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GFP–anillin marks proliferating cells in human iPS- and ES cell lines.(a) Scheme of the lentiviral vector: black triangle, mutation in 3′ LTR, leading to self inactivation (SIN vector); CAG, combination of early enhancer element of CMV (cytomegalovirus) promoter and chicken β-actin promoter; cPPT, central polypurine tract; eGFP-anillin, enhanced green fluorescent protein fused to anillin; gag, truncated group-specific antigen sequence; SA, splice acceptor sites; LTR, long terminal repeat; Psi, packaging signal; RRE, rev response element; SD, splice donor sites; WPRE, post-transcriptional regulatory element of woodchuck hepatitis virus. (b) Stainings of lentivirus CAG–eGFP–anillin-transduced human H9 ESCs and human iPS cells for the proliferation markers Ki-67, pHH3 and Aurora-B kinase (all three red). eGFP–anillin (green) has M-phase-specific localization in the nucleus, cytoplasm (open arrowhead), contractile ring (solid arrowhead) and midbodies (arrows); Ki-67 stains nucleoli in human cells; nuclei are stained with Hoechst dye (blue). Scale bars, 10 μm. (c) Analysis of cell cycle kinetics of human iPS and ESCs with time-lapse microscopy. Data are shown as mean±s.e.m. from n=22 (iPS) and n=21 (H9) cells.
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Current approaches to monitor and quantify cell division in live cells, and reliably distinguish between acytokinesis and endoreduplication, are limited and complicate determination of stem cell pool identities. Here we overcome these limitations by generating an in vivo reporter system using the scaffolding protein anillin fused to enhanced green...
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Endoreduplication is the process by which the nuclear genome is repeatedly replicated without mitotic cell division, resulting in nuclei that contain numerous additional genome copies. Endoreduplication occurs widely throughout Eucarya and is particularly common in angiosperms and insects. Although endoreduplication is an important process in the t...
Significance
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Citations
... Tagged cell populations were enriched by FACS, and microscopy was used to determine if their localization was consistent with prior studies ( Figure 2C). As expected, the fluorescent signal from H2B histone was nuclear (Viana et al., 2023), β-actin formed filaments and was enriched at the cortex (Roberts et al., 2017;Viana et al., 2023), α-tubulin was cytosolic and enriched in filaments and mitotic spindles (Roberts et al., 2017;Viana et al., 2023), anillin was nuclear and enriched in the furrow of dividing cells (Hesse et al., 2012;Husser et al., 2022), RhoA was cytosolic and weakly enriched at the cortex (Husser et al., 2022;Yonemura et al., 2004), keratin 18 formed cortical filaments (Maurer et al., 2008), SOX2 was nuclear (Allencell.org; Strebinger et al., 2019), Nestin formed distinct filaments (Kuang et al., 2019), and β-3-tubulin was weakly expressed and cytosolic (Guo et al., 2010;Turaç et al., 2013). ...
... This sensitivity has not been previously reported in human or in mouse stem cells, likely because imaging conditions are rarely reported and vary with different setups. In addition, timelapse imaging often involves the use of over-expressing transgenes or dyes, which generate higher fluorescent signal intensity and enable the use of optical settings with lower laser power and exposure time (Chaigne et al., 2021;Hesse et al., 2012;Roberts et al., 2017). Since most proteins are expressed weakly in the endogenous context, improvements in fluorophore brightness, imaging conditions, or image processing are required. ...
Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG2 1-10 ) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG2 11 ). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.
... This sensitivity has not been previously reported in human or in mouse stem cells, likely because imaging conditions are rarely reported and vary with different setups. In addition, timelapse imaging often involves the use of over-expressing transgenes or dyes, which generate higher fluorescent signal intensity and enable the use of optical settings with lower laser power and exposure time (Chaigne et al., 2021 ;Hesse et al., 2012 ;Roberts et al., 2017 ). Since most proteins are expressed weakly in the endogenous context, improvements in fluorophore brightness, imaging conditions or image processing are required. ...
Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.
... It is well known that in the late mitotic (M) phase of the cell cycle, with the activity of spindle microtubules, the centromeres split longitudinally, and chromatids unscrew, the cell morphological structure changes rapidly, which is closely related to the periodic changes of various biochemical and nuclear physiology in the cell. 26,27 Therefore, we used cell synchronization assays to block the cells in the M phase and observed through immunofluorescence that the nuclear entry of GPR162 was significantly increased, and there was a small amount of co-localization with γH2AX (Fig. 3l, m). Therefore, we speculated that GPR162 entered the nucleus in large quantities during the M phase and directly participated in the DNA damage process. ...
