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//CELL DIVISION AND THE CELL CYCLE

CELL DIVISION AND THE CELL CYCLE

During prenatal development, most cells undergo repeated division as the body grows in size and complexity. As cells mature, they differentiate structurally and functionally. Some cells, such as neurones, lose the ability to divide. Others may persist throughout the lifetime of the individual as replication-competent stem cells, e.g. cells in the haemopoietic tissue of bone marrow. Many stem cells divide infrequently, but give rise to daughter cells that undergo repeated cycles of mitotic division as transit (or transient) amplifying cells. Their divisions may occur in rapid succession, as in cell lineages with a short lifespan and similarly fast turnover and replacement time. Transit amplifying cells are all destined to differentiate and ultimately to die and be replaced, unlike the population of parental stem cells, which self-renews.

Patterns and rates of cell division within tissues vary considerably. In many epithelia, such as the crypts between intestinal villi, the replacement of damaged or effete cells by division of stem cells can be rapid. Rates of cell division may also vary according to demand, as occurs in the healing of wounded skin, in which cell proliferation increases to a peak and then returns to the normal replacement level (see Ch. 7). The rate of cell division is tightly coupled to the demand for growth and replacement. Where this coupling is faulty, tissues either fail to grow or replace their cells, or they can overgrow, producing neoplasms.

The cell cycle is the period of time between the birth of a cell and its own division to produce two daughter cells. It generally lasts a minimum of 12 hours, but in most adult tissues can be considerably longer, and is divided into four distinct phases, which are known as G1, S, G2 and M. The combination of G1, S and G2 phases is known as interphase. M is the mitotic phase. G1 is the period when cells respond to growth factors directing the cell to initiate another cycle; once made, this decision is irreversible. It is also the phase in which most of the molecular machinery required to complete another cell cycle is generated. Cells that retain the capacity for proliferation, but which are no longer dividing, have entered a phase called G0 and are described as quiescent even though they may be quite active physiologically. Growth factors can stimulate quiescent cells to leave G0 and re-enter the cell cycle, whereas the proteins encoded by certain tumour suppressor genes (e.g. the gene mutated in retinoblastoma, Rb) block the cycle in G1. DNA synthesis (replication of the genome) occurs during S phase, at the end of which the DNA content of the cell has doubled. During G2, the cell prepares for division; this period ends with the onset of chromosome condensation and breakdown of the nuclear membrane. The times taken for S, G2 and M are similar for most cell types, and occupy 6–8, 2–4 and 1–2 hours respectively. In contrast, the duration of G1 shows considerable variation, sometimes ranging from less than 2 hours in rapidly dividing cells, to more than 100 hours within the same tissue.

Cell cycle progression is driven in part by changes in the activity of cyclin-dependent protein kinases, CDKs (protein kinases which are activated by binding of a cyclin subunit). Each cell cycle stage is characterized by the activity of one or more CDK-cyclin pairs. Transitions between cell cycle stages are triggered by highly specific proteolysis of the cyclins and other key components. The targets for proteolysis are marked for destruction by E3 ubiquitin ligases, which decorate them with polymers of the small protein ubiquitin, a sign for recognition by the proteasome. To give one example, the transition from G2 to mitosis is driven by activation of CDK1 by its partners the A- and B-type cyclins: the characteristic changes in cellular structure that occur as cells enter mitosis are largely driven by phosphorylation of proteins by active CDK1-cyclin A and CDK1-cyclin B (further details are beyond the scope of this book). Cells exit from mitosis when the anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase, marks the cyclins for destruction. The APC/C also triggers for destruction another protein whose function is to protect the cohesion between sister chromatids. An analogous process of protein phosphorylation coupled with targeted destruction drives the transition between G1 and S phase as cells commit to another cycle of proliferation.

There are important checkpoints in the cell cycle at which progress will be arrested if, for instance, DNA replication or mitotic spindle assembly and chromosome attachment are incomplete. Negative regulation systems also operate to delay cell cycle progression when DNA has been damaged by radiation or chemical mutagens. Cells with checkpoint defects, such as loss of the protein p53, which is a major negative control element in the division cycle of all cells, are commonly associated with the development of malignancy. Cells lacking one of the critical checkpoint functions are able to progress through the cycle carrying defects, thus increasing the probability that further abnormalities will accumulate in their progeny. The p53 gene is an example of a tumour suppressor gene. For further reading, see Blow & Tanaka (2005); Pollard & Earnshaw (2007).

MITOSIS AND MEIOSIS

Mitosis is the process that results in the distribution of identical copies of the parent cell genome to the two daughter somatic cells. In meiosis, the divisions immediately before the final production of gametes halve the number of chromosomes to the haploid number, so that at fertilization the diploid number is restored. Moreover, meiosis includes a phase in which exchange of genetic material occurs between homologous chromosomes. This allows a reassortment of genes to take place, which means that the daughter cells differ from the parental cell in both their precise genetic sequence and their haploid state. Mitosis and meiosis are alike in many respects, and differ principally in chromosomal behaviour during the early stages of cell division. In meiosis, two divisions occur in succession, without an intervening S phase. Meiosis I is distinct from mitosis, whereas meiosis II is more like mitosis.

