Recent Advances in Nuclear Electrophysiology - Fig. 2 - Smallest

Click image to go to larger resolution.

Fig. 2. Use of fluorescence microscopy to determine the state of ion and macromolecular conduction of nuclear pores. (a, b) Tests for NHT are performed with probes (e.g. Alexa Fluor-Dextrans, Molecular Probes) to determine the relevance of the leak or shunt current and the dimension of the gap. In a, the fluorescent probe passes through the gap between the nucleus and the tube wall but does not enter the nucleus. Therefore, the shunt is too great to assume it negligible (i.e. R_shunt << R_NE). In b, the gap is of small dimensions and the molecular interactions between the NE and the tube wall are strong enough to produce a tight seal. Therefore, the shunt is negligible (i.e. R_shunt >> R_NE). (c, d) Test for patch-clamp are performed with similar probes. When the NPCs are closed or plugged, panel c, the probes cannot enter the nucleus because the NPCs are the only direct route for nucleocytoplasmic transport. Therefore, any ion channel activity recorded derives from the ONM. When the NPCs are open and unplugged (i.e. there is no macromolecular translocation), panel d, the probes go inside the nucleus. Therefore, the ion channel activity derives from NPCs. Under these conditions, the NPCs outside the pipette provide a bypass route that makes the nucleoplasmic side of the NE virtually connected to the bath electrode. Panels e and f demonstrate the use of macromolecular probes (e.g. B-phycoerythrin, Molecular Probes, conjugated to the NLS of the SV40 large T antigen, Sigma Chemical). A macromolecular probe is placed outside the nucleus. Whether or not the molecules enter the nucleus, will indicate that the NPCs are or ar not capable of macromolecular transport.