Use of Xenopus laevis oocyte nuclei and nuclear envelopes in nucleocytoplasmic transport studies.

Abstract

In this chapter, two techniques for the analysis of transport through the nuclear pore complex are described. In the first technique, nuclei isolated manually from Xenopus laevis oocytes are used to measure the import kinetics of fluorescent substrates by confocal fluorescence microscopy. In the second technique, referred to as optical single transporter recording (OSTR), isolated Xenopus oocyte nuclei, perforated nuclei, or isolated nuclear envelopes are tightly bound to planar transparent substrates containing arrays of nanoscopic-to-microscopic cavities. Transport through membrane patches spanning these cavities is recorded by confocal microscopy. By these means, the transport through single nuclear pore complexes or populations of pore complexes can be quantitatively measured.