Ultra Structure of the Nuclear Envelope
The elegant structure of the eukaryotic nucleus, which is disconnected from the cytoplasm by the nuclear envelope, was recognized a long time ago. The model formed the basis of thoughtful investigations into the dynamic functional reorganization of the nuclear envelope and its components during varying states of the cell cycle. The nuclear envelope consists of an inner and an outer membrane, nuclear pore complexes, and the underlying nuclear lamina, a filamentous scaffold structure formed by lamins. The inner membrane is linked to the lamina and chromatin by its integral membrane proteins, such as lamin B receptor, emerin, and various isoforms of lamina-associated polypeptides 1 and 2. The outer membrane is continuous with the endoplasmic reticulum, and associating protein constituents include ribosome-binding protein, the transmembrane 'stretchability sensor' protein, and protein targeting factor.
The nuclear envelope is highly dynamic during the cell cycle. At prophase, it first becomes morphologically transformed which is accompanied by phosphorylation of its core components including lamins, inner membrane proteins, and nuclear pore complexes, leading to their disassembly. Conventionally, nuclear assembly is viewed as a process of reformation of the disrupted nuclear envelope leading to nuclear compartmentalization at the end of mitosis. As such, nuclear reassembly after sister chromatid separation requires a timely coordinated and dephosphorylation-dependent association of lamin-binding proteins and lamins with chromosomal proteins anchoring the nuclear envelope to chromatin, and its relatives, and, at later stages, chromatin, and protein phosphatase. Adhering and vesicular fusion of the primed and lamina-free endoplasmic reticulum membranes to the chromatin surface precede nuclear pore complex insertion into the nuclear envelope. There are two membranes, and periodic electron-dense lamins between these two membranes. The enclosing structure consists of a double afferent layer, a porous granular coat, and spongy fibers radiating from it.
The nuclear envelope (NE) is a subcellular structure that surrounds, encloses, and protects eukaryotic genomic material within the nucleus. Assembly and maintenance of nuclear envelope architecture are critically important since the NE is involved in regulating molecule transport between the nucleoplasm and cytoplasm, as well as in mediating the interaction between chromatin and multiple cellular processes. Malfunction of the nuclear envelope causes chromatin misregulation that gives rise to degenerative diseases (Foisner, 2003). There are two concentric bilayers composing the NE. The outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum (ER) and is the site for connection with the cytoskeleton at the outer surface of the nucleus. The inner nuclear membrane (INM) is linked to the chromatin by a nuclear lamin filamentous scaffold structure formed by the lamina A/C and lamina B1/B2 proteins. The inner and outer membranes (OM) are joined at sites where the nuclear pore complex (NPC) is inserted into the double membrane. Although the overall architecture of the nuclear envelope is understood, precise molecular details of interaction between chromatin and the NE bilayer and filaments remain poorly understood. Maintaining an intact nuclear envelope is essential for cellular homeostasis. The nuclear envelope (NE) forms a barrier between the cytoplasm and nucleoplasm. The NE is comprised of the inner nuclear membrane (INM), the outer nuclear membrane (ONM), and the perinuclear space (PNS), and is associated with the nuclear pore complexes (NPC) that regulate nucleocytoplasmic transport. The nuclear lamin pore complexes (NLP) are involved in NE structure and chromatin organization. Chromatin tethering to the nuclear envelope facilitates the regulation of gene expression and formation of transcription factories (Bozler et al., 2014). There are significant ongoing research efforts focused on building models of NE structure, composition, and function, at the ultra-structural level by high resolution imaging approaches. These efforts have revealed the nanoscale organization of membrane and filamentous components working together to maintain the NE structure. Advances have also been made in understanding how chromatin structure and function is regulated by interactions with the NE. In addition, efforts are ongoing to determine how misregulation of processes at the NE leads to disease. Collectively, these studies are providing fundamental insight into the biological roles of the NE and chromatin structures underlying cellular functions.
The NE is composed of the outer nuclear membrane (ONM) and the inner nuclear membrane (INM): two lipid bilayers separated by a perinuclear space that connects to the ER. IM leads to perinuclear heterochromatin and regulates the transcription state of genes. OM contains NPCs, where nucleocytoplasmic transport takes place. The lipid bilayers are inseparable from membrane proteins, which guide local membrane curvature. The consensus opinion regarding NPC primary membrane curvature is that it is generated by Nups’ integration to the bilayer as wedges. However, the NP is a multi-step assembly process in which essential Nups arrive first and, with lateral membrane tension, generate the deformation. How this step occurs and the role of lipids remain unclear (W. A. Peeters et al., 2022). The INM and ONM are, respectively, the inner and outer lipid bilayers of the NE. NPCs are embedded in the NE determined primarily by structural Nups which shape the membrane into a large pore. That the Nups and other membrane proteins first enter the NE bilayers widely leads to increasing membrane curvature and pore formation is a consensus view. However, a multi-step NPC assembly model proposes that the NE pores are assembled first with a ring structure, and that core proteins minimally bind to the pore.
Despite the advances in our understanding of the NE and its constituents, it is likely that there are still many unknowns in the field. Nevertheless, recent technical advances have led to a new understanding of the diversity and complexity of the proteins that constitute the NE and NPCs, as well as the molecular machinery that is involved in organelle biogenesis and shaping. Several methods for identifying integral membrane proteins and examining their topology and structure have also matured and been applied to the NE and NPC. Although many questions still remain to be answered, a new picture has emerged that reveals the NE and NPC as complex, elaborately structured, and highly dynamic assemblies. Building on the methods that have transformed the field, an outline is provided of the challenges that lie ahead in answering the many biological and biophysical questions that are raised by the current understanding of these remarkable structures.
Until recently, the structure and biology of the NE and NPC were largely characterized and explored through classical methods. These powerful approaches stemmed from a robust understanding of the NE and NPC, but many of the details at the molecular level remained unclear, including the identification of their constituents. The construction of the NE is a fundamental biological process that is central to the preservation of species and the evolution of life on Earth. It has many consequences for human development and wellbeing that are not fully understood. Overall, the NE is a complex, organized, and dynamic structure that performs a myriad of contextual tasks.
While considerable information has been gained about the organization of the NE and NPC scaffolds, little is known about the spatial organization and interactions of the membrane proteins embedded in the two bilayers. The proteins of the NE remain poorly defined in general, but cell-type-specific inclusion in, or exclusion from, the NE has been documented for some. It is known that NE proteins interact with transmembrane proteins of the ER, the latter being involved in NPC assembly and anchoring. Several proteins have been described to link chromatin remodeling to nuclear transport, spacing between pore complexes, and nuclear motor function, but only a few direct connections with chromatin, RNA, and transcription factors have been made otherwise. The question of how chromatin is organized presents powerful scientific challenges to describe the spatial properties of chromatin.
The nuclear envelope (NE) is a double-membrane structure surrounding the interphase nucleus. It consists of the inner nuclear membrane (INM), linking proteins on the inner nuclear membrane (INMPs) and underlying lamins forming the nuclear lamina, outer nuclear membrane (ONM), and nuclear pore complexes (NPCs) spanning the nuclear membranes, through which transport of RNAs and proteins occurs. The NE is continuous with the endoplasmic reticulum (ER) and thus connected with membrane trafficking pathways. Linkage between chromatin and the NE provides structural support by anchoring the chromatin to the nuclear lamina and enabling transcriptional silencing of heterochromatin/TADs at the nuclear periphery (Foisner, 2003). The two membrane layers are punctuated with large NPCs formed by tens of different nucleoporins (NUPs), which create a diffusion barrier for proteins larger than approximately 40 kDa.
Recent studies identified additional structures of the NE that are more complex than a simple double membrane layer. For example, a membrane structure in an onion skin cell comprising sheets of the inner nuclear membrane (INM), extensive membrane invaginations from the outer nuclear membrane (ONM), and everted vesicles at the ONM was observed at this larger scale. In mammalian cells, a variety of nucleoplasmic reticulum (NR) structures, which are internal invaginations and convolutions of the NE, were identified. These structures comprise membrane structures continuous with the NE and membrane proteins such as NUP 88 (Bozler et al., 2014). However, their role is poorly understood since even fixed FRAP experiments indicated that GTP-Rab5 localization to NR is highly dynamic. Tethering of cytoplasmic proteins to the NE membrane may increase their local concentrations thousands of times, which could enhance associations with highly abundant chromatin proteins to form stable complexes. Though the factors involved in long-range chromosome/NM associations are largely unknown, one model proposes that these associations occur in parallel with interactions with NUPs and the NPC. The NE has varied roles in nuclear structure and functioning beyond a simple barrier, creating a highly complex and specific interface between the cytoplasm and nucleoplasm.
