Chromatin organization and its interactions are essential for biological processes, such as DNA repair, transcription, and DNA replication. Detailed cytogenetics data on chromatin conformation, and the arrangement and mutual positioning of chromosome territories in interphase nuclei are still widely missing in plants. In this study, level of chromatin condensation in interphase nuclei of rice (Oryza sativa) and the distribution of chromosome territories (CTs) were analyzed. Super-resolution, stimulated emission depletion (STED) microscopy showed different levels of chromatin condensation in leaf and root interphase nuclei. 3D immuno-FISH experiments with painting probes specific to chromosomes 9 and 2 were conducted to investigate their spatial distribution in root and leaf nuclei. Six different configurations of chromosome territories, including their complete association, weak association, and complete separation, were observed in root meristematic nuclei, and four configurations were observed in leaf nuclei. The volume of CTs and frequency of their association varied between the tissue types. The frequency of association of CTs specific to chromosome 9, containing NOR region, is also affected by the activity of the 45S rDNA locus. Our data suggested that the arrangement of chromosomes in the nucleus is connected with the position and the size of the nucleolus.

• Klíčová slova
• 3D immuno-FISH, chromosome painting, chromosome territory, microscopy, rice, spatial organization,
• Publikační typ
• časopisecké články MeSH

Telomere repeat binding proteins (TRBs) belong to a family of proteins possessing a Myb-like domain which binds to telomeric repeats. Three members of this family (TRB1, TRB2, TRB3) from Arabidopsis thaliana have already been described as associated with terminal telomeric repeats (telomeres) or short interstitial telomeric repeats in gene promoters (telo-boxes). They are also known to interact with several protein complexes: telomerase, Polycomb repressive complex 2 (PRC2) E(z) subunits and the PEAT complex (PWOs-EPCRs-ARIDs-TRBs). Here we characterize two novel members of the TRB family (TRB4 and TRB5). Our wide phylogenetic analyses have shown that TRB proteins evolved in the plant kingdom after the transition to a terrestrial habitat in Streptophyta, and consequently TRBs diversified in seed plants. TRB4-5 share common TRB motifs while differing in several others and seem to have an earlier phylogenetic origin than TRB1-3. Their common Myb-like domains bind long arrays of telomeric repeats in vitro, and we have determined the minimal recognition motif of all TRBs as one telo-box. Our data indicate that despite the distinct localization patterns of TRB1-3 and TRB4-5 in situ, all members of TRB family mutually interact and also bind to telomerase/PRC2/PEAT complexes. Additionally, we have detected novel interactions between TRB4-5 and EMF2 and VRN2, which are Su(z)12 subunits of PRC2.

• Klíčová slova
• PEAT, PRC2, TERT, TRB, Telomere repeat binding, Telomeric,
• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• fylogeneze MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• proteiny vázající telomery genetika   metabolismus   MeSH
• půda MeSH
• telomerasa * genetika   metabolismus   MeSH
• telomery genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny huseníčku * MeSH
• proteiny vázající telomery MeSH
• půda MeSH
• telomerasa * MeSH

Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images.Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.

• Klíčová slova
• 3D organization, Nucleus, RNA Pol II, SIM, STED imaging, chromatin, chromosome, crossovers, image analysis, meiosis, metaphase, mitosis, nuclear bodies, nuclear speckles, oligo FISH, pachytene, quantification, segmentation, spatial distribution, transcription factories,
• MeSH
• buněčné jádro * MeSH
• chromatin * MeSH
• fluorescenční mikroskopie MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• průběh práce MeSH
• zelené fluorescenční proteiny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin * MeSH
• zelené fluorescenční proteiny MeSH

The evolution of eukaryotic genomes is accompanied by fluctuations in chromosome number, reflecting cycles of chromosome number increase (polyploidy and centric fissions) and decrease (chromosome fusions). Although all chromosome fusions result from DNA recombination between two or more nonhomologous chromosomes, several mechanisms of descending dysploidy are exploited by eukaryotes to reduce their chromosome number. Genome sequencing and comparative genomics have accelerated the identification of inter-genome chromosome collinearity and gross chromosomal rearrangements and have shown that end-to-end chromosome fusions (EEFs) and nested chromosome fusions (NCFs) may have played a more important role in the evolution of eukaryotic karyotypes than previously thought. The present review aims to summarize the limited knowledge on the origin, frequency, and evolutionary implications of EEF and NCF events in eukaryotes and especially in land plants. The interactions between nonhomologous chromosomes in interphase nuclei and chromosome (mis)pairing during meiosis are examined for their potential importance in the origin of EEFs and NCFs. The remaining open questions that need to be addressed are discussed.

• MeSH
• genomika MeSH
• karyotyp MeSH
• meióza MeSH
• molekulární evoluce * MeSH
• polyploidie * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH