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The Nuclear Envelope During Cell Division

Introduction

Nuclear Envelope and its Proteins

The nuclear envelope (NE) is a highly specialised membrane that outlines the nucleus [1] and is a key physical compartment that defines eukaryotic cells. It has been described as a highly organised and regulated double membrane that compartmentalises the cell’s genome. [2]

It is composed of two concentric membranes, the outer nuclear membrane and the inner nuclear membrane, which are joined by Nuclear Pore Complexes (NPC) that span both membranes. [3] The outer membrane is continuous with the membrane of the Endoplasmic Reticulum (ER), [4] while the inner membrane is attached to the lamina and chromatin of the nucleus. [3]

The NE serves to maintain the structure of the nucleus and its position in the cell by providing anchoring sites for its attachment to the cell’s cytoskeleton. [3] More importantly it serves to separate nuclear and cytoplasmic activities,[5] including transcription from translation, protecting genetic material from the highly metabolic environment of the cytoplasm. [6] It acts as a selective barrier between the cytoplasm and nuclear contents, with the NPCs contributing to this diffusion barrier by regulating the passage of proteins, RNA and ribonuleoprotein complexes in and out of the nucleus. [5] In addition, more recent studies have shown the inner membrane proteins to play various important roles in the function of the nucleus including chromatin organization, gene expression and DNA metabolism. [2]

Changes to the NE’s structure occur at the onset of mitosis, these changes are very slight in lower eukaryotes and in higher eukaryotes result in the complete disassembly of the nuclear membrane. [7] This complete or partial breakdown is necessary in order to form the mitotic spindle on condensed chromosomes. [5] Higher eukaryotes must consequently reassemble the NE around the genetic material of the daughter cells each time a cell divides in order to re-establish the nuclear compartment. [2] This page aims to explore the process of the breakdown and reassembly of the NE and its role in cell division.

Links: Cell Nucleus

Historical Background

Timeline of Historical Research On The Nuclear Envelope

Time Discovery
1913 The Nuclear Envelope was described as a double membrane that encloses the genome by G.L Kite. [8]
1950 First EM study on the NE by Callan and Tomlin revealed that they contained pores. Furthermore they reported the nuclear envelope having two layers, a continuous internal layer and an outer porous layer. [9] [10]
1955 Watson reported that the nuclear envelope is continuous with the endoplasmic reticulum by carrying out an electron microscopy study of thin sections of interphase cells. He hypothesised that there were two communicating pathways between the cytoplasm and the nucleus; the perinuclear space and the cavities of the ER and the the NPCs. [11]
1967 Gall and co-workers discovered that NPCs had an octagonal structure. They did this by using negative staining on the NE from starfish, frogs and newts. [12]
1989 Hans Ris set in motion research of the function and structure of NPCs after developing high resolution electron micrographs. These micrographs were able to reveal the existence of nucleoporins. [13]
Nuclear Pore Complex
1992 A study was carried out using high resolution scanning electron microscopy (HRSEM) and the findings revealed a fibrous lattice attached to the Nuclear Pore Complex Basket. [14]
High resolution scanning EM of the Nuclear Envelope Lattice
1994 The discovery of mutations in a gene encoding for nuclear proteins being the causative agent of Emery-Dreifuss muscular dystrophy. This lead to the idea of laminopathies, human diseases caused by abnormal functioning of NE proteins. [15]
1996 The study carried out by MR Paddy and co-workers revealed the rearrangements the nuclear lamina undergoes and furthermore they observed that this process takes place in conjunction with the movements of centrosomes and microtubules. While these dynamics were observed via the technique of Time-resolved, two-component, three-dimensional fluorescence light microscopy imaging,the study did not cast a light on the significance of these structural dynamics. [16]
1998 It was hypothesised that the nuclear lamina plays an important role in the nuclear disassembly during mitosis by P.Collas through the investigation of mitotic extract from sea urchins. [17]
2000s 2000 and onwards studies on the specific mechanism and functions of the nuclear envelope as well as its role and interaction with other processes and factors such as proteins were investigated. The NE was also observed in conjunction with viruses and abnormal cells leading to a broader understanding of the normal and abnormal functioning of the NE. [18] [19]

Structure of the Nuclear Envelope

Structural Components of the Nuclear Envelope


The (NE)is an important structure that covers the nucleus with a double membrane. The prominent constituents of the NE include the nuclear lamina, NPCs and nuclear membranes [20] . The nuclear membrane is made up of distinctive but unified domains: the outer nuclear membrane (ONM) and the inner nuclear membrane (INM) [21] It is the small pore membranes that join the INM with the outer ONM together and the double membrane is separated by an intermembrane space. Moreover, the NE forms a partition between the nucleus and the cytoplasm and also allows attachment sites for structures such as the cytoskeleton to the nuclear periphery [21] [22]

Outer Nuclear Membrane

Structurally continuous with and comparable in composition to the peripheral endoplasmic reticulum is the outer nuclear membrane. [23] [21] As reviewed in Lynn et al, (1984), it is thought that the outer membrane could be a site of membrane-bound synthesis of glycoproteins. Ribosomes are also found on the cytoplasmic surface of the outer nuclear membrane which is continous with the ER. Since there is a significant similarity between the endoplasmic reticulum and the outer nuclear membrane, it is thought that NE is a more elaborate form of ER. [24] However, the outer nuclear membrane also has sites that assist with nuclear positioning.[25]

