Quantification of Proliferating and Mitotically Active Retinal Cells in Mice by Flow Cytometry.

Adult mammals lack the ability to regenerate retinal neurons after injury. However, in previous studies from this lab, topical application of the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, has been associated with an increase in the number of retinal neurons in adult murine models both in the presence and absence of injury to the retina. Additionally, studies assaying mitotic markers have shown a substantial increase in the amount of mitotically active and proliferating cells with the topical application of the alpha7 nAChR agonist. However, these previous studies were performed using fluorescent immunolabeling and subsequent confocal microscopy, thus limiting the number of antibodies that can be multiplexed. As a result, we have developed a flow cytometry method that allows for the multiplexing and analysis of multiple external and internal markers in dissociated retinal cells. In this paper, a step-by-step protocol is described for the labeling of multiple retinal cell types such as retinal ganglion cells, rod photoreceptors, and Müller glia, concurrently with Müller glia-derived progenitor cells that arise after treatment with PNU-282987. Key features • Neurogenesis in the adult mammalian retina. • Flow cytometry of retinal cells. • PNU-282987-induced mitotic activity in the retina. • Dissociation of the retina for flow cytometry analysis. Graphical overview Schematic demonstrating the protocol for preparation of retinal cells for flow cytometry analysis. (A) Adult mice (3-6 months) are subjected to topical PBS eyedrop treatment containing DMSO (control groups) or PNU-282987 (experimental groups). Both eyedrop treatments contain 1 mg/mL of BrdU to label proliferating cells. After treatment, mice are euthanized, and retinae are harvested for dissociation using papain. (B) Dissociated retina cells are fixed and permeabilized before aliquots are taken for cell counts on a hemocytometer. After determining the number of cells present, conjugated antibodies and unconjugated primary antibodies are added at the appropriate dilutions. Fluorescent secondary antibodies are added for markers that are unconjugated. Cells are then subjected to flow cytometric analysis using a BD LSRFortessa.

Enfortumab vedotin-induced cutaneous eruption: Ring mitotic figures as a distinctive histopathologic feature.

Enfortumab vedotin (EV), a nectin-4-binding agent that affects microtubules, has become standard therapy for advanced urothelial carcinoma. The agent, now given in combination with pembrolizumab, frequently induces cutaneous reactions. Here, we report a severe EV-induced cutaneous eruption. A 58-year-old woman with metastatic urothelial carcinoma developed a rash after receiving simultaneous first doses of EV and pembrolizumab. The eruption began on the flank and spread to involve her trunk and extremities with prominent involvement of folds, including the axillae and medial thighs. Skin biopsy revealed extensive vacuolar alteration of the basal epidermis and numerous epidermal keratinocytic mitotic figures, often suprabasilar, including ring and "starburst" forms. The findings supported a diagnosis of EV-induced eruption. With EV cessation and systemic corticosteroids, the rash resolved over a few weeks. Pembrolizumab was restarted as monotherapy, and the patient's cancer showed a significant radiographic treatment response at 3 months. An emerging literature of small series and case reports, largely from oncologic literature, presents the histopathology of EV-induced cutaneous eruption as a vacuolar interface dermatitis with the inconsistently reported feature of arrested mitotic figures. This case study demonstrates distinctive clinical and histopathologic features of EV-induced eruption, which may inform dermatologic and oncologic management.

Unravelling the unusual: chromosome elimination, nondisjunction and extra pollen mitosis characterize the B chromosome in wild sorghum.

