Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry.

Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.

Important role and underlying mechanism of non­SMC condensin I complex subunit G in tumours (Review).

At present, the incidence of tumours is increasing on a yearly basis, and tumourigenesis is usually associated with chromosomal instability and cell cycle dysregulation. Moreover, abnormalities in the chromosomal structure often lead to DNA damage, further exacerbating gene mutations and chromosomal rearrangements. However, the non­SMC condensin I complex subunit G (NCAPG) of the structural maintenance of chromosomes family is known to exert a key role in tumour development. It has been shown that high expression of NCAPG is closely associated with tumour development and progression. Overexpression of NCAPG variously affects chromosome condensation and segregation during cell mitosis, influences cell cycle regulation, promotes tumour cell proliferation and invasion, and inhibits apoptosis. In addition, NCAPG has been associated with tumour cell stemness, tumour resistance and recurrence. The aim of the present review was to explore the underlying mechanisms of NCAPG during tumour development, with a view towards providing novel targets and strategies for tumour therapy, and through the elucidation of the mechanisms involved, to lay the foundation for future developments in health.

Entangling and disentangling mitotic chromosomes.

The DNA topological challenges generated by cellular manipulation of extremely long DNA fibers remain poorly understood. In this issue of Molecular Cell, Hildebrand et al.1 describe how mitotic chromosomes are self entangled and that disentanglement requires TOP2 activity in late mitosis.

Shining light on DHX9: UV-induced stress granules illuminate protective mechanisms for daughter cell resilience.

In a recent article in Cell, Zhou et al. investigate the origins, composition, and biological consequences of UV-induced stress granules. They find that UV-induced stress granules are triggered by the formation of RNA-protein crosslinks, uniquely contain DHX9 as a marker, form during mitosis independently of translation repression, and are enriched in intron-containing RNAs and splicing factors. Moreover, UV-induced granules contain double-stranded RNA (dsRNA) and trigger a dsRNA response. This work identifies a mechanism for resolving UV-damaged RNA and broadens the types of cytosolic "stress granules" that form.

Functions of HP1 in preventing chromosomal instability.

Chromosomal instability (CIN), caused by errors in the segregation of chromosomes during mitosis, is a hallmark of many types of cancer. The fidelity of chromosome segregation is governed by a sophisticated cellular signaling network, one crucial orchestrator of which is Heterochromatin protein 1 (HP1). HP1 dynamically localizes to distinct sites at various stages of mitosis, where it regulates key mitotic events ranging from chromosome-microtubule attachment to sister chromatid cohesion to cytokinesis. Our evolving comprehension of HP1's multifaceted role has positioned it as a central protein in the orchestration of mitotic processes.

Could the mitotic count improve personalized prognosis in melanoma patients?

A number of studies have indicated that the mitotic rate may be a predictive factor for poor prognosis in melanoma patients. The aim of this study was to investigate whether the mitotic rate is associated with other prognostic clinical and anatomopathological characteristics. After adjusting for other anatomopathological characteristics, we then verified the prognostic value of the number of mitoses, determining in which population subgroup this variable may have greater prognostic significance on 3-year mortality. The Veneto Cancer Registry (Registro Tumori del Veneto-RTV), a high-resolution population-based dataset covering the regional population of approximately 4.9 million residents, served as the clinical data source for the analysis. Inclusion criteria included all incident cases of invasive cutaneous malignant melanoma recorded in the RTV in 2015 (1,050 cases) and 2017 (1,205 cases) for which the number of mitoses was available. Mitotic classes were represented by Kaplan-Meier curves for short-term overall survival. Cox regression calculated hazard ratios in multivariable models to evaluate the independent prognostic role of different mitotic rate cut-offs. The results indicate that the mitotic rate is associated with other survival prognostic factors: the variables comprising the TNM stage (e.g., tumor thickness, ulceration, lymph node status and presence of metastasis) and the characteristics that are not included in the TNM stage (e.g., age, site of tumor, type of morphology, growth pattern and TIL). Moreover, this study demonstrated that, even after adjusting for these prognostic factors, mitoses per mm2 are associated with higher mortality, particularly in T2 patients. In conclusion, these findings revealed the need to include the mitotic rate in the histological diagnosis because it correlates with the prognosis as an independent factor. The mitotic rate can be used to develop a personalized medicine approach in the treatment and follow-up monitoring of melanoma patients.

