal mitotic delays. APC/C deficiency is a strong driver of KIF18A dependency, and genetically engineering hyperploidy and low APC/C activity synergistically generates strong KIF18A dependence in otherwise insensitive cells. KIF18A-dependent cells exhibit SAC:APC/C imbalance, including slow metaphase-to-anaphase transition, long overall mitotic duration, and delayed cyclin B1 degradation Sensitivity to depletion of APC/C genes is the strongest correlate for KIF18A dependency in the 550+ cell line Broad DepMap cancer database. A fraction of cancer cells are highly dependent on kinesin KIF18A function because of alterations in the ratio of APC/C and spindle assembly checkpoint (SAC) signals. [ABSTRACT FROM AUTHOR]

SETDB2 is a H3K9 histone methyltransferase required for accurate chromosome segregation. Its H3K9 histone methyltransferase activity was reported to be associated with chromosomes during metaphase. Here, we confirm that SETDB2 is required for mitosis and accurate chromosome segregation. However, these functions are independent of its histone methyltransferase activity. Further analysis showed that SETDB2 can interact with BUBR1, and is required for CDC20 binding to BUBR1 and APC/C complex and CYCLIN B1 degradation. The ability of SETDB2 to regulate the binding of CDC20 to BUBR1 or APC/C complex, and stabilization of CYCLIN B1 are also independent of its histone methyltransferase activity. These results suggest that SETDB2 interacts with BUBR1 to promote binding of CDC20 to BUBR1 and APC3, then degrades CYCLIN B1 to ensure accurate chromosome segregation and mitosis, independently of its histone methyltransferase activity. [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.

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.

Abstract Spindle assembly checkpoint (SAC) is a crucial safeguard mechanism of mitosis fidelity that ensures equal division of duplicated chromosomes to the two progeny cells. Impaired SAC can lead to chromosomal instability (CIN), a well‐recognized hallmark of cancer that facilitates tumor progression; paradoxically, high CIN levels are associated with better therapeutic response and prognosis. However, the mechanism by which CIN determines tumor cell survival and therapeutic response remains poorly understood. Here, using a cross‐omics approach, YY2 is identified as a mitotic regulator that promotes SAC activity by activating the transcription of budding uninhibited by benzimidazole 3 (BUB3), a component of SAC. While both conditions induce CIN, a defect in YY2/SAC activity enhances mitosis and tumor growth. Meanwhile, hyperactivation of SAC mediated by YY2/BUB3 triggers a delay in mitosis and suppresses growth. Furthermore, it is revealed that YY2/BUB3‐mediated excessive CIN causes higher cell death rates and drug sensitivity, whereas residual tumor cells that survived DNA damage‐based therapy have moderate CIN and increased drug resistance. These results provide insights into the role of SAC activity and CIN levels in influencing tumor cell survival and drug response, as well as suggest a novel anti‐tumor therapeutic strategy that combines SAC activity modulators and DNA‐damage agents.

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.

ABSTRACT An interaction between human papillomavirus 16 (HPV16) E2 and the cellular proteins TopBP1 and BRD4 is required for E2 plasmid segregation function. The E2-TopBP1 interaction promotes increased mitotic E2 protein levels in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by HPV16 (HFK + HPV16). SIRT1 deacetylation reduces E2 protein stability and here we demonstrate that increased E2 acetylation occurs during mitosis in a TopBP1 interacting-dependent manner, promoting E2 mitotic stabilization. p300 mediates E2 acetylation and acetylation is increased due to E2 switching off SIRT1 function during mitosis in a TopBP1 interacting-dependent manner, confirmed by increased p53 stability and acetylation on lysine 382, a known target for SIRT1 deacetylation. SIRT1 can complex with E2 in growing cells but is unable to do so during mitosis due to the E2-TopBP1 interaction; SIRT1 is also unable to complex with p53 in mitotic E2 wild-type cells but can complex with p53 outside of mitosis. E2 lysines 111 and 112 are highly conserved residues across all E2 proteins and we demonstrate that K111 hyper-acetylation occurs during mitosis, promoting E2 interaction with Topoisomerase 1 (Top1). We demonstrate that K112 ubiquitination promotes E2 proteasomal degradation during mitosis. E2-TopBP1 interaction promotes mitotic acetylation of CHK2, promoting phosphorylation and activation of the DNA damage response (DDR). The results present a new model in which the E2-TopBP1 complex inactivates SIRT1 during mitosis, and activates the DDR. This is a novel mechanism of HPV16 activation of the DDR, a requirement for the viral life cycle.IMPORTANCEHuman papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. While there are prophylactic vaccines that will significantly alleviate HPV disease burden on future generations, there are currently no anti-viral strategies available for the treatment of HPV cancers. To generate such reagents, we must understand more about the HPV life cycle, and in particular about viral-host interactions. Here, we describe a novel mitotic complex generated by the HPV16 E2 protein interacting with the host protein TopBP1 that controls the function of the deacetylase SIRT1. The E2-TopBP1 interaction disrupts SIRT1 function during mitosis in order to enhance acetylation and stability of viral and host proteins. We also demonstrate that the E2-TopBP1 interaction activates the DDR. This novel complex is essential for the HPV16 life cycle and represents a novel anti-viral therapeutic target.

ABSTRACT Herpesvirus genomes are maintained as extrachromosomal plasmids within the nuclei of infected cells. Some herpesviruses persist within dividing cells, putting the viral genome at risk of being lost to the cytoplasm during mitosis because karyokinesis (nuclear division) requires nuclear envelope breakdown. Oncogenic herpesviruses (and papillomaviruses) avoid genome loss during mitosis by tethering their genomes to cellular chromosomes, thereby ensuring viral genome uptake into newly formed nuclei. These viruses use viral proteins with DNA- and chromatin-binding capabilities to physically link viral and cellular genomes together in a process called tethering. The known viral tethering proteins of human papillomavirus (E2), Epstein-Barr virus (EBNA1), and Kaposi’s sarcoma-associated herpesvirus (LANA) each contain two independent domains required for genome tethering, one that binds sequence specifically to the viral genome and another that binds to cellular chromatin. This latter domain is called a chromatin tethering domain (CTD). The human cytomegalovirus UL123 gene encodes a CTD that is required for the virus to productively infect dividing fibroblast cells within the S phase of the cell cycle, presumably by tethering the viral genome to cellular chromosomes during mitosis. The CTD-containing UL123 gene product that supports S-phase infections is the IE19 protein. Here, we define two motifs in IE19 required for S-phase infections: an N-terminal triple lysine motif and a C-terminal nucleosome-binding motif within the CTD.IMPORTANCEThe IE19 protein encoded by human cytomegalovirus (HCMV) is required for S-phase infection of dividing cells, likely because it tethers the viral genome to cellular chromosomes, thereby allowing them to survive mitosis. The mechanism through which IE19 tethers viral genomes to cellular chromosomes is not understood. For human papillomavirus, Epstein-Barr virus, and Kaposi’s sarcoma-associated herpesvirus, viral genome tethering is required for persistence (latency) and pathogenesis (oncogenesis). Like these viruses, HCMV also achieves latency, and it modulates the properties of glioblastoma multiforme tumors. Therefore, defining the mechanism through which IE19 tethers viral genomes to cellular chromosomes may help us understand, and ultimately combat or control, HCMV latency and oncomodulation.

