Apoptosis

Cell proliferation for renewal and growth is a process of self-evident physiological significance. Less evident, but no less important for body functions and health, is the process of programmed cell death called apoptosis. A few examples of apoptosis will illustrate its significance.

Most T lymphocytes originating in the thymus have the ability to attack and destroy body components and would cause serious damage if they entered the blood circulation. Inside the thymus, these T lymphocytes receive signals that activate the apoptotic program encoded in their chromosomes. They are destroyed by apoptosis before leaving the thymus (see Chapter 14: Lymphoid Organs).

Medical Application

Most cells of the body can activate their apoptotic program when major changes occur in their DNA for example, just before a tumor appears, when a number of mutations have already accumulated in the DNA. In this way, apoptosis prevents the proliferation of malignant cells that develop as a result of accumulated mutations in the DNA. To form a clone and develop into a tumor, the malignant cell needs to deactivate the genes that control the apoptotic process.

Apoptosis was first discovered in developing embryos, where programmed cell death is an essential process for shaping the embryo (morphogenesis). Later investigators observed that apoptosis is also a common event in the tissues of normal adults.

In apoptosis, the cell and its nucleus become compact, decreasing in size. At this stage the apoptotic cell shows a dark-stained nucleus (pyknotic nucleus), easily identified with the light microscope (Figure 3-24). Next, the chromatin is cut into pieces by DNA endonucleases. During apoptosis the cell shows cytoplasmic large vesicles (blebs) that detach from the cell surface (Figure 3-25). These detached fragments are contained within the plasma membrane, which is changed in such a way that all cell remnants are readily engulfed, or phagocytosed, mainly by macrophages. However, in macrophages the apoptotic fragments do not elicit the synthesis of the molecules that triggers the inflammatory process (see below).

Figure 3-24

Section of a mammary gland from an animal whose lactation was interrupted for 5 days. Note atrophy of the epithelial cells and dilation of the alveolar lumen, which contains several detached cells in the process of apoptosis, as seen from the nuclear alterations. PT stain. Medium magnification.

Figure 3-25

Electron micrograph of a cell in apoptosis showing that its cytoplasm is undergoing a process of fragmentation in blebs that preserve their plasma membranes. These blebs are phagocytosed by macrophages without eliciting an inflammatory reaction. No cytoplasmic substances are released into the extracellular space.

Medical Application The accidental death of cells, a pathological process, is called necrosis. Necrosis can be caused by microorganisms, viruses, chemicals, and other harmful agents. Necrotic cells swell; their organelles increase in volume; and finally they burst, releasing their contents into the extracellular space. Macrophages engulf the debris of necrotic cells by phagocytosis and then secrete molecules that activate other immunodefensive cells to promote inflammation.

References Cooper GM: The Cell: A Molecular Approach. ASM Press/Sinauer Associates, Inc., 1997. Doye V, Hurt E: From nucleoporins to nuclear pore complexes. Curr Opin Cell Biol 1997;9:401. [PMID: 9159086] Duke RC et al: Cell suicide in health and disease. Sci Am 1996;275(6):48. Fawcett D: The Cell, 2nd ed. Saunders, 1981. Goodman SR: Medical Cell Biology. Lippincott, 1994. Jordan EG, Cullis CA (editors): The Nucleolus. Cambridge University Press, 1982. Kornberg RD, Klug A: The nucleosome. Sci Am 1981;244:52. [PMID: 7209486] Krstíc RV: Ultrastructure of the Mammalian Cell. Springer-Verlag, 1979. Lloyd D et al: The Cell Division Cycle. Academic Press, 1982. Mélèse T, Xue Z: The nucleolus: an organelle formed by the act of building a ribosome. Curr Opin Cell Biol 1995;7:319. [PMID: 15900607] Trent RJ: Molecular Medicine. An Introductory Text for Students. Churchill Livingstone, 1993. Watson JD et al: Recombinant DNA, 2nd ed. Scientific American Books, 1992.

The Cell Cycle

Mitosis is the visible manifestation of cell division, but other processes, not so easily observed with the light microscope, play a fundamental role in cell multiplication. Principal among these is the phase in which DNA replicates. This process can be analyzed by introducing labeled radioactive DNA precursors (eg, [³H]thymidine) into the cell and tracing them by means of biochemical and autoradiographic methods. DNA replication has been shown to occur during interphase, when no visible phenomena of cell division can be seen with the microscope. This alternation between mitosis and interphase, known as the cell cycle, occurs in all tissues with cell turnover. A careful study of the cell cycle reveals that it can be divided into two stages: mitosis, consisting of the four phases already described (prophase, metaphase, anaphase, and telophase), and interphase (Figures 3-20 and 3-21).

