NPHP proteins are binding partners of nucleoporins at the base of the primary cilium


Autoři: T. Lynne Blasius aff001;  Daisuke Takao aff001;  Kristen J. Verhey aff001
Působiště autorů: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America aff001
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222924

Souhrn

Cilia are microtubule-based organelles that protrude from the surface of eukaryotic cells to generate motility and to sense and respond to environmental cues. In order to carry out these functions, the complement of proteins in the cilium must be specific for the organelle. Regulation of protein entry into primary cilia has been shown to utilize mechanisms and components of nuclear gating, including nucleoporins of the nuclear pore complex (NPC). We show that nucleoporins also localize to the base of motile cilia on the surface of trachea epithelial cells. How nucleoporins are anchored at the cilium base has been unclear as transmembrane nucleoporins, which anchor nucleoporins at the nuclear envelope, have not been found to localize at the cilium. Here we use the directed yeast two-hybrid assay to identify direct interactions between nucleoporins and nephronophthisis proteins (NPHPs) which localize to the cilium base and contribute to cilium assembly and identity. We validate NPHP-nucleoporin interactions in mammalian cells using the knocksideways assay and demonstrate that the interactions occur at the base of the primary cilium using bimolecular fluorescence complementation. We propose that NPHP proteins anchor nucleoporins at the base of primary cilia to regulate protein entry into the organelle.

Klíčová slova:

Cell motility – DNA-binding proteins – Mitochondria – Protein interactions – Yellow fluorescent protein – Cilia – Yeast two-hybrid assays – Nuclear membrane


Zdroje

1. Anvarian Z, Mykytyn K, Mukhopadhyay S, Pedersen LB, Christensen ST (2019) Cellular signalling by primary cilia in development, organ function and disease. Nat Rev Nephrol 15: 199–219. doi: 10.1038/s41581-019-0116-9 30733609

2. Elliott KH, Brugmann SA (2019) Sending mixed signals: Cilia-dependent signaling during development and disease. Dev Biol 447: 28–41. doi: 10.1016/j.ydbio.2018.03.007 29548942

3. Mitchison HM, Valente EM (2017) Motile and non-motile cilia in human pathology: from function to phenotypes. J Pathol 241: 294–309. doi: 10.1002/path.4843 27859258

4. Braun DA, Hildebrandt F (2017) Ciliopathies. Cold Spring Harb Perspect Biol 9:a028191. doi: 10.1101/cshperspect.a028191 27793968

5. Reiter JF, Leroux MR (2017) Genes and molecular pathways underpinning ciliopathies. Nat Rev Mol Cell Biol 18: 533–547. doi: 10.1038/nrm.2017.60 28698599

6. Garcia-Gonzalo FR, Reiter JF (2017) Open Sesame: How Transition Fibers and the Transition Zone Control Ciliary Composition. Cold Spring Harb Perspect Biol 9: a028134. doi: 10.1101/cshperspect.a028134 27770015

7. Jensen VL, Leroux MR (2017) Gates for soluble and membrane proteins, and two trafficking systems (IFT and LIFT), establish a dynamic ciliary signaling compartment. Curr Opin Cell Biol 47: 83–91. doi: 10.1016/j.ceb.2017.03.012 28432921

8. Goncalves J, Pelletier L (2017) The Ciliary Transition Zone: Finding the Pieces and Assembling the Gate. Mol Cells 40: 243–253. doi: 10.14348/molcells.2017.0054 28401750

9. Lu L, Madugula V (2018) Mechanisms of ciliary targeting: entering importins and Rabs. Cell Mol Life Sci 75: 597–606. doi: 10.1007/s00018-017-2629-3 28852774

10. Johnson CA, Malicki JJ (2019) The Nuclear Arsenal of Cilia. Dev Cell 49: 161–170. doi: 10.1016/j.devcel.2019.03.009 31014478

11. McClure-Begley TD, Klymkowsky MW (2017) Nuclear roles for cilia-associated proteins. Cilia 6: 8. doi: 10.1186/s13630-017-0052-x 28560031

12. Floch AG, Palancade B, Doye V (2014) Fifty years of nuclear pores and nucleocytoplasmic transport studies: multiple tools revealing complex rules. Methods Cell Biol 122: 1–40. doi: 10.1016/B978-0-12-417160-2.00001-1 24857723

