An Introduction to Molecular Biology/Nucleus

The nucleus was the first organelle to be discovered. The probably oldest preserved drawing dates back to the early microscopist Antonie van Leeuwenhoek (1632 – 1723). He observed a "Lumen", the nucleus, in the red blood cells of salmon. Unlike mammalian red blood cells, those of other vertebrates still possess nuclei. The nucleus was also described by Franz Bauer in 1804 and in 1831 by Scottish botanist Robert Brown in a talk at the Linnean Society of London. Brown was studying orchids under microscope when he observed an opaque area, which he called the areola or nucleus, in the cells of the flower's outer layer. He did not suggest a potential function.

In 1838, Matthias Schleiden proposed that the nucleus plays a role in generating cells, thus he introduced the name "Cytoblast" (cell builder). He believed that he had observed new cells assembling around "cytoblasts". Franz Meyen was a strong opponent of this view, having already described cells multiplying by division and believing that many cells would have no nuclei. The idea that cells can be generated de novo, by the "cytoblast" or otherwise, contradicted work by Robert Remak (1852) and Rudolf Virchow (1855) who decisively propagated the new paradigm that cells are generated solely by cells ("Omnis cellula e cellula").

The function of the nucleus remained unclear. Between 1876 and 1878, Oscar Hertwig published several studies on the fertilization of sea urchin eggs, showing that the nucleus of the sperm enters the oocyte and fuses with its nucleus. This was the first time it was suggested that an individual develops from a (single) nucleated cell. This was in contradiction to Ernst Haeckel's theory that the complete phylogeny of a species would be repeated during embryonic development, including generation of the first nucleated cell from a "Monerula", a structureless mass of primordial mucus ("Urschleim"). Therefore, the necessity of the sperm nucleus for fertilization was discussed for quite some time. However, Hertwig confirmed his observation in other animal groups, e.g., amphibians and molluscs. Eduard Strasburger produced the same results for plants (1884). This paved the way to assign the nucleus an important role in heredity. In 1873, August Weismann postulated the equivalence of the maternal and paternal germ cells for heredity. The function of the nucleus as carrier of genetic information became clear only later, after mitosis was discovered and the Mendelian rules were rediscovered at the beginning of the 20th century; the chromosome theory of heredity was developed.

Nucleus


The nucleus (pl. nuclei; from Latin nucleus or nuculeus, meaning kernel) is a membrane enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material (DNA), organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these chromosomes are the cell's nuclear genome. The function of the nucleus is to maintain the integrity of these genes and to control the activities of the cell by regulating gene expression — the nucleus is therefore the control center of the cell. The main structures making up the nucleus are the nuclear envelope, a double membrane that encloses the entire organelle and separates its contents from the cellular cytoplasm, and the nuclear lamina, a meshwork within the nucleus that adds mechanical support, much like the cytoskeleton supports the cell as a whole. Because the nuclear membrane is impermeable to most molecules, nuclear pores are required to allow movement of molecules across the envelope. These pores cross both of the membranes, providing a channel that allows free movement of small molecules and ions. The movement of larger molecules such as proteins is carefully controlled, and requires active transport regulated by carrier proteins. Nuclear transport is crucial to cell function, as movement through the pores is required for both gene expression and chromosomal maintenance. Although the interior of the nucleus does not contain any membrane-bound subcompartments, its contents are not uniform, and a number of subnuclear bodies exist, made up of unique proteins, RNA molecules, and particular parts of the chromosomes. The best known of these is the nucleolus, which is mainly involved in the assembly of ribosomes. After being produced in the nucleolus, ribosomes are exported to the cytoplasm where they translate mRNA.

The nucleoskeleton
The nucleoskeletal framework that remains insoluble after treatment of nuclei with non-ionic detergents, followed by nuclease treatment and high salt extraction to remove chromatin and soluble proteins, is generally termed the nuclear matrix. This nucleoskeleton consists of two parts namely, the nuclear lamina and a network of intricately structured fibres connected to the lamina and distributed throughout the nuclear volume. These highly structured fibre assemblies have been shown to be attached to an underlying network of 10 nm core filaments. The nuclear lamins A, B and C are the major structural components of the peripheral lamina. Additional proteins that connect the lamina to the nuclear envelope and the heterochromatin are clustered at the nuclear periphery. Other nuclear matrix-associated components like hnRNP complexes, newly transcribed full-length mRNA, RNA polymerase I and II, various transcription factors and spliceosomal complexes are positioned on the underlying network of branched 10 nm filaments that is connected to the nuclear lamina. These associations might be dynamic and allow for considerable plasticity in nuclear architecture and function. Furthermore, numerous studies have suggested the presence of an organizing structure such as the nuclear matrix to coordinate the spatial regulation of DNA synthesis. The major candidate proteins that are likely to comprise the nucleoskeleton are lamins and nuclear actin.

