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The morphology of the nuclei represents most of the clinical pathological information, and nuclei segmentation is a vital step in current automated histopathological image analysis. Supervised machine learning-based segmentation models have already achieved outstanding performance with sufficiently precise human annotations. Nevertheless, outlining such labels on numerous nuclei is extremely professional needing and time consuming. Automatic nuclei segmentation with minimal manual interventions is highly needed to promote the effectiveness of clinical pathological researches. Semi-supervised learning greatly reduces the dependence on labeled samples while ensuring sufficient accuracy. In this paper, we propose a Multi-Edge Feature Fusion Attention Network (MEFFA-Net) with three feature inputs including image, pseudo-mask and edge, which enhances its learning ability by considering multiple features. Only a few labeled nuclei boundaries are used to train annotations on the remaining mostly unlabeled data. The MEFFA-Net creates more precise boundary masks for nucleus segmentation based on pseudo-masks, which greatly reduces the dependence on manual labeling. The MEFFA-Block focuses on the nuclei outline and selects features conducive to segment, making full use of the multiple features in segmentation. Experimental results on public multi-organ databases including MoNuSeg, CPM-17 and CoNSeP show that the proposed model has the mean IoU segmentation evaluations of 0.706, 0.751, and 0.722, respectively. The model also achieves better results than some cutting-edge methods while the labeling work is reduced to 1/8 of common supervised strategies. Our method provides a more efficient and accurate basis for nuclei segmentations and further quantifications in pathological researches., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Nuclear IGF1R has been linked to poor outcome in cancer. We recently showed that nuclear IGF1R phosphorylates PCNA and increases DNA damage tolerance. In this paper we aimed to describe this mechanism in cancer tissue as well as in cancer cell lines. In situ proximity ligation assay identified frequent IGF1R and PCNA colocalization in many cancer types. IGF1R/PCNA colocalization was more frequently increased in tumor cells than in adjacent normal, and more prominent in areas with dysplasia and invasion. However, the interaction was often lost in tumors with poor response to neoadjuvant treatment and most metastatic lesions. In two independent cohorts of serous ovarian carcinomas and oropharyngeal squamous cell carcinomas, stronger IGF1R/PCNA colocalization was significantly associated with a higher overall survival. Ex vivo irradiation of ovarian cancer tissue acutely induced IGF1R/PCNA colocalization together with γH2AX-foci formations. In vitro, RAD18 mediated mono-ubiquitination of PCNA during replication stress was dependent on IGF1R kinase activity. DNA fiber analysis revealed that IGF1R activation could rescue stalled DNA replication forks, but only in cancer cells with baseline IGF1R/PCNA interaction. We believe that the IGF1R/PCNA interaction is a basic cellular mechanism to increase DNA stress tolerance during proliferation, but that this mechanism is lost with tumor progression in conjunction with accumulated DNA damage and aberrant strategies to tolerate genomic instability. To exploit this mechanism in IGF1R targeted therapy, IGF1R inhibitors should be explored in the context of concomitant induction of DNA replication stress as well as in earlier clinical stages than previously tried., Competing Interests: The authors have declared that no competing interests exist.

A wide variety of methods have been proposed in protein subnuclear localization to improve the prediction accuracy. However, one important trend of these means is to treat fusion representation by fusing multiple feature representations, of which, the fusion process takes a lot of time. In view of this, this paper novelly proposed a method by combining a new single feature representation and a new algorithm to obtain good recognition rate. Specifically, based on the position-specific scoring matrix (PSSM), we proposed a new expression, correlation position-specific scoring matrix (CoPSSM) as the protein feature representation. Based on the classic nonlinear dimension reduction algorithm, kernel linear discriminant analysis (KLDA), we added a new discriminant criterion and proposed a dichotomous greedy genetic algorithm (DGGA) to intelligently select its kernel bandwidth parameter. Two public datasets with Jackknife test and KNN classifier were used for the numerical experiments. The results showed that the overall success rate (OSR) with single representation CoPSSM is larger than that with many relevant representations. The OSR of the proposed method can reach as high as 87.444% and 90.3361% for these two datasets, respectively, outperforming many current methods. To show the generalization of the proposed algorithm, two extra standard datasets of protein subcellular were chosen to conduct the expending experiment, and the prediction accuracy by Jackknife test and Independent test is still considerable.

X-ray and γ-ray photons have been widely used for studying radiobiological effects of ionizing radiations. Photons are indirectly ionizing radiations so they need to set in motion electrons (which are a directly ionizing radiation) to perform the ionizations. When the photon dose decreases to below a certain limit, the number of electrons set in motion will become so small that not all cells in an "exposed" cell population can get at least one electron hit. When some cells in a cell population are not hit by a directly ionizing radiation (in other words not irradiated), there will be rescue effect between the irradiated cells and non-irradiated cells, and the resultant radiobiological effect observed for the "exposed" cell population will be different. In the present paper, the mechanisms underlying photon interactions in radiobiological experiments were studied using our developed NRUphoton computer code, which was benchmarked against the MCNP5 code by comparing the photon dose delivered to the cell layer underneath the water medium. The following conclusions were reached: (1) The interaction fractions decreased in the following order: 16O > 12C > 14N > 1H. Bulges in the interaction fractions (versus water medium thickness) were observed, which reflected changes in the energies of the propagating photons due to traversals of different amount of water medium as well as changes in the energy-dependent photon interaction cross-sections. (2) Photoelectric interaction and incoherent scattering dominated for lower-energy (10 keV) and high-energy (100 keV and 1 MeV) incident photons. (3) The fractions of electron ejection from different nuclei were mainly governed by the photoelectric effect cross-sections, and the fractions from the 1s subshell were the largest. (4) The penetration fractions in general decreased with increasing medium thickness, and increased with increasing incident photon energy, the latter being explained by the corresponding reduction in interaction cross-sections. (5) The areas under the angular distribution curves of photons exiting the medium layer and subsequently undergoing interactions within the cell layer became smaller for larger incident photon energies. (6) The number of cells suffering at least one electron hit increased with the administered dose. For larger incident photon energies, the numbers of cells suffering at least one electron hit became smaller, which was attributed to the reduction in the photon interaction cross-section. These results highlighted the importance of the administered dose in radiobiological experiments. In particular, the threshold administered doses at which all cells in the exposed cell array suffered at least one electron hit might provide hints on explaining the intriguing observation that radiation-induced cancers can be statistically detected only above the threshold value of ~100 mSv, and thus on reconciling controversies over the linear no-threshold model.

