THE CELL NUCLEUS 

Department Of General Histology

Introduction

The nucleus contains a blueprint for all cell structures and activities encoded in the DNA of the chromosomes. It also contains the molecular machinery to replicate its DNA and to synthesize and process all types of RNA. Macromolecular transfer between the nuclear and cytoplasmic compartments is regulated. Because functional ribosomes do not occur in the nucleus, no proteins are produced there. The numerous protein molecules needed for the activities of the nucleus are imported from the cytoplasm.

Nuclei of large, active cells

Liver cells (hepatocytes) have large, well-stained nuclei located in the center of the cytoplasm. One or more nucleoli are seen inside each nucleus, indicating intense protein synthesis by these cells. Most of the chromatin is light-staining or euchromatic, with small areas of more darkly stained heterochromatin scattered throughout the nucleus and just inside the nuclear envelope. This superficial heterochromatin allows the boundary of the organelle to be seen more easily by light microscopy. One cell here has two nuclei, which is fairly common in the liver. X500. Pararosaniline–toluidine blue.

Structural components of the nucleus.

(a): TEM of a typical cell nucleus clearly shows the electron-dense heterochromatin (HC) and the more diffuse euchromatin (EC). The arrows indicate the nucleolus-associated heterochromatin around the nucleolus (NU). Arrowheads indicate areas where the perinuclear space between the two membranes of the nuclear envelope is clearly seen. Just inside the nuclear envelope is a thin electron-dense region containing the nuclear lamina and more heterochromatin.

(b): Schematic representation of a cell nucleus shows that the nuclear envelope is made of two membranes separated by the perinuclear space. The outer membrane has ribosomes bound to it and is continuous with the ER. The two membranes fuse at many places to form nuclear pores. Heterochromatin clumps (HC) are associated with the meshwork of the nuclear lamina just inside the nuclear envelope, whereas the euchromatin (EC) appears dispersed in the interior of the nucleus. The nucleolus contains distinct regions called the pars granulosa (G) and the pars fibrosa (F).

Relationship of nuclear envelope to the rough ER

Nuclear laminaThe nuclear lamina is formed from a class of intermediate filaments proteins, the lamins, which assemble as a lattice adjacent to the inner nuclear membrane. When the nuclear envelope disperses during early prophase of cell division, at least some lamin proteins remain attached to the membrane fragments and reassembly of the nuclear lamina immediately after cell division facilitates re-formation of the nuclear envelopes of the two new nuclei. The nuclear lamina also contains binding sites for chromatin, helping to organize this material in the nucleus. Chromatin is not present at the openings through the nuclear envelope called nuclear pore complexes.

Nuclear pores

•(a): Section through the nuclear envelope and the two-membrane structure of the nuclear envelope clearly. The electron-dense proteins that make up the nuclear pore complexes can also be seen (arrows). Immediately beneath the nuclear envelope is the nuclear lamina and heterochromatin, material that is not present however at the nuclear pores. •(b): Tangential section through a nuclear envelope shows the electron-dense nuclear pore complexes (arrows) and the electron-lucent patches in the peripheral heterochromatin which represent the areas just inside the pores.

Cryofracture of nuclear envelop showing nuclear poresElectron micrograph obtained by freeze-fracture of an intestinal cell shows the two components of the nuclear envelope and the nuclear pores. The fracture plane occurs partly between the two nuclear envelope membranes (left) but mostly just inside the envelope with the chromatin falling away. The size and distribution of the nuclear pore complexes are clearly seen. The same nuclear pore complexes can mediate both the import and export of macromolecules between the nucleus and cytoplasm using tightly controlled processes in each direction.

Components of a nucleosome

Nucleosome is a structure that produces the initial organization of free double-stranded DNA into chromatin. Each nucleosome has an octomeric core complex made up of four types of histones, two copies each of H2A, H2B, H3, and H4. Around this core is wound DNA approximately 150 base pairs in length. One H1 histone is located outside the DNA on the surface of each nucleosome. DNA associated with nucleosomes in vivo thus resembles a long string of beads. Nucleosomes are very dynamic structures, with H1 loosening and DNA unwrapping at least once every second to allow other proteins, including transcription factors and enzymes, access to the DNA.

Sex chromatin

Either X chromosome in cells from females can undergo inactivation and clumping to form heterochromatic sex chromatin. Morphologic features of sex chromatin can be seen in human female epithelial cells lining the mouth and neutrophils. Left: In the oral epithelial cells, heterochromatic sex chromatin appears as a small granule adhering to the nuclear envelope. These superficial buccal cells lining the cheeks are frequently used to study sex chromatin or as a very convenient source of nucleated cells for DNA analyses. Right: In neutrophils, chromatin often has the shape of a drumstick projecting from the multilobed nucleus that is unique to these cells. The genetically inactive, heterochromatic X chromosome comprising the sex chromatin is sometimes called a Barr body, after the cytologist who first discovered it in the cells of females.