1.Nuclear envelope:

The nuclear envelope (NE) (also known as the perinuclear envelope, nuclear membrane, nucleolemma or karyotheca) is a double lipid bilayer that encloses the genetic material in eukaryotic cells. The nuclear envelope also serves as the physical barrier, separating the contents of the nucleus (DNA in particular) from the cytosol (cytoplasm). Many nuclear pores are inserted in the nuclear envelope, which facilitate and regulate the exchange of materials (proteins such as transcription factors, and RNA) between the nucleus and the cytoplasm.Nuclear membranes is composed of a lipid bilayer. The outer membrane is continuous with the rough endoplasmic reticulum while the inner nuclear membrane is the primary residence of several inner nuclear membrane proteins. The outer and inner nuclear membrane are fused at the site of nuclear pore complexes. The structure of the membrane also consists of ribosomes.The space between the two membranes that make up the nuclear envelope is called the perinuclear space (also called the perinuclear cisterna, NE Lumen), and is usually about 20 - 40 nm wide.The inner nuclear membrane is connected to the nuclear lamina.

Nuclear envelope breakdown during mitosis

Nuclear envelope breakdown and reassembly in mitosis. At the end of G2, the activation of cyclin-dependent kinases, including CDK1, triggers entry into mitotic prophase. The nuclear membrane breaks down, and the NE-associate proteins either translocate to kinetochores, distribute with the fragmented ER networks, or dissolve in the cytoplasm. During NE reassembly in anaphase, SUN1 and LAP2 first appear around the condensed chromatin, though at different regions. The nuclear lamins then join the nuclear periphery in telophase. This figure illustrates the important roles played by the NE and the nuclear lamina in normal mitosis. Chi et al. Journal of Biomedical Science 2009.[1] During prophase in mitosis, the chromatids begin condensing to form chromosomes, and the nuclear envelope breaks down and is retracted into the mitotic endoplasmic reticulum. At metaphase, the nuclear envelope has been completely disassembled and absorbed by the ER allowing the chromosomes to be put together by spindle fibers attached to each chromosome at the kinetochore. Other eukaryotes such as yeast undergo closed mitosis, where the chromosomes segregate within the nuclear envelope, which then buds as the three daughter cells divide. In the process of mitosis, the nuclear envelope is degraded during prometaphase. Without this step, the nuclear material would be unable to separate into two nuclei, and by extension, two cells. At the end of anaphase however, the chromosomes are now separated, and each set is in its respective half of the parent cell. In order to protect the genetic material, the nuclear membrane must re-form at this stage. This is done using membrane from the endoplasmic reticulum and proteins called lamins that guide the new envelope. (Alberts) Like some of the other organelles in the cell, the endoplasmic reticulum is composed of a phospholipid bilayer. This thin membrane is very flexible, and portions can be pinched off to form new organelles, vesicles, or in this case, nuclei. In 2007, researchers discovered that pieces of the ER break off and merge to form the nuclear envelope. However, there must be a structural element that brings these vesicles together, because the nucleus is much more structurally complicated than the ER. This element is a layer of lamin between the chromatid and the nuclear envelope itself, known as the lamina. Lamins are long fibrous proteins. In prometaphase, they are phosphorylated (a phosphate group is added), causing the protein to change shape and lose its structural properties. This is what causes the breaking down of the nuclear membrane. However, towards the end of anaphase, the existing lamin is dephosphorylated, and even more is produced. Once the chromosomes have separated, the lamina begins to form again. Sometime at the beginning of mitosis, ends of the endoplasmic reticulum bound to the DNA, using lamin A receptor. (Duband-Goulet and Courvalin) Once the lamina begins to re-form, it forces tubules of ER to form a network across the surface of the chromatid. (Anderson and Hetzer) These tubes eventually are flattened out and merged, forming a solid nuclear membrane. It is not certain what mechanisms cause this to happen. The ER and the LBR eventually detach themselves from the DNA. In a mature cell, the lamina forms a continuous layer just inside the nuclear envelope, and is attached to the nucleus by protein known as emerin.

2.Nuclear pores:

Nuclear pores are large protein complexes that cross the nuclear envelope, which is the double membrane surrounding the eukaryotic cell nucleus. There are about on average 2000 nuclear pore complexes in the nuclear envelope of a vertebrate cell, but it varies depending on cell type and the stage in the life cycle. The proteins that make up the nuclear pore complex are known as nucleoporins. About half of the nucleoporins typically contain either an alpha solenoid or a beta-propeller fold, or in some cases both as separate structural domains. The other half show structural characteristics typical of "natively unfolded" proteins, i.e. they are highly flexible proteins that lack ordered secondary structure.

