The following points highlight the three main constituents of nucleus. The constituents are: 1. Nuclear Envelope 2. Nucleolus 3. Nucleoplasm.
Constituent # 1. Nuclear Envelope:
Under the electron microscope the nuclear envelope appears to consist of two membranes, the outer and the inner nuclear membranes, separated by a perinuclear space of 20 nm (Fig. 2.65). Each of the two membranes of the nuclear envelope appears to have trilaminar unit membrane structure of 7 – 10 nm thickness.
The outer nuclear membrane communicates with endoplasmic reticulum at several points and has ribosomes on the outer side.
The nuclear envelope is perforated by many circular apertures called nuclear pores. Each nuclear pore shows the presence of an electron- dense ring or cylinder called the annulus. The actual opening of the nuclear pore is thus confined to the cavity of the annulus. The annulus extends both into the cytoplasm and the nucleoplasm.
The annulus typically consists of eight subunits arranged in radial symmetry around the periphery of the pore. The subunits have been variously interpreted as micro-cylinders, filaments, spheres or ovoid’s.
A central ribonucleoprotein granule of 10-15 nm size may be present in some pore complexes and may be absent in adjacent ones. On the inner side of the nuclear envelope of many cell types is present fibrous material which has been called the fibrous lamina which extends into the nucleoplasm.
The inner nuclear membrane is lined on the inner surface by the nuclear lamina, which is a protein fibrous network of 30-100 nm thick that connects the inner nuclear membrane with chromatin. It is composed of three principal extrinsic membrane proteins — lamins A, B and C. The lamins are made up of two parts — one is rod like tail of 52 nm long and the other is two globular heads at one end.
Lamins are highly similar in sequence and structure with the cytoplasmic intermediate filament. Inner nuclear membrane contains lamin B receptors that binds to lamin B; and lamin A and C bind with lamin B and with interactions between the lamina and chromatin (Fig. 2.66).
The nuclear lamina and its polypeptide carry out the following functions:
(a) Regulating assembly and disassembly of the nuclear membrane during cell division. De-polymerization of lamina due to phosphorylation at late prophase causes the nuclear envelope to disassemble into a number of small vesicles. At telophase de-phosphorylation and polymerisation of lamins into fibrous network along with fusion of small nuclear vesicles causes reassembly of nuclear envelope.
(b) Attachment of chromatin to the nuclear envelope.
(c) Helps to form micronuclei when the cells are left for a long time in colchicine.
Nuclear Pore Complex:
The nuclear pore is a large complex structure of 125 million Daltons with 120 nm diameter and 50 nm thickness. Electron micrograph has shown that nuclear pore complexes have an eight-fold symmetry. Pore complex consists of annuli and a structure is formed from a set of large protein granules arranged in octagonal patterns.
The hole in the centre of each complex often appears to be plugged by a large central granule. Eight radial spokes also extends from plug to rings (Fig. 2.67 & 2.68).
During 1990s, significant progress has been made towards better understanding of the structure and function of tine nuclear pore. New pore protein have been identified, cloned, mutants isolated and detailed mechanism of nucleocytoplasmic transfer has been proposed.
Further, the pore has been reconstituted in vitro, a number of signal sequences and one or more signal sequence receptors have been identified and a new ‘basket like structure’ has been attached to the inner side of the nuclear pore.
It consists of four separate elements:
(i) Scaffold, which included majority of the pore,
(ii) Transporter, the central hub which carries out active transport (both import and export) of proteins and RNAs,
(iii) Short thick filaments attached to the cytoplasmic side of the pore,
(iv) A basket attached to the nucleocytoplasmic side of the pore (Fig. 2.69).
The scaffold is a stack of three closely apposed rings — cytoplasmic ring, nucleocytoplasmic ring and a central ring of thick spokes.
The spokes of central ring are attached to the transporter on the inner side and to the nucleocytoplasmic and cytoplasmic rings on the outer side. Interspersed between the spokes are aqueous channels, 9 nm wide, which allow diffusion of proteins and metabolites between the nucleus and the cytoplasm.
The transporter is a proteinaceous ring, 36-38 nm in diameter and consists of two irises of eight arms each. The two irises are assumed to be stacked atop one another and open sequentially, each like the diaphragm of a camera, to let a nuclear protein or RNA pass through from the nucleus to the cytoplasm. On the cytoplasmic side of the pore, thick filaments of 3.3 nm in diameter, extend into the cytoplasm.
On the nuclear side, a large basket like structure is found, which consists of eight filaments of 100 nm long, extending from nucleocytoplasmic ring of the pore and meeting a smaller ring of 60 nm in diameter within the nucleus. This basket plays an important role in RNA export.
The function of nuclear pore complex is the nucleocytoplasmic transport mediated through a number of proteins, called nucleoporin (NUP).’ The nuclear pore complex has a passive diffusion channel and also can diffuse many substances by active process using energy or signal sequence mediated by carrier molecules.