13. Gorlich D, Kutay U (1999) Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol 15: 607–660. doi: 10.1146/annurev.cellbio.15.1.607 10611974

14. Schmidt HB, Gorlich D (2016) Transport Selectivity of Nuclear Pores, Phase Separation, and Membraneless Organelles. Trends Biochem Sci 41: 46–61. doi: 10.1016/j.tibs.2015.11.001 26705895

15. Breslow DK, Koslover EF, Seydel F, Spakowitz AJ, Nachury MV (2013) An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier. J Cell Biol 203: 129–147. doi: 10.1083/jcb.201212024 24100294

16. Endicott SJ, Brueckner M (2018) NUP98 Sets the Size-Exclusion Diffusion Limit through the Ciliary Base. Curr Biol 28: 1643–1650. doi: 10.1016/j.cub.2018.04.014 29731308

17. Kee HL, Dishinger JF, Blasius TL, Liu CJ, Margolis B, Verhey KJ. (2012) A size-exclusion permeability barrier and nucleoporins characterize a ciliary pore complex that regulates transport into cilia. Nat Cell Biol 14: 431–437. doi: 10.1038/ncb2450 22388888

18. Lin YC, Niewiadomski P, Lin B, Nakamura H, Phua SC, Jiao J, et al. (2013) Chemically inducible diffusion trap at cilia reveals molecular sieve-like barrier. Nat Chem Biol 9: 437–443. doi: 10.1038/nchembio.1252 23666116

19. Christie M, Chang CW, Rona G, Smith KM, Stewart AG, Takeda AA, et al. (2016) Structural Biology and Regulation of Protein Import into the Nucleus. J Mol Biol 428: 2060–2090. doi: 10.1016/j.jmb.2015.10.023 26523678

20. Matsuura Y (2016) Mechanistic Insights from Structural Analyses of Ran-GTPase-Driven Nuclear Export of Proteins and RNAs. J Mol Biol 428: 2025–2039. doi: 10.1016/j.jmb.2015.09.025 26519791

21. Baade I, Kehlenbach RH (2018) The cargo spectrum of nuclear transport receptors. Curr Opin Cell Biol 58: 1–7. doi: 10.1016/j.ceb.2018.11.004 30530239

22. Dishinger JF, Kee HL, Jenkins PM, Fan S, Hurd TW, Hammond JW, et al. (2010) Ciliary entry of the kinesin-2 motor KIF17 is regulated by importin-beta2 and RanGTP. Nat Cell Biol 12: 703–710. doi: 10.1038/ncb2073 20526328

23. Fan S, Whiteman EL, Hurd TW, McIntyre JC, Dishinger JF, Liu CJ, et al. (2011) Induction of Ran GTP drives ciliogenesis. Mol Biol Cell 22: 4539–4548. doi: 10.1091/mbc.E11-03-0267 21998203

24. Funabashi T, Katoh Y, Michisaka S, Terada M, Sugawa M, Nakayama K. (2017) Ciliary entry of KIF17 is dependent on its binding to the IFT-B complex via IFT46-IFT56 as well as on its nuclear localization signal. Mol Biol Cell 28: 624–633. doi: 10.1091/mbc.E16-09-0648 28077622

25. Han Y, Xiong Y, Shi X, Wu J, Zhao Y, Jiang J. (2017) Regulation of Gli ciliary localization and Hedgehog signaling by the PY-NLS/karyopherin-beta2 nuclear import system. PLoS Biol 15: e2002063. doi: 10.1371/journal.pbio.2002063 28777795

26. Hurd TW, Fan S, Margolis BL (2011) Localization of retinitis pigmentosa 2 to cilia is regulated by Importin beta2. J Cell Sci 124: 718–726. doi: 10.1242/jcs.070839 21285245

27. Madugula V, Lu L (2016) A ternary complex comprising transportin1, Rab8 and the ciliary targeting signal directs proteins to ciliary membranes. J Cell Sci 129: 3922–3934. doi: 10.1242/jcs.194019 27633000

28. Torrado B, Grana M, Badano JL, Irigoin F (2016) Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-alpha/beta1-Independent. PLoS One 11: e0162033. doi: 10.1371/journal.pone.0162033 27579771