Assembly of the NPC

As the NPC controls access to the genome, it is essential that it exists in large amounts in areas of the cell cycle where plenty of transcription is necessary. For example, cycling mammalian and yeast cells double the amount of NPC in the nucleus between the G1 and G2 phase of cell Mitosis. And oocytes accumulate large numbers of NPCs to prepare for the rapid mitosis that exists in the early stages of development. Interphase cells must also keep up a level of NPC generation to keep the levels of NPC in the cell constant as some may get damaged. Some cells can even increase the NPC numbers due to increased transcriptional demand.

Theories of assembly So how are these vast proteins complexes assembled? As the immunodepletion of certain protein complexes, such as the Nup 107–160 complex, leads to the formation of poreless nuclei, it seems likely that the Nup complexes are involved in fusing the outer membrane of the nuclear envelope with the inner and not that the fusing of the membrane begins the formation of the pore. There are several ways that this could lead to the formation of the full NPC. One possibility is that as a protein complex it binds to the chromatin. It is then inserted into the double membrane close to the chromatin. This, in turn, leads to the fusing of that membrane. Around this protein complex others eventually bind forming the NPC. This method is possible during every phases of mitosis as the double membrane is present around the chromatin before the membrane fusion proteins complex can insert. Post mitotic cells could form a membrane first with pores being inserted into after formation. Another model for the formation of the NPC is the production of a prepore as a start as opposed to a single protein complex. This prepore would form when several Nup complexes come together and bind to the chromatin. This would have the double membrane form around it in during mitotic reassembly. Possible prepore structures have been observed on chromatin before nuclear envelope(NE) formation using electron microscopy. During the interphase of the cell cycle the formation of the prepore would happen within the nucleus, each component being transported in through existing NPCs. These Nups would bind to an importin, once formed, preventing the assembly of a prepore in the cytoplasm. Once transported into the nucleus Ran GTP would bind to the importin and cause it to release the cargo. This Nup would be free to from a prepore. The binding of importins has at least been shown to bring Nup 107 and the Nup 153 nucleoporins into the nucleus. NPC assembly is a very rapid process yet defined intermediate states occur which leads to the idea that this assembly occurs in a stepwise fashion. A third possible method of NPC assembly is splitting. This method seems to be tailor made for NPC formation during the interphase. It happens when more protomers are added on to an existing NPC. The eightfold symmetry of the NPC has been shown to have a degree of plasticity and will allow this. Eventually enough protomers will add and allow a new NPC to split off the original. This method of NPC assembly can only happen during the interphase of the cell cycle.

Disassembly of NPC During mitosis the NPC appears to disassemble in stages. Peripheral nucleoporins such as the Nup 153 Nup 98 and Nup 214 disassociate from the NPC. The rest, which can be considered a scaffold proteins remain stable, as cylindrical ring complexes within the nuclear envelope. This disassembly of the NPC peripheral groups is largely thought to be phosphate driven, as several of these nucleoporins are phosphorylated during the stages of mitosis. However, the enzyme involved in the phosphorlyation is unknown in vivo. In metazoans (which undergo open mitosis) the NE degrades quickly after the loss of the peripheral Nups. The reason for this may be due to the change in the NPC’s architecture. This change may make the NPC more permeable to enzymes involved in the degradation of the NE such as cytoplasmic tubulin, as well as allowing the entry of key mitotic regulator proteins.

It was shown, in fungi that undergo closed mitosis (where the nucleus does not degrade), that the change of the permeability barrier of the NE was due to changes with in the NPC and is what allows the entry of mitotic regulators. In Aspergillus nidulans the NPC composition appears to be effected by the mitotic kinase NIMA, possibly by phosphorylating the nucleoporins Nup98 and Gle2/Rae1. This remodelling seems to allow the proteins complex cdc2/cyclinB enter the nucleus as well as many other proteins such as soluble tubulin. The NPC scaffold remains intact throughout the whole closed mitosis. This seems to preserve the integrity of the NE.

3.Nuclear lamina:

The nuclear lamina is a dense (~30 to 100 nm thick) fibrillar network inside the nucleus of a eukaryotic cell. It is composed of intermediate filaments and membrane associated proteins. Besides providing mechanical support, the nuclear lamina regulates important cellular events such as DNA replication and cell division. Additionally, it participates in chromatin organization and it anchors the nuclear pore complexes embedded in the nuclear envelope.The nuclear lamina is associated with the inner face of the bilayer nuclear envelope whereas the outer face stays continuous with the endoplasmic reticulum.