Figure 3-20

Phases of the cell cycle in bone tissue. The G₁ phase (presynthesis) varies in duration, which depends on many factors, including the rate of cell division in the tissue. In bone tissue, G₁ lasts 25 h. The S phase (DNA synthesis) lasts about 8 h. The G₂-plus-mitosis phase lasts 2.5-3 h. (The times indicated are courtesy of RW Young.)

Figure 3-21

The four phases of the cell cycle. In G₁ the cell either continues the cycle or enters a quiescent phase called G₀. From this phase, most cells can return to the cycle, but some stay in G₀ for a long time or even for their entire lifetime. The checking or restriction point (R) in G₁ stops the cycle under conditions unfavorable to the cell. When the cell passes this restriction point, it continues the cycle through the synthetic phase (S) and the G₂ phase, originating two daughter cells in mitosis (M) except when interrupted by another restriction point (not shown) in G₂.

Interphase is itself divided into three phases: G₁ (presynthesis), S (DNA synthesis), and G₂ (post-DNA duplication). The sequence of these phases and the approximate times involved are illustrated in Figures 3-20 and 3-21. The S phase is characterized by the synthesis of DNA and the beginning of the duplications of the centrosomes with their centrioles. During the G₁ phase, there is an intense synthesis of RNA and proteins, including proteins that control the cell cycle, and the cell volume, previously reduced to one-half by mitosis, is restored to its normal size. In cells that are not continuously dividing, the activities of the cell cycle may be temporarily or permanently suspended. Cells in such a state (eg, muscle, nerve) are referred to as being in the G₀ phase.

Regulation of the mammalian cell cycle is complex. It is known that cultured cells deprived of serum stop proliferating and arrest in G₀. The essential components provided by serum are highly specific proteins called growth factors, which are required only in very low concentrations.

Medical Application

Some growth factors are being used in medicine. One example is erythropoietin, which stimulates proliferation, differentiation, and survival of red blood cell precursors in the bone marrow.

The cell cycle is also regulated by a variety of signals that inhibit progression through the cycle. DNA damage arrests the cell cycle not only in G₂ but also at a checkpoint in G₁ (Figure 3-21). G₁ arrest may permit the damage to be repaired before the cell enters S phase, where the damaged DNA would be replicated. In mammalian cells, arrest at the G1 checkpoint is mediated by the action of a protein known as p53. The gene encoding p53 is often mutated in human cancers, thus reducing the cell's ability to repair damaged DNA. Inheritance of damaged DNA by daughter cells results in an increased frequency of mutations and general instability of the genome, which may contribute to the development of cancer.

Processes that occur during the G₂ phase include the accumulation of energy to be used during mitosis, the synthesis of tubulin to be assembled in mitotic microtubules, and the synthesis of chromosomal nonhistone proteins. In G₂ there is also a checkpoint at which the cell remains until all DNA synthesized with defects is corrected. In G₂ there is an accumulation of the protein complex maturation promoting factor (MPF) that induces the beginning of mitosis, the condensation of the chromosomes, the rupture of the nuclear envelope, and other events related to mitosis.

Medical Application

Rapidly growing tissues (eg, intestinal epithelium) frequently contain cells in mitosis, whereas slowly growing tissues do not. The increased number of mitotic figures and abnormal mitoses in tumors is an important characteristic that distinguishes malignant from benign tumors. The organism has elaborate regulatory systems that control cell reproduction by either stimulating or inhibiting mitosis. Normal cell proliferation and differentiation are controlled by a group of genes called protooncogenes; altering the structure or expression of these genes promotes the production of tumors. Altered protooncogenes are present in tumor-producing viruses and are probably derived from cells. Altered oncogene activity can be induced by a change in the DNA sequence (mutation), an increase in the number of genes (gene amplification), or gene rearrangement, in which genes are relocated near an active promoter site. Altered oncogenes have been associated with several tumors and hematological neoplasia. Proteins that stimulate mitotic activity in various cell types include nerve growth factor, epithelial growth factor, fibroblast growth factor, and precursors of erythrocyte growth factor (erythropoietin); there is an extensive and rapidly growing list of these proteins (see Chapter 13: Hematopoiesis).

