NPC Structure in Model Organisms: Transmission Electron Microscopy and Immunogold Labeling Using High-Pressure...

The nuclear pore complex (NPC) is a large elaborate structure embedded within the nuclear envelope, and intimately linked to the cytoskeleton, nucleoskeleton, and chromatin. Many different cargoes pass through its central channel and along the membrane at its periphery. The NPC is dismantled and reassembly, fully or partially, every cell cycle. In post-mitotic cells it consists of a combination of hyper-stable and highly dynamic proteins. Because of its size, dynamics, heterogeneity and integration, it is not possible to understand its structure and molecular function by any one, or even several, methods. For decades, and to this day, thin section transmission electron microscopy (TEM) has been a central tool for understanding the NPC, its associations, dynamics and role in transport as it can uniquely answer questions concerning fine structural detail within a cellular context. Using immunogold labeling specific components can also be identified within the ultrastructural context. Model organisms such as Saccharomyces cerevisiae are also central to NPC studies but have not been used extensively in structural work. This is because the cell wall presents difficulties with structural preservation and processing for TEM. In recent years, high-pressure freezing and freeze substitution have overcome these problems, as well as opened up methods to combine immunogold labeling with detailed structural analysis. Other model organisms such as the worm Caenorhabditis elegans and the plant Arabidopsis thaliana have been underused for similar reasons, but with similar solutions, which we present here. There are also many advantages to using these methods, adapted for use in mammalian systems, due to the instant nature of the initial fixation, to capture rapid processes such as nuclear transport, and preservation of dynamic membranes.