Our understanding of how chromosomes structurally organize and dynamically interact has been revolutionized through the lens of long-chain polymer physics. Major protein contributors to chromosome structure and dynamics are condensin and cohesin that stochastically generate loops within and between chains, and entrap proximal strands of sister chromatids. In this paper, we explore the ability of transient, protein-mediated, gene-gene crosslinks to induce clusters of genes, thereby dynamic architecture, within the highly repeated ribosomal DNA that comprises the nucleolus of budding yeast. We implement three approaches: live cell microscopy; computational modeling of the full genome during G1 in budding yeast, exploring four decades of timescales for transient crosslinks between 5kbp domains (genes) in the nucleolus on Chromosome XII; and, temporal network models with automated community (cluster) detection algorithms applied to the full range of 4D modeling datasets. The data analysis tools detect and track gene clusters, their size, number, persistence time, and their plasticity (deformation). Of biological significance, our analysis reveals an optimal mean crosslink lifetime that promotes pairwise and cluster gene interactions through “flexible” clustering. In this state, large gene clusters self-assemble yet frequently interact (merge and separate), marked by gene exchanges between clusters, which in turn maximizes global gene interactions in the nucleolus. This regime stands between two limiting cases each with far less global gene interactions: with shorter crosslink lifetimes, “rigid” clustering emerges with clusters that interact infrequently; with longer crosslink lifetimes, there is a dissolution of clusters. These observations are compared with imaging experiments on a normal yeast strain and two condensin-modified mutant cell strains. We apply the same image analysis pipeline to the experimental and simulated datasets, providing support for the modeling predictions. [ABSTRACT FROM AUTHOR]

The process of Nuclei detection in high-grade breast cancer images is quite challenging in the case of image processing techniques due to certain heterogeneous characteristics of cancer nuclei such as enlarged and irregularly shaped nuclei, highly coarse chromatin marginalized to the nuclei periphery and visible nucleoli. Recent reviews state that existing techniques show appreciable segmentation accuracy on breast histopathology images whose nuclei are dispersed and regular in texture and shape; however, typical cancer nuclei are often clustered and have irregular texture and shape properties. This paper proposes a novel segmentation algorithm for detecting individual nuclei from Hematoxylin and Eosin (H&E) stained breast histopathology images. This detection framework estimates a nuclei saliency map using tensor voting followed by boundary extraction of the nuclei on the saliency map using a Loopy Back Propagation (LBP) algorithm on a Markov Random Field (MRF). The method was tested on both whole-slide images and frames of breast cancer histopathology images. Experimental results demonstrate high segmentation performance with efficient precision, recall and dice-coefficient rates, upon testing high-grade breast cancer images containing several thousand nuclei. In addition to the optimal performance on the highly complex images presented in this paper, this method also gave appreciable results in comparison with two recently published methods-Wienert et al. (2012) and Veta et al. (2013), which were tested using their own datasets., Competing Interests: The authors have declared that no competing interests exist.

Structural analysis of biological membranes is important for understanding cell and sub-cellular organelle function as well as their interaction with the surrounding environment. Imaging of whole cells in three dimension at high spatial resolution remains a significant challenge, particularly for thick cells. Cryo-transmission soft X-ray microscopy (cryo-TXM) has recently gained popularity to image, in 3D, intact thick cells (∼10μm) with details of sub-cellular architecture and organization in near-native state. This paper reports a new tool to segment and quantify structural changes of biological membranes in 3D from cryo-TXM images by tracking an initial 2D contour along the third axis of the microscope, through a multi-scale ridge detection followed by an active contours-based model, with a subsequent refinement along the other two axes. A quantitative metric that assesses the grayscale profiles perpendicular to the membrane surfaces is introduced and shown to be linearly related to the membrane thickness. Our methodology has been validated on synthetic phantoms using realistic microscope properties and structure dimensions, as well as on real cryo-TXM data. Results demonstrate the validity of our algorithms for cryo-TXM data analysis. [ABSTRACT FROM AUTHOR]

Pap test involves searching of morphological changes in cervical squamous epithelial cells by pathologists or cytotechnologists to identify potential cancerous cells in the cervix. Nuclear membrane irregularity is one of the morphological changes of malignancy. This paper proposes two novel techniques for the evaluation of nuclear membrane irregularity. The first technique, namely, penalty-driven smoothing analysis, introduces different penalty values for nuclear membrane contour with different degrees of irregularity. The second technique, which can be subdivided into mean- or median-type residual-based analysis, computes the number of points of nuclear membrane contour that deviates from the mean or median of the nuclear membrane contour. Performance of the proposed techniques was compared to three state-of-the-art techniques, namely, radial asymmetric, shape factor, and rim difference. Friedman and post hoc tests using Holm, Shaffer, and Bergmann procedures returned significant differences for all the three classes, i.e., negative for intraepithelial lesion or malignancy (NILM) versus low grade squamous intraepithelial lesion (LSIL), NILM versus high grade squamous intraepithelial lesion (HSIL), and LSIL versus HSIL when the span value equaled 3 was employed with linear penalty function. When span values equaled 5, 7, and 9, NILM versus LSIL and HSIL showed significant differences regardless of the penalty functions. In addition, the results of penalty-driven smoothing analysis were comparable with those of other state-of-the-art techniques. Residual-based analysis returned significant differences for the comparison among the three diagnostic classes. Findings of this study proved the significance of nuclear membrane irregularity as one of the features to differentiate the different diagnostic classes of cervical squamous epithelial cells. [ABSTRACT FROM AUTHOR]

This paper presents ChroTeMo, a tool for chromosome territory modelling, accompanied by ChroTeVi–a chromosome territory visualisation software that uses the data obtained by ChroTeMo. These tools have been developed in order to complement the molecular cytogenetic research of interphase nucleus structure in a model grass Brachypodium distachyon. Although the modelling tool has been initially created for one particular species, it has universal application. The proposed version of ChroTeMo allows for generating a model of chromosome territory distribution in any given plant or animal species after setting the initial, species-specific parameters. ChroTeMo has been developed as a fully probabilistic modeller. Due to this feature, the comparison between the experimental data on the structure of a nucleus and the results obtained from ChroTeMo can indicate whether the distribution of chromosomes inside a nucleus is also fully probabilistic or is subjected to certain non-random patterns. The presented tools have been written in Python, so they are multiplatform, portable and easy to read. Moreover, if necessary they can be further developed by users writing their portions of code. The source code, documentation, and wiki, as well as the issue tracker and the list of related articles that use ChroTeMo and ChroTeVi, are accessible in a public repository at Github under GPL 3.0 license. [ABSTRACT FROM AUTHOR]

Cell nuclei detection in fluorescent microscopic images is an important and time consuming task in a wide range of biological applications. Blur, clutter, bleed through and partial occlusion of nuclei make individual nuclei detection a challenging task for automated image analysis. This paper proposes a novel and robust detection method based on the active contour framework. Improvement over conventional approaches is achieved by exploiting prior knowledge of the nucleus shape in order to better detect individual nuclei. This prior knowledge is defined using a dictionary based approach which can be formulated as the optimization of a convex energy function. The proposed method shows accurate detection results for dense clusters of nuclei, for example, an F-measure (a measure for detection accuracy) of 0.96 for the detection of cell nuclei in peripheral blood mononuclear cells, compared to an F-measure of 0.90 achieved by state-of-the-art nuclei detection methods.