Cell proliferation is usually regulated by precise mechanisms that can, when necessary, stimulate or retard mitosis according to the needs of the organism. Several factors (eg, chemical substances, certain types of radiation, viral infections) can induce DNA damage, mutation, and abnormal cell proliferation that bypass normal regulatory mechanisms for controlled growth and result in the formation of tumors.

The term tumor, initially used to denote any localized swelling in the body caused by inflammation or abnormal cell proliferation, is now usually used as a synonym for neoplasm (Gr. neos, new, + plasma, thing formed). Neoplasm can be defined as an abnormal mass of tissue formed by uncoordinated cell proliferation. Neoplasms are either benign or malignant according to their characteristics of slow growth and no invasiveness (benign) or rapid growth and great capacity to invade other tissues and organs (malignant). Cancer is the common term for all malignant tumors (Figures 3-22 and 3-23).

Figure 3-22

Section of a malignant epithelial skin tumor (squamous cell carcinoma). An increase in the number of cells in mitosis and diversity of nuclear morphology are signs of malignancy. PT stain. Medium magnification.

Figure 3-23

Section of a fast-growing malignant epithelial skin tumor showing an increased number of cells in mitosis and great diversity of nuclear morphology. H&E stain. Medium magnification.

References

Cooper GM: The Cell: A Molecular Approach. ASM Press/Sinauer Associates, Inc., 1997.

Doye V, Hurt E: From nucleoporins to nuclear pore complexes. Curr Opin Cell Biol 1997;9:401. [PMID: 9159086]

Duke RC et al: Cell suicide in health and disease. Sci Am 1996;275(6):48.

Fawcett D: The Cell, 2nd ed. Saunders, 1981.

Goodman SR: Medical Cell Biology. Lippincott, 1994.

Jordan EG, Cullis CA (editors): The Nucleolus. Cambridge University Press, 1982.

Kornberg RD, Klug A: The nucleosome. Sci Am 1981;244:52. [PMID: 7209486]

Krstíc RV: Ultrastructure of the Mammalian Cell. Springer-Verlag, 1979.

Lloyd D et al: The Cell Division Cycle. Academic Press, 1982.

Mélèse T, Xue Z: The nucleolus: an organelle formed by the act of building a ribosome. Curr Opin Cell Biol 1995;7:319. [PMID: 15900607]

Trent RJ: Molecular Medicine. An Introductory Text for Students. Churchill Livingstone, 1993.

Watson JD et al: Recombinant DNA, 2nd ed. Scientific American Books, 1992.

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 phase between two mitoses is called interphase, during which the nucleus appears as it is normally observed in microscopic preparations. The process of mitosis is subdivided into phases to facilitate its study (Figures 3-15, 3-16, and 3-17).

Figure 3-15

Phases of mitosis.

Figure 3-16

Photomicrograph of cultured cells to show cell division. Picrosirius–hematoxylin stain. Medium magnification. A: Interphase nuclei. Note the chromatin and nucleoli inside each nucleus. B: Prophase. No distinct nuclear envelope, no nucleoli. Condensed chromosomes. C: Metaphase. The chromosomes are located in a plate at the cell equator. D: Late anaphase. The chromosomes are located in both cell poles, to distribute the DNA equally between the daughter cells.

Figure 3-17

Images obtained with a confocal laser scanning microscope from cultured cells. An interphase nucleus and several nuclei are in several phases of mitosis. DNA appears red, and microtubules in the cytoplasm are blue. Medium magnification. A: Interphase. A nondividing cell. B: Prophase. The blue structure over the nucleus is the centrosome. Note that the chromosomes are becoming visible because of their condensation. The cytoplasm is acquiring a round shape typical of cells in mitosis. C: Metaphase. The chromosomes are organized in an equatorial plane. D: Anaphase. The chromosomes are pulled to the cell poles through the activity of microtubules. E: Early telophase. The two sets of chromosomes have arrived at the cell poles to originate the two daughter cells, which will contain sets of chromosomes similar to those in the mother cell. F: Telophase. The cytoplasm is being divided by a constriction in the cell equator. Note that the daughter cells are round and smaller than the mother cell. Soon they will increase in size and become elongated. (Courtesy of R Manelli-Oliveira, R Cabado, and G Machado-Santelli.)