Inner Nuclear Membrane

The Inner Nuclear membrane (INM), neighbouring with the endoplasmic reticulum membrane, is connected with the lamina and the chromatin [26]. It is in close contact with the genetic material of the nucleus and has various proteins attached to it. These proteins are manufactured on the rough endoplasmic reticulum and have important implication in human diseases which will be discussed later. [27] In recent years there has been an increase in the number of discoveries of various INM proteins mainlydue to the proteomic and computational approaches. [28] One such group of proteins include transmembrane proteins that are associated with the INM and they usually interact with chromatin and/or lamina. [29]

It is thought that these transmemberane proteins direct the chromatin to the membrane; this is essential during the reformation of the NE after mitosis. [29] Furthermore, important intermediate proteins called lamins are attached with one another to form 10nm-diameter filaments and these lamins make up an important network called the nuclear lamina. This structure, the Nuclear Lamina, is important because it not only supplies rigidity to the nucleus but also has other roles in the cell [30] [31] Ulbert et al.(2006), through their research on transmemberane proteins, have illustrated that interaction of Lem2 with chromatin or lamins are crucial to the maintenance of NE. Ulbert et al. (2006) have also demonstrated that (from the interaction of Lem2 and its partners) that this transmemberane protein has an essential function in the structural integrity of NE . Furthermore, a reduction in the transmemberane protein leads to aggregation of cells with deformed nuclei and ultimately cell death. [29]

Nuclear Pore Complexes

Schematic view of a model adopted for the NPC/NE system

One of the most essential components of the NE are the Nuclear Pore Complexes (NPCs) which are embedded in the nuclear envelope and they are necessary for proper cell functioning since they control the entrance and exit of macromolecules between the nucleus to the cytoplasm. [32] [33] The NPCs are composed of numerous copies of ~30 nucleoporins and span the nuclear envelope at junctions of the inner and outer nuclear membranes. [34] [35] The NPC has an estimate mass of 125MD in vertebrates and in a yeast, the NPC is found to have approximately 100nm ringed-shaped diameter. The NPC also has a central channel that is about 30nm in diameter. [34]

Moreover, as reviewed in DeGrasse et al. (2009), the NPC in eukaryotes have eight spokes around the central tube which serves as the medium for the bidirectional movement of macromolecules. [36] As reviewed in Akey (1985), it is thought that the NPCs have structural plasticity as its flexible nuclear pore diameter assists with the movement of specific macromolecules. [37]


Links: Structure of the NPCs

The Different Forms of Mitosis

Open and Closed Mitosis and Virtual Nuclear Envelope Breakdown

Comparing Open and Closed Mitosis

Typically eukaryotic cells undergo one of the two forms of mitosis; higher eukaryotes (metazoans) go through open mitosis, while lower eukaryotes including yeast and other types of fungi undergo closed mitosis. [38] The distinction between open and closed mitosis can be made by focusing on the behaviour of the NE which separates the nuclear contents from the cytoplasm and is split to form daughter nuclei. [39] Open mitosis is so named because the nuclear envelope completely breaks down at the transition from G2 stage to M stage of the cell cycle [38] and the nuclear content, including the genetic material, is “open” to mix with cytoplasmic macromolecules [40] until the nuclear envelope is reassembled after chromosomal segregation during telophase. [38] [40] In contrast, during closed mitosis the nuclear envelope remains intact and mitosis continues within the nucleus resulting in the fission of the NE after chromosomal segregation. [39]

In open mitosis the entire NE, including the NPCs, breakdown allowing the attachment of the spindles to the chromosomes [5] as well as their interaction with cytoplasmic macromolecules needed for mitosis. This means that the transport of molecules in and out of the nucleus through NPCs is not required during open mitosis. [41] In contrast, the NE of eukaryotes undergoing closed mitosis remains intact, with the continual function of its NPCs that are important in maintaining the connection between the nucleus and cytoplasm so that tubulin and proteins, that are necessary for the regulation of entry into mitosis, are allowed to enter the nucleus. [38]

The importance of the maintenance of the NPCs in closed mitosis is evident in a study conducted by Louk et.al (2002) where fluorescent microscopy was used to analyse the NE and NPCs of the yeast Saccharomyces cerevisiae. They found a link between components of yeast NPCs and the mitotic checkpoint machinery, showing that the Mad1p and Mad2p proteins, required for the establishment of the spindle checkpoint, were mostly found at NPCs throughout the cell cycle. They observed that once the spindle checkpoint was activated, a build up of Mad2p at the kinetochores occurred which coincided with the hyperphosphorylation of Mad1p mediated by the nucleoporin sub-complex Nup53. These observations led Louk and his peers to suggest a model where the association of Mad1p to Nup53p-containing NPCs traps Mad2p until it is primed for release by the spindle checkpoint activation. In addition they suggest that the bond between Nup53p and Mad1p has a role in the aid of transport through the NE. [41]


The variant forms of mitosis occurring in higher and lower eukaryotic organisms

In addition, it has been established that in order for a eukaryotic cell to undergo mitosis successfully microtubules need to gain access to the chromosomes so they can be evenly distributed among the daughter cells. As previously explained, this is made possible through the breakdown of the NE in open mitosis. Under closed mitotic conditions, where the nuclear envelope remains intact, the spindle poles or microtubule organising centres (from which the microtubules arise) are embedded in the NE, hence, the microtubules form within the nuclear compartment and so are able to gain access to the kinetochores of the chromosomes. [42]

