Chapter 9.

Cell division as a part of cell reproduction

9.1 Cell cycle

Cell division is the process through which a mother cell generates two new daughter cells. This process is only the last step of a larger process cell reproduction that in turn is an aspect of the reproduction as general property of living matter (that also includes the reproduction of individuals and of species). Before division all cellular components are doubled during biochemical reproduction. The cells take ions and small substances required for synthesis of simple organic substances (micromolecules) that will be further used in synthesis of RNA, proteins and DNA. DNA synthesis (DNA replication) is the most complex process that results in doubling the DNA amount in order to send the same genetic information to the two daughter cells. As consequence, the chromosomes made of a single chromatid turn into double chromatid chromosomes. The biochemical reproduction occurs in interphase between two consecutive cell divisions. These cyclic processes are parts of cell cycle. The cell cycle is defined as period of time between the end of a cell division and the end of the following division (of the daughter cells). Thus, the cell cycle has two main parts: an interphase and a cell division. The interphase is divided into: G1 phase, S phase and G2 phase. The S (synthetic) phase is the period when synthesis of DNA occurs, the G1 (gap no DNA synthesis) is before the S phase, and the G2 (another gap) is after the S phase. In G1 the DNA amount remains constant: the cell is diploid with 2n single-chromatid chromosomes. In the S phase the amount of DNA is doubled (each DNA molecule of each chromosome is copied) and at the end of S phase the cell has 2n double-chromatid chromosomes. In the G2 phase the amount of DNA is the same as in the S phase. During division of diploid cells (mitosis) the quantity of DNA is halved in the daughter cells because the double-chromatid chromosomes are split in two single-chromatid chromosomes. A complete cell cycle comprises 4 phases: G1, S, G2 and M (mitosis) and may have a variable length depending on species and tissue. Generally G1 is very variable, the S period is of 7-8 hours, G2 is quite constant, of 3-4 hours, and the mitosis is very constant (1 hour). There are cells with very different cell cycles, according to their different G1 phases. Some cells have very short cycles, of 14-16 hours (epithelial cells in intestinal mucosa or epidermis, bone marrow cells, cells in sperm germinal line, malignant cells etc.). These tissues have two distinct cellular compartments, a proliferative one that consists of dividing cells and a nonproliferative one that contains reserve cells stem cells. When some cells die, like
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erythrocytes and enterocytes, their loss is compensated by the stem cells that activate under the action of specific stimuli. There are cells that leave the cell cycle, and from G1 enter in a phase named G0 in which they can remain for years. If the G0 cell is stimulated to divide with mitogenic factors like growth factors, estrogen hormones etc., the cell can return back to G1 and will continue the rest of the cell cycle. Examples of such cells are: endocrine cells, fibroblasts and liver cells that can divide rapidly after hepatic injuries. Finally, there are cells in the human body that do not divide any more after the birth. These cells are stopped in G1 for the entire rest of their life: neurons, muscle cells and some cells from stomach. It was recently observed that neurons can be stimulated to divide in certain conditions with importance in treatment of neurodegenerative diseases like Alzheimers disease (senile dementia), or of neuronal loss after cerebral strokes.

Control of the cell cycle Progression of the cell in the cell cycle depends on the pass over certain restriction points located in the different phases of the cell cycle. The study of this process is very important because any deregulation of the cell cycle drives the cells to malignancy. Understanding of this process may lead to new methods to cure the cancer since the malignant cells have rapid cell cycles escaped from any control. In the cell cycle there are control points like barriers that dont open if the previous phase was not completed. The first control point is named R or START and is in G1, at the derivation to G0. In certain conditions, especially in the presence of stimuli like growth factors, the cell can continue the cell cycle. If the requested stimuli are not present, the cell enters into G0, where it can stay for a long time, and will return to the cell cycle when it will be stimulated. The second point is placed at the end of G1, before the DNA synthesis begins. If the DNA is damaged, the cell is stopped at this point until the damages are repaired in order to avoid an amplification of errors in DNA. The third control point is in the S phase, where the replicated DNA is verified. The fourth point is in G2. The cell can pass over this point only if it has an adequate size and the DNA is completely replicated. In mitosis it is a fifth control point that can be outrun if all chromosomes are linked on the mitotic spindle fibers. The mechanism involved in these control points is represented by phosphorylation of certain proteins by enzymes called kinases. The cell cycle kinases have a catalytic subunit but such a kinase is activated only when attached on another protein called cyclin. These
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enzymes are known as cyclin-dependent kinases (cdks). The cyclins are proteins whose concentration fluctuates (varies in a cyclic manner) during the cell cycle. There are families of cdks numbered form 1 to 7, and families of cyclins as well, named A, B, D and E. The cdks specifically associate with the corresponding cyclins for a particular control point. For example, at the transition G1-S cdk 2 or 3 associate with cyclin E; in the S phase, cdk1 and 2 with cyclin A; in G2 and M cdk 1 with cyclin B. In the different phases of the cell cycle the concentration of different cyclins increases and activates one cdk or another.