Proper plant growth and development require spatial coordination of cell divisions. Two unrelated microtubule-binding proteins, TANGLED1 (TAN1) and AUXIN-INDUCED IN ROOT CULTURES9 (AIR9), are together required for normal growth and division plane orientation in Arabidopsis (Arabidopsis thaliana). The tan1 air9 double mutant has synthetic growth and division plane orientation defects, while single mutants lack obvious defects. Here we show that the division site-localized protein, PHRAGMOPLAST ORIENTING KINESIN1 (POK1), was aberrantly lost from the division site during metaphase and telophase in the tan1 air9 mutant. Since TAN1 and POK1 interact via the first 132 amino acids of TAN1 (TAN11-132), we assessed the localization and function of TAN11-132 in the tan1 air9 double mutant. TAN11-132 rescued tan1 air9 mutant phenotypes and localized to the division site during telophase. However, replacing six amino-acid residues within TAN11-132, which disrupted the POK1-TAN1 interaction in the yeast-two-hybrid system, caused loss of both rescue and division site localization of TAN11-132 in the tan1 air9 mutant. Full-length TAN1 with the same alanine substitutions had defects in phragmoplast guidance and reduced TAN1 and POK1 localization at the division site but rescued most tan1 air9 mutant phenotypes. Together, these data suggest that TAN1 and AIR9 are required for POK1 localization, and yet unknown proteins may stabilize TAN1-POK1 interactions.

Histone acetylation levels are reduced during mitosis. To study the mitotic regulation of H3K9ac, we used an array of inhibitors targeting specific histone deacetylases. We evaluated the involvement of the targeted enzymes in regulating H3K9ac during all mitotic stages by immunofluorescence and immunoblots. We identified HDAC2, HDAC3, and SIRT1 as modulators of H3K9ac mitotic levels. HDAC2 inhibition increased H3K9ac levels in prophase, whereas HDAC3 or SIRT1 inhibition increased H3K9ac levels in metaphase. Next, we performed ChIP-seq on mitotic-arrested cells following targeted inhibition of these histone deacetylases. We found that both HDAC2 and HDAC3 have a similar impact on H3K9ac, and inhibiting either of these two HDACs substantially increases the levels of this histone acetylation in promoters, enhancers, and insulators. Altogether, our results support a model in which H3K9 deacetylation is a stepwise process-at prophase, HDAC2 modulates most transcription-associated H3K9ac-marked loci, and at metaphase, HDAC3 maintains the reduced acetylation, whereas SIRT1 potentially regulates H3K9ac by impacting HAT activity.

Abstract Mitosis is a critical criterion for meningioma grading. However, pathologists’ assessment of mitoses is subject to significant inter-observer variation due to challenges in locating mitosis hotspots and accurately detecting mitotic figures. To address this issue, we leverage digital pathology and propose a computational strategy to enhance pathologists’ mitosis assessment. The strategy has two components: (1) A depth-first search algorithm that quantifies the mathematically maximum mitotic count in 10 consecutive high-power fields, which can enhance the preciseness, especially in cases with borderline mitotic count. (2) Implementing a collaborative sphere to group a set of pathologists to detect mitoses under each high-power field, which can mitigate subjective random errors in mitosis detection originating from individual detection errors. By depth-first search algorithm (1) , we analyzed 19 meningioma slides and discovered that the proposed algorithm upgraded two borderline cases verified at consensus conferences. This improvement is attributed to the algorithm’s ability to quantify the mitotic count more comprehensively compared to other conventional methods of counting mitoses. In implementing a collaborative sphere (2) , we evaluated the correctness of mitosis detection from grouped pathologists and/or pathology residents, where each member of the group annotated a set of 48 high-power field images for mitotic figures independently. We report that groups with sizes of three can achieve an average precision of 0.897 and sensitivity of 0.699 in mitosis detection, which is higher than an average pathologist in this study (precision: 0.750, sensitivity: 0.667). The proposed computational strategy can be integrated with artificial intelligence workflow, which envisions the future of achieving a rapid and robust mitosis assessment by interactive assisting algorithms that can ultimately benefit patient management.

Abstract Objective The centrosome is universally recognized as the microtubule organizing center of animal cells, but emerging evidence suggests that it has other important functions including primary cilia formation, DNA damage checkpoints, and cell cycle progression. Despite this, the role of individual components of the centrosome remains unclear. Previous studies suggest that one component, centriolin, has an important function in cytokinesis and cell cycle progression, although its exact role in these processes is not known. To determine how centriolin influences the progression through the cell cycle, we sought to identify interacting partners that may be involved in regulating its function. Results This study provides evidence that the ubiquitin E3 ligase HectD1 binds to centriolin and that this association likely accounts for our observation that HectD1 co-localizes with centriolin at the centrosome during mitosis. In addition to its centrosomal localization, we also show that the expression of HectD1 fluctuates throughout the cell cycle, with the highest levels during mitosis, coinciding with a marked reduction in centriolin expression. We propose that the interaction between HectD1 and centriolin may be necessary for normal cell cycle progression and we speculate that this function may involve HectD1-mediated degradation of centriolin.

Abstract Background The maintenance of spindle pole integrity is essential for spindle assembly and chromosome segregation during mitosis. However, the underlying mechanisms governing spindle pole integrity remain unclear. Methods ENSA was inhibited by siRNA or MKI-2 treatment and its effect on cell cycle progression, chromosome alignment and microtubule alignment was observed by immunohistochemical staining and western blotting. PP2A-B55α knockdown by siRNA was performed to rescue the phenotype caused by ENSA inhibition. The interaction between ENSA and Aurora A was detected by in situ PLA. Furthermore, orthotopic implantation of 4Tl-luc cancer cells was conducted to confirm the consistency between the in vitro and in vivo relationship of the ENSA-Aurora A interaction. Results During mitosis, p-ENSA is localized at the spindle poles, and the inhibition of ENSA results in mitotic defects, such as misaligned chromosomes, multipolar spindles, asymmetric bipolar spindles, and centrosome defects, with a delay in mitotic progression. Although the mitotic delay caused by ENSA inhibition was rescued by PP2A-B55α depletion, spindle pole defects persisted. Notably, we observed a interaction between ENSA and Aurora A during mitosis, and inhibition of ENSA reduced Aurora A expression at the mitotic spindle poles. Injecting MKI-2-sensitized tumors led to increased chromosomal instability and downregulation of the MASTL-ENSA-Aurora A pathway in an orthotopic breast cancer mouse model. Conclusions These findings provide novel insights into the regulation of spindle pole integrity by the MASTL-ENSA-Aurora A pathway during mitosis, highlighting the significance of ENSA in recruiting Aurora A to the spindle pole, independent of PP2A-B55α.

