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Nucleolar localization and identification of nuclear/nucleolar localization signals of the calmodulin-binding protein nucleomorphin during growth and mitosis in Dictyostelium

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Abstract

The calmodulin-binding protein nucleomorphin isoform NumA1 is a nuclear number regulator in Dictyostelium that localizes to intra-nuclear patches adjacent to the nuclear envelope and to a lesser extent the nucleoplasm. Earlier studies have shown similar patches to be nucleoli but only three nucleolar proteins have been identified in Dictyostelium. Here, actinomycin-D treatment caused the loss of NumA1 localization, while calcium and calmodulin antagonists had no effect. In keeping with a nucleolar function, NumA1 moved out of the presumptive nucleoli during mitosis redistributing to areas within the nucleus, the spindle fibers, and centrosomal region before re-accumulating in the presumptive nucleoli at telophase. Together, these data verify NumA1 as a true nucleolar protein. Prior to this study, the dynamics of specific nucleolar proteins had not been determined during mitosis in Dictyostelium. FITC-conjugated peptides equivalent to presumptive nuclear localization signals within NumA1 localized to nucleoli indicating that they also act as nucleolar localization signals. To our knowledge, these represent the first precisely defined nucleolar localization signals as well as the first nuclear/nucleolar localization signals identified in Dictyostelium. Together, these results reveal that NumA1 is a true nucleolar protein and the only nucleolar calmodulin-binding protein identified in Dictyostelium. The possible use of nuclear/nucleolar localization signal-mediated drug targeting to nucleoli is discussed.

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Abbreviations

BAPTA:

1,2-Bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid

CaM:

Calmodulin

CBP4a:

Calcium-binding protein 4a

eif6:

Eukaryotic translation initiation factor 6

hsp32:

Heat shock protein 32

NLS:

Nuclear localization signal

NoLS:

Nucleolar localization signal

NPM1:

Nucleophosmin

References

  • Arap W, Pasqualini R, Ruoslahti E (1998) Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279:377–380

    Article  PubMed  CAS  Google Scholar 

  • Balbo A, Bozzaro S (2006) Cloning of Dictyostelium eIF6 (p27BBP) and mapping its nucle(ol)ar localization subdomains. Eur J Cell Biol 85:1069–1078

    Article  PubMed  CAS  Google Scholar 

  • Benichou J-C, Quiviger B, Ryter A (1983) Cytochemical study of the nucleolus of the slime mold Dictyostelium discoideum. J Ultrastruc Res 84:60–66

    Article  CAS  Google Scholar 

  • Birbach A, Bailey ST, Ghosh S, Schmid JA (2004) Cytosolic, nuclear and nucleolar localization signals determine subcellular distribution and activity of the NF-κB-inducing kinase NIK. J Cell Sci 117:3615–3624

    Article  PubMed  CAS  Google Scholar 

  • Cappuccinelli P, Fighetti M, Rubino S (1979) A mitotic inhibitor for chromosomal studies in slime moulds. J Gen Microbiol 5:25–27

    Google Scholar 

  • Catalano A, O’Day DH (2008) Calmodulin-binding proteins in the model organism Dictyostelium. A complete & critical review. Cell Signal 20:277–291

    Article  PubMed  CAS  Google Scholar 

  • Catalano A, Luo W, Wang Y, O’Day DH (2010) Synthesis and biological activity of peptides equivalent to the IP22 repeat motif found in proteins from Dictyostelium and Mimivirus. Peptides 31:1799–1805

    Article  PubMed  CAS  Google Scholar 

  • Cha H, Hancock C, Dangi S, Maiguel D, Carrier F, Shapiro P (2004) Phosphorylation regulates nucleophosmin targeting to the centrosome during mitosis as detected by cross-reactive phosphorylation-specific MKK1/MKK2 antibodies. Biochem J 378:857–865

    Article  PubMed  CAS  Google Scholar 

  • Chan WC, White PD (2000) Fmoc solid phase peptide synthesis: a practical approach. Oxford University Press, New York

    Google Scholar 

  • Charlesworth MC, Parish RW (1975) The isolation of nuclei and basic nucleoproteins from the cellular slime mold Dictyostelium discoideum. Eur J Biochem 54:307–316

    Article  PubMed  CAS  Google Scholar 

  • Collavoli A, Hatcher CJ, He J, Okin D, Deo R, Basson CT (2003) TBX5 nuclear localization is mediated by dual cooperative intramolecular signals. J Mol Cell Cardiol 35:1191–1195

    Article  PubMed  CAS  Google Scholar 

  • Dang CV, Lee WMF (1989) Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HSP70, and HIV Tat proteins. J Biol Chem 264:18019–18023

