Advertisement

Using FRET to Study RanGTP Gradients in Live Mouse Oocytes

  • Julien DumontEmail author
  • Marie-Hélène Verlhac
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 957)

Abstract

Oocytes are extremely large cells that have to coordinate accurate chromosome segregation, asymmetric cytoplasm partitioning together with their own development as fertilizable gametes. For this, they undergo both global (cell cycle progression related) and local changes. It is therefore essential to be able to monitor local changes as they take place in live maturing oocytes. We describe here a method to follow RanGTP gradients using FRET technology in vivo.

Key words

RanGTPase Ratiometric FRET Live imaging Mouse Oocyte 

Notes

Acknowledgments

This work was supported by grants from the Ligue Nationale Contre le Cancer (EL/2009/LNCC/MHV) and from the Agence Nationale pour la Recherche (ANR08-BLAN-0136-01) to MHV, and by grants from Agence Nationale pour la Recherche (ANR-RPDOC-005-01) and Fondation pour la Recherche Médicale (FRM “Amorçage Jeunes Equipes”) to JD.

References

  1. 1.
    Deng M, Suraneni P, Schultz RM, Li R (2007) The RanGTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes. Dev Cell 12:301–308PubMedCrossRefGoogle Scholar
  2. 2.
    Dumont J, Million K, Sunderland K, Rassinier P, Lim H, Leader B, Verlhac M-H (2007) Formin-2 is required for spindle migration and for late steps of cytokinesis in mouse oocytes. Dev Biol 301:254–265PubMedCrossRefGoogle Scholar
  3. 3.
    Halet G, Carroll J (2007) Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes. Dev Cell 12:309–317PubMedCrossRefGoogle Scholar
  4. 4.
    Longo FJ, Chen D-Y (1985) Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus. Dev Biol 107:382–394PubMedCrossRefGoogle Scholar
  5. 5.
    Verlhac M-H, Lefebvre C, Guillaud P, Rassinier P, Maro B (2000) Asymmetric division in mouse oocytes: with or without Mos. Curr Biol 10:1303–1306PubMedCrossRefGoogle Scholar
  6. 6.
    Azoury J, Verlhac M-H, Dumont J (2009) Actin filaments: key players in the control of asymmetric divisions in mouse oocytes. Biol Cell 101:69–78PubMedCrossRefGoogle Scholar
  7. 7.
    Maro B, Johnson MH, Webb M, Flach G (1986) Mechanism of polar body formation in the mouse oocyte: an interaction between the chromosomes, the cytoskeleton and the plasma membrane. J Embryol Exp Morphol 92:11–32PubMedGoogle Scholar
  8. 8.
    Dumont J, Petri S, Pellegrin F, Terret M-E, Bohnsack MT, Rassinier P, Georget V, Kalab P, Gruss OJ, Verlhac M-H (2007) A centriole- and RanGTP-independent spindle assembly pathway in meiosis I of vertebrate oocytes. J Cell Biol 176:295–305PubMedCrossRefGoogle Scholar
  9. 9.
    Bischoff FR, Krebber H, Smirnova E, Dong W, Ponstingl H (1995) Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1. EMBO J 14:705–715PubMedGoogle Scholar
  10. 10.
    Saitoh H, Dasso M (1995) The RCC1 protein interacts with Ran, RanBP1, hsc70, and a 340-kDa protein in Xenopus extracts. J Biol Chem 270:10658–10663PubMedCrossRefGoogle Scholar
  11. 11.
    Wilken N, Senecal JL, Scheer U, Dabauvalle MC (1995) Localization of the Ran-GTP binding protein RanBP2 at the cytoplasmic side of the nuclear pore complex. Eur J Cell Biol 68:211–219PubMedGoogle Scholar
  12. 12.
    Lounsbury KM, Macara IG (1997) Ran-binding protein 1 (RanBP1) forms a ternary complex with Ran and karyopherin beta and reduces Ran GTPase-activating protein (RanGAP) inhibition by karyopherin beta. J Biol Chem 272:551–555PubMedCrossRefGoogle Scholar
  13. 13.
    Pu RT, Dasso M (1997) The balance of RanBP1 and RCC1 is critical for nuclear assembly and nuclear transport. Mol Biol Cell 8:1955–1970PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Plafker K, Macara IG (2000) Facilitated nucleocytoplasmic shuttling of the Ran binding protein RanBP1. Mol Cell Biol 20:3510–3521PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Carazo-Salas RE, Guarguaglini G, Gruss OJ, Segref A, Karsenti E, Mattaj IW (1999) Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation. Nature 400:178–181PubMedCrossRefGoogle Scholar
  16. 16.
    Kalab P, Pu RT, Dasso M (1999) The Ran GTPase regulates mitotic spindle assembly. Curr Biol 9:481–484PubMedCrossRefGoogle Scholar
  17. 17.
    Nachury MV, Maresca TJ, Salmon WC, Waterman-Storer CM, Heald R, Weis K (2001) Importin beta is a mitotic target of the small GTPase Ran in spindle assembly. Cell 104:95–106PubMedCrossRefGoogle Scholar
  18. 18.
    Wiese C, Wilde A, Moore MS, Adam SA, Merdes A, Zheng Y (2001) Role of importin-beta in coupling Ran to downstream targets in microtubule assembly. Science 291:653–656PubMedCrossRefGoogle Scholar
  19. 19.
    Yi K, Unruh JR, Deng M, Slaughter BD, Rubinstein B, Li R (2011) Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes. Nat Cell Biol 13:1252–1258PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Kalab P, Pralle A, Isacoff EY, Heald R, Weis K (2006) Analysis of a RanGTP-regulated gradient in mitotic somatic cells. Nature 440:697–701PubMedCrossRefGoogle Scholar
  21. 21.
    Tsurumi C, Hoffmann S, Geley S, Graeser R, Polanski Z (2004) The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes. J Cell Biol 167:1037–1050PubMedCrossRefGoogle Scholar
  22. 22.
    Reis A, Chang HY, Levasseur M, Jones KT (2006) APCcdh1 activity in mouse oocytes prevents entry into the first meiotic division. Nat Cell Biol 8:539–540PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Lemaire P, Gurdon JB (1994) A role for cytoplasmic determinants in mesoderm patterning: cell-autonomous activation of the goosecoid and Xwnt-8 genes along the dorsoventral axis of early Xenopus embryos. Development 120:1191–1199PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Institut Jacques Monod, CNRS, UMR 7592Univ Paris DiderotParisFrance
  2. 2.Collège de FranceCenter for Interdisciplinary Research in Biology (CIRB), UMR-CNRS7241/INSERM-U1050ParisFrance
  3. 3.Memolife Laboratory of Excellence and Paris Science LettreParisFrance

Personalised recommendations