Semi-closed Mitosis

Classification of mitosis in eukaryotes into open or closed forms can be limiting as some organisms have been found to have varying extents of nuclear envelope breakdown (NEBD) and the timing of the breakdown can also be atypical of open mitosis. [39] [38] For example, a study conducted by Paddy et.al. (1996) on early Drosophila embryos, using time-resolved 3D fluorescence light microscopy imaging, showed that there was an abnormally long period during mitosis where a large fraction of lamins remained intact and localised around the periphery of the nucleus. This semi-disassembled envelope persisted long into metaphase spindle formation and eventually the lamins dispersed just before chromosomal segregation. They also observed an extensive series of structural rearrangements in the lamina which appeared to be linked to or driven by the movements of chromosomes and spindle fibres. Paddy and peers (1996) state that this behaviour isn’t characteristic of neither open nor closed mitosis but instead appears to be of an intermediate form, [16] one of semi-closed mitosis until after metaphase. [39]

The Nuclear Envelope At the Onset of Mitosis

Just before the onset of mitosis the nuclear envelope begins to undergo processes that will eventually lead to the tearing and breakdown of the NE. This involves centrosome migration and microtubule interactions with the NE. Preparation for the breakdown of the NE occurs in late G2/early prophase and involves an early structural deformation at particular sites of the NE. [43]

The review by Guttinger et.al explains that the indentations which form around the centrosomes (microtubule organising centre) are induced by the pulling forces of the microtubules that have attached to the outer surface of the NE. These stretching forces ultimately lead to the development of fenestrations on the distal side of the nucleus and are observed in the fragmentation of the nuclear lamina. [44] The microtubule motor protein, dynein is required to participate in this process and it associates with the NE at the end of the G2 stage of interphase. [7] The interactions of the microtubules with the NE facilitate NEBD as well as the rupturing of NE. [44] Observations made in a particular study give evidence as to the involvement of microtubules at the entry into mitosis, this includes: the placement of sister chromosomes and microtubule asters directly at two indentations of the NE during early and mid-prophase, the correlation between microtubule growth toward the NE and the deepening of the nuclear indentations that ultimately lead to the fenestrations in the NE, and lastly the absence of focal nuclear indentations in cells with experimentally induced disassembled microtubules [45]

Moreover chromatin condensation occurs before NEBD and the rate of condensation increases by three-fold after the NE is made permeable. This supports the ideas that changes in the NE constitute changes of other elements in cell division [44]

A study conducted by Raaijmakers JA and colleagues further found that the microtubule motor protein kinesin-5 (Eg5) is also important in centrosome separation. Furthermore the study showed that with the absence of Eg5 the cell still undergoes mitotic activity. They discovered that the cell uses the MT motor protein dynein to carry out centrosome separation. [46]

Breakdown of the Nuclear Envelope

Nuclear Envelope Breakdown and Reassembly During Open Mitosis

The breakdown of the NE only occurs in higher eukaryotes/metazoans like plants and animals. The entry of these types of cells into mitosis (prophase) is defined by the condensation of chromatin and the events leading up to the loss of compartmentalisation of the nuclear content [42] [2] and it not only involves the removal of the inner and outer nuclear membranes but also the disassembly of the underlying lamina and NPCs. [44] “By prometaphase, the NE has largely dispersed and the nuclear contents are released into the cytoplasm.” [42]

Metazoans form spindle microtubules which are located in the cytoplasm and are vital in mitosis for the segregation of chromosomes, therefore the disassembly of the NE is required in order for these microtubules to gain access to the kinetochores of the chromosomes. [44]

The precise mechanism by which the NE is broken down has not been established,[43] as studies have revealed various high order eukaryotic species to have different ways in which their NE disassembles; therefore there are a number of proposed methods by which this occurs. In addition the order in which the different components of the nuclear envelope are disassembled is also unclear.

What is clear, however, is the involvement of mitotic kinases in the breakdown of the nuclear envelope. Such kinases as the Maturation/Mitosis Promoting Factor (MPF) containing cyclin-dependant kinase 1 (CDK1), also known as p34, are responsible for the phosphorylation of NE proteins, which eliminates protein-protein interactions, and the consequent disintegration and dispersal of all major NE structural components. [43]

Reviews such as those by Kutay and Hetzer (2008) describe the breakdown of the NE as involving three key processes: the disassembly of NPCs and the subsequent increase in nuclear envelope permeability, the depolymerisation and consequent breakdown of the nuclear lamina and the disassociation of the NE from the chromatin. [7] The breakdown culminates in the dispersal of most of the disassociated components into the ER. [47] It has also been suggested that NE vesiculation and spindle microtubule mechanism are other contributing methods for NE disassembly. [43]

Microtubule Involvement in Nuclear Envelope Breakdown

Microtubules not only function in the separation of chromosome pairs but studies of mammalian cells using multicolour confocal 4D imaging have also shown growing microtubules to contribute to the breakdown of the NE, particularly through their role in membrane indentation and consequent fragmentation. [45] [47] As mentioned in the previous section, the forces exerted by microtubules onto the NE are suggested to result in its deformation as well as its stretching in preparation for its consequent breakdown.