Cell division
There are two types of cell division: direct division (or amitosis), and indirect division with two forms mitosis and meiosis. The direct division is specific for prokaryotes. They dont have mitotic apparatus and the cell body splits into two equal parts. The direct division was also described in tumor cells or in regenerating tissues where the divisions are rapid; it is possible that the mitotic apparatus cannot be observed because of the high speed of division. The indirect division is specific for eukaryotes. It has synchronous cytoplasmic and nuclear changes that drive to equal distribution of genetic material to the daughter cells.

9.2 Mitosis

Mitosis is an indirect division specific for diploid cells. By mitosis two daughter cells with the same number of chromosomes as the mother cell are formed. A cell with 2n chromosomes divides into two cells with 2n chromosomes each. In the classic description, the mitosis has 4 phases: prophase, metaphase, anaphase and telophase. Between prophase and metaphase another phase, prometaphase was described, and the division ends with cytokinesis that is cytoplasmic division. During prophase, cytoplasmic and nuclear changes are produced. In cytoplasm the mitotic apparatus is formed after the depolymerization of the interphase microtubules. Centrioles replicate and two pairs of centrioles are formed. They move towards the opposite cell poles and between them new polymerized microtubules assemble mitotic apparatus (mitotic spindle after its aspect). Around each pair of centrioles microtubules form asters. Inside nucleus chromatin condenses and form thicker fibers (spirem), and nucleolus reduces and disappears.
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In prometaphase the nuclear envelope disassembles and the mitotic apparatus continues to expand. The chromosomes are attached to the fibers of the mitotic spindle and start to move to the middle of the cell. In metaphase the chromosomes are arranged at equatorial plaque (metaphase plaque) with their long axis perpendicular on the spindle microtubules. At the end of the metaphase each chromosome splits longitudinally into two chromatids that will form two unichromatidic chromosomes. In anaphase the two sets of newly separated chromosomes migrate along the mitotic spindle towards the opposite poles. Telophase begins when the chromosomes arrive at the cell poles. The nuclear envelope reassembles around each of the two sets of chromosomes. In parallel, the chromosomes decondense to form the spirem and later on the two forms of chromatin (hetero- and euchromatin). The nucleolus reappears. The mitotic apparatus disassembles, each pairs of centrioles becomes a future cell centre, and finally the cell splits into two cells. The latter process is cytokinesis; during this process a groove in the plasmalemma advances until the membrane will be completely strangulated and the two cells separate. There are four types of mitosis according to the resemblance of the daughter cells with the mother cell and in between: 1. homotypic mitosis the daughter cells are similar to the mother cell 2. heterotypic mitosis the daughter cells are similar, but are more differentiated (more mature) than the mother cell (differentiation mitosis). 3. asymmetric mitosis only one daughter cell is similar to the mother cell while the other is different. 4. dedifferentiation mitosis the daughter cells are younger, more non-differentiated than the mother cell; it is specific for lymphocytes. In a tissue the mitosis is triggered by general factors, such as temperature, light, hypophyseal and thyroidian hormones, estrogens, vitamins etc.; intracellular factors, like changes in the nuclear-cytoplasmic ratio and nucleolar-nuclear ratio; intercellular factors, especially the ratio between the mature cells, very old cells, and dividing cells.

9.3 Molecular mechanisms of mitosis

1. Replication of centrioles occurs in prophase when the two centrioles of the centrosome separate and on each one is synthesized a new, perpendicular centriole. There is evidence that a particular type of RNA centriolar RNA is involved in this process.