The prophase of mitosis is characterized by the gradual coiling of nuclear chromatin (uncoiled chromosomes), giving rise to several individual rod- or hairpin-shaped bodies (coiled chromosomes) that stain intensely. At the end of prophase, the nuclear envelope is broken by phosphorylation (addition of PO₄^3-) of the nuclear lamina proteins, originating vesicles that remain in the cytoplasm. The centrosomes with their centrioles separate, and a centrosome migrates to each pole of the cell. The duplication of the centrosomes and centrioles starts in the interphase, before mitosis. Simultaneously with centrosome migration, the microtubules of the mitotic spindle appear between the two centrosomes, and the nucleolus disintegrates.

During metaphase, chromosomes, due to the activity of microtubules, migrate to the equatorial plane of the cell, where each divides longitudinally to form two chromosomes called sister chromatids. The chromatids attach to the microtubules of the mitotic spindle (Figures 3-18 and 3-19) at an electron-dense, DNA protein plaque, the kinetochore (Gr. kinetos, moving, + chora, central region), located close to the centromere (Gr. kentron, center, + meros, part) of each chromatid.

Figure 3-18

Electron micrograph of a section of a rooster spermatocyte in metaphase. The figure shows the two centrioles in each pole, the mitotic spindle formed by microtubules, and the chromosomes in the equatorial plane. The arrows show the insertion of microtubules in the centromeres. Reduced from x19,000. (Courtesy of R McIntosh.)

Figure 3-19

Electron micrograph of the metaphase of a human lung cell in tissue culture. Note the insertion of microtubules in the centromeres (arrows) of the densely stained chromosomes. Reduced from x50,000. (Courtesy of R McIntosh.)

In anaphase, the sister chromatids separate from each other and migrate toward the opposite poles of the cell, pulled by microtubules. Throughout this process, the centromeres move away from the center, pulling the remainder of the chromosome along. The centromere is the constricted region of a mitotic chromosome that holds the two sister chromatids together until the beginning of anaphase.

Telophase is characterized by the reappearance of nuclei in the daughter cells. The chromosomes revert to their semidispersed state, and the nucleoli, chromatin, and nuclear envelope reappear. While these nuclear alterations are taking place, a constriction develops at the equatorial plane of the parent cell and progresses until the cytoplasm and its organelles are divided in two. This constriction is produced by microfilaments of actin associated with myosin that accumulate in a beltlike shape beneath the cell membrane.

Most tissues undergo constant cell turnover because of continuous cell division and the ongoing death of cells. Nerve tissue and cardiac muscle cells are exceptions, since they do not multiply postnatally and therefore cannot regenerate. The turnover rate of cells varies greatly from one tissue to another rapid in the epithelium of the digestive tract and the epidermis and slow in the pancreas and the thyroid gland.

References

Cooper GM: The Cell: A Molecular Approach. ASM Press/Sinauer Associates, Inc., 1997.

Doye V, Hurt E: From nucleoporins to nuclear pore complexes. Curr Opin Cell Biol 1997;9:401. [PMID: 9159086]

Duke RC et al: Cell suicide in health and disease. Sci Am 1996;275(6):48.

Fawcett D: The Cell, 2nd ed. Saunders, 1981.

Goodman SR: Medical Cell Biology. Lippincott, 1994.

Jordan EG, Cullis CA (editors): The Nucleolus. Cambridge University Press, 1982.

Kornberg RD, Klug A: The nucleosome. Sci Am 1981;244:52. [PMID: 7209486]

Krstíc RV: Ultrastructure of the Mammalian Cell. Springer-Verlag, 1979.

Lloyd D et al: The Cell Division Cycle. Academic Press, 1982.

Mélèse T, Xue Z: The nucleolus: an organelle formed by the act of building a ribosome. Curr Opin Cell Biol 1995;7:319. [PMID: 15900607]

Trent RJ: Molecular Medicine. An Introductory Text for Students. Churchill Livingstone, 1993.

Watson JD et al: Recombinant DNA, 2nd ed. Scientific American Books, 1992.

Nucleolus

The nucleolus is a spherical structure (Figure 3-13) that is rich in rRNA and protein. It is usually basophilic when stained with hematoxylin and eosin. As seen with the electron microscope, the nucleolus consists of three distinct components: (1) From one to several pale-staining regions are composed of nucleolar organizer DNA sequences of bases that code for rRNA (Figure 3-14). In the human genome, five pairs of chromosomes contain nucleolar organizers. (2) Closely associated with the nucleolar organizers are densely packed 5- to 10-nm ribonucleoprotein fibers that comprise the pars fibrosa, which consists of primary transcripts of rRNA genes. (3) The pars granulosa consists of 15- to 20-nm granules (maturing ribosomes; see Figure 3-14). Proteins, synthesized in the cytoplasm, become associated with rRNAs in the nucleolus; ribosome subunits then migrate into the cytoplasm. Heterochromatin is often attached to the nucleolus (nucleolus-associated chromatin), but the functional significance of the association is not known. The rRNAs are synthesized and modified inside the nucleus. In the nucleolus they receive proteins and are organized into small and large ribosomal subunits, which migrate to the cytoplasm through the nuclear pores.