Observations on dividing mammalian cells made using quantitative 4D imaging in conjunction with fluorescence photobleaching techniques showed that the stretching of the NE led to the “formation of holes in the NE at the site of maximal tension” and hence can be thought of as a tearing of the envelope. These tearing sites were found to be located opposite the force producing centres, the centrosomes and after this tearing the NE collapsed releasing the tension produced by the stretch. [43]

Other studies have found that microtubule action is not essential for the breakdown of the NE. One study used Nocodazole, which inhibits the assembly of microtubules, and found that even though the extension of microtubules had ceased, the disassembly of the NE continued, however, it was noted that upon the addition of this inhibitor, the breakdown rate of the envelope was slowed and disordered and so they therefore concluded that microtubules aid but are not pivotal to the deconstruction of the NE. [47]

The breakdown of the NE has been proposed to be the catalyst which causes both the accelerated condensation of the chromosomes, as a result of the loss of the balance of forces that maintained the spherical integrity of the nucleus; and the dissociation of lamins, inner nuclear membrane proteins and NPCs through the activity of multiple kinases that enter the nuclear space. [43]

The Disassembly of the Nuclear Pore Complexes

NPCs of yeast cells using various imaging techniques.

One of the initial steps in the breakdown of the nucleus is the disassembly of the NPCs through the loss of nucleoporins (Nups), [2] which are removed in the form of nucleoporin sub-complexes and are released into the mitotic cytoplasm. [48]

It has been observed that this process is very rapid in living mammalian cells being completed within minutes [49] and removes the permeability barrier of the NE resulting in the mixing of nuclear and cytoplasmic components. [50] The process of removing nucleoporins from NPCs is achieved through phosphorylation of their proteins which involves the activity of a number of kinases, mainly CDK1 with the help of members of the NIMA-related kinase (Nek) family [50] [51]

Kenetic analysis of single living, dividing cells using confocal-time-lapse microscopy has revealed that the disassembly of NPCs occurs faster than their assembly after cell division and in addition it has uncovered that the removal of the majority of nucleoporins occurs in a synchronous matter except for one which is lost very early on in mitosis. [49]

Studies of living mammalian cells and starfish oocytes reveal that the first nucleoporin to clearly disassociate from the NPCs is Nup98. [49] [52] Nup98 is a peripheral nucleoporin that is found symmetrically on both sides (i.e. cytoplasmic and nucleoplasmic sides) of the NPC with GLFG-type Phe-Gly (FG) repeats that are thought to be important in the formation of the NPC permeability barrier. [50] [48]

Studies conducted by Laurell and peers (2011) aimed to explore the role of phosophorylation in NPC disassembly, they found that the hyperphosphorylation of 13 different phosphosites (phosphorylation sites) in human GLFG Nup93 is required for successful dissociation of Nup98 from NPCs and is a very important step in the breakdown of NPCs. The mitotic kinases which were identified as being responsible for this hyperphosorylation include CDK1, Nek kinases and Plk1 and they proposed that the involvement of multiple kinases could be a mechanism which ensures that only those cells which are entering mitosis undergo NPC disassembly. They also discovered that mutations of phosphorylation sites on Nup98 lead to the slowing down of NPC disassembly and inhibition of the vital kinase CDK1 resulted in the cessation of NPC breakdown, therefore it was concluded that the hyperphosphorylation of Nup98 is the rate-limiting step needed in mitotic NPC disassembly to initiate the breakdown of the NE selectivity barrier [48]

The removal of Nup98 from the complex is proposed to be the trigger for the wave of nucleoprin dissociations from NPCs that follows. [44] It is suggested that the order in which nucleoporins are phosphorylated and removed may reflect their position within the NPCs, such that those on the periphery are solubilised first as they are more accessible to the mitotic kinases, this is followed by the more internal nucleoporins. [53] This is consistent with the dissociation of the first nucleoporin, Nup98, which is located on the periphery of the NPC.

Separate studies conducted on live starfish oocytes and live mammalian cells reveal Nup153 as another nucleoporin which dissociates early on in the disassembly process of NPCs. This is followed by the removal of Nup214 (situated on the cytoplasmic side of the complex) and Nup133 which occurs during the middle of the disassembly process, this precedes the detachment of the Nup107-160 complex, leaving the Nup62 complex (nup50 and Nup58) and POM121, which are the longest remaining core components of the NPC and the last to be removed. [52] [49]

The Depolymerisation of the Nuclear Lamina

The effects of mutation on phosphorylation sites of Lamin B1

The NE is stabilized by the underlying tight meshwork of intermediate filaments known as the nuclear lamina, which is disassembled after the NE is broken down. [43]

The nuclear lamina is composed of A-, B1- and B2-type lamin polypeptides that are associated with the inner nuclear membrane [45] and serve as anchoring sites for chromatin during interphase. During the disassembly of the nuclear envelope these lamins undergo reversible depolymerisation into monomers which are dispersed in the cytoplasm, the reversibility has been suggested to regulate the NE disassembly and reassembly. [54]

The depolymerisation and consequent deconstruction of the lamina is achieved through the phosphorylation of the three key lamins by the main kinase in NE breakdown, CDK1. [43] The necessity of depolymerisation by CDK1 is shown in a study where mutations in the sites of phosphorylation on the lamins, prevent the disassembly of the nuclear lamina during mitosis. [55] PKC-βII kinase has also been identified to phosphorylate B-type lamins in various species during mitosis. [56] [57]

Experiments on the three different types of lamins revealed that their disassembly pattern differs from one another, showing that the A-type depolymerises quite early on in mitosis (during early/mid-prophase). [45] Additionally, these lamin residues are no longer membrane associated, i.e. they have become disassociated from the inner nuclear membrane, as well as being depolymerised into their monomeric form and released into the cytoplasm. [58] [57] In contrast, most of the B-type lamin appears to be focally deformed and somewhat fragmented, yet continues to be associated with their inner nuclear membrane anchorage sites until prometaphase. This layer of B-type lamins lining the NE in early mitotic cells is proposed to function in sustaining the membrane’s elasticity and form until prometaphase when they dissociate and disperse into the ER. [45] [57] The majority of the depolymerised lamin residues are recycled to form the lamina of daughter cells, hence highlighting the reversibility of the depolymerisation of the lamins. [58]