The nuclear envelope (NE) consists of an outer nuclear membrane (ONM) and an inner nuclear membrane (INM). Together, they form a continuous double membrane, which surrounds the nucleus and separates the nucleoplasm from the cytoplasm. The ONM is composed of a lipid bilayer similar to the membrane of the rough endoplasmic reticulum (W. A. Peeters et al., 2022). The ONM and INM are linked at regions where large proteinaceous structures called nuclear pore complexes (NPCs) are embedded in the membrane. NPCs perforate the NE and mediate the transport of macromolecules into and out of the nucleus. Exposed to the cytoplasm, the ONM contains large ribonucleoprotein (RNP) complexes that initiate transcription and RNA splicing. INM proteins bind chromatin to facilitate gene repression and regulation. The nuclear pore is formed by a mixture of integral membrane proteins, which form a basket-like structure projecting into the nucleus, and fibrils projecting into the cytoplasm.
The data indicate that the NPC develops from an initial narrow pore that widens and elongates as the basket forms and cytoplasmic fibrils emerge. In parallel, the membrane becomes increasingly curved and is separated by a greater distance. These changes are accompanied by a decrease in the vertical density of the basket as it acquires its final morphology. Multiple human diseases, including cancer, diabetes, cardiac dysfunction, and neurodegeneration, have been linked to mislocalization of genetic material resulting from pore complex malfunction. Exploring the molecular mechanisms involved in the formation of the NPC hole in the nuclear envelope may provide insight into therapeutic strategies for ameliorating the defects in membrane organization and pore complexity found in these diseases. However, these pore assembly mechanisms remain insufficiently understood.
Ultra-structure studies using electron microscopy have revealed that the nuclear pore is formed by an elaborate arrangement of multiple integral membrane proteins. At the periphery of the pore, they form a ring structure that is continuous with the outer and inner nuclear membranes and, in vertebrate cells, encapsulates a mass of thinner filaments projecting into the cytoplasm and a basket structure protruding into the nucleus. The dimensions of the pore at the membrane construction site are more than 20% smaller than the dimensions of the mature pore, suggesting the presence of membrane fission and expansion processes during pore maturation.
The inner nuclear membrane (INM) is an integral membrane bilayer that encloses the nucleus and is involved in regulating the composition and behavior of the nucleoplasm. Within the nucleoplasm resides the nuclear lamina, a fibrous meshwork of lamins A, B, and C that function in maintaining the shape of the nucleus and anchoring chromatin. The INM is a site of processing for nascent ribosomal RNA, has a proteome composition that undergoes reversible changes during the cell cycle, and is involved in the execution of DNA-damage repair. It is studded with an array of transmembrane glycoproteins with a range of sizes and structures. These proteins are distributed asymmetrically between the INM and the outer nuclear membrane (ONM), giving the nuclear envelope (NE) a distinct identity.
The INM and ONM are intimately connected at sites where the large nuclear pore complexes (NPCs) reside. Pore complexes are selectively permeable channels that allow the regulated exchange of RNA and proteins between the nucleus and cytoplasm. The NPCs form intricate structures composed of multiple copies of 30 different proteins, or nucleoporins (Nups). Structurally, they consist of a central channel through which cargo moves and cytoplasmic and nuclear coat scaffolds. The coating serves to stabilize the NPC structure and recruit additional Nups that are involved in cargo recognition and translocation.
Membrane proteins are essential components of the NE. They include membrane-spanning proteins such as lamina-associated polypeptides (LAP) 1 and 2, emerin, the lamin B receptor (LBR) and several integral proteins with unknown function. It is thought that these integral proteins link the membrane bilayer to chromatin and the nuclear lamina through protein-protein interactions and extensive cytoskeletal networks that mediate directional motion. For several of these membrane proteins, such as emerin, lamin B receptor, and LAP 1 and 2, the accessory proteins binding involve several domains, including a transmembrane segment, an alpha/beta domain, and an Ig-like module.
Ultrastructure of perinuclear space was studied by a jigsaw approach (E. Shaiken & R. Opekun, 2014) that was used to visualize the modular structures: perinuclear web (PNW), actin-organizing center (AOC), and actin cap (AC). PNW mAb broad-spectrum epitope allowed visualization of a novel braided structure surrounding the isolated core nuclei. It is highly suggested that the nuclear structure consists of about 100 PNW proteins. The modular structure contains a three-dimensional meshwork of rapidly exchanging proteins that contribute to the viscoelasticity of the PNW formation. The PNW constraint at least partially restricts the nuclear diffusion of COPE and eIFa. Knockdown of PNW proteins STMN3 and PRKRIP1 increased the motion of evolutionally conserved transcriptional factors at the perinucleus within the spatio-temporal extent of the PNW structure expansion, resulting in decreased motion conformity upon disturbances. It was concluded that the perinuclear space comprises not only the nuclear envelope but also modular assemblies of diverse proteins, the barrier of the perinuclear space, and its dynamic studies in live cells.
More importantly, combination of PVP staining with MNase was successfully applied not only to visualize the thin membranes in the nucleus but also to study the interaction between compact chromatin and the nuclear structures. At least 90% of the chromatin was rapidly extracted in live cells, suggesting that the chromatin is not compacted by the physical barrier but rather by the biochemical level. Accordingly, the histone-modifying enzymes were relocated to the nuclear domes-proposed sites for transcriptional regulation depletion of which resulted in enhanced chromatin anchored at the surface of the nuclear structure. It is hypothesized that, to regulate transcription, chromatin would translocate to the cap to accommodate modifications and be balanced by the return of chromatin de-modified at surface-wide chromatin remodeling.
Nuclear pores are based on an annular scaffold that has 8-fold rotational symmetry with a central aqueous transport channel that connects the nuclear and cytoplasmic compartments. Fibrous appendages project from both the cytoplasmic and nucleoplasmic faces of the nuclear pores, contributing to the formation of a distinctive nuclear basket. These fibrous appendages function as binding sites for a range of factors involved in transport and gene expression (Stewart, 2022).
Nuclear pores are constructed from multiple copies of ~30 different nucleoporin proteins. A number of these nucleoporins contain long intrinsically disordered domains that protrude into the central channel, generating a dense meshwork that impedes the diffusion of macromolecules between the nuclear and cytoplasmic compartments. The densely packed natively unfolded chains of the FG-nucleoporins generate a barrier to the diffusion of macromolecules and also function as transient binding sites for nuclear transport factors that mediate the active import and/or export of a range of macromolecules. Interactions between the transport factors and the FG-nucleoporins enable them to enter the nuclear pore transport channel and carry protein and RNA cargoes between the nuclear and cytoplasmic compartments.
The molecular crowding in the nuclear pore transport channel is increased by the considerable number of transport factors present.
Cryoelectron tomography and helical reconstruction have provided glimpses of the NPC architecture in situ, particularly in budding yeast. The acquisition of hybrid models consisting of the low-frequency backbone structure of the NPC by cryo-electron tomography with the high-resolution structures of its constituent proteins by X-ray crystallography and cryo-electron microscopy has revealed the surficial structure of the NPC at very high resolutions (H. Lin & Hoelz, 2019). Furthermore, both cryo-electron tomography and unbiased 3D classification have revealed the dynamic configurations of the transporter-loaded, RB-loaded, and NE-anchored NPCs. A wealth of structural, biophysical, and logics has unraveled the molar masses, symmetry, switching and modularity, and the channel/composite of the NPC, as well as the transport and anchoring mechanisms of the NPC. As a consequence, an expected desire for nearly 30 years has been answered: elucidation of the structures of the mammalian NPC and its transport-unloaded and anchoring forms at unprecedented resolutions (Dultz et al., 2022). Recent studies have shifted the focus to the outer field of the NPC: the NE and the remnant NPCs attached to the NE membrane. The role of the ribosomes, RBs, nucleoplasmic FLNA, and PG90 in regulating NE subsiding and NPC fragmentation has been shown. Global heterogeneity in the distribution of NPCs and various gradients in the density of numbers and sizes of the NPCs in the arches of patterning NPCs have been revealed. In situ evidence suggests that the cytoplasmic FG-NUPs, widely assumed to be the exclusive transmembrane anchors, are an evolutionarily conserved component of the NE. Overall, the ultra-structures of the NPC and NE have quantitative and qualitative breadth and depth at an unprecedented level.
The ESC and TS constituent structures characterize the transport from the nuclear envelope to the cytoplasm or vice versa. In eukaryotes, the nucleocytoplasmic transport is mainly mediated by the nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE), which ensure selective translocation of macromolecules between the nucleus and the cytoplasm. NPCs are octagonally symmetrical structures assembled from soluble proteins called nucleoporins (Nups), which are found in roughly ~30 different Nups nodes. The pores formed by the Nups have a passive diffusion role for molecules of mass ≤ 40–60 kDa, whereas the transport of larger materials involves a selective energy-dependent mechanism. The majority of the performed studies concern conventional NPCs and pore structures, and very few evaluations have been made according to the transport outside the conventionally described manner (S. Keuenhof et al., 2023). The study of NE-budding-based transport is still explored. Various biological findings imply that a considerable amount of nucleocytoplasmic transport takes place outside NPCs, even in higher eukaryotes where NPCs are abundantly present.