Several orders of chromatin packing are believed to occur during condensation of chromatin during mitotic prophase, although the protein associations involved at each stage are not well understood. Genetic activity is almost completely shut down during this process and histones are chemically modified in various ways. The top drawing shows the 2-nm DNA double helix, followed by the association of DNA with histones to form 11-nm filaments of nucleosomes connected by the DNA ("beads on a string"). Nucleosomes on the DNA then interact in a manner not well understood to form a more compact 30-nm fiber. Through further condensation, filaments with diameters of 300 nm and 700 nm are formed. The highly folded loops of chromatin at these stages are stabilized by interactions with protein complexes made of condensins which eventually make up a central framework at the long axis of each chromatid. The bottom drawing shows a metaphase chromosome, which exhibits the maximum packing of DNA. The chromosome consists of two chromatids held together at a narrow point called the centromere.

Karyotyping

Human karyotype preparations are made by staining and then photographing the chromosomes of cells disrupted after mitotic arrest with colchicine. Nuclei are chosen for analysis in which the individual chromosomes are maximally condensed. The individual chromosomes are cut from the photograph and pasted together in various ways for study. With certain stains each chromosome has a particular pattern of banding that facilitates its identification and shows the relationship of the banding pattern to genetic anomalies. The 22 pairs of autosomes are numbered in order of decreasing size; the pair of X and Y chromosomes differ in both size and morphology

Nucleoli

Primary oocytes are very large cells with very large round euchromatic nuclei. The cells are actively increasing in volume, synthesizing much protein and many ribosomes, and each nucleus has one well-developed, intensely basophilic nucleolus. The strong basophilia reflects the high concentration of rRNA being processes in this small region of nucleoplasm. Other cells may each have one large nucleolus or a few smaller nucleoli. All are involved in transcription and processing of rRNA. Primary oocytes arrest for a prolonged period during prophase of the first meiotic division, when the chromosomes have already begun to condense. Parts of the condensed chromosomes are seen as the stained material in the sectioned nuclei shown here. Meiosis in oocytes will proceed just before they are ovulated

Regions within a nucleolus

Different regions of the nucleolus can often be seen in sections of cell nuclei examined by TEM. The major parts of the nucleolus identified in this way are one or more pale-staining regions containing nucleolar organizer (NO) DNA—sequences of bases coding for rRNA. In the human genome, five pairs of chromosomes contain nucleolar organizers. Closely associated with the nucleolar organizers are densely packed 5- to 10-nm ribonucleoprotein fibers of the pars fibrosa (PF), which consists of primary rRNA transcripts. The pars granulose (PG) consists of 15- to 20-nm granules that represent maturing ribosomal subunits. Proteins, synthesized in the cytoplasm, become associated with rRNAs in the nucleolus. The resulting ribosomal subunits are then exported to the cytoplasm. A small amount of heterochromatic nucleolus-associated chromatin (NAC) is also sometimes part of the nucleolus, but its functional significance is unknown.

Cell Division

Cell division, or mitosis (Gr. mitos, a thread), can be observed with the light microscope. During this process, the parent cell divides, and each of the daughter cells receives a chromosomal set identical to that of the parent cell. Essentially, a longitudinal duplication of the chromosomes takes place, and these chromosomes are distributed to the daughter cells. The period between mitoses is called interphase, during which the DNA is replicated and the nucleus appears as it is most commonly seen in histological preparations. The process of mitosis is subdivided into four phases

Phases of mitosis

Chromosomal changes during mitosis are easily seen and most commonly studied in large cultured cells or in the large cells in the very early embryos of invertebrates or primitive vertebrates after sectioning. Shown here are cells in sections of a fish blastodisc. a. During the relatively long prophase the centrosomes move to opposite poles, the nuclear envelope fragments, and chromosomes condense and become visible. Having undergone DNA replication, each chromosome consists of two chromatids joined at their centromere regions by a kinetochore protein complex. b. At the short metaphase the chromosomes have become aligned at the equatorial plate as a result of their attachments to the dynamic microtubules which run from the kinetochores to the centrosomes. c. During anaphase the kinetochores come apart and the chromatids (now called chromosomes themselves) are pulled on microtubules toward the two centrosomes. d. In telophase the cell pinches in two by constriction of bundled actin filaments in the cell cortex and the chromosomes decondense, transcription resumes, nucleoli reappear, and the nuclear lamina and nuclear envelopes reassemble

In the lining of the small intestine, many mitotic transit amplifying cells, progeny of nearby stem cells that are not yet fully differentiated, can be found in the area above the most basal region of the intestinal crypts. Condensed chromosomes of cells in late anaphase and telophase phase can be distinguished

Metaphase cells in a gland of proliferating uterine endometrium

Telophase cells in the esophagus lining.