 Architucture of Nuclear Lamina

The nuclear lamina consists of two main components, lamins and nuclear lamin associated membrane proteins. The lamins are type V intermediate filaments which can be categorized as either A-type (lamin A, C) or B-type(lamin B1, B2) according to homology in sequence, biochemical properties and cellular localization during the cell cycle. Type V intermediate filaments differ from cytoplasmic intermediate filaments in the way that they have an extended rod domain (42 amino acid longer), that they all carry a nuclear localization signal (NLS) at their C-terminus and that they display typical tertiary structures. Lamin polypeptides have an almost complete α-helical conformation with multiple α-helical domains separated by non-α-helical linkers that are highly conserved in length and amino acid sequence. Both the C-terminus and the N- terminus are non α-helical, with the C-terminus displaying a globular structure. Their molecular weight ranges from 60 to 80 kilodaltons (kDa). In the amino acid sequence of nuclear lamins, there are also two phosphoacceptor sites present, flanking the central rod domain. A phosphorylation event at the onset of mitosis leads to a conformational change which causes the disassembly of the nuclear lamina. In the vertebrate genome, lamins are encoded by three genes. By alternative splicing, at least seven different polypeptides (splice variants) are obtained, some of which are specific for germ cells and play an important role in the chromatin reorganisation during meiosis. Not all organisms have the same number of lamin encoding genes; Drosophila melanogaster for example has only 2 genes, whereas Caenorhabditis elegans has only one. The presence of lamin polypeptides is an exclusive property of Metazoan organisms. Plants or single-cell Eukaryotic organisms such as Saccharomyces cerevisiae lack lamins. The nuclear lamin-associated membrane proteins are either integral or peripheral membrane proteins. The most important are lamin associated polypeptide 1 and 2 (LAP1, LAP2), emerin, lamin B-receptor (LBR), otefin and MAN1. Due to their positioning within or their association with the inner membrane, they mediate the attachment of the nuclear lamina to the nuclear envelope.

Function of nuclear lamin

The nuclear lamina is assembled by interactions of two lamin polypeptides in which the α-helical regions are wound around each other to form a two stranded α-helical coiled-coil structure, followed by a head-to-tail association of the multiple dimers. The linearly elongated polymer is extended laterally by a side-by-side association of polymers, resulting in a 2D structure underlying the nuclear envelope. Next to providing mechanical support to the nucleus, the nuclear lamina plays an essential role in chromatin organization, cell cycle regulation, DNA replication, cell differentiation and apoptosis.

Chromatin organization The non-random organization of the genome strongly suggests that the nuclear lamina plays a role in chromatin organization. Indeed, it has been shown that lamin polypeptides have an affinity for binding chromatin through their α-helical (rod like) domains at specific DNA sequences called matrix attachment regions (MAR). A MAR has a length of approximately 300–1000 bp and has a high A/T content. Lamin A and B can also bind core histones through a sequence element in their tail domain.

Cell cycle regulation At the onset of mitosis, (prophase, prometaphase) the cellular machinery is engaged in the disassembly of various cellular components including structures such as the nuclear envelope, the nuclear lamina and the nuclear pore complexes. This nuclear breakdown is necessary to allow the mitotic spindle to interact with the (condensed) chromosomes and to bind them at their kinetochores. These different disassembly events are initiated by the cyclin B/Cdk1 protein kinase complex (MPF). Once this complex is activated, the cell is forced into mitosis, by the subsequent activation and regulation of other protein kinases or by direct phosphorylation of structural proteins involved in this cellular reorganisation. After phosphorylation by cyclin B/Cdk1, the nuclear lamina depolymerises and B-type lamins stay associated with the fragments of the nuclear envelope whereas A-type lamins remain completely soluble throughout the remaining of the mitotic phase. The importance of the nuclear lamina breakdown at this stage is underlined by experiments where inhibition of the disassembly event leads to a complete cell cycle arrest. At the end of mitosis, (anaphase, telophase) there is a nuclear reassembly which is highly regulated in time, starting with the association of 'skeletal' proteins on the surface of the still partially condensed chromosomes, followed by nuclear envelope assembly. Novel nuclear pore complexes are formed through which nuclear lamins are actively imported by use of their NLS. This typical hierarchy raises the question whether the nuclear lamina at this stage has a stabilizing role or some regulative function, for it is clear that it plays no essential part in the nuclear membrane assembly around chromatin.