Figure 3-13

Photomicrograph of two primary oocytes, each with its pale cytoplasm and round, dark-stained nucleus. In each nucleus the nucleolus, very darkly stained, is clearly seen. The sectioned chromosomes are also seen, because they are condensed. These cells stopped at the first meiotic division. Meiosis will proceed just before ovulation (extrusion of the oocyte from the ovary; see Chapter 22: The Female Reproductive System).

Figure 3-14

Electron micrograph of a nucleolus. The nucleolar organizer DNA (NO), pars fibrosa (PF), pars granulosa (PG), nucleolus-associated chromatin (NAC), nuclear envelope (NE), and cytoplasm (C) are shown.

Medical Application

Large nucleoli are encountered in embryonic cells during their proliferation, in cells that are actively synthesizing proteins, and in rapidly growing malignant tumors. The nucleolus disperses during the prophase of cell division but reappears in the telophase stage of mitosis.

Nuclear Matrix

The nuclear matrix is the component that fills the space between the chromatin and the nucleoli in the nucleus. It is composed mainly of proteins (some of which have enzymatic activity), metabolites, and ions. When its nucleic acids and other soluble components are removed, a continuous fibrillar structure remains, forming the nucleoskeleton. The fibrous lamina of the nuclear envelope is part of the nuclear matrix. The nucleoskeleton probably contributes to the formation of a protein base to which DNA loops are bound.

References

Cooper GM: The Cell: A Molecular Approach. ASM Press/Sinauer Associates, Inc., 1997.

Doye V, Hurt E: From nucleoporins to nuclear pore complexes. Curr Opin Cell Biol 1997;9:401. [PMID: 9159086]

Duke RC et al: Cell suicide in health and disease. Sci Am 1996;275(6):48.

Fawcett D: The Cell, 2nd ed. Saunders, 1981.

Goodman SR: Medical Cell Biology. Lippincott, 1994.

Jordan EG, Cullis CA (editors): The Nucleolus. Cambridge University Press, 1982.

Kornberg RD, Klug A: The nucleosome. Sci Am 1981;244:52. [PMID: 7209486]

Krstíc RV: Ultrastructure of the Mammalian Cell. Springer-Verlag, 1979.

Lloyd D et al: The Cell Division Cycle. Academic Press, 1982.

Mélèse T, Xue Z: The nucleolus: an organelle formed by the act of building a ribosome. Curr Opin Cell Biol 1995;7:319. [PMID: 15900607]

Trent RJ: Molecular Medicine. An Introductory Text for Students. Churchill Livingstone, 1993.

Watson JD et al: Recombinant DNA, 2nd ed. Scientific American Books, 1992.

Chromatin

Chromatin, in nondividing nuclei, is in fact the chromosomes in a different degree of uncoiling. According to the degree of chromosome condensation, two types of chromatin can be distinguished with both the light and electron microscopes (Figures 3-2 and 3-4, in previous post). Heterochromatin (Gr. heteros, other, + chroma, color), which is electron dense, appears as coarse granules in the electron microscope and as basophilic clumps in the light microscope. Euchromatin is the less coiled portion of the chromosomes, visible as a finely dispersed granular material in the electron microscope and as lightly stained basophilic areas in the light microscope. The proportion of heterochromatin to euchromatin accounts for the light-to-dark appearance of nuclei in tissue sections as seen in light and electron microscopes. The intensity of nuclear staining of the chromatin is frequently used to distinguish and identify different tissues and cell types in the light microscope.

Chromatin is composed mainly of coiled strands of DNA bound to basic proteins (histones); its structure is schematically presented in Figure 3-5 (in previous post). The basic structural unit of chromatin is the nucleosome (Figure 3-9), which consists of a core of four types of histones: two copies each of histones H2A, H2B, H3, and H4, around which are wrapped 166 DNA base pairs. An additional 48-base pair segment forms a link between adjacent nucleosomes, and another type of histone (H1 or H5) is bound to this DNA. This organization of chromatin has been referred to as "beads-on-a-string." Nonhistone proteins are also associated with chromatin, but their arrangement is less well understood.