There is considerable interest in cell biology in determining whether, and to what extent, the spatial arrangement of nuclear objects affects nuclear function. A common approach to address this issue involves analyzing a collection of images produced using some form of fluorescence microscopy. We assume that these images have been successfully pre-processed and a spatial point pattern representation of the objects of interest within the nuclear boundary is available. Typically in these scenarios, the number of objects per nucleus is low, which has consequences on the ability of standard analysis procedures to demonstrate the existence of spatial preference in the pattern. There are broadly two common approaches to look for structure in these spatial point patterns. First a spatial point pattern for each image is analyzed individually, or second a simple normalization is performed and the patterns are aggregated. In this paper we demonstrate using synthetic spatial point patterns drawn from predefined point processes how difficult it is to distinguish a pattern from complete spatial randomness using these techniques and hence how easy it is to miss interesting spatial preferences in the arrangement of nuclear objects. The impact of this problem is also illustrated on data related to the configuration of PML nuclear bodies in mammalian fibroblast cells.

Generally, effect of fragmentation per se on biodiversity has not been separated from the effect of habitat loss. In this paper, using nDNA and cpDNA SSRs, we studied genetic diversity of Castanopsis sclerophylla (Lindl. & Paxton) Schotty populations and decoupled the effects of habitat loss and fragmentation per se. We selected seven nuclear and six cpDNA microsatellite loci and genotyped 460 individuals from mainland and island populations, which were located in the impoundment created in 1959. Number of alleles per locus of populations in larger habitats was significantly higher than that in smaller habitats. There was a significant relationship between the number of alleles per locus and habitat size. Based on this relationship, the predicted genetic diversity of an imaginary population of size equaling the total area of the islands was lower than that of the global population on the islands. Re-sampling demonstrated that low genetic diversity of populations in small habitats was caused by unevenness in sample size. Fisher's α index was similar among habitat types. These results indicate that the decreased nuclear and chloroplast genetic diversity of populations in smaller habitats was mainly caused by habitat loss. For nuclear and chloroplast microsatellite loci, values of F(ST) were 0.066 and 0.893, respectively, and the calculated pollen/seed dispersal ratio was 162.2. When separated into pre-and post-fragmentation cohorts, pollen/seed ratios were 121.2 and 189.5, respectively. Our results suggest that habitat loss explains the early decrease in genetic diversity, while fragmentation per se may play a major role in inbreeding and differentiation among fragmented populations and later loss of genetic diversity.

PTEN is a critical gene involved in the regulation of many cellular processes. The product of this gene has dual phosphatase activity and is able to dephosphorylate the 5' end of the phosphatidylinositol (3,4,5)-trisphosphate. Within the cellular nucleus, this protein has been associated with regulation of the expression of many genes, although the mechanism of this regulation remains unclear. In this paper, two specific oligonucleotide aptamers were developed and selected, using the SELEX procedure, according to their ability to detect the PTEN protein in different subcellular compartments of neurons. While one aptamer was able to detect PTEN in the nucleus, the other recognized PTEN in the cytoplasm. The recognition pattern of PTEN by both aptamers was confirmed using antibodies in western blots of the proteins purified from mouse cerebellar homogenates and subcellular fractions. Additionally, we demonstrated that the two aptamers recognized different epitopes of the target peptide. The results presented here could not be fully explained by the canonical phosphatase structure of PTEN, suggesting the existence of different conformations of phosphatase in the nucleus and the cytoplasm.

In order to preserve genetic information in stress conditions, bacterial DNA is organized into higher order nucleoid structure. In this paper, with the help of Atomic Force Microscopy, we show the different structural changes in mycobacterial nucleoid at different points of growth in the presence of different concentrations of glucose in the medium. We also observe that in Mycobacterium smegmatis, two different Dps proteins (Dps1 and Dps2) promote two types of nucleoid organizations. At the late stationary phase, under low glucose availability, Dps1 binds to DNA to form a very stable toroid structure. On the other hand, under the same condition, Dps2-DNA complex forms an incompletely condensed toroid and finally forms a further stable coral reef structure in the presence of RNA. This coral reef structure is stable in high concentration of bivalent ion like Mg(2+).

Accepting the hypothesis that cancers are self-organizing, opportunistic systems, it is crucial to understand the collective behavior of cancer cells in their tumorous heterogeneous environment. In the present paper, we ask the following basic question: Is this self-organization of tumor evolution reflected in the manner in which malignant cells are spatially distributed in their heterogeneous environment? We employ a variety of nontrivial statistical microstructural descriptors that arise in the theory of heterogeneous media to characterize the spatial distributions of the nuclei of both benign brain white matter cells and brain glioma cells as obtained from histological images. These descriptors, which include the pair correlation function, structure factor and various nearest neighbor functions, quantify how pairs of cell nuclei are correlated in space in various ways. We map the centroids of the cell nuclei into point distributions to show that while commonly used local spatial statistics (e.g., cell areas and number of neighboring cells) cannot clearly distinguish spatial correlations in distributions of normal and abnormal cell nuclei, their salient structural features are captured very well by the aforementioned microstructural descriptors. We show that the tumorous cells pack more densely than normal cells and exhibit stronger effective repulsions between any pair of cells. Moreover, we demonstrate that brain gliomas are organized in a collective way rather than randomly on intermediate and large length scales. The existence of nontrivial spatial correlations between the abnormal cells strongly supports the view that cancer is not an unorganized collection of malignant cells but rather a complex emergent integrated system.

Heat shock factor 1 is the key transcription factor of the heat shock response. Its function is to protect the cell against the deleterious effects of stress. Upon stress, HSF1 binds to and transcribes hsp genes and repeated satellite III (sat III) sequences present at the 9q12 locus. HSF1 binding to pericentric sat III sequences forms structures known as nuclear stress bodies (nSBs). nSBs represent a natural amplification of RNA pol II dependent transcription sites. Dynamics of HSF1 and of deletion mutants were studied in living cells using multi-confocal Fluorescence Correlation Spectroscopy (mFCS) and Fluorescence Recovery After Photobleaching (FRAP). In this paper, we show that HSF1 dynamics modifications upon heat shock result from both formation of high molecular weight complexes and increased HSF1 interactions with chromatin. These interactions involve both DNA binding with Heat Shock Element (HSE) and sat III sequences and a more transient sequence-independent binding likely corresponding to a search for more specific targets. We find that the trimerization domain is required for low affinity interactions with chromatin while the DNA binding domain is required for site-specific interactions of HSF1 with DNA. [ABSTRACT FROM AUTHOR]

