From live-cell imaging to scanning electron microscopy (SEM): the use of green fluorescent protein (GFP) as a common label.

The identification and characterization of many biological substructures at high resolution requires the use of electron microscopy (EM) technologies. Scanning electron microscopy (SEM) allows the resolution of cellular structures to approximately 3 nm and has facilitated the direct visualization of macromolecular structures, such as nuclear pore complexes (NPCs), which are essential for nucleo-cytoplasmic molecular trafficking. However, SEM generates only static images of fixed samples and therefore cannot give unambiguous information about protein dynamics. The investigation of active processes and analysis of protein dynamics has greatly benefited from the development of molecular biology techniques whereby vectors can be generated and transfected into tissue culture cells for the expression of specific proteins tagged with a fluorescent moiety for real-time light microscopy visualization. As light microscopy is limited in its powers of resolution relative to electron microscopy, it has been important to adapt a protocol for the processing of samples for real-time imaging by conventional light microscopy with protein labels that can also be identified by SEM. This allows correlation of dynamic events with high resolution molecular and structural identification. This method describes the use of GFP for tracking the dynamic distribution of NPC components in real-time throughout the cell cycle and for high resolution immuno-SEM labeling to determine localization at the nanometer level.

Scanning electron microscopy of nuclear structure.

Accessing internal structure and retaining relative three dimensional (3D) organization within the nucleus has always proved difficult in the electron microscope. This is due to the overall size and largely fibrous nature of the contents, making large scale 3D reconstructions difficult from thin sections using transmission electron microscopy. This chapter brings together a number of methods developed for visualization of nuclear structure by scanning electron microscopy (SEM). These methods utilize the easily accessed high resolution available in field emission instruments. Surface imaging has proved particularly useful to date in studies of the nuclear envelope and pore complexes, and has also shown promise for internal nuclear organization, including the dynamic and radical reorganization of structure during cell division. Consequently, surface imaging in the SEM has the potential to make a significant contribution to our understanding of nuclear structure.

Nuclear membrane disassembly and rupture.

The nuclear envelope consists of two membranes traversed by nuclear pore complexes. The outer membrane is continuous with the endoplasmic reticulum. At mitosis nuclear pore complexes are dismantled and membranes disperse. The mechanism of dispersal is controversial: one view is that membranes feed into the endoplasmic reticulum, another is that they vesiculate. Using Xenopus egg extracts, nuclei have been assembled and then induced to breakdown by addition of metaphase extract. Field emission scanning electron microscopy was used to study disassembly. Strikingly, endoplasmic reticulum-like membrane tubules form from the nuclear surface after the addition of metaphase extracts, but vesicles were also observed. Microtubule inhibitors slowed but did not prevent membrane removal, whereas Brefeldin A, which inhibits vesicle formation, stops membrane disassembly, suggesting that vesiculation is necessary. Structures that looked like coated buds were observed and buds were labelled for beta-COP. We show that nuclear pore complexes are dismantled and the pore closed prior to membrane rupturing, suggesting that rupturing is an active process rather than a result of enlargement of nuclear pores.

Actin- and protein-4.1-containing filaments link nuclear pore complexes to subnuclear organelles in Xenopus oocyte nuclei.

We imaged the interiors of relatively intact Xenopus oocyte nuclei by field emission scanning electron microscopy (feSEM) and visualized a network of filaments that attach to nuclear pore complexes and extend throughout the nucleus. Within the nucleus, these 'pore-linked filaments' (PLFs) were embedded into spherical structures 100 nm to approximately 5 microm in diameter. A subset of spheres was identified as Cajal bodies by immuno-gold labeling; the rest were inferred to be nucleoli and snurposomes both of which are abundant in Xenopus oocyte nuclei. Most PLFs were independent of chromatin. The thickness of a typical PLF was 40 nm (range, approximately 12-100 nm), including the 4 nm chromium coat. PLFs located inside the nucleus merged, bundled and forked, suggesting architectural adaptability. The PLF network collapsed upon treatment with latrunculin A, which depolymerizes actin filaments. Jasplakinolide, which stabilizes actin filaments, produced PLFs with more open substructure including individual filaments with evenly-spaced rows of radially projecting short filaments. Immuno-gold labeling of untreated oocyte nuclei showed that actin and protein 4.1 each localized on PLFs. Protein 4.1-gold epitopes were spaced at approximately 120 nm intervals along filaments, and were often paired ( approximately 70 nm apart) at filament junctions. We suggest that protein 4.1 and actin contribute to the structure of a network of heterogeneous filaments that link nuclear pore complexes to subnuclear organelles, and discuss possible functions for PLFs in nuclear assembly and intranuclear traffic.

Yeast nuclear pore complexes have a cytoplasmic ring and internal filaments.

The nuclear pore complex (NPC) controls transport of macromolecules across the nuclear envelope. It is large and complex but appears to consist of only approximately 30 different proteins despite its mass of > 60MDa. Vertebrate NPC structure has been analyzed by several methods giving a comprehensive architectural model. Despite our knowledge of yeast nucleoporins, structural data is more limited and suggests the basic organization is similar to vertebrates, but may lack some peripheral and other components. Using field emission scanning electron microscopy to probe NPC structure we found that the yeast, like higher eukaryotic, NPCs contain similar peripheral components. We can detect cytoplasmic rings and evidence of nucleoplasmic rings in yeasts. A filamentous basket is present on the nucleoplasmic face and evidence for cytoplasmic filaments is shown. We observed a central structure, possibly the transporter, that which may be linked to the cytoplasmic ring by internal filaments. Immuno-gold labeling suggested that Nup159p may be attached to the cytoplasmic ring, whereas Nup116p may be associated, partly, with the cytoplasmic filaments. Analysis of a Nup57p mutant suggested a role in maintaining the stability of cytoplasmic components of the NPC. We conclude that peripheral NPC components appear similar in yeasts compared to higher organisms and present a revised model for yeast NPC structural composition.

Concentration of Ran on chromatin induces decondensation, nuclear envelope formation and nuclear pore complex assembly.

Nuclear envelope (NE) formation can be studied in a cell-free system made from Xenopus eggs. In this system, NE formation involves the small GTPase Ran. Ran associates with chromatin early in nuclear assembly and concentration of Ran on inert beads is sufficient to induce NE formation. Here, we show that Ran binds to chromatin prior to NE formation and recruits RCC1, the nucleotide exchange factor that generates Ran-GTP. In extracts prepared by high-speed centrifugation, increased concentrations of Ran are sufficient to induce chromatin decondensation and NE assembly. Using field emission in-lens scanning electron microscopy (FEISEM), we show that Ran promotes the formation of smoothed membranes and the assembly of nuclear pore complexes (NPCs). In contrast, RanT24N, a mutant that fails to bind GTP and inhibits RCC1, does not support efficient NE assembly, whereas RanQ69L, a mutant locked in a GTP-bound state, permits some membrane vesicle recruitment to chromatin, but inhibits vesicle fusion and NPC assembly. Thus, binding of Ran to chromatin, followed by local generation of Ran-GTP and GTP hydrolysis by Ran, induces chromatin decondensation, membrane vesicle recruitment, membrane formation and NPC assembly. We propose that the biological activity of Ran is determined by its targeting to structures such as chromatin as well as its guanine nucleotide bound state.