Background: The centrosome is one of the principal cell hubs, where numerous proteins important for intracellular regulatory processes are concentrated. One of them, serine-threonine kinase 6, alias Aurora A, is involved in centrosome duplication and mitotic spindle formation and maintenance. Methods: Long-term vital observations of cells, immunofluorescence analysis of protein localization, synchronization of cells at different phases of the cell cycle, Western blot analysis of protein content were used in the work. Results: In this study, we investigated the dynamics of Aurora A protein accumulation and degradation in the XL2 Xenopus cell line during its 28-hour cell cycle. Using Western blot and immunofluorescence analyses, we demonstrated that Aurora A disappeared from the centrosome within one hour following mitosis and was not redistributed to other cell compartments. Using double Aurora A/Bromodeoxyuridine immunofluorescence labeling of the cells with precisely determined cell cycle stages, we observed that Aurora A reappeared in the centrosome during the S-phase, which was earlier than reported for all other known proteins with mitosis-specific centrosomal localization. Moreover, Aurora A accumulation in the centrosomal region and centrosome separation were asynchronous in the sister cells. Conclusions: The reported data allowed us to hypothesize that Aurora A is one of the primary links in coordinating centrosome separation and constructing the mitotic spindle.

The centrosome of the amoebozoan model Dictyostelium discoideum provides the best-established model for an acentriolar centrosome outside the Opisthokonta. Dictyostelium exhibits an unusual centrosome cycle, in which duplication is initiated only at the G2/M transition and occurs entirely during the M phase. Little is known about the role of conserved centrosomal kinases in this process. Therefore, we have generated knock-in strains for Aurora (AurK), CDK1, cyclin B, Nek2, and Plk, replacing the endogenous genes with constructs expressing the respective green fluorescent Neon fusion proteins, driven by the endogenous promoters, and studied their behavior in living cells. Our results show that CDK1 and cyclin B arrive at the centrosome first, already during G2, followed by Plk, Nek2, and AurK. Furthermore, CDK1/cyclin B and AurK were dynamically localized at kinetochores, and AurK in addition at nucleoli. The putative roles of all four kinases in centrosome duplication, mitosis, cytokinesis, and nucleolar dynamics are discussed.

The kinetochore links chromosomes to spindle microtubules to drive chromosome segregation at cell division. While we know nearly all mammalian kinetochore proteins, how these give rise to the strong yet dynamic microtubule attachments required for function remains poorly understood. Here, we focus on the Astrin-SKAP complex, which localizes to bioriented kinetochores and is essential for chromosome segregation but whose mechanical role is unclear. Live imaging reveals that SKAP depletion dampens the movement and decreases the coordination of metaphase sister kinetochores and increases the tension between them. Using laser ablation to isolate kinetochores bound to polymerizing versus depolymerizing microtubules, we show that without SKAP, kinetochores move slower on both polymerizing and depolymerizing microtubules and that more force is needed to rescue microtubules to polymerize. Thus, in contrast to the previously described kinetochore proteins that increase the grip on microtubules under force, Astrin-SKAP reduces the grip, increasing attachment dynamics and force responsiveness and reducing friction. Together, our findings suggest a model where the Astrin-SKAP complex effectively "lubricates" correct, bioriented attachments to help preserve them.

During mitosis and meiosis in the majority of eukaryotes, centromeric chromatin comprised of CENP-A nucleosomes and their reader CENP-C recruits components of the outer kinetochore to build an interface with spindle microtubules.1,2 One exception is C. elegans oocyte meiosis, where outer kinetochore proteins form cup-like structures on chromosomes independently of centromeric chromatin.3 Here, we show that the nucleoporin MEL-28 (ortholog of human ELYS) and CENP-CHCP-4 act in parallel to recruit outer kinetochore components to oocyte meiotic chromosomes. Unexpectedly, co-inhibition of MEL-28 and CENP-CHCP-4 resulted in chromosomes being expelled from the meiotic spindle prior to anaphase onset, a more severe phenotype than what was observed following ablation of the outer kinetochore.4,5 This observation suggested that MEL-28 and the outer kinetochore independently link chromosomes to spindle microtubules. Consistent with this, the chromosome expulsion defect was observed following co-inhibition of MEL-28 and the microtubule-coupling KNL-1/MIS-12/NDC-80 (KMN) network of the outer kinetochore. Use of engineered mutants showed that MEL-28 acts in conjunction with the microtubule-binding NDC-80 complex to keep chromosomes within the oocyte meiotic spindle and that this function likely involves the Y-complex of nucleoporins that associate with MEL-28; by contrast, the ability to dock protein phosphatase 1, shared by MEL-28 and KNL-1, is not involved. These results highlight nuclear pore-independent functions for a conserved nucleoporin and explain two unusual features of oocyte meiotic chromosome segregation in C. elegans: centromeric chromatin-independent outer kinetochore assembly, and dispensability of the outer kinetochore for constraining chromosomes in the acentrosomal meiotic spindle.

Regulation of microtubule dynamics by microtubule-associated proteins (MAPs) is essential for mitotic spindle assembly and chromosome segregation. Altered microtubule dynamics, particularly increased microtubule growth rates, were found to be a contributing factor for the development of chromosomal instability, which potentiates tumorigenesis. The MAP XMAP215/CKAP5 is the only known microtubule growth factor, and whether other MAPs regulate microtubule growth in cells is unclear. Our recent in vitro reconstitution experiments have demonstrated that Cytoskeleton-Associated Protein 2 (CKAP2) increases microtubule nucleation and growth rates, and here, we find that CKAP2 is also an essential microtubule growth factor in cells. By applying CRISPR-Cas9 knock-in and knock-out (KO) as well as microtubule plus-end tracking live cell imaging, we show that CKAP2 is a mitotic spindle protein that ensures faithful chromosome segregation by regulating microtubule growth. Live cell imaging of endogenously labeled CKAP2 showed that it localizes to the spindle during mitosis and rapidly shifts its localization to the chromatin upon mitotic exit before being degraded. Cells lacking CKAP2 display reduced microtubule growth rates and an increased proportion of chromosome segregation errors and aneuploidy that may be a result of an accumulation of kinetochore-microtubule misattachments. Microtubule growth rates and chromosome segregation fidelity can be rescued upon ectopic CKAP2 expression in KO cells, revealing a direct link between CKAP2 expression and microtubule dynamics. Our results unveil a role of CKAP2 in regulating microtubule growth in cells and provide a mechanistic explanation for the oncogenic potential of CKAP2 misregulation. [ABSTRACT FROM AUTHOR]

