Phase separation forms membraneless compartments, including heterochromatin "domains" and repair foci. Pericentromeric heterochromatin mostly comprises repeated sequences prone to aberrant recombination. In Drosophila cells, "safe" homologous recombination (HR) repair of these sequences requires their relocalization to the nuclear periphery before Rad51 recruitment and strand invasion. How this mobilization initiates is unknown, and the contribution of phase separation is unclear. Here, we show that Nup98 nucleoporin is recruited to repair sites before relocalization by Sec13 or Nup88, and downstream of the Smc5/6 complex and heterochromatin protein 1 (HP1). Remarkably, Nup98 condensates are immiscible with HP1 condensates, and they are required and sufficient to mobilize repair sites and exclude Rad51, thus preventing aberrant recombination while promoting HR repair. Disrupting this pathway results in heterochromatin repair defects and widespread chromosome rearrangements, revealing an "off-pore" role for nucleoporins and phase separation in nuclear dynamics and genome integrity in a multicellular eukaryote.
- MeSH
- Chromosomal Proteins, Non-Histone metabolism genetics MeSH
- Drosophila melanogaster * genetics metabolism MeSH
- DNA Breaks, Double-Stranded MeSH
- Heterochromatin * genetics metabolism MeSH
- Chromobox Protein Homolog 5 MeSH
- Nuclear Pore Complex Proteins * metabolism genetics MeSH
- Cell Cycle Proteins metabolism genetics MeSH
- Drosophila Proteins * metabolism genetics MeSH
- Recombinational DNA Repair * MeSH
- Rad51 Recombinase * metabolism genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Alzheimer's disease (AD) is a progressive brain disorder characterized by extracellular amyloid-β (Aβ) plaques, intracellular neurofibrillary tangles formed by hyperphosphorylated Tau protein and neuroinflammation. Previous research has shown that obesity and type 2 diabetes mellitus, underlined by insulin resistance (IR), are risk factors for neurodegenerative disorders. In this study, obesity-induced peripheral and central IR and inflammation were studied in relation to AD-like pathology in the brains and periphery of APP/PS1 mice, a model of Aβ pathology, fed a high-fat diet (HFD). APP/PS1 mice and their wild-type controls fed either a standard diet or HFD were characterized at the ages of 3, 6 and 10 months by metabolic parameters related to obesity via mass spectroscopy, nuclear magnetic resonance, immunoblotting and immunohistochemistry to quantify how obesity affected AD pathology. The HFD induced substantial peripheral IR leading to central IR. APP/PS1-fed HFD mice had more pronounced IR, glucose intolerance and liver steatosis than their WT controls. The HFD worsened Aβ pathology in the hippocampi of APP/PS1 mice and significantly supported both peripheral and central inflammation. This study reveals a deleterious effect of obesity-related mild peripheral inflammation and prediabetes on the development of Aβ and Tau pathology and neuroinflammation in APP/PS1 mice.
- MeSH
- Alzheimer Disease * etiology MeSH
- Amyloid beta-Peptides MeSH
- Diabetes Mellitus, Type 2 * MeSH
- Diet, High-Fat adverse effects MeSH
- Insulin Resistance * MeSH
- Mice MeSH
- Neuroinflammatory Diseases MeSH
- Inflammation MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an 'experiment of nature' to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.
- MeSH
- B-Lymphocytes * MeSH
- Cell Differentiation MeSH
- DNA-Binding Proteins * deficiency genetics MeSH
- Homeodomain Proteins * genetics MeSH
- Immune Tolerance MeSH
- Nuclear Proteins * deficiency MeSH
- Humans MeSH
- Lymphocyte Count MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, N.I.H., Intramural MeSH
The nuclear lamina is the main component of the nuclear cytoskeleton that maintains the integrity of the nucleus. However, it represents a natural barrier for viruses replicating in the cell nucleus. The lamina blocks viruses from being trafficked to the nucleus for replication, but it also impedes the nuclear egress of the progeny of viral particles. Thus, viruses have evolved mechanisms to overcome this obstacle. Large viruses induce the assembly of multiprotein complexes that are anchored to the inner nuclear membrane. Important components of these complexes are the viral and cellular kinases phosphorylating the lamina and promoting its disaggregation, therefore allowing virus egress. Small viruses also use cellular kinases to induce lamina phosphorylation and the subsequent disruption in order to facilitate the import of viral particles during the early stages of infection or during their nuclear egress. Another component of the nuclear cytoskeleton, nuclear actin, is exploited by viruses for the intranuclear movement of their particles from the replication sites to the nuclear periphery. This study focuses on exploitation of the nuclear cytoskeleton by viruses, although this is just the beginning for many viruses, and promises to reveal the mechanisms and dynamic of physiological and pathological processes in the nucleus.
