Mitotic Nuclei Research Articles

Mitotic and Meiotic Functions for the SUMOylation Pathway in the Caenorhabditis elegans Germline.

Meiosis is a highly regulated process, partly due to the need to break and then repair DNA as part of the meiotic program. Post-translational modifications are widely used during meiotic events to regulate steps such as protein complex formation, checkpoint activation, and protein attenuation. In this paper, we investigate how proteins that are obligatory components of the SUMO (small ubiquitin-like modifier) pathway, one such post-translational modification, affect the Caenorhabditis elegans germline. We show that UBC-9, the E2 conjugation enzyme, and the C. elegans homolog of SUMO, SMO-1, localize to germline nuclei throughout prophase I. Mutant analysis of smo-1 and ubc-9 revealed increased recombination intermediates throughout the germline, originating during the mitotic divisions. SUMOylation mutants also showed late meiotic defects including defects in the restructuring of oocyte bivalents and endomitotic oocytes. Increased rates of noninterfering crossovers were observed in ubc-9 heterozygotes, even though interfering crossovers were unaffected. We have also identified a physical interaction between UBC-9 and DNA repair protein MRE-11. ubc-9 and mre-11 null mutants exhibited similar phenotypes at germline mitotic nuclei and were synthetically sick. These phenotypes and genetic interactions were specific to MRE-11 null mutants as opposed to RAD-50 or resection-defective MRE-11. We propose that the SUMOylation pathway acts redundantly with MRE-11, and in this process MRE-11 likely plays a structural role.

Microfluidic Encapsulation of Demembranated Sperm Nuclei in Xenopus Egg Extracts.

The cell-free nature of Xenopus egg extract makes it a uniquely tractable experimental model system. The extract, effectively unconfined cytoplasm, allows the direct and relatively straight-forward addition of purified proteins and other reagents, a characteristic that renders the system amenable to many biochemical and cell biological manipulations. Accessibility to the system also facilitates the direct physical manipulation and probing of biological structures, in turn enabling mechanical properties of intracellular assemblies and organelles, such as the mitotic spindle and nucleus, to be measured. Recently, multiphase microfluidics have been combined with Xenopus egg extracts to encapsulate discrete cytoplasmic volumes. Described here is a protocol detailing the use of multiphase microfluidic devices to encapsulate sperm nuclei within extract droplets of defined size and shape. This protocol can also be applied more generally to encapsulation of microbeads and other particles.

Effects of the isoflavone genistein in early life stages of the Senegalese sole, Solea senegalensis: role of the Survivin and proliferation versus apoptosis pathways

BackgroundPhytochemical flavonoids are widely distributed in the environment and are derived from many anthropogenic activities. The isoflavone genistein is a naturally occurring compound found in soya products that are habitual constituents of the aquafeeds. This isoflavone possesses oestrogenic biological activity and also apoptotic properties. The present study has been performed to determine the effects of the genistein in the early life stages of the flatfish Senegalese sole during the first month of larval life, and it is focused especially at the metamorphosis, analysing the expression transcript levels and the immunohistochemical protein patterns implicated in the cell proliferation and apoptosis pathways (proliferation cellular/PCNA, anti-apoptosis Survivin/BIRC-5, death receptors/Fas, and Caspases).ResultsThe isoflavone genistein induced some temporal disrupting effects in several pro-apoptotic signalling pathways (Fas, CASP-6) at both genistein doses (3 mg/L and 10 mg/L), with increased Fas transcripts and also decreasing CASP-6 mRNA expression levels during metamorphic and post-metamorphic stages of the Senegalese sole. On the other hand, the anti-apoptotic BIRC-5 expression levels were weakly down-regulated with both the highest and lowest doses, but all of these imbalances were stabilised to the baseline levels. In early life stages of the controls, the constitutive basal transcript levels were temporarily and differentially expressed, reaching the highest levels at the pre-metamorphosis phase, as especially in endotrophic larvae (i.e. BIRC-5 mRNA), as well as in the metamorphic (i.e. CASP-6 mRNA) and post-metamorphic stages (i.e. Fas mRNA). In general, through development, continuous and progressive increases in the protein patterns of cell proliferation-PCNA (e.g. mitotic nuclei), anti-apoptotic Survivin (e.g. haematopoietic system, brain, digestive system, gills) and CASP-2 and -6 (e.g. brain, gills, kidney, digestive system, vascular systems, among others) have been immunohistochemically detected. Besides, both the controls and genistein exposed larvae displayed parallel immunostaining protein patterns in the different organ-systems and tissues.ConclusionsThe transcriptional imbalances observed in the studied genes (BIRC-5, CASP-6, Fas) were only temporarily induced, and apparently no changes in the immunohistochemical protein patterns were detected. Thus, the isoflavone genistein caused not harmful effects in the development and metamorphosis of the Senegalese sole exposed to chronic environmentally relevant concentrations (3 and 10 mg/L).