The mitosis to meiosis transition requires dynamic changes in gene expression, but whether and how the mitotic transcriptional machinery is regulated during this transition is unknown. In budding yeast, SBF and MBF transcription factors initiate the mitotic gene expression program. Here, we report two mechanisms that work together to restrict SBF activity during meiotic entry: repression of the SBF-specific Swi4 subunit through LUTI-based regulation and inhibition of SBF by Whi5, a functional homolog of the Rb tumor suppressor. We find that untimely SBF activation causes downregulation of early meiotic genes and delays meiotic entry. These defects are largely driven by the SBF-target G1 cyclins, which block the interaction between the central meiotic regulator Ime1 and its cofactor Ume6. Our study provides insight into the role of SWI4LUTI in establishing the meiotic transcriptional program and demonstrates how the LUTI-based regulation is integrated into a larger regulatory network to ensure timely SBF activity. [ABSTRACT FROM AUTHOR]

Mammalian preimplantation embryos often contend with aneuploidy that arose either by the inheritance of meiotic errors from the gametes, or from mitotic mis-segregation events that occurred following fertilization. Regardless of the origin, mis-segregated chromosomes become encapsulated in micronuclei (MN) that are spatially isolated from the main nucleus. Much of our knowledge of MN formation comes from dividing somatic cells during tumorigenesis, but the error-prone cleavage-stage of early embryogenesis is fundamentally different. One unique aspect is that cellular fragmentation (CF), whereby small subcellular bodies pinch off embryonic blastomeres, is frequently observed. CF has been detected in both in vitro and in vivo-derived embryos and likely represents a response to chromosome mis-segregation since it only appears after MN formation. There are multiple fates for MN, including sequestration into CFs, but the molecular mechanism(s) by which this occurs remains unclear. Due to nuclear envelope rupture, the chromosomal material contained within MN and CFs becomes susceptible to double stranded-DNA breaks. Despite this damage, embryos may still progress to the blastocyst stage and exclude chromosome-containing CFs, as well as non-dividing aneuploid blastomeres, from participating in further development. Whether these are attempts to rectify MN formation or eliminate embryos with poor implantation potential is unknown and this review will discuss the potential implications of DNA removal by CF/blastomere exclusion. We will also extrapolate what is known about the intracellular pathways mediating MN formation and rupture in somatic cells to preimplantation embryogenesis and how nuclear budding and DNA release into the cytoplasm may impact overall development. [ABSTRACT FROM AUTHOR]

During cell division, precise and regulated distribution of cellular material between daughter cells is a critical step and is governed by complex biochemical and biophysical mechanisms. To achieve this, membraneless organelles and condensates often require complete disassembly during mitosis. The biophysical principles governing the disassembly of condensates remain poorly understood. Here, we used a physical biology approach to study how physical and material properties of the nucleolus, a prominent nuclear membraneless organelle in eukaryotic cells, change during mitosis and across different scales. We found that nucleolus disassembly proceeds continuously through two distinct phases with a slow and reversible preparatory phase followed by a rapid irreversible phase that was concurrent with the nuclear envelope breakdown. We measured microscopic properties of nucleolar material including effective diffusion rates and binding affinities as well as key macroscopic properties of surface tension and bending rigidity. By incorporating these measurements into the framework of critical phenomena, we found evidence that near mitosis surface tension displays a power-law behavior as a function of biochemically modulated interaction strength. This two-step disassembly mechanism maintains structural and functional stability of nucleolus while enabling its rapid and efficient disassembly in response to cell cycle cues. [ABSTRACT FROM AUTHOR]

Subcellular location and activation of Tank Binding Kinase 1 (TBK1) govern precise progression through mitosis. Either loss of activated TBK1 or its sequestration from the centrosomes causes errors in mitosis and growth defects. Yet, what regulates its recruitment and activation on the centrosomes is unknown. We identified that NAK-associated protein 1 (NAP1) is essential for mitosis, binding to and activating TBK1, which both localize to centrosomes. Loss of NAP1 causes several mitotic and cytokinetic defects due to inactivation of TBK1. Our quantitative phosphoproteomics identified numerous TBK1 substrates that are not only confined to the centrosomes but are also associated with microtubules. Substrate motifs analysis indicates that TBK1 acts upstream of other essential cell cycle kinases like Aurora and PAK kinases. We also identified NAP1 as a TBK1 substrate phosphorylating NAP1 at S318 to promote its degradation by the ubiquitin proteasomal system. These data uncover an important distinct function for the NAP1-TBK1 complex during cell division. [ABSTRACT FROM AUTHOR]

In this paper, the biological function of PLK-1, the correlation between PLK-1 and tumors, and the latest research progress on PLK-1 inhibitors under study are reviewed, in order to provide references for the research and development of PLK-1 inhibitors. [ABSTRACT FROM AUTHOR]

The preservation of gene expression patterns that define cellular identity throughout the cell division cycle is essential to perpetuate cellular lineages. However, the progression of cells through different phases of the cell cycle severely disrupts chromatin accessibility, epigenetic marks, and the recruitment of transcriptional regulators. Notably, chromatin is transiently disassembled during S-phase and undergoes drastic condensation during mitosis, which is a significant challenge to the preservation of gene expression patterns between cell generations. This article delves into the specific gene expression and chromatin regulatory mechanisms that facilitate the preservation of transcriptional identity during replication and mitosis. Furthermore, we emphasize our recent findings revealing the unconventional role of yeast centromeres and mitotic chromosomes in maintaining transcriptional fidelity beyond mitosis. [ABSTRACT FROM AUTHOR]

There is dynamic and bidirectional regulation of cell metabolism and cell cycle progression that is incompletely understood. Changes to cellular metabolism, including redox state, can directly affect the activity of cell cycle regulators and signaling pathways to impact cell cycle control. Mitochondrial metabolism changes dramatically during the cell cycle and most acutely during mitosis. Cells adjust cell cycle duration and growth rate to couple cell size to cell cycle progression and maintain uniform cell size across generations. Disruption of protein synthesis or DNA replication can drive cells to exit the cell cycle and become quiescent. The relationship between metabolism and cell cycle progression is complex and bidirectional. Cells must rewire metabolism to meet changing biosynthetic demands across cell cycle phases. In turn, metabolism can influence cell cycle progression through direct regulation of cell cycle proteins, through nutrient-sensing signaling pathways, and through its impact on cell growth, which is linked to cell division. Furthermore, metabolism is a key player in mediating quiescence–proliferation transitions in physiologically important cell types, such as stem cells. How metabolism impacts cell cycle progression, exit, and re-entry, as well as how these processes impact metabolism, is not fully understood. Recent advances uncovering mechanistic links between cell cycle regulators and metabolic processes demonstrate a complex relationship between metabolism and cell cycle control, with many questions remaining. [ABSTRACT FROM AUTHOR]