    PubMed  CAS  Google Scholar 

  • Fey P, Kowal AS, Gaudet P, Pilcher KE, Chisholm RL (2007) Protocols for growth and development of Dictyostelium discoideum. Nat Protoc 2:1307–1316

    Article  PubMed  CAS  Google Scholar 

  • Gas N, Escande ML, Stevens BJ (1985) Immunolocalization of the 100 kDa nucleolar protein during the mitotic cycle in CHO cells. Biol Cell 53:209–218

    PubMed  CAS  Google Scholar 

  • Gaudet P, Pilcher KE, Fey P, Chisholm RL (2007) Transformation of Dictyostelium discoideum with plasmid DNA. Nat Protoc 2:1317–1324

    Article  PubMed  CAS  Google Scholar 

  • Ginisty H, Sicard H, Roger B, Bouvet P (1999) Structure and functions of nucleolin. J Cell Sci 112:761–772

    PubMed  CAS  Google Scholar 

  • Grisendi S, Mecucci C, Falini B, Pandolfi PP (2006) Nucleophosmin and cancer. Nat Rev Cancer 6:493–505

    Article  PubMed  CAS  Google Scholar 

  • Guerriero V Jr, Rowley DR, Means AR (1981) Production and characterization of an antibody to myosin light chain kinase and intracellular localization of the enzyme. Cell 27:449–458

    Article  PubMed  CAS  Google Scholar 

  • Hagedorn M, Neuhaus EM, Soldati T (2006) Optimized fixation and immunofluorescence staining methods for Dictyostelium cells. Methods Mol Biol 346:327–338

    PubMed  CAS  Google Scholar 

  • Hebert MD, Matera AG (2000) Self-association of coilin reveals a common theme in nuclear body localization. Mol Biol Cell 11:4159–4171

    PubMed  CAS  Google Scholar 

  • Henderson JE, Amizuka N, Warshawsky H, Biasotto D, Lanske BMK, Goltzman D, Karaplis A (1995) Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death. Mol Cell Biol 15:4064–4075

    PubMed  CAS  Google Scholar 

  • Hussain S, Benavente SB, Nascimento E, Dragoni I, Kurowski A, Gillich A, Humphreys P, Frye M (2009) The nucleolar RNA methyltransferase Misu (NSun2) is required for mitotic spindle stability. J Cell Biol 186:27–40

    Article  PubMed  CAS  Google Scholar 

  • Kakuk A, Friedlander E, Vereb G Jr, Lisboa D, Bagossi P, Toth G, Gergely P, Vereb G (2008) Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230. Exp Cell Res 314:2376–2388

    Article  PubMed  CAS  Google Scholar 

  • Kawashima K, Sameshima M, Izawa M (1979) Isolation of nucleoli from the cellular slime mold Dictyostelium discoideum, strain A-3. Cell Struct Funct 4:183–191

    Article  CAS  Google Scholar 

  • Kieffer-Kwon P, Martianov I, Davidson I (2004) Cell-specific nucleolar localization of TBP-related factor 2. Mol Biol Cell 15:4356–4368

    Article  PubMed  CAS  Google Scholar 

  • Kitanishi-Yumura T, Fukui Y (1987) Reorganization of microtubules during mitosis in Dictyostelium: dissociation from MTOC and selective assembly/disassembly in situ. Cell Motil Cytoskeleton 8:106–117

    Article  Google Scholar 

  • Kubota S, Siomi H, Satoh T, Endo S, Maki M, Hatanaka M (1989) Functional similarity of HIV-I rev and HTLV-I rex proteins: identification of a new nucleolar-targeting signal in rev protein. Biochem Biophys Res Commun 162:963–970

    Article  PubMed  CAS  Google Scholar 

  • Lam YW, Trinkle-Mulcahy L, Lamond AI (2005) The nucleolus. J Cell Sci 118:1335–1337

    Article  PubMed  CAS  Google Scholar 

  • Lim MJ, Wang XW (2006) Nucleophosmin and human cancer. Cancer Detect Prev 30:481–490

    Article  PubMed  CAS  Google Scholar 

  • Lohrum MAE, Ashcroft M, Kubbutat MHG, Vousden KH (2000) Identification of a cryptic nucleolar-localization signal in MDM2. Nat Cell Biol 2:179–181

    Article  PubMed  CAS  Google Scholar 

  • Maeda Y, Takeuchi I (1969) Cell differentiation and fine structures in the development of the cellular slime molds. Dev Growth Differ 11:232–245