The B chromosomes exhibit diverse behaviour compared with conventional genetic models. The capacity of the B chromosome either to accumulate or to be eliminated in a tissue-specific manner is dependent on biological processes related to aberrant cell division(s), but here yet remains compatible with normal development. We studied B chromosome elimination in Sorghum purpureosericeum embryos through cryo-sections and demonstrated the B chromosome instability during plant growth using flow cytometry, molecular markers and fluorescent in situ hybridization techniques. Consequently, using B chromosome-specific probes we revealed the non-Mendelian inheritance of B chromosomes in developing pollen. We disclosed that the occurrence of the B chromosome is specific to certain tissues or organs. The distribution pattern is mainly caused by an extensive elimination that functions primarily during embryo development and persists throughout plant development. Furthermore, we described that B chromosome accumulation can occur either by nondisjunction at first pollen mitosis (PMI) or the initiation of extra nuclear division(s) during pollen development. Our study demonstrates the existence of a not-yet-fully described B chromosome drive process, which is likely under the control of the B chromosome.

PP2A-B55 phosphatase counteracts Ki-67-dependent chromosome individualization during mitosis.

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.

FOXM1 requires IDH1 for late genes expression in mitotic cells.

Isocitrate dehydrogenase 1 (IDH1) is a metabolic enzyme that converts isocitrate to α-ketoglutarate in cells. However, research on IDH1 is more focused on the metabolite D-2-hydroxyglutarate than the cellular roles of the IDH1 protein. Metabolic enzymes can moonlight by participating in diverse cellular processes in cancer cells. This moonlighting function of the metabolic enzymes can contribute to changes in gene expression. It is unknown whether IDH1 associates with any transcription factor. We asked whether IDH1 coordinates with forkhead box protein M1 (FOXM1) in mitotic cells to regulate late genes expression. We found that depletion of IDH1 reduces canonical FOXM1-target expression in mitotic cells. Also, IDH1 binds to FOXM1 and a subset of MuvB proteins, Lin-9 and Lin-54, in mitotic cells. Based on these observations, we suggest that IDH1 coordinates with FOXM1 in mitotic cells to regulate late genes expression.

Plasticity of the mitotic spindle in response to karyotype variation.

Karyotypes, composed of chromosomes, must be accurately partitioned by the mitotic spindle for optimal cell health. However, it is unknown how underlying characteristics of karyotypes, such as chromosome number and size, govern the scaling of the mitotic spindle to ensure accurate chromosome segregation and cell proliferation. We utilize budding yeast strains engineered with fewer chromosomes, including just two "mega chromosomes," to study how spindle size and function are responsive to, and scaled by, karyotype. We determined that deletion and overexpression of spindle-related genes are detrimental to the growth of strains with two chromosomes, suggesting that mega chromosomes exert altered demands on the spindle. Using confocal microscopy, we demonstrate that cells with fewer but longer chromosomes have smaller spindle pole bodies, fewer microtubules, and longer spindles. Moreover, using electron tomography and confocal imaging, we observe elongated, bent anaphase spindles with fewer core microtubules in strains with mega chromosomes. Cells harboring mega chromosomes grow more slowly, are delayed in mitosis, and a subset struggle to complete chromosome segregation. We propose that the karyotype of the cell dictates the microtubule number, type, spindle pole body size, and spindle length, subsequently influencing the dynamics of mitosis, such as the rate of spindle elongation and the velocity of pole separation. Taken together, our results suggest that mitotic spindles are highly plastic ultrastructures that can accommodate and adjust to a variety of karyotypes, even within a species.

Primary cell cultures from the single-chromosome ant Myrmecia croslandi.

The number of chromosomes varies tremendously across species. It is not clear whether having more or fewer chromosomes could be advantageous. The probability of non-disjunction should theoretically decrease with smaller karyotypes, but too long chromosomes should enforce spatial constraint for their segregation during the mitotic anaphase. Here, we propose a new experimental cell system to acquire novel insights into the mechanisms underlying chromosome segregation. We collected the endemic Australian ant Myrmecia croslandi, the only known species with the simplest possible karyotype of a single chromosome in the haploid males (and one pair of chromosomes in the diploid females), since males are typically haploid in hymenopteran insects. Five colonies, each with a queen and a few hundreds of workers, were collected in the Canberra district (Australia), underwent karyotype analysis to confirm the presence of a single pair of chromosomes in worker pupae, and were subsequently maintained in the laboratory in Paris (France). Starting from dissociated male embryos, we successfully conducted primary cell cultures comprised of single-chromosome cells. This could be developed into a unique model that will be of great interest for future genomic and cell biology studies related to mitosis.