Transcription represents a central aspect of gene expression with RNA polymerase machineries (RNA Pol) driving the synthesis of RNA from DNA template molecules. In eukaryotes, a total of three RNA Pol enzymes generate the plethora of RNA species and RNA Pol II is the one transcribing all protein-coding genes. A high number of cis- and trans-acting factors orchestrates RNA Pol II-mediated transcription by influencing the chromatin recruitment, activation, elongation, and/or termination steps. The levels of DNA accessibility, defining open-euchromatin versus close-heterochromatin, delimits RNA Pol II activity as well as the encounter with other factors acting on chromatin such as the DNA replication or DNA repair machineries. The stage of the cell cycle highly influences RNA Pol II activity with mitosis representing the major challenge. In fact, there is a massive inhibition of transcription during the mitotic entry coupled with chromatin dissociation of most of the components of the transcriptional machinery. Mitosis, as a consequence, highly compromises the transcriptional memory and the perpetuation of cellular identity. Once mitosis ends, transcription levels immediately recover to define the cell fate and to safeguard the proper progression of daughter cells through the cell cycle. In this review, we evaluate our current understanding of the transcriptional repression associated with mitosis with a special focus on the molecular mechanisms involved, on the potential function behind the general repression, and on the transmission of the transcriptional machinery into the daughter cells. We finally discuss the contribution that errors in the inheritance of the transcriptional machinery across mitosis might play in stem cell aging. [ABSTRACT FROM AUTHOR]

Simple Summary: Performing a mitosis count (MC) is essential in grading canine Soft Tissue Sarcoma (cSTS) and canine Perivascular Wall Tumours (cPWTs), although it is subject to inter- and intra-observer variability. To enhance standardisation, an artificial intelligence mitosis detection approach was investigated. A two-step annotation process was utilised with a pre-trained Faster R-CNN model, refined through veterinary pathologists' reviews of false positives, and subsequently optimised using an F1-score thresholding method to maximise accuracy measures. The study achieved a best F1-score of 0.75, demonstrating competitiveness in the field of canine mitosis detection. Performing a mitosis count (MC) is the diagnostic task of histologically grading canine Soft Tissue Sarcoma (cSTS). However, mitosis count is subject to inter- and intra-observer variability. Deep learning models can offer a standardisation in the process of MC used to histologically grade canine Soft Tissue Sarcomas. Subsequently, the focus of this study was mitosis detection in canine Perivascular Wall Tumours (cPWTs). Generating mitosis annotations is a long and arduous process open to inter-observer variability. Therefore, by keeping pathologists in the loop, a two-step annotation process was performed where a pre-trained Faster R-CNN model was trained on initial annotations provided by veterinary pathologists. The pathologists reviewed the output false positive mitosis candidates and determined whether these were overlooked candidates, thus updating the dataset. Faster R-CNN was then trained on this updated dataset. An optimal decision threshold was applied to maximise the F1-score predetermined using the validation set and produced our best F1-score of 0.75, which is competitive with the state of the art in the canine mitosis domain. [ABSTRACT FROM AUTHOR]

Recently, the diarylpentanoid BP-M345 (5) has been identified as a potent in vitro growth inhibitor of cancer cells, with a GI50 value between 0.17 and 0.45 µM, showing low toxicity in non-tumor cells. BP-M345 (5) promotes mitotic arrest by interfering with mitotic spindle assembly, leading to apoptotic cell death. Following on from our previous work, we designed and synthesized a library of BP-M345 (5) analogs and evaluated the cell growth inhibitory activity of three human cancer cell lines within this library in order to perform structure–activity relationship (SAR) studies and to obtain compounds with improved antimitotic effects. Four compounds (7, 9, 13, and 16) were active, and the growth inhibition effects of compounds 7, 13, and 16 were associated with a pronounced arrest in mitosis. These compounds exhibited a similar or even higher mitotic index than BP-M345 (5), with compound 13 displaying the highest antimitotic activity, associated with the interference with mitotic spindle dynamics, inducing spindle collapse and, consequently, prolonged mitotic arrest, culminating in massive cancer cell death by apoptosis. [ABSTRACT FROM AUTHOR]

As part of the central nervous system (CNS), the retina senses light and also conducts and processes visual impulses. The damaged development of the retina not only causes visual damage, but also leads to epilepsy, dementia and other brain diseases. Recently, we have reported that copper (Cu) overload induces retinal developmental defects and down-regulates microtubule (MT) genes during zebrafish embryogenesis, but whether the down-regulation of microtubule genes mediates Cu stress induced retinal developmental defects is still unknown. In this study, we found that microtubule gene stmn4 exhibited obviously reduced expression in the retina of Cu overload embryos. Furthermore, stmn4 deficiency (stmn4−/−) resulted in retinal defects similar to those seen in Cu overload embryos, while overexpression of stmn4 effectively rescued retinal defects and cell apoptosis occurred in the Cu overload embryos and larvae. Meanwhile, stmn4 deficient embryos and larvae exhibited reduced mature retinal cells, the down-regulated expression of microtubules and cell cycle-related genes, and the mitotic cell cycle arrests of the retinal cells, which subsequently tended to apoptosis independent on p53. The results of this study demonstrate that Cu stress might lead to retinal developmental defects via down-regulating expression of microtubule gene stmn4, and stmn4 deficiency leads to impaired cell cycle and the accumulation of retinal progenitor cells (RPCs) and their subsequent apoptosis. The study provides a certain referee for copper overload in regulating the retinal development in fish. [ABSTRACT FROM AUTHOR]

Stable transmission of genetic information during cell division requires faithful chromosome segregation. Mounting evidence has demonstrated that polo-like kinase 1 (PLK1) dynamics at kinetochores control correct kinetochore–microtubule attachments and subsequent silencing of the spindle assembly checkpoint. However, the mechanisms underlying PLK1-mediated silencing of the spindle checkpoint remain elusive. Here, we identified a regulatory mechanism by which PLK1-elicited zeste white 10 (ZW10) phosphorylation regulates spindle checkpoint silencing in mitosis. ZW10 is a cognate substrate of PLK1, and the phosphorylation of ZW10 at Ser12 enables dynamic ZW10–Zwint1 interactions. Inhibition of ZW10 phosphorylation resulted in misaligned chromosomes, while persistent expression of phospho-mimicking ZW10 mutant caused premature anaphase, in which sister chromatids entangled as cells entered anaphase. These findings reveal the previously uncharacterized PLK1–ZW10 interaction through which dynamic phosphorylation of ZW10 fine-tunes accurate chromosome segregation in mitosis. [ABSTRACT FROM AUTHOR]

In eukaryotes, microtubule polymers are essential for cellular plasticity and fate decisions. End-binding (EB) proteins serve as scaffolds for orchestrating microtubule polymer dynamics and are essential for cellular dynamics and chromosome segregation in mitosis. Here, we show that EB1 forms molecular condensates with TIP150 and MCAK through liquid–liquid phase separation to compartmentalize the kinetochore–microtubule plus-end machinery, ensuring accurate kinetochore–microtubule interactions during chromosome segregation in mitosis. Perturbation of EB1–TIP150 polymer formation by a competing peptide prevents phase separation of the EB1-mediated complex and chromosome alignment at the metaphase equator in both cultured cells and Drosophila embryos. Lys220 of EB1 is dynamically acetylated by p300/CBP-associated factor in early mitosis, and persistent acetylation at Lys220 attenuates phase separation of the EB1-mediated complex, dissolves droplets in vitro , and harnesses accurate chromosome segregation. Our data suggest a novel framework for understanding the organization and regulation of eukaryotic spindle for accurate chromosome segregation in mitosis. [ABSTRACT FROM AUTHOR]