Embryonic development and cell differentiation The presence of lamins in embryonic development is readily observed in various model organisms such as Xenopus laevis, the chick and mammals. In Xenopus laevis, five different types were identified which are present in different expression patterns during the different stages of the embryonic development. The major types are LI and LII, which are considered homologs of lamin B1 and B2. LA are considered homologous to lamin A and LIII as a B-type lamin. A fourth type exists and is germ cell specific. In the early embryonic stages of the chick, the only lamins present are B-type lamins. In further stages, the expression pattern of lamin B1 decreases and there is a gradual increase in the expression of lamin A. Mammalian development seems to progress in a similar way. In the latter case as well it is the B-type lamins that are expressed in the early stages. Lamin B1 reaches the highest expression level, whereas the expression of B2 is relatively constant in the early stages and starts to increase after cell differentiation. With the development of the different kinds of tissue in a relatively advanced developmental stage, there is an increase in the levels of lamin A and lamin C. These findings would indicate that in its most basic form, a functional nuclear lamina requires only B-type lamins.

DNA replication Various experiments show that the nuclear lamina plays a part in the elongation phase of DNA replication. It has been suggested that lamins provide a scaffold, essential for the assembly of the elongation complexes, or that it provides an initiation point for the assembly of this nuclear scaffold. Not only nuclear lamina associated lamins are present during replication, but free lamin polypeptides are present as well and seem to have some regulative part in the replication process.

Apoptosis Apoptosis, basically to be considered as cellular suicide is of the highest importance in homeostasis of tissue and in defending the organism against invasive entry of viruses or other pathogens. Apoptosis is a highly regulated process in which the nuclear lamina is disassembled in an early stage. In contrast to the phosphorylation-induced disassembly during mitosis, the nuclear lamina is degraded by proteolytic cleavage, and both the lamins and the nuclear lamin-associated membrane proteins are targeted. This proteolytic activity is performed by members of the caspase-protein family who cleave the lamins after aspartic acid (Asp) residues.

Chromosomes


In a series of experiments beginning in the mid-1880s, Theodor Boveri gave the definitive demonstration that chromosomes are the vectors of heredity. His two principles were the continuity of chromosomes and the individuality of chromosomes. It is the second of these principles that was so original. Wilhelm Roux suggested that each chromosome carries a different genetic load. Boveri was able to test and confirm this hypothesis. Aided by the rediscovery at the start of the 1900s of Gregor Mendel's earlier work, Boveri was able to point out the connection between the rules of inheritance and the behaviour of the chromosomes. Boveri influenced two generations of American cytologists: Edmund Beecher Wilson, Walter Sutton and Theophilus Painter were all influenced by Boveri (Wilson and Painter actually worked with him). In his famous textbook The Cell in Development and Heredity, Wilson linked together the independent work of Boveri and Sutton (both around 1902) by naming the chromosome theory of inheritance the "Sutton-Boveri Theory" (the names are sometimes reversed). Ernst Mayr remarks that the theory was hotly contested by some famous geneticists: William Bateson, Wilhelm Johannsen, Richard Goldschmidt and T.H. Morgan, all of a rather dogmatic turn-of-mind. Eventually, complete proof came from chromosome maps in Morgan's own lab. The cell nucleus contains the majority of the cell's genetic material, in the form of multiple linear DNA molecules organized into structures called chromosomes.A chromosome is an organized structure of DNA and protein that is found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions. The word chromosome comes from the Greek χρῶμα (chroma, colour) and σῶμα (soma, body) due to their property of being very strongly stained by particular dyes.

Telomere A telomere is a region of repetitive DNA at the end of a chromosome, which protects the end of the chromosome from deterioration. Its name is derived from the Greek nouns telos "end" and merοs "part". The telomere regions deter the degradation of genes near the ends of chromosomes by allowing for the shortening of chromosome ends, which necessarily occurs during chromosome replication.

The total number of chromosomes (including sex chromosomes) present in a cell nucleus of different organisms are described below in the tables.

DNA packaging Prokaryotes do not possess nuclei. Instead, their DNA is organized into a structure called the nucleoid. The nucleoid is a distinct structure and occupies a defined region of the bacterial cell. This structure is, however, dynamic and is maintained and remodeled by the actions of a range of histone-like proteins, which associate with the bacterial chromosome. In archaea, the DNA in chromosomes is even more organized, with the DNA packaged within structures similar to eukaryotic nucleosomes. Bacterial chromosomes tend to be tethered to the plasma membrane of the bacteria. In molecular biology application, this allows for its isolation from plasmid DNA by centrifugation of lysed bacteria and pelleting of the membranes (and the attached DNA). Prokaryotic chromosomes and plasmids are, like eukaryotic DNA, generally supercoiled. The DNA must first be released into its relaxed state for access for transcription, regulation, and replication.



