Chapter six - Nuclear Pore Complex: Biochemistry and Biophysics of Nucleocytoplasmic Transport in Health and Disease

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Abstract

Nuclear pore complexes (NPCs) are the gateways connecting the nucleoplasm and cytoplasm. This structures are composed of over 30 different proteins and 60–125 MDa of mass depending on type of species. NPCs are bilateral pathways that selectively control the passage of macromolecules into and out of the nucleus. Molecules smaller than 40 kDa diffuse through the NPC passively while larger molecules require facilitated transport provided by their attachment to karyopherins. Kinetic studies have shown that approximately 1000 translocations occur per second per NPC. Maintaining its high selectivity while allowing for rapid translocation makes the NPC an efficient chemical nanomachine. In this review, we approach the NPC function via a structural viewpoint. Putting together different pieces of this puzzle, this chapter confers an overall insight into what molecular processes are engaged in import/export of active cargos across the NPC and how different transporters regulate nucleocytoplasmic transport. In the end, the correlation of several diseases and disorders with the NPC structural defects and dysfunctions is discussed.

Introduction

The nucleus and cytoplasm are home to a myriad of processes vital to the cell. These processes are dependent upon shuttling of various macromolecules between these two environments (Kau et al., 2004). Such transport phenomena occur through nanopores called nuclear pore complexes (NPCs). NPCs are embedded in the nuclear envelop (NE), where the internal and external membranes fuse (Fig. 6.1) (Peters, 2009a).

Different studies have shown that the NPC structure resembles an hourglass (Peters, 2009a) or a donut (Stoffler et al., 2003) shape but has an octagonal radial symmetry around its central axis (Frenkiel-Krispin et al., 2010, Miao and Schulten, 2009, Wolf and Mofrad, 2008, Yang and Musser, 2006) as well as a pseudo-twofold symmetry across the NE (Frenkiel-Krispin et al., 2010, Miao and Schulten, 2009). This structure comprises eight centered cylindrical frameworks, each called a spoke, which enclose a central channel. This channel is sandwiched between the cytoplasmic and nuclear rings (Fig. 6.1; Akey and Radermacher, 1993, Wolf and Mofrad, 2008). In addition, eight cytoplasmic filaments emanate from the cytoplasmic ring to the cytoplasm and eight nuclear filaments, which are branched from the nuclear ring, join each other in the nuclear side of the NPC, shaping a basket-like structure known as the nuclear basket (Fig. 6.1; Elad et al., 2009). Various microscopic studies have shown that the overall length of the NPC including its extended cytoplasmic and nuclear filaments reaches 150–200 nm, while its external diameter is around 100–125 nm. The radius of the cytoplasmic and nuclear sides of this channel is 60–70 nm, and it narrows to about 25–45 nm at the center (Adam, 2001, Brohawn and Schwartz, 2009, DeGrasse et al., 2009, Kau et al., 2004, Schwartz, 2005, Stoffler et al., 2003, Yang and Musser, 2006) (Fig. 6.1). Nevertheless, the opening diameter of this channel has been estimated to be approximately 10 nm (Shulga and Goldfarb, 2003). In addition to the central channel, some peripheral channels with diameters of about 8 nm reportedly exist, which contribute to transport of ions and small proteins (Frenkiel-Krispin et al., 2010, Kramer et al., 2007, Stoffler et al., 2003).