Accurate chromosome segregation in mitosis depends on kinetochores that connect centromeric chromatin to spindle microtubules. Centromeres are captured by individual microtubules via a kinetochore constitutive centromere-associated network (CCAN) during chromosome segregation. CCAN contains 16 subunits, including CENP-W and CENP-T. However, the molecular recognition and mitotic regulation of the CCAN assembly remain elusive. Here, we revealed that CENP-W binds to the histone fold domain and an uncharacterized N-terminal region of CENP-T. Aurora B phosphorylates CENP-W at threonine 60, which enhances the interaction between CENP-W and CENP-T to ensure robust metaphase chromosome alignment and accurate chromosome segregation in mitosis. These findings delineate a conserved signaling cascade that integrates protein phosphorylation with CCAN integrity for the maintenance of genomic stability. [ABSTRACT FROM AUTHOR]

Mitosis cell counting from histopathology image is one of the important process as a part of proliferative activity for cancer grading. It provides a level of progression and estimate the aggressiveness of particular diseases. Unfortunately, manual mitosis counting evaluation is a tedious, very labor intensive, and challenging task in analysing grade of a particular cancer. Since breast cancer recurrence and metastasis are intrinsically related to mortality, it is critical to predict the recurrence and metastasis risk of an individual patient, which is essential for adjutant therapy and early intervention. The aim of this study is to propose supervised model to solve the problem of mitosis detection and reduced the time lapse of process compared to the manual counting. We formulate a Low-Rank Matrix Recovery (LRMR) model to find salient regions from microscopy images and extract candidate patch sequences, which potentially contain mitosis cells. Once the patches are segmented, a novel Sparse Patch based Mitosis Handcrafted Feature (SMHCF) are extracted and based on the feature values a eXtreme Gradient Boosting (XGBoost) model are used to classify the mitosis and non-mitosis cells and count of the each cells. Our method is compared with the manual counting values and other state of the art methods. In that performances of our model achieved with better precision is 0.971, recall is 0.960, F1-score is 0.963 rates. [ABSTRACT FROM AUTHOR]

Cancer cells evolve to be refractory to the intrinsic programmed cell death mechanisms, which ensure cellular tissue homeostasis in physiological conditions. Chemotherapy using cytotoxic drugs seeks to eliminate cancer cells but spare non-cancerous host cells by exploring a likely subtle difference between malignant and benign cells. Presumably, chemotherapy agents achieve efficacy by triggering programmed cell death machineries in cancer cells. Currently, many major solid tumors are treated with chemotherapy composed of a combination of platinum agents and taxanes. Platinum agents, largely cisplatin, carboplatin, and oxaliplatin, are DNA damaging agents that covalently form DNA addicts, triggering DNA repair response pathways. Taxanes, including paclitaxel, docetaxel, and cabazitaxel, are microtubule stabilizing drugs which are often very effective in purging cancer cells in clinical settings. Generally, it is thought that the stabilization of microtubules by taxanes leads to mitotic arrest, mitotic catastrophe, and the triggering of apoptotic programmed cell death. However, the precise mechanism(s) of how mitotic arrest and catastrophe activate the caspase pathway has not been established. Here, we briefly review literature on the involvement of potential cell death mechanisms in cancer therapy. These include the classical caspase-mediated apoptotic programmed cell death, necroptosis mediated by MLKL, and pore forming mechanisms in immune cells, etc. In particular, we discuss a newly recognized mechanism of cell death in taxane-treatment of cancer cells that involves micronucleation and the irreversible rupture of the nuclear membrane. Since cancer cells are commonly retarded in responding to programmed cell death signaling, stabilized microtubule bundle-induced micronucleation and nuclear membrane rupture, rather than triggering apoptosis, may be a key mechanism accounting for the success of taxanes as anti-cancer agents. [ABSTRACT FROM AUTHOR]

The life cycles of plants are characterized by significant alternations between the diploid sporophytic and the haploid gametophytic generations. Meiosis and fertilization are the prerequisites for achieving the alternation of generations. Diploid sporophytes undergo meiosis to produce haploid gametes, and male-female gametes fuse (double fertilization) to revert to the diploid sporophyte. The formation and development of male gametophytes are crucial for the alternation of generations in higher plants. During the long evolution of plants from aquatic to terrestrial, the way of sexual reproduction has undergone subversive innovations. From sperm swimming for fertilization to relying on the precise delivery of pollen tubes to female gametes for the fusion of the male-female gametes, higher requirements are placed on the male gametophytes' development and fertilization process. The formation of male gametophytes has undergone significant changes to meet the needs for delivery and transportation of the male gametes. With the emergence of more and more evidence, the molecular mechanism underlying male gametophytes' development, especially the initiation and specialization of germline cells has been better understood. Focusing on the latest studies, we reviewed and elucidated the critical proteins and factors involved in male gametophyte formation, highlighting the decisive role of auxin in archesporial cell specialization and the importance of microspore maturation in pre-mitosis, and analyzed the molecular mechanisms underlying male gametogenesis, with a view to providing insights for further exploration of male gametophytes formation in the future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mitotic cell division is tightly monitored by checkpoints that safeguard the genome from instability. Failures in accurate chromosome segregation during mitosis can cause numerical aneuploidy, which was hypothesized by Theodor Boveri over a century ago to promote tumorigenesis. Recent interrogation of pan-cancer genomes has identified unexpected classes of chromosomal abnormalities, including complex rearrangements arising through chromothripsis. This process is driven by mitotic errors that generate abnormal nuclear structures that provoke extensive yet localized shattering of mis-segregated chromosomes. Here, we discuss emerging mechanisms underlying chromothripsis from micronuclei and chromatin bridges, as well as highlight how this mutational cascade converges on the DNA damage response. A fundamental understanding of these catastrophic processes will provide insight into how initial errors in mitosis can precipitate rapid cancer genome evolution. Chromothripsis drives rapid genome evolution through the extensive shattering of mis-segregated chromosomes. This review discusses the cell cycle checkpoints that protect against mitotic errors, how mis-segregated chromosomes entrapped in abnormal nuclear structures undergo shattering, and the DNA damage response mechanisms that reassemble shattered chromosomes to generate complex cancer genome rearrangements. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To determine the association between apoptosis and the histopathological parameters that define squamous intraepithelial lesion of the uterine cervix. MATERIALS AND METHODS: Observational, cross-sectional, and relational study, by histopathological study of cervical biopsies with colposcopic suspicion of squamous intraepithelial lesion received at the Department of Pathology in the years 2018-2021. The following parameters were evaluated: apoptosis, koilocytes, number of mitoses, mitosis height, atypical mitosis, chromatin, basaloid atypia, nucleolus, and intraepithelial neutrophils. With the SPSS software, Pearson's Chi-square, Phi, Cramer's V and probability ratio for apoptosis were calculated with each of the parameters. Subsequently, the association and the measurement of the strength of association between apoptosis and the histopathological diagnosis of the studied cases were determined. RESULTS: A total of 624 glass slides were studied. A significant and relatively strong association was observed between apoptosis and bi-multinucleations, chromatin alterations, increased mitosis, and mitosis above the suprabasal layer, and a significant and moderate association between apoptosis and koilocytes, basaloid atypia, atypical mitosis, overlap and nuclear density. A significant and strong association was demonstrated between apoptosis and histopathological diagnosis of high-grade squamous intraepithelial lesion (Cramer's V: 701, p <.001) and a significant but weak and negative association with low-grade intraepithelial lesion (Phi: -.263, p <.001). CONCLUSIONS: Apoptosis is strongly associated with high-grade squamous intraepithelial lesion, and weakly and negatively associated with low-grade lesion. [ABSTRACT FROM AUTHOR]

