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Quantitative Proteomics and Dynamic Imaging of the Nucleolus Reveal Distinct Responses to UV and Ionizing Radiation

2011; Elsevier BV; Volume: 10; Issue: 10 Linguagem: Inglês

10.1074/mcp.m111.009241

1535-9484

Henna M. Moore, Baoyan Bai, François‐Michel Boisvert, Leena Latonen, Ville Rantanen, Jeremy C. Simpson, Rainer Pepperkok, Angus I. Lamond, Marikki Laiho,

RNA and protein synthesis mechanisms

The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner. The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner. The nucleolus forms around hundreds of repeats of ribosomal DNA (rDNA) 1The abbreviations used are:rDNAribosomal DNACFPcyan fluorescent proteinCOcorrelation coefficiencyDFCdense fibrillar centerDRB5,6-dichloro-1-beta-D-ribofuranosylbenzimidazoledsRNAdouble-strand RNAECGFPenhanced cyan green fluorescent proteinFBLfibrillarinFCfibrillar centerFPfluorescent proteinGCgranular componentIRionizing radiationNHEJnonhomologous end-joiningNPMnucleophosminRNA pol IRNA polymerase IRNA pol IIRNA polymerase IIrRNAribosomal RNASSUsmall subunitTEMtransmission electron microscopyUBFupstream binding factorYFPyellow fluorescent protein. genes and comprises a complex set of proteins, ribosomal RNA (rRNA) and hundreds of small nucleolar RNA (snoRNA) species. Its key function is in ribosome subunit production. In higher eukaryotes, the nucleolus is organized in distinct substructures corresponding to fibrillar centers (FC), dense fibrillar component (DFC), and granular component (GC) (1Olson M.O. Dundr M. 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These, and other studies on specific pathways, have provided evidence of the relevance of the nucleolus, or nucleolus-mediated processes in the regulation of tumor suppressor and oncogene activities, cell-cycle regulation, signal recognition particle assembly, modification of small RNAs, control of aging and modulation of telomerase function (reviewed in 6Olson M.O. Dundr M. The moving parts of the nucleolus.Histochem. Cell Biol. 2005; 123: 203-216Crossref PubMed Scopus (180) Google Scholar, 10Boisvert F.M. van Koningsbruggen S. Navascués J. Lamond A.I. The multifunctional nucleolus.Nat. Rev. Mol. Cell Biol. 2007; 8: 574-585Crossref PubMed Scopus (1122) Google Scholar, 18Ruggero D. Pandolfi P.P. Does the ribosome translate cancer?.Nat. Rev. Cancer. 2003; 3: 179-192Crossref PubMed Scopus (774) Google Scholar, 19Grisendi S. Mecucci C. Falini B. Pandolfi P.P. Nucleophosmin and cancer.Nat. Rev. Cancer. 2006; 6: 493-505Crossref PubMed Scopus (661) Google Scholar). Furthermore, interference of rDNA transcription by pol I, the general pol II transcription machinery, or cellular stress caused by various substances including cytotoxic drugs, DNA intercalating agents, proteasomal stress, or viruses, cause reorganization of the nucleolar proteins and structures, and nucleolar dysfunction (3Sirri V. Urcuqui-Inchima S. Roussel P. Hernandez-Verdun D. Nucleolus: The fascinating nuclear body.Histochem. Cell Biol. 2008; 129: 13-31Crossref PubMed Scopus (304) Google Scholar, 20Rubbi C.P. Milner J. Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses.EMBO J. 2003; 22: 6068-6077Crossref PubMed Scopus (648) Google Scholar, 21Drygin D. Rice W.G. Grummt I. The RNA polymerase I transcription machinery: An emerging target for the treatment of cancer.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 131-156Crossref PubMed Scopus (313) Google Scholar, 22Hiscox J.A. RNA viruses: Hijacking the dynamic nucleolus.Nat. Rev. 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DNA double-strand break repair: All's well that ends well.Annu. Rev. Genet. 