Nucleolus
The nucleolus is a discrete densely stained structure found in the nucleus. It is non-membrane bound structure, and is sometimes called a suborganelle. It forms around tandem repeats of rDNA, DNA coding for ribosomal RNA (rRNA). These regions are called nucleolar organizer regions (NOR). The main roles of the nucleolus are to synthesize rRNA and assemble ribosomes. The structural cohesion of the nucleolus depends on its activity, as ribosomal assembly in the nucleolus results in the transient association of nucleolar components, facilitating further ribosomal assembly, and hence further association. This model is supported by observations that inactivation of rDNA results in intermingling of nucleolar structures.

Splicing speckles or SC35 speckles
Sometimes referred to as interchromatin granule clusters (IGCs) or as splicing-factor compartments, speckles are rich in splicing snRNPs and other splicing proteins necessary for pre-mRNA processing. Because of a cell's changing requirements, the composition and location of these bodies changes according to mRNA transcription and regulation via phosphorylation of specific proteins.

Cajal bodies or coiled bodies
A nucleus typically contains between 1 and 10 compact structures called Cajal bodies or coiled bodies (CB), whose diameter measures between 0.2 µm and 2.0 µm depending on the cell type and species. When seen under an electron microscope, they resemble balls of tangled thread and are dense foci of distribution for the protein coilin. CBs are involved in a number of different roles relating to RNA processing, specifically small nucleolar RNA (snoRNA) and small nuclear RNA (snRNA) maturation, and histone mRNA modification.

Nuclear localization signal (NLS)
A nuclear localization signal or sequence (NLS) is an amino acid sequence which acts like a 'tag' on the exposed surface of a protein. This sequence is used to target the protein to the cell nucleus through the Nuclear Pore Complex and to direct a newly synthesized protein into the nucleus via its recognition by cytosolic nuclear transport receptors. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines. Different nuclear localized proteins may share the same NLS. An NLS has the opposite function of a nuclear export signal, which targets proteins out of the nucleus.

A. Classical NLSs Classical Nuclear localization signals can be further classified as either monopartite or bipartite. The first NLS to be discovered was the sequence PKKKRKV in the SV40 Large T-antigen (a monopartite NLS). The NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK, is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids, separated by a spacer of about 10 amino acids. Both signals are recognized by importin α. Importin α contains a bipartite NLS itself, which is specifically recognized by importin β. The latter can be considered the actual import mediator. Chelsky et al. proposed the consensus sequence K-K/R-X-K/R for monopartite NLSs. A Chelsky sequence may, therefore, be part of the downstream basic cluster of a bipartite NLS. Makkerh et al. carried out comparative mutagenesis on the nuclear localisation signals of SV40 T-Antigen (monopartite), C-myc (monopartite) and nucleoplasmin (bipartite), and showed amino acid features common to all three. Notably the role of neutral and acidic amino acids was shown for the first time in contributing to the efficiency of the NLS.

B. Non-classical NLSs

There are many other types of NLS, such as the acidic M9 domain of hnRNP A1, the sequence KIPIK in yeast transcription repressor Matα2, and the complex signals of U snRNPs. Most of these NLSs appear to be recognized directly by specific receptors of the importin β family without the intervention of an importin α-like protein. A signal that appears to be specific for the massively produced and transported ribosomal proteins, seems to come with a specialized set of importin β-like nuclear import receptors. Recently a class of NLSs known as PY-NLSs has been proposed, originally by Lee et al. This PY-NLS motif, so named because of the proline-tyrosine amino acid pairing in it, allows the protein to bind to Importin β2 (also known as transportin or karyopherin β2), which then translocates the cargo protein into the nucleus. The structural basis for the binding of the PY-NLS contained in Importin β2 has been determined and an inhibitor of import designed.

