In this article we will discuss about the cell division in eukaryotes.
Eukaryotic cell division involves two major events:
(a) The division of nucleus into two daughter nuclei (karyokinesis), and
(b) The division of cytoplasm to produce two daughter cells, each cell normally having a single daughter nucleus (cytokinesis).
In eukaryotes there are two types of cell division, mitosis and meiosis. Mitosis produces daughter nuclei identical with their parent nuclei. It is involved in growth and repair of tissues. In contrast, meiosis reduces the chromosome number to half, and is responsible for the production of spores (plants) and gametes (animals).
The term mitosis was given by Flemming in 1882. It is the mode of nuclear division that produces two daughter nuclei each of which contains the same number and kind of chromosomes and is genetically identical to the other and to the parent nucleus from which it was produced.
This division is responsible for the growth and repair in the somatic tissues of organisms. In plants, megaspore and microspore mother cells are produced by mitosis; further, the microspores and megaspores produce male and female gametes, respectively, through mitosis. In animals the spermatocytes and oocytes are produced by mitosis, and they undergo meiosis to produce spermatozoa and ova, respectively.
Cell Cycle:
The life cycle of an individual cell consists of two main phases, the “interphase” and the “mitotic phase” (M phase).
Interphase is very long in duration and has been divided into three phases:
(1) G1 phase (gap-1 period) or pre-DNA synthesis phase,
(2) S phase (DNA synthesis phase), and
(3) G2 phase (gap-2 period) or post-DNA synthesis phase.
Auto-radiographic experiments using 3H-thymidine have shown that in the mitotic cells of higher organisms, DNA is synthesized during the middle part of the interphase called the “S period”. Cells move from G1 to S to G2 and then divide to produce daughter cells. The division phase (mitotic phase) itself is of very short duration (Fig. 9.2).
Certain cells do not enter the cell cycle and resemble the G1 phase, but actually they are not in G1 phase. Such cells are called to be in “GO” state. However, in certain conditions some cell types in the GO state can be stimulated to enter the cell cycle. There are two control points during the cell cycle.
The first control point is the transition of the cell from G1 to S phase and the second control point is the transition from G2 to mitotic phase (M). Both these transition points have regulators.
Method of study of cell cycle:
The cells cultured in vitro are incubated with the radioactive DNA precursor 3H-thymidine for a short period of 15 minutes. Radioactive medium is withdrawn; cells are washed and incubated again in a nonradioactive medium. Then they are examined at various intervals for the presence of label over the chromosomes, as they move into metaphase.
The cells synthesizing DNA (cells in S phase) become labelled with 3H-thymidine. The labelled cells proceed through G2 into mitosis; labelled metaphases begin to appear and the frequency rises to almost 100%.
As the un-labelled cells in G1 come to metaphase, the frequency of labelled metaphases drops to almost zero. As the labelled cells enter the second cycle, the frequency of labelled metaphases again rises. From the frequency curve of metaphase figures labelled at different intervals (Fig. 9.3), the period of different stages is determined as follows:
1. Average duration of cell cycle or generation time (G1 + S + G2 + M) is estimated as the interval between 50% points on the two successive ascending curves.
2. The period of DNA replication (S phase) is estimated by measuring the interval between 50% points on the ascending and descending curve.
3. The interval between the time of labelling (time zero) and the time when 50% of metaphase figures labelled mades the G2 period which includes the actual G2 period plus prophase and metaphase stages.
4. The Gl period is estimated by subtracting the sum of G2 and 5 from the generation time. However, this period includes anaphase, telophase and actual G1 period.
5. Actual G1 and G2 periods are determined by subtracting the durations of various mitotic phases from G1 and G2 durations estimated above.
6. Duration of various phases of mitosis are computed from mitotic index and the generation time.
The cell cycle duration varies in different species (Table 9.1). It varies in different cells depending on the cell type within the same population and on functional condition of the cell (Table 9.2). Temperature also causes variation in the cell cycle duration. The S and G2 periods are much less variable than Gl. In mammalian cells, the S period varies between 6 and 10 h, while G2 varies between 2 and 5 h.
G1 Period:
It is the pre-DNA replication period of interphase, i.e., the period between preceding telophase and the S phase. Its duration is affected by both physiological and environmental factors. It varies among different cell types and also within the same cell population, from few hours to few days, e.g., from few hours to 3 days in mammary tumors of mouse, 4.5 h in Chinese hamster cells cultured in vitro, 4 h in macronucleus of prozoan Euplotes eurystomus.