Key message: Imbalanced chromosomes and cell cycle arrest, along with down-regulated genes in DNA damage repair and sperm cell differentiation, caused pollen abortion in synthetic allodiploid Brassica juncea hybrids. Interspecific hybridization is considered to be a major pathway for species formation and evolution in angiosperms, but the occurrence of pollen abortion in the hybrids is common, prompting us to recheck male gamete development in allodiploid hybrids after the initial combination of different genomes. Here, we investigated the several key meiotic and mitotic events during pollen development using the newly synthesised allodiploid B. juncea hybrids (AB, 2n = 2× = 18) as a model system. Our results demonstrated the partial synapsis and pairing of non-homologous chromosomes concurrent with chaotic spindle assembly, affected chromosome assortment and distribution during meiosis, which finally caused difference in genetic constitution amongst the final tetrads. The mitotic cell cycle arrest during microspore development resulted in the production of anucleate pollen cells. Transcription analysis showed that sets of key genes regulating cyclin (CYCA1;2 and CYCA2;3), DNA damage repair (DMC1, NBS1 and MMD1), and ubiquitin–proteasome pathway (SINAT4 and UBC) were largely downregulated at the early pollen meiosis stages, and those genes involved in sperm cell differentiation (DUO1, PIRL1, PIRL9 and LBD27) and pollen wall synthesis (PME48, VGDH11 and COBL10) were mostly repressed at the late pollen mitosis stages in the synthetic allodiploid B. juncea hybrids (AB). In conclusion, this study elucidated the related mechanisms affecting pollen fertility during male gametophyte development at the cytological and transcriptomic levels in the synthetic allodiploid B. juncea hybrids. [ABSTRACT FROM AUTHOR]

The c‐Jun N‐terminal kinase‐associated leucine zipper protein (JLP), a scaffold protein of mitogen‐activated protein kinase signaling pathways, is a multifunctional protein involved in a variety of cellular processes. It has been reported that JLP is overexpressed in various types of cancer and is expected to be a potential therapeutic target. However, whether and how JLP overexpression affects non‐transformed cells remain unknown. Here, we aimed to investigate the effect of JLP overexpression on chromosomal stability in human non‐transformed cells and the mechanisms involved. We found that aneuploidy was induced in JLP‐overexpressed cells. Moreover, we established JLP‐inducible cell lines and observed an increased frequency of chromosome missegregation, reduced time from nuclear envelope breakdown to anaphase onset, and decreased levels of the spindle assembly checkpoint (SAC) components at the prometaphase kinetochore in cells overexpressing the wild‐type JLP. In contrast, we observed that a point mutant JLP lacking the ability to interact with dynein light intermediate chain 1 (DLIC1) failed to induce chromosomal instability. Our results suggest that overexpression of the wild‐type JLP facilitates premature SAC silencing through interaction with DLIC1, leading to aneuploidy. This study provides a novel insight into the mechanism through which JLP overexpression is associated with cancer development and progression. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we explored the assembly of the mitotic spindle in silkworm ovary-derived BmN4 cells, with a specific focus on the role played by a key protein called BmAurora B. Using advanced visualization techniques for cell structures, we examined the architecture and dynamics of spindle microtubules during the early stages of BmN4 cell division. Our investigation showed the formation of a distinctive multipolar square-shaped mitotic spindle, crucial for the accurate segregation of holocentric chromosomes. Intentionally reducing the levels of BmAurora B led to a decrease in the number and size of spindle poles, altering the spindle structure. This change could potentially impact the even distribution of chromosomes between daughter cells. Furthermore, we observed distinct patterns of BmAurora B expression in male and female gonads. This information holds significance as it provides insights into the fundamental processes of cell division in holocentric species. Understanding these mechanisms can contribute to enhancing silk production and advancing silkworm biotechnology. Due to Aurora B's identification as a cancer cell marker and its association with multipolar spindles in some cancer cells, this research can contribute to broader cancer biology knowledge and may open avenues for advances in cancer diagnostics and therapeutics. Silkworm ovary-derived BmN4 cells rely on chromatin-induced spindle assembly to form microtubule-based square mitotic spindles that ensure accurate segregation of holocentric chromosomes during cell division. The chromosome passenger protein Aurora B regulates chromosomal condensation and segregation, spindle assembly checkpoint activation, and cytokinesis; however, its role in holocentric organisms needs further clarification. This study examined the architecture and dynamics of spindle microtubules during prophase and metaphase in BmN4 cells and those with siRNA-mediated BmAurora B knockdown using immunofluorescence labeling. Anti-α-tubulin and anti-γ-tubulin antibodies revealed faint γ-tubulin signals colocalized with α-tubulin in early prophase during nuclear membrane rupture, which intensified as prophase progressed. At this stage, bright regions of α-tubulin around and on the nuclear membrane surrounding the chromatin suggested the start of microtubules assembling in the microtubule-organizing centers (MTOCs). In metaphase, fewer but larger γ-tubulin foci were detected on both sides of the chromosomes. This resulted in a distinctive multipolar square spindle with holocentric chromosomes aligned at the metaphase plate. siRNA-mediated BmAurora B knockdown significantly reduced the γ-tubulin foci during prophase, impacting microtubule nucleation and spindle structure in metaphase. Spatiotemporal BmAurora B expression analysis provided new insights into the regulation of this mitotic kinase in silkworm larval gonads during gametogenesis. Our results suggest that BmAurora B is crucial for the formation of multipolar square spindles in holocentric insects, possibly through the activation of γ-tubulin ring complexes in multiple centrosome-like MTOCs. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor-1 (HIF-1) is the key transcriptional mediator of the cellular response to hypoxia and is also involved in cancer progression. Regulation of its oxygen-sensitive HIF-1α subunit involves post-translational modifications that control its stability, subcellular localization, and activity. We have previously reported that phosphorylation of the HIF-1α C-terminal domain by ERK1/2 promotes HIF-1α nuclear accumulation and stimulates HIF-1 activity while lack of this modification triggers HIF-1α nuclear export and its association with mitochondria. On the other hand, modification of the N-terminal domain of HIF-1α by CK1δ impairs HIF-1 activity by obstructing the formation of a HIF-1α/ARNT heterodimer. Investigation of these two antagonistic events by expressing double phospho-site mutants in HIF1A−/− cells under hypoxia revealed independent and additive phosphorylation effects that can create a gradient of HIF-1α subcellular localization and transcriptional activity. Furthermore, modification by CK1δ caused mitochondrial release of the non-nuclear HIF-1α form and binding to microtubules via its N-terminal domain. In agreement, endogenous HIF-1α could be shown to co-localize with mitotic spindle microtubules and interact with tubulin, both of which were inhibited by CK1δ silencing or inhibition. Moreover, CK1δ expression was necessary for equal partitioning of mother cell-produced HIF-1α to the daughter cell nuclei at the end of mitosis. Overall, our results suggest that phosphorylation by CK1δ stimulates the association of non-nuclear HIF-1α with microtubules, which may serve as a means to establish a symmetric distribution of HIF-1α during cell division under low oxygen conditions. [ABSTRACT FROM AUTHOR]

