Visualizing Microtubule Networks During Drosophila Oogenesis Using Fixed and Live Imaging.
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.
Institut Jacques Monod, UMR 7592 - CNRS, Université Paris Diderot, 15 rue Hélène Brion, Bât Buffon, 75205, Paris, France.
Kevin
K
Legent
Nicolas
N
Tissot
Antoine
A
Guichet
D000818:Animals; D003599:Cytoskeleton / Q000235:genetics / Q000648:ultrastructure*; D004331:Drosophila melanogaster / Q000235:genetics; D005260:Female; D008853:Microscopy; D008870:Microtubules / Q000235:genetics / Q000648:ultrastructure*; D008967:Molecular Biology / Q000379:methods*; D009866:Oogenesis / Q000235:genetics*