Nuclear Egress of Herpesviruses

Teresa Hellberg , ... Thomas C. Mettenleiter , in Advances in Virus Research, 2016

2.1 The Nuclear Envelope

The NE is essential in maintaining the unique biochemical identity of nucleus and cytoplasm. Two concentric lipid bilayers, the ONM and the INM which are separated by the perinuclear space (PNS), act as a strong physical barrier. The ONM and INM, which do not significantly differ in lipid composition and phospholipid mobility (Schindler, Holland, & Hogan, 1985), are connected at annular junctions, where NPCs are inserted. Despite the similarity in lipid composition, the INM and ONM are biochemically distinct (Hetzer, 2010). While the ONM is continuous and functionally interrelated with the rough endoplasmic reticulum (RER), the INM contains its own set of integral membrane proteins, which provide docking sites for lamins, the host cell chromatin and, via interaction with ONM proteins, the cytoskeleton (Crisp & Burke, 2008).

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Organizational Cell Biology

Y. Mimura , N. Imamoto , in Encyclopedia of Cell Biology, 2016

Nuclear membrane

The nuclear membrane is comprised of two phospholipid bilayers. The membrane facing the cytoplasm is termed the outer nuclear membrane (ONM), and the membrane facing the nucleoplasm is termed the INM. The ONM continuously connects to the ER, and its surface, like that of the ER, is decorated with ribosomes. The INM accumulates specific sets of proteins collectively termed INM proteins, and A-type and B-type lamins construct the nuclear lamina underneath the INM. Many INM proteins, either directly or through association with lamins, interact with chromatin. The ONM is fused with the INM at sites termed pore membrane at which the NPC is present. The NE functions as the physical barrier that protects the nuclear interior from the cytoplasmic activities ( Figure 1).

Figure 1. Structure of metazoan nuclear envelope (NE). Schematic representation of the overview of NE. The nuclear pore complex is embedded in the NE composed of the inner nuclear membrane (INM) and the outer nuclear membrane (ONM). INM proteins containing LEM proteins, SUN1/2 and Lamin B receptor (LBR) exclusively localized to the INM. SUN1/2 and Nesprin form linker of nucleoskeleton and cytoskeleton (LINC) complex via SUN–KASH interaction, and connect the nucleoskelton to cytoskeleton through their interaction. Heterochromatin is accumulated underneath the INM via the interaction with INM proteins and the nuclear lamina.

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International Review of Cell and Molecular Biology

Roberto Bravo , ... Sergio Lavandero , in International Review of Cell and Molecular Biology, 2013

2.1.2 Nuclear Envelope

The NE is a highly specialized and selectively permeable double membrane that surrounds the genetic material of the cell. Electron microscopic analysis revealed early on that NE and ER form a continuous structure (Watson, 1955), sharing some common components, while others, like nuclear pore-forming nucleoporins (NUPs), are specific for the NE. Other important components are the transmembrane proteins of the NE, which interact with laminin, a protein localized in the internal surface of the NE, and with chromatin, thereby participating in anchorage of the genetic material and in gene regulation. On the other hand, KASH domain-containing proteins are small transmembrane proteins at the external face of the NE that associate with cytoskeletal proteins, thus determining the shape and positioning of the NE (Hetzer, 2010).

The NE is a highly dynamic structure, which is modulated during different stages of the cell cycle. In stage G2, the nucleus must duplicate its size by not only increasing its volume, but also by duplicating its NE proteins; during mitosis, specifically in prophase, the NE disintegrates. The processes that lead to dismantling of the NE are not fully understood, however, these events are preceded by loss of NUPs and transfer of the NE proteins to the mitotic ER. Following anaphase, NE restructuring occurs and terminates in complete morphological and functional restructuring of the nucleus. Our understanding of the NE restoration process is controversial, as there are two leading theories. The first suggests that the NE is fragmented into small vesicles that are not degraded completely during mitosis and subsequently merge to restructure the NE. The second theory proposes that the ER differentiates into the NE, which is supported by the mitotic ER structure and enriched in NE proteins (Hetzer, 2010).