Utilization of NE budding as a new form of nucleocytoplasmic transport to vesicularize molecules transported from the nucleus to the cytoplasm has been investigated using live cell imaging, electron microscopy, and machine-learning-based analysis of 4D information in living samples. The membranous ESC structures are revealed to bud directly from the nucleoplasm, shedding new light on multivesicular endosome formation and vesicle transportation mechanisms. As an ultimate proof of the transport mechanism, the role of NE budding in the molecular translocation of ESCs into the cytoplasm has been inconveniently assessed by trap-and-release editing in real-time in living embryos (Stewart, 2022).
The nuclear pore complex is capable of selective transport of macromolecules. It enables the passive diffusion of small molecules. Probably the most sophisticated structures in eukaryotic cells, nuclear pore complexes consist of roughly 30 types of proteins (nucleoporins) that form a basket-like structure, spanning the nuclear envelope and permeating into both the nucleoplasm and cytosol (Y. H. Lim et al., 2008). Computed tomography reconstructions have shown that the yeast nuclear pore complex has an octagonal symmetrical architecture with distinct cytoplasmic, nuclear and central domains leading to a basket-like structure extending into the nucleoplasm. 8-fold rotational symmetry was built into the reconstruction. Inside the nuclear pore, the filling furthermore forms a curved meshwork. Consistent with a helical arrangement of protofilaments, subunits once removed from the axis protruding into the center distinguish adjacent structures.
At first, this structure appears both simple and complex. It is made solely of proteins and the possible central hole, when viewed face on, seems sparse. Very rapid equilibration can occur between the cytoplasm and nucleoplasm if there is no barrier, and experimental work has determined good estimates for the pore radius for rapid equilibration. At the same time, it can be shown that the edge of the complex is 25–40 nm across. Assuming this edge is composed of homopolymeric rings of one or two nucleoporins producing a continuous density along the edge of the face, such a structure would appear to be very dense considering that nucleoporins can be linked to one another in the Megadalton range. Common wisdom dictates that the fold, dimension or filling of the homopolymeric rings ought to dictate these properties. Recent nano-scale structures of adjacent macromolecules which can transport a viral genome are illustrative. They account for how everything happens along the rim with high density and, when suitably timed, none at all.
Specifically, the pore is filled with protein structures, some of which appear to be bent filamentous materials possibly resembling the Cascades of jets from exploding stars — which the face on image also resembles — making modelling this view difficult. Some of these protein structures are plausibly ordered in rows with repeating spacing, and bent rather than straight consistent with a helical arrangement of density producing in a radial fashion a network with the observed mesh size. Given such dimensions, each filament could be roughly a few nm thick with possible intrinsic flexibility. As a network of differing density, it could be transparent to some structures, excluding others, without the need for any gating mechanism.
The nuclear envelope serves as a physical and functional barrier that maintains the separation of transcriptionally distinct compartments. This barrier is made up of double membranes connected by nuclear pore complexes: large protein assemblies embedded in the nuclear envelope that mediate nucleocytoplasmic transport. The nuclear lamina is a thin fiberous meshwork of lamin A, B and C proteins located adjacent to the inner nuclear membrane, providing structural support to the nucleus. The organization and maintenance of the nuclear architecture is further regulated by a variety of anchoring proteins that are embedded in the inner nuclear membrane (INM) and display cytoplasmic domains that interact with the cytoskeleton. There is emerging evidence supporting a more direct role for the anchoring proteins in the assembly or organization of NE and the lamina (A Dittmer & Misteli, 2011). Proteins whose integral membrane domains span the INM include the lamina-associated polypeptide 2 (LAP2), the emerin protein and the nesprin protein superfamily (Foisner, 2003). Each family of membrane-spanning proteins interacts with distinct nuclear lamins or incomplicated combinations of them and thus tether the lamina and associated chromatin to the INM. Several human diseases, called laminopathies, are associated with mutations either in lamins themselves or in any of the diverse anchoring proteins. These result in cell or tissue specific defects that provide a link between nuclear structure, nuclear organization and cellular functions. However, among the wide variety of anchoring proteins and membrane domains, most focus has been on the LAP2/emerin/myein (LEM) structure family.
Most eukaryotic cells contain a nuclear envelope (NE), which consists of a double membrane structure spanning the nuclear pore complexes and is widely regarded as the gateway to the nucleus (A Dittmer & Misteli, 2011); (Vahabikashi et al., 2022). Most eukaryotic cells contain a nuclear envelope (NE), which consists of a double membrane structure spanning the nuclear pore complexes and is widely regarded as the gateway to the nucleus. Interspersed on the inner nuclear membrane, nuclear pore complexes (NPCs) provide a regulated gateway between the interior of a cell and its nucleus. Juxtaposed to the nucleoplasmic surface of the inner nuclear membrane is the nuclear lamina (NL), a network of intermediate filaments that comprises type V nuclear lamins and links peripheral heterochromatin to the nucleoskeleton in metazoan cells. The ensuing compilation elucidates lamina structure and biophysical properties, membrane-less compartments involved in regulatory roles, and receptors that mediate mechanotransduction from the nuclear envelope to signaling pathways regulating cell function and fate.
The NE of most eukaryotic cells is a double membranous organelle that separates the nucleoplasm from the cytoplasm. It consists of a 10 to 100 nm thick inner nuclear membrane and outer nuclear membrane, molecular bent and flattened shapes, and distinctive subsets of integral membrane proteins on the inner and outer nuclear membranes. The NE is powered by the nuclear pore complexes (NPCs) composed of at least 30 different nucleoporins that span the full depth of the NE to bridge the inner and outer nuclear membranes. The pore membrane is composed of a ~120 nm diameter and ~50 nm thick annular structure that contains eightfold symmetrically arranged spokes embedded in the pore membrane; the spokes are composed of transmembrane nucleoporins assembled through long coiled-coil regions on the outer membrane and cytoplasmic arc-like structures that enable gating of nuclear transport. This composition was well characterized by cryoelectron tomography in several cell types, including rat liver and Xenopus laevis oocytes. But the inner membrane of NPCs has remained elusive, primarily due to the technical difficulties in resolving structures near the densely packed nuclear lamina.
Ever since bacteria were first observed to compartmentalise their genetic material by membrane early in the 19th century, the evolution of a nucleus as a double membrane compartment has been viewed as one of the major hallmarks of the evolution of eukaryotes (Bozler et al., 2014). The nuclear envelope is a prominent structure with profound implications for genome organization and global gene expression in eukaryotes. It does not only provide a compartment for nuclear ribonucleoprotein (RNP) synthesis and processing, but it also plays a key role in the organization of the genome. The nuclear envelope harbours the nuclear pore complexes that mediate all macromolecule transport across the nuclear compartment. Also providing a physical platform for active transcription and processing of gene products in adjacent nuclear speckles. The recent advances in live cell fluorescence microscopy and advances in protein immunostaining techniques have allowed for the visualization and examination of the nuclear envelope’s substructure and its interaction with other nuclear components in unprecedented detail.
The well-described physical structures of the double membrane layer and nuclear structural filaments, such as nuclear lamins, remain the predominant platforms for interaction between chromatin and the cytoplasm. These two membrane layers are connected through integral membrane proteins and are punctuated with the nuclear pore complex that facilitates bidirectional macromolecule movement between nucleoplasm and cytoplasm. The internal structural support of the nucleus is provided in large part by the nuclear lamina, a meshwork of lamin A- and B-type polymers. Studies of nuclear structure have exposed tissue-specific changes in the three-dimensional architecture of the nuclear envelope, revealing intricate patterns of tunneling and branching of the nuclear envelope within the nuclear space. The higher order arrangement of the nuclear envelope has been given the name nucleoplasmic reticulum (NR). The importance of these NR structures for chromosome partitioning, nuclear transport, gene expression, or other cellular processes are not well understood. It has become clear that the nuclear envelope and its three-dimensional shape can profoundly impact genome organization and gene expression.