Mitosis

New DNA is synthesized during the S phase of the cell cycle interphase. This means that the amount of DNA in diploid cells has doubled to the tetraploid value by the onset of mitosis, although the chromosome number is still diploid. During mitosis, this amount is halved between the two daughter cells, so that DNA quantity and chromosome number are diploid in both cells. The cellular changes that achieve this distribution are conventionally divided into four phases called prophase, metaphase, anaphase and telophase (Fig. 1.16, Fig. 1.17).

  

Fig. 1.16  The stages in mitosis, including the appearance and distribution of the chromosomes.

  

Fig. 1.17  Immunofluorescence images of stages in mitosis in human carcinoma cells in culture. A, Metaphase, with spindle microtubules (green), the microtubule-stabilizing protein (HURP; red) and chromosomal DNA (blue). B, Anaphase, with spindle microtubules (green), the central spindle (Aurora-B kinase, red) and segregated chromosomes (blue). C, Late anaphase, with spindle microtubules (green), the central spindle (Plk1 kinase, red, appearing yellow where co-localized with microtubule protein) and segregated chromosomes (blue).
(By courtesy of Dr Herman Silljé, Max-Planck-Institute fur Biochemie, Martinsried, Germany.)

Prophase

During prophase, the strands of chromatin, which are highly extended during interphase, shorten, thicken and resolve themselves into recognizable chromosomes. Each chromosome is made up of duplicate chromatids (the products of DNA replication) joined at their centromeres. Outside the nucleus, the two centriole pairs begin to separate, and move towards opposite poles of the cell. Parallel microtubules are assembled between them to create the mitotic spindle, and others radiate to form the asters, which come to form the spindle poles. As prophase proceeds, the nucleoli disappear, and the nuclear membrane suddenly disintegrates to release the chromosomes, an event that marks the end of prophase.

Prometaphase–metaphase

As the nuclear membrane disappears, the spindle microtubules extend into the central region of the cell, attaching to the chromosomes which subsequently move towards the equator of the spindle (prometaphase). The grouping of chromosomes at the spindle equator is called the metaphase or equatorial plate. The chromosomes, attached at their centromeres, appear to be arranged in a ring when viewed from either pole of the cell, or to lie linearly across this plane when viewed from above. Cytoplasmic movements during late metaphase effect the approximately equal distribution of mitochondria and other organelles around the cell periphery.

Anaphase

By the end of metaphase every chromosome consists of a pair of sister chromatids attached to opposing spindle poles by bundles of microtubules associated with the centromeres. The onset of anaphase begins with the proteolytic cleavage of a key subunit of a complex known as cohesin, which holds the replicated sister chromatids together. This cleavage releases the cohesion between sister chromatids, which each then move towards opposite spindle poles as the microtubule bundles attached to the centromeres shorten and move polewards. At the end of anaphase the sister chromatids are grouped at either end of the cell, and both clusters are diploid in number. An infolding of the cell equator begins, and deepens during telophase as the cleavage furrow.

Telophase

During telophase the nuclear membranes reform, beginning with the association of membranous vesicles with the surface of the chromosomes. Later, after the vesicles have fused and the nuclear envelope is complete, the chromosomes decondense and the nucleoli reform. At the same time, cytoplasmic division, which usually begins in early anaphase, continues until the new cells separate, each with its derived nucleus. The spindle remnant now disintegrates. While the cleavage furrow is active, a peripheral band or belt of actin and myosin appears in the constricting zone: contraction of this band is responsible for furrow formation.

Failure of disjunction of chromatids, so that sister chromatids pass to the same pole, may sometimes occur. Of the two new cells, one will have more, and the other fewer, chromosomes than the diploid number. Exposure to ionizing radiation promotes non-disjunction and may, by chromosomal damage, inhibit mitosis altogether. A typical symptom of radiation exposure is the failure of rapidly dividing epithelia to replace lost cells, with consequent ulceration of the skin and mucous membranes. Mitosis can also be disrupted by chemical agents, particularly colchicine, taxol and their derivatives. These compounds either disassemble spindle microtubules or interfere with their dynamics. As a result, mitosis is arrested in metaphase. Taxol and its derivatives are widely used in the treatment of breast cancer. Colchicine is widely used for the treatment of gout, but the mechanism is not known and may have nothing to do with mitotic regulation.

Meiosis

There are two cell divisions during meiosis (Fig. 1.18). Details of this process differ at a cellular level for male and female lineages.

  

Fig. 1.18  The stages in meiosis, depicted by two pairs of maternal and paternal homologues (dark and pale colours). DNA and chromosome complement changes and exchange of genetic information between homologues are indicated.

Meiosis I

Prophase I

Meiotic prophase I is a long and complex phase that differs considerably from mitotic prophase and is customarily divided into five substages, called leptotene, zygotene, pachytene, diplotene and diakinesis (see Pollard & Earnshaw 2007).