Dynamic localization of the chromosomal passenger complex in trypanosomes is controlled by the orphan kinesins KIN-A and KIN-B.

The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess unique kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles nor how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in Trypanosoma brucei, the kinetoplastid parasite that causes African sleeping sickness. KIN-A and KIN-B form a scaffold for the assembly of the remaining CPC subunits. We show that the C-terminal unstructured tail of KIN-A interacts with the KKT8 complex at kinetochores, while its N-terminal motor domain promotes CPC translocation to spindle microtubules. Thus, the KIN-A:KIN-B complex constitutes a unique 'two-in-one' CPC localization module, which directs the CPC to kinetochores from S phase until metaphase and to the central spindle in anaphase. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora kinases in early eukaryotes.

The Golgi checkpoint: Golgi unlinking during G2 is necessary for spindle formation and cytokinesis.

Entry into mitosis requires not only correct DNA replication but also extensive cell reorganization, including the separation of the Golgi ribbon into isolated stacks. To understand the significance of pre-mitotic Golgi reorganization, we devised a strategy to first block Golgi segregation, with the consequent G2-arrest, and then force entry into mitosis. We found that the cells forced to enter mitosis with an intact Golgi ribbon showed remarkable cell division defects, including spindle multipolarity and binucleation. The spindle defects were caused by reduced levels at the centrosome of the kinase Aurora-A, a pivotal spindle formation regulator controlled by Golgi segregation. Overexpression of Aurora-A rescued spindle formation, indicating a crucial role of the Golgi-dependent recruitment of Aurora-A at the centrosome. Thus, our results reveal that alterations of the pre-mitotic Golgi segregation in G2 have profound consequences on the fidelity of later mitotic processes and represent potential risk factors for cell transformation and cancer development.

Structural basis for the phase separation of the chromosome passenger complex.

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

Mitosis detection, fast and slow: Robust and efficient detection of mitotic figures.

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).

Domain generalization across tumor types, laboratories, and species - Insights from the 2022 edition of the Mitosis Domain Generalization Challenge.

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 F1 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.

Mechanobiology of the nucleus during the G2-M transition.

Cellular behavior is continuously influenced by mechanical forces. These forces span the cytoskeleton and reach the nucleus, where they trigger mechanotransduction pathways that regulate downstream biochemical events. Therefore, the nucleus has emerged as a regulator of cellular response to mechanical stimuli. Cell cycle progression is regulated by cyclin-CDK complexes. Recent studies demonstrated these biochemical pathways are influenced by mechanical signals, highlighting the interdependence of cellular mechanics and cell cycle regulation. In particular, the transition from G2 to mitosis (G2-M) shows significant changes in nuclear structure and organization, ranging from nuclear pore complex (NPC) and nuclear lamina disassembly to chromosome condensation. The remodeling of these mechanically active nuclear components indicates that mitotic entry is particularly sensitive to forces. Here, we address how mechanical forces crosstalk with the nucleus to determine the timing and efficiency of the G2-M transition. Finally, we discuss how the deregulation of nuclear mechanics has consequences for mitosis.

Identifying and characterising promising small molecule inhibitors of kinesin spindle protein using ligand-based virtual screening, molecular docking, molecular dynamics and MM­GBSA calculations.