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The Nuclear Lamina and Genome Organization

Marie-Cécile Gaillard , Karen L. Reddy , in Nuclear Architecture and Dynamics, 2018

14.3 The Lamins Directly Interact With INM Proteins

NE transmembrane proteins (NETs) of the INM are a substantial part of the integrated interface of the INM/lamina. Well-known INM proteins like lamin B receptor (LBR), Lap2β, LEM-2, and MAN1 (among others) link the INM to the underlying nuclear lamina and other nucleoplasmic proteins, including chromatin modifiers (Fig. 14.1) (Ellenberg et al., 1997; Moir et al., 2000b; Wilson and Foisner, 2010). Indeed, these INM proteins require interactions with the lamina network to remain enriched at the INM displaying diffusion to the ONM and ER upon lamin depletion (reviewed in Wilson and Foisner, 2010). More recently, studies have indicated that mammals encode large repertories of hundreds of cell-type restricted NET proteins (Wilson and Berk, 2010; Wong et al., 2014). Seminal studies analyzing the NE proteome in specific tissues, as the liver, muscle, and blood leukocytes, identified 1037 NETs, expanding the previously identified NET proteome substantially (Korfali et al., 2012; Schirmer et al., 2003; Korfali et al., 2010). Interestingly, these NETs revealed a high degree of tissue specificity in NE protein composition with only 16% of identified transmembrane proteins shared between the three tissue types (Korfali et al., 2010, 2012; Wilkie et al., 2011). Subsequent studies using immunofluorescence have validated both the cell-type specificity and the NE localization of many of these proteomically identified proteins. The cell-type specificity of these proteins is also correlated with annotated protein complexes reported previously by the Johns Hopkins Human Protein Reference Database (Wong et al., 2014; http://www.thehpp.org/; http://pandeylab.igm.jhmi.edu/). The surprising and cell-type-specific complexity of the NETs, and therefore ONM and INM/lamina composition, illustrates how a ubiquitous structure, the nuclear lamina, by its proteomic interactome can have diverse and cell-type-specific functions (reviewed in Wong et al., 2014).

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Nuclear Envelope, Transport

J.D. Aitchison , M.P. Rout , in Encyclopedia of Genetics, 2001

Structure of the Nuclear Envelope

The NE consists of two continuous, distinct parallel membranes, the inner and outer nuclear membranes, enclosing a perinuclear space ( Figure 1). The outer nuclear membrane and perinuclear space are continuous with the endoplasmic reticulum, and share its functions. The inner nuclear membrane is compositionally distinct from the outer nuclear membrane and in many cells is lined with a fibrous nuclear lamina on its nucleoplasmic face. The nuclear lamina is a lattice-like sheet of variable width, consisting mainly of polymers of filamentous lamin proteins (which are related to intermediate filament proteins), and is thought to contribute to the structural integrity of the NE.

Figure 1. The nuclear pore complex.

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International Review of Cell and Molecular Biology

Sarah Cohen , ... Nelly Panté , in International Review of Cell and Molecular Biology, 2012

Abstract

The nuclear envelope (NE) is a vital structure that separates the nucleus from the cytoplasm. Because the NE is such a critical cellular barrier, many viral pathogens have evolved to modulate its permeability. They do this either by breaching the NE or by disrupting the integrity and functionality of the nuclear pore complex (NPC). Viruses modulate NE permeability for different reasons. Some viruses disrupt NE to deliver the viral genome into the nucleus for replication, while others cause NE disruption during nuclear egress of newly assembled capsids. Yet, other viruses modulate NE permeability and affect the compartmentalization of host proteins or block the nuclear transport of host proteins involved in the host antiviral response. Recent scientific advances demonstrated that other viruses use proteins of the NPC for viral assembly or disassembly. Here we review the ways in which various viruses affect NE and NPC during infection.