Differential RPA-1 and RAD-51 recruitment in vivo throughout the C. elegans germline, as revealed by laser microirradiation.

Studies of the repair pathways associated with DNA double strand breaks (DSBs) are numerous, and provide evidence for cell-cycle specific regulation of homologous recombination (HR) by the regulation of its associated proteins. Laser microirradiation is a well-established method to examine in vitro kinetics of repair and allows for live-imaging of DSB repair from the moment of induction. Here we apply this method to whole, live organisms, introducing an effective system to analyze exogenous, microirradiation-induced breaks in the Caenorhabditis elegans germline. Through this method we observed the sequential kinetics of the recruitment of ssDNA binding proteins RPA-1 and RAD-51 in vivo. We analyze these kinetics throughout different regions of the germline, and thus throughout a range of developmental stages of mitotic and meiotic nuclei. Our analysis demonstrates a largely conserved timing of recruitment of ssDNA binding proteins to DSBs throughout the germline, with a delay of RAD-51 recruitment at mid-pachytene nuclei. Microirradiated nuclei are viable and undergo a slow kinetics of resolution. We observe RPA-1 and RAD-51 colocalization for hours post-microirradiation throughout the germline, suggesting that there are mixed RPA-1/RAD-51 filaments. Finally, through live imaging analysis we observed RAD-51 foci movement with low frequency of coalescence.

Optical clearing of small intestine for three-dimensional visualization of cellular proliferation within crypts.

New methods on optical clearing provide a valuable alternative to traditional physical section histology. Optical clearing allows investigation of relatively large tissue samples at histological resolution while maintaining the three-dimensional architecture of the intact system. There is significant potential for applying optical clearing to gastrointestinal tissues. In particular, intestinal crypts contain high concentrations of stem cells, making these structures especially important for research on cellular proliferation in the intestinal epithelium. The objective of our study is to demonstrate an optical clearing method that is easy to implement and is compatible with mitotic fluorescent labeling. The optical clearing method we present utilizes a Triton/DMSO delipidization step followed by refractive index matching, rendering the tissue nearly transparent. We use EdU click chemistry to fluorescently label mitotic cell nuclei. Our results demonstrate successful clearing of jejunal samples with readily visible EdU staining by means of confocal microscopy. Three-dimensional reconstruction of labeled samples reveals preservation of intestinal cytoarchitecture including muscular, submucosal, and mucosal layers. Additionally, the morphology of intestinal crypts and individual EdU-positive mitotic nuclei are visible in sharp detail within their intact three-dimensional organization. In summary, we present an optical clearing method that is easy to implement and has the potential to provide more accurate assessment of cellular proliferation within the gastrointestinal tract in both healthy and disease states.

Two-phase deep convolutional neural network for reducing class skewness in histopathological images based breast cancer detection

Different types of breast cancer are affecting lives of women across the world. Common types include Ductal carcinoma in situ (DCIS), Invasive ductal carcinoma (IDC), Tubular carcinoma, Medullary carcinoma, and Invasive lobular carcinoma (ILC). While detecting cancer, one important factor is mitotic count – showing how rapidly the cells are dividing. But the class imbalance problem, due to the small number of mitotic nuclei in comparison to the overwhelming number of non-mitotic nuclei, affects the performance of classification models.This work presents a two-phase model to mitigate the class biasness issue while classifying mitotic and non-mitotic nuclei in breast cancer histopathology images through a deep convolutional neural network (CNN). First, nuclei are segmented out using blue ratio and global binary thresholding. In Phase-1 a CNN is then trained on the segmented out 80×80 pixel patches based on a standard dataset. Hard non-mitotic examples are identified and augmented; mitotic examples are oversampled by rotation and flipping; whereas non-mitotic examples are undersampled by blue ratio histogram based k-means clustering. Based on this information from Phase-1, the dataset is modified for Phase-2 in order to reduce the effects of class imbalance.The proposed CNN architecture and data balancing technique yielded an F-measure of 0.79, and outperformed all the methods relying on specific handcrafted features, as well as those using a combination of handcrafted and CNN-generated features.