Leptotene stage

During leptotene, homologous chromosomes (maternal and paternal copies of the same chromosome), which were replicated in a preceding S phase and each consist of sister chromatids joined at the centromere (see above), locate one another within the nucleus, and the process of genetic recombination is initiated. Cytologically, chromosomes begin to condense, appearing as individual threads that are attached via their telomeres to the nuclear membrane. They often show characteristic beading throughout their length.

Zygotene stage

During zygotene, the homologous chromosomes initiate a process known as synapsis, during which they become intimately associated with one another. Synapsis may begin near the telomeres at the inner surface of the nuclear membrane, and during this stage the telomeres often cluster to one side of the nucleus (a stage known as the bouquet because the chromosomes resemble a bouquet of flowers). The pairs of synapsed homologues, also known as bivalents, are linked together by a highly structured fibrillar band, the synaptonemal complex.

The sex chromosomes also synapse during zygotene. In males, with distinct X and Y chromosomes, synapsis involves a short region of shared DNA sequence known as the pseudoautosomal region. The unpaired sex-specific regions adopt a special hypercondensed structure known as the sex vesicle.

Chromosome behaviour in meiosis is intimately linked with the process of genetic recombination. This begins during leptotene, as homologous chromosomes first locate one another at a distance. Synapsis is thought to represent the completion of recombination, as sites of genetic exchange are turned into specialized structures known as chiasmata. Chiasmata are topological ‘knots’ that hold homologous chromosomes together.

Pachytene stage

When synapsis is complete for all chromosomes, the cell is said to be in pachytene. Each bivalent looks like a single structure, but is actually two pairs of sister chromatids held together by the synaptonemal complex. Genetic recombination is completed at this point, with sites where it has occurred (usually one per chromosome arm) appearing as nodules in the center of the synaptonemal complex.

Diplotene stage

During diplotene, the synaptonemal complex disassembles, and pairs of homologous chromosomes, now much shortened, separate except where crossing over has occurred (chiasmata). At least one chiasma forms between each homologous pair, exchanging maternal and paternal sequences, and up to five have been observed. In the ovaries, primary oocytes become diplotene by the fifth month in utero and each remains at this stage until the period before ovulation (up to 50 years).

Diakinesis

Diakinesis is the prometaphase of the first meiotic division. The chromosomes, still as bivalents, become even shorter and thicker. They gradually attach to the spindle and become aligned at a metaphase plate. In eggs, the spindle forms without centrosomes. Microtubules first nucleate and are stabilized near the chromosomes, and the action of various motor molecules eventually sorts them into a bipolar spindle. Perhaps surprisingly, this spindle is as efficient a machine for chromosome segregation as the spindle of mitotic cells with centrosomes at the poles.

Metaphase I

Metaphase I resembles mitotic metaphase, except that the bodies attaching to the spindle microtubules are bivalents, not single chromosomes. These become arranged so that the homologous pairs occupy the equatorial plane of the spindle. The centromeres of each pair of sister chromatids function as a single unit, facing a single spindle pole. Homologous chromosomes are pulled towards opposite spindle poles, but are held paired at the spindle midzone by chiasmata. Thus, recombination is essential for the mechanics of the meiosis I division – without it the chromosomes cannot align properly, and errors in chromosome segregation (known as non-disjunction) lead to the production of aneuploid progeny. Most human aneuploid embryos are non-viable, and this is the major cause of fetal loss (spontaneous abortion), particularly during the first trimester of pregnancy in humans. The most common form of viable aneuploid progeny in humans is Down syndrome (trisomy for chromosome 21), which is relatively rare in young mothers, but exhibits a dramatic increase with maternal age.

Anaphase and telophase I

Anaphase I of meiosis begins with the release of cohesion between the arms of sister chromatids, much as it does during mitosis. In meiosis, this ‘unties’ the topological knots of the chiasmata, and allows homologous chromosomes to separate and migrate to opposite spindle poles. As positioning of bivalent pairs is random, assortment of maternal and paternal chromosomes in each telophase nucleus is also random. Critically, sister centromeres, and thus chromatids, do not separate during anaphase I.

During meiosis I, cytoplasmic division occurs by specialized mechanisms. In females, the division is highly asymmetric, producing one egg and one tiny cell known as a polar body. In males, the process does not go to full completion, resulting in production of spermatocytes that remain joined by small cytoplasmic bridges.

Meiosis II

Meiosis II commences after only a short interval during which no DNA synthesis occurs. The centromeres of sister chromatids remain paired, but rotate so that each one can face an opposite spindle pole. Onset of anaphase II is triggered by loss of cohesion between the centromeres, as it is in mitosis. This second division is more like mitosis, in that chromatids separate during anaphase, but, unlike mitosis, the separating chromatids are genetically different (the result of genetic recombination). Cytoplasmic division also occurs and thus, in the male, four haploid cells result from meiosis I and II.

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