2006; 40: 363-383Crossref PubMed Scopus (623) Google Scholar). UV and IR activate damage sensors ATR (ataxia-telangiectasia mutated and RAD3-related) and ATM (ataxia-telangiectasia mutated) kinase cascades, respectively. ATR and ATM kinases evoke distinct signaling cascades leading to diverse cellular responses like cell cycle arrest, DNA damage repair and apoptosis (30Durocher D. Jackson S.P. DNA-PK, ATM and ATR as sensors of DNA damage: Variations on a theme?.Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (446) Google Scholar). Although UV-caused DNA lesions inhibit pol I and halt pol II driven transcription elongation, IR is not considered to directly interfere with the transcription machineries. However, there is evidence that IR inhibits RNA pol I activity (31Kruhlak M. Crouch E.E. Orlov M. Montaño C. Gorski S.A. Nussenzweig A. Misteli T. Phair R.D. Casellas R. The ATM repair pathway inhibits RNA polymerase I transcription in response to chromosome breaks.Nature. 2007; 447: 730-734Crossref PubMed Scopus (222) Google Scholar). Although UV leads to nucleoplasmic relocalization of several nucleolar proteins (NPM, Ki67, HRad17, WRN) (32Chang M.S. Sasaki H. Campbell M.S. Kraeft S.K. Sutherland R. Yang C.Y. Liu Y. Auclair D. Hao L. Sonoda H. Ferland L.H. Chen L.B. HRad17 colocalizes with NHP2L1 in the nucleolus and redistributes after UV irradiation.J. Biol. Chem. 1999; 274: 36544-36549Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 33Al-Baker E.A. Boyle J. Harry R. Kill I.R. A p53-independent pathway regulates nucleolar segregation and antigen translocation in response to DNA damage induced by UV irradiation.Exp. Cell Res. 2004; 292: 179-186Crossref PubMed Scopus (33) Google Scholar, 34Blander G. Zalle N. Daniely Y. Taplick J. Gray M.D. Oren M. DNA damage-induced translocation of the werner helicase is regulated by acetylation.J. Biol. Chem. 2002; 277: 50934-50940Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), there are only few reports on nucleolar proteins relocalizing after IR. Daniely et al. (35Daniely Y. Dimitrova D.D. Borowiec J.A. Stress-dependent nucleolin mobilization mediated by p53-nucleolin complex formation.Mol. Cell. Biol. 2002; 22: 6014-6022Crossref PubMed Scopus (142) Google Scholar) showed that nucleolin is relocalized to nucleoplasm after IR but not UV damage. Interestingly, the nucleolar proteome contains several key factors involved in sensing the DNA damage or DNA repair processes, including the ATM, ATR, BLM, MRE11, PARP1, TOPBP1, WRN, XRCC1 and Ku70/80 proteins. Whether they participate in the surveillance and repair of rDNA or are redistributed to chromatin upon DNA damage is currently not known. Here, we provide a systematic analysis of the nucleolar responses to physiologically relevant DNA damaging agents, i.e. UV and IR, utilizing a combination of cellular imaging and quantitative proteomics. We demonstrate extensive damage specific responses of functionally-related groups of nucleolar proteins. WS1 skin fibroblasts (CRL-1502, ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), nonessential amino acids, and penicillin-streptomycin. U2OS osteosarcoma cells (HTB-96, ATCC) were maintained in DMEM supplemented with 15% FCS. Open reading frames, cloned into expression vectors generating fluorescent fusion proteins (FP) with cyan or yellow FP (CFP and YFP, respectively), were derived from the EMBL-DKFZ Protein Localization Project resource (http://www.dkfz.de/LIFEdb/). Stable U2OS cell lines were generated by cotransfecting FP-expression constructs and pCDNeo selection marker using Lipofectamine (Invitrogen). Following a 2-week selection in the presence of G418, stable cell colonies were isolated and verified for the expression of the CFP or YFP fusion proteins. Cells were maintained in DMEM supplemented with 15% FCS and 1 mg/ml of G418. All cells were kept at +37 °C in a humidified atmosphere containing 5% CO2. Chemicals used were actinomycin D (Sigma) and 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) (Sigma). All other cell