IDermatoPath - a novel software tool for mitosis detection in H&E-stained tissue sections of malignant melanoma.

Malignant Melanoma (MM) is characterized by a growing incidence and a high malignant potential. Besides well-defined prognostic factors such as tumour thickness and ulceration, the Mitotic Rate (MR) was included in the AJCC recommendations for diagnosis and treatment of MM. In daily routine, the identification of a single mitosis can be difficult on haematoxylin and eosin slides alone. Several studies showed a big inter- and intra-individual variability in detecting the MR in MM even by very experienced investigators, thus raising the question for a computer-assisted method. The objective was to develop a software system for mitosis detection in MM on H&E slides based on machine learning for diagnostic support. We developed a computer-aided staging support system based on image analysis and machine learning on the basis of 59 MM specimens. Our approach automatically detects tumour regions, identifies mitotic nuclei and classifies them with respect to their diagnostic relevance. A convenient user interface enables the investigator to browse through the proposed mitoses for fast and efficient diagnosing. A quantitative evaluation on manually labelled ground truth data revealed that the tumour region detection yields a medium spatial overlap index (dice coefficient) of 0.72. For the mitosis detection, we obtained high accuracies of above 83%. On the technical side, the developed iDermatoPath software tool provides a novel approach for mitosis detection in MM, which can be further improved using more training data such as dermatopathologist annotations. On the practical side, a first evaluation of the clinical utility was positive, albeit this approach provides most benefit for difficult cases in a research setting. Assuming all slides to be digitally processed and reported in the near future, this method could become a helpful additional tool for the pathologist.

Quantifying growing versus non-growing ovarian follicles in the mouse

BackgroundA standard histomorphometric approach has been used for nearly 40 years that identifies atretic (e.g., dying) follicles by counting the number of pyknotic granulosa cells (GC) in the largest follicle cross-section. This method holds that if one pyknotic granulosa nucleus is seen in the largest cross section of a primary follicle, or three pyknotic cells are found in a larger follicle, it should be categorized as atretic. Many studies have used these criteria to estimate the fraction of atretic follicles that result from genetic manipulation or environmental insult. During an analysis of follicle development in a mouse model of Fragile X premutation, we asked whether these ‘historical’ criteria could correctly identify follicles that were not growing (and could thus confirmed to be dying).MethodsReasoning that the fraction of mitotic GC reveals whether the GC population was increasing at the time of sample fixation, we compared the number of pyknotic nuclei to the number of mitotic figures in follicles within a set of age-matched ovaries.ResultsWe found that, by itself, pyknotic nuclei quantification resulted in high numbers of false positives (improperly categorized as atretic) and false negatives (improperly categorized intact). For preantral follicles, scoring mitotic and pyknotic GC nuclei allowed rapid, accurate identification of non-growing follicles with 98% accuracy. This method most often required the evaluation of one follicle section, and at most two serial follicle sections to correctly categorize follicle status. For antral follicles, we show that a rapid evaluation of follicle shape reveals which are intact and likely to survive to ovulation.ConclusionsCombined, these improved, non-arbitrary methods will greatly improve our ability to estimate the fractions of growing/intact and non-growing/atretic follicles in mouse ovaries.

The appressorium of the rice blast fungus Magnaporthe oryzae remains mitotically active during post-penetration hyphal growth

To investigate the mitotic dynamics of an appressorium, we used live-cell confocal imaging of a fluorescence-based mitotic reporter strain of Magnaporthe oryzae. We present evidence that the M. oryzae appressorium remains viable and mitotically active well after host penetration. These results suggest the potential roles of the appressorium during post-penetration proliferation of invasive hyphae. Our studies also revealed that a mitotic appressorial nucleus undergoes extreme constriction and elongation as it migrates through the penetration peg in a manner analogous to mitosis during cell-to-cell movement of invasive hyphae. Understanding the mechanisms underlying these pathogen-specific nuclear dynamics may provide new targets for disease control.