The kinesin spindle protein (Eg5) is a mitotic protein that plays an essential role in the formation of the bipolar spindles during the mitotic phase. Eg5 protein controls the segregation of the chromosomes in mitosis which renders it a vital target for cancer treatment. In this study our approach to identifying novel scaffold for Eg5 inhibitors is based on targeting the novel allosteric pocket (α4/α6/L11). Extensive computational techniques were applied using ligand-based virtual screening and molecular docking by two approaches, MOE and AutoDock, to screen a library of commercial compounds. We identified compound 8-(3-(1H-imidazol-1-ylpropylamino)-3-methyl-7-((naphthalen-3-yl)methyl)-1H-purine-2, 6 (3H,7H)-dione (compound 5) as a novel scaffold for Eg5 inhibitors. This compound inhibited cancer cell Eg5 ATPase at 2.37 ± 0.15 µM. The molecular dynamics simulations revealed that the identified compound formed stable interactions in the allosteric pocket (α4/α6/L11) of the receptor, indicating its potential as a novel Eg5 inhibitor.

RNAP II antagonizes mitotic chromatin folding and chromosome segregation by condensin.

Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin is stalled by transcription, the impact of transcription on condensin, which is enriched at highly expressed genes in many species, remains unclear. Using degrons of Rpb1 or the torpedo nuclease Dhp1XRN2 to either deplete or displace RNAPII on chromatin in fission yeast metaphase cells, we show that RNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cis and hinders its ability to fold mitotic chromatin and to support chromosome segregation, consistent with the stalling of a loop extruder. Transcription termination by Dhp1 limits such a hindrance. Our results shed light on the integrated functioning of condensin, and we argue that a tight control of transcription underlies mitotic chromosome assembly by loop-extruding condensin.

A mitotic stopwatch determines cell fate.

Surveillance of mitotic timing prevents amplification of damaged cells.

The Drosophila neuroblast polarity cycle at a glance.

Drosophila neural stem cells, or neuroblasts, rapidly proliferate during embryonic and larval development to populate the central nervous system. Neuroblasts divide asymmetrically to create cellular diversity, with each division producing one sibling cell that retains the neuroblast fate and another that differentiates into glia or neurons. This asymmetric outcome is mediated by the transient polarization of numerous factors to the cell cortex during mitosis. The powerful genetics and outstanding imaging tractability of the neuroblast make it an excellent model system for studying the mechanisms of cell polarity. This Cell Science at a Glance article and the accompanying poster explore the phases of the neuroblast polarity cycle and the regulatory circuits that control them. We discuss the key features of the cycle - the targeted recruitment of proteins to specific regions of the plasma membrane and multiple phases of highly dynamic actomyosin-dependent cortical flows that pattern both protein distribution and membrane structure.

Broken chromosomes heading into mitosis: More than one way to patch a flat tire.

A cell dealing with a broken chromosome in mitosis is like a driver dealing with a flat tire on the highway: damage repair must occur under non-ideal circumstances. Mitotic chromosome breaks encounter problems related to structures called micronuclei. These aberrant nuclei are linked to cell death, mutagenesis, and cancer. In the last few years, a flurry of studies illuminated two mechanisms that prevent mitotic problems related to micronuclei. One mechanism prevents micronuclei from forming during mitosis and involves DNA Polymerase Theta, a DNA repair regulator that patches up broken mitotic chromosomes. A second mechanism is activated after micronuclei form and then rupture, and involves CIP2A and TOPBP1 proteins, which patch micronuclear fragments to promote their subsequent mitotic segregation. Here, we review recent progress in this field of mitotic DNA damage and discuss why multiple mechanisms exist. Future studies in this exciting area will reveal new DNA break responses and inform therapeutic strategies.

Nek2A prevents centrosome clustering and induces cell death in cancer cells via KIF2C interaction.