- MeSH
- Actins metabolism MeSH
- Cell Nucleus metabolism MeSH
- Cytoskeleton genetics metabolism MeSH
- Species Specificity MeSH
- Host-Pathogen Interactions * MeSH
- Nuclear Lamina metabolism MeSH
- Nuclear Envelope metabolism MeSH
- Lamins metabolism MeSH
- Humans MeSH
- Disease Susceptibility * MeSH
- Gene Expression Regulation, Viral MeSH
- Virus Replication MeSH
- Virus Diseases etiology metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
METTL16 methyltransferase is responsible for the methylation of N6-adenosine (m6A) in several RNAs. In mouse cells, we showed that the nuclear distribution of METTL16 is cell cycle-specific. In the G1/S phases, METTL16 accumulates to the nucleolus, while in the G2 phase, the level of METTL16 increases in the nucleoplasm. In metaphase and anaphase, there is a very low pool of the METTL16 protein, but in telophase, residual METTL16 appears to be associated with the newly formed nuclear lamina. In A-type lamin-depleted cells, we observed a reduction of METTL16 when compared with the wild-type counterpart. However, METTL16 does not interact with A-type and B-type lamins, but interacts with Lamin B Receptor (LBR) and Lap2α. Additionally, Lap2α depletion caused METTL16 downregulation in the nuclear pool. Furthermore, METTL16 interacted with DDB2, a key protein of the nucleotide excision repair (NER), and also with nucleolar proteins, including TCOF, NOLC1, and UBF1/2, but not fibrillarin. From this view, the METTL16 protein may also regulate the transcription of ribosomal genes because we observed that the high level of m6A in 18S rRNA appeared in cells with upregulated METTL16.
- Publication type
- Journal Article MeSH
BACKGROUND: Tumors can influence peripheral immune macroenvironment, thereby creating opportunities for non-invasive serum/plasma immunobiomarkers for immunostratification and immunotherapy designing. However, current approaches for immunobiomarkers' detection are largely quantitative, which is unreliable for assessing functional peripheral immunodynamics of patients with cancer. Hence, we aimed to design a functional biomarker modality for capturing peripheral immune signaling in patients with cancer for reliable immunostratification. METHODS: We used a data-driven in silico framework, integrating existing tumor/blood bulk-RNAseq or single-cell (sc)RNAseq datasets of patients with cancer, to inform the design of an innovative serum-screening modality, that is, serum-functional immunodynamic status (sFIS) assay. Next, we pursued proof-of-concept analyses via multiparametric serum profiling of patients with ovarian cancer (OV) with sFIS assay combined with Luminex (cytokines/soluble immune checkpoints), CA125-antigen detection, and whole-blood immune cell counts. Here, sFIS assay's ability to determine survival benefit or malignancy risk was validated in a discovery (n=32) and/or validation (n=699) patient cohorts. Lastly, we used an orthotopic murine metastatic OV model, with anti-OV therapy selection via in silico drug-target screening and murine serum screening via sFIS assay, to assess suitable in vivo immunotherapy options. RESULTS: In silico data-driven framework predicted that peripheral immunodynamics of patients with cancer might be best captured via analyzing myeloid nuclear factor kappa-light-chain enhancer of activated B cells (NFκB) signaling and interferon-stimulated genes' (ISG) responses. This helped in conceptualization of an 'in sitro' (in vitro+in situ) sFIS assay, where human myeloid cells were exposed to patients' serum in vitro, to assess serum-induced (si)-NFκB or interferon (IFN)/ISG responses (as active signaling reporter activity) within them, thereby 'mimicking' patients' in situ immunodynamic status. Multiparametric serum profiling of patients with OV established that sFIS assay can: decode peripheral immunology (by indicating higher enrichment of si-NFκB over si-IFN/ISG responses), estimate survival trends (si-NFκB or si-IFN/ISG responses associating with negative or positive prognosis, respectively), and coestimate malignancy risk (relative to benign/borderline ovarian lesions). Biologically, we documented dominance of pro-tumorigenic, myeloid si-NFκB responseHIGHsi-IFN/ISG responseLOW inflammation in periphery of patients with OV. Finally, in an orthotopic murine metastatic OV model, sFIS assay predicted the higher capacity of chemo-immunotherapy (paclitaxel-carboplatin plus anti-TNF antibody combination) in achieving a pro-immunogenic peripheral milieu (si-IFN/ISG responseHIGHsi-NFκB responseLOW), which aligned with high antitumor efficacy. CONCLUSIONS: We established sFIS assay as a novel biomarker resource for serum screening in patients with OV to evaluate peripheral immunodynamics, patient survival trends and malignancy risk, and to design preclinical chemo-immunotherapy strategies.
