2013 Group 5 Project
- 1 The Nuclear Envelope During Cell Division
- 1.1 Introduction
- 1.2 Historical Background
- 1.3 Structure of the Nuclear Envelope
- 1.4 The Nuclear Envelope At the Onset of Mitosis
- 1.5 Breakdown of the Nuclear Envelope
- 1.6 Mitotic Functions of Nuclear Envelope Components
- 1.7 Reformation of the Nuclear Envelope
- 1.8 Open vs. Closed/Semi-closed Mitosis
- 1.9 Abnormalities in Nuclear Envelope Breakdown and Reformation
- 1.10 Current and Future Research
- 1.11 Glossary
- 1.12 Images
- 1.13 References
The Nuclear Envelope During Cell Division
The nuclear envelope is a highly specialised membrane that outlines the nucleus  and is a key physical compartment that defines eukaryotic cells. Hetzer’s review (2010) describes it as a highly organised and regulated double membrane that compartmentalises the cell’s genome. 
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.  The outer membrane is continuous with the membrane of the Rough Endoplasmic Reticulum (rER),  while the inner membrane is attached to the lamina (directly beneath the inner membrane) and chromatin of the nucleus. 
The nuclear envelope 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.  More importantly it serves to separate nuclear and cytoplasmic activities, including transcription from translation, protecting genetic material from the highly metabolic environment of the cytoplasm.  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.  In addition, Hetzer’s has pointed out in his review article (2010) that 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. 
Changes to the nuclear envelope’s structure occur at the onset of mitosis, these changes are very slight in lower eukaryotes and in vertebrate cells result in the complete disassembly of the nuclear membrane,  this complete or partial breakdown is necessary in order to form the mitotic spindle on condensed chromosomes.  Higher eukaryotes must consequently reassemble nuclear envelopes round the genetic material of the daughter cells each time a cell divides in order to re-establish the nuclear compartment.  This page aims to explore the process of the breakdown and reassembly of the nuclear envelope and its role in cell division.
Timeline of Historical Research On The Nuclear Envelope
|1950s||The Nuclear envelope was discovered as a memebrane that separated the nucleus from the cytoplasm.|
Structure of the Nuclear Envelope
The nuclear envelope (NE) is an important structure that covers the nucleus with a double membrane. The prominent constituents of the NE include the nuclear lamina, nuclear pore complexes (NPCs) and nuclear membranes  . The nuclear membrane is made up of distinctive but unified domains: the outer nuclear membrane and the inner nuclear membrane  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  
Outer Nuclear Membrane
Inner Nuclear Membrane
The Inner Nuclear membrane (INM), neighbouring with the endoplasmic reticulum membrane, is connected with the lamina and the chromatin . 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.  In recent years there has been an increase in the number of discoveries of various INM proteins mainlydue to the proteomic and computational approaches.  One such group of proteins include transmembrane proteins that are associated with the INM and they usually interact with chromatin and/or lamina. 
It is thought that these transmemberane proteins direct the chromatin to the membrane; this is essential during the reformation of the NE after mitosis.  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   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. 
Nuclear Pore Complexes
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.   The NPCs are composed of numerous copies of ~30 nucleoporins and span the nuclear envelope at junctions of the inner and outer nuclear membranes.   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. 
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.  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. 
Figure 1. Schematic view of model adopted for the NPC/NE system
The Nuclear Envelope At the Onset of Mitosis
Once the cell reaches the M phase of interphase mitotic activity takes place. At the onset of mitosis the nuclear envelope begins to undergo processes that eventually lead to NEBD. This involves centrosome migration and NE tearing.
The microtubule asters form at the centrosomes causing the tearing of the NE. During this important process of centrosome migration and NE tearing the Microtubule (MT) motor protein, dynein is required to participate in this process. Furthermore this MT motor protein associates with the NE at the end of the G2 stage of interphase . These interactions with the MT and the NE facilitate NEBD as well as the rupturing of NE. Moreover chromatin condensation occurs before NEBD and the rate of condensation increases by three-fold after the NE is made permeable. This supports that changes in the nuclear envelope constitute changes of other factors of cell division .
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 .
The nuclear envelope then breaks down through the disassembly of the nuclear pore complexes, the depolymerisation of the Nuclear Lamina, the disassociation of the Nuclear Envelope from chromatin and the dispersal of Nuclear Envelope components into the Endoplasmic Reticulum
Breakdown of the Nuclear Envelope
The breakdown of the nuclear envelope only occurs in higher eukaryotes/metazoans like plants and animals. The entry of a cell into mitosis (prophase) is defined by this loss of compartmentalisation of the nuclear content  and it not only involves the removal of the inner and outer nuclear membranes but also the disassembly of the underlying lamina and NPCs.  These organisms form spindle microtubules which are located in the cytoplasm and are vital in mitosis for the segregation of chromosomes, therefore the disassembly of the nuclear envelope is required in order for these microtubules to gain access to the kinetochores of the chromosomes. 
The precise mechanism by which the nuclear envelope is broken down has not been established, therefore there are a number of proposed methods by which this occurs.
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 B and cyclin-dependant kinase (CDK1), also known as p34, are responsible for the phosphorylation of nuclear envelope proteins, which eliminates protein-protein interactions, and the consequent disintegration and dispersal of all major nuclear envelope structural components. 
Reviews such as those by Kutay and Hetzer (2008) describe the breakdown of the nuclear envelope 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 then the disassociation of the nuclear envelope from the chromatin.  It has also been suggested that nuclear envelope vesiculation and spindle microtubule piercing are other contributing methods for nuclear envelope disassembly. 
