Nuclear bodies are membraneless organelles that play important roles in genome functioning. A specific type of nuclear bodies known as interphase prenucleolar bodies (iPNBs) are formed in the nucleoplasm after hypotonic stress from partially disassembled nucleoli. iPNBs are then disassembled, and the nucleoli are reformed simultaneously. Here, we show that diffusion of B23 molecules (also known as nucleophosmin, NPM1) from iPNBs, but not fusion of iPNBs with the nucleoli, contributes to the transfer of B23 from iPNBs to the nucleoli. Maturation of pre-ribosomal RNAs (rRNAs) and the subsequent outflow of mature rRNAs from iPNBs led to the disassembly of iPNBs. We found that B23 transfer was dependent on the synthesis of pre-rRNA molecules in nucleoli; these pre-rRNA molecules interacted with B23 and led to its accumulation within nucleoli. The transfer of B23 between iPNBs and nucleoli was accomplished through a nucleoplasmic pool of B23, and increased nucleoplasmic B23 content retarded disassembly, whereas B23 depletion accelerated disassembly. Our results suggest that iPNB disassembly and nucleolus assembly might be coupled through RNA-dependent exchange of nucleolar proteins, creating a highly dynamic system with long-distance correlations between spatially distinct processes.
- Klíčová slova
- B23, Interphase prenucleolar body, NPM1, Nuclear bodies, Nucleolus, Pre-rRNA, Self-organization,
- MeSH
- adenosintrifosfát metabolismus MeSH
- buněčné jadérko metabolismus MeSH
- difuze MeSH
- fyziologický stres MeSH
- HeLa buňky MeSH
- interfáze MeSH
- intranukleární inkluzní tělíska metabolismus MeSH
- lidé MeSH
- nukleofosmin MeSH
- posttranskripční úpravy RNA MeSH
- RNA metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- adenosintrifosfát MeSH
- NPM1 protein, human MeSH Prohlížeč
- nukleofosmin MeSH
- RNA MeSH
We studied epigenetics, distribution pattern, kinetics, and diffusion of proteins recruited to spontaneous and γ-radiation-induced DNA lesions. We showed that PML deficiency leads to an increased number of DNA lesions, which was accompanied by changes in histone signature. In PML wt cells, we observed two mobile fractions of 53BP1 protein with distinct diffusion in spontaneous lesions. These protein fractions were not detected in PML-deficient cells, characterized by slow-diffusion of 53BP1. Single particle tracking analysis revealed limited local motion of 53BP1 foci in PML double null cells and local motion 53BP1 foci was even more reduced after γ-irradiation. However, radiation did not change co-localization between 53BP1 nuclear bodies and interchromatin granule-associated zones (IGAZs), nuclear speckles, or chromocenters. This newly observed interaction pattern imply that 53BP1 protein could be a part of not only DNA repair, but also process mediated via components accumulated in IGAZs, nuclear speckles, or paraspeckles. Together, PML deficiency affected local motion of 53BP1 nuclear bodies and changed composition and a number of irradiation-induced foci. J. Cell. Biochem. 117: 2583-2596, 2016. © 2016 Wiley Periodicals, Inc.
- Klíčová slova
- 53BP1 PROTEIN, DNA REPAIR, HISTONE MODIFICATIONS, MICROSCOPY, PHOTOBLEACHING, PML BODIES,
- MeSH
- 53BP1 metabolismus MeSH
- akutní promyelocytární leukemie metabolismus patologie radioterapie MeSH
- fluorescenční protilátková technika MeSH
- intranukleární inkluzní tělíska metabolismus patologie účinky záření MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- nádorové buňky kultivované MeSH
- oprava DNA fyziologie účinky záření MeSH
- poškození DNA fyziologie účinky záření MeSH
- vztah dávky záření a odpovědi MeSH
- western blotting MeSH
- záření gama škodlivé účinky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 53BP1 MeSH
- TP53BP1 protein, human MeSH Prohlížeč
Studies on fixed samples or genome-wide analyses of nuclear processes are useful for generating snapshots of a cell population at a particular time point. However, these experimental approaches do not provide information at the single-cell level. Genome-wide studies cannot assess variability between individual cells that are cultured in vitro or originate from different pathological stages. Immunohistochemistry and immunofluorescence are fundamental experimental approaches in clinical laboratories and are also widely used in basic research. However, the fixation procedure may generate artifacts and prevents monitoring of the dynamics of nuclear processes. Therefore, live-cell imaging is critical for studying the kinetics of basic nuclear events, such as DNA replication, transcription, splicing, and DNA repair. This review is focused on the advanced microscopy analyses of the cells, with a particular focus on live cells. We note some methodological innovations and new options for microscope systems that can also be used to study tissue sections. Cornerstone methods for the biophysical research of living cells, such as fluorescence recovery after photobleaching and fluorescence resonance energy transfer, are also discussed, as are studies on the effects of radiation at the individual cellular level.