- MeSH
- Survival Analysis MeSH
- Immunotherapy methods MeSH
- Humans MeSH
- Mice MeSH
- Ovarian Neoplasms drug therapy genetics mortality MeSH
- NF-kappa B metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: African annual killifishes (Nothobranchius spp.) are adapted to seasonally desiccating habitats (ephemeral pools), surviving dry periods as dormant eggs. Given their peculiar life history, geographic aspects of their diversity uniquely combine patterns typical for freshwater taxa (river basin structure and elevation gradient) and terrestrial animals (rivers acting as major dispersal barriers). However, our current knowledge on fine-scale inter-specific and intra-specific genetic diversity of African annual fish is limited to a single, particularly dry region of their distribution (subtropical Mozambique). Using a widespread annual killifish from coastal Tanzania and Kenya, we tested whether the same pattern of genetic divergence pertains to a wet equatorial region in the centre of Nothobranchius distribution. RESULTS: In populations of Nothobranchius melanospilus species group across its range, we genotyped a part of mitochondrial cytochrome oxidase subunit 1 (COI) gene (83 individuals from 22 populations) and 10 nuclear microsatellite markers (251 individuals from 16 populations). We found five lineages with a clear phylogeographic structure but frequent secondary contact. Mitochondrial lineages were largely congruent with main population genetic clusters identified on microsatellite markers. In the upper Wami basin, populations are isolated as a putative Nothobranchius prognathus, but include also a population from a periphery of the middle Ruvu basin. Other four lineages (including putative Nothobranchius kwalensis) coexisted in secondary contact zones, but possessed clear spatial pattern. Main river channels did not form apparent barriers to dispersal. The most widespread lineage had strong signal of recent population expansion. CONCLUSIONS: We conclude that dispersal of a Nothobranchius species from a wet part of the genus distribution (tropical lowland) is not constrained by main river channels and closely related lineages frequently coexist in secondary contact zones. We also demonstrate contemporary connection between the Ruvu and Rufiji river basins. Our data do not provide genetic support for existence of recently described cryptic species from N. melanospilus complex, but cannot resolve this issue.
- MeSH
- Killifishes genetics MeSH
- Ecosystem * MeSH
- Phylogeny MeSH
- Phylogeography MeSH
- Genetic Variation * MeSH
- Genetic Drift MeSH
- Microsatellite Repeats MeSH
- DNA, Mitochondrial genetics MeSH
- Genetics, Population MeSH
- Rivers MeSH
- Electron Transport Complex IV genetics MeSH
- Fresh Water MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Tanzania MeSH
During meiosis, homologous chromosomes undergo a dramatic movement in order to correctly align. This is a critical meiotic event but the molecular properties of this 'chromosomal dance' still remainunclear. We identified DEB-1 - an orthologue of mammalian vinculin - as a new component of the mechanistic modules responsible for attaching the chromosomes to the nuclear envelope as apart of the LINC complex. In early meiotic nuclei of C. elegans, DEB-1 is localized to the nuclear periphery and alongside the synaptonemal complex of paired homologues. Upon DEB-1 depletion, chromosomes attached to SUN-1 foci remain highly motile until late pachytene. Although the initiation of homologue pairing started normally, irregularities in the formation of the synaptonemal complex occur, and these results in meiotic defects such as increased number of univalents at diakinesis and high embryonic lethality. Our data identify DEB-1 as a new player regulating chromosome dynamics and pairing during meiotic prophase I.