The Disassembly of the Nuclear Pore Complexes
One of the initial steps in the breakdown of the nucleus is the disassembly of the NPCs through the loss of nucleoporins (Nups),  which are removed in the form of nucleoporin sub-complexes and are released into the mitotic cytoplasm. 
It has been observed that this process is very rapid in living mammalian cells being completed within minutes  and removes the permeability barrier of the nuclear envelope resulting in the mixing of nuclear and cytoplasmic components.  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  
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. 
Studies of living mammalian cells and starfish oocytes reveal that the first nucleoporin to clearly disassociate from the NPCs is Nup98.   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.  
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 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 nuclear envelope selectivity barrier 
The removal of Nup98 from the complex is proposed to be the trigger for the wave of nucleoprin dissociations from NPCs that follows.  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.  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.  
The Depolymerisation of the Nuclear Lamina
The nuclear envelope is stabilized by the underlying tight meshwork of intermediate filaments known as the nuclear lamina. The removal of this network occurs during the breakdown of the nuclear envelope.  The nuclear lamina is composed of A- and B-type lamin polypeptides that are associated with the inner nuclear membrane  as well as serving 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 nuclear envelope disassembly and reassembly. 
The depolymerisation and consequent deconstruction of the lamina is achieved through the phosphorylation of the three key lamins by the main kinase in nuclear envelope breakdown, CDK1.  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. 
The Disassociation of the Nuclear Envelope From Chromatin
The Dispersal of Nuclear Envelope Components into the Endoplasmic Reticulum
Mitotic Functions of Nuclear Envelope Components
There is substantial evidence to suggest that the nuclear envelope (NE) is reabsorbed into the endoplasmic reticulum (ER)  which, as stated earlier is continuous border with the NE   . It has been postulated that the NE proteins are dispersed through the ER during nuclear replication.  Using fluorescence time-lapse microscopy in living cells NE and Inner Nuclear Membrane Proteins (INM’s) have been found throughout the ER during mitosis  . Ultimately the true nature of ER activity in reference to NE breakdown (NEBD) is unknown .
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,  other NUPS have a variety of roles in assembly and spindle formation of microtubules. 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. 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. 
Reformation of the Nuclear Envelope
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.  The exact mechanisms involved in NE reconstruction are unappreciatively complex and thus the precise process of NE assembly is not yet fully understood.  The majority of studies on NE have been carried out in a cell free extract based on the Xenopus Laevis egg extracts.  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.  The NE reconstruction occurs at the end of mitosis and it is characterised with the immediate build-up of membranes near the chromatin.  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. 
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.  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.  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.   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. 
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.  
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.  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.  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  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 fusion of membrane vesicles and ER membranes consequently leads to coating and covering of chromatin to result in the double membrane of the NE. 
Reassembly of the NPC during NE reformation
It is argued that a complete closure of the NE cannot take place if the NPCs are not incorporated into the fusing membranes  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.  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   As reviewed inPrunske and Ullman  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.  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  It was illustrated by Gerace and Blobel (1980)  that the nuclear lamina breaks apart during the prophase stage. Furthermore, according to Chaudhary and Courvalin (1993)  , 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. 
Open vs. Closed/Semi-closed Mitosis
Mitosis is the process by which eukaryotic cells divide to form two equal daughter cells each with a copy of its genome.  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.  The distinction between open and closed mitosis can be made by focusing on the behaviour of the nuclear envelope which separates the nuclear contents from the cytoplasm and is split to form daughter nuclei.  Open mitosis is so named because the nuclear envelope completely breaks down at the transition from G2 to M stage of the cell cycle  and the nuclear content, including the genetic material, is “open” to mix with cytoplasmic macromolecules  until the nuclear envelope is reassembled after chromosomal segregation during telophase/G1.   In contrast, during closed mitosis the nuclear envelope remains intact and mitosis continues within the nucleus resulting in the fission of the nuclear envelope after chromosomal segregation. 
However, 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 and the timing of the breakdown can also be atypical of open mitosis.   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 microtubules. 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,  one of semi-open mitosis until after metaphase. 
In open mitosis the entire nuclear envelope, including the NPCs, breakdown allowing the formation of the spindles on the chromosomes  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.  In contrast, the nuclear envelope 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. 
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 nuclear envelope 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. These proteins were previously found to be associated with nucleoporin sub-complexes made up of Nup53p, Nup59p and Nup170p. Further investigation conducted in this study showed that the association of Mad proteins with NPCs particularly required Mad1p and that Nup53p was responsible for ensuring the connection between the Mad1p-Mad2p complex and the NPCs. 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 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 nuclear envelope. 
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 and provide valuable insight to the function of lamins (product of the LMNA gene) and the function of the NE (lamins main target) .
Complications Resulting from Laminopathies
Alterations to the nuclear lamins has been linked to a wide variety of disease . Research compiled by Sullivan et al. 1998 showed that mutations in the LMNA gene lead to skeletal muscle disease inside two months of life. 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 . More research suggests that the cytoskeleton could also be affected by mutation of the cytoskeleton, which disrupts inter and intracellular transport .
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. Moreover the event of NE rupturing can be observed in cancerous cells and thus the study of the NE in cancer cells may offer an explanation or a perspective on the treatment 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 . 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.
More to be added
Lamina: a dense network of fibres composed of intermediate filaments made up of lamin proteins and lamin-associated proteins 
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 
Rough Endoplasmic Reticulum (rER):
- Evans D, Hutchison C & Bryant J (2004) The Nuclear Envelope, Garland Science/BIOS Scientific Publishers, New York
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- Dr Mark Hill 2013, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G