The transport of macromolecules into the cell nucleus occurs through nuclear pore complexes (NPCs) and is mediated by cellular receptors. Recently, a novel mechanism of nuclear entry, in which actin polymerization provides a propulsive force driving the transport through the NPC, has been proposed. This mechanism is used by the nucleocapsid from baculovirus, one of the largest viruses to replicate in the nucleus of their host cells, which crosses the NPC and enters the nucleus independently of cellular receptors. The baculovirus nucleocapsid contains a protein that hijacks the cellular actin polymerization machinery to assemble actin filaments that propel the nucleocapsid through the host cell cytoplasm. In this study, we functionalized carbon nanotubes by covalently attaching a protein domain responsible for inducing actin polymerization and investigated their nuclear entry. We found that the functionalized carbon nanotubes were able to enter the cell nucleus under permissive conditions for actin polymerization, but not when this process was inhibited. We conclude that the mechanical force generated by actin polymerization can drive cargo entry into the cell nucleus. Our results support a novel force-driven mechanism for molecular entry into the cell nucleus. [ABSTRACT FROM AUTHOR]

The nuclear pore complex (NPC) forms a gateway for nucleocytoplasmic transport. The outer ring protein complex of the NPC (the Nup107-160 subcomplex in humans) is a key component for building the NPC. Nup107-160 subcomplexes are believed to be symmetrically localized on the nuclear and cytoplasmic sides of the NPC. However, in S. pombe immunoelectron and fluorescence microscopic analyses revealed that the homologous components of the human Nup107-160 subcomplex had an asymmetrical localization: constituent proteins spNup132 and spNup107 were present only on the nuclear side (designated the spNup132 subcomplex), while spNup131, spNup120, spNup85, spNup96, spNup37, spEly5 and spSeh1 were localized only on the cytoplasmic side (designated the spNup120 subcomplex), suggesting the complex was split into two pieces at the interface between spNup96 and spNup107. This contrasts with the symmetrical localization reported in other organisms. Fusion of spNup96 (cytoplasmic localization) with spNup107 (nuclear localization) caused cytoplasmic relocalization of spNup107. In this strain, half of the spNup132 proteins, which interact with spNup107, changed their localization to the cytoplasmic side of the NPC, leading to defects in mitotic and meiotic progression similar to an spNup132 deletion strain. These observations suggest the asymmetrical localization of the outer ring spNup132 and spNup120 subcomplexes of the NPC is necessary for normal cell cycle progression in fission yeast. [ABSTRACT FROM AUTHOR]

Evidence is rapidly mounting that transposable element (TE) expression and replication may impact biology more widely than previously thought. This includes potential effects on normal physiology of somatic tissues and dysfunctional impacts in diseases associated with aging, such as cancer and neurodegeneration. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by the use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, reporter constructs demonstrating engineered long interspersed nuclear element (LINE-1; L1) mobilization have been in use for quite some time, and strategies similar to L1 retrotransposition reporter assays have been developed to report replication of Ty1 elements in yeast and mouse intracisternal A particle (IAP) long terminal repeat (LTR) retrotransposons in cultivated cells. We describe a novel approach termed cellular labeling of endogenous retrovirus replication (CLEVR), which reports replication of the gypsy element within specific cells in vivo in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly. We also demonstrate that the gypsy-CLEVR replication rate is increased when the short interfering RNA (siRNA) silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR retrotransposons, endogenous retroviruses (ERVs), and exogenous retroviruses. [ABSTRACT FROM AUTHOR]

In Drosophila it has recently been demonstrated that a spindle matrix in the form of a membrane-less macromolecular assembly embeds the microtubule-based spindle apparatus. In addition, two of its constituents, Megator and Chromator, were shown to function as spatial regulators of spindle checkpoint proteins. However, whether the spindle matrix plays a wider functional role in spatially regulating cell cycle progression factors was unknown. Here using a live imaging approach we provide evidence that a number of key cell cycle proteins such as Cyclin B, Polo, and Ran co-localize with the spindle matrix during mitosis. Furthermore, prevention of spindle matrix formation by injection of a function blocking antibody against the spindle matrix protein Chromator results in cell cycle arrest prior to nuclear envelope breakdown. In such embryos the spatial dynamics of Polo and Cyclin B enrichment at the nuclear rim and kinetochores is abrogated and Polo is not imported into the nucleus. This is in contrast to colchicine-arrested embryos where the wild-type dynamics of these proteins are maintained. Taken together these results suggest that spindle matrix formation may be a general requirement for the localization and proper dynamics of cell cycle factors promoting signaling events leading to cell cycle progression. [ABSTRACT FROM AUTHOR]

Most known giant viruses, i.e., viruses producing giant virions, parasitize amoebae and other unicellular eukaryotes. Although they vary in the level of dependence on host nuclear functions, their virions self-assemble in the host cell’s cytoplasm. Here we report the discovery of a new prototype of giant virus infecting epidermal cells of the marine arrow worm Adhesisagitta hispida. Its 1.25 μm-long virions self-assemble and accumulate in the host cell’s nucleus. Conventional transmission electron microscopy reveals that the virions have a unique bipartite structure. An ovoid nucleocapsid, situated in a broad “head” end of the virion is surrounded by a thin envelope. The latter extends away from the head to form a voluminous conical “tail” filled with electron-dense extracapsidular material. The 31nm-thick capsid wall has a distinctive substructure resulting from a patterned arrangement of subunits; it bears no ultrastructural resemblance to the virion walls of other known giant viruses. The envelope self-assembles coincident with the capsid and remotely from all host membranes. We postulate that transmission to new hosts occurs by rupture of protruding virion-filled nuclei when infected arrow worms mate. Future genomic work is needed to determine the phylogenetic position of this new virus, which we have provisionally named Meelsvirus. [ABSTRACT FROM AUTHOR]

Tardigrades represent an invertebrate phylum with no circulatory or respiratory system. Their body cavity is filled with free storage cells of the coelomocyte-type, which are responsible for important physiological functions. We report a study comparing the ultrastructure of storage cells in anhydrobiotic and hydrated specimens of the eutardigrade Richtersius coronifer. We also analysed the effect of temperature stress on storage cell structure. Firstly, we verified two types of ultrastructurally different storage cells, which differ in cellular organelle complexity, amount and content of reserve material and connection to oogenetic stage. Type I cells were found to differ ultrastructurally depending on the oogenetic stage of the animal. The main function of these cells is energy storage. Storage cells of Type I were also observed in the single male that was found among the analysed specimens. The second cell type, Type II, found only in females, represents young undifferentiated cells, possibly stem cells. The two types of cells also differ with respect to the presence of nucleolar vacuoles, which are related to oogenetic stages and to changes in nucleolic activity during oogenesis. Secondly, this study revealed that storage cells are not ultrastructurally affected by six months of desiccation or by heating following this desiccation period. However, heating of the desiccated animals (tuns) tended to reduce animal survival, indicating that long-term desiccation makes these animals more vulnerable to heat stress. We confirmed the degradative pathways during the rehydration process after desiccation and heat stress. Our study is the first to document two ultrastructurally different types of storage cells in tardigrades and reveals new perspectives for further studies of tardigrade storage cells. [ABSTRACT FROM AUTHOR]

