Advertisement

Visualizing Microtubule Networks During Drosophila Oogenesis Using Fixed and Live Imaging

  • Kevin Legent
  • Nicolas Tissot
  • Antoine GuichetEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1328)

Abstract

The microtubule cytoskeleton is a plastic network of polarized cables. These polymers of tubulin provide orientated routes for the dynamic transport of cytoplasmic molecules and organelles, through which cell polarity is established and maintained. The role of microtubule-mediated transport in the asymmetric localization of axis polarity determinants, in the Drosophila oocyte, has been the subject of extensive studies in the past years. However, imaging the distribution of microtubule fibers in a large cell, where vitellogenesis ensures the uptake of a thick and hazy yolk, presents a series of technical challenges. This chapter briefly reviews some of these aspects and describes two methods designed to circumvent these difficulties. We provide a detailed protocol for the visualization by immunohistochemistry of the three-dimensional organization of tubulin cables in the oocyte. Additionally, we detail the stepwise procedure for the live imaging of microtubule dynamics and network remodeling, using fluorescently labeled microtubule-associated proteins.

Keywords

Microtubule Tubulin MAP +TIP Drosophila Oogenesis Live imaging Dynamics Jupiter EB1 

Notes

Acknowledgments

The manuscript was improved by the critical comments of Véronique Brodu and Alain Debec. This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Ligue Nationale Contre le Cancer (LNCC, Grant RS11/75-34 to A.G.), and the Fondation ARC pour la Recherche sur le Cancer (ARC, Grant SL220100601358 to A.G.). K.L. is a fellow of the LNCC (GB/MA/IQ-10594). N.T. is a fellow of France-BioImaging.