The NPC, as the largest nanomachine of the cell, is a bilateral, selective filter for a variety of molecules, transporting them quite rapidly without completely opening or closing the gateway structure (Kapon et al., 2008). Small molecules and ions (Mr ~ 20–40 kDa, diameter ~ 5–9 nm) travel through the NPC via passive diffusion, whereas larger molecules (Mr > 40 kDa up to ~ 25 MDa, diameter of up to ~ 40 nm) such as ribosomes, RNAs, and some proteins can only be transported through an active mechanism regulated by transporters (Miao and Schulten, 2009, Yang and Musser, 2006). It is observed that the size of the entering molecule affects the transport rate through the NPC (Moussavi-Baygi et al., 2011). A sharp drop in the transport rate is expected for cargo complexes with radii larger than the channel radius (Peters, 2005). Some researchers reported that passive diffusion and facilitated transport are not coupled and occur through different pathways (Naim et al., 2007). Nevertheless, recent studies provided evidence to refute this hypothesis, suggesting that passive and facilitated transports take place through the same channel in the NPC (Mohr et al., 2009). Studies of microscopy indicate that the NPC structure dynamically changes its conformation in response to chemical or physical effectors, such as alterations in calcium ion concentration, Co2, and ATP in the cell environment (Erickson et al., 2006, Oberleithner et al., 2000, Rakowska et al., 1998).

Proteomics analyses show that the NPC is a supra molecule made up of approximately 30 types of proteins named nucleoporins (Nups) (Fig. 6.2) (Hetzer, 2010, Walde and Kehlenbach, 2010). Due to the eightfold symmetry of the NPC structure, Nups have been observed in sets of multiple (8–48) copies in yeast and mammalian cells (Bednenko et al., 2003b). Therefore, the total number of Nups per NPC is estimated to be approximately 500–1000 (Peters, 2005, Sorokin et al., 2007). Some Nups are symmetrically found in both cytoplasmic and nuclear sides, some are only present on one side, and others make up the central framework (Strawn et al., 2004, Zeitler and Weis, 2004). This structure also possesses some mobile Nups that have different activities during each period of the cell cycle (Hou and Corces, 2010, Terry and Wente, 2009, Walde and Kehlenbach, 2010). The structure of the NPC is composed of three concentric layers (Fig. 6.1; Lusk et al., 2007, Suntharalingam and Wente, 2003, Walde and Kehlenbach, 2010): (1) The FG repeat layer is the innermost layer of the NPC and is directly exposed to cargo undergoing transport; this layer coats the channel, facilitating the active transport of cargos (Terry and Wente, 2009). (2) The membrane layer is the outermost layer that anchors the NPC to the NE. (3) The scaffold layer is located between the above-mentioned layers, forming the structure of the NPC (Lusk et al., 2007). These layers are composed of various Nups (Fig. 6.2). The first layer is composed of phenylalanine–glycine-rich repeat domains such as FxFG, GLFG, PxFG, and SxFG. These Nups have flexible structures and are spread over the peripheral and central parts of the NPC (Denning et al., 2003, Peleg and Lim, 2010, Zeitler and Weis, 2004) (Fig. 6.1). It is reported that FG domains include some one-third of the Nups, and FG repeat domains account for 12–20% of the NPC mass (Devos et al., 2006, Frey and Gorlich, 2007). Although the cargo complex transport mechanism through the NPC remains unsolved, several models, such as virtual gate, selective phase, reduction of dimensionality, reversible FG, and forest model (Frenkiel-Krispin et al., 2010, Moussavi-Baygi et al., 2011, Peleg and Lim, 2010, Suntharalingam and Wente, 2003), have been proposed, each elucidating a transport mechanism through this proteinaceous structure in a distinct way.

The flexible domains of these Nups play a key role in the passage of cargos along the nucleotransport pathway via their low affinity with cargo. Molecules that travel through the NPC by binding to FG repeats have significantly higher transport rates than those without attachment to FG repeats, given their similar size and shapes (Ribbeck and GoÈrlich, 2001). Additionally, FG-Nups are equipped with coiled coil domains, which anchor them to the NPC bulk (Devos et al., 2006). The membrane layer of the NPC is composed of integral membrane proteins, which have transmembrane helices. Generally, these structures are equipped with α-helical domains to anchor them to the NE and with Cadherin-fold domains to reinforce the NE against excessive lateral movements. The only nondynamic Nups of the NPC are those existing in the membrane layer (Bednenko et al., 2003b). Aside from Nups containing FG, coiled coil, and transmembrane domains, all other Nups are part of the scaffold (the third) layer and include approximately 1/2 of all Nups (Walde and Kehlenbach, 2010). These Nups have β-propeller and α-solenoid structures and a significant percentage of the NPC mass is composed of these Nups, with approximately one-third of Nups containing α-solenoid domains (Devos et al., 2006, Rout et al., 2000). In addition, some Nups include specific structural motifs such as “Zinc finger domains” and motifs connected to RNA (Cassola and Frasch, 2009, Yaseen and Blobel, 1999).