But in many cases, G1. e.g., yeast or G1 and G2 both, e.g., grasshopper neuro-blasts and bacteria are absent, and DNA synthesis occurs from telophase to metaphase. G1 prepares the cell for the S phase so that synthesis of RNA and protein occurs during this period.
Near the end of G1 period, the beginning of S period is triggered by some event. However, the molecular basis of this event is not clearly known. The commitment point to lead the cell into S phase is called “restriction point” (in animal cells) or “START” (in yeast cells).
S Period:
Activation occurs in G1 and the cell enters the S phase. Replication of DNA occurs in this period and it can be traced by the use of labelled DNA precursor 3H-thymidine. The S phase cell contains a regulator of DNA replication called “S phase activator”. However, the molecular basis of this activator is unknown.
In general, the S period is longer than G1 and G2 periods. For most of the mammalian cell types it varies between 6 and 10 hours with most values distributed around 7-8 hours. It may become very long in some cases, for example, the S period is 30 h in the ear epidermis of mouse and ranges from 9 to 30 h in its mammary gland cells.
In Vicia faba root tip cells, the cell cycle is 31 h with the S period of 6 h. In plants, S period of root meristem cells of several species is related to the amount of DNA per cell but the length of S period is the same in diploid and colchicine-induced tetraploid cells within a given tissue.
In many cases, heterochromatin completes DNA replication after that of euchromatin. The period of DNA synthesis in embryonic cells is shorter where the chromosomes are less condensed than in adult tissues.
In most organisms and cells types, the replication of DNA is an asynchronous process as summarised below:
(a) Different chromosomes of the complement and different loci within the same chromosome replicate their DNA at different points of time during the cell S period and the sequence of this replication is under genetic control.
(b) The sum of the times taken by the individual asynchronous chromosomal loci to duplicate their DNA makes up the duration of S period. Therefore, the G2 period may begin at different points of time for the different chromosomes or chromosome segments of the genome.
(c) Heterochromatic sex chromosomes and heterochromatic segments of the autosomes complete DNA replication after the euchromatic chromosome segments.
(d) In case of mammalian cells in culture, the pattern of DNA replication is consistent over cell generations. However, the replication pattern of a particular part of chromosome may be changed during cell differentiation.
The initiation of DNA replication requires the synthesis of specific proteins and RNA. Inhibitors of DNA replication are the most effective when applied during G1; once a cell starts replicating DNA, it becomes relatively insensitive to their inhibitory effects.
The activity of enzyme thymidine kinase appears at the beginning of replication and disappears when replication is finished; it has therefore, been proposed that this enzyme is involved in the initiation of DNA replication.
G2 Period:
Post DNA synthesis or G2 period is not much affected by the physiological, environmental or ploidy conditions of cells. Generally it varies from 2 to 5 h. The alteration in the periods of G2 and S may occur during the process of cell differentiation.
In the mouse spermatogonia (cell cycle 27-30.5 h), the G2 period becomes reduced from 14 to 4.5 h, while the S period is increased from 7 to 14.5 h during the process of differentiation from the stem cell (type A spermatogonium) to the mature (type B) spermatogonium. RNA and protein syntheses occur in this period. In most of the organisms, centriole replication is completed by the end of G2 period and two pairs of centriole are visible at one side of nuclear envelope.
The chromosomes are in duplicated state in this period. Transition into and out of mitosis (M phase) is controlled by the enzyme M phase kinase. In animal cells, M phase kinase is activated by dephosphorylation of two amino acid residues (threonine-14 and tyrosine-15) located at ATP binding site and by phosphorylating a residue (threonine-167) somewhere else. This phenomenon leads the entry of the cell into “mitosis”.
RNA and Protein synthesis:
The sequence of gene transcription during cell division cycle plays a major role in its regulation. In higher organisms, the rate of RNA synthesis increases continuously from G1 through S into the G2 and then drops to zero at metaphase, and it resumes again at telophase.
There is continuous increase in the rate of protein synthesis from G1 to G2 and thus the pattern is similar to that of RNA. However, protein synthesis continues during metaphase and anaphase, but at a much lower rate than that during interphase and prophase.