    Article  PubMed  CAS  Google Scholar 

  • Maggi LB Jr, Weber JD (2006) Nucleolar adaptation in human cancer. Cancer Invest 23:599–608

    Article  Google Scholar 

  • Mijatovic T, De Neve N, Gailly P, Mathieu V, Haibe-Kains B, Bontempi G, Lapeira J, Decaestecker C, Facchini V, Kiss R (2008) Nucleolus and c-Myc: potential targets of cardenolide-mediated antitumor activity. Mol Cancer Ther 7:1285–1296

    Article  PubMed  CAS  Google Scholar 

  • Misteli T (2001) Protein dynamics: implications for nuclear architecture and gene expression. Science 291:843–847

    Article  PubMed  CAS  Google Scholar 

  • Moerman AM, Klein C (1998) Dictyostelium discoideum Hsp32 is a resident nucleolar heat-shock protein. Chromosoma 107:145–154

    Article  PubMed  CAS  Google Scholar 

  • Montanaro L, Trere D, Derenzini M (2008) Nucleolus, ribosomes, and cancer. Am J Pathol 173:301–310

    Article  PubMed  CAS  Google Scholar 

  • Myre MA, O’Day DH (2002) Nucleomorphin. A novel, acidic, nuclear calmodulin-binding protein from Dictyostleium that regulates nuclear number. J Biol Chem 277:19735–19744

    Article  PubMed  CAS  Google Scholar 

  • Myre MA, O’Day DH (2004a) Dictyostelium calcium-binding protein 4a interacts with nucleomorphin, a BRCT-domain protein that regulates nuclear number. Biochem Biophys Res Commun 322:665–671

    Article  PubMed  CAS  Google Scholar 

  • Myre MA, O’Day DH (2004b) Dictyostelium nucleomorphin is a member of the BRCT-domain family of cell cycle checkpoint proteins. Biochim Biophys Acta 1675:192–197

    PubMed  CAS  Google Scholar 

  • Myre MA, O’Day DH (2005) An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostetlium. Biochem Biophys Res Commun 332:157–166

    Article  PubMed  CAS  Google Scholar 

  • Ochs R, Lischwe M, O’Leary P, Busch H (1983) Localization of nucleolar phosphoproteins B23 and C23 during mitosis. Exp Cell Res 146:139–149

    Article  PubMed  CAS  Google Scholar 

  • Ohta Y, Ohba T, Miyamoto E (1990) Ca2+/calmodulin-dependent protein kinase II: Localization in the interphase nucleus and the mitotic apparatus of mammalian cells. Proc Natl Acad Sci USA 87:5341–5345

    Article  PubMed  CAS  Google Scholar 

  • Peterson T, Tsai RYL (2009) In search of nonribosomal nucleolar protein function and regulation. J Cell Biol 184:771–776

    Article  Google Scholar 

  • Prado A, Ramos I, Frehlick LJ, Muga A, Ausio J (2004) Nucleoplasmin: a nuclear chaperone. Biochem Cell Biol 82:437–445

    Article  PubMed  CAS  Google Scholar 

  • Robertson R (2007) Hand it to the nucleolus. J Cell Biol 85:52–52

    Google Scholar 

  • Roos U-P, De Brabander M, De Mey J (1984) Indirect immunofluorescence of microtubules in Dictyostelium discoideum. Exp Cell Res 151:183–193

    Article  PubMed  CAS  Google Scholar 

  • Russo G, Ricciardelli G, Pietropaolo C (1997) Different domains cooperate to target the human ribosomal L7a protein to the nucleus and to the nucleoli. J Biol Chem 272:5229–5235

    Article  PubMed  CAS  Google Scholar 

  • Sansam CL, Wells KS, Emeson RB (2003) Modulation of RNA editing by functional nucleolar sequestration of ADAR2. Proc Natl Acad Sci USA 100:14018–14023

    Article  PubMed  CAS  Google Scholar 

  • Schally AV, Nagy A (1999) Cancer chemotherapy based on targeting of cytotoxic peptide conjugates to their receptors on tumors. Eur J Endocrinol 141:1–14

    Article  PubMed  CAS  Google Scholar 

  • Siomi H, Shida H, Nam SH, Nosaka T, Maki M, Hatanaka M (1988) Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing. Cell 55:197–209

    Article  PubMed  CAS  Google Scholar 

  • Sirri V, Urcuqui-Inchima S, Roussel P, Hernandez-Verdun D (2008) Nucleolus: the fascinating nuclear body. Histochem Cell Biol 129:13–31