The Disassociation of the Nuclear Envelope From Chromatin

The majority of the NE has been disassembled by prometaphase, however, studies like one conducted by Beaudouin et.al (2002) revealed that there are some fragments of the envelope, containing LAP2β (lamin protein), B1 type lamin and POM121 (NPC protein), that remain attached to the chromosomes. These fragments were observed to disassociate from the chromosomes and “migrate” towards the centrosomes in a vectorial manner and in a way that resembled microtubule motor-dependant stop-and-start movement, hence, leading to the conclusion that after the breakdown of the NE, microtubules continue their involvement in the disassociation of NE structures by pulling the remaining chromosome-associated fragments in a minus-ended-direction towards the centrosomes, completing the disassembly of the NE. [43]

The Dispersal of Nuclear Envelope Components

Dynamics of integral membrane proteins of the NE during mitosis

Two different methods have been proposed for the dispersal of NE structures, one involves the encapsulation of NE proteins in NE-derived vesicles and the other involves the dispersal of the envelope components into the ER. [59]


Vesiculation

Studies on Xenopus eggs and starfish eggs, as well as some mammalian cells, had shown the nuclear membrane to be budding off as vesicles which were released into the cytoplasm. [60] [59] [61] It has been reported that the B-type lamins remained attached to these vesicles while the A-type were dissociated and released into the cytoplasm in some organisms. [62] These vesicles are then recycled for the reassembly of the NE after cell division. [47]


Distribution into the Endoplasmic Reticulum

Dispersion of LAP2, gp210 and Man.II into the ER

More recent studies of mammalian cells have shown the ER to remain intact and to be responsible for taking in the disassembled nuclear membranes and their proteins. [63] This is made possible by the previous disassembly of the lamina and the phosphorylation of the inner-membrane proteins and those associated with the chromatin. [44]

As mentioned previously, the outer nuclear membrane of the NE is continuous with the ER. This close association allows the ER to play an important part in the breakdown of the NE. During interphase the ER has a dominant sheet-like layered appearance, and in some eukaryotes, this is dramatically reorganised to form branched tubular networks that will function in the absorption of NE structures. [63]

The fate of nuclear membranes after NE breakdown was examined in a study conducted on cultured mammalian cells using light and electron microscope immunolocalisation. This study specifically tracked the movement of several integral nuclear membrane proteins using immunofluorescence staining, these proteins included LAP1 and LAP2 of the inner nuclear membrane, and the NPC protein gp210. These three nuclear envelope markers are representative of almost all NE membrane proteins and were found to become dispersed throughout the ER after the breakdown of the envelope in prometaphase. It was observed that they remained there until they became recycled to re-form the NEs of the daughter cells in telephase. [59]


Links: Dissociation of lamin filaments | Vesiculation of the nuclear membranes | NPC architecture and nucleoporin localization

Mitotic Functions of Nuclear Envelope Components

As mentioned above there is substantial evidence to suggest that the NE is reabsorbed into the ER [64] which is continuous border with the NE [65]69 70. It has been postulated that the NE proteins are dispersed through the ER during nuclear replication. [66] Using fluorescence time-lapse microscopy in living cells NE and Inner Nuclear Membrane Proteins (INM’s) have been found throughout the ER during mitosis [64] [67]. Ultimately the true nature of ER activity in reference to NE breakdown (NEBD) is unknown [2].

What we know more about are the functions of membrane proteins in mitosis. Nucleoporins (NUPS) are involved in the assembly and function of the nuclear pore, [68] other NUPS have a variety of roles in assembly and spindle formation of microtubules [69]. Nuclear lamins have been show to facilitate intermediate filament formation, and it has been indicated that spindle-associated membrane protein 1 (SAMP1) in functionally connected with the cytoskeleton[70]. Unfortunately research has not determined the exact mechanisms for these nuclear membrane proteins, however deficiencies in these products result in irregularity of cellular function and replication. [2]

Reformation of the Nuclear Envelope

Reformation of the NE membrane in Vivo

In higher cells, the membranes of the NE (which breaks down during prophase) have the remarkable ability to reassemble themselves at the end of mitosis in the two daughter cells. The organization and control of NE re-formation is imperative to proper cell functioning. [71][72] The exact mechanisms involved in NE reconstruction are unappreciatively complex and thus the precise process of NE assembly is not yet fully understood. [73] The majority of studies on NE have been carried out in a cell free extract based on the Xenopus Laevis egg extracts. [74] The process of NE reformation is not the same in different types of eukaryotic cells; in higher eukaryotes open mitosis occurs which is marked by significant alterations to nuclear structural design. [5] The NE reconstruction occurs at the end of mitosis and it is characterised with the immediate build-up of membranes near the chromatin. [71] Studies have shown that this initial process occurs in just minutes, however, the successive growth and stabilisation of the NE tends to take more than an hour. [64]


According to Ulbert et al. (2005), membrane attachment can occur with no energy and the membrane vesicles have a somewhat degree of freedom as to where they attach on the chromatin. Though the de-condensation of the chromatin is crucial before the NE assembly can occur because it is thought that de-condensation allows for the binding sites of the chromatin to be adequately presented to the membrane vesicles. [71] The attraction and thus subsequent attachment of the membrane vesicles to the binding sites relies heavily upon transmemberane proteins and is further assisted by mitotic phosphorylation. [75][76] For example, lamina-associated polypeptide 2β and lamin B receptor (LBR) are two important tansmembrane proteins that have been clearly found in vitro to assist with the binding of the decondensed chromatin. [71] [77] Collase et al. (1996) showed the crucial role of LBR in binding of ER to chromatin. Their experiments on Sea Urchin showed that in order for an ER-derviced vesicle to bind to chromatin during the NE reconstruction (in vitro), it needed the assistance of LBR. [78]

