Nuclear transport kinetics in microarrays of nuclear envelope patches.

ABSTRACT

Optical Single Transporter Recording (OSTR) is a technique for analyzing membrane transport kinetics at high sensitivity, selectivity, and spatial resolution. Cellular membranes are firmly attached to microarrays of small test compartments (TCs) with diameters between approximately 0.1 and 100 microm and depths between approximately 10 and 100 microm. This permits to generate either "small" membrane patches containing few transporters or "large" patches containing many transporters. Transport of substrates across membrane patches is recorded by confocal microscopy. The present article reviews recent applications of OSTR to the nuclear pore complex (NPC). The results show that the transport functions of the NPC, previously studied almost exclusively in intact and permeabilized cells, are conserved in isolated nuclei and can be fully reconstituted in purified nuclear envelopes by addition of recombinant transport factors. This opens new avenues to the analysis of nuclear transport including the export of nucleic-acid-protein and ribosomal particles.