Background. Melanoma is the malignant proliferation of melanocytes associated with aggressive behavior. The objective of this study was to determine the histological variables of cutaneous melanoma. Methods: Observational, cross-sectional, descriptive, retrospective study carried out with reports of pathologies with a diagnosis of cutaneous melanoma in a pathology laboratory in Cali between 2016-2021. The variables were age, sex, location, subtype, Breslow thickness, ulceration, margins, mitosis, lymphovascular invasion, neurotropism, tumoral regression, Clark level and tumor infiltration by lymphocytes. Results: One hundred and six reports were obtained and 54 were excluded due to duplication. A descriptive analysis was made on the 52 records that were included, the mean age was 61 years, with a higher frequency in women with 55.8%. Of the 33 cases where the histological subtype was specified, the most frequent was superficial extension with 66.6%, followed by acral lentiginous with 18.1% and nodular with 15.2%. The most frequent location was in the extremities (61.5%); the most common Breslow was IV (34.6%), and the most frequent Clark was IV (34.6%). Ulceration was in 40.4%. The nodular subtype was the most aggressive presentation where 100% presented Breslow IV. Conclusions. The most common subtype of melanoma was that of superficial extension. In our population, the second most frequent was the acral lentiginous subtype, which was always located on the extremities. More than 50% of the melanomas had Breslow greater than or equal to III, which affects the prognosis. [ABSTRACT FROM AUTHOR]

We describe the time‐evolution of a tumor spheroid without necrotic core using the quasi‐stationary reaction–diffusion approach. We assume that inhibitor and nutrient supplies are time‐dependent and oscillating by incorporating continuous almost periodic functions in the model. The functional form of the intensity of the mitosis S(σ,β)$S(\sigma ,\beta)$ is supposed to be the product of two linear functions depending on the nutrient concentration σ and of the inhibitor concentration, β, respectively. Under some mild conditions, we give criteria predicting two possible global asymptotic limits: either the tumor oscillates converging towards a stable global almost periodic solution or the tumor shrinks as time increases. [ABSTRACT FROM AUTHOR]

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Oral epithelial dysplasia (OED) is diagnosed and graded using a range of histological features, making grading subjective and challenging. Mitotic counting and phosphohistone-H3 (PHH3) staining have been used for the prognostication of various malignancies; however, their importance in OED remains unexplored. This study conducts a quantitative analysis of mitotic activity in OED using both haematoxylin and eosin (H&E)-stained slides and immunohistochemical (IHC) staining for PHH3. Specifically, the diagnostic and prognostic importance of mitotic number, mitotic type and intra-epithelial location is evaluated. Whole slide images (WSI) of OED (n = 60) and non-dysplastic tissue (n = 8) were prepared for analysis. Five-year follow-up data was collected. The total number of mitosis (TNOM), mitosis type and intra-epithelial location was manually evaluated on H&E images and a digital mitotic count performed on PHH3-stained WSI. Statistical associations between these features and OED grade, malignant transformation and OED recurrence were determined. Mitosis count increased with grade severity (H&E: p < 0.005; IHC: p < 0.05), and grade-based differences were seen for mitosis type and location (p < 0.05). The ratio of normal-to-abnormal mitoses was higher in OED (1.61) than control (1.25) and reduced with grade severity. TNOM, type and location were better predictors when combined with histological grading, with the most prognostic models demonstrating an AUROC of 0.81 for transformation and 0.78 for recurrence, exceeding conventional grading. Mitosis quantification and PHH3 staining can be an adjunct to conventional H&E assessment and grading for the prediction of OED prognosis. Validation on larger multicentre cohorts is needed to establish these findings. [ABSTRACT FROM AUTHOR]

Simple Summary: The aim of this study was to evaluate a new microscopic parameter (atypical mitotic figures) in canine cutaneous mast cell tumors regarding the ability to predict patient survival (prognosis). Mast cell tumors are one of the most common skin tumors in dogs. Counting the number of tumor cells undergoing division (mitotic figures) is of prognostic importance for this tumor type. While normal cells have a highly regulated cell division (normal mitotic figures), tumor cells might exhibit errors during chromosome separation into daughter cells. These errors can be identified microscopically as atypical mitotic figures and are described as a malignancy criterion for some human tumors. In this study, we have shown that a high number of atypical mitotic figures in canine cutaneous mast cell tumors is predictive of shorter patient survival. As compared to enumerating all mitotic figures, increased numbers of atypical mitotic figures (≥3 per 2.37 mm2) had a higher specificity for tumor-related death. These findings should be validated in future studies. Cell division through mitosis (microscopically visible as mitotic figures, MFs) is a highly regulated process. However, neoplastic cells may exhibit errors in chromosome segregation (microscopically visible as atypical mitotic figures, AMFs) resulting in aberrant chromosome structures. AMFs have been shown to be of prognostic relevance for some neoplasms in humans but not in animals. In this study, the prognostic relevance of AMFs was evaluated for canine cutaneous mast cell tumors (ccMCT). Histological examination was conducted by one pathologist in whole slide images of 96 cases of ccMCT with a known survival time. Tumor-related death occurred in 11/18 high-grade and 2/78 low-grade cases (2011 two-tier system). The area under the curve (AUC) was 0.859 for the AMF count and 0.880 for the AMF to MF ratio with regard to tumor-related mortality. In comparison, the AUC for the mitotic count was 0.885. Based on our data, a prognostically meaningful threshold of ≥3 per 2.37 mm2 for the AMF count (sensitivity: 76.9%, specificity: 98.8%) and >7.5% for the AMF:MF ratio (sensitivity: 76.9%, specificity: 100%) is suggested. While the mitotic count of ≥ 6 resulted in six false positive cases, these could be eliminated when combined with the AMF to MF ratio. In conclusion, the results of this study suggests that AMF enumeration is a prognostically valuable test, particularly due to its high specificity with regard to tumor-related mortality. Additional validation and reproducibility studies are needed to further evaluate AMFs as a prognostic criterion for ccMCT and other tumor types. [ABSTRACT FROM AUTHOR]