culture reagents were obtained from Invitrogen. Cells were treated with ultraviolet C radiation (UVC) (35 J/m2) (254 nm UVC light bulbs, Stratalinker) or IR (1 or 10 Gy) (137Cs γ-ray source, BioBeam 8000; STS, Braunschweig, Germany). NPM-enhanced cyan green fluorescent protein (ECGFP) fusion protein was generated by excision of NPM1 cDNA from B23-GFP (a kind gift from Dr. M. Olson, University of Mississippi Medical Center, MS, ref. 36Dundr M. Misteli T. Olson M.O. The dynamics of postmitotic reassembly of the nucleolus.J. Cell Biol. 2000; 150: 433-446Crossref PubMed Scopus (207) Google Scholar) and ligation to ECGFP-pRSETb (a kind gift from Dr. A. Miyawaki, Brain Science Institute, RIKEN, Saitama, Japan, ref. 37Sawano A. Miyawaki A. Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis.Nucleic Acids Res. 2000; 28: E78Crossref PubMed Scopus (302) Google Scholar). The construct was further subcloned to pCDNA3.1+ (Invitrogen) to yield NPM-ECGFP. Cells were fixed with 3.5% paraformaldehyde followed by permeabilization with 0.5% Nonidet P-40. Following primary antibodies were used: mouse anti-nucleophosmin (NPM) (Zymed Laboratories Inc., South San Francisco, CA), rabbit anti-fibrillarin (FBL) (Abcam, Cambridge, MA), rabbit anti-UBF (H-300, Santa Cruz, Santa Cruz, CA), mouse anti-DDX56 (M03, Abnova), mouse anti-Ku70 (3C3.11, Santa Cruz), rabbit anti-AATF/Che-1 (Bethyl, Montgomery, TX), and rabbit anti-nucleostemin (H-270, Santa Cruz). Antibodies were detected with secondary antibodies conjugated to Alexa 488 or 594 (Molecular Probes, Eugene, OR) and nuclei were counterstained with Hoechst 33258. The fluorochromes were visualized with Zeiss Axioplan 2 Imaging MOT (Jena) equipped with 20x/0.5NA or 40x/0.75NA Plan-Neofluar objectives and Chroma 31000v2, Chroma 41001, and Chroma 41004 filters. Images were captured with Zeiss AxioCam HRm 14-bit grayscale CCD camera and AxioVision program version 4.6. Confocal imaging was performed with Zeiss LSM510 META microscope equipped with 63x/1.25NA Plan-Neofluar or 63x/1.4NA Plan-Apochromat objectives, diode, argon and HeNe lasers. Emissions were detected with the following filter settings: BP 420–480 for Hoechst and BP 505–530 for Alexa 488. HFT405/488/543 was used as dichroic beam splitter and NFT545 as emission splitter. WS1 cells were harvested by pelleting and fixed by 2.5% glutaraldehyde. The specimen was postfixed by 1% osmium tetroxide for 1 h in room temperature, dehydrated in graded ethanol and embedded in epoxy resin LX-112. Ultrathin sections were cut at 60 nm using Reichert-Jung ultra-microtome and stained by uranyl acetate and lead citrate in Leica EMstain automatic staining unit under standard protocols. Uranyl acetate and lead citrate increase resolution and contrast of cellular structures, such as nucleoli because of affinity to nucleic acids and protein. The sections were observed under Jeol JEM 1400 TEM at 80 KV. Electron micrographs were taken with Olympus-SIS Morada digital camera. All images are obtained at 5000 × magnification. U2OS cells were plated on 8-well Lab-Tek Chambered coverglass (Nunc). After reaching 50–80% confluency, cells were treated with UV, IR, or left untreated after which nuclei were stained with Hoechst 33342. Prior imaging, the culture medium was changed to DMEM without phenol red. Phase contrast was used for autofocusing. FP-proteins were detected using filter sets for DAPI (Semrock 5060B), CFP, and YFP (Chroma 52017; single excitation filters, double emission filter) or GFP and mRFP (Chroma 52022; single excitation filters, double emission filter) and images were captured every 10 min for 16 h using Zeiss/Intelligent Imaging Innovations (3i) - Stallion HIS live cell imaging system mounted on Zeiss Axiovert 100 with 20x/0.50NA Plan-Neofluar objective and equipped with humidified chambered heating stage and CO2 source. Cells in control