... Cell synchronization The double cell cycle blockade analysis was carried out exactly as stated previously. 26 Treatment with 50 ng/ml Nocodazole for 12 h synchronized A549 cells to mitosis, after which the cells were released in new media for 3.5 h. The M phase is then followed by double blocking. ...
In the treatment of most malignancies, radiotherapy plays a significant role. However, the resistance of cancer cells to ionizing radiation (IR) is the main reason for the failure of radiotherapy, which causes tumor recurrence and metastasis. In this study, we confirmed that GPR162, an orphan receptor in the G-protein-coupled receptor family, acted as a novel radiotherapy sensitizer by interacting with the stimulator of interferon genes (STING), which targeted DNA damage responses, activated IRF3, accelerated the activation of type I interferon system, promoted the expression of chemokines including CXCL10 and CXCL4, and inhibited the occurrence and development of tumors. Interestingly, the activation of STING by overexpression of GPR162 was independent of the classical pathway of cGAS. STING inhibitors could resist the antitumor effect of overexpression of GPR162 in IR-induced mouse models. In addition, most solid tumors showed low expression of GPR162. And the higher expression of GPR162 indicated a better prognosis in patients with lung adenocarcinoma, liver cancer, breast cancer, etc. In summary, these results suggested that GPR162 may serve as a potential sensitizer of radiotherapy by promoting radiotherapy-induced STING-IFN production and increasing the expression of chemokines including CXCL10 and CXCL4 in DNA damage response, providing an alternative strategy for improving cancer radiotherapy.
... ICC demonstrated typical, speckled, G2 phase-associated pHH3 staining in mature adipocytes, with no bright M-phase-associated staining (Fig. 2b,c) 30,31 . Similarly, anillin, a protein that relocates to the cytoplasm and cell membrane during mitosis 32 , was never observed outside adipocyte nuclei (Fig. 2b,c). Moreover, no adipocytes with mitotic nuclei or condensed chromosomes were identified throughout the study as evaluated by DAPI staining, despite confocal analyses of more than 500,000 adipocytes. ...
... The α-MHC-H2B-mCh/CAG-eGFP-anillin double transgenic mouse line was produced by crossing the α-MHC-H2BmCh-mice with the CAG-eGFP-anillin-mice [20][21][22]. The double transgenic mice (α-MHC-H2BmCh-mice/CAG-eGFP-anillin) were maintained on a mixed genetic background (CD-1 × C57BL/6Ncr × 129S6/SvEvTac). ...
Aims
Metabolic switching during heart development contributes to postnatal cardiomyocyte (CM) cell cycle exit and loss of regenerative capacity in the mammalian heart. Metabolic control has potential for developing effective CM proliferation strategies. We sought to determine whether lactate dehydrogenase A (LDHA) regulated CM proliferation by inducing metabolic reprogramming.
Methods and results
LDHA expression was high in P1 hearts and significantly decreased during postnatal heart development. CM-specific LDHA knockout mice were generated using CRISPR/Cas9 technology. CM-specific LDHA knockout inhibited CM proliferation, leading to worse cardiac function and a lower survival rate in the neonatal apical resection model. In contrast, CM-specific overexpression of LDHA promoted CM proliferation and cardiac repair post-MI. The α-MHC-H2B-mCh/CAG-eGFP-anillin system was used to confirm the proliferative effect triggered by LDHA on P7 CMs and adult hearts. Metabolomics, proteomics and Co-IP experiments indicated that LDHA-mediated succinyl coenzyme A reduction inhibited succinylation-dependent ubiquitination of thioredoxin reductase 1 (Txnrd1), which alleviated ROS and thereby promoted CM proliferation. In addition, flow cytometry and western blotting showed that LDHA-driven lactate production created a beneficial cardiac regenerative microenvironment by inducing M2 macrophage polarization.
Conclusions
LDHA-mediated metabolic reprogramming promoted CM proliferation by alleviating ROS and inducing M2 macrophage polarization, indicating that LDHA might be an effective target for promoting cardiac repair post-MI.