The nuclear envelope (NE) is biochemically and morphologically highly asymmetric. The inner nuclear membrane (INM) consists of integral membrane proteins, which are frequently linked to, or directly associated with, the underlying nuclear lamina, a filamentous scaffold structure formed by lamins. In contrast to the protein-rich INM, the outer nuclear membrane (ONM) harbors ribosomes, consists of membrane proteins primarily associated with the cytoskeleton, and is directly linked to the rough endoplasmic reticulum. The ONM and the inner membrane are linked by the nuclear pore complexes (NPCs). The NE is a highly dynamic compartment that undergoes profound structural and biochemical rearrangements during the cell cycle (Foisner, 2003). Although the genetics and structure of the NE are conserved, species-specific features mediate distinct mechanisms of NE disassembly and reassembly in different organisms. The NE is disassembled upon phosphorylation of its core components during prophase. In mammalian cells, the NE is torn apart by a dynein-driven microtubule-dependent mechanism that actively pulls apart a fragile ONM by exerting tens of piconewtons of force on the NE. Consequently, the NE becomes highly defective, leading to the redistribution of integral membrane proteins to the endoplasmic reticulum (ER) and aberrant mitotic kinetochores in human cells. The global reorganization of the mitotic NE provides a platform for the phosphorylation-driven remodeling of the NPC, and the local recruitment of the Nup107/160 complex facilitates the disassembly of the NPC.
During anaphase, the NE is reassembled at multiple sites via the recruitment of membrane bearing integral membrane proteins of the INM and ONM, which could also involve vesicular intermediates. Nuclear reassembly after sister chromatid separation requires a timely coordinated and phosphorylation-dependent association of lamin-binding proteins and the lamins with chromosomal proteins as well as nuclear envelope vesicles. Genetic and biochemical studies have revealed the various chromatin-binding domains in the lamina proteins, such as the newly discovered LEM domain. The rapid recruitment of lamina proteins to the dephosphorylated chromatin surface is essential for the formation of the nuclear lamina and is interconnected with the interaction of the chromatin-binding domain in lamina proteins with chromatin. Deciphering how the NE is formed after mitosis and how its dynamics are controlled during interphase is a long-standing question in cell biology. Recent advancements in this area have identified new mechanisms by which the NPC components and the Otp1 protein organize the spatial and temporal distribution of the ONM and the INM. These findings not only provide insights into how giant organelles are formed via a localized growth mode but also lay the groundwork for future studies on the NE dynamics in response to developmental cues and environmental signals.
The nuclear envelope is a distinct organelle structurally and functionally different from other cytoplasmic organelles. It is formed of two membranes joined and assembled at nuclear pore complexes (NPCs). The inner and outer membrane faces the nucleoplasm and the cytoplasm, respectively, and has a diverse collection of proteins involved in transport. The inner nuclear membrane binds proteins that anchor chromatin, provide stability via a mesh network of intermediate filaments and associate with protein complexes that control the life of chromatin, such as replication, response to stress and DNA repair.
The envelope allows for the compartmentalization of signaling and transcription within the nucleus and protects fragile chromatin from physical and chemical states in the cytoplasm. Gradual disassembly of the nuclear envelope must occur during cell division to release mitotic chromosomes into the cytoplasm. Breakdown of the nuclear lamina (major quartz of the nuclear envelope) has been shown to occur via phosphorylation of lamins by the cdc2-cyclin mitotic-factor and disruption of lamin dimers/trimers. Phosphorylated lamins and lamina fragments remain dispersed and do not reassociate during anaphase (Robbins & K. Gonatas, 1964). Separation of nuclear cup membranes in the polar relays around the chromosomes also occur. Nup358 is essential for NEBD in other cell lines and is targeted to the cytoplasm before NPC disassembly (Salina et al., 2003).
Key events in the formation of the nuclear envelope around condensing chromosomes in the daughter cells have been imaged in living tissue. One hour following completion of anaphase, the NPC protein Nup358 begins to market the condensing daughter chromosomes until cytoplasmic microtubules contact them. Assembly of the shell occurs slowly by a process that also involves Nup153 and Nup62. The formation of a new nuclear envelope, an ongoing research topic, is fundamental to eukaryotic cell biology and is in every cell cycle of every eukaryote except the yeast may be. The NEBD ensures that chromatin is competent for the action of chromosome segregating and organizing apparatus. Breakdown of the nuclear envelope (NE) at the prophase to prometaphase transition allows cytoplasmic access to chromatin, which needs to be decompacted, marked for mitosis and associated with kinetochores, spindle localized protein complexes that mediate chromosome segregation. In many non-dividing cells, the nuclear envelope re-assembles around the daughter nuclei, but little is known regarding how, when and where after cell division this occurs.
Following karyokinesis, a series of coordinated events lead to the reformation of the NE, thus encircling the DNA again. Following breakdown there are some predictable and well understood cytoplasmic states/machinery and a nuclear chromatin-associated state that is poorly understood (Liu & Pellman, 2020). There are well understood sets of biophysical mechanisms by which membranes can remodel as well as ubiquitously conserved membrane events like clathrin-mediated endocytosis and scission. The machinery that acts on chromatin is largely uncharacterized, although there are some known players. Most eukaryotic chromatin fuses after mitosis and then expands into distinct nuclear domains, however NE invagination is poorly understood and appears to be non-ubiquitously conserved and involve divergence away from common ancestors. Mechanistic models for changes in chromatin topological state that propagate over a minutes/hours timescale are still in their infancy.
Despite the above limitations some intuitive, predictive understanding does exist. For example, vesicle arrangement is established at by centrifugal forces due to chromatin density wave propagation associated with expansion and fusion. By analogy, cytoplasmic states can be predicted from intact NE functions and structure. This is a time-limited cue, however, and once karyotypic interactions and configurations remodelling becomes harder to predict – for instance, the NPC invade the growing NE and regulate recruitments. Moreover, many events rely on the NE returning to an intact state prior to other downstream fates occurring and efficient-resolution prediction. Effectively understanding/introspection into how/why dynamics slow, stop, are pruned and/or negotiated is one major challenge going forwards.
The current knowledge regarding the role of the nuclear envelope in genome organization spans a wide range of topics. As the barrier between transcriptionally active chromatin and the cytoplasm, the nuclear envelope is the primary site for interactions with chromatin. Importantly, there appears to be a baseline level of interactions that defines the broad spatial understanding of the genome. In addition to these “classic” interactions, there are chromatin–nuclear envelope interactions that are more dynamic in nature and are important for cellular processes, such as transcription and replication. Differences in the specific nuclear proteins used by these varying types of chromatin interactions likely reflects the broad functional diversity or distinct classes of chromatin anchoring mechanisms.
The nuclear envelope and heterochromatin provide a structural framework for the timely and regulated expression of transcriptionally silent genes (Burla et al., 2020). In bilateral animals, the nuclear envelope contains a “lock” that binds and promotes transcriptional repression of DANAs. This structure is composed entirely of the membrane-spanning emerin protein and functions both as a structural anchor for the heterochromatin bank and a scaffold for silencing factors. As a regulatory hub of silenced chromatin, the nuclear envelope is located adjacent to and provides a barrier against promoters because interactions on this boundary should be minimized so that transcriptional reallocation to neighbouring active chromatin regions can occur smoothly.
Recent data are available on the modulation of 3D genome organization that raises questions regarding whether a similar mechanism at the nuclear envelope with a more expansive view of chromatin–nuclear envelope interactions has evolved. The teleological understanding of these studies is that localization at the nuclear envelope and formation of a heterochromatic network represents key strategies for chromatin organization across diverse groups of metazoans (Bozler et al., 2014). When tethered to a bacterial artificial chromosome on the lamina, algorithmically calculated average contact probabilities show that the chromatin tether correctly predicts contact probabilities for most experimental resolutions. Moreover, the inclusion of additional nuclear periphery components in computational modelling produces more finely tuned predictions, indicating the importance of selectively modelling the nuclear envelope. When the tether approaches the nuclear pore complex, chromatin stretching becomes evident, induced by a greater pulling of the chromatin away from the encapsulating volume. Thus, not only are substantial forces exerted onto the chromatin upon anchoring at the nuclear envelope, but the physical properties of this structure can also capture the diversity of chromatin organization that is observed in mammals and fly cells. Overall, this offers an explanation for observations regarding the structure of the nuclear envelope and its relationship with chromatin.
Many aspects of gene regulation and transcription occur at the nuclear envelope (NE). Several diseases including cancer and muscular dystrophy arise from mutations in components of the NE. Pioneering studies of budding yeast helped mechanistically define a role for nuclear pore complexes (NPCs) in gene activation. In animal cells, the composition of NPCs is dynamic and altered in several ways during development, differentiation, and disease. Recent studies began to determine the functional consequences of these changes on genome output. NPC components directly regulate gene expression through mechanisms similar to those defined in yeast, while other NPC proteins affect genes indirectly by building micron-scale channels in the NE that sequester regulatory factors and transcriptional activators (A. D'Angelo, 2018).
