Except for a small amount of DNA in mitochondria and chloroplasts, cellular DNA is enclosed in the nucleus by the double membrane of the nuclear envelope (NE). In a diploid human cell nucleus there are 46 chromosomes, each chromosome containing one DNA molecule. The total length of the 46 DNA molecules in each cell is about 2 meters. Histones are the most abundant proteins in the nucleus. Four histones (H2A, H2B, H3, and H4) each form a homodimer, and four homodimers associate into an octamer. The DNA is 1.65 times wound around a histone octamer. The resulting "beads-on-a-string" configuration represents the most open form of chromosomal DNA. Numerous additional proteins participate in further compaction of chromosomal DNA. The most compact state of each chromosomal DNA is achieved during the second phase of the cell division cycle (metaphase chromosomes).
After the completion of cell division, chromosomal DNA is again decondensed, but not uniformly. Instead, along each chromosomal DNA, more-condensed regions (heterochromatin) alternate with less-condensed (euchromatin) ones. The length and position of stretches of hetero- and euchromatin along each chromosomal DNA are likely to vary from cell type to cell type. Moreover, in each cell type, hetero- and euchromatic regions of each chromosome are thought to occupy distinct territories in the approximately spherical space of the nucleus. Despite the constraints of packaging, euchromatin regions remain highly flexible.
The NE and its associated structures, particularly the inner NE membrane and the NE-embedded NPCs, play a major role in the organization of chromatin. Euchromatin is found subjacent to NPCs, whereas heterochromatin is located in inter-NPC domains.
NPCs are the only route for macromolecular traffic between the nucleus and the cytoplasm.
In recent years, our laboratory has contributed to understanding of the composition of the NPC, its structure, and the traffic across it. We have isolated several of the soluble transport factors, have biochemically characterized them, and have analyzed the structure of some by x-ray crystallography. In addition, we have isolated the first proteins of the NPC and termed them collectively nucleoporins (nups). We have also described specific integral membrane proteins that are located in the pore membrane domain of the NE, the sharply bent membrane connecting the inner and outer membrane of the NE, and termed these integral membrane proteins "pore membrane proteins" (poms). Poms function to anchor nups to the 100-nm-diameter circular opening in the NE in which the NPC is embedded. A yeast nucleus contains about 200 NPCs; an average mammalian nucleus 2,000; and an amphibian oocyte 20,000,000.
In cells undergoing open mitosis, NPCs undergo reversible mitotic disassembly into distinct subcomplexes. Reversible disassembly appears to be regulated by reversible phosphorylation at numerous nup sites. NPCs are doubled in S phase of the cell cycle, but little is known about how they are assembled into the NE.
The NPC is composed of 27 nups and is anchored to the membrane by three distinct poms. Many of the nups are predicted to contain more than one structural module. Besides the natively unfolded phenylalanine-glycine repeat module (FG repeats), there are predicted β propellers, solenoids, and coiled-coil modules.
The NPC is a pseudosymmetric structure with an eightfold nucleocytoplasmic axis and a twofold axis in the plane of the membrane. A cylinder, about 40 nm high, with an internal diameter of about 40 nm, represents the central transport channel. This cylinder is anchored to the pom by eight spokes. About half of the nups contain FG repeats that, like tentacles of a jellyfish, extend for more than 100 nm into the cytoplasm and the nucleoplasm, thereby entropically occluding access to the openings of the central transport channel. The FG repeats serve as docking sites for transport factors and transport substrates. Electron microscopic studies of transport have revealed that the NPC is a highly flexible structure.
Some of our present efforts are focused on structural biology (x-ray crystallography and cryo-electron microscopy), with the long-term goal of obtaining an atomic-resolution structure of the core of the NPC. We are also doing x-ray crystallographic analyses of histone-modifying enzymes and the proteins to which they bind. In other efforts we use traditional methods in cell biology and genetics (using mammalian cells, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) to learn more about the chromatin-organizing role of NPCs. Moreover, we work on integral membrane proteins of the inner and outer membrane that form protein patches in the NE that link to specific regions of heterochromatin on the nuclear side and to microtubules, actin, and intermediate filaments on the cytoplasmic side. These interactions appear to mechanically facilitate large-scale movements of chromatin.