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.

Institute of Biophysics ; Academy of Sciences of the Czech Republic ; Brno Czech Republic

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Eppink B, Essers J, Kannar R. Interplay and quality of DNA damage repair mechanism In: Rippe K, ed. Genome organization and function in the cell nucleus. Weinheim, Germany: Wiley-VCH, 2012:395-415

Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature 2009; 461:1071-8; PMID:19847258; http://dx.doi.org/10.1038/nature08467 PubMed DOI PMC

Nagy Z, Soutoglou E. DNA repair: easy to visualize, difficult to elucidate. Trends Cell Biol 2009; 19:617-29; PMID:19819145; http://dx.doi.org/10.1016/j.tcb.2009.08.010 PubMed DOI

Wang H, Zeng ZC, Perrault AR, Cheng X, Qin W, Iliakis G. Genetic evidence for the involvement of DNA ligase IV in the DNA-PK-dependent pathway of non-homologous end joining in mammalian cells. Nucleic Acids Res 2001; 29:1653-60; PMID:11292837; http://dx.doi.org/10.1093/nar/29.8.1653 PubMed DOI PMC

Chen L, Trujillo K, Sung P, Tomkinson AE. Interactions of the DNA ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase. J Biol Chem 2000; 275:26196-205; PMID:10854421; http://dx.doi.org/10.1074/jbc.M000491200 PubMed DOI

Bubulya PA, Spector DL. “On the move”ments of nuclear components in living cells. Exp Cell Res 2004; 296:4-11; PMID:15120987; http://dx.doi.org/10.1016/j.yexcr.2004.03.018 PubMed DOI

Sustackova G, Kozubek S, Stixova L, Legartova S, Matula P, Orlova D, Bártová E. Acetylation-dependent nuclear arrangement and recruitment of BMI1 protein to UV-damaged chromatin. J Cell Physiol 2012; 227:1838-50; PMID:21732356; http://dx.doi.org/10.1002/jcp.22912 PubMed DOI

Shav-Tal Y, Blechman J, Darzacq X, Montagna C, Dye BT, Patton JG, Singer RH, Zipori D. Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition. Mol Biol Cell 2005; 16:2395-413; PMID:15758027; http://dx.doi.org/10.1091/mbc.E04-11-0992 PubMed DOI PMC

Moore HM, Bai B, Boisvert FM, Latonen L, Rantanen V, Simpson JC, Pepperkok R, Lamond AI, Laiho M. Quantitative proteomics and dynamic imaging of the nucleolus reveal distinct responses to UV and ionizing radiation. Mol Cell Proteomics 2011; 10:M111 009241; PMID:21778410; http://dx.doi.org/10.1074/mcp.M111.009241 PubMed DOI PMC

Stixova L, Hruskova T, Sehnalova P, Legartova S, Svidenska S, Kozubek S, Bártová E. Advanced microscopy techniques used for comparison of UVA- and gamma-irradiation-induced DNA damage in the cell nucleus and nucleolus. Folia Biol (Praha) 2014; 60 Suppl 1:76-84; PMID:25369346 PubMed

Horakova AH, Bartova E, Galiova G, Uhlirova R, Matula P, Kozubek S. SUV39h-independent association of HP1 β with fibrillarin-positive nucleolar regions. Chromosoma 2010; 119:227-41; PMID:20033197; http://dx.doi.org/10.1007/s00412-009-0252-2 PubMed DOI

Stixova L, Sehnalova P, Legartova S, Suchankova J, Hruskova T, Kozubek S, Sorokin DV, Matula P, Raška I, Kovařík A, et al.. HP1beta-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs. Epigenetics Chromatin 2014; 7:39; PMID:25587355; http://dx.doi.org/10.1186/1756-8935-7-39 PubMed DOI PMC

Yuan X, Feng W, Imhof A, Grummt I, Zhou Y. Activation of RNA polymerase I transcription by cockayne syndrome group B protein and histone methyltransferase G9a. Mol Cell 2007; 27:585-95; PMID:17707230; http://dx.doi.org/10.1016/j.molcel.2007.06.021 PubMed DOI