The NE consists of two membranes that are connected and penetrated by the NPC. The cytoplasmic nuclear envelope (cNE) is complexed with the NPC, a large molecular machine that is assembled from ∼30 different proteins collectively termed nucleoporins. Many of the nucleoporins are composed of long phenylalanine glycine (FG)-repeat containing intrinsically disordered regions. This organization, along with a cytoplasmic filament basket structure, conveys a selective barrier and facilitates the nuclear transport of RNA and proteins. The so-called nuclear basket scaffold is built from coiled-coil nucleoporins that link a large number of nuclear basket nucleoporins that are also intrinsically disordered. Their composition in animal cells is more variable than their yeast orthologs. Differences in nucleoporins within the nuclear basket appear to reflect the misspecification of the NPC in the nematode worm C. elegans, while differences in nucleoporins reported between various species and cell types suggest that nucleoporins have evolved varied functions (Bozler et al., 2014). The scaffold anchors chromatin to the NE, while there exist connections between nucleoporins and chromatin through different protein partners, which transmit spatial information persuading transcription outcome.
Prior to the mid-Paris Communes, a 400 W electric incandescent lamp was placed in the cuboid workplace of 268 m3 volume. In January, the test glass bubble was opened and the bulk of the initial volume of inert gas 950 m3 at 1 bar and +273 c. Conclusively, either O2 at 60 m3 or N2 at 60 m3 was subsequently injected while simultaneously removing the existing gases at 50 m3/min by two exhaust pipes: one was opened straight to the atmosphere while the other was connected with a rotary brush vacuum pump system. After 30 min of injection, all lamps remained transparent, signifying the removal of O2 or N2 in a significant quantity. However, at the end of all injections, one ruptured while two continued burning for over 80 and 120 min, respectively. Baseline spectra of the burning lamp from 5 min to over 60 min had, besides an electron beam, similar double-peaked peaks 2148 and 2184, suggestive of their being common to the burnt gases produced both in the combustion reactions of either activated C or the methane-contained N gas. Peaks at 1058 and 1070 were indexable to -C-C- or -C- =C-, and at 674 and 695 to in-plane wagging vibrations of the C- =O bonds. Because the excitation pattern was identical in continuous laser-induced combustion of a carbon-blacked seed bulb in air, burning ejects of this and all failing lamps unambiguously turned out being O2-containing gas entirely produced by the combustion of the ignited carbon, while the transparent bulb of the recovered lamp remarkably still held N2 containing an atomic ratio of O2: N2: C: H=0.0542:100:0.0258:1.0811. The effectiveness or ineffectiveness of any relevant environment in a closed low-frequency gas-discharge system was thus evaluative by the transparent operator of a failed self-metered double-parameter combustor, burster, and detector of the insert gases.
The outer nuclear membrane of most eukaryotic cells is attached to the rough endoplasmic reticulum, developing complex folded structures that extend deep into the cytoplasm in secretory and metabolic cell types such as pancreatic acinar cells and hepatocytes or projecting finger-like processes in epithelial retinal cells (Wiener et al., 1965). The nucleus is situated 3 μ in diameter in the central region of the syncytium of oocytic follicle cells in Drosophila, where it is surrounded by an apical cytoplasmic region with numerous ribosomes, parallel bundles of filaments, and glandular membrane-enclosed vesicles transporting proteins from the basal membrane to the cell surface. Unlike follicle cells, Drosophila muscle cells contain an extensive nucleoplasmic reticulum without direct continuity between the nucleus and the smooth endoplasmic reticulum (Bozler et al., 2014). The nuclear envelope is continuous with the rough endoplasmic reticulum in the cardiomyocytes of various vertebrates. The nuclear envelope forms processes that taper toward the nucleus and branch extensively, which were named nucleoplasmic reticulum. In rat cardiac muscle, the ribosome-decorated nuclear envelope is continuous with the rough endoplasmic reticulum but develop processes that taper toward the nucleus, forming a dumbbell shape around the pore zone, and branch extensively. The irregularities of the cardiomyocyte nuclear envelope occur in the region where the membrane is highly rippled. Importantly, nuclei were often found with rod-like invaginations folding upon themselves.
The nuclear envelope (NE) is a double membrane structure that surrounds the eukaryotic nucleus, separating nuclear content from cy promoted by progeroid mutations. The mammalian inner nuclear membrane (INM) consists of integral membrane proteins (IMPs) and lipids that are uniquely maintained in the INM by active mechanisms. Nuclear lamins are the major component of the underlying nuclear lamina, a proteinaceous filamentous meshwork underneath the inner membrane (Foisner, 2003). In mammals, lamins A, B1, and B2, proteins of ~225, ~70, and ~64 kDa in size respectively, are expressed from single genes (LMNA, LMNB1, and LMNB2) with different isoforms. Upon degradation, head–tail residues of lamin proteins are cleaved off which leads to mislocalized and/or enlarged/irregular nuclei with perinuclear accumulation of Crm1 cargoes as a result of mounting nuclear transport dysfunction. The mammalian outer nuclear membrane (ONM) is continuous with endoplasmic reticulum (ER) membranes. Like the INM, the ONM consists of IMPs and lipid bilayers. ER exit sites (ERES) are enriched for IMPs for assembling vesicles budding off from the ER. Many ERES are functionally coupled with INM proteins in maturing INM vesicles. Loss of lamins A and C leads to nuclear shape deformities linked to loss of decorin. Indeed, decorin degradation induces nuclear shape deformity in primary human fibroblasts and modulates lapdynamics. The NE is composed of the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) separated by the perinuclear space (PNS) and studded with nuclear pore complexes (NPCs) that span the nuclear envelope and regulate nucleocytoplasmic transport of macromolecules (W. A. Peeters et al., 2022). The NPCs are highly curved membrane areas populated by a large number of proteins (FG-NUPs) forming a gel-like barrier in the PNS, which regulates selective transport of macromolecules to and from the nucleus. INM proteins are functionally linked to nucleocytoplasmic transport through direct binding to nucleocytoplasmic transport receptors (NTRs) and chromatin organization and positioning. The organization of INM proteins remains poorly understood and only a single model describing the INM model of a mammalian adipocyte has been reported. The security of the NE and NPCs is essential for maintaining cellular homeostasis and spatial organization of genomes.
The primary items, structures, and components of the entire eukaryotic cell are the nucleus and the nuclear envelope. Whole cell shape and movement, cytoskeletal organization, intercellular division, aggregation of components, gene transcription and expression, and so on, the nuclear structure and its dynamic properties are thought to play critical roles. The nucleus has a structure in which the chromatin and the other components containing nuclear bodies are surrounded by the NE (Goto et al., 2021). The NE consists of the INM, ONM, nuclear lamina, and nuclear pore complexes (NPCs). The nuclear lamina is a fibrillar meshwork structure underlying the INM, which has been previously observed in plant cells by electron microscopy. As noted above, the animal nuclear envelope was analyzed after a mild detergent treatment to remove cytoplasmic constituents, and then INM and ONM could be discriminated clearly by TEM in the onion epidermal cells. It should be noted that the experiments were performed under conditions that very usually damage cell structures and that so many experimentally induced expression systems have apparently crashed due to usage. The experiments with whole living cells should thus be emphasized because they can provide invaluable data and techniques that are difficult to achieve under destructive treatments. The vendors demonstrated precise 3D view of an intact NE on living plant cells, depicting its discontinuities, dynamic comings and goings of nucleoplasmic and nuclear envelope, which could be used as a good standard of surveillance for plant cell nuclei. Plant cells have unique characterizations, including a rigid cell wall, chloroplasts, large central vacuoles, and importantly, an additional layer in NE called the nuclear-associated cytoplasmic bodies (NACBs). Although such cell systems are often regarded as hard fields in biomedical studies, a handful of examples available for nanoparticles in organelle targeting were cited in this article. In contrast to established animal cells, neural stem cells, and so on, it is still a long shot for trans-kingdom efficacious targeting of eukaryotic organelles. Yet, with ongoing electrifying designs and gene blueprints for proteins of desire, it is believed that bioengineering technologies would shepherd into domains less traveled undercover.