Figure 3-9

Schematic representation of a nucleosome. This structure consists of a core of four types of histones (two copies of each) H2A, H2B, H3, and H4 and one molecule of H1 or H5 located outside the DNA filament.

The next higher order of organization of chromatin is the 30-nm fiber (Figure 3-10). In this structure, nucleosomes become coiled around an axis, with six nucleosomes per turn, to form the 30-nm chromatin fiber. There are higher orders of coiling, especially in the condensation of chromatin during mitosis and meiosis.

Figure 3-10

The orders of chromatin packing believed to exist in the metaphase chromosome. Starting at the top, the 2-nm DNA double helix is shown; next is the association of DNA with histones to form filaments of nucleosomes of 11 nm and 30 nm. Through further condensation, filaments with diameters of 300 nm and 700 nm are formed. Finally, the bottom drawing shows a metaphase chromosome, which exhibits the maximum packing of DNA.

The chromatin pattern of a nucleus has been considered a guide to the cell's activity. In general, cells with light nuclei are more active than those with condensed, dark nuclei. In light-stained nuclei (with few heterochromatin clumps), more DNA surface is available for the transcription of genetic information. In dark-stained nuclei (rich in heterochromatin), the coiling of DNA makes less surface available.

Careful study of the chromatin of mammalian cell nuclei reveals a heterochromatin mass that is frequently observed in female cells but not in male cells. This chromatin clump is the sex chromatin and is one of the two X chromosomes present in female cells. The X chromosome that constitutes the sex chromatin remains tightly coiled and visible, whereas the other X chromosome is uncoiled and not visible. Evidence suggests that the sex chromatin is genetically inactive. The male has one X chromosome and one Y chromosome as sex determinants; the X chromosome is uncoiled, and therefore no sex chromatin is visible. In human epithelial cells, sex chromatin appears as a small granule attached to the nuclear envelope. The cells lining the internal surface of the cheek are frequently used to study sex chromatin. Blood smears are also often used, in which case the sex chromatin appears as a drumsticklike appendage to the nuclei of the neutrophilic leukocytes (Figure 3-11).

Figure 3-11

Morphological features of sex chromatin in human female oral (buccal) epithelium and in a polymorphonuclear leukocyte. In the epithelium, sex chromatin appears as a small, dense granule adhering to the nuclear envelope. In the leukocyte, it has a drumstick shape.

Medical Application

The study of sex chromatin discloses the genetic sex in patients whose external sex organs do not permit assignment of gender, as in hermaphroditism and pseudohermaphroditism. Sex chromatin helps the study of other anomalies involving the sex chromosomes—eg, Klinefelter syndrome, in which testicular abnormalities, azoospermia (absence of spermatozoa), and other symptoms are associated with the presence of XXY chromosomes.

The study of chromosomes progressed considerably after the development of methods that induce cells to divide, arrest mitotic cells during metaphase, and cause cell rupture. Mitosis can be induced by phytohemagglutinin (in cell cultures) and can be arrested in metaphase by colchicine. Cells are immersed in a hypotonic solution, which causes swelling, after which cells are flattened and broken between a glass slide and a coverslip.

The pattern of chromosomes obtained in a human cell after staining is illustrated in Figure 3-12. In addition to the X and Y sex chromosomes, the remaining chromosomes are customarily grouped according to their size and morphological characteristics into 22 successively numbered pairs.

Figure 3-12

Human karyotype preparation made by means of a banding technique. Each chromosome has a particular pattern of banding that facilitates its identification and also the relationship of the banding pattern to genetic anomalies. The chromosomes are grouped in numbered pairs according to their morphological characteristics.

Medical Application

The number and characteristics of chromosomes encountered in an individual are known as the karyotype (Figure 3-12). The study of karyotypes has revealed chromosomal alterations associated with tumors, leukemias, and several types of genetic diseases.

The development of techniques that reveal segmentation of chromosomes in transverse, differentially stained bands permitted a more precise identification of individual chromosomes and the study of gene deletions and translocations. These techniques are based mainly on the study of chromosomes previously treated with saline or enzyme solution and stained with fluorescent dyes or Giemsa's blood-staining technique. In situ hybridization is also a valuable technique for localizing DNA sequences (genes) in chromosomes.

References

Cooper GM: The Cell: A Molecular Approach. ASM Press/Sinauer Associates, Inc., 1997.

Doye V, Hurt E: From nucleoporins to nuclear pore complexes. Curr Opin Cell Biol 1997;9:401. [PMID: 9159086]

Duke RC et al: Cell suicide in health and disease. Sci Am 1996;275(6):48.