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Nuclear Envelope, Transport

H.J. Worman , in Brenner's Encyclopedia of Genetics (Second Edition), 2013

Structure of the Nuclear Envelope

The nuclear envelope (NE) represents the boundary of the interphase nucleus. It is composted of two morphologically distinct parallel membranes, the inner and outer nuclear membranes, enclosing a perinuclear space ( Figure 1 ). The outer nuclear membrane and perinuclear space are continuous with the endoplasmic reticulum. The inner nuclear membrane is compositionally different from the outer nuclear membrane, and in mammals ∼60 transmembrane proteins are concentrated in this membrane. The inner nuclear membrane of most metazoans is lined on its nucleocytoplasmic face with a meshwork of intermediate filament proteins called lamins ( Figure 1 ). In humans, three genes, LMNA, LMNB1, and LMNB2, encode lamins. The mediators of transport across the NE are the nuclear pore complexes (NPCs), proteinaceous assemblies embedded within pore membranes that connect the inner and outer nuclear membranes at numerous points. Most NPC proteins are termed nucleoporins, which in humans are encoded by ∼30 different genes.

Figure 1. The nuclear envelope and nuclear lamina. The nuclear envelope surrounds the nucleus. It is composed of the nuclear membranes, nuclear pore complexes, and nuclear lamina. The inner and outer nuclear membranes are separated by the perinuclear space, which is continuous with the endoplasmic reticulum. Approximately 60 transmembrane proteins are concentrated in the inner nuclear membrane; examples shown are MAN1, lamina-associated polypeptide-1 (LAP1), a SUN protein, lamina-associated polypeptide-2β (LAP2β), lamin B receptor (LBR), emerin, and a nesprin-1 isoform. Some proteins, such as large nesprin-2 isoforms, concentrate in the outer nuclear membrane by binding to SUN proteins within the perinuclear space. Proteins secreted into the endoplasmic reticulum, such as torsinA (TOR1A), can reach the perinuclear space; a disease-causing variant of TOR1A concentrates in the perinuclear space. The nuclear lamina is a meshwork of intermediate filaments on the inner aspect of the inner nuclear membrane and is composed of proteins called lamins (schematic of lamin protein shown is in the lower inset; not to scale). Lamins have α-helical rod domains conserved among intermediate filament proteins and head and tail domains that vary in sequence among members of the intermediate filament protein family. Within the tail domain, lamins contain a nuclear localization signal (NLS) and an immunoglobulin-like fold (Ig fold). Most lamins contain at their carboxyl termini a CAAX motif that acts as a signal to trigger a series of chemical reactions leading to modification by farnesylation.

 Reproduced with permission from Dauer WT and Worman HJ (2009) The nuclear envelope as a signaling node in development and disease. Developmental Cell 17: 626–638.

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Loss of Nuclear Envelope Integrity in Aging and Disease

Joke Robijns , ... Winnok H. De Vos , in International Review of Cell and Molecular Biology, 2018

Abstract

The nuclear envelope (NE) serves as a central organizing unit for the eukaryotic cell. By virtue of its highly selective, semipermeable barrier function, the NE shields the enclosed genetic material, while at the same time ensuring its regulated transcription, replication, and repair. The NE has long been considered to only dismantle during mitosis. However, in recent years it has become clear that in a variety of pathologies, NE integrity becomes compromised during interphase as well. Loss of NE integrity, or briefly NE stress, is manifested in various ways, ranging from a gradual reduction in nucleocytoplasmic transport function, to selective loss and degradation of NE components, and finally to catastrophic rupture events that provoke abhorrent molecular fluxes between the nucleus and cytoplasm. Although cells manage to cope with such forms of NE stress, the different insults to nuclear compartmentalization alter gene regulation and jeopardize genome stability. Hence, loss of NE integrity is emerging as a broad-spectrum pathogenic mechanism. In this review, we discuss the relevance of nuclear compartmentalization and the loss thereof in aging and disease development.