Generally, the number of NPCs on the NE is independent of the nucleus’ surface area and DNA volume. The number of NPCs per nucleus varies significantly for different species, environmental conditions, cell activities, and periods of the cell life cycle (Gerace and Burke, 1988). For example, yeast barely have 200 NPCs per nucleus, while the number of NPCs per nucleus for vertebrates is on average 2000–5000 (10–20 pores/μm2) (Fabre and Hurt, 1997). Large nuclei of Xenopus oocytes are home to as many as 5 × 107 NPCs (over 60 pores/μm2) (Gorlich and Kutay, 1999). While NPCs are seen to exist throughout the cell cycle, the number of these pores doubles in dividing cells during interphase and before mitosis and they reach a maximum number in the S-phase of the cell cycle (D'Angelo et al., 2006). It has also been shown that some hormones increase the number and density of NPCs (Miller et al., 1991). Apparently, an increased number of NPCs provides some cells with resistance to chemotherapy (Lim et al., 2008). Experiments clearly indicate an increase in the number of NPCs in embryonic stem cells (ES) as they differentiate into proliferative cardiomyocytes. Therefore, differentiation of embryonic stem cells could be traced to changes in the number of NPCs. Differentiation of ES in cardiac progeny is likely associated with the structural and functional remodeling of the NPC (Lim et al., 2008, Perez-Terzic et al., 2007). A comparison between isolated cardiomyocytes from heart and ES-originated cells indicates no difference in dimensions of the NPCs. Nonetheless, it is reported that the number of NPCs in cells isolated from the heart is greater than those originating from stem cells. Also, increased transport activity of the NPC is reportedly observed in stem cell-derived cardiomyocytes (Perez-Terzic et al., 2003).

The overall architecture of the NPC is conserved among the eukaryotic cells (Brohawn and Schwartz, 2009) and this conservation is established in the last eukaryotic common ancestor (DeGrasse et al., 2009). It is likely that the NPC has been a structural part of another organism like archea (Bapteste et al., 2005). Also, it is speculated that NPCs are the chimaeras from endomembrane and mitosis-related chromatin-associated proteins (Cavalier-Smith, 2010). Investigations conducted on yeast and mammalian cells indicate that functions and localization of their NPCs are similar even though their sequences are not exactly conserved (Bapteste et al., 2005, Kiseleva et al., 2004, Neumann et al., 2006, Yasuhara et al., 2009). Interestingly, a large number of Nups are conserved among all known eukaryotic cells, but many of them are specific to certain cells (Frenkiel-Krispin et al., 2010, Neumann et al., 2006). As a result, it is claimed that the overall shape and size of the NPC has been conserved through evolution (Alber et al., 2007, Yasuhara et al., 2009); however, some structural distinctions are observable among NPCs of different species (Elad et al., 2009). Structural analyses illustrate some differences in the location of Nups as well as their number of copies among the NPCs of different species. In contrast to related species, though, NPCs of distinct species have low structural homology (Frenkiel-Krispin et al., 2010).

Indeed, transport through the NPC and the NPC components provides a broad range of vital functions for the cell. Comparative proteomic analysis of the NPC predicts some unexpected functions for this massive complex (Elad et al., 2009). Generally, this structure could affect indispensible cellular functions, such as gene expression, DNA damage and repair, aging, apoptosis, and even determination of cell differentiation and fate (Batrakou et al., 2009, Mishra et al., 2010, Nagai et al., 2008, Nakano et al., 2010, Yasuhara et al., 2009, Yasuhara et al., 2007, Wolf and Mofrad, 2009). As a result, any structural defect or malfunction in this key regulator could cause different diseases or even death. Since understanding the NPC structure is essential to deciphering its function, many scientists have been engaged in structural studies of this large complex during the past few years and various approaches have been exploited to investigate the role of the NPC in different diseases with the hope to find remedial solutions. In this review, we first examine and discuss how different molecules are transported through the NPC while the dysfunctions caused by the NPC structural defects will be described at the end.