The spatial architecture of signaling pathways and the interaction with cell size and morphology are complex, but little understood. With the advances of single cell imaging and single cell biology, it becomes crucial to understand intracellular processes in time and space. Activation of cell surface receptors often triggers a signaling cascade including the activation of membrane-attached and cytosolic signaling components, which eventually transmit the signal to the cell nucleus. Signaling proteins can form steep gradients in the cytosol, which cause strong cell size dependence. We show that the kinetics at the membrane-cytosolic interface and the ratio of cell membrane area to the enclosed cytosolic volume change the behavior of signaling cascades significantly. We suggest an estimate of average concentration for arbitrary cell shapes depending on the cell volume and cell surface area. The normalized variance, known from image analysis, is suggested as an alternative measure to quantify the deviation from the average concentration. A mathematical analysis of signal transduction in time and space is presented, providing analytical solutions for different spatial arrangements of linear signaling cascades. Quantification of signaling time scales reveals that signal propagation is faster at the membrane than at the nucleus, while this time difference decreases with the number of signaling components in the cytosol. Our investigations are complemented by numerical simulations of non-linear cascades with feedback and asymmetric cell shapes. We conclude that intracellular signal propagation is highly dependent on cell geometry and, thereby, conveys information on cell size and shape to the nucleus. [ABSTRACT FROM AUTHOR]

The repeated rDNA array gives rise to the nucleolus, an organelle that is central to cellular processes as varied as stress response, cell cycle regulation, RNA modification, cell metabolism, and genome stability. The rDNA array is also responsible for the production of more than 70% of all cellular RNAs (the ribosomal RNAs). The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1 while the 45S rDNA arrays reside on the short arm of five human acrocentric chromosomes. These critical genome elements have remained unassembled and have been excluded from all Hi-C analyses to date. Here we built the first high resolution map of 5S and 45S rDNA array contacts with the rest of the genome combining over 15 billion Hi-C reads from several experiments. The data enabled sufficiently high coverage to map rDNA-genome interactions with 1MB resolution and identify rDNA-gene contacts. The map showed that the 5S and 45S arrays display preferential contact at common sites along the genome but are not themselves sufficiently close to yield 5S-45S Hi-C contacts. Ribosomal DNA contacts are enriched in segments of closed, repressed, and late replicating chromatin, as well as CTCF binding sites. Finally, we identified functional categories whose dispersed genes coalesced in proximity to the rDNA arrays or instead avoided proximity with the rDNA arrays. The observations further our understanding of the spatial localization of rDNA arrays and their contribution to the architecture of the cell nucleus. [ABSTRACT FROM AUTHOR]

Salinity is one of the most prominent abiotic factors, which greatly influence reproduction, development, growth, physiological and metabolic activities of fishes. Spotted sea bass (Lateolabrax maculatus), as a euryhaline marine teleost, has extraordinary ability to deal with a wide range of salinity changes. However, this species is devoid of genomic resources, and no study has been conducted at the transcriptomic level to determine genes responsible for salinity regulation, which impedes the understanding of the fundamental mechanism conferring tolerance to salinity fluctuations. Liver, as the major metabolic organ, is the key source supplying energy for iono- and osmoregulation in fish, however, little attention has been paid to its salinity-related functions but which should not be ignored. In this study, we perform RNA-Seq analysis to identify genes involved in salinity adaptation and osmoregulation in liver of spotted sea bass, generating from the fishes exposed to low and high salinity water (5 vs 30ppt). After de novo assembly, annotation and differential gene expression analysis, a total of 455 genes were differentially expressed, including 184 up-regulated and 271 down-regulated transcripts in low salinity-acclimated fish group compared with that in high salinity-acclimated group. A number of genes with a potential role in salinity adaptation for spotted sea bass were classified into five functional categories based on the gene ontology (GO) and enrichment analysis, which include genes involved in metabolites and ion transporters, energy metabolism, signal transduction, immune response and structure reorganization. The candidate genes identified in L. maculates liver provide valuable information to explore new pathways related to fish salinity and osmotic regulation. Besides, the transcriptomic sequencing data supplies significant resources for identification of novel genes and further studying biological questions in spotted sea bass. [ABSTRACT FROM AUTHOR]

Planctomycetes are distinguished from other Bacteria by compartmentalization of cells via internal membranes, interpretation of which has been subject to recent debate regarding potential relations to Gram-negative cell structure. In our interpretation of the available data, the planctomycete Gemmata obscuriglobus contains a nuclear body compartment, and thus possesses a type of cell organization with parallels to the eukaryote nucleus. Here we show that pore-like structures occur in internal membranes of G.obscuriglobus and that they have elements structurally similar to eukaryote nuclear pores, including a basket, ring-spoke structure, and eight-fold rotational symmetry. Bioinformatic analysis of proteomic data reveals that some of the G. obscuriglobus proteins associated with pore-containing membranes possess structural domains found in eukaryote nuclear pore complexes. Moreover, immunogold labelling demonstrates localization of one such protein, containing a β-propeller domain, specifically to the G. obscuriglobus pore-like structures. Finding bacterial pores within internal cell membranes and with structural similarities to eukaryote nuclear pore complexes raises the dual possibilities of either hitherto undetected homology or stunning evolutionary convergence. [ABSTRACT FROM AUTHOR]

An unresolved issue in structural biology is how the encapsulated lens removes membranous organelles to carry out its role as a transparent optical element. In this ultrastructural study, we establish a mechanism for nuclear elimination in the developing chick lens during the formation of the organelle-free zone. Day 12–15 chick embryo lenses were examined by high-resolution confocal light microscopy and thin section transmission electron microscopy (TEM) following fixation in 10% formalin and 4% paraformaldehyde, and then processing for confocal or TEM as described previously. Examination of developing fiber cells revealed normal nuclei with dispersed chromatin and clear nucleoli typical of cells in active ribosome production to support protein synthesis. Early signs of nuclear degradation were observed about 300 μm from the lens capsule in Day 15 lenses where the nuclei display irregular nuclear stain and prominent indentations that sometimes contained a previously undescribed macromolecular aggregate attached to the nuclear envelope. We have termed this novel structure the nuclear excisosome. This complex by confocal is closely adherent to the nuclear envelope and by TEM appears to degrade the outer leaflet of the nuclear envelope, then the inner leaflet up to 500 μm depth. The images suggest that the nuclear excisosome separates nuclear membrane proteins from lipids, which then form multilamellar assemblies that stain intensely in confocal and in TEM have 5 nm spacing consistent with pure lipid bilayers. The denuded nucleoplasm then degrades by condensation and loss of structure in the range 600 to 700 μm depth producing pyknotic nuclear remnants. None of these stages display any classic autophagic vesicles or lysosomes associated with nuclei. Uniquely, the origin of the nuclear excisosome is from filopodial-like projections of adjacent lens fiber cells that initially contact, and then appear to fuse with the outer nuclear membrane. These filopodial-like projections appear to be initiated with a clathrin-like coat and driven by an internal actin network. In summary, a specialized cellular organelle, the nuclear excisosome, generated in part by adjacent fiber cells degrades nuclei during fiber cell differentiation and maturation. [ABSTRACT FROM AUTHOR]