    Article  PubMed  CAS  Google Scholar 

  • Song Z, Wu M (2005) Identification of a novel nucleolar localization signal and a degradation signal in surviving-delta Ex3: a potential link between nucleolus and protein degradation. Oncogene 24:2723–2734

    Article  PubMed  CAS  Google Scholar 

  • Stegh AH, Schickling O, Ehret A, Scaffidi C, Peterhansel C, Hofmann TG, Grummt I, Krammer PH, Peter ME (1998) DEDD, a novel death effector domain-containing protein, targeted to the nucleolus. EMBO J 17:5974–5986

    Article  PubMed  CAS  Google Scholar 

  • Tao W, Levine AJ (1999) P19ARF stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2. Proc Natl Acad Sci USA 96:6937–6941

    Article  PubMed  CAS  Google Scholar 

  • Theodore M, Kawai Y, Yang J, Kleshchenko Y, Reddy SP, Villalta F, Arinze IJ (2008) Multiple nuclear localization signals function in the nuclear import of the transcription factor Nrf2. J Biol Chem 283:14176–14176

    Google Scholar 

  • Thorogate R, Torok K (2007) Role of Ca2+activation and bilobal structure of calmodulin in nuclear and nucleolar localization. Biochem J 402:71–80

    Article  PubMed  CAS  Google Scholar 

  • Torgan CE, Daniels MP (2006) Calcineurin localization in skeletal muscle offers insights into potential new targets. J Histochem Cytochem 54:119–128

    Article  PubMed  CAS  Google Scholar 

  • Ueki N, Kondo M, Seki N, Yano K, Oda T, Masuho Y, Muramatsu M-A (1998) NOLP: identification of a novel human nucleolar protein and determination of sequence requirements for its nucleolar localization. Biochem Biophys Res Commun 252:97–102

    Article  PubMed  CAS  Google Scholar 

  • Visintin R, Amon A (2000) The nucleolus: the magician’s hat for cell cycle tricks. Curr Opin Cell Biol 12:372–377

    Article  PubMed  CAS  Google Scholar 

  • Wang Y-H, Chen Y-H, Lu J-H, Tsai H-J (2005) A 23-amino acid motif spanning the basic domain targets zebrafish myogenic regulatory factor Myf5 into nucleolus. DNA Cell Biol 24:651–660

    Article  PubMed  Google Scholar 

  • Wong EC, Saffitz JE, McDonald JM (1991) Association of calmodulin with isolated nuclei from rat hepatocytes. Biochem Biophys Res Commun 181:1548–1556

    Article  PubMed  CAS  Google Scholar 

  • Xue Z, Melese T (1994) Nucleolar proteins that bind NLSs: a role in nuclear import or ribosome biogenesis? Trends Cell Biol 4:414–417

    Article  PubMed  CAS  Google Scholar 

  • Yamaguchi H, Morita T, Amagai A, Maeda Y (2005) Changes in spatial and temporal localization of Dictyostelium homologues of TRAP1 and GRP94 revealed by immunoelectron microscopy. Exp Cell Res 303:415–424

    Article  PubMed  CAS  Google Scholar 

  • Yao YL, Yang WM (2005) Nuclear proteins: promising targets for cancer drugs. Curr Cancer Drug Targets 5:595–610

    Article  PubMed  CAS  Google Scholar 

  • Yun C, Wang Y, Mukhopadhyay D, Backlund P, Kolli N, Yergey A, Wilkinson KD, Dasso M (2008) Nucleolar protein B23/nucleophosmin regulates the vertebrate SUMO pathway through SENP3 and SENP5 proteases. J Cell Biol 183:589–595

    Article  PubMed  CAS  Google Scholar 

  • Zhang Y, Xiong Y (1999) Mutations in human ARF exon 2 disrupt its nucleolar localization and impair its ability to block nuclear export of MDM2 and p53. Mol Cell 3:579–591

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Natural Sciences and Engineering Research Council of Canada grant (DDD; A6807).We would like to thank Dr. Yali Wang, Wei Luo, David Xiao, and Dr. Sheng Cai for the synthesis and conjugation of all peptides used in this study.

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Authors and Affiliations

  1. Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, M5S 3G5, Canada

    Andrew Catalano & Danton H. O’Day

  2. Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road. N, Mississauga, ON, L5L 1C6, Canada

    Danton H. O’Day

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  1. Andrew Catalano
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Correspondence to Danton H. O’Day.

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Catalano, A., O’Day, D.H. Nucleolar localization and identification of nuclear/nucleolar localization signals of the calmodulin-binding protein nucleomorphin during growth and mitosis in Dictyostelium . Histochem Cell Biol 135, 239–249 (2011). https://doi.org/10.1007/s00418-011-0785-3

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