29. Hampoelz B, Andres-Pons A, Kastritis P, Beck M (2019) Structure and Assembly of the Nuclear Pore Complex. Annu Rev Biophys 48: 515–536. doi: 10.1146/annurev-biophys-052118-115308 30943044

30. Lin DH, Hoelz A (2019) The Structure of the Nuclear Pore Complex (An Update). Annu Rev Biochem.

31. Del Viso F, Huang F, Myers J, Chalfant M, Zhang Y, Reza N, et al. (2016) Congenital Heart Disease Genetics Uncovers Context-Dependent Organization and Function of Nucleoporins at Cilia. Dev Cell 38: 478–492. doi: 10.1016/j.devcel.2016.08.002 27593162

32. Endicott SJ, Basu B, Khokha M, Brueckner M (2015) The NIMA-like kinase Nek2 is a key switch balancing cilia biogenesis and resorption in the development of left-right asymmetry. Development 142: 4068–4079. doi: 10.1242/dev.126953 26493400

33. Takao D, Dishinger JF, Kee HL, Pinskey JM, Allen BL, Verhey KJ. (2014) An assay for clogging the ciliary pore complex distinguishes mechanisms of cytosolic and membrane protein entry. Curr Biol 24: 2288–2294. doi: 10.1016/j.cub.2014.08.012 25264252

34. Takao D, Wang L, Boss A, Verhey KJ (2017) Protein Interaction Analysis Provides a Map of the Spatial and Temporal Organization of the Ciliary Gating Zone. Curr Biol 27: 2296–2306. doi: 10.1016/j.cub.2017.06.044 28736169

35. Mollet G, Silbermann F, Delous M, Salomon R, Antignac C, Saunier S. (2005) Characterization of the nephrocystin/nephrocystin-4 complex and subcellular localization of nephrocystin-4 to primary cilia and centrosomes. Hum Mol Genet 14: 645–656. doi: 10.1093/hmg/ddi061 15661758

36. Gupta GD, Coyaud E, Goncalves J, Mojarad BA, Liu Y, Wu Q, et al. (2015) A Dynamic Protein Interaction Landscape of the Human Centrosome-Cilium Interface. Cell 163: 1484–1499. doi: 10.1016/j.cell.2015.10.065 26638075

37. Sang L, Miller JJ, Corbit KC, Giles RH, Brauer MJ, Otto EA, et al. (2011) Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways. Cell 145: 513–528. doi: 10.1016/j.cell.2011.04.019 21565611

38. Fukui H, Shiba D, Asakawa K, Kawakami K, Yokoyama T (2012) The ciliary protein Nek8/Nphp9 acts downstream of Inv/Nphp2 during pronephros morphogenesis and left-right establishment in zebrafish. FEBS Lett 586: 2273–2279. doi: 10.1016/j.febslet.2012.05.064 22687244

39. Lin DH, Stuwe T, Schilbach S, Rundlet EJ, Perriches T, Mobbs G, et al. (2016) Architecture of the symmetric core of the nuclear pore. Science 352: aaf1015. doi: 10.1126/science.aaf1015 27081075

40. Apelt L, Knockenhauer KE, Leksa NC, Benlasfer N, Schwartz TU, Stelzl U. (2016) Systematic Protein-Protein Interaction Analysis Reveals Intersubcomplex Contacts in the Nuclear Pore Complex. Mol Cell Proteomics 15: 2594–2606. doi: 10.1074/mcp.M115.054627 27194810

41. Robinson MS, Sahlender DA, Foster SD (2010) Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria. Dev Cell 18: 324–331. doi: 10.1016/j.devcel.2009.12.015 20159602

42. Robinson MS, Hirst J (2013) Rapid inactivation of proteins by knocksideways. Curr Protoc Cell Biol 61: 11–17.

43. Stavru F, Nautrup-Pedersen G, Cordes VC, Gorlich D (2006) Nuclear pore complex assembly and maintenance in POM121- and gp210-deficient cells. J Cell Biol 173: 477–483. doi: 10.1083/jcb.200601002 16702234

44. Schwartz M, Travesa A, Martell SW, Forbes DJ (2015) Analysis of the initiation of nuclear pore assembly by ectopically targeting nucleoporins to chromatin. Nucleus 6: 40–54. doi: 10.1080/19491034.2015.1004260 25602437