The nuclear envelope is a structure thought by many to be ubiquitous in eukaryotes, delimiting the cellular nucleus, with a diameter of 100 to 200 angstroms, comprising inner and outer membranes separated by a lumen of the diameter about 50 angstroms, and ascribed with multiple functions arising from its regulation of nuclear transport. However, the nuclear envelope is absent in some phylogenetically-characterized eukaryotes, including diplomonads, jakobids, licea and entamoebae; the nucleus in these protozoan cells is described as a karyosome, surrounded by a karyomatrix containing dispersed eukaryotic ribosomes, DNA and proteins. Due to the absence of a nuclear envelope, the karyosomes in the above taxa are not invested and are assumed to be in the same cytoplasmic phase - the cytoplasm of all extant eukaryotes - while strictly packaging of the nuclear compartment by a nuclear envelope is assumed in all other characterized eukaryotes (T. Moore & H. McAlear, 1963). Some workers have confidently denied any evolutionary relationship between cladistically-defined eukaryota with and without a nuclear envelope, viewing the last common ancestor of eukaryotes as a “cell type” without a nuclear envelope, which has been exactly followed by some archaeplastid and excavate taxa, an event regarded as evolutionary retrogresion. Others point out that divergently-evolved organisms with a nuclear envelope have independently and equally retrogressed, losing their nuclear envelope without any systematic pattern established. Understanding the ubiquity as well as absence of the nuclear envelope in eukaryote evolution requires determining whether entrants into these different categories and conversely organisms without a nuclear envelope have been defined in terms of a suite of apomorphic features and invested with a particular karyosystem. Although elements of universal cytology understandable across all phylogenetic lines exist, karyologies compatible with evolutionary history replete with retrogresion, have evolved recently, convergently and independently, a viewpoint recently articulated in a review of the karyology of uplanctonic dinoflagellates with an apocryphal analysis of evolutionary specializations.
Nuclear envelope (NE) undergoes significant alterations during programmed cell death that precedes nuclear degenerations (ND) and apoptosis of specialized or intentional neurodegeneration in cultured cells. The NE is a double-membrane structure surrounding the nucleus. Neurite injury was induced by the mechanical cut of specific neurons using a laser-ablation method in vitro. NE collapse and NEs' loss at the injury sites occurred prior to ND and apoptosis. Injured neurites underwent swelling and vacuolation, which were followed by collagenase-sensitive plunging degeneration and nuclear condensation. NE disruption in neurites was not an early event. Brain-derived neurotrophic factor (BDNF) application could block early occurrences of NDs. Besides, NDs were initially seen at the proximal side of an injured axon, moved retrogradiently, and characterized as severe swelling and subsequently apoptotic degeneration. It is plausible that alterations in the NE are sequential necrotically-triggered events of supervaricosity. No NE alterations were recognized in depolarization-triggered so-called secondary die-back degeneration. Translocation of nucleoplasmic proteins into the cytoplasm was seen in scanning EM observations in cultured neurons exposed to excitotoxicity. Inhibition of Ca2+/calmodulin-dependent protein kinase II could remarkably ameliorate morphologic alterations including nuclear shrinkage in vitro (Janin & Gache, 2018). Nuclear envelope (NE) and nuclear pore complexes (NPCs) play fundamental roles in the organization of the nucleus and spatial and temporal regulation of signaling within it. Accounting for almost one-third of the mass of the NE, integral membrane proteins form components of the NPC and exhibiting vital functions usually coupled to their folding within the endoplasmic reticulum. Genetic defects in such proteins can cause aberrations in NE and NPC structure/function that affect cellular homeostasis and eventuate in tissue injury and disease. In addition to such structural roles, other integral membrane proteins function as channels that control nucleocytoplasmic transport by gating. Transport defects, either by overactivity or by loss of function, compromise cellular homeostasis and promote injury and disease. Investigators have failed to establish cellular systems in which to follow the specific protein import/export pathways involved and the consequent translocation of signaling to/from the nucleus. Such cellular systems would allow for greater understanding of how such pathways are coordinated with the myriad of other molecular and cellular events which underpin health and promote disease (Fichtman et al., 2019).
Progeria has become a paradigm for studying aging. This extremely rare disease occurs in less than one in eight million births and manifests a premature aging phenotype as children grow, with symptoms including growth failure, loss of body fat, alopecia, skin changes, joint stiffness, and cardiovascular disease. Almost all cases of progeria are due to a single point mutation in the LMNA gene that encodes the A-type lamins, nuclear envelope proteins with multiple functions in nuclear structure and organization, chromatin dynamics, and genome integrity. The most common mutation results in the deletion of 50 nucleotides, producing the most studied progeria-associated protein: progerin. Progerin highlights important cellular functions of the nuclear lamina, such as progerin accumulation in non-dividing cells, cellular senescence, nuclear ruptures, changes in chromatin organization, loss of heterochromatin, loss of mechanical properties and elasticity, and uneven distribution of lamina-associated proteins. These alterations have been linked to nuclear pore complex dysfunction and changes in nucleocytoplasmic transport. Nuclear pore complexes are composed of 30 distinct proteins and are the only channel for transport molecules larger than 60 kDa. They maintain nuclear homeostasis by regulating nuclear import and export of proteins and RNAs. Cellular enlargement and chromatin decondensation observed in nuclear pore complex mutants have been proposed to explain premature aging symptoms. Defects in nucleocytoplasmic transport and differential effects of mutant lamins were observed on assembly-disassembly dynamics of GFP-nuclear pore complexes in HGPS cells.
Mutations in several nucleoporins have been linked to several disorders, which exhibit aging-related phenotypes. Identifying early and late cells with abnormal nucleoporins may provide valuable insights into the underlying mechanisms of HGPS. Examining nuclear pore complex composition over time in mammalian cell lines exogenously expressing progerin may clarify unexplained aspects of progerin cytotoxicity. Overall, while pathogenic mechanisms underpinning premature aging in HGPS are incompletely understood, changes in the nucleocytoplasmic transport are widely seen in HGPS and in other age-related diseases. The disruption of the nuclear envelope and nuclear pore complex due to cellular stresses is viewed as a novel common mechanism of aging that warrants further investigation in the study of progerin and its interactions with other proteins.
Muscular dystrophies are a group of diseases characterized by muscular weakness and wasting, caused by degeneration and regeneration of muscle fibers that eventually replaces muscle with fibrosis and fat. The underlying disease mechanisms are poorly understood and many forms of the disease are untreatable. Two widely known forms, Duchenne muscular dystrophy and congenital muscular dystrophy, belong to separate categories of the disease but are anticipated, from genetic and histological study, to share a common defect in a component of the dystrophin-associated protein complex which is localized at the sarcolemma of myofibers. In addition, mutations in several genes encoding lamina-associated proteins have been implicated in muscular dystrophies.
A mutation in the SUN fragment of SUN1/SUN2 has recently been shown to cause an Emery-Dreifuss form of muscular dystrophy. On the other hand, reports of other mutations in lamin A/C, an inner nuclear membrane protein, have also been shown to cause muscular dystrophy with concurrently altered shapes of the nuclear envelope. It is, therefore, of interest to figure out how a defect in a protein targeting the NE thereby participating in the lamina affects nuclear lamination and shape to contribute to dysregulation of the NE by either one or both NE defects.
In addition to their roles in providing structural mechanical strength and rigidity to the NE and nuclei, lamins and proteins associated with the lamina or the NE are also critical for regulating chromatin organization and gene expression. It is, therefore, possible that mutations in these proteins alter gene regulation so that muscle and brain identity genes are aberrantly expressed in non-muscle or glial cells through a mechanism coupled with dysregulation of the NE (Meinke et al., 2014). Several of the lamina-associated structural proteins have been shown to be targeted to the NE independently from either lamins. Hence it is likely that effects on nuclear and NE morphology by an alteration of the lamina is propagated to dysregulation of the NE in a manner not involving lamins. As such, direct targeting of the nuclear morphogenic machinery by the defect in a protein involved in the lamina should also be considered in a future study.
Although preliminary, findings of nuclear changes in neoplasia are thought to have implications for the understanding of malignant transformation as these changes reflect alterations of both nuclear envelope and nuclear material structural integrity. Alterations of the nuclear envelope components, namely undulations of the inner leaflet of the NE, loss of so-called “bolts" area of chromatin apposition to the lamina, and irregular nuclear lamina structure can be revealed by conventional transmission electron microscopy. The loss of normal lamina structure manifested as accumulation of aggregates in large fissure-like spaces between the inner and outer lipid bilayer membranes and in mitotic cells was associated with abnormalities of lamina-stabilizing filaments being long, dense and intermixed with fibrous proteins. Cachexia was associated with large primitive nuclei and increased nuclear pores number in breast carcinoma and with foci of non-homogeneous-laminar NE in mammary carcinomas. The latter model in follow-up studies provided evidence that alterations of nuclear envelope associated with cytoplasmic and genomic structural changes and necrosis have implications for malignancy. In contrast to data suggesting that a dysfunction of lamina-recycling vesicular transport machinery underlies nuclear lamina breakdown in transit from quiescent to rapidly dividing cells, strong association existed between necrotic cancer growth and multi-fusion of vesicular cytoplasmic compartments, general impairment of cytoplasmic structure, and cytoskeletal degradation. Also, while there was no obvious relation between nuclear alterations and either mitosis indexing or any gradient of growth rates along the growth-inhibitory interpillar sheet, it was shown by appropriate pharmacological manipulations that combined perturbations of either cellular level energetics or cytoskeleton structure have profound and reciprocal effects on the speed and form of changes in NE integrity, in lamina structure and in chromatin and nucleolar organization indicative of temporal multi-pathway applicability of overall NE disorganization and of neoplastic transformation, which is not bounded to specific genic mutations or types of dysregulation.