Eukaryotic Chromosome Replication:
The mechanism of eukaryotic chromosome replication was studied by Taylor and coworkers in 1957, using the root tip cells of Vicia faba. The cells were labelled with 3H-thymidine during S phase; they were then allowed to grow in the absence of this radioactive thymidine. At the metaphase immediately following the exposure to –1H-thymidine, both the chromatids of each chromosome were labelled.
At the next metaphase, however, only one chromatid was labelled, while at the third metaphase, only half of the chromosomes were labelled in one chromatid only (Fig. 9.4). These results indicated that each chromosome in G1 consists of two complementary subunits of replication, i.e., two complementary strands of a single DNA molecule; these remain intact during successive replications.
In the S phase, the two subunits (the two strands of a DNA molecule) separate from each other and each of them synthesizes a new subunit. Thus each chromatid receives an original subunit and a new subunit; this is similar to the “semiconservative” mode of DNA replication. It suggests that each chromatid consists of a single (unineme) continuous length of DNA double helix (Fig. 9.4).
Regulation of Mitotic Phase (M phase):
A protein known as “M phase kinase” is responsible for mitotic phase. Its activation and inactivation regulates the mitosis.
The main events during mitotic phase are given below (these events are reversible):
(i) Chromatin is condensed and chromosome become visible,
(ii) Nuclear lamina is degraded into its constituent proteins called “lamins”.
(iii) Nuclear envelope breaks down,
(iv) Microtubules are dissociated and reconstructed into a spindle,
(v) Actin filaments are reorganized for cytokinesis.
M Phase Kinase:
M phase kinase is a dimer that is composed of two subunits called “P34” and “cyclin. These two subunits have different functions.
(a) Subunit P34 (34,000 Dalton subunit):
It is the catalytic subunit which phosphorylates serine and threonine residues in target proteins. Target proteins are HI histone, lamins, nucleolin myosis light chain, SW15 and certain others. Stimulation of mitosis or meiosis occurs due to phosphorylation of the above substrates.
H1 histone is the major substrate for phosphorylation and therefore, “HI kinase activity” is used as a routine assay for “M phase kinase”. Phosphorylation of HI histone results into chromosome condensation. During the early mitotic phase, phosphorylation of lamins leads to the dissolution of nuclear lamina and breakdown of nuclear envelope.
Phosphorylation of nucleolin occurs in early M phase and it inhibits the synthesis of ribosomes. Thus phosphorylation of different proteins by M phase kinase is required for “mitotic organization” and dephosphorylation is required for the “interphase organization” of the cell.
In animal cells, M phase kinase is activated by:
(i) Dephosphorylation of two amino acid residues, threonine-14 and tyrosine-15 located at ATP binding sites of p34, and
(ii) Phosphorylation of one amino acid residue (threonine-161) located elsewhere in p34. This phosphorylation occurs in G2. This event leads to the cell to enter into the mitotic phase (Fig. 9.5). Phosphate group is removed at the end of mitosis.
(b) Cyclin:
It is a regulatory subunit of M phase kinase. Cyclins are of two types, ‘A cyclins’ The “B cyclins” can be divided into B1 and B2 subtypes in mammals and frogs. In frogs, cyclin B is an inducer of meiosis-I and is provided as a maternal product. The “A cyclin” is produced de novo. Degradation of A cyclin occurs before the degradation of B cyclin.
Thus the M phase kinase can be divided into two generation forms; (1) p34 Cyclin A, and (2) p34 Cyclin B. The B cyclins are also phosphorylated in mitosis, but result of this phosphorylation is not known. Presence of cyclin is necessary for the functioning of M phase kinase.
This subunit is destroyed abruptly by proteolytic enzymes during mitosis. M phase kinase becomes inactivated due to destruction of cyclin. The inactivation causes the daughter cells to leave mitosis (Fig. 9.5).
Mitotic Phase (M Phase):
The mitotic phase can be divided into:
(1) Prophase,
(2) Metaphase
(3) Anaphase, and
(4) Telophase.
These stages vary in duration (Table 9.2), and are described below:
Prophase:
During the prophase, progressive coiling or condensation and folding of chromosomes begins and they become visible as thread-like structures. Each chromosome consists of two chromatids called sister chromatids which become visible clearly by the mid-prophase (Fig. 9.6). During early prophase, the two chromatids are twisted about each other in relational or plectonemic coils.