Metaphase in the basal layer of epidermis. Mitotic figures are normally difficult to identify in most animal tissues, both because they are rare and because the various cell shapes and locations seldom allow specific phases of mitosis to be seen clearly. Most commonly, mitotic figures in organs appear simply as nuclei with clumped, darkly stained chromatin. X400. H&E.

Mitotic cells in adult tissues

(a): In the lining of the small intestine, many mitotic transit amplifying cells, progeny of nearby stem cells that are not yet fully differentiated, can be found in the area above the most basal region of the intestinal crypts. Condensed chromosomes of cells in late anaphase and telophase phase can be distinguished.

(b): Metaphase cells in a gland of proliferating uterine endometrium.

(c): Telophase cells in the esophagus lining.

(d): Metaphase in the basal layer of epidermis. Mitotic figures are normally difficult to identify in most animal tissues, both because they are rare and because the various cell shapes and locations seldom allow specific phases of mitosis to be seen clearly. Most commonly, mitotic figures in organs appear simply as nuclei with clumped, darkly stained chromatin.

The Cell Cycle

The cell cycle

Control of the cell cycleOne factor determining the time a cell spends in G1 is the cell's state of differentiation, or how much time it spends expressing gene products specific to its cell type before resuming DNA replication. Differentiating cells in growing tissues may have very long G1 periods and such cells are often said to be "in a G0 phase" of the cell cycle. From this phase many differentiated cells can return to the cycle, but some stay in G0 for a long time or even for their entire lifetime. Entry into each phase of the cell cycle is controlled by proteins called cyclins and cyclin-dependent kinases which phosphorylate/activate many proteins needed for phase-specific functions. Cyclin activity produces an important restriction point (R) late in G1 and a similar G2/M checkpoint which are important for the maintenance of chromosome stability and cell viability. These control points stop the cycle under conditions unfavorable to the cell and help insure that neither the DNA replication nor the mitotic phases occurs prematurely. For example, at the G2/M checkpoint the cell pauses while enzymes insure that all DNA has been replicated properly.

Stem cells

In rapidly growing adult tissues and perhaps in other tissues there are slowly dividing populations of stem cells. Stem cells divide asymmetrically, producing one cell that remains as a stem cell and another which becomes committed to a differentiative pathway but divides a few more times at a more rapid rate. Such cells have been termed "transit amplifying cells," each of which eventually stops dividing and becomes fully differentiated.

Mitosis

Mitosis and meiosis share many aspects of chromatin condensation and separation, but differ in various key ways. As chromosomal condensation begins in meiosis, the two homologous maternal and paternal chromosomes physically align in synapsis and regions are exchanged during crossing over or recombination.

Meiosis

This is followed by two meiotic divisions with no intervening S phase. Mitosis produces two diploid cells which are the same genetically. Meiosis with its two successive cell divisions produces four haploid cells. During meiotic crossing over, new combinations of genes arise so that every haploid cell is genetically unique.

Apoptosis

Less evident, but no less important than cell proliferation for body functions, is the process of cell suicide or programmed cell death called apoptosis (Gr. apo, off + ptosis, a falling). Apoptosis is a highly regulated cellular activity that occurs rapidly and produces small membrane-enclosed apoptotic bodies, which quickly undergo phagocytosis by neighboring cells or macrophages specialized for debris removal. Unlike cells undergoing necrosis as a result of accidental injury, apoptotic cells do not rupture and release none of their contents. This difference is highly significant because release of cellular components causes a rapid series of local reactions and immigration of leukocytes in an elaborate reaction called an inflammatory response. Such a response is unwanted when cells are routinely eliminated following DNA damage or as part of a normal development process. These routine cell eliminations therefore occur rapidly and without repercussions by apoptosis.

With their condensed nuclear chromatin, such cells may superficially resemble some mitotic cells. Shown here are apoptotic cells (A) in epithelium of a villus from the lining of the small intestine

In a corpus luteum beginning to undergo involution

Epithelium of a uterine endometrial gland at the onset of menstruation

Late apoptosis—formation of apoptotic bodies

TEM of a cell in late apoptosis shows that during this process the cell's shape changes radically and large cytoplasmic vesicles (blebs) are formed. These detach from the cell and often separate one from another, but remain contained within plasma membrane so that no cytoplasmic contents are released into the extracellular space. The membrane surrounding such apoptotic bodies is changed in such a way that the blebs are recognized by neighboring cells or macrophages and are very rapidly phagocytosed. The rapid formation and engulfment of such blebs without their disruption allow apoptosis to occur without eliciting an inflammatory reaction.

Thank you for attention!