Foltankova V, Matula P, Sorokin D, Kozubek S, Bartova E. Hybrid detectors improved time-lapse confocal microscopy of PML and 53BP1 nuclear body colocalization in DNA lesions. Microsc Microanal 2013; 19:360-9; PMID:23410959; http://dx.doi.org/10.1017/S1431927612014353 PubMed DOI

Dundr M. Nuclear bodies: multifunctional companions of the genome. Curr Opin Cell Biol 2012; 24:415-22; PMID:22541757; http://dx.doi.org/10.1016/j.ceb.2012.03.010 PubMed DOI PMC

Chenouard N, Smal I, de Chaumont F, Maska M, Sbalzarini IF, Gong Y, Cardinale J, Carthel C, Coraluppi S, Winter M, et al.. Objective comparison of particle tracking methods. Nat Methods 2014; 11:281-9; PMID:24441936; http://dx.doi.org/10.1038/nmeth.2808 PubMed DOI PMC

Stixova L, Bartova E, Matula P, Danek O, Legartova S, Kozubek S. Heterogeneity in the kinetics of nuclear proteins and trajectories of substructures associated with heterochromatin. Epigenetics Chromatin 2011; 4:5; PMID:21418567; http://dx.doi.org/10.1186/1756-8935-4-5 PubMed DOI PMC

Stixova L, Matula P, Kozubek S, Gombitova A, Cmarko D, Raska I, Bártová E. Trajectories and nuclear arrangement of PML bodies are influenced by A-type lamin deficiency. Biol Cell 2012; 104:418-32; PMID:22443097; http://dx.doi.org/10.1111/boc.201100053 PubMed DOI

Branzei D, Foiani M. Regulation of DNA repair throughout the cell cycle. Nat Rev Mol Cell Biol 2008; 9:297-308; PMID:18285803; http://dx.doi.org/10.1038/nrm2351 PubMed DOI

Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 2004; 73:39-85; PMID:15189136; http://dx.doi.org/10.1146/annurev.biochem.73.011303.073723 PubMed DOI

Antoniali G, Lirussi L, Poletto M, Tell G. Emerging roles of the nucleolus in regulating the DNA damage response: the noncanonical DNA repair enzyme APE1/Ref-1 as a paradigmatical example. Antioxid Redox Signal 2014; 20:621-39; PMID:23879289; http://dx.doi.org/10.1089/ars.2013.5491 PubMed DOI PMC

Kleeff J, Kornmann M, Sawhney H, Korc M. Actinomycin D induces apoptosis and inhibits growth of pancreatic cancer cells. Int J Cancer 2000; 86:399-407; PMID:10760829; http://dx.doi.org/10.1002/(SICI)1097-0215(20000501)86:3%3c399::AID-IJC15%3e3.0.CO;2-G PubMed DOI

Verheij M, Bartelink H. Radiation-induced apoptosis. Cell Tissue Res 2000; 301:133-42; PMID:10928286; http://dx.doi.org/10.1007/s004410000188 PubMed DOI

Mischo HE, Hemmerich P, Grosse F, Zhang S. Actinomycin D induces histone gamma-H2AX foci and complex formation of gamma-H2AX with Ku70 and nuclear DNA helicase II. J Biol Chem 2005; 280:9586-94; PMID:15613478; http://dx.doi.org/10.1074/jbc.M411444200 PubMed DOI

Bartova E, Kozubek S, Kozubek M, Jirsova P, Lukasova E, Skalnikova M, Buchnícková K. The influence of the cell cycle, differentiation and irradiation on the nuclear location of the abl, bcr and c-myc genes in human leukemic cells. Leuk Res 2000; 24:233-41; PMID:10739005; http://dx.doi.org/10.1016/S0145-2126(99)00174-5 PubMed DOI

Boulon S, Westman BJ, Hutten S, Boisvert FM, Lamond AI. The nucleolus under stress. Mol Cell 2010; 40:216-27; PMID:20965417; http://dx.doi.org/10.1016/j.molcel.2010.09.024 PubMed DOI PMC