Proposed Models of Nuclear Envelope Reformation

Although many would argue that in open mitosis the majority of the NE is sourced from ER, there is nonetheless, no single mechanism that is unanimously considered to be the correct representation of the processes that occur in NE reassembly. [79] [5]

The first model proposes that the re-organisation of the endoplasmic reticulum, near the end stage of cell division, encapsulates the chromosomes to form the NE in the absence of any assistance from the membrane vesicle fusions. [80] It suggests that the binding of the ER tubules to the de-condensed chromatin surface (with the help of specific proteins), is pursued by the straightening of these membranes against the surface of the chromatin and thus fully surrounding it. The residual ‘holes’ that subsequently form in the membrane are filled through the inclusion of NPCs. [73] Membrane fusion has little role in this proposed model as its main role if to replenish and repair the ER. Recent study conducted by Andreson et al. (2007) on this particular mechanism of NE reformation propose that proteins of the ER such as reticulon 4a (Rtn4a), are mainly accountable for binding to chromatin [81] . Their studies further predicts that nuclear membrane proteins of the NE are scattered in the ER during cell division in animals.

The second model primarily suggests that reassembly of the NE occurs through the binding of the membrane vesicles, which formed when the NE was breaking down, to chromosome surfaces. This association is thought to come about as a result of interactions between the chromosomes and both lamins and integral membrane proteins of the vesicles. The vesicles then fuse around individual chromosomes creating a double membrane around them and then these in turn fuse together to create a single nucleus. [6] In addition, fusion of the membrane vesicles and ER membranes consequently leads to coating and covering of chromatin to result in the double membrane of the NE. [73]

Reassembly of the NPC during NE reformation

NPC Formation at the End of Mitosis


It is argued that a complete closure of the NE cannot take place if the NPCs are not incorporated into the fusing membranes [73] The nucleoporin POM121 is vital to bringing the assembly of the NE with that of the NPCs. The study conducted by Antonin et al. (2005) illustrated that the POM121 is necessary in order for membrane vesicles to fuse during NE re-assembly. [75] Further studies have shown that in addition to POM121, Nup107 complex is mandatory in order for the nuclear pores to be successfully incorporated into the NE during its reformation [75] [82] As reviewed in Prunske and Ullman [73] there is a strong interaction between Nup107 complex and POM121 as they somewhat direct membrane recruitment, membrane fusion and NPC assembly.

The Nuclear Lamina during NE reformation

The Nuclear Lamina is a mesh-like network of fibres on the inner surface of the inner nuclear membrane that has mainly been studied by indirect immunofluorescence and by confocal microscopy. [83] As reviews in Brian et al. (1986) the nuclear lamina is thought to play a pivotal role in nuclear envelope reassembly and that the process is affected by dephosphorylation. Lamins are associated with the re-organisation of the chrmoatins in nuclear envelope reformation reactivities [84] It was illustrated by Gerace and Blobel (1980) [58] that the nuclear lamina breaks apart during the prophase stage. Furthermore, according to Chaudhary and Courvalin (1993) [85] , the polypeptides, lamins A and C were found to be soluble whereas lamin B was associated with mitotic membranes . It was further observed that lamins A, B, and C transform into the nuclear lamina during late mitosis. Lamins continue to enter the nucleus via the nuclear pores. [86]


Links: The breakdown and re-formation of the NE during mitosis | Reformation of the NE from membrane vesicles

Abnormalities in Nuclear Envelope Breakdown and Reformation

In recent years diseases linked to the LMNA gene have come to the forefront of cell biology. These diseases collectively known as laminopathies include; dilated cardiomyopathy with variable muscular dystrophy, mandibulolacral dysplasia and Hutchinson-Gilford progeria syndrome. All of these diseases are linked to mutations or alternate splicing of the LMNA gene (Lamin A) and provide valuable insight to the function of lamins (product of the LMNA gene) and the function of the NE (lamins main target) [87]. As of today no human diseases have been linked to mutations in Lamin B. One study inserted a gene trap into the Lamin B1 gene which lead to mouse death at birth. The mice presented with abnormal tissue in their skeletal muscle as well as poor nuclear morphology. The study concluded the B1 Lamin is crucial in proliferation and embryogenesis [88].

Complications Resulting from Laminopathies

Alterations to the nuclear lamins has been linked to a wide variety of disease [87]. Research compiled by Sullivan et al. 1998 showed that mutations in the LMNA gene lead to skeletal muscle disease inside two months of life. [89]A hypothesis for the mechanism behind this disease is that nuclear lamins (in this case type A lamins) play a role in providing structural support for the nuclear envelope, a defective lamina could lead the cell unable or less able to support its NE [90]. More research suggests that the cytoskeleton could also be affected by mutation of the cytoskeleton, which disrupts inter and intracellular transport[87] .