- MeSH
- Caenorhabditis elegans genetics MeSH
- Chromosomes genetics MeSH
- Meiosis genetics MeSH
- Chromosome Pairing genetics MeSH
- Vinculin genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Biomolecule (lipid and protein) oxidation products formed in plant cells exposed to photooxidative stress play a crucial role in the retrograde signaling and oxidative damage. The oxidation of biomolecules initiated by reactive oxygen species is associated with formation of organic (alkyl, peroxyl and alkoxyl) radicals. Currently, there is no selective and sensitive technique available for the detection of organic radicals in plant cells. Here, based on the analogy with animal cells, immuno-spin trapping using spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to image organic radicals in Arabidopsis leaves exposed to high light. Using antibody raised against the DMPO nitrone adduct conjugated with the fluorescein isothiocyanate, organic radicals were imaged by confocal laser scanning microscopy. Organic radicals are formed predominantly in the chloroplasts located at the periphery of the cells and distributed uniformly throughout the grana stack. Characterization of protein radicals by standard immunological techniques using anti-DMPO antibody shows protein bands with apparent molecular weights of 32 and 34 kDa assigned to D1 and D2 proteins and two protein bands below the D1/D2 band with apparent molecular weights of 23 and 18 kDa and four protein bands above the D1/D2 band with apparent molecular weights of 41, 43, 55 and 68 kDa. In summary, imaging of organic radicals by immuno-spin trapping represents selective and sensitive technique for the detection of organic radicals that might help to clarify mechanistic aspects on the role of organic radicals in the retrograde signaling and oxidative damage in plant cell.
- MeSH
- Cyclic N-Oxides chemistry MeSH
- Electron Spin Resonance Spectroscopy MeSH
- Lipids chemistry isolation & purification MeSH
- Oxidation-Reduction MeSH
- Oxidative Stress drug effects MeSH
- Hydrogen Peroxide chemistry MeSH
- Peroxides chemistry MeSH
- Proteins chemistry MeSH
- Reactive Oxygen Species chemistry MeSH
- Spin Trapping * MeSH
- Spin Labels MeSH
- Free Radicals chemistry isolation & purification MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
During interphase, the chromosomes of eukaryotes decondense and they occupy distinct regions of the nucleus, called chromosome domains or chromosome territories (CTs). In plants, the Rabl's configuration, with telomeres at one pole of nucleus and centromeres at the other, appears to be common, at least in plants with large genomes. It is unclear whether individual chromosomes of plants adopt defined, genetically determined addresses within the nucleus, as is the case in mammals. In this study, the nuclear disposition of alien rye and barley chromosomes and chromosome arm introgressions into wheat while using 3D-FISH in various somatic tissues was analyzed. All of the introgressed chromosomes showed Rabl's orientation, but their relative positions in the nuclei were less clear. While in most cases pairs of introgressed chromosomes occupied discrete positions, their association (proximity) along their entire lengths was rare, and partial association only marginally more frequent. This arrangement is relatively stable in various tissues and during various stages of the cell cycle. On the other hand, the length of a chromosome arm appears to play a role in its positioning in a nucleus: shorter chromosomes or chromosome arms tend to be located closer to the centre of the nucleus, while longer arms are more often positioned at the nuclear periphery.
- MeSH
- Cell Nucleus MeSH
- Chromatin genetics MeSH
- Chromosomes, Plant * MeSH
- In Situ Hybridization, Fluorescence * methods MeSH
- Interphase * genetics MeSH
- Hordeum genetics MeSH
- Image Processing, Computer-Assisted MeSH
- Flow Cytometry MeSH
- Triticum genetics MeSH
- Secale genetics MeSH
- Publication type
- Journal Article MeSH