Nuclear pore complex assembly through the cell cycle: regulation and membrane organization

In eukaryotes, all macromolecules traffic between the nucleus and the cytoplasm through nuclear pore complexes (NPCs), which are among the largest supramolecular assemblies in cells. Although their composition in yeast and metazoa is well characterized, understanding how NPCs are assembled and form the pore through the double membrane of the nuclear envelope and how both processes are controlled still remains a challenge. Here, we summarize what is known about the biogenesis of NPCs throughout the cell cycle with special focus on the membrane reorganization and the regulation that go along with NPC assembly.     

Classical nuclear localization signals: definition, function, and interaction with importin alpha

The best understood system for the transport of macromolecules between the cytoplasm and the nucleus is the classical nuclear import pathway. In this pathway, a protein containing a classical basic nuclear localization signal (NLS) is imported by a heterodimeric import receptor consisting of the beta-karyopherin importin beta, which mediates interactions with the nuclear pore complex, and the adaptor protein importin alpha, which directly binds the classical NLS. Here we review recent studies that have advanced our understanding of this pathway and also take a bioinformatics approach to analyze the likely prevalence of this system in vivo. Finally, we describe how a predicted NLS within a protein of interest can be confirmed experimentally to be functionally important.     

In and around the nucleus

Studies of how the eukaryotic nucleus is functionally organized have led to the realization that nuclei are incredibly dynamic. Many nuclear structures are actually by products of a large steady-state flux of macromolecules through a given domain. A recent conference in the south of France on Nuclear Structure and Dynamics brought together scientists with diverse perspectives on the nucleus to try to provide a more coherent picture of the nucleus's dynamic organization and how this architecture is entwined with epigenetic control of gene expression.     

Assembly,organization, and function of the COPII coat

A full mechanistic understanding of how secretory cargo proteins are exported from the endoplasmic reticulum for passage through the early secretory pathway is essential for us to comprehend how cells are organized, maintain compartment identity, as well as how they selectively secrete proteins and other macromolecules to the extracellular space. This process depends on the function of a multi-subunit complex, the COPII coat. Here we describe progress towards a full mechanistic understanding of COPII coat function, including the latest findings in this area. Much of our understanding of how COPII functions and is regulated comes from studies of yeast genetics, biochemical reconstitution and single cell microscopy. New developments arising from clinical cases and model organism biology and genetics enable us to gain far greater insight in to the role of membrane traffic in the context of a whole organism as well as during embryogenesis and development. A significant outcome of such a full understanding is to reveal how the machinery and processes of membrane trafficking through the early secretory pathway fail in disease states.     

Enzymatic synthesis and modification of polymers in nonaqueous solvents

Enzymes catalyse several reactions that are difficult to perform with chemical catalysts and that are important in the synthesis and modification of different polymers in organic solvents. In enzyme-based synthesis, alteration of the reaction medium can have a significant influence on the molecular weight, polydispersity, yield and architecture of the polymers that are produced. Modification of these macromolecules for industrial applications requires an understanding of the different reaction strategies involved.     

Ion hydration: Implications for cellular function, polyelectrolytes, and protein crystallization

Only oppositely charged ions with matching absolute free energies of hydration spontaneously form inner sphere ion pairs in free solution [K.D.Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34 (2004) 300-311.]. We approximate this with a Law of Matching Water Affinities which is used to examine the issues of (1) how ions are selected to be compatible with the high solubility requirements of cytosolic components; (2) how cytosolic components tend to interact weakly, so that association or dissociation can be driven by environmental signals; (3) how polyelectrolytes (nucleic acids) differ from isolated charges (in proteins); (4) how ions, osmolytes and polymers are used to crystallize proteins; and (5) how the "chelate effect" is used by macromolecules to bind ions at specific sites even when there is a mismatch in water affinity between the ion and the macromolecular ligands.     