... On basis of our previous research, the actin-binding protein Anillin has been verified anomalously overexpressed in HCC tumor tissues and cells and induces tumor cell division and growth. Anillin is also a pivotal regulating gene triggering cell mitosis through modulating cytokinesis, which is the final step of cell division and determines the ploidy status of hepatocytes physiologically [13,14]. In brief, as described by Zhu, et.al, the binuclear polyploid hepatocytes compose the main population of the polyploid hepatocytes under normal physiological conditions, and depletion of Anillin in hepatocytes could theoretically reduce binuclear hepatocytes and facilitate tumorigenesis consequentially [15]. ...
Background: Short-term recurrence of hepatocellular carcinoma (HCC) after radical operation results in poor prognosis and short overall survival period. Assessment of the post-operational recurrence risk could provide an advantage for preventing lethal progress by assisting to set up necessarily individual adjuvant treatment. Here, on basis of our previous research on the ploidy status of hepatocytes, we detected the expression of Anillin, a pivotal regulating gene of depolyploidization in para-cancerous hepatocytes, and discussed the relation between its alternative expression and short-term recurrence of HCC patients after radical operation.
Methods: One hundred and twenty-one specimens of the para-cancerous tissues from a cohort of HCC patients were collected. The RT-qPCR assay and the Immunohistochemistry assay were conducted for a thorough profile of Anillin expression, along with the analysis of patients' information from the GEO database. The clinicopathological para-maters of the patients were detected to determine the relationship between Anillin in the para--cancerous and the tumor relapse. Specimens treated with HE staining were examined for the precise count of the binuclear polyploid hepatocytes, and the karyoplasmic ratio was calculated.
Results: Anillin was verified raised in the para-cancerous tissues of the patients who relapsed. The raising of Anillin in para-cancerous tissue is related to poor clinicopathologic features of HCC patients and short-term recurrence. Binuclear polyploid hepatocytes were reduced along with a higher expression of Anillin, and the proportion of high karyoplasmic ratio hepatocytes was significantly decreased.
Conclusion: The raising of Anillin is associated with the depolyploidization of hepatocytes in the representation of losing binuclear hepatocytes and reducing proportion reduction of high karyoplasmic ratio hepatocytes. Measuring Anillin in para-cancerous tissue, along with the pathomorphological examination may provide us a strategy for screening high risk of HCC recurrence, and help to design individual adjuvant treatment post-operation.
... Based on the cell cycle-dependent subcellular location, particularly to the contractile ring and midbody during cytokinesis, Anillin serves as a potential substrate for creating transgenics to identify proliferating cells. Hesse and colleagues generated transgenic mice (CAG-eGFP-Anillin) that ubiquitously expressed a chimeric of the mouse Anillin cDNA fused to the C-terminus of eGFP under the control of the CAG promoter [59] (Figure 5). The adult CAG-eGFP-Anillin mice underwent cryoinjuries of the left ventricular or permanent LAD artery ligation myocardial infarctions, and eGFP-anillin-expressing cells were quantified. ...
... Similar to the above-described transgenic mice, eGFP-Anillin mice label all cycling cells and require careful co-localization of the eGFP-Anillin to cardiomyocytes to avoid the potential overestimation of cycling events. terminus of eGFP under the control of the CAG promoter [59] ( Figure 5). The adult CAG-eGFP-Anillin mice underwent cryoinjuries of the left ventricular or permanent LAD artery ligation myocardial infarctions, and eGFP-anillin-expressing cells were quantified. ...
Adult mammalian cardiomyocytes demonstrate scarce cycling and even lower proliferation rates in response to injury. Signals that enhance cardiomyocyte proliferation after injury will be groundbreaking, address unmet clinical needs, and represent new strategies to treat cardiovascular diseases. In vivo methods to monitor cardiomyocyte proliferation are critical to addressing this challenge. Fortunately, advances in transgenic approaches provide sophisticated techniques to quantify cardiomyocyte cycling and proliferation.
... For this purpose, we have generated mouse G4 ES cells, in which the CAG promoter drives the inducible overexpression of Cx43. The CAG promoter was chosen because of its strong expression in muscle cells [34,40,41] at all stages of mouse embryonic development and after birth. The inducibility of the expression cassette is provided by a loxP flanked stop cassette with three SV40 polyadenylation signals that can be removed by Cre protein activity ( Figure 1a). ...