2. Formation of the mitotic spindle and the molecular mechanism of chromosomes movement. The interphase network of microtubules disassembles in prophase and after the centrioles replication new microtubules are synthesized around each pair of centrioles; they arrange to form two asters and the mitotic spindle, between the asters. Like in any other previously presented situation the end of the microtubules is captured in the pericentriolar material and the +end is located distally. Actually, during the cell division three distinct sets of microtubules can be described: a) aster microtubules around each pair of centrioles; together with the centrioles they constitute the asters (look like stars); b) polar microtubules that lie between the opposite pairs of centrioles and the equatorial region of the mitotic spindle, where they overlap and establish lateral interactions at the level of their +ends; c) kinetochore microtubules are attached with the end to the pericentriolar material and with the +end to chromosomes, on their centromeres. On the two sides of a centromere there are two specialized structures called kinetochores. The kynetochore consists of three regions (like disks) with different chemical composition and aspect: an internal disk containing chromatin with a different level of condensation; an intermediary disk and an external disk on which the kynetochore microtubules anchor. All these disks are interconnected by linker fibers. The microtubules of the mitotic spindle associate (sometimes in high numbers over 1,000) to constitute fibers. The movements of chromosomes are based on several types of motor molecules interacting with the above mentioned microtubules in various types of fibers: a) KRPs (Kinesin Related Proteins) are proteins located on the kynetochore outer disk. They have tails inserted in that disk and globular heads able to hydrolyze ATP and to undergo conformational changes and consequently to step on microtubules of kynetochore fibers from their +end to the end towards centrioles.
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b) +KRPs anchored in the pericentriolar material with their tails and the globular heads step on the same kynetochore fibers, towards the +end (to kynetochore). In parallel, the depolymerization of these fibers near the centrioles pair occurs (they shorten) and the chromosomes are pulled apart towards the poles of the mitotic spindle. c) +KRPs attached with the tails to a polar fiber near the microtubules +end (at the equator) and that step with their globular heads towards the +end of another polar fiber originated from the opposite pole. There are such +KRPs on the polar fibers from the both poles. Their activity, together with a polymerization (at the +end) of the polar fibers, is responsible for migration of the two pairs of centrioles towards the two poles. This molecular mechanism participates indirectly to the movement of chromosomes towards the poles. d) Cytosolic dyneins inserted in plasmalemma with their tails interact through their heads with the aster fibers. Since they step from the +end to the end, they pull the two pairs of centrioles toward the plasmalemma.

3. Nuclear envelope disassembles in prometaphase by the phosphorylation of lamins A and C in the nuclear lamina (intermediate nuclear filaments). The two lamins become soluble while lamin B remains attached to the inner nuclear membrane. The entire nuclear envelope remained without support turns into small vesicles that surround the mitotic spindle. In telophase the lamins A and C are de-phosphorylated and the nuclear envelope is rebuilt.

4. Cytoplasm is separated during cytokinesis by an actin-myosin ring assembled in the equatorial region of the cell, on the inner side of plasmalemma. This is a contractile ring that also depolymerizes and pulls the membrane inside the cell. At the cells surface a groove is observed during the process.

5. An accumulation of membranes as numerous extensions on the cell surface is observed before division. These microvilosities that increase the surface area of the plasmalemma represent in fact a reserve of coating membranes for the daughter cells that eventually will have together a bigger surface than that of the mother cell.

9.4. Meiosis

Meiosis is an indirect cell division which generates haploid (n) germ cells (ovum and spermatozoon) with a number of chromosomes reduced to half (23 in humans). Meiosis has an important significance because haploid gametes will fuse and form a diploid (2n) zygote (with 46 chromosomes in humans), in order to maintain constant the characteristic number of chromosomes for a species. If the gametes were diploid cells, the zygote would be tetraploid and not able to live. During meiosis, a single round of DNA replication is followed by two divisions, termed meiosis I and meiosis II and the doubled chromosomes separate twice. In meiosis I the chromosomes formed by two chromatids dont split like in mitosis but the entire chromosomes are equally distributed to the daughter cells. From each pair of homologous chromosomes, one chromosome goes to one daughter cell and the other chromosome to the other cell. This separation results in haploid daughter cells, with only one set of chromosomes (and a half number of chromosomes). In meiosis II, the chromosomes made of two chromatids are longitudinally split (as in mitosis) and the chromatids will be equally distributed to the daughter cells. These daughter cells will be haploid, but unlike their mother cell, they will have unichomatidic chromosomes. The final result of the meiosis is the generation of 4 haploid gametes from each diploid mother cell.