While mutations in Lamins result in very rare diseases there is research to suggest that the study of these conditions can enlighten us on other diseases and processes of aging [87] . It has been postulated that polymorphic variants of these genes be predispositions to more common conditions like insulin resistance, diabetes mellitus, and metabolic syndrome [91] [92] [93]

Current and Future Research

The NE is an important structure of the cell that is now being profusely investigated by scientists. This is partly due to the lack of knowledge we have on the processes carried out with association with the NE. There is incomplete knowledge regarding the breakdown of the NE. While the scientific community now knows certain aspects of NEBD we have still yet to draw the boundaries of the overall process.

Disease

Nuclear rupturing as well as abnormal morphologies of the Nucleus is a well known characteristic of cancer cells. Thus the study of the NE in oncotic cells may offer an explanation or a perspective on the treatment or prevention of cancer. The research article ‘Transient nuclear envelope rupturing during interphase in human cancer cells’ explores the rupturing of the nuclear envelope in cancer cells using various techniques such as cell culture, siRNA transfection as well as live and confocal imaging and the use of the electron microscope. Cancer cells can be diagnosed by the presence of nuclear envelope invaginations and extrusions. However despite this clinical pattern it is unclear why changes in structure of the nuclear envelope is present in cancer cells. The findings of the paper illustrate that the nuclear lamina, intermediate filaments that provide support to the nuclear envelope, was not properly formed in cancer cells that had ruptured nuclear envelopes. Furthermore it was found that nuclear envelope rupture occurred when there was an entrapment of cytoplasmic in the nuclear interior [94]. As there is a clinical correlation between abnormal nuclear envelope and cancer cells it is important to gain an understanding into the causation of this relationship.

Mislocalization of nuclear and cytoplasmic components

Moreover C.D Capo-chichi and co-workers investigated the expression of NE proteins A/C in ovarian cancer.The investigation uses techniques such as siRNA and immunofluorescence microscopy to look at nuclear morphology in conjunction with flow cytometry to investigate cellular DNA content. The study found that the proteins lamin A/C are absent in 47% of ovarian cancer cells which indicates that there may be an underlying relationship between the absence of these proteins and the occurrence of ovarian cancer. The study concludes that the loss of proteins lamin A/C may lead to a morphological change in the nuclear envelope and that this then leads to the diagnostic characteristics of cancer. [95]

There is a substantial amount of research going into this area as mutations in genes coding for NE proteins have been shown to cause not just cancer but a range of human diseases. As the scientific community lacks the understanding of the processes involved in a normally functioning cell to become abnormal and cancerous this area of research has become prevalent investigation in the scientific community.


Vesicles

A popular direction that future research is interested in is the role vesicles have in the reformation or re-assembly of the NE. The study conducted on the NE assembly by Q.Lu and co-workers discovered that NLS(Nuclear localization sequence) proteins act as docking sites for nucleoporins and NE precursor membrane vesicles through importin-α and -β. [96]. As the mechanisms behind NE breakdown and assembly are not fully understood the research area has significant prospect in future research of the NE.

Glossary

Cell cycle: a series of events that occur in cells leading up to their division and replication. It's divided into two main parts, Interphase and a Mitotic or M phase. Interphase is further subdivided into three phases including: G1, S and G2. Each phase has different events occurring within them

Cell division: an essential process that occurs in almost all living things. It is characterised by the separation of a cell into two daughter cells. In metazoans, it involves separation of the nucleus and of the cytoplasm

Centromere: Confined portion of a mitotic chromosome where sister chromatids are attached and from which kinetochore fibers extend toward a spindle pole. It is essential for correct chromosome segregation during mitosis

Chromatin:it is made up of histones, non-histone proteins and DNA which condenses to form eukaryotic chromosomes

Chromosome: a single molecule of DNA which is highly organised and tightly packed around proteins (histones)

Chromosomal segregation: process by which chromosomes or chromosome pairs align then separate to opposite ends of the cell

Closed Mitosis: one of the forms of mitosis where the nuclear envelope remains intact during cell division and it occurs in lower eukaryotes

Cytoskeleton: a network of protein filaments and tubules located in the cytoplasm of cells and serves to maintain a cell's shape and internal structure

Depolymerization: process by which a polymer broken up into its component monomers

Disease: A pathological condition of an organism which disrupts the normal bodily functions that often leads to feeling of weakness, and usually associated with symptoms.

Dynein: Minus end-directed motor protein that uses energy in the form of ATP and converts it to mechanical energy for movement. It functions to transport cell cargo along microtubules towards its minus-end. It has an important function in cell division as well

Endoplasmic Reticulum (ER): an organelle of eukaryotic cells that is made up of an interconnected network of membranes and is classified into two types, Rough and Smooth ER. It's located near the nucleus, with part of it being continuous with the nuclear envelope (rough ER)

Eukaryotes: almost all organisms (except viruses and Prokaryotes) are considered to be this. A eukaryote is an organism that is characterised by having one or more cells and a membrane-bound nucleus and organelles

Fluorescence microscopy: a cell biology technique that uses a high intensity light source from a specialised microscope and this excites a fluorescent specimens, resulting in the emission of a lower energy light of longer wavelength that produces a magnified image

G2 stage: this stage of the cell cycle is the third subphase of interphase preceding mitosis. During this phase rapid cell growth and protein synthesis is occurring and the cell prepares for cell division

Hyperphosphorylation: a state that occurs when phosphorylation sites are fully saturated/phosphorylated

Inner nuclear membrane:it is the innermost of the two nuclear membranes. It has binding sites for nuclear lamina and the chromatin on its internal face.