experiments were viable throughout the incubation and divided at the expected rate. Raw data on each image capture were extracted and analyzed by creating an image analysis application called "CellGrain" (https://wiki.helsinki.fi/display/∼[email protected]/Cellgrain+Download+Page) in the analysis framework Anduril (38Ovaska K. Laakso M. Haapa-Paananen S. Louhimo R. Chen P. Aittomäki V. Valo E. Núñez-Fontarnau J. Rantanen V. Karinen S. Nousiainen K. Lahesmaa-Korpinen A.M. Miettinen M. Saarinen L. Kohonen P. Wu J. Westermarck J. Hautaniemi S. Large-scale data integration framework provides a comprehensive view on glioblastoma multiforme.Genome Med. 2010; 2: 65Crossref PubMed Scopus (138) Google Scholar). Data on each image capture were analyzed by first identifying nuclei by Hoechst staining and to record nuclear intensities on GFP/CFP/YFP or red channels. Nuclear recognition was based on thresholding, watershedding, and removing objects smaller than 100 pixels. Nuclei were tracked to unambiguously identify changes in individual cells over time. Nucleoli were identified using a constant size local maxima finder, which finds small intensity areas brighter than immediate surroundings, and the nucleolar intensities were recorded. Background level for each image was set as 5% percentile intensity outside nuclei and all measured intensity values were subtracted with the background. Each video in the analysis contained 40 to 200 cells. Data from UV and IR treated cells were normalized to control experiments to exclude possible intensity changes of the FPs during imaging. Two-Way ANOVA analysis was applied to address statistical changes over time for each FP as compared with the control. Student's two-tailed t test was used for statistical analysis for fixed time point analyses. Nucleoli were isolated as previously described (13Andersen J.S. Lam Y.W. Leung A.K. Ong S.E. Lyon C.E. Lamond A.I. Mann M. Nucleolar proteome dynamics.Nature. 2005; 433: 77-83Crossref PubMed Scopus (941) Google Scholar), lysed into Laemmli sample buffer supplemented with dithiotreitol (DTT) and sonicated briefly. Loading was normalized according to number of nucleoli in each sample. To obtain total cellular lysates, cells were scraped, solubilized in urea-Tris buffer (9 m urea, 75 mm Tris-HCl [pH 7.5] and 1 mm DTT) and sonicated. Protein concentration was determined using Bio-Rad Bradford protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein were loaded into 9% SDS-PAGE and transferred into nitrocellulose membrane (Trans-Blot, Transfer Medium, Bio-Rad). Immunoblotting was carried out using anti-nucleophosmin (NPM) (Zymed Laboratories Inc.) and anti-fibrillarin (Abcam) antibodies followed by secondary antibodies conjugated to biotin and streptavidin conjugated to horseradish peroxidase (HRP), after which the signals were detected using enhanced chemiluminescence (ECL, Amersham Biosciences Life Sciences). WS1 cells were cultivated for at least five passages in custom-made DMEM (Biowest) where arginine and lysine were replaced either by standard amino acids (Arg0, A8094; Lys0, L8662, Sigma; light) or by isotope-labeled amino acids (Arg6, CLM-2265 and Lys4, DLM-2640, Cambridge Isotope Laboratories; medium), or (Arg10, CNLM-539 and Lys8, CNLM-291 Cambridge Isotope Lab; heavy) and supplemented with 10% dialyzed FCS (Invitrogen) and penicillin-streptomycin. Cells were treated with UV, IR, or left untreated, and harvested at different times. Cells grown in light, medium, and heavy-isotope containing media treated at the indicated times were pooled and nucleoli were isolated as previously described (13Andersen J.S. Lam Y.W. Leung A.K. Ong S.E. Lyon C.E. Lamond A.I. Mann M. Nucleolar proteome dynamics.Nature. 