Detection of mitotic nuclei in breast histopathology images using localized ACM and Random Kitchen Sink based classifier.

The exact measure of mitotic nuclei is a crucial parameter in breast cancer grading and prognosis. This can be achieved by improving the mitotic detection accuracy by careful design of segmentation and classification techniques. In this paper, segmentation of nuclei from breast histopathology images are carried out by Localized Active Contour Model (LACM) utilizing bio-inspired optimization techniques in the detection stage, in order to handle diffused intensities present along object boundaries. Further, the application of a new optimal machine learning algorithm capable of classifying strong non-linear data such as Random Kitchen Sink (RKS), shows improved classification performance. The proposed method has been tested on Mitosis detection in breast cancer histological images (MITOS) dataset provided for MITOS-ATYPIA CONTEST 2014. The proposed framework achieved 95% recall, 98% precision and 96% F-score.

Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans.

Homologous recombination (HR) repairs cytotoxic DNA double-strand breaks (DSBs) with high fidelity. Deficiencies in HR result in genome instability. A key early step in HR is the search for and invasion of a homologous DNA template by a single-stranded RAD-51 nucleoprotein filament. The Shu complex, composed of a SWIM domain-containing protein and its interacting RAD51 paralogs, promotes HR by regulating RAD51 filament dynamics. Despite Shu complex orthologs throughout eukaryotes, our understanding of its function has been most extensively characterized in budding yeast. Evolutionary analysis of the SWIM domain identified Caenorhabditis elegans sws-1 as a putative homolog of the yeast Shu complex member Shu2. Using a CRISPR-induced nonsense allele of sws-1, we show that sws-1 promotes HR in mitotic and meiotic nuclei. sws-1 mutants exhibit sensitivity to DSB-inducing agents and fail to form mitotic RAD-51 foci following treatment with camptothecin. Phenotypic similarities between sws-1 and the two RAD-51 paralogs rfs-1 and rip-1 suggest that they function together. Indeed, we detect direct interaction between SWS-1 and RIP-1 by yeast two-hybrid assay that is mediated by the SWIM domain in SWS-1 and the Walker B motif in RIP-1 Furthermore, RIP-1 bridges an interaction between SWS-1 and RFS-1, suggesting that RIP-1 facilitates complex formation with SWS-1 and RFS-1 We propose that SWS-1, RIP-1, and RFS-1 compose a C. elegans Shu complex. Our work provides a new model for studying Shu complex disruption in the context of a multicellular organism that has important implications as to why mutations in the human RAD51 paralogs are associated with genome instability.

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay.

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.

Concurrent BCL2/MYC Gene Amplification Associates with Increased DNA G-Quadruplex Formation in DLBCL

Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of diseases consisting of gene translocations, amplifications, and mutations. G-quadruplex (G4) secondary DNA structures arising from at least 4 contiguous runs of guanines may facilitate this genomic instability. The rapidly progressing G4 field has demonstrated their involvement in nuclear processes, particularly replication and transcription. We previously demonstrated that DNA secondary structures within the MYC and BCL2 promoter regions serve as therapeutic targets for modulating oncogene expression. However, the prevalence and molecular associations of G4 formation within lymphoma tissues is unknown. In previous studies, cancer tissues, such as stomach and liver carcinomas, displayed higher G4 frequencies compared to their benign counterparts using a novel antibody. Here, we characterize the incidence of G4 structures within a highly annotated cohort of DLBCL.We successfully adapted a manual cell-based assay for G4 detection with a FLAG-tagged single-chain Fv antibody (BG4) to develop an automated colorimetric immunohistochemistry (IHC) protocol on a BenchMark® XT instrument (VMSI, Tucson AZ) for G4 visualization in DLBCL formalin-fixed paraffin-embedded tissues (FFPET). A non-malignant tonsil biopsy was used for IHC optimization and we observed BG4 positive staining of B-cells within normal germinal centers, mantle zones, and interfollicular regions (Figure 1A). We then applied our novel IHC technique to 86 DLBCL FFPET, evaluated the presence of G4 formation within a 150 cell count, and analyzed for associations with MYC and BCL2 genetic abnormalities (translocation, amplification, and chromosome polysomy), cell-of-origin (COO), and patient outcome collected in previous studies (Valentino et al, AJCP 2013; Kendrick et al, Human Path 2014; Lenz et al, NEJM 2008). The human tissues and clinical data for this study were kindly provided by the Lymphoma/Leukemia Molecular Profiling Project and use was approved from the University of Arizona Institutional Review Board in accordance with the Declaration of Helsinki.Overall, G4 formation was detected in 85% (73/86) of the DLBCL FFPET (Figure 1B-D) with intense staining observed in mitotic nuclei (Figure 1C, inset). DLBCL with MYC amplification with or without chromosome 8 polysomy significantly associated with an increased presence of G4s (12/23 cases, P =0.02 and 19/23 cases, P =0.005, respectively). Notably, elevated G4 formation was also observed in DLBCL cases with dual BCL2/MYC amplification (6/18 cases, P =0.02), but not in BCL2 amplification positive only cases (26/54 cases, P =0.49). Amplification was defined as >44 gene copies according to previous publications. Only 2/24 cases were MYC translocation positive, both with low BG4 staining (0% and 4% positive cells), and 14/86 cases harbored a BCL2 translocation; however, BG4 staining did not differ from BCL2 translocation negative cases (46% v. 54% average positive cells, P =0.45). There was a trend (P =0.11) for a higher incidence of G4 formation in ABC-DLBCL (33/36 cases) in comparison to GCB-DLBCL (27/35 cases) with ABC cases also trending towards an increase in average positive cells (54% v. 39%, P =0.10). We then assessed whether BG4 staining correlated with overall survival by initially stratifying the patients into quartiles. We observed that the 3rd and 4th quartiles displayed steeper curves and thus we used a 75% cut-off threshold to dichotomize the patients. DLBCL cases with ≥75% BG4 positive cells tend to have worse survival (log rank test; P =0.08).For the first time, we demonstrate G4 formation within DLBCL FFPET and that this phenomenon associates with concurrent gene amplification of two highly genetically altered and prognostically important oncogenes in DLBCL, BCL2 and MYC, as well as MYC amplification alone. G4 formation, if left unresolved by helicases, may lead to DNA replication errors resulting in increased genome instability and gene amplification. We are currently investigating this hypothesis by correlating PIF1, XBP, and XBD helicase expression with G4 prevalence. Our work greatly advances this field by providing a novel tool to directly measure G4 DNA within lymphoma tissue. With this new methodology, we can address the biological and clinical significance of these structures in contributing to the genomic instability and aggressiveness of DLBCL. [Display omitted] DisclosuresRimsza:Nanostring: Patents & Royalties: U.S. Patent Application 61/900, 553; "Methods for Selecting and Treating Lymphoma Types"; Ventana Medical Systems, Inc.: Consultancy; Celgene: Consultancy.

Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina.

The human retina does not replace lost or damaged neurons, ultimately causing vision impairment. In contrast, zebrafish are capable of regenerating lost neurons. Understanding the mechanisms that regulate retinal regeneration in these organisms will help to elucidate approaches to stimulate a similar response in humans. In the damaged zebrafish retina, Müller glia dedifferentiate and proliferate to generate neuronal progenitor cells (NPCs) that differentiate into the lost neurons. We show that the nuclei of Müller glia and NPCs migrate apically and basally in phase with the cell cycle. This migration is facilitated by the actin cytoskeleton and Rho-associated coiled-coil kinases (Rocks). We demonstrate that Rock function is required for sufficient proliferation and the regeneration of photoreceptors, likely via regulating nuclear migration.

Defining multiple, distinct, and shared spatiotemporal patterns of DNA replication and endoreduplication from 3D image analysis of developing maize (Zea mays L.) root tip nuclei.