Muratani M, Gerlich D, Janicki SM, Gebhard M, Eils R, Spector DL. Metabolic-energy-dependent movement of PML bodies within the mammalian cell nucleus. Nat Cell Biol 2002; 4:106-10; PMID:11753375; http://dx.doi.org/10.1038/ncb740 PubMed DOI

Scott M, Boisvert FM, Vieyra D, Johnston RN, Bazett-Jones DP, Riabowol K. UV induces nucleolar translocation of ING1 through two distinct nucleolar targeting sequences. Nucleic Acids Res 2001; 29:2052-8; PMID:11353074; http://dx.doi.org/10.1093/nar/29.10.2052 PubMed DOI PMC

Platani M, Goldberg I, Swedlow JR, Lamond AI. In vivo analysis of Cajal body movement, separation, and joining in live human cells. J Cell Biol 2000; 151:1561-74; PMID:11134083; http://dx.doi.org/10.1083/jcb.151.7.1561 PubMed DOI PMC

Tvarusko W, Bentele M, Misteli T, Rudolf R, Kaether C, Spector DL, Gerdes HH, Eils R. Time-resolved analysis and visualization of dynamic processes in living cells. Proc Natl Acad Sci U S A 1999; 96:7950-5; PMID:10393928; http://dx.doi.org/10.1073/pnas.96.14.7950 PubMed DOI PMC

Sorokin DV, Tektonidis M, Rohr K, Matula P. Non-rigid Contour-based Temporal Registration of 2D Cell Nuclei Images Using the Navier Equation. In IEEE International Symposium on Biomedical Imaging: From Nano to Macro. Beijing: IEEE, 2014:746-749; ISBN 978-1-4673-1959-1.

Bartova E, Foltankova V, Legartova S, Sehnalova P, Sorokin DV, Suchankova J, Kozubek S. Coilin is rapidly recruited to UVA-induced DNA lesions and gamma-radiation affects localized movement of Cajal bodies. Nucleus 2014; 5; PMID:24859326; http://dx.doi.org/10.4161/nucl.29229 PubMed DOI PMC

Dundr M, Hebert MD, Karpova TS, Stanek D, Xu H, Shpargel KB, Meier UT, Neugebauer KM, Matera AG, Misteli T. In vivo kinetics of Cajal body components. J Cell Biol 2004; 164:831-42; PMID:15024031; http://dx.doi.org/10.1083/jcb.200311121 PubMed DOI PMC

Olson MO, Dundr M. The moving parts of the nucleolus. Histochem Cell Biol 2005; 123:203-16; PMID:15742198; http://dx.doi.org/10.1007/s00418-005-0754-9 PubMed DOI

Rubbi CP, Milner J. Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses. EMBO J 2003; 22:6068-77; PMID:14609953; http://dx.doi.org/10.1093/emboj/cdg579 PubMed DOI PMC

Olson MO. Sensing cellular stress: another new function for the nucleolus? Sci STKE 2004; 2004:pe10; PMID:15026578 PubMed

Lukas C, Savic V, Bekker-Jensen S, Doil C, Neumann B, Pedersen RS, Grøfte M, Chan KL, Hickson ID, Bartek J, et al.. 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress. Nat Cell Biol 2011; 13:243-53; PMID:21317883; http://dx.doi.org/10.1038/ncb2201 PubMed DOI

Lukas C, Falck J, Bartkova J, Bartek J, Lukas J. Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat Cell Biol 2003; 5:255-60; PMID:12598907; http://dx.doi.org/10.1038/ncb945 PubMed DOI

Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H, et al.. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 2008; 132:487-98; PMID:18267078; http://dx.doi.org/10.1016/j.cell.2007.12.033 PubMed DOI

Matula P, Matula P, Kozubek M, Dvorak V. Fast point-based 3-D alignment of live cells. IEEE Trans Image Process 2006; 15:2388-96; PMID:16900692; http://dx.doi.org/10.1109/TIP.2006.875209 PubMed DOI

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