- Klíčová slova
- Live-cell studies, chromatin, fluorescent proteins, microscopy, tissue sections,
- MeSH
- mikroskopie metody trendy MeSH
- patologie metody MeSH
- počítačové zpracování obrazu metody trendy MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Tracking motile cells in time-lapse series is challenging and is required in many biomedical applications. Cell tracks can be mathematically represented as acyclic oriented graphs. Their vertices describe the spatio-temporal locations of individual cells, whereas the edges represent temporal relationships between them. Such a representation maintains the knowledge of all important cellular events within a captured field of view, such as migration, division, death, and transit through the field of view. The increasing number of cell tracking algorithms calls for comparison of their performance. However, the lack of a standardized cell tracking accuracy measure makes the comparison impracticable. This paper defines and evaluates an accuracy measure for objective and systematic benchmarking of cell tracking algorithms. The measure assumes the existence of a ground-truth reference, and assesses how difficult it is to transform a computed graph into the reference one. The difficulty is measured as a weighted sum of the lowest number of graph operations, such as split, delete, and add a vertex and delete, add, and alter the semantics of an edge, needed to make the graphs identical. The measure behavior is extensively analyzed based on the tracking results provided by the participants of the first Cell Tracking Challenge hosted by the 2013 IEEE International Symposium on Biomedical Imaging. We demonstrate the robustness and stability of the measure against small changes in the choice of weights for diverse cell tracking algorithms and fluorescence microscopy datasets. As the measure penalizes all possible errors in the tracking results and is easy to compute, it may especially help developers and analysts to tune their algorithms according to their needs.
The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011 ). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase.
- Klíčová slova
- DNA damage, UBF1, live cells, nucleolus, nuncleoli tracking,
- MeSH
- apoptóza účinky záření MeSH
- buněčné jadérko účinky záření MeSH
- buněčné linie MeSH
- buněčný cyklus účinky záření MeSH
- G2 fáze účinky záření MeSH
- genetická transkripce MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- poškození DNA účinky záření MeSH
- transkripční iniciační komplex Pol1 - proteiny genetika metabolismus MeSH
- ultrafialové záření MeSH
- výpočetní biologie MeSH
- záření gama škodlivé účinky MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- transcription factor UBF MeSH Prohlížeč
- transkripční iniciační komplex Pol1 - proteiny MeSH
BACKGROUND: The repair of spontaneous and induced DNA lesions is a multistep process. Depending on the type of injury, damaged DNA is recognized by many proteins specifically involved in distinct DNA repair pathways. RESULTS: We analyzed the DNA-damage response after ultraviolet A (UVA) and γ irradiation of mouse embryonic fibroblasts and focused on upstream binding factor 1 (UBF1), a key protein in the regulation of ribosomal gene transcription. We found that UBF1, but not nucleolar proteins RPA194, TCOF, or fibrillarin, was recruited to UVA-irradiated chromatin concurrently with an increase in heterochromatin protein 1β (HP1β) level. Moreover, Förster Resonance Energy Transfer (FRET) confirmed interaction between UBF1 and HP1β that was dependent on a functional chromo shadow domain of HP1β. Thus, overexpression of HP1β with a deleted chromo shadow domain had a dominant-negative effect on UBF1 recruitment to UVA-damaged chromatin. Transcription factor UBF1 also interacted directly with DNA inside the nucleolus but no interaction of UBF1 and DNA was confirmed outside the nucleolus, where UBF1 recruitment to DNA lesions appeared simultaneously with cyclobutane pyrimidine dimers; this occurrence was cell-cycle-independent. CONCLUSIONS: We propose that the simultaneous presence and interaction of UBF1 and HP1β at DNA lesions is activated by the presence of cyclobutane pyrimidine dimers and mediated by the chromo shadow domain of HP1β. This might have functional significance for nucleotide excision repair.
- Klíčová slova
- DNA repair, DNA-damage response, Irradiation, Live-cell studies, Nucleolus, UBF1,
- Publikační typ
- časopisecké články MeSH
Cajal bodies are important nuclear structures containing proteins that preferentially regulate RNA-related metabolism. We investigated the cell-type specific nuclear distribution of Cajal bodies and the level of coilin, a protein of Cajal bodies, in non-irradiated and irradiated human tumor cell lines and embryonic stem (ES) cells. Cajal bodies were localized in different nuclear compartments, including DAPI-poor regions, in the proximity of chromocenters, and adjacent to nucleoli. The number of Cajal bodies per nucleus was cell cycle-dependent, with higher numbers occurring during G2 phase. Human ES cells contained a high coilin level in the nucleoplasm, but coilin-positive Cajal bodies were also identified in nuclei of mouse and human ES cells. Coilin, but not SMN, recognized UVA-induced DNA lesions, which was cell cycle-independent. Treatment with γ-radiation reduced the localized movement of Cajal bodies in many cell types and GFP-coilin fluorescence recovery after photobleaching was very fast in nucleoplasm in comparison with GFP-coilin recovery in DNA lesions. By contrast, nucleolus-localized coilin displayed very slow fluorescence recovery after photobleaching, which indicates very slow rates of protein diffusion, especially in nucleoli of mouse ES cells.
- Klíčová slova
- Cajal bodies, DNA repair, chromatin, coilin, nucleolus, nucleus,
- MeSH
- buněčné jádro genetika metabolismus účinky záření MeSH
- buněčné linie MeSH
- buňky K562 MeSH
- Cajalova tělíska genetika metabolismus účinky záření MeSH
- DNA genetika účinky záření MeSH
- G2 fáze genetika MeSH
- HeLa buňky MeSH
- jaderné proteiny genetika metabolismus MeSH
- lidé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- ultrafialové záření škodlivé účinky MeSH
- záření gama škodlivé účinky MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
- jaderné proteiny MeSH
- p80-coilin MeSH Prohlížeč
- rekombinantní fúzní proteiny MeSH
- zelené fluorescenční proteiny MeSH