Spatiotemporal patterns of DNA replication have been described for yeast and many types of cultured animal cells, frequently after cell cycle arrest to aid in synchronization. However, patterns of DNA replication in nuclei from plants or naturally developing organs remain largely uncharacterized. Here we report findings from 3D quantitative analysis of DNA replication and endoreduplication in nuclei from pulse-labeled developing maize root tips. In both early and middle S phase nuclei, flow-sorted on the basis of DNA content, replicative labeling was widely distributed across euchromatic regions of the nucleoplasm. We did not observe the perinuclear or perinucleolar replicative labeling patterns characteristic of middle S phase in mammals. Instead, the early versus middle S phase patterns in maize could be distinguished cytologically by correlating two quantitative, continuous variables, replicative labeling and DAPI staining. Early S nuclei exhibited widely distributed euchromatic labeling preferentially localized to regions with weak DAPI signals. Middle S nuclei also exhibited widely distributed euchromatic labeling, but the label was preferentially localized to regions with strong DAPI signals. Highly condensed heterochromatin, including knobs, replicated during late S phase as previously reported. Similar spatiotemporal replication patterns were observed for both mitotic and endocycling maize nuclei. These results revealed that maize euchromatin exists as an intermingled mixture of two components distinguished by their condensation state and replication timing. These different patterns might reflect a previously described genome organization pattern, with “gene islands” mostly replicating during early S phase followed by most of the intergenic repetitive regions replicating during middle S phase.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-015-0364-4) contains supplementary material, which is available to authorized users.

LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms.

CHK1 is a multifunctional protein kinase integral to both the cellular response to DNA damage and control of the number of active replication forks. CHK1 inhibitors are currently under investigation as chemopotentiating agents due to CHK1's role in establishing DNA damage checkpoints in the cell cycle. Here, we describe the characterization of a novel CHK1 inhibitor, LY2606368, which as a single agent causes double-stranded DNA breakage while simultaneously removing the protection of the DNA damage checkpoints. The action of LY2606368 is dependent upon inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Loss of the CHK1-dependent DNA damage checkpoints permits cells with damaged DNA to proceed into early mitosis and die. The majority of treated mitotic nuclei consist of extensively fragmented chromosomes. Inhibition of apoptosis by the caspase inhibitor Z-VAD-FMK had no effect on chromosome fragmentation, indicating that LY2606368 causes replication catastrophe. Changes in the ratio of RPA2 to phosphorylated H2AX following LY2606368 treatment further support replication catastrophe as the mechanism of DNA damage. LY2606368 shows similar activity in xenograft tumor models, which results in significant tumor growth inhibition. LY2606368 is a potent representative of a novel class of drugs for the treatment of cancer that acts through replication catastrophe.

Inhibition of p38 mitogen-activated protein kinase phosphorylation decreases H₂O₂-induced apoptosis in human lens epithelial cells.

Oxidative damage resulting from ROS is a known causal factor for cataractogenesis. The mitogen-activated protein kinases (MAPK) pathway plays an important role in the apoptosis of HLE cells. The purpose of this study was to investigate the role of phosphorylated p38 mitogen-activated protein kinase in H2O2-induced apoptosis in cultured human lens epithelial (HLE) cells. The effect of SB203580 on HLE cells treated with H2O2 was determined by various assays. Cell viability was monitored by the MTT assay. The rates of apoptosis and ROS generation were determined by flow cytometric analysis. The numbers of mitotic and apoptotic cell nuclei were determined after staining with Hoechst 33342. The protein level of phospho-p38 was measured using western blot analysis. SB203580 reduced H2O2-induced cellular apoptosis and inhibited the generation of reactive oxygen species (ROS); it also delayed the progression of H2O2-induced opacification of lenses. The level of p-p38 was increased when cells were exposed to H2O2 and significantly SB203580-inhibited phosphorylation of p38. The p38MAPK pathway plays an important role in H2O2-induced apoptosis of HLE cells. The study demonstrates that activation of p38MAPK plays an important role in H2O2-induced apoptosis of HLE cells. SB203580 may potentially be exploited as a useful tool for cataract prevention.