Connexins (Cx) are a large family of membrane proteins that can form intercellular connections, so-called gap junctions between adjacent cells. Cx43 is widely expressed in mammals and has a variety of different functions, such as the propagation of electrical conduction in the cardiac ventricle. Despite Cx43 knockout models, many questions regarding the biology of Cx43 in health and disease remain unanswered. Herein we report the establishment of a Cre-inducible Cx43 overexpression system in murine embryonic stem (ES) cells. This enables the investigation of the impact of Cx43 overexpression in somatic cells. We utilized a double reporter system to label Cx43-overexpressing cells via mCherry fluorescence and exogenous Cx43 via fusion with P2A peptide to visualize its distribution pattern. We proved the functionality of our systems in ES cells, HeLa cells, and 3T3-fibroblasts and demonstrated the formation of functional gap junctions based on dye diffusion and FRAP experiments. In addition, Cx43-overexpressing ES cells could be differentiated into viable cardiomyocytes, as shown by the formation of cross striation and spontaneous beating. Analysis revealed faster and more rhythmic beating of Cx43-overexpressing cell clusters. Thus, our Cx43 overexpression systems enable the investigation of Cx43 biology and function in cardiomyocytes and other somatic cells.
... Similarly, increased anillin expression in an epithelial cell line increased stemness properties (66,67). Interestingly, pluripotent cells of mouse embryos, Drosophila testes, and zebrafish retina all have higher anillin expression, while senescent human fibroblasts and cervical cancer cells have decreased anillin expression (133,(140)(141)(142)(143)(144). This correlation in stem-like properties is interconnected with differentiation state. ...
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers due to low therapeutic response rates and poor prognoses. Majority of patients present with symptoms post metastatic spread, which contributes to its overall lethality as the 4th leading cause of cancer-related deaths. Therapeutic approaches thus far target only one or two of the cancer specific hallmarks, such as high proliferation rate, apoptotic evasion, or immune evasion. Recent genomic discoveries reveal that genetic heterogeneity, early micrometastases, and an immunosuppressive tumor microenvironment contribute to the inefficacy of current standard treatments and specific molecular-targeted therapies. To effectively combat cancers like PDAC, we need an innovative approach that can simultaneously impact the multiple hallmarks driving cancer progression. Here, we present the mechanical properties generated by the cell’s cortical cytoskeleton, with a spotlight on PDAC, as an ideal therapeutic target that can concurrently attack multiple systems driving cancer. We start with an introduction to cancer cell mechanics and PDAC followed by a compilation of studies connecting the cortical cytoskeleton and mechanical properties to proliferation, metastasis, immune cell interactions, cancer cell stemness, and/or metabolism. We further elaborate on the implications of these findings in disease progression, therapeutic resistance, and clinical relapse. Manipulation of the cancer cell’s mechanical system has already been shown to prevent metastasis in preclinical models, but it has greater potential for target exploration since it is a foundational property of the cell that regulates various oncogenic behaviors.
... To distinguish between these variations of the cell cycle and authentic cell division, we have established a test system based on double transgenic αMHC-H2BmCherry/CAG-eGFPanillin mice. αMHC-H2BmCherry labels CM nuclei by the red fluorescent protein mCherry, whereas eGFP-anillin enables to monitor cell cycle activity and progression with high spatial and temporal resolution in M-phase (Hesse et al., 2012). This double transgenic system enables to unequivocally identify CM nuclei and cell cycle variations (Raulf et al., 2015;Hesse et al., 2018). ...
Cardiovascular disease is a leading cause of death in the Western world. The exploration of strategies to enhance the regenerative capacity of the mammalian heart is therefore of great interest. One approach is the treatment of isolated transgenic mouse cardiomyocytes (CMs) with potentially cell cycle-inducing substances and assessment if this results in atypical cell cycle activity or authentic cell division. This requires the tedious and cost intensive manual analysis of microscopy images. Recent advances have led to an increasing use of deep learning (DL) algorithms in cellular image analysis. While developments in image or single-cell classification are well advanced, multi-cell classification in crowded image scenarios remains a challenge. This is reinforced by typically smaller dataset sizes in such laboratory-specific analyses. In this paper, we propose a modular DL-based image analysis pipeline for multi-cell classification of mononuclear and binuclear CMs in confocal microscopy imaging data. We trisect the pipeline structure into preprocessing, modelling and postprocessing. We perform semantic segmentation to extract general image features, which are further analyzed in postprocessing. In total, we conduct 173 experiments. We benchmark 18 encoder-decoder model architectures, perform hyperparameter optimization across 28 runs, and conduct 127 experiments to evaluate dataset-related effects. The results show that our approach has great potential for automating specific cell culture analyses even with small datasets.