Light and fluorescence microscopy approaches can be used to characterize the nuclear envelope at very different levels in terms of the spatial resolution achieved. The ultrastructure of the nuclear envelope is best viewed using high-performance lens or electron microscopy (EM) approaches. When sections or replicas are examined in a transmission electron microscope, ultra-thin sections, i.e. sections < 70 nm, allow to visualize ultrastructural details of the nuclear pore complexes (NPCs) in epon-embedded, chemically fixed, and contrasted materials. As an alternative to EM sections, replicas can be employed. While carbon replicas yield only poor contrast and a low likelihood of visualizing only the nuclear lamina, metal – typically platinum – replicas provide excellent contrast at the expense of better preservation and a more complicated preparation protocol. Cryo-related techniques interested the observation of thin film vitrified sections. A wider variety of strategies was implemented to analyze such samples. Cryo scanning electron microscopy (cryo-SEM) made first use of the cellular array and preparation strategy developed for electron tomography. While sections of 300 nm thickness may nowadays be achieved under optimized conditions, tomographic reconstructions of cryo-SEM allow to limit information to a maximum of 45 nm. Substantial parts of the specimen can be examined but information regarding the 3D nature of the nuclear envelope, higher order arrangements of lamina-associated chromatin, and access to fluorescent probes was not detailed.
Cryo electron tomography of detergent-extracted nuclear envelopes (NEs) delivered information regarding the ultra structure and the polymerization of imported in the NE surrounding the nuclear pore complexes (NPCs), et al. (Bouchet-Marquis et al., 2018). 3D reconstructions obtained from frozen-hydrated sections of a filamentous fungus and CE-TEM of vitreous sections allowed depicting the complex and previously unknown arrangement of the nuclear envelope and growth sectors. The main difficulty of these studies lied in the performance of zero-loss imaging, a focus criterion and in controlling the stage drift over time scales of hours. Combined with averaged template-based approaches combined with direct electron detectors lead to very revealing 3D reconstructions of Saccharomyces cerevisiae NEs. The relevance of using a warm lamp to irradiate frozen samples, with a bombarding e-beam resulting from the pre-exposure images recorded on a top coherently detected transmission of electron microscope was also presented.
Cryo-electron tomography of vitreous sections would be an excellent venue to detail how these elements assemble, operate as a structure, modify their structure and interact with the nucleus and the entire cellular cubicle. No special care is needed to ensure that the specimens remain vitrified, as they are scrutinized at ???C. Many new types of microscopes targeting different niches (medium throughput, cryo etc.) should improve site accessibility to a wider number of users and maximize the effort in visualizing and understanding how these components contribute to cellular functions. Expected advances in detection technologies should limit the risk of losing resolution as the number of access points increases. The use of a smaller pixel size and because of an increase in investment in better optics, cameras with larger effective pixel sizes as a consequence, should help improve cystic fibrosis. All imaging modes used in electron cryo-tomography studies are needed to understand how NEs acquire their plasticity either during normal cellular operations or through structural remodeling. These studies can provide vital information about how NEs work, as well as on the etiology of diseases about which little is currently known.
The nuclear envelope consists of two membranes, i.e., an outer and an inner nuclear membrane, which are elaborate double membranes contiguous with the rough endoplasmic reticulum. A space, termed the perinuclear space, exists between the two membranes. The inner nuclear membrane, which differs in protein composition from the outer nuclear membrane, is supported by a thin lattice, the nuclear lamina composed of subunit proteins termed lamins. Nuclear pore complexes (or NPCs) are embedded in the nuclear envelope at intervals of about 100 nm. NPCs are bent octagonal complex structures composed of a large number of proteins termed nucleoporins (Nups). With nuclear basket structures protruding from the nuclear side, the NPC forms a non-ferritin or annular channel about a 120 Å wide through which only small, water-soluble molecules can freely diffuse, whereas larger molecules, e.g., ribonucleoproteins and proteins bound to RNAs, require the nuclear pore transport mechanism and, hence, export and/or import factors to traverse through the NPCs. The overwhelming majority of these transport factors are soluble proteins.
Some of the approaches used to study the nuclear envelope are described below. Initially examined were fixed and embedded cells using ultrathin sections cut with a diamond knife. Negatively stained to enhance the contrast with uranyl acetate, lead citrate was applied to non-membrane materials like chromatin. Serial ultrathin sections of chemically fixed cells were cut without any pre-treatment and stained by standard uranyl acetate and lead citrate methods. Staining was needed to improve the contrast, which was more effective in negative stains than in contrast-enhancing stains. The regulation of structure and distribution of NPCs by applying an agent like α-amanitin, which blocks transcription, was investigated. Subsequent freezing and freeze-fracture replica methods permitted the observation of changes in the structures of the nuclear envelope after the addition of physiological agents to living cells. Each analysis, combined with biochemical fractionation of cellular components, immunodiffusion, immunolocalization and gel electrophoresis, demonstrated how distinct nuclear pore nutrients, biogenesis, transport, or NPCs, and their nucleoporins were compartmentalized.
With the past centuries advancements in microscopy, significant alterations have seen divergent imaging instruments. During the 20th century, polarized light microscopy and phase contrast microscopy were exemplary achievements. The latter allowed non-invasive observation of unstained biological material. Increased interest in cellular organization and function resulted in enhanced resolution and thus increased appreciation of the intricacy of subcellular structures/components and their importance in maintaining proper cell function. Modern light microscopy procedures allow localization tracking of specific molecules and macromolecules within living cells. These capabilities have been afforded by the biophysical advances with regard to the development of non-toxic fluorescent compounds. Most objects reside within inhomogeneous refractive-index media rendering some degradation of the obtainable resolution (Bouchet-Marquis et al., 2018). For instance, resolution becomes diffraction-limited of around 200 nm in aqueous solutions for visible light corresponding to 300-800 THz energy region. The resolution range of experimental designs includes atomic resolution achieved with EELS-STEM, about 0.5-1 picometer, and up to 1-5 Å for crystallography and coherent high-energy x-ray studies up to ffemto. Based on the biological structures and applications, various XM designs have been developed and employed. For instance, analysis of nuclear pore complexes, halide and noble gas stable isotopes and ions bound to NUP358 were studied using Cl and X-ray fluorescence imaging and confocal XRF. These studies demonstrated that they are composed of aluminal spoke complex situated between a nuclear and a cytoplasmic ring, on which, filaments are attached interacting with cargos, thus facilitating their approach and transit through the complex. The observed nuclear pore complexes appear as 100-nm-wide aperture in the nuclear envelope in agreement with earlier studies. The tomography of vitreous sections probably represents the most powerful tool at the present moment. It combines the advantage of vitreous section with the possibility of high-resolution reconstruction into a 3-D model of the section. A resolution better than 6 nm can presently be obtained on thin vitreous layers of cells.
Biochemical approaches to study the NP staining method are also of particular interest. Improved imaging quality can be achieved using biochemical strategies to limit imaging to the NP staining itself while inhibiting staining of the supporting phospholipid bilayers. Dried, cryo-dried, and cryo-fixed plastic embedded tissues were treated with organic solvents and/or with detergents to remove cytoplasmic staining. Cleaning treatments were first developed on artificial tissues made of proteins and sugars, prior to application to tissues. These approaches may be of use for TON treatment to study the 3D ultra structure of NPCs in various species and tissues.
Although the use of chemical fixation is often unavoidable for the sake of preserving tissue shape, it is known to alter the occlusion of the phenylamine rings in the TOR molecule, resulting in diffusion of the DAB compounds into other cellular compartments. Furthermore, fixation can result in higher background staining by non-specific reactions between aldehydes and amines that are present as free amine radicals on proteins. Fixation can be further complicated when chemical fixation is followed by immunostaining. Recent methods to improve this fixation step have consisted of genetically eliminating amine-containing residues not involved in protein function and folding, homogeneous fixation, and the use of diazolidinylurea. Therefore, alternative methods of fixation that minimize tissue distortion and amine groups would be welcome. Arresting enzymatic and diffusion processes shortly after flooding living tissues has previously been shown to prevent protein diffusion through phospholipid membranes. Preliminary attempts to analyze the potential of using FLASH treatment for fixation purposes have proven promising for limiting the leakage of DAB products from NP staining.