45. Kerppola TK (2008) Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu Rev Biophys 37: 465–487. doi: 10.1146/annurev.biophys.37.032807.125842 18573091

46. Pascual-Garcia P, Capelson M (2019) Nuclear pores in genome architecture and enhancer function. Curr Opin Cell Biol 58: 126–133. doi: 10.1016/j.ceb.2019.04.001 31063899

47. Raices M, D'Angelo MA (2017) Nuclear pore complexes and regulation of gene expression. Curr Opin Cell Biol 46: 26–32. doi: 10.1016/j.ceb.2016.12.006 28088069

48. Chatel G, Fahrenkrog B (2011) Nucleoporins: leaving the nuclear pore complex for a successful mitosis. Cell Signal 23: 1555–1562. doi: 10.1016/j.cellsig.2011.05.023 21683138

49. Nakano H, Wang W, Hashizume C, Funasaka T, Sato H, Wong RW. (2011) Unexpected role of nucleoporins in coordination of cell cycle progression. Cell Cycle 10: 425–433. doi: 10.4161/cc.10.3.14721 21270521

50. Mossaid I, Fahrenkrog B (2015) Complex Commingling: Nucleoporins and the Spindle Assembly Checkpoint. Cells 4: 706–725. doi: 10.3390/cells4040706 26540075

51. Borlido J, D'Angelo MA (2014) Nup62: a novel regulator of centrosome integrity and function. Cell Cycle 13: 14. doi: 10.4161/cc.27299 24270860

52. Ori A, Banterle N, Iskar M, Andres-Pons A, Escher C, Khanh Bui H, et al. (2013) Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines. Mol Syst Biol 9: 648. doi: 10.1038/msb.2013.4 23511206

53. Hayama R, Rout MP, Fernandez-Martinez J (2017) The nuclear pore complex core scaffold and permeability barrier: variations of a common theme. Curr Opin Cell Biol 46: 110–118. doi: 10.1016/j.ceb.2017.05.003 28624666

54. Liu HL, De Souza CP, Osmani AH, Osmani SA (2009) The three fungal transmembrane nuclear pore complex proteins of Aspergillus nidulans are dispensable in the presence of an intact An-Nup84-120 complex. Mol Biol Cell 20: 616–630. doi: 10.1091/mbc.E08-06-0628 19019988

55. Kim SJ, Fernandez-Martinez J, Sampathkumar P, Martel A, Matsui T, Tsuruta H, et al. (2014) Integrative structure-function mapping of the nucleoporin Nup133 suggests a conserved mechanism for membrane anchoring of the nuclear pore complex. Mol Cell Proteomics 13: 2911–2926. doi: 10.1074/mcp.M114.040915 25139911

56. Vollmer B, Schooley A, Sachdev R, Eisenhardt N, Schneider AM, Sieverding C, et al. (2012) Dimerization and direct membrane interaction of Nup53 contribute to nuclear pore complex assembly. EMBO J 31: 4072–4084. doi: 10.1038/emboj.2012.256 22960634

57. Drin G, Casella JF, Gautier R, Boehmer T, Schwartz TU, Antonny B. (2007) A general amphipathic alpha-helical motif for sensing membrane curvature. Nat Struct Mol Biol 14: 138–146. doi: 10.1038/nsmb1194 17220896

58. Shiba D, Manning DK, Koga H, Beier DR, Yokoyama T (2010) Inv acts as a molecular anchor for Nphp3 and Nek8 in the proximal segment of primary cilia. Cytoskeleton 67: 112–119. doi: 10.1002/cm.20428 20169535

59. Hoff S, Halbritter J, Epting D, Frank V, Nguyen TM, van Reeuwijk J, et al. (2013) ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3. Nat Genet 45: 951–956. doi: 10.1038/ng.2681 23793029

60. Czarnecki PG, Gabriel GC, Manning DK, Sergeev M, Lemke K, Klena NT, et al. (2015) ANKS6 is the critical activator of NEK8 kinase in embryonic situs determination and organ patterning. Nat Commun 6: 6023. doi: 10.1038/ncomms7023 25599650

61. Nakajima Y, Kiyonari H, Mukumoto Y, Yokoyama T (2018) The Inv compartment of renal cilia is an intraciliary signal-activating center to phosphorylate ANKS6. Kidney Int 93: 1108–1117. doi: 10.1016/j.kint.2017.11.016 29395339