Intermediate filaments: one of the principle elements pf the cytoskeleton of the cell. It has a diameter of ~10nm and function in providing mechanical strength to cells and tissues

Interphase: longest phase of the cell cycle where the cell spends the majority of its time and where it's performing the majority of its functions including gene expression and prepares for cell division

Kinase: an enzyme that catalyzes the transfer of phosphate groups from a donor, such as ATP or ADP, to a specific acceptor molecule

Kinetochore: a protein structure that forms on chromatids and is where the spindle fibers attach during cell division to pull sister chromatids appart

Lamina: a dense network of fibres composed of intermediate filaments made up of lamin proteins and lamin-associated proteins

Lamin A/C is an LMNA protein that is associated with the lamina of the nuclear envelope.

M stage: or Mitotic phase is the stage in the cell cycle where the cell undergoes mitosis and divides into two identical daughter cells

Metaphase: the third stage of mitosis where the chromosomes align in the center of the cell at the metaphase plate ready for segregation and the spindles are fully developed

Metazoans: multicellular animals whose cells are arranged in layers or groups to form specialised tissues or organs. They make up the majority of the animal kingdom

Microtubules: fibrous, hollow rods that function as support structures for the cell shape. Also used to move organelles around and aggregate to form spindles that segregate the chromosomes

Microtubule aster: microtubules arranged in a star array around each centrosome during mitosis

Microtubule organising center (Centrosome): organelle in eukaryotic cells from which microtubules organise themselves into tubular structures which in turn are arranged in a specific pattern

Mitosis: one of the types of cell division where a non-germ cell divides its chromosomes, cytoplasm, membranes and organelles evenly among its two identical daughter cells. This division produces daughter cells that are genetically identical to the parent cell

Mitosis promoting factor: a protein containing Cyclin-dependant Kinase (CDK1) that stimulates the progression into the M phase of the cell cycle by phosphorylating multiple proteins needed during mitosis

Mitotic checkpoint machinery: a complex composed of many proteins that functions to inhibit the anaphase-promoting complex, stopping the advance into anaphase

Monomer: a molecule that is capable of reacting with the same or different types of molecules to form a polymer

Nocodazole: an agent that inhibits the polymerisation and hence the formation of microtubules

Nek: NimA related kinases, a family of kinases involved in cell cycle regulation in eukaryotic cells

Nuclear envelope: A double-layered membrane that envelopes the nucleus of motozoan cells. separating the materials of the nucleus from the cytoplams

Nuclear lamina: it is made up of lamin filaments and it can be found on the inner surface of the INM in eukaryotic cells. It not only provides mechanical support for the cell but also regulates essential cellular events such as cell division

Nuclear Pore Complex (NPC): Very large protein complexes spanning through the outer nuclear and inner nuclear membranes creating gated channels that allow for transport of substances between the cytoplasm and nucleus

Nuclear Pore Complex Basket: A region of the Nuclear Pore Complex which extends into the nucleoplasm.

Nucleoporin: protein building block of nuclear pore complexes

Nucleus: is a relatively big membrane-bounded organelle that is found in eukaryotic cells and contains DNA which are organized into chromosomes. Additionally, the creation and processing of RNA and ribosome assembly occur in the nucleus

Oncotic Cells are cells that undergo oncotic necrosis, programmed cell death.

Open Mitosis: one of the forms of mitosis where the nuclear envelope is completely disassembled and removed from the chromatin. This occurs in the somatic cells of higher eukaryotes

Organelle: a structure that is bound by a membrane and normally found in the cytoplasm of multi-cellular organisms

Outer nuclear membrane: it is the outermost of the two nuclear membranes and it is continuous with the endoplasmic reticulum

Phosphorylation: action of adding a phosphate group to an organic compound

PKC-βII: a Protein Kinase C isoform that controls the function ofother proteins through phosphorylation

Plk-1: Polo-like Kinase 1 is an enzyme involved in phosphorylation of NPCs

Prometaphase: the stage in mitosis following prophase and preceding metaphase where the nuclear envelope breaks down and the microtubules attach to the kinetochores of chromosomes

Prophase: stage in mitosis where the chromatin condenses into chromosomes becoming visible

Protein: is made up of amino acids that are joined together in a specific order to make a polymer of a certain length. Proteins make up the essential structural components in cells and take part in nearly all cellular activities

Plasma membrane: this is the membrane that encapsulates the entire cell and isolates it from the external environment. The plasma membrane is made up of a typical phospholipid bilayer and associated proteins

Rate-limiting step: the slowest step of a pathway or reaction that determines the rate of reaching the desired outcome

Reformation: the act of reforming or the state of being reformed

Ribonuleoprotein:is a nucleoprotein that consist of RNA, that is it is an association that combines ribonucleic acid and protein together

Saccharomyces cerevisiae: a species of yeast

Semi-closed Mitosis: a form of mitosis, occurring in selected higher eukaryotes, where the nuclear envelope only partially dissociates

Spindle fibers: form from aggregates of microtubules and are resposible for separating chromosomes into daughter cells during cell division

Telophase: the final or penultimate stage in cell division where the duplicated genetic material has been segregated into the separating daughter cells and a the nuclear compartment/envelope forms around the genetic material of each daughter cell. Cytokenesis will then split the cells and their cytoplasm.

Transcription: the process by which a DNA sequence is transferred to messenger RNA (mRNA), which is complementary strand of nucleic acids (with only uracil replacing thymine)that will exit the nucleus through the NPCs. This is the first step of gene expression

Translation: the process in which the mRNA is converted to an amino acid chain or polypeptide, through the action of ribosomes, which will later fold to form a protein

Tubulin: a globular protein that is the main component of microtubules in living cells

Images

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2013 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7

Dr Mark Hill 2013, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G