Use of intact Xenopus oocytes in nucleocytoplasmic transport studies.

Abstract

Because of its large nucleus, the Xenopus laevis oocyte offers an excellent system to study nucleocytoplasmic transport. This system, in combination with electron microscopy, has provided much of our insight into the mechanisms of nuclear import and export. In a typical experiment, the nuclear transport substrate is first labeled with colloidal gold, and the resulting complex is injected into the cytoplasm (to study nuclear import) or the nucleus (to study nuclear export) of Xenopus oocytes. The oocytes are then fixed, dehydrated, infiltrated, and embedded into an epoxy resin. Following resin polymerization, thin sections of oocyte nuclei are obtained and examined under an electron microscope. Subsequent evaluation of the position and distribution of the gold-labeled substrate reveals whether the substrate has undergone nuclear import (or export) and the position of rate-limiting events. This chapter describes in detail the protocols for performing electron microscopy import assays with Xenopus oocytes and presents some data illustrating the types of experiments possible using this system.