CG10126, A Calcium‐Binding Microtubule‐Associated Protein, Promotes Mitosis during Drosophila Development

The Epidermal Growth Factor Receptor (EGFR) is a receptor tyrosine kinase that regulates signaling pathways critical for development and proliferation in Drosophila. EGFR activity is also highly upregulated in a number of human cancers, making identification of its downstream targets important for understanding both basic cellular processes and disease states. We have identified CG10126, a Drosophila homolog of the human protein Calcyphosine‐like, as a transcriptional target of EGFR signaling. Calcyphosine encodes a calcium‐binding protein regulated in several human cancers, but little is known of its function. Here we examine the role of CG10126 during Drosophila development. We expressed RNA interference constructs at various points in development. Reducing the level of CG10126 in eye imaginal discs produced adults with small eyes, while reducing its level ubiquitously in embryos was generally lethal. To further examine the role of CG10126, we used mass spectrometry to identify the binding partners of CG10126 in the presence or absence of calcium. Specifically in the presence of calcium, CG10126 bound α‐ and β‐tubulin and several spindle assembly proteins, suggesting that it may influence formation of the mitotic spindle. Consistent with this, embryos with reduced CG10126 show a diminished number of mitotic nuclei and abnormal spindle structures. Calcium is indispensable for cell cycle regulation; our results suggest that calcium waves present during mitosis may enable CG10126 to influence microtubule dynamics and spindle assembly. Together, our studies suggest that CG10126, a downstream target of EGFR signaling, is a microtubule‐associated protein that affects cell proliferation by regulating mitotic spindle formation.

Chromatin Association with the Nuclear Envelope Supports Stable Nuclear Mechanics

Although the nuclear lamina is considered to be the primary mechanical defense of the nucleus, lamins are part of an integrated network of lipids, proteins, and chromatin. Here, we isolate the contribution of chromatin to nuclear mechanics by employing fission yeast, which lack a nuclear lamina. We have combined a quantitative imaging platform capable of measuring 3D nuclear contours in vivo with an in vitro optical tweezers assay to probe the mechanical properties of S. pombe nuclei. In live cells, we find that association of chromatin with the inner nuclear membrane (INM) through integral membrane proteins is required for a normal mechanical response to microtubule (MT) forces. Increasing loss of integral INM proteins results in highly deformable nuclei specifically in response to exogenous MT forces. These nuclei also show a decreased capacity to recover from mechanical stress. Using optical tweezers, we find that nuclei lacking integral INM proteins are less stiff than wild type nuclei and have increased chromatin flow, particularly when force is applied at rates that recapitulate the kinetics of MT dynamics in vivo. Wild type mitotic nuclei, in which chromatin is globally released from the INM, are extremely soft and also display increased chromatin flow compared to interphase nuclei. Interestingly, decreasing the chromatin to nuclear volume ratio without altering the association of chromatin with the INM has only a slight effect on stiffness and does not alter chromatin flow. Together, these findings suggest that association of chromatin with the nuclear envelope underlies nuclear stiffness. Further, release of chromatin from the INM allows chromatin to flow into MT-dependent fluctuations of the nuclear envelope, leading to larger, longer-lasting nuclear deformations. Lastly, these results suggest that chromatin association with the lamina may contribute to the mechanical behavior of metazoan nuclei.

Developmentally arrested Austrofundulus limnaeus embryos have changes in post-translational modifications of histone H3.

Although vertebrate embryogenesis is typically a continuous and dynamic process, some embryos have evolved mechanisms to developmentally arrest. The embryos of Austrofundulus limnaeus, a killifish that resides in ephemeral ponds, routinely enter diapause II (DII), a reversible developmental arrest promoted by endogenous cues rather than environmental stress. DII, which starts at 24-26 days post-fertilization and can persist for months, is characterized by a significant decline in heart rate and an arrest of development and differentiation. Thus, A. limnaeus is a unique model to study epigenetic features associated with embryonic arrest. To investigate chromosome structures associated with mitosis or gene expression, we examined the post-translational modifications of histone H3 (phosphorylation of serine 10, mono-, di- and tri-methylation of lysine 4 or 27) in preDII, DII and postDII embryos. As seen by microscopy analysis, DII embryos have a significant decrease in the H3S10P marker for mitotic nuclei and an inner nuclear membrane localization of the H3K27me2 marker associated with silencing of gene expression. ELISA experiments reveal that the levels of methylation at H3K4 and H3K27 are significantly different between preDII, DII and postDII embryos, indicating that there are molecular differences between embryos of different chronological age and stage of development. Furthermore, in DII embryos relative to preDII embryos, there are differences in the level of H3K27me3 and H3K4me3, which may reflect critical chromatin remodeling that occurs prior to arrest of embryogenesis. This work helps lay a foundation for chromatin analysis of vertebrate embryo diapause, an intriguing yet greatly understudied phenomenon.