2005; 433: 77-83Crossref PubMed Scopus (941) Google Scholar). Two biological replicates were included for each treatment. Nucleoli were directly resuspended in Laemmli sample buffer supplemented with β-mercaptoethanol and boiled. Nucleolar proteins were separated by one-dimensional SDS-PAGE (4–12% Bis-Tris gel, BioRad) and visualized by colloidal Coomassie staining (Novex, Invitrogen). The entire protein gel lanes were excised and cut into six slices each. Proteins were reduced in 10 mm DTT and alkylated in 50 mm iodoacetamide. Every slice was subjected to in-gel digestion with trypsin and tryptic peptides were extracted by 1% formic acid, acetonitrile, lyophilized in a SpeedVac, and resuspended in 1% formic acid as previously described (17Boisvert F.M. Lam Y.W. Lamont D. Lamond A.I. A quantitative proteomics analysis of subcellular proteome localization and changes induced by DNA damage.Mol. Cell. Proteomics. 2010; 9: 457-470Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Trypsin digested peptides were separated using an Ultimate U3000 (Dionex Corporation) nanoflow LC-system as in (17Boisvert F.M. Lam Y.W. Lamont D. Lamond A.I. A quantitative proteomics analysis of subcellular proteome localization and changes induced by DNA damage.Mol. Cell. Proteomics. 2010; 9: 457-470Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Mass spectrometry was conducted essentially as in reference (17Boisvert F.M. Lam Y.W. Lamont D. Lamond A.I. A quantitative proteomics analysis of subcellular proteome localization and changes induced by DNA damage.Mol. Cell. Proteomics. 2010; 9: 457-470Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) using a LTQ Orbitrap XL (Thermo Fisher Scientific Inc) via a nano ES ion source (Proxeon Biosystems). Data were acquired using the Xcalibur software. Quantification was performed using MaxQuant version 1.0.13.13 (17Boisvert F.M. Lam Y.W. Lamont D. Lamond A.I. A quantitative proteomics analysis of subcellular proteome localization and changes induced by DNA damage.Mol. Cell. Proteomics. 2010; 9: 457-470Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 39Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9150) Google Scholar). The peak list generated by MaxQuant was searched using Mascot version 2.2.2 (Matrix Sciences, London, UK) as the database search engine for peptide identifications against the International Protein Index human protein database version 3.37 containing 69,290 proteins, to which 175 commonly observed contaminants and all the reversed sequences had been added (39Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9150) Google Scholar). Mass tolerance was set to 7 ppm and MS/MS mass tolerance was 0.5 Da. Enzyme was set to trypsin with no proline restriction (trypsin/p) with two missed cleavages. Carbamidomethylation of cysteine was searched as a fixed modification, and N-acetyl protein and oxidation of methionine were searched as variable modifications. Identification of proteins was set to a false discovery rate of 1%. To achieve reliable identifications, all proteins were accepted based on the criteria that the number of forward hits in the database was at least 100-fold higher than the number of reverse database hits, thus resulting in a false discovery rate of 1%. A minimum of two peptides was quantified for each protein. Protein isoforms and proteins that cannot be distinguished based on the peptides identified are grouped. Protein intensity values were converted to LOG2 scale to facilitate the comparison of the biological repeats. MaxQuant-indicated contaminations were excluded from the analysis. Only proteins that were identified in both biological repeats were included in further analysis. To minimize the effect of outliers, protein ratios were calculated as the means of the biological repeats. The variability of the biological repeats was defined as the standard deviation (S.D.). To minimize the variability, only proteins with S.D. less than average S.D. were considered for the