Meiosis I has the same phases as mitosis, but the most important events occur during prophase I. Prophase I has essential 3 processes: - condensation of chromatin with formation of meiotic chromosome with a special structure. It consists of a protein axis and chromatin is organized in loops on one side of the axis. Each chromosome is attached to the nuclear envelope with its extremities. - conjugation of homologous chromosomes. The homologous chromosomes (maternal and paternal) recognize each other and start an exact and intimate pairing like closing a zipper with the aid of axial proteins. As a result of conjugation, the homologous genes (alleles genes encoding the same character) are paired face to face. The structure between the two chromosomes is the synaptonemal complex (synapsis). Here, large recombination nodules appear at certain intervals and mediate the chromosomal exchanges. Association of the two homologous
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chromosomes results in a structure called bivalent chromosome or tetrade (contains four chromatids two maternal and two paternal chromatids). - crossing-over. One chromatid (maternal) crosses the homologous chromatid (paternal) in one or several points and the DNA-ases and DNA-ligases from the recombination nodules cut the chromatids at the points of crossing-over and inversely splice the fragments. As a result, some paternal fragments will be included in the maternal chromatid and vice-versa. The other two chromatids, one paternal and one maternal dont undergo crossing-over, and remain pure. This process is very important because the gametes will contain, after the crossing-over, genes from both parents, and the children will resemble not only with the parents but also with the grandparents. The non-homologous sex chromosomes X and Y have some regions of homology where they undergo synapsis and crossing-over, so the X chromosome can take genes from the Y chromosome. In prometaphase I the nuclear envelope disassembles, and the mitotic spindle is formed. In metaphase I the chromosomes are arranged in the metaphase plaque and at the end of this phase they separate and each chromosome with two chromatids goes to one pole of the mitotic spindle. In anaphase I the chromosomes with two chromatids migrate towards the poles of the mitotic spindle. In telophase I the chromsomes arrive to poles, the chromatin decondenses, the nuclear envelope is remade, and the cell is split in two cells through cytokinesis. The daughter cells have only a half of the chromosomes of the mother cell, so they are haploid.

Meiosis II is similar to mitosis but haploid cells (with bichromatidic chromosomes) will divide in two other haploid cells (with unichromatidic chromosomes).

The meiosis in women may last between 12-50 years. The prophase I is the longest and in this phase mutations can occur, under the influence of mutagen factors. Therefore, frequency of chromosomal anomalies increases with age of mother. Primordial germ cells, gonocytes form in ovaries oogonia that divide through mitoses until the third month of the intrauterine life and become primary oocytes. The primary oocytes begin their first meiotic division and stop in prophase I until puberty. At birth the ovary has 2 millions primary oocytes that degenerate and at puberty only about 300,000 remain. Starting with the puberty, every month for the next about 40
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years, a primary oocyte completes the meiosis and generates an ovum. The primary oocyte undergoes meiosis I (asymmetric) and produces a big cell named the secondary oocyte and a small, totally different one named the first polar body. In the next division named meiosis II the secondary oocyte forms the ovum and another small polar body. The first polar body divides and forms two second polar bodies. In men, the primary germ cells migrate to testis during embryogenesis and are included in the seminiferous tubules; these immature germ cells are called spermatogonia and at puberty, they divide through mitosis and after differentiation they form primary spermatocytes. These cells enter in the meiosis I and form secondary spermatocytes, which in meiosis II will form spermatids and these will transform in sperm cells. The cycle of spermatogenesis is completed in approximate 24 days (of which the prophase I is of 14 days) and continues until an advanced age. The spermatogenesis may be also influenced by environmental factors such as air or water pollution, stress, alcohol (even in small amounts), that result in a decreased number of sperm cells, or/and a reduced mobility. In both sexes, from the initial diploid mother cell 4 haploid cells result after the two meioses. The 4 haploid cells have different sets of genes compared to each other or compared to the mother cells. This is why each ovum and spermatozoon is unique and after gametes fusion, genetic different organisms are made. As a very important consequence, each organism is unique, excepting the monozygote twins that have identical genetic material but are different only because of the influence of environment.