(a) Import of nuclear proteins through nuclear pore involves the formation of NLS- Protein-lmportin complex (NLS = nuclear localization sequence).
(b) Export of RNA from the nucleus across the pore is mediated through NES-Rev protein (NES = nuclear export sequence).
(c) Export followed by reimport of 5SrRNA and UsnRNA occurs through the nuclear pore by the protein with NES like sequence.
Functionof Nuclear Envelope:
The nuclear envelope is an interface between nucleus and the cytoplasm. It serves to separate the genetic component of the cell (the chromosomes) from the protein-synthesis machinery (ribosome and ER). It thus provides protection to DNA against the mutagenic effects of cytoplasmic enzymes.
It is concerned with nucelocytoplasmic exchange, attachment of structural elements to the cytoplasm, attachment of nuclear components, contribution to other cell membranes and electron transport activity.
Constituent # 2. Nucleolus:
Nucleolus is usually a-spheroidal body situated within the nucleus, either in a central or peripheral position. It is found in close association with the nucleolar organizer region of two or more chromosomes (Fig. 2.70). One or many nucleoli may be present in a nucleus.
Ultrastructure of Nucleolus:
The ultra-structure of the nucleolus shows four chief components: an amorphous matrix, nucleolar associated chromatin, fibrils and granules (Fig. 2.71).
The matrix or pars amorpha of the nucleolus is homogenous. It contains scattered granules and fibrils.
Chromatin associated with the nucleolus contains DNA which serves as a template for rRNA synthesis. Surrounding the nucleolus like a shell is perinucleolar chromatin. Projecting into the nucleolus from the perinucleolar chromatin are septa like trabeculae, called intra-nucleolar chromatin.
The fibrils are 80-100Å in diameter and constitute pars fibrosa. They contain RNA and are probably the precursors of the granules. Granules are of 150 – 200Å diameter which constitute the pars granulosa. These granules contain protein, RNA and are precursors of ribosomes.
The granules appear to be vesicles with a light central core and a dense peripheral structure. They are connected together by a thin filament, forming a structure (the primary nucleolonema) resembling a string of beads. The primary nucleolonema undergoes folding to form the secondary nucleolonema.
Function of Nucleolus:
The nucleolus is one of the most active sites of RNA synthesis and the source of ribosomal RNA (rRNA). The chromatin in the nucleolus contains genes or ribosomal DNA (rDNA) for coding ribosomal RNA. The fibrils represent the origin of ribosomal RNA, and the granules the next stage. The granules in turn are the precursors of ribosomes (Fig. 2.72).
In eukaryotes the site of synthesis of most of the ribosomal RNA (rRNA) is the nucleolus. The nucleolar organizer contains many copies of ribosomal DNA (repetitive DNA). Several distinct types of rRNA have been isolated from cells.
Of these only four classes, namely 28S, IBS, 5.8S and 5S have been found in the ribosomes. The other types are intermediate stages in the formation of the RNA of the ribosomes. Nucleolar DNA transcribes 45S precursor which on processing results 28S, 18S and 5.8S RNA (Fig. 2.73). 5S RNA is transcribed outside the nucleolus.
Ribosomal proteins are synthesized in the cytoplasm and trans-located to the nucleus where they become associated with RNA. Structural core proteins first associate with 45S RNA to form ribonucleoprotein particles. Other proteins are probably bound later.
Ribonucleoprotein particles on processing ultimately form 40S and 605 subunits of the ribosome. In prokaryotes, the DNA transcribes 30S rRNA precursors which are trimmed to form 16S, 23S, 5S RNA. These become associated with protein to form 30S and 50S subunits of the ribosome (Fig. 2.74).
Constituent # 3. Nucleoplasm:
The transparent, semisolid, granular and slightly acidophilic ground substance of the nucleus is known as nucleoplasm. The nuclear components, such as the chromatin threads and the nucleolus, remain suspended in the nucleoplasm. It is composed mainly of nucleoproteins, but it also contains various inorganic and organic substances such as nucleic acid, proteins, enzymes and other minerals.
Nuclear Reticulum (Chromatin):
The thread-like elongated filamentous structures of nucleoplasm which take readily the basic stains are known as chromatin fibres. It is a complex of DNA and protein. Chromatin fibres are observed only at interphase stage. During cell division these chromatin fibres become thick, short thread-like structure known as chromosomes.
Two types of chromatin materials have been recognized. The darkly stained condensed region of the chromatin is known as heterochromatin which is supposed to be metabolically and genetically inert. The light stained region of the chromatin fibre is known as euchromatin. This region contains metabolically active DNA and, therefore, is genetically very important.
Function of Nuclear Reticulum (Chromatin):
Chromatin forms the basic constituent of the chromosomes, the site of hereditary material—the genes. It contains DNA which produces different types of RNA and controls synthesis of enzymes involved in different metabolic processes.