Fawcett D: The Cell, 2nd ed. Saunders, 1981.

Goodman SR: Medical Cell Biology. Lippincott, 1994.

Jordan EG, Cullis CA (editors): The Nucleolus. Cambridge University Press, 1982.

Kornberg RD, Klug A: The nucleosome. Sci Am 1981;244:52. [PMID: 7209486]

Krstíc RV: Ultrastructure of the Mammalian Cell. Springer-Verlag, 1979.

Lloyd D et al: The Cell Division Cycle. Academic Press, 1982.

Mélèse T, Xue Z: The nucleolus: an organelle formed by the act of building a ribosome. Curr Opin Cell Biol 1995;7:319. [PMID: 15900607]

Trent RJ: Molecular Medicine. An Introductory Text for Students. Churchill Livingstone, 1993.

Watson JD et al: Recombinant DNA, 2nd ed. Scientific American Books, 1992.

Nuclear Envelope

Electron microscopy shows that the nucleus is surrounded by two parallel membranes separated by a narrow space (40-70 nm) called the perinuclear cisterna (Figures 3-2 and 3-4). Together, the paired membranes and the intervening space make up the nuclear envelope. Closely associated with the internal membrane of the nuclear envelope is a protein structure called the fibrous lamina (Figure 3-4), which helps to stabilize the nuclear envelope. The fibrous lamina is composed of three main proteins called lamins A, B, and C. In nondividing cells, chromosomes are associated with the fibrous lamina (Figure 3-5). The pattern of association is regular from cell to cell within a tissue, supporting the conclusion that chromosomes have a definite localization within the nucleus. Polyribosomes are attached to the outer membrane, showing that the nuclear envelope is a part of the endoplasmic reticulum. Proteins synthesized in the polyribosomes attached to the nuclear envelope are temporarily segregated in the perinuclear cisterna. At sites at which the inner and outer membranes of the nuclear envelope fuse, there are gaps, the nuclear pores (Figures 3-6 and 3-7), that provide controlled pathways between the nucleus and the cytoplasm. The pores are not open but show an octagonal pore complex made of more than 100 proteins (Figure 3–8). Because the nuclear envelope is impermeable to ions and molecules of all sizes, the exchange of substances between the nucleus and the cytoplasm is made only through the nuclear pores. Ions and molecules with a diameter up to 9 nm pass freely through the nuclear pore without consuming energy. But molecules and molecular complexes larger than 9 nm are transported by an active process, mediated by receptors, which uses energy from adenosine triphosphate (ATP) and takes place in two stages. First, proteins with one or several nuclear signal locations become attached to specific cytosolic proteins, originating a complex, which is temporarily attached to the nuclear pore complex without using energy. In the second stage, proteins with nuclear signal locations are transferred to the nucleus, using energy from ATP, and the cytosolic protein remains in the cytoplasm. At least part of the ATP energy may be utilized to open the nuclear pore complex to make the passage of large molecules possible. Less is known about the transfer of molecules and molecular complexes, some as large as ribosome subunits, from the nucleus to the cytoplasm.

Figure 3-4

Electron micrograph of a nucleus, showing the heterochromatin (HC) and euchromatin (EC). Unlabeled arrows indicate the nucleolus-associated chromatin around the nucleolus (NU). Arrowheads indicate the perinuclear cisterna. Underneath the cisterna is a layer of heterochromatin, the main component of the so-called nuclear membrane seen under the light microscope. x26,000.

Figure 3-5

Illustration showing the structure, the localization, and the relationship of the nuclear lamina with chromosomes. The drawing also shows that the nuclear pore complex is composed of two protein rings in an octagonal organization. From the cytoplasmic ring, long filaments penetrate the cytosol, and from the intranuclear ring arise filaments that constitute a basketlike structure. The presence of the central cylindrical granule in the nuclear pore is not universally accepted.

Figure 3-6

Electron micrographs of nuclei showing their envelopes composed of two membranes and the nuclear pores (arrows). A, B: Transverse sections; C: A tangential section. Chromatin, frequently condensed below the nuclear envelope, is not usually seen in the pore regions. x80,000.

Figure 3-7

Electron micrograph obtained by cryofracture of a rat intestine cell, showing the two components of the nuclear envelope and the nuclear pores. (Courtesy of P Pinto da Silva.)

Figure 3-8

Simplified representation of two nuclear pore complexes. In this model, the final nuclear portion is seen to be a more continuous structure, in the shape of a ring.