As the number of questions about NE function, morphogenesis, and disease continues to grow, techniques have emerged and matured that will push the field forward. Some likely fruitful directions include: Developing new models and new techniques to analyze NE morphogenesis, such as live-cell imaging techniques, and using new experimental organisms, such as zebrafish and Drosophila. To better understand the initial establishment of the NE, it may be advantageous to screen for morphogenesis mutants using plasma membrane “wedge” assays to visualize Node-to-embryo transitions. This might uncover the full range of pertinently unexpected dynamics uncovered in cultured cells and weld the study of embryonic development and NE research more tightly together. As diverse, low-dimensional, self-replicating, and self-propagating quasi-crystal systems become easier to access. Their analysis might yield new views of the assembly of membraneless organelles, as assembly pathways based on 2D symmetry might bridge between disparate morphologies diverse.
Constructing rationally designed energy landscapes of increasing complexity to induce NE-sized structures nucleating from simple building blocks might help shed light on both spontaneous lipid bilayer formation and vesicle budding mechanisms. Understanding ANE function, disease, and morphogenesis will require integrating efforts across many fields (Bozler et al., 2014). Cell biologists might quantify NE architecture using the approaches developed by materials scientists to classify and monitor self-assembled crystal morphology. Evolutionary game theory might tackle neurodegenerative diseases involving membraneless organelle formation and use field theoretical techniques to compare relative stabilities and shapes of low-dimensional tessellations and quasi-crystal networks.
The nucleocytoplasmic barrier that separates the eukaryote nucleus from the cytoplasm is formed by a nuclear envelope (NE) composed of an inner nuclear membrane (INM) and an outer nuclear membrane (ONM) projecting into the cytoplasm. The structural, functional, and biophysical properties of the NE are defined by the membrane bilayers and the integral membrane proteins, and the structure of the NE is coordinated with timing and sub-cellular locations of the membrane proteins. The structural backbone of the NE, on which membranes and membrane proteins are assembled, is the nuclear lamina that is composed of filamentous lamins, soluble intermediates, and assembly factors (Foisner, 2003). The NE is dynamically remodeled in response to extracellular stimuli, or deregulated in some pathological conditions like cancer, differentiation, and development, via changes to the structure of the NE membranes or nuclear lamina. Lamins and nuclear pore complexes (NPCs) undergo complex and multisteps disassembly and reassembly processes during mitosis.
In NE dynamics and assembly, consistent methods of protein-attribution (dis)assembly labeling and mass spectrometry have been developed to identify and track membrane, PANE and lamina components during cell cycle, and quantitative models are built with Monte Carlo-simulations using these data with NE models. The NE consists of double phospholipid bilayers with an associated nuclear lamina and abundant NPCs, and its integrity has to be maintained while facilitating the rapid adaptation of cellular processes, including gene expression and signal transduction. Although there has been significant progress recently in understanding the components, structure, and influences on the NE, key factors controlling the dynamics and assembly of the distinct NE ultrastructures at the molecular level and its roles in regulating cellular processes remain largely unknown.
Cell nuclei can vary greatly in structure and appearance, due to differences in cell types, microenvironment and internal physiological stimulus. Portholes (nuclear pore complexes (NPCs)), which perforate the nuclear envelope (NE) are conserved structures essential for the controlled movement of macromolecules into and out of the nucleus. The NPCs comprise over thirty unique proteins termed nucleoporins (Nups) (Shahin et al., 2016). Thousands of NPCs in each cell are coupled with NI, oligomeric complexes of which are assembled into two-fold symmetry barrels composed of massive Nups. Importantly, the FG-rich Nups are fundamentally floppy and dilute gel-like and form a selective barrier that blocks the path of most macromolecules with a diameter greater than 5-9 nm. Despite the gaping 60-120 nm holes in the NI, the exclusion mechanism of the NPCs has created several long-standing conundrums. Many assembly factors bound to nups and the organelle provide insight into the relationships between NPC biogenesis and Nup folding events. On the other hand, the central channel diameter varies upon binding or release of transport cargo.
The nuclear envelope (NE) forms a barrier between the nuclear environment and the cytoplasm. It consists of two phospholipid bilayers: the inner nuclear membrane (INM) and outer nuclear membrane (ONM) (M. Drozdz et al., 2017). Despite the highly ordered structure of the NE, cell nuclei can rapidly undergo structural changes in response to different stimuli, including differentiation, replication, and cell migration or in pathological situations such as ageing or cancer. An example of alterations to the NE that significantly change its morphology is the formation of the nucleoplasmic reticulum (NR). NRs can be composed of only INM (ONM absence is indicated by the label INM#) or both INM and ONM (NR and ONM#). Prominent NR structures can be found in many blueprints, tissues, and species both under normal conditions and during pathological states of the cells or tissues, which suggests cells can regulate its formation (before they are genetically reprogrammed) and function. Despite the numerous reports on the presence and persistence of NR structures, their exact function remains incompletely understood. There is nevertheless some evidence suggesting the NR may play a role in: 1. providing structural support for the large and spherical nucleus, 2. insignaling between the cytoplasm and nucleoplasm.
There is an incredible structure to the cell in the form of the nuclear envelope. The thickness of the membranes is considerable, even at 12-15 nm, so measurements must be interpreted with caution. It is likely that localized changes in both the nuclear inner and outer membranes occur. Such membrane variations can play an active role in studies of both the nuclear structure and function. The attachments between the membranes appear to define some structure in the nucleus. These variations along the nuclear envelope vary in strength and persistence. There are many theories about the structure and pathologies of the nuclear envelope based on such sheets, linked as a ladder. However, it remains to be shown whether or not their separation relates to changes in the behavior of the chromatin. The nuclear pores are robust and highly symmetrical, opening in only a small area of the membrane. They do not form chains of pores. These pores remain liquid-like and expandable according to molecular simulations. Super resolution images show that the pores give rise to extensive meshes on the nuclear surface suggesting that some chromatin directly attaches to this mesh. The complexity of the X-Y coordinates of the visualized chromatin appears to be reduced at the boundaries of these pores, suggesting that they may help organize the chromatin. The basic morphology of the nuclear envelope, chromatin, and nuclear pore complexes appear quite similar between scanning EM and cryo-electron tomography images, despite a significant difference in sectioning strategies.
The interactions of the nuclear envelope with the cytoplasm, nuclear pore complexes, chromatin, and membranes have been studied in great detail. The predicted mechanical properties of the interior nuclear envelope and lamina filaments may play an active role in genome organization and function. The comprehensive structure of the cell nucleus is as yet unresolved. The combination of a reliable high-throughput imaging system, image analysis, atomic structure construction, and predictions provide a prototype system to study the nuclear structure in an energetic way. A comprehensive analysis of the nuclear envelope alone can generate many new insights about chromatin, membranes, and associated proteins which will highlight their specific structure within this complex yet dynamic environment.
The nuclear envelope (NE) divides the nucleus from the cytoplasm and functions to prevent non-specific transport events into or out of it. In eukaryotes, a nuclear lamina lies on the inner-face of the NE and is thought to provide the structure for NPC insertion. The NPCs then provide the only route for material transport between the nucleus and cytoplasm. The NE contains unique lipids, but it remains largely uncharacterized how the common membrane structures can achieve such unique properties and forms, or how they are modified at the NPCs. A lipid point of view, which considers the self-organization of lipids in terms of curvature generation, energy minimization, interaction energies, or membrane insertion, can be employed in the interpretation of NE structure and dynamics.
Mammalian cells have an NE that consists of two fused lipid bilayers and their associated insoluble proteins. Together these forms a quasi-spherical nuclear compartment that is separated from the cytoplasmic compartment containing water, ions, and soluble proteins. Except at the NPCs, the NE bilayers merge into a flat and smooth surface that is 10 times more continuous and two times smoother than the inner and outer membranes of phagosomes. The NE bilayers curve gradually towards the nuclear pore entrances. Near the nuclear pore entrances, the individual bilayers converge within a 10 nm wide neck. The narrow neck widens into a maneuverable 150 nm deep membrane chimney and further merges at the pore insert. Starting below a 130 nm greater curvature threshold, the bilayers become continuous at the pore insert (W. A. Peeters et al., 2022). This NE ultrastructure ensures stable enrichment of Nups and efficient NPC function, as well as maintaining long-term homeostasis for the nucleus.
As in yeast and frogs, NPCs in mammals appear to arise from preexisting domes and they expand after engulfment of cytoplasmic Nups. The integration of new membrane materials at chimneys further enhances this enlargement process. In this sense, domes and chimneys are decisive morphogenic elements for generating new pore complexes. To assist continued stability and expansion of the NE, the NE assembly process should also reform lumen and membranes. Although that aspect is less well understood, the gradual versal evolution of the pore membrane morphology possesses important implications for finding bilayer remodeling catalysts at the bilayer necks or along the neck length (Bozler et al., 2014).
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