Nuclear export signal (NES)
A nuclear export signal (NES) is a short amino acid sequence of 4 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex. It has the opposite effect of a nuclear localization signal, which targets a protein located in the cytoplasm for import to the nucleus. The NES is recognized and bound by exportins. In silico analysis of known NESs found the most common spacing of the hydrophobic residues to be LxxxLxxLxL, where "L" is a hydrophobic residue (often leucine) and "x" is any other amino acid; the spacing of these hydrophobic residues may be explained by examination of known structures that contain an NES, as the critical residues usually lie in the same face of adjacent secondary structures within a protein, which allows them to interact with the exportin[1]. Ribonucleic acid (RNA) are composed of nucleotides, and thus, lack the nuclear export signal to move out of the nucleus. As a result, most forms of RNA will bind to a protein molecule to form a ribonucleoprotein complex to be exported from the nucleus. Nuclear export first begins with the binding of Ran-GTP (a G-protein) to exportin. This causes a shape change in exportin, increasing its affinity for the export cargo. Once the cargo is bound, the Ran-exportin-cargo complex moves out of the nucleus through the nuclear pore. GTPase activating proteins (GAPs) then hydrolyze the Ran-GTP to Ran-GDP, and this causes a shape change and subsequent exportin release. Once no longer bound to Ran, the exportin molecule loses affinity for the nuclear cargo as well, and the complex falls apart. Exportin and Ran-GDP are recycled to the nucleus separately, and guanine exchange factor (GEF) in the nucleus switches the GDP for GTP on Ran.

Role of Ran GTPase in nuclear transport during interphase
Ran (RAs-related Nuclear protein) is a small 25Kda protein that is involved in transport into and out of the cell nucleus during interphase and also involved in mitosis. It is a member of the Ras superfamily.Ran exists in the cell in two nucleotide-bound forms: GDP-bound and GTP-bound. RanGDP is converted into RanGTP through the action of RCC1(regulator of chromosome condensation 1), the nucleotide exchange factor for Ran. RCC1 is also known as RanGEF (Ran Guanine nucleotide Exchange Factor). Ran's intrinsic GTPase-activity is activated through interaction with Ran GTPase activating protein (RanGAP), facilitated by complex formation with Ran-binding protein (RanBP). GTPase-activation leads to the conversion of RanGTP to RanGDP, thus closing the Ran cycle.

Ran can diffuse freely within the cell, but because RCC1 and RanGAP are located in different places in the cell, the concentration of RanGTP and RanGDP differs locally as well, creating concentration gradients that act as signals for other cellular processes. RCC1 is bound to chromatin and therefore located inside the nucleus. RanGAP is cytoplasmic in yeast and bound to the nuclear envelope in plants and animals. In mammalian cells, it is SUMO modified and attached to the cytoplasmic side of the nuclear pore complex via interaction with the nucleoporin RanBP2 (Nup358). This difference in location of the accessory proteins in the Ran cycle leads to a high RanGTP to RanGDP ratio inside the nucleus and an inversely low RanGTP to RanGDP ratio outside the nucleus. In addition to a gradient of the nucleotide bound state of Ran, there is a gradient of the protein itself, with a higher concentration of Ran in the nucleus than in the cytoplasm. Cytoplasmic RanGDP is imported into the nucleus by the small protein NTF2 (Nuclear Transport Factor 2), where RCC1 can then catalyze exchange of GTP for GDP on Ran.

Ran is involved in the transport of proteins across the nuclear envelope by interacting with karyopherins and changing their ability to bind or release cargo molecules. Cargo proteins containing a nuclear localization signal (NLS) are bound by importins and transported into the nucleus. Inside the nucleus, RanGTP binds to importin and releases the import cargo. Cargo that needs to get out of the nucleus into the cytoplasm binds to exportin in a ternary complex with RanGTP. Upon hydrolysis of RanGTP to RanGDP outside the nucleus, the complex dissociates and export cargo is released.

Importin

Importin also play important role in nuclear transport. Importin is a type of protein that moves other protein molecules into the nucleus by binding to a specific recognition   sequence, called the nuclear localization signal (NLS). Importin is classified as a karyopherin. Importin has two subunits, importin α and importin β. Of these, importin α binds to the NLS of the protein to be imported to the nucleus whereas importin β helps in the docking of the importin heterodimer-bound protein to the nuclear pore complex. The NLS-Importin α-Importin β trimer dissociates after binding to Ran GTP inside the nucleus

Function of nucleus

The main function of the cell nucleus is to control gene expression and mediate the replication of DNA during the cell cycle. The nucleus provides a site for genetic transcription that is segregated from the location of translation in the cytoplasm, allowing levels of gene regulation that are not available to prokaryotes.