Unlike normal cells, cancer cells frequently exhibit supernumerary centrosomes, leading to formation of multipolar spindles that can trigger cell death. Nevertheless, cancer cells with supernumerary centrosomes escape the deadly consequences of unequal segregation of genomic material by coalescing their centrosomes into two poles. This unique trait of cancer cells presents a promising target for cancer therapy, focusing on selectively attacking cells with supernumerary centrosomes. Nek2A is a kinase involved in mitotic regulation, including the centrosome cycle, where it phosphorylates linker proteins to separate centrosomes. In this study, we investigated if Nek2A also prevents clustering of supernumerary centrosomes, akin to its separation function. Reduction of Nek2A activity, achieved through knockout, silencing, or inhibition, promotes centrosome clustering, whereas its overexpression results in inhibition of clustering. Significantly, prevention of centrosome clustering induces cell death, but only in cancer cells with supernumerary centrosomes, both in vitro and in vivo. Notably, none of the known centrosomal (e.g., CNAP1, Rootletin, Gas2L1) or non-centrosomal (e.g., TRF1, HEC1) Nek2A targets were implicated in this machinery. Additionally, Nek2A operated via a pathway distinct from other proteins involved in centrosome clustering mechanisms, like HSET and NuMA. Through TurboID proximity labeling analysis, we identified novel proteins associated with the centrosome or microtubules, expanding the known interaction partners of Nek2A. KIF2C, in particular, emerged as a novel interactor, confirmed through coimmunoprecipitation and localization analysis. The silencing of KIF2C diminished the impact of Nek2A on centrosome clustering and rescued cell viability. Additionally, elevated Nek2A levels were indicative of better patient outcomes, specifically in those predicted to have excess centrosomes. Therefore, while Nek2A is a proposed target, its use must be specifically adapted to the broader cellular context, especially considering centrosome amplification. Discovering partners such as KIF2C offers fresh insights into cancer biology and new possibilities for targeted treatment.

Automatic detection of cell-cycle stages using recurrent neural networks.

Mitosis is the process by which eukaryotic cells divide to produce two similar daughter cells with identical genetic material. Research into the process of mitosis is therefore of critical importance both for the basic understanding of cell biology and for the clinical approach to manifold pathologies resulting from its malfunctioning, including cancer. In this paper, we propose an approach to study mitotic progression automatically using deep learning. We used neural networks to predict different mitosis stages. We extracted video sequences of cells undergoing division and trained a Recurrent Neural Network (RNN) to extract image features. The use of RNN enabled better extraction of features. The RNN-based approach gave better performance compared to classifier based feature extraction methods which do not use time information. Evaluation of precision, recall, and F-score indicates the superiority of the proposed model compared to the baseline. To study the loss in performance due to confusion between adjacent classes, we plotted the confusion matrix as well. In addition, we visualized the feature space to understand why RNNs are better at classifying the mitosis stages than other classifier models, which indicated the formation of strong clusters for the different classes, clearly confirming the advantage of the proposed RNN-based approach.

A fast-acting lipid checkpoint in G1 prevents mitotic defects.

Lipid synthesis increases during the cell cycle to ensure sufficient membrane mass, but how insufficient synthesis restricts cell-cycle entry is not understood. Here, we identify a lipid checkpoint in G1 phase of the mammalian cell cycle by using live single-cell imaging, lipidome, and transcriptome analysis of a non-transformed cell. We show that synthesis of fatty acids in G1 not only increases lipid mass but extensively shifts the lipid composition to unsaturated phospholipids and neutral lipids. Strikingly, acute lowering of lipid synthesis rapidly activates the PERK/ATF4 endoplasmic reticulum (ER) stress pathway that blocks cell-cycle entry by increasing p21 levels, decreasing Cyclin D levels, and suppressing Retinoblastoma protein phosphorylation. Together, our study identifies a rapid anticipatory ER lipid checkpoint in G1 that prevents cells from starting the cell cycle as long as lipid synthesis is low, thereby preventing mitotic defects, which are triggered by low lipid synthesis much later in mitosis.