| Nuclear pore complexes (NPCs) are large macromolecular protein complexes that traverse the nuclear envelope and form the only known route of transport between the cytoplasmic and nuclear spaces. Previous studies have indicated that the activation of the calcium releasing channels, inositol 1, 4, 5-trisphosphate (IP3) receptors, within the nuclear envelope leads to conformational changes in the NPC. However, other calcium releasing channels, such as, ryanodine (Ry) receptors were not investigated. The presence of Ry receptors were detected in Xenopus laevis nuclear envelopes, which had previously gone undetected. Atomic force microscopy (AFM) was utilized to measure conformational changes in the NPC upon activation of Ry receptors within the nuclear envelope. These results indicate that activation of either IP3 or Ry receptors alter the conformation of the NPC. While it has been suggested that these changes can modify NPC permeability, it has yet to be directly related to function. The conformational change may alter access to phenylalanine-glycine (FG) rich repeat sites within the lumen of the pore, which have been shown to interact with transported cargo. Near-field scanning optical microscopy (NSOM) will allow for the direct correlation between NPC topography and fluorescently labeled proteins. However, concerns revolve around heating effects that take place near the aperture of the fiber optic NSOM probe that may damage biological samples. A ratiometric thermochromic polymer was utilized to measure the precise amount of sample heating that occurs beneath the aperture of a NSOM probe. Both AFM and NSOM provide high-resolution structural information of the NPC, but dynamic measurements are required to unravel the mechanisms involved in cyto-nuclear trafficking. The development of a single molecule nuclear transport assay provides a convenient method to visualize dynamics at the NPC. A novel micropatterning method using Langmuir-Blodgett (LB) films and cyanoacrylate fuming was developed as a rapid method for patterning substrates with controlled lateral and vertical features. This micropatterning method can be utilized in the development of an assay to probe NPC dynamics. Unraveling the mechanisms by which NPCs regulate nuclear translocation will provide new insights for future studies involving drug development, gene therapy, and viral invasion of the nucleus. |