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Nuclear Mechanics & Genome Regulation

Zhixia Zhong , ... Kris Noel Dahl , in Methods in Cell Biology, 2010

B Endogenous Protein Localization at the INM Versus the ONM

Nuclear envelope membranes and their enclosed lumenal space are continuous with the endoplasmic reticulum, but nevertheless represent three highly specialized functional domains: the ONM domain, the INM domain, and the "pore membrane" domain around each NPC. Proteins that localize at the pore membrane domain are readily distinguished by punctate fluorescent staining that overlaps with NPC markers, as recently (and surprisingly) shown for SUN1 in HeLa cells (Liu et al., 2007). One can also deduce the subnuclear envelope localization of candidate proteins by double or triple labeling with antibodies against NPC proteins located on either the cytoplasmic (e.g., Nup358 or Nup214) or nucleoplasmic (e.g., Nup153 or Nup98) side of the NPC, or marker proteins specific to the INM (e.g., emerin, MAN1) or nuclear envelope lumen (Schermelleh et al., 2008). However, these methods require very high spatial sensitivity in imaging.

Localization can be determined unambiguously by other methods including transmission electron microscopy of immuno-gold-labeled samples (not described here), or the following classic method that distinguishes ONM and INM localization based on indirect immunofluorescence staining of cells made permeable using either Triton-X100 (dissolves all membranes) or Digitonin (affects the plasma membrane but not interior or nuclear membranes).

Digitonin is a steroid glycoside that binds cholesterol and other β-hydroxysterols that are highly enriched in the plasma membrane, relative to intracellular membranes (Fiskum et al., 1980). Antibodies incubated with digitonin-permeabilized cells have access only to epitopes that face the cytoplasm, including proteins on the ONM. Proteins located at the INM, nuclear envelope lumen, or nucleoplasm are detected only in Triton-permeabilized cells (see Fig. 3).

Fig. 3. Altered membrane permeablization allows visualization of INM versus ONM proteins. Digitonin permeabilizes the plasma membrane but not the nuclear membrane, whereas Triton X-100 permeabilizes all membranes. During immunocytochemistry, this allows for altered antibody accessibility to different regions of the nucleus. INM proteins cannot be detected with digitonin permeabilization (A and A′) but can be detected with Triton X-100 treatment (B and B′). ONM protein can be detected with both digitonin (C and C′) and Triton X-100 (D and D′) treatment. This has been shown functionally with the nucleoskeleton protein lamin A/C which is inside the nucleus, and the ONM protein Nesprin-4 (A′ through D′).

 Adapted from Roux et al. (2009).

To apply the digitonin method to fixed cells, incubate a second set of fixed cells 15   min (on ice) in PBS/0.004% digitonin (reported range is 0.002–0.005%), rather than PBS/Triton, and then continue as described in Section I.

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Mitosis and Cytokinesis

In Cell Biology (Third Edition), 2017

Nuclear Envelope Disassembly in Prometaphase

Nuclear envelope disassembly involves the removal of two membrane bilayers coupled with disassembly of the nuclear pores and the fibrous nuclear lamina meshwork that underlies the inner bilayer (Fig. 44.6). Phosphorylation causes the nucleoporin Nup98 to dissociate from nuclear pores. This removes the permeability barrier between nucleus and cytoplasm. Phosphorylation of other proteins causes the pore to disassemble to soluble subcomplexes. Phosphorylation of the nuclear lamins at two sites flanking the coiled-coil causes the lamina network to disassemble into subunits. Interaction between microtubules and dynein associated with the nuclear envelope can rip holes in the envelope, although this is not required for nuclear envelope disassembly.

Nuclear envelope membranes are dispersed in the cytoplasm from prometaphase until telophase (Fig. 44.6 ), but the mechanism is not settled. Some experiments suggest that the nuclear membranes break up into small vesicles that disperse in the cytoplasm. Other experiments suggest that the nuclear envelope is absorbed into the endoplasmic reticulum, which remains as an extensive tubular (or flattened cisternal network—another source of discussion) throughout mitosis. Further experiments are required to answer this question, and both mechanisms could contribute. Lamin B remains associated with the dispersed nuclear envelope, whereas lamins A and C and many proteins of the nuclear pore complexes disperse as soluble subunits.

During prophase, kinetochores transform from nondescript balls of condensed chromatin into structures on the surface of the chromosomes. By early prometaphase, the characteristic trilaminar disk structure (see Fig. 8.19) can be seen. Each sister chromatid has a kinetochore. Sister kinetochores are located on opposite faces of the mitotic chromosome.

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