Disassembly of the cone-shaped HIV-1 capsid in target cells is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. Here we report a novel strategy to visualize HIV-1 uncoating using a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed), which is specifically packaged into virions through the high-avidity binding to capsid (CA). Single virus imaging reveals that CypA-DsRed remains associated with cores after permeabilization/removal of the viral membrane and that CypA-DsRed and CA are lost concomitantly from the cores in vitro and in living cells. The rate of loss is modulated by the core stability and is accelerated upon the initiation of reverse transcription. We show that the majority of single cores lose CypA-DsRed shortly after viral fusion, while a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is dependent on reverse transcription. Uncoating occurs both in the cytoplasm and at the nuclear membrane. Our novel imaging assay thus enables time-resolved visualization of single HIV-1 uncoating in living cells, and reveals the previously unappreciated spatio-temporal features of this incompletely understood process. [ABSTRACT FROM AUTHOR]

Interleukin-1 mediates inflammation and stress response through nuclear activity of p38α. Although IL-1 receptor is not degraded, p38α activation is transient. IL-1 also causes cell migration and EMT by modulating cell-cell junctions. Although molecules involved in p38 activation are known, mechanism of the transient nuclear response and its basal activity remains unknown. By mathematical modeling of IL1/p38 signaling network, we show that IL-1 induces robust p38α activation both in the nucleus and in the cytoplasm/membrane. While nuclear response consists of an acute phase, membrane response resembles a step change. Following stimulation, p38α activity returns to a basal level in absence of receptor degradation. While nuclear pulse is controlled by MKP1 through a negative feedback to pp38, its basal activity is controlled by both TAB1 and MKP1 through a positive feedback loop. Our model provides insight into the mechanism of p38α activation, reason for its transient nuclear response, and explanation of the basal activity of MKK3/6 and p38α, which has been experimentally observed by other groups. [ABSTRACT FROM AUTHOR]

For efficient polarity and migration, cells need to regulate the magnitude and spatial distribution of actin assembly. This process is coordinated by reciprocal interactions between the actin cytoskeleton and mechanical forces. Actin polymerization-based protrusion increases tension in the plasma membrane, which in turn acts as a long-range inhibitor of actin assembly. These interactions form a negative feedback circuit that limits the magnitude of membrane tension in neutrophils and prevents expansion of the existing front and the formation of secondary fronts. It has been suggested that the plasma membrane directly inhibits actin assembly by serving as a physical barrier that opposes protrusion. Here we show that efficient control of actin polymerization-based protrusion requires an additional mechanosensory feedback cascade that indirectly links membrane tension with actin assembly. Specifically, elevated membrane tension acts through phospholipase D2 (PLD2) and the mammalian target of rapamycin complex 2 (mTORC2) to limit actin nucleation. In the absence of this pathway, neutrophils exhibit larger leading edges, higher membrane tension, and profoundly defective chemotaxis. Mathematical modeling suggests roles for both the direct (mechanical) and indirect (biochemical via PLD2 and mTORC2) feedback loops in organizing cell polarity and motility—the indirect loop is better suited to enable competition between fronts, whereas the direct loop helps spatially organize actin nucleation for efficient leading edge formation and cell movement. This circuit is essential for polarity, motility, and the control of membrane tension. [ABSTRACT FROM AUTHOR]

In the present study, the combination of the micronucleus test with analysis of the activity of the rRNA genes in mutagen-treated Hordeum vulgare (barley) by maleic hydrazide (MH) cells was performed. Simultaneously fluorescence in situ hybridization (FISH) with 25S rDNA as probes and an analysis of the transcriptional activity of 35S rRNA genes with silver staining were performed. The results showed that transcriptional activity is always maintained in the micronuclei although they are eliminated during the next cell cycle. The analysis of the transcriptional activity was extended to barley nuclei. MH influenced the fusion of the nucleoli in barley nuclei. The silver staining enabled detection of the nuclear bodies which arose after MH treatment. The results confirmed the usefulness of cytogenetic techniques in the characterization of micronuclei. Similar analyses can be now extended to other abiotic stresses to study the response of plant cells to the environment. [ABSTRACT FROM AUTHOR]

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm. [ABSTRACT FROM AUTHOR]

Cardiac glycosides (CGs), inhibitors of Na+/K+-ATPase (NKA), used clinically to treat heart failure, have garnered recent attention as potential anti-cancer and anti-viral agents. A high-throughput phenotypic screen designed to identify modulators of promyelocytic leukemia protein (PML) nuclear body (NB) formation revealed the CG gitoxigenin as a potent activator of PML. We demonstrate that multiple structurally distinct CGs activate the formation of PML NBs and induce PML protein SUMOylation in an NKA-dependent fashion. CG effects on PML occur at the post-transcriptional level, mechanistically distinct from previously described PML activators and are mediated through signaling events downstream of NKA. Curiously, genomic deletion of PML in human cancer cells failed to abrogate the cytotoxic effects of CGs and other apoptotic stimuli such as ceramide and arsenic trioxide that were previously shown to function through PML in mice. These findings suggest that alternative pathways can compensate for PML loss to mediate apoptosis in response to CGs and other apoptotic stimuli. [ABSTRACT FROM AUTHOR]

