Nuclear pore assembly

Dabauvalle. M.-C., Loos, K., and Scheer, U. (1990). Identification of a soluble precursor complex essential for nuclear pore assembly in vitro. Chromosoma 100, 56-66. 

In the last few years, a large number of in vitro and in vivo studies have suggested that membrane curvature plays a major role in regulating multiple cellular phenomena, ° including vesicular trafficking, nuclear pore assembly or autophagocytosis. 

Replicative cycles of representative DNA and RNA viruses. A. Replicative cycle of a herpesvirus, an example of a DNA virus. 1. Attachment. 2. Membrane fusion. 3. Release of viral DNA through nuclear pores. 4. Transcription of viral mRNA. 5. Synthesis of viral proteins by host cell s ribosomes. 6. Replication of viral DNA by viral polymerases. 7. Assembly of virus particles. 

The nuclear envelope is perforated with huge macromolecular assemblies of 30 different proteins that form nuclear pore complexes with a central channel of 25-30 nm in diameter. This channel allows proteins smaller than 30 kDa to passively traverse the outer and inner nuclear membranes. Larger proteins are actively transported across the nuclear envelope and contain nuclear localization signal (NLS) sequence motifs. These signals consist of one or two clusters of four or five basic residues localized usually within the polypeptide chain. The import of proteins with NLS through the channel is facilitated by the carrier heterodimer of importin-a ( > (Gorlich and Kutay 1999 Pemberton and Paschal... 

Molecular communication between the nucleus and the cytosol requires the movement of macromolecules through nuclear pores. RNA molecules synthesized in the nucleus are exported to the cytosol. Ribosomal proteins synthesized on cytosolic ribosomes are imported into the nucleus and assembled into 60S and 40S ribosomal subunits in the nucleolus completed subunits are then exported back to the cytosol. A variety of nuclear proteins (RNA and DNA polymerases, histones, topo-isomerases, proteins that regulate gene expression, and so forth) are synthesized in the cytosol and imported into the nucleus. This traffic is modulated by a complex system of molecular signals and transport proteins that is gradually being elucidated. 

In telophase, nuclei for each daughter cell form at the two poles, and the mitotic spindle apparatus disappears. Furthermore, nuclear membranes, nuclear lamina, nuclear pores, and nucleoli are reformed. The cell is now ready for cytokinesis, which is physical division of the cytoplasm. The cytoplasm divides as actin/myosin filaments contract and pinch off the plasma membrane, which results in two daughter cells that enter into Go or Gi for another round of division. The main checkpoint that exists during M phase in mammalian cells is the spindle checkpoint it is in place to ensure proper microtubule assembly, proper cell division, and that each daughter cell receives one copy of DNA. 

Aguilera, A. 2005, Cotranscriptional mRNP assembly From the IVX.A to the nuclear pore. Curr. Opin. (iell Biol. 17 242-].50,... 

Fig. 10.21. The nuclear pore complex. The approximately 100 different polypeptide chains of the nuclear pore complex form an assembly of 8 spokes attached to two ring structures (a cytoplasmic ring in the outer nuclear membrane and a nuclear ring through the inner membrane) with a transporter plug in the center. Small molecules, ions, and proteins with less than a 50-kDa mass passively diffuse through the pore in either direction. However, RNAs and most proteins are too large to diffuse through, and are actively transported in a process that requires energy, is selective for the molecule transported, is unidirectional, and can be regulated.

Transient PPIs are many and varied. The enumeration of their involvement in so many vital biological functions has reached daunting levels. There are thus many examples, including the recruitment and assembly of the transcription complex, protein transport across membranes, chaperonin-catalyzed protein folding, and the recycling of subcellular structures during the cell cycle. Such recycling includes that of microtubules, the spindle apparatus, the nuclear pore complex, and the nuclear lamina. 

Guam, T., Muller, S., Klier, G., Pante, N., Blevitt, J. M., Haner, M., Paschal, B., Aebi, U., and Gerace, L. (1995). Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex. Mol. Biol. Cell 1591-1603. Harris, J. R. (1977). Fractionation of the nuclear envelope. In Methodological Surveys in Biochemistry. Membranous Elements and Movement of Molecules (E. Reid, ed.), Vol. 6, pp. 245-250. Horwood, Chichester. 

Stewart, M., and Garkson, W. D. (1996). Nuclear pores and macromolecular assemblies involved in nucleocytoplasmic transport. Curr. Opin. Struct. BioL 6,162-165. 

Goldberg, M. W., Wiese, C., Allen, T. D., and Wilson, K. L. (1997). Dimples, pores, star rings and thin rings on growing nuclear envelopes Evidence for structural intermediates in nuclear pore complex assembly. J. Cell. Sci. 110, 409-420. 

Reassembly of nuclei can be followed most easily by phase-contrast microscopy. During the course of the assembly reaction clusters of chromosomes will be observed to fuse into single masses of chromatin, which will eventually become spherical in shape as decondensation progresses. At this point, thin-section electron microscopy should reveal the presence of a continuous double membrane studded with nuclear pore complexes surrounding each chromatin mass. Assembly of individual nuclear envelope components such as nuclear lamins or nuclear pore complex proteins can best be analyzed by immunofluorescence microscopy (Burke and Gerace, 1986). This is performed as follows. 

Macaulay, C and Forbes, D. J. (1996). Assembly of the nuclear pore Biochemically distinct steps revealed with NEM, GTPyS and BAPTA. J. Cell Biol 132, 5-20. 

Meier, E Miller, B., and Forbes, D. J. (1995). Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation. J. Cell Biol 129, 1459-1472. 

Figure 10. AFM measurements of nuclear pore complex (NPC) conformation in the < > 0 (A) and closed (B) configuration. Nuclear pores are 12S MDa protein assemblies that control transport across the nuclear envelope. The outer diameter of each NPC is 120 nm. Several groups have found evidence for a calcium dependent displacement of the central granule in the pore region from recessed (A), to extended (B), following calcium release. (Fahrenkrog, Stoffler et al. 2001 Moore-Nichols, Amott et ai. 2002 Perez-Teizic, Pyle et al. 1996 Stoffler, Feja et al. 2003 Wang and Clapham 1999) Repriced with permission from (Vickery et al. 1999). Copyright 2000 Biophysical Society.

Dabauvalle MC, Scheer U (1991) Assembly of nuclear pore complexes in Xenopus egg extract. Biol Cell 72 25-29... 

Each cell nucleus contains one or more dense nucleoli, regions that are rich in RNA and may contain 10-20% of the total RNA of cells. Nucleoli are sites of synthesis and of temporary storage of ribosomal RNA, which is needed for assembly of ribosomes. The nuclear envelope is a pair of membranes, usually a few tens of nanometers apart, that surround the nucleus. The two membranes of the pair separate off a thin perinuclear space (Fig. 1-7). The membranes contain "pores" -130 ran in diameter with a complex structure (see Fig. 27-8).38/39 There is a central channel -42 ran in diameter, which provides a route for controlled passage of RNA and other large molecules from the nucleus into the cytoplasm and also from the cytoplasm to the nucleus. Smaller -10 nm channels allow passive diffusion of ions and small molecules. 

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