This review is composed of two major parts. In the first part (2 What Drives Cargo Transport Through the NPC?, 3 Nucleocytoplasmic Transport Pathway), we will discuss briefly how different molecules (Fig. 6.3) orchestrate the exquisite process of nucleocytoplasmic transport (NCT). Since some cargos need to form complexes with specific molecules, termed transporters, in order to be able to pass through the NPC, we will first explain the definition of transporters along with signal sequences on the cargo transporter proteins required to identify a protein. Section 2.1 will focus mainly on the structure of important transporters, such as importin α (impα) and importin β (impβ), and interactions between cargos and these transporters, which lead to the formation of a nucleus entering cargo complex in the cytoplasm. In Section 2.2, we will follow the pathway of a typical active cargo through the NPC, while Section 2.3 will take a glance at some newly deciphered molecular structures, such as Ran, Nup50 (Nup2p), and their interaction with the entering cargo complexes, which leads to dissociation of the complex in the nuclear basket. Next, in Section 2.4, the recycling pathway of transporters to the cytoplasm and the molecular structure of cellular apoptosis susceptibility molecule (CAS), which is highly engaged in this process, will be explained. Section 2.5 will examine the Ran cycle (hydrolysis process of GTP of RanGTP and replacement of GTP with GDP in RanGDP), the molecules involved in this cycle, and their contribution to NCT. Finally, in Section 3, we conclude this part of the review by summarizing the nucleocytoplasmic pathway mechanism. The second part of this review is dedicated to diseases and disorders linked to NPC structural and functional defects, for example cancer, nervous system and autoimmune disorders, and cardiac and infectious diseases will be discussed.

Section snippets

What Drives Cargo Transport Through the NPC?

The major task of the NPC is control and regulation of the traffic of macromolecules into and out of the nucleus. When considering the bidirectional transport of cargos from the cytoplasm to the nucleus and vice versa, on average, as many as 1000 transports are observed through an NPC per second (Fahrenkrog et al., 2004, Peters, 2005), with this high-throughput rate of cargo translocation achieved via transporters. The most important category of transporters is known as the β karyopherin

Nucleocytoplasmic Transport Pathway

The main NCT pathways are shown in Fig. 6.19. Nuclear pore complexes mediate bidirectional transport of various macromolecules. At first, in the cytoplasm, the NLS-carrying cargo through interaction with impα binds to the impα/impβ complex to form an import complex, which then docks the cytoplasmic filaments. This complex passes across the NPC toward the nucleus via weak interactions with FG-Nups. Inside the nucleus, the imported complex attaches to the nuclear basket Nups such as Nup50 (Nup2).

NPC and Diseases

Structural changes in Nups and carriers or defects in transport pathways leading to nuclear or cytoplasmic overaccumulation of materials are correlated with a number of diseases, such as cancer, immune system disorders, and nervous system diseases. Sometimes, an alteration in the transport mechanism is the reason behind these types of diseases; while they could also caused by conformational changes or malfunctions appear as a result of an NPC disorder. A thorough understanding of the relation

Conclusion

The purpose of this review was to examine the nucleocytoplasmic pathways, interaction of molecules taking part in transport processes, and NPC-related diseases. In the past few years, major scientific efforts have been made to express and analyze the sophisticated structure of the NPC, the bilateral NPC pathways, and the roles played by molecules involved in the NCT process, which are critical to understanding how this super-efficient nanomachine works and how it may potentially control the

Acknowledgments

The authors thank members of Molecular Cell Biomechanics Laboratory, especially Dr. Mohammad Azimi, for fruitful discussions and editorial assistance.

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