During central nervous system (CNS) development neural stem cells (Neuroblasts, NBs) have to acquire an identity appropriate to their location. In thoracic and abdominal segments of Drosophila, the expression pattern of Bithorax-Complex Hox genes is known to specify the segmental identity of NBs prior to their delamination from the neuroectoderm. Compared to the thoracic, ground state segmental units in the head region are derived to different degrees, and the precise mechanism of segmental specification of NBs in this region is still unclear. We identified and characterized a set of serially homologous NB-lineages in the gnathal segments and used one of them (NB6-4 lineage) as a model to investigate the mechanism conferring segment-specific identities to gnathal NBs. We show that NB6-4 is primarily determined by the cell-autonomous function of the Hox gene Deformed (Dfd). Interestingly, however, it also requires a non-cell-autonomous function of labial and Antennapedia that are expressed in adjacent anterior or posterior compartments. We identify the secreted molecule Amalgam (Ama) as a downstream target of the Antennapedia-Complex Hox genes labial, Dfd, Sex combs reduced and Antennapedia. In conjunction with its receptor Neurotactin (Nrt) and the effector kinase Abelson tyrosine kinase (Abl), Ama is necessary in parallel to the cell-autonomous Dfd pathway for the correct specification of the maxillary identity of NB6-4. Both pathways repress CyclinE (CycE) and loss of function of either of these pathways leads to a partial transformation (40%), whereas simultaneous mutation of both pathways leads to a complete transformation (100%) of NB6-4 segmental identity. Finally, we provide genetic evidences, that the Ama-Nrt-Abl-pathway regulates CycE expression by altering the function of the Hippo effector Yorkie in embryonic NBs. The disclosure of a non-cell-autonomous influence of Hox genes on neural stem cells provides new insight into the process of segmental patterning in the developing CNS. [ABSTRACT FROM AUTHOR]

Testosterone (T) and related androgens are performance enhancing drugs (PEDs) abused by some athletes to gain competitive advantage. To monitor unauthorized androgen abuse, doping control programs use mass spectrometry (MS) to detect androgens, synthetic anabolic-androgenic steroids (AASs) and their metabolites in an athlete’s urine. AASs of unknown composition will not be detected by these procedures. Since AASs achieve their anabolic effects by activating the Androgen Receptor (AR), cell-based bioassays that measure the effect of a urine sample on AR activity are under investigation as complementary, pan-androgen detection methods. We evaluated an AR BioAssay as a monitor for androgen activity in urine pre-treated with glucuronidase, which releases T from the inactive T-glucuronide that predominates in urine. AR BioAssay activity levels were expressed as ‘T-equivalent’ concentrations by comparison to a T dose response curve. The T-equivalent concentrations of androgens in the urine of hypogonadal participants supplemented with T (in whom all androgenic activity should arise from T) were quantitatively identical to the T measurements conducted by MS at the UCLA Olympic Analytical Laboratory (0.96 ± 0.22). All 17 AASs studied were active in the AR BioAssay; other steroids were inactive. 12 metabolites of 10 commonly abused AASs, which are used for MS monitoring of AAS doping because of their prolonged presence in urine, had reduced or no AR BioAssay activity. Thus, the AR BioAssay can accurately and inexpensively monitor T, but its ability to monitor urinary AASs will be limited to a period immediately following doping in which the active AASs remain intact. [ABSTRACT FROM AUTHOR]

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy. [ABSTRACT FROM AUTHOR]

Hexamerins are insect storage proteins abundantly secreted by the larval fat body into the haemolymph. The canonical role of hexamerins consists of serving as an amino acid reserve for development toward the adult stage. However, in Apis mellifera, immunofluorescence assays coupled to confocal laser-scanning microscopy, and high-throughput sequencing, have recently shown the presence of hexamerins in other organs than the fat body. These findings have led us to study these proteins with the expectation of uncovering additional functions in insect development. We show here that a honeybee hexamerin, HEX 110, localizes in the cytoplasm and nucleus of ovarian cells. In the nucleus of somatic and germline cells, HEX 110 colocalized with a nucleolar protein, fibrillarin, suggesting a structural or even regulatory function in the nucleolus. RNase A provoked the loss of HEX 110 signals in the ovarioles, indicating that the subcellular localization depends on RNA. This was reinforced by incubating ovaries with pyronin Y, a RNA-specific dye. Together, the colocalization with fibrillarin and pyronin Y, and the sensitivity to RNase, highlight unprecedented roles for HEX110 in the nucleolus, the nuclear structure harbouring the gene cluster involved in ribosomal RNA production. However, the similar patterns of HEX 110 foci distribution in the active and inactive ovaries of queens and workers preclude its association with the functional status of these organs. [ABSTRACT FROM AUTHOR]

The perinucleolar compartment (PNC) is a subnuclear stucture forming predominantly in cancer cells; its prevalence positively correlates with metastatic capacity. Although several RNA-binding proteins have been characterized in PNC, the molecular function of this compartment remains unclear. Here we demonstrate that the cyclin–dependent kinase 13 (CDK13) is a newly identified constituent of PNC. CDK13 is a kinase involved in the regulation of gene expression and whose overexpression was found to alter pre-mRNA processing. In this study we show that CDK13 is enriched in PNC and co-localizes all along the cell cycle with the PNC component PTB. In contrast, neither the cyclins K and L, known to associate with CDK13, nor the potential kinase substrates accumulate in PNC. We further show that CDK13 overexpression increases PNC prevalence suggesting that CDK13 may be determinant for PNC formation. This result linked to the finding that CDK13 gene is amplified in different types of cancer indicate that this kinase can contribute to cancer development in human. [ABSTRACT FROM AUTHOR]

Background: Recent studies have shown that heterogeneous nuclear ribonucleoprotein U (hnRNP U), a component of the hnRNP complex, contributes to stabilize the kinetochore-microtubule interaction during mitosis. CENP-W was identified as an inner centromere component that plays crucial roles in the formation of a functional kinetochore complex. Results: We report that hnRNP U interacts with CENP-W, and the interaction between hnRNP U and CENP-W mutually increased each other’s protein stability by inhibiting the proteasome-mediated degradation. Further, their co-localization was observed chiefly in the nuclear matrix region and at the microtubule-kinetochore interface during interphase and mitosis, respectively. Both microtubule-stabilizing and microtubule-destabilizing agents significantly decreased the protein stability of CENP-W. Furthermore, loss of microtubules and defects in microtubule organization were observed in CENP-W-depleted cells. Conclusion: Our data imply that CENP-W plays an important role in the attachment and interaction between microtubules and kinetochore during mitosis. [ABSTRACT FROM AUTHOR]

Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and ultimately affect viral replication. [ABSTRACT FROM AUTHOR]

Plants have evolved a considerable number of intrinsic tolerance strategies to acclimate to ambient temperature increase. However, their molecular mechanisms remain largely obscure. Here we report a DEAD-box RNA helicase, TOGR1 (Thermotolerant Growth Required1), prerequisite for rice growth themotolerance. Regulated by both temperature and the circadian clock, its expression is tightly coupled to daily temperature fluctuations and its helicase activities directly promoted by temperature increase. Located in the nucleolus and associated with the small subunit (SSU) pre-rRNA processome, TOGR1 maintains a normal rRNA homeostasis at high temperature. Natural variation in its transcript level is positively correlated with plant height and its overexpression significantly improves rice growth under hot conditions. Our findings reveal a novel molecular mechanism of RNA helicase as a key chaperone for rRNA homeostasis required for rice thermotolerant growth and provide a potential strategy to breed heat-tolerant crops by modulating the expression of TOGR1 and its orthologs. [ABSTRACT FROM AUTHOR]