THE NUCLEAR PERMEABILITY, INTRACELLULAR DISTRIBUTION, AND DIFFUSION OF INULIN IN THE AMPHIBIAN OOCYTE

[³H]Inulin (mol wt ≈ 5,500) solutions are microinjected into the cytoplasm of mature oocytes of Rana pipiens and the subsequent movement of the solute recorded by quantitative ultralow temperature autoradiography. The autoradiographs show transient cellular diffusion gradients, the influence of the nucleus on these gradients, and the nuclear:cytoplasmic distribution of inulin. Analysis leads to the following conclusions: (a) Inulin diffuses in cytoplasm at about 3 x 10^-6 cm²/s, or one-fifth as rapidly as in water. Most of this decrease is attributable to the increased tortuosity of the diffusional path due to the presence of inclusions and macromolecules. (b) The nuclear envelope is very permeable to inulin; its resistance to inulin's passage is similar to that of cytoplasm. The envelope appears to play a negligible role in regulating the nucleocytoplasmic movement of solutes smaller than macromolecules, (c) Inulin concentrates in the nucleus to four times its cytoplasmic level; this is attributed to solute exclusion from cytoplasmic water. Evidence is presented that among hydrophilic solutes the degree of exclusion increases with molecular size. The potential significance of cytoplasmic exclusion processes to understanding secretion and the intracellular movement of macromolecules is briefly discussed.     

Molecular trafficking across the nuclear pore complex.

The nuclear pore complex is the gateway for protein and RNA transport between the cytoplasm and nucleus. Recent work has characterized signals and components involved in nuclear import of macromolecules and has described mechanisms for transport regulation. Advances in understanding the structure of the pore complex are starting to provide a framework for interpreting the biochemistry of nuclear import. Information on the export of RNA from the nucleus is only beginning to emerge.     

Mapping the amino acid properties of constituent nucleoporins onto the yeast nuclear pore complex

Visualization of molecular structures aids in the understanding of structural and functional roles of biological macromolecules. Macromolecular transport between the cell nucleus and cytoplasm is facilitated by the nuclear pore complex (NPC). The ring structure of the NPC is large and contains several distinct proteins (nucleoporins) which function as a selective gate for the passage of certain molecules into and out of the nucleus. In this note we demonstrate the utility of a python code that allows direct mapping of the physiochemical properties of the constituent nucleoporins on the scaffold of the yeast NPC׳s cytoplasmic view. We expect this tool to be useful for researchers to visualize the NPC based on their physiochemical properties and how it alters when specific mutations are introduced in one or more of the nucleoporins. The code developed using Python is available freely from the authors.     

The nuclear pore complex mystery and anomalous diffusion in reversible gels

The exchange of macromolecules between the cytoplasm and the nucleus of eukaryotic cells takes place through the nuclear pore complex (NPC), which contains a selective permeability barrier. Experiments on the physical properties of this barrier appear to be in conflict with current physical understanding of the rheology of reversible gels. This paper proposes that the NPC gel is anomalous and characterized by connectivity fluctuations. It develops a simplified model to demonstrate the possibility of enhanced diffusion constants of macromolecules trapped in such a gel.     

Aaron Klug and the revolution in biomolecular structure determination

Aaron Klug's group was one of the first to use a combination of X-ray diffraction and electron microscopy to study the structures of macromolecules. He helped to provide the intellectual framework for understanding the self-assembly of regular viruses and developed methods for analyzing their three-dimensional structures from electron microscope images, as well as the structures of helical polymers. He and his coworkers established the basic features of chromatin organization, including the structure of the repeating units (nucleosomes) and how they are stacked together. He studied a variety of molecules that interact with DNA or RNA, including disks of tobacco mosaic virus protein, a tRNA and a ribozyme, and also discovered the zinc-finger motif in nucleic acid-binding proteins. Thus, he has played a major part in developing the ideas and techniques that established structural molecular biology as an exciting new science during the second half of the twentieth century.     

Nucleocytoplasmic transport of macromolecules.