References

  1. 1.
    Gross SP (2004) Hither and yon: a review of bi-directional microtubule-based transport. Phys Biol 1:R1–R11CrossRefPubMedGoogle Scholar
  2. 2.
    Janke C (2014) The tubulin code: molecular components, readout mechanisms, and functions. J Cell Biol 206:461–472PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Marx A, Muller J, Mandelkow EM et al (2006) Interaction of kinesin motors, microtubules, and MAPs. J Muscle Res Cell Motil 27:125–137CrossRefPubMedGoogle Scholar
  4. 4.
    Gardner MK, Zanic M, Howard J (2013) Microtubule catastrophe and rescue. Curr Opin Cell Biol 25:14–22PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    St. Johnston D, Ahringer J (2010) Cell polarity in eggs and epithelia: parallels and diversity. Cell 141:757–774CrossRefPubMedGoogle Scholar
  6. 6.
    Sugioka K, Sawa H (2012) Formation and functions of asymmetric microtubule organization in polarized cells. Curr Opin Cell Biol 24:517–525CrossRefPubMedGoogle Scholar
  7. 7.
    Siegrist SE, Doe CQ (2007) Microtubule-induced cortical cell polarity. Genes Dev 21:483–496CrossRefPubMedGoogle Scholar
  8. 8.
    Steinhauer J, Kalderon D (2006) Microtubule polarity and axis formation in the Drosophila oocyte. Dev Dyn 235:1455–1468CrossRefPubMedGoogle Scholar
  9. 9.
    Spradling A (1993) Developmental genetics of oogenesis. In: Martinez-Arias A, Bate M (eds) The development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 1–70Google Scholar
  10. 10.
    Horne-Badovinac S, Bilder D (2005) Mass transit: epithelial morphogenesis in the Drosophila egg chamber. Dev Dyn 232:559–574CrossRefPubMedGoogle Scholar
  11. 11.
    Kugler JM, Lasko P (2009) Localization, anchoring and translational control of oskar, gurken, bicoid and nanos mRNA during Drosophila oogenesis. Fly (Austin) 3:15–28CrossRefGoogle Scholar
  12. 12.
    Theurkauf WE, Smiley S, Wong ML et al (1992) Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport. Development 115:923–936PubMedGoogle Scholar
  13. 13.
    Guichet A, Peri F, Roth S (2001) Stable anterior anchoring of the oocyte nucleus is required to establish dorsoventral polarity of the Drosophila egg. Dev Biol 237:93–106CrossRefPubMedGoogle Scholar
  14. 14.
    Januschke J, Gervais L, Dass S et al (2002) Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation. Curr Biol 12:1971–1981CrossRefPubMedGoogle Scholar
  15. 15.
    Januschke J, Gervais L, Gillet L et al (2006) The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte. Development 133:129–139CrossRefPubMedGoogle Scholar
  16. 16.
    Zhao T, Graham OS, Raposo A et al (2012) Growing microtubules push the oocyte nucleus to polarize the Drosophila dorsal-ventral axis. Science 336:999–1003PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Berleth T, Burri M, Thoma G et al (1988) The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo. EMBO J 7:1749–1756PubMedCentralPubMedGoogle Scholar
  18. 18.
    Ephrussi A, Dickinson LK, Lehmann R (1991) Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell 66:37–50CrossRefPubMedGoogle Scholar
  19. 19.
    Riechmann V, Ephrussi A (2001) Axis formation during Drosophila oogenesis. Curr Opin Genet Dev 11:374–383CrossRefPubMedGoogle Scholar
  20. 20.
    Neuman-Silberberg FS, Schupbach T (1993) The Drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGF alpha-like protein. Cell 75:165–174CrossRefPubMedGoogle Scholar
  21. 21.
    Parton RM, Hamilton RS, Ball G et al (2011) A PAR-1-dependent orientation gradient of dynamic microtubules directs posterior cargo transport in the Drosophila oocyte. J Cell Biol 194:121–135PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Belaya K, St. Johnston D (2011) Using the mRNA-MS2/MS2CP-FP system to study mRNA transport during Drosophila oogenesis. In: Gerst JE (ed) RNA detection and visualization. Methods and protocols. Humana Press, Hatfield, pp 265–283CrossRefGoogle Scholar
  23. 23.
    Zimyanin VL, Belaya K, Pecreaux J et al (2008) In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization. Cell 134:843–853PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Becalska AN, Gavis ER (2009) Lighting up mRNA localization in Drosophila oogenesis. Development 136:2493–2503PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Parton RM, Valles AM, Dobbie IM et al (2010) Live cell imaging in Drosophila melanogaster. Cold Spring Harb Protoc 2010:pdb.top75CrossRefPubMedGoogle Scholar
  26. 26.
    Pizon V, Iakovenko A, Van Der Ven PF et al (2002) Transient association of titin and myosin with microtubules in nascent myofibrils directed by the MURF2 RING-finger protein. J Cell Sci 115:4469–4482CrossRefPubMedGoogle Scholar
  27. 27.
    Hudson AM, Cooley L (2014) Methods for studying oogenesis. Methods 68:207–217PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Grieder NC, de Cuevas M, Spradling AC (2000) The fusome organizes the microtubule network during oocyte differentiation in Drosophila. Development 127:4253–4264PubMedGoogle Scholar
  29. 29.
    Baffet AD, Benoit B, Januschke J et al (2012) Drosophila tubulin-binding cofactor B is required for microtubule network formation and for cell polarity. Mol Biol Cell 23:3591–3601PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Karpova N, Bobinnec Y, Fouix S et al (2006) Jupiter, a new Drosophila protein associated with microtubules. Cell Motil Cytoskeleton 63:301–312CrossRefPubMedGoogle Scholar
  31. 31.
    Rebollo E, Sampaio P, Januschke J et al (2007) Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells. Dev Cell 12:467–474CrossRefPubMedGoogle Scholar
  32. 32.
    Van Doren M, Williamson AL, Lehmann R (1998) Regulation of zygotic gene expression in Drosophila primordial germ cells. Curr Biol 8:243–246CrossRefPubMedGoogle Scholar
  33. 33.
    Prasad M, Jang AC, Starz-Gaiano M et al (2007) A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging. Nat Protoc 2:2467–2473CrossRefPubMedGoogle Scholar
  34. 34.
    Haack T, Bergstralh DT, St. Johnston D (2013) Damage to the Drosophila follicle cell epithelium produces “false clones” with apparent polarity phenotypes. Biol Open 2:1313–1320PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Small J, Rottner K, Hahne P et al (1999) Visualising the actin cytoskeleton. Microsc Res Tech 47:3–17CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Institut Jacques MonodUMR 7592 – CNRS, Université Paris DiderotParisFrance

Personalised recommendations