Exploring nuclear pore complex molecular architecture by immuno-electron microscopy using Xenopus oocytes.

Xenopus oocytes are large in size and perfectly suited for microinjection experiments. Their nuclei, which can be readily isolated manually, are characterized by an extremely high density of nuclear pore complexes (NPCs). Therefore, Xenopus oocytes are an excellent system to study NPC structure and molecular architecture, as well as nucleocytoplasmic transport on an ultrastructural level. A wide range of electron microscopy (EM) techniques can be employed to do so and thin-sectioning immuno-EM has been proven to be a powerful tool in this context. NPCs are composed of multiple copies of a set of about 30 different nucleoporins, which are often large, multidomain proteins. Their complex organization within NPCs can be unraveled by using domain-specific antibodies to individual nucleoporins in combination with microinjection and expression of epitope-tagged nucleoporins. Here, we describe the immuno-EM methods using Xenopus oocyte that allow for precise ultrastructural localization of nucleoporins within the structure of the NPC.