Nucleocytoplasmic transport is a complex process that consists of the movement of numerous macromolecules back and forth across the nuclear envelope. All macromolecules that move in and out of the nucleus do so via nuclear pore complexes that form large proteinaceous channels in the nuclear envelope. In addition to nuclear pores, nuclear transport of macromolecules requires a number of soluble factors that are found both in the cytoplasm and in the nucleus. A combination of biochemical, genetic, and cell biological approaches have been used to identify and characterize the various components of the nuclear transport machinery. Recent studies have shown that both import to and export from the nucleus are mediated by signals found within the transport substrates. Several studies have demonstrated that these signals are recognized by soluble factors that target these substrates to the nuclear pore. Once substrates have been directed to the pore, most transport events depend on a cycle of GTP hydrolysis mediated by the small Ras-like GTPase, Ran, as well as other proteins that regulate the guanine nucleotide-bound state of Ran. Many of the essential factors have been identified, and the challenge that remains is to determine the exact mechanism by which transport occurs. This review attempts to present an integrated view of our current understanding of nuclear transport while highlighting the contributions that have been made through studies with genetic organisms such as the budding yeast, Saccharomyces cerevisiae.     

Dynamics of nuclear pore complex organization through the cell cycle

In eukaryotic cells, all macromolecules that traffic between the nucleus and the cytoplasm cross the double nuclear membrane through nuclear pore complexes (NPCs). NPCs are elaborate gateways that allow efficient, yet selective, translocation of many different macromolecules. Their protein composition has been elucidated, but how exactly these nucleoporins come together to form the pore is largely unknown. Recent data suggest that NPCs are composed of an extremely stable scaffold on which more dynamic, exchangeable parts are assembled. These could be targets for molecular rearrangements that change nuclear pore transport properties and, ultimately, the state of the cell.     

Nuclear RNA export in yeast.

Eukaryotic cells massively exchange macromolecules (proteins and RNAs) between the nucleus and cytoplasm through the nuclear pore complexes. Whereas a mechanistic picture emerges of how proteins are imported into and exported from the nucleus, less is known about nuclear exit of the different classes of RNAs. However, the yeast Saccharomyces cerevisiae offers an experimental system to study nuclear RNA export in vivo and thus to genetically dissect the different RNA export machineries. In this review, we summarize our current knowledge and recent progress in identifying components involved in nuclear RNA export in yeast.     

Misfolded proteins and human diseases

Though protein folding is a regular phenomenon inside the cellular milieu, slight differences in the folding pattern of proteins may lead to disease-causing forms. Many diseases have been identified that are caused by these misfolded macromolecules and a considerable amount of focus is still directed towards better understanding of the processes that lead to these misfolded structures. Further progress in the field of how soluble proteins begin to misfold and how resultant oligomers begin dysfunction offers exciting prospects for specific molecular intervention.     

Microtubules form by progressively faster tubulin accretion,not by nucleation–elongation

Microtubules are dynamic polymers that play fundamental roles in all eukaryotes. Despite their importance, how new microtubules form is poorly understood. Textbooks have focused on variations of a nucleation–elongation mechanism in which monomers rapidly equilibrate with an unstable oligomer (nucleus) that limits the rate of polymer formation; once formed, the polymer then elongates efficiently from this nucleus by monomer addition. Such models faithfully describe actin assembly, but they fail to account for how more complex polymers like hollow microtubules assemble. Here, we articulate a new model for microtubule formation that has three key features: (1) microtubules initiate via rectangular, sheet-like structures that grow faster the larger they become; (2) the dominant pathway proceeds via accretion, the stepwise addition of longitudinal or lateral layers; and (3) a “straightening penalty” to account for the energetic cost of tubulin’s curved-to-straight conformational transition. This model can quantitatively fit experimental assembly data, providing new insights into biochemical determinants and assembly pathways for microtubule nucleation.     

Nuclear inositide signalling -- expansion, structures and clarification

The extent and content of this review issue highlights how our understanding of lipid signalling in the nucleus has grown, both in what we actually know, and the breadth of signalling pathways that we now have to consider. Here, a few key issues with regard to nuclear inositide signalling are briefly addressed.