62. Otto EA, Schermer B, Obara T, O'Toole JF, Hiller KS, Mueller AM, et al. (2003) Mutations in INVS encoding inversin cause nephronophthisis type 2, linking renal cystic disease to the function of primary cilia and left-right axis determination. Nat Genet 34: 413–420. doi: 10.1038/ng1217 12872123

63. Wiegering A, Dildrop R, Kalfhues L, Spychala A, Kuschel S, Lier JM, et al. (2018) Cell type-specific regulation of ciliary transition zone assembly in vertebrates. EMBO J 37: e97791. doi: 10.15252/embj.201797791 29650680

64. Shiba D, Yamaoka Y, Hagiwara H, Takamatsu T, Hamada H, Yokoyama T. (2009) Localization of Inv in a distinctive intraciliary compartment requires the C-terminal ninein-homolog-containing region. J Cell Sci 122: 44–54. doi: 10.1242/jcs.037408 19050042

65. Warburton-Pitt SR, Jauregui AR, Li C, Wang J, Leroux MR, Barr MM. (2012) Ciliogenesis in Caenorhabditis elegans requires genetic interactions between ciliary middle segment localized NPHP-2 (inversin) and transition zone-associated proteins. J Cell Sci 125: 2592–2603. doi: 10.1242/jcs.095539 22393243

66. Watanabe D, Saijoh Y, Nonaka S, Sasaki G, Ikawa Y, Yokoyama T, et al. (2003) The left-right determinant Inversin is a component of node monocilia and other 9+0 cilia. Development 130: 1725–1734. doi: 10.1242/dev.00407 12642479

67. Tsuji T, Matsuo K, Nakahari T, Marunaka Y, Yokoyama T (2016) Structural basis of the Inv compartment and ciliary abnormalities in Inv/nphp2 mutant mice. Cytoskeleton 73: 45–56. doi: 10.1002/cm.21264 26615802

68. Warburton-Pitt SR, Silva M, Nguyen KC, Hall DH, Barr MM (2014) The nphp-2 and arl-13 genetic modules interact to regulate ciliogenesis and ciliary microtubule patterning in C. elegans. PLoS Genet 10: e1004866. doi: 10.1371/journal.pgen.1004866 25501555

69. Morgan D, Eley L, Sayer J, Strachan T, Yates LM, Craighead AS, et al. (2002) Expression analyses and interaction with the anaphase promoting complex protein Apc2 suggest a role for inversin in primary cilia and involvement in the cell cycle. Hum Mol Genet 11: 3345–3350. doi: 10.1093/hmg/11.26.3345 12471060

70. Hua K, Ferland RJ (2017) Fixation methods can differentially affect ciliary protein immunolabeling. Cilia 6: 5. doi: 10.1186/s13630-017-0045-9 28352462

71. Mansfeld J, Guttinger S, Hawryluk-Gara LA, Pante N, Mall M, Galy V, et al. (2006) The conserved transmembrane nucleoporin NDC1 is required for nuclear pore complex assembly in vertebrate cells. Mol Cell 22: 93–103. doi: 10.1016/j.molcel.2006.02.015 16600873

72. D'Angelo MA, Gomez-Cavazos JS, Mei A, Lackner DH, Hetzer MW (2012) A change in nuclear pore complex composition regulates cell differentiation. Dev Cell 22: 446–458. doi: 10.1016/j.devcel.2011.11.021 22264802

73. Fakhro KA, Choi M, Ware SM, Belmont JW, Towbin JA, Lifton RP, et al. (2011) Rare copy number variations in congenital heart disease patients identify unique genes in left-right patterning. Proc Natl Acad Sci U S A 108: 2915–2920. doi: 10.1073/pnas.1019645108 21282601

74. Horie C, Suzuki H, Sakaguchi M, Mihara K (2002) Characterization of signal that directs C-tail-anchored proteins to mammalian mitochondrial outer membrane. Mol Biol Cell 13: 1615–1625. doi: 10.1091/mbc.01-12-0570 12006657

75. Saka Y, Hagemann AI, Piepenburg O, Smith JC (2007) Nuclear accumulation of Smad complexes occurs only after the midblastula transition in Xenopus. Development 134: 4209–4218. doi: 10.1242/dev.010645 17959720


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