Cell compartmentalization The nuclear envelope allows the nucleus to control its contents, and separate them from the rest of the cytoplasm where necessary. This is important for controlling processes on either side of the nuclear membrane. In some cases where a cytoplasmic process needs to be restricted, a key participant is removed to the nucleus, where it interacts with transcription factors to downregulate the production of certain enzymes in the pathway. This regulatory mechanism occurs in the case of glycolysis, a cellular pathway for breaking down glucose to produce energy. Hexokinase is an enzyme responsible for the first the step of glycolysis, forming glucose-6-phosphate from glucose. At high concentrations of fructose-6-phosphate, a molecule made later from glucose-6-phosphate, a regulator protein removes hexokinase to the nucleus, where it forms a transcriptional repressor complex with nuclear proteins to reduce the expression of genes involved in glycolysis. In order to control which genes are being transcribed, the cell separates some transcription factor proteins responsible for regulating gene expression from physical access to the DNA until they are activated by other signaling pathways. This prevents even low levels of inappropriate gene expression. For example, in the case of NF-κB-controlled genes, which are involved in most inflammatory responses, transcription is induced in response to a signal pathway such as that initiated by the signaling molecule TNF-α, binds to a cell membrane receptor, resulting in the recruitment of signalling proteins, and eventually activating the transcription factor NF-κB. A nuclear localisation signal on the NF-κB protein allows it to be transported through the nuclear pore and into the nucleus, where it stimulates the transcription of the target genes. The compartmentalization allows the cell to prevent translation of unspliced mRNA. Eukaryotic mRNA contains introns that must be removed before being translated to produce functional proteins. The splicing is done inside the nucleus before the mRNA can be accessed by ribosomes for translation. Without the nucleus, ribosomes would translate newly transcribed (unprocessed) mRNA, resulting in misformed and nonfunctional proteins.

Gene expression Gene expression first involves transcription, in which DNA is used as a template to produce RNA. In the case of genes encoding proteins, that RNA produced from this process is messenger RNA (mRNA), which then needs to be translated by ribosomes to form a protein. As ribosomes are located outside the nucleus, mRNA produced needs to be exported. Since the nucleus is the site of transcription, it also contains a variety of proteins that either directly mediate transcription or are involved in regulating the process. These proteins include helicases, which unwind the double-stranded DNA molecule to facilitate access to it, RNA polymerases, which synthesize the growing RNA molecule, topoisomerases, which change the amount of supercoiling in DNA, helping it wind and unwind, as well as a large variety of transcription factors that regulate expression.

Processing of pre-mRNA Newly synthesized mRNA molecules are known as primary transcripts or pre-mRNA. They must undergo post-transcriptional modification in the nucleus before being exported to the cytoplasm; mRNA that appears in the cytoplasm without these modifications is degraded rather than used for protein translation. The three main modifications are 5' capping, 3' polyadenylation, and RNA splicing. While in the nucleus, pre-mRNA is associated with a variety of proteins in complexes known as heterogeneous ribonucleoprotein particles (hnRNPs). Addition of the 5' cap occurs co-transcriptionally and is the first step in post-transcriptional modification. The 3' poly-adenine tail is only added after transcription is complete. RNA splicing, carried out by a complex called the spliceosome, is the process by which introns, or regions of DNA that do not code for protein, are removed from the pre-mRNA and the remaining exons connected to re-form a single continuous molecule. This process normally occurs after 5' capping and 3' polyadenylation but can begin before synthesis is complete in transcripts with many exons. Many pre-mRNAs, including those encoding antibodies, can be spliced in multiple ways to produce different mature mRNAs that encode different protein sequences. This process is known as alternative splicing, and allows production of a large variety of proteins from a limited amount of DNA.

Facts to be remembered
Red blood cell of human doesn't contain nucleus.

Intestinal parasites in the genus Giardia which have two nuclei per cell.

Some species of protozoa and some fungi in mycorrhizaehave polynucleated cells.

During mitosis the NPC appears to disassemble in stages. Peripheral nucleoporins such as the Nup 153 Nup 98 and Nup 214 disassociate from the NPC. The rest, which can be considered a scaffold proteins remain stable, as cylindrical ring complexes within the nuclear envelope. This disassembly of the NPC peripheral groups is largely thought to be phosphate driven, as several of these nucleoporins are phosphorylated during the stages of mitosis. However, the enzyme involved in the phosphorlyation is unknown in vivo.

In metazoans (which undergo open mitosis) the NE degrades quickly after the loss of the peripheral Nups. The reason for this may be due to the change in the NPC’s architecture. This change may make the NPC more permeable to enzymes involved in the degradation of the NE such as cytoplasmic tubulin, as well as allowing the entry of key mitotic regulator proteins.

The table given below lists the numbers of chromosomes in various plants, animals, protists, and other living organisms, given as the diploid number (2n)

Question time
What do you know about nuclear membrane?

What is the function of nucleolus?

What is a chromosome?

Design an experiment which indicate that a particular protein is going into nucleus?

How will you determine that a particular protein X is localizing in to the nucleus not in cytoplasm?

What are importins?

What due understand with NLS?

What is the difference between active and passive transport?