Chromosome compaction is triggered by an autonomous DNA-binding module within condensin.

The compaction of chromatin into mitotic chromosomes is essential for faithful transmission of the genome during cell division. In eukaryotes, chromosome morphogenesis is regulated by the condensin complex, though the exact mechanism used to target condensin to chromatin and initiate condensation is not understood. Here, we reveal that condensin contains an intrinsically disordered region (IDR) that modulates its association with chromatin in early mitosis and exhibits phase separation. We describe DNA-binding motifs within the IDR that, upon deletion, inflict striking defects in chromosome condensation and segregation, ill-timed condensin turnover on chromatin, and cell death. Importantly, we demonstrate that the condensin IDR can impart cell cycle regulatory functions when transferred to other subunits within the complex, indicating its autonomous nature. Collectively, our study unveils the molecular basis for the initiation of chromosome condensation in early mitosis and how this process ultimately promotes genomic stability and faultless cell division.

FOXM1/DEPDC1 feedback loop promotes hepatocarcinogenesis and represents promising targets for cancer therapy.

Forkhead box M1 (FOXM1) is a key regulator of mitosis and is identified as an oncogene involved in several kinds of human malignancies. However, how it induces carcinogenesis and related therapeutic approaches remains not fully understood. In this study, we aimed to identify a regulatory axis involving FOXM1 and its target gene DEP domain containing 1 (DEPDC1) and investigate their biological functions. FOXM1 bound to the promoter and transcriptionally induced DEPDC1 expression, in turn, DEPDC1 physically interacted with FOXM1, promoted its nuclear translocation, and reinforced its transcriptional activities. The FOXM1/DEPDC1 axis was indispensable for cancer cells, as evidenced by the fact that DEPDC1 rescued cell growth inhibition caused by FOXM1 knockdown, and silencing DEPDC1 efficiently attenuated tumor growth in a murine hepatocellular carcinoma model. Furthermore, strong positive associations between FOXM1/DEPDC1 axis and poor clinical outcome were observed in human hepatocellular carcinoma samples, further indicating their significance for hepatocarcinogenesis. Finally, we attempted to exploit immunotherapy approaches to target the FOXM1/DEPDC1 axis. Several HLA-A24:02-restricted T-cell epitopes targeting FOXM1 or DEPDC1 were identified through bioinformatic analysis. Then, T cell receptor (TCR)-engineered T cells targeting FOXM1262-270 or DEPDC1294-302 were successfully established and proved to efficiently eradicate tumor cells. Our findings highlight the significance of the FOXM1/DEPDC1 axis in the process of oncogenesis and indicate their potential as immunotherapy targets.

The expression profiles of piRNAs and their interacting Piwi proteins in cellular model of renal development: Focus on Piwil1 in mitosis.

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

The thrombin receptor (PAR1) is associated with microtubules, mitosis and process formation in glioma cells.

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.

Mechanical stress during confined migration causes aberrant mitoses and c-MYC amplification.

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

FAM110A promotes mitotic spindle formation by linking microtubules with actin cytoskeleton.

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

Proteomic analysis reveals a PLK1-dependent G2/M degradation program and a role for AKAP2 in coordinating the mitotic cytoskeleton.