References Cooper GM: The Cell: A Molecular Approach. ASM Press/Sinauer Associates, Inc., 1997. Doye V, Hurt E: From nucleoporins to nuclear pore complexes. Curr Opin Cell Biol 1997;9:401. [PMID: 9159086] Duke RC et al: Cell suicide in health and disease. Sci Am 1996;275(6):48. Fawcett D: The Cell, 2nd ed. Saunders, 1981. Goodman SR: Medical Cell Biology. Lippincott, 1994. Jordan EG, Cullis CA (editors): The Nucleolus. Cambridge University Press, 1982. Kornberg RD, Klug A: The nucleosome. Sci Am 1981;244:52. [PMID: 7209486] Krstíc RV: Ultrastructure of the Mammalian Cell. Springer-Verlag, 1979. Lloyd D et al: The Cell Division Cycle. Academic Press, 1982. Mélèse T, Xue Z: The nucleolus: an organelle formed by the act of building a ribosome. Curr Opin Cell Biol 1995;7:319. [PMID: 15900607] Trent RJ: Molecular Medicine. An Introductory Text for Students. Churchill Livingstone, 1993. Watson JD et al: Recombinant DNA, 2nd ed. Scientific American Books, 1992.

Introduction of The Cell Nucleus

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 the three types of RNA : ribosomal (rRNA), messenger (mRNA), and transfer (tRNA). Mitochondria have a small DNA genome and produce RNAs to be used in this organelle, but the genome is so small that it is not sufficient even for the mitochondrion itself. On the other hand, the nucleus does not produce proteins; the numerous protein molecules needed for the activities of the nucleus are imported from the cytoplasm.

The nucleus frequently appears as a rounded or elongated structure, usually in the center of the cell (Figure 3-1). Its main components are the nuclear envelope, chromatin (Figures 3-2 and 3-3), nucleolus, and nuclear matrix. The size and morphological features of nuclei in a specific normal tissue tend to be uniform. In contrast, the nuclei in cancer cells have an irregular shape, variable size, and atypical chromatin patterns.

Figure 3-1

Liver cells (hepatocytes). Several dark-stained nuclei are shown. Note the apparent nuclear membrane consisting mainly of a superficial condensation of chromatin. Several nucleoli are seen inside the nuclei, suggesting intense protein synthesis. One hepatocyte contains two nuclei. Pararosaniline–toluidine blue (PT) stain. Medium magnification.

Figure 3-2

Schematic representation of a cell nucleus. The nuclear envelope is composed of two membranes of the endoplasmic reticulum, enclosing a perinuclear cisterna. Where the two membranes fuse, they form nuclear pores. Ribosomes are attached to the outer nuclear membrane. Heterochromatin clumps are associated with the nuclear lamina, whereas the euchromatin (EC) appears dispersed in the interior of the nucleus. In the nucleolus, note the associated chromatin, heterochromatin (Hc), the pars granulosa (G), and the pars fibrosa (F).

Figure 3-3

 

Three-dimensional representation of a cell nucleus showing the distribution of the nuclear pores, the heterochromatin (dark regions), the euchromatin (light regions), and a nucleolus. Note that there is no chromatin closing the pores. The number of nuclear pores varies greatly from cell to cell.

References Cooper GM: The Cell: A Molecular Approach. ASM Press/Sinauer Associates, Inc., 1997. Doye V, Hurt E: From nucleoporins to nuclear pore complexes. Curr Opin Cell Biol 1997;9:401. [PMID: 9159086] Duke RC et al: Cell suicide in health and disease. Sci Am 1996;275(6):48. Fawcett D: The Cell, 2nd ed. Saunders, 1981. Goodman SR: Medical Cell Biology. Lippincott, 1994. Jordan EG, Cullis CA (editors): The Nucleolus. Cambridge University Press, 1982. Kornberg RD, Klug A: The nucleosome. Sci Am 1981;244:52. [PMID: 7209486] Krstíc RV: Ultrastructure of the Mammalian Cell. Springer-Verlag, 1979. Lloyd D et al: The Cell Division Cycle. Academic Press, 1982. Mélèse T, Xue Z: The nucleolus: an organelle formed by the act of building a ribosome. Curr Opin Cell Biol 1995;7:319. [PMID: 15900607] Trent RJ: Molecular Medicine. An Introductory Text for Students. Churchill Livingstone, 1993. Watson JD et al: Recombinant DNA, 2nd ed. Scientific American Books, 1992.