Microtubules play multiple roles in a wide range of cellular phenomena, including cell polarity establishment and chromosome segregation. A number of microtubule regulators have been identified, including microtubule-associated proteins and kinases, and knowledge of these factors has contributed to our molecular understanding of microtubule regulation of each relevant cellular process. The known regulators, however, are insufficient to explain how those processes are linked to one another, underscoring the need to identify additional regulators. To find such novel mechanisms and microtubule regulators, we performed a screen that combined genetics and microscopy for fission yeast mutants defective in microtubule organization. We isolated approximately 900 mutants showing defects in either microtubule organization or the nuclear envelope, and these mutants were classified into 12 categories. We particularly focused on one mutant, kis1, which displayed spindle defects in early mitosis. The kis1 mutant frequently failed to assemble a normal bipolar spindle. The responsible gene encoded a kinetochore protein, Mis19 (also known as Eic1), which localized to the interface of kinetochores and spindle poles. We also found that the inner kinetochore proteins Mis6/CENP-I and Cnp1/CENP-A were delocalized from kinetochores in the kis1 cells and that kinetochore-microtubule attachment was defective. Another mutant, mis6, also displayed similar spindle defects. We conclude that Kis1 is required for inner kinetochore organization, through which Kis1 ensures kinetochore-microtubule attachment and spindle integrity. Thus, we propose an unexpected relationship between inner kinetochore organization and spindle integrity. [ABSTRACT FROM AUTHOR]

Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture. [ABSTRACT FROM AUTHOR]

We present a computational model of transcription factor motion that explains both the observed rapid target finding of transcription factors, and how this motion influences protein and genome structure. Using the Smoldyn software, we modelled transcription factor motion arising from a combination of unrestricted 3D diffusion in the nucleoplasm, sliding along the DNA filament, and transferring directly between filament sections by intersegmental transfer. This presents a fine-grain picture of the way in which transcription factors find their targets two orders of magnitude faster than 3D diffusion alone allows. Eukaryotic genomes contain sections of nucleosome free regions (NFRs) around the promoters; our model shows that the presence and size of these NFRs can be explained as their acting as antennas on which transcription factors slide to reach their targets. Additionally, our model shows that intersegmental transfer may have shaped the quaternary structure of transcription factors: sequence specific DNA binding proteins are unusually enriched in dimers and tetramers, perhaps because these allow intersegmental transfer, which accelerates target site finding. Finally, our model shows that a ‘hopping’ motion can emerge from 3D diffusion on small scales. This explains the apparently long sliding lengths that have been observed for some DNA binding proteins observed in vitro. Together, these results suggest that transcription factor diffusion dynamics help drive the evolution of protein and genome structure. [ABSTRACT FROM AUTHOR]

The fast-paced development of nanotechnology needs the support of effective safety testing. We have developed a screening platform measuring simultaneously several cellular parameters for exposure to various concentrations of nanoparticles (NPs). Cell lines representative of different organ cell types, including lung, endothelium, liver, kidney, macrophages, glia, and neuronal cells were exposed to 50 nm amine-modified polystyrene (PS-NH2) NPs previously reported to induce apoptosis and to 50 nm sulphonated and carboxyl-modified polystyrene NPs that were reported to be silent. All cell lines apart from Raw 264.7 executed apoptosis in response to PS-NH2 NPs, showing specific sequences of EC50 thresholds; lysosomal acidification was the most sensitive parameter. Loss of mitochondrial membrane potential and plasma membrane integrity measured by High Content Analysis resulted comparably sensitive to the equivalent OECD-recommended assays, allowing increased output. Analysis of the acidic compartments revealed good cerrelation between size/fluorescence intensity and dose of PS-NH2 NPs applied; moreover steatosis and phospholipidosis were observed, consistent with the lysosomal alterations revealed by Lysotracker green; similar responses were observed when comparing astrocytoma cells with primary astrocytes. We have established a platform providing mechanistic insights on the response to exposure to nanoparticles. Such platform holds great potential for in vitro screening of nanomaterials in heighthroughput format. [ABSTRACT FROM AUTHOR]

Introduction: Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different. Results: Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell – along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex. Summary: We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance. [ABSTRACT FROM AUTHOR]

Fluorescence recovery after photobleaching (FRAP) is a widely used imaging technique for measuring protein dynamics in live cells that has provided many important biological insights. Although FRAP presumes that the conversion of a fluorophore from a bright to a dark state is irreversible, GFP as well as other genetically encoded fluorescent proteins now in common use can also exhibit a reversible conversion known as photoswitching. Various studies have shown how photoswitching can cause at least four different artifacts in FRAP, leading to false conclusions about various biological phenomena, including the erroneous identification of anomalous diffusion or the overestimation of the freely diffusible fraction of a cellular protein. Unfortunately, identifying and then correcting these artifacts is difficult. Here we report a new characteristic of an organic fluorophore tetramethylrhodamine bound to the HaloTag protein (TMR-HaloTag), which like GFP can be genetically encoded, but which directly and simply overcomes the artifacts caused by photoswitching in FRAP. We show that TMR exhibits virtually no photoswitching in live cells under typical imaging conditions for FRAP. We also demonstrate that TMR eliminates all of the four reported photoswitching artifacts in FRAP. Finally, we apply this photoswitching-free FRAP with TMR to show that the chromatin decondensation following UV irradiation does not involve loss of nucleosomes from the damaged DNA. In sum, we demonstrate that the TMR Halo label provides a genetically encoded fluorescent tag very well suited for accurate FRAP experiments. [ABSTRACT FROM AUTHOR]

The nuclear export of proteins is regulated largely through the exportin/CRM1 pathway, which involves the specific recognition of leucine-rich nuclear export signals (NESs) in the cargo proteins, and modulates nuclear–cytoplasmic protein shuttling by antagonizing the nuclear import activity mediated by importins and the nuclear import signal (NLS). Although the prediction of NESs can help to define proteins that undergo regulated nuclear export, current methods of predicting NESs, including computational tools and consensus-sequence-based searches, have limited accuracy, especially in terms of their specificity. We found that each residue within an NES largely contributes independently and additively to the entire nuclear export activity. We created activity-based profiles of all classes of NESs with a comprehensive mutational analysis in mammalian cells. The profiles highlight a number of specific activity-affecting residues not only at the conserved hydrophobic positions but also in the linker and flanking regions. We then developed a computational tool, NESmapper, to predict NESs by using profiles that had been further optimized by training and combining the amino acid properties of the NES-flanking regions. This tool successfully reduced the considerable number of false positives, and the overall prediction accuracy was higher than that of other methods, including NESsential and Wregex. This profile-based prediction strategy is a reliable way to identify functional protein motifs. NESmapper is available at . [ABSTRACT FROM AUTHOR]

Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation. [ABSTRACT FROM AUTHOR]