Ubiquitination is an essential regulator of cell division. The kinase Polo-like kinase 1 (PLK1) promotes protein degradation at G2/M phase through the E3 ubiquitin ligase Skp1-Cul1-F box (SCF)ßTrCP. However, the magnitude to which PLK1 shapes the mitotic proteome is uncharacterized. Combining quantitative proteomics with pharmacologic PLK1 inhibition revealed a widespread, PLK1-dependent program of protein breakdown at G2/M. We validated many PLK1-regulated proteins, including substrates of the cell-cycle E3 SCFCyclin F, demonstrating that PLK1 promotes proteolysis through at least two distinct E3 ligases. We show that the protein-kinase-A-anchoring protein A-kinase anchor protein 2 (AKAP2) is cell-cycle regulated and that its mitotic degradation is dependent on the PLK1/ßTrCP signaling axis. Expression of a non-degradable AKAP2 mutant resulted in actin defects and aberrant mitotic spindles, suggesting that AKAP2 degradation coordinates cytoskeletal organization during mitosis. These findings uncover PLK1's far-reaching role in shaping the mitotic proteome post-translationally and have potential implications in malignancies where PLK1 is upregulated.

D-MAINS: A Deep-Learning Model for the Label-Free Detection of Mitosis, Apoptosis, Interphase, Necrosis, and Senescence in Cancer Cells.

BACKGROUND: Identifying cells engaged in fundamental cellular processes, such as proliferation or living/death statuses, is pivotal across numerous research fields. However, prevailing methods relying on molecular biomarkers are constrained by high costs, limited specificity, protracted sample preparation, and reliance on fluorescence imaging. METHODS: Based on cellular morphology in phase contrast images, we developed a deep-learning model named Detector of Mitosis, Apoptosis, Interphase, Necrosis, and Senescence (D-MAINS). RESULTS: D-MAINS utilizes machine learning and image processing techniques, enabling swift and label-free categorization of cell death, division, and senescence at a single-cell resolution. Impressively, D-MAINS achieved an accuracy of 96.4 ± 0.5% and was validated with established molecular biomarkers. D-MAINS underwent rigorous testing under varied conditions not initially present in the training dataset. It demonstrated proficiency across diverse scenarios, encompassing additional cell lines, drug treatments, and distinct microscopes with different objective lenses and magnifications, affirming the robustness and adaptability of D-MAINS across multiple experimental setups. CONCLUSIONS: D-MAINS is an example showcasing the feasibility of a low-cost, rapid, and label-free methodology for distinguishing various cellular states. Its versatility makes it a promising tool applicable across a broad spectrum of biomedical research contexts, particularly in cell death and oncology studies.

An aggregated mitochondrial distribution in preimplantation embryos disrupts nuclear morphology, function, and developmental potential.

A dispersed cytoplasmic distribution of mitochondria is a hallmark of normal cellular organization. Here, we have utilized the expression of exogenous Trak2 in mouse oocytes and embryos to disrupt the dispersed distribution of mitochondria by driving them into a large cytoplasmic aggregate. Our findings reveal that aggregated mitochondria have minimal impact on asymmetric meiotic cell divisions of the oocyte. In contrast, aggregated mitochondria during the first mitotic division result in daughter cells with unequal sizes and increased micronuclei. Further, in two-cell embryos, microtubule-mediated centering properties of the mitochondrial aggregate prevent nuclear centration, distort nuclear shape, and inhibit DNA synthesis and the onset of embryonic transcription. These findings demonstrate the motor protein-mediated distribution of mitochondria throughout the cytoplasm is highly regulated and is an essential feature of cytoplasmic organization to ensure optimal cell function.

Interaction of the tumor suppressor SMAD4 and WNT signaling in progression to oral squamous cell carcinoma.

SMAD4 is a tumor suppressor mutated or silenced in multiple cancers, including oral cavity squamous cell carcinoma (OSCC). Human clinical samples and cell lines, mouse models and organoid culture were used to investigate the role that SMAD4 plays in progression from benign disease to invasive OSCC. Human OSCC lost detectable SMAD4 protein within tumor epithelium in 24% of cases, and this loss correlated with worse progression-free survival independent of other major clinical and pathological features. A mouse model engineered for KrasG12D expression in the adult oral epithelium induced benign papillomas, however the combination of KrasG12D with loss of epithelial Smad4 expression resulted in rapid development of invasive carcinoma with features of human OSCC. Examination of regulatory pathways in 3D organoid cultures of SMAD4+ and SMAD4- mouse tumors with Kras mutation found that either loss of SMAD4 or inhibition of TGFß signaling upregulated the WNT pathway and altered the extracellular matrix. The gene signature of the mouse tumor organoids lacking SMAD4 was highly similar to the gene signature of human head and neck squamous cell carcinoma. In summary, this work has uncovered novel mechanisms by which SMAD4 acts as a tumor suppressor in OSCC. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cytogenetic and Molecular Effects of Kaolin's Foliar Application in Grapevine (Vitis vinifera L.) under Summer's Stressful Growing Conditions.

Grapevine varieties from "Douro Superior" (NE Portugal) experience high temperatures, solar radiation, and water deficit during the summer. This summer's stressful growing conditions induce nucleic acids, lipids, and protein oxidation, which cause cellular, physiological, molecular, and biochemical changes. Cell cycle anomalies, mitosis delay, or cell death may occur at the cellular level, leading to reduced plant productivity. However, the foliar application of kaolin (KL) can mitigate the impact of abiotic stress by decreasing leaf temperature and enhancing antioxidant defence. Hence, this study hypothesised that KL-treated grapevine plants growing in NE Portugal would reveal, under summer stressful growing conditions, higher progression and stability of the leaf mitotic cell cycle than the untreated (control) plants. KL was applied after veraison for two years. Leaves, sampled 3 and 5 weeks later, were cytogenetically, molecularly, and biochemically analysed. Globally, integrating these multidisciplinary data confirmed the decreased leaf temperature and enhanced antioxidant defence of the KL-treated plants, accompanied by an improved regularity and completion of the leaf cell cycle relative to the control plants. Nevertheless, the KL efficacy was significantly influenced by the sampling date and/or variety. In sum, the achieved results confirmed the hypothesis initially proposed.

The endoplasmic reticulum connects to the nucleus by constricted junctions that mature after mitosis.

Junctions between the endoplasmic reticulum (ER) and the outer membrane of the nuclear envelope (NE) physically connect both organelles. These ER-NE junctions are essential for supplying the NE with lipids and proteins synthesized in the ER. However, little is known about the structure of these ER-NE junctions. Here, we systematically study the ultrastructure of ER-NE junctions in cryo-fixed mammalian cells staged in anaphase, telophase, and interphase by correlating live cell imaging with three-dimensional electron microscopy. Our results show that ER-NE junctions in interphase cells have a pronounced hourglass shape with a constricted neck of 7-20 nm width. This morphology is significantly distinct from that of junctions within the ER network, and their morphology emerges as early as telophase. The highly constricted ER-NE junctions are seen in several mammalian cell types, but not in budding yeast. We speculate that the unique and highly constricted ER-NE junctions are regulated via novel mechanisms that contribute to ER-to-NE lipid and protein traffic in higher eukaryotes.

Not just binary: embracing the complexity of nuclear division dynamics.

Cell division presents a challenge for eukaryotic cells: how can chromosomes effectively segregate within the confines of a membranous nuclear compartment? Different organisms have evolved diverse solutions by modulating the degree of nuclear compartmentalization, ranging from complete nuclear envelope breakdown to complete maintenance of nuclear compartmentalization via nuclear envelope expansion. Many intermediate forms exist between these extremes, suggesting that nuclear dynamics during cell division are surprisingly plastic. In this review, we highlight the evolutionary diversity of nuclear divisions, focusing on two defining characteristics: (1) chromosome compartmentalization and (2) nucleocytoplasmic transport. Further, we highlight recent evidence that nuclear behavior during division can vary within different cellular contexts in the same organism. The variation observed within and between organisms underscores the dynamic evolution of nuclear divisions tailored to specific contexts and cellular requirements. In-depth investigation of diverse nuclear divisions will enhance our understanding of the nucleus, both in physiological and pathological states.