In plectonemic coiling, the sister chromatids are intertwined around each other so that they cannot be separated from each other without rotation; this type of coiling occurs in prophase of mitosis (Fig. 9.7).
There is another type of coiling called par-anemic coiling; in this case, the sister chromatids are not twisted round each other but the coils of one sister chromatid are slipped into those of the other so that they can be easily separated without rotation.
Such coiling occurs in the prophase of meiosis. At the end of prophase, the nuclear membrane begins to break down and the pieces of nuclear envelope disappear into the cytoplasm and become a part of the endoplasmic reticulum.
The nucleolus also disappears and its both fibrillar and granular components disperse throughout the cytoplasm. Some authors are of the view that nuclear envelope originates from the endoplasmic reticulum. However, in protozoa and fungi, the nuclear membrane remains intact throughout the entire mitotic division.
Metaphase:
At the beginning of metaphase, the nuclear membrane is completely broken down and nucleolus has disappeared. The two sister chromatids of each chromosome are held together at the centromere. The centromere provides the site for attachment of spindle microtubules, which is mediated by two specialized structures called kinetochores.
Kinetochores are attached to the opposite sides of a centromere; they are disc shaped structure ca. 200 nm in thickness and composed of layers of granular or fibrillar material, and face in the opposite direction (Fig. 9.8).
Therefore, the centromere of one sister chromatid becomes attached to the spindle fibre originating from one pole, while the centromere of the other sister chromatid gets attached to that from the other pole.
For convenience, metaphase may be divided into:
(i) Prometaphase, and
(ii) Metaphase.
During prometaphase, chromosomes begin to move towards an equilibrium position, half way between the two poles (Fig. 9.6). As soon as the chromosomes have achieved the equilibrium position, the stage is called metaphase.
The movement of chromosomes during the prometaphase has been divided by Darlington into three sub-stages, namely (a) chromosome congression, (b) chromosome orientation, and (c) chromosome distribution.
Cinematographic studies have revealed that individual chromosomes move towards the pole first, then make a turn and finally arrive at the equator. When the organization of spindle is prevented by colchicine, chromosomes are not able to move to the equator; this suggests that spindle fibres are required for chromosomes movement.
Centromeres are oriented in such a way that their chromosomes are more or less evenly distributed on the equatorial plate. The chromosomes are not always distributed randomly but they may be subject to specific arrangements on the equatorial plate. All the chromosomes are arranged on a flat plane celled the metaphase plate, midway between the two poles of the spindle.
Chromosome coiling is the maximum at metaphase; the chromosomes appear shorter and thicker than in other stages. Relational coiling is no longer present and the sister chromatids lie side-by side (Fig. 9.6). This stage is of intermediate duration between prophase and anaphase.
At the end of metaphase, the centromere becomes functionally double and separation of sister chromatids occurs. It has been suggested that the two chromatids are held together by an un-replicated region of DNA (its replication was not completed in S phase).
Therefore, DNA replication must occur before the chromatids can separate. Experiments using inhibitors of DNA replication at this stage have indicated that the replication of centromeric DNA may be responsible for the chromosome separation.
However, the factors responsible for delaying the DNA replication of the centromeric region from S phase until the end of metaphase and beginning of anaphase have not be identified.
Anaphase:
Anaphase is the shortest of all the mitotic stages; it begins just when the mutual attachment between the sister chromatids is lost. The centromeres of the two sister chromatics of each chromosome at the metaphase plate separate from each other and the chromatids begin to move towards the opposite poles. (Fig. 9.6).
These chromatids may now be called daughter chromosomes. The centromeres lead the way during chromosome movement at anaphase indicating that the chromosomes are being pulled by the microtubules attached to them. The two arms of each chromatid drag behind forming different shapes such as, V, J or rod, depending on the position of the centromere on the chromosome.
Simultaneous with the movement of chromosomes towards poles, the mitotic spindle also moves away from each other. Thus both the types of movement are responsible for the separation of the two sets of chromosomes from each other (Fig. 9.9).
Telophase:
When the chromosomes reach the opposite poles of the spindle, telophase is considered to begin. During this phase, the chromosomes uncoil, become very long and form an indistinguishable mass of chromatin. Nucleoli are reformed at the nucleolar organizer region of the concerned chromosomes, the spindle apparatus is broken down, and DNA transcription is resumed.
Membrane vesicles start to condense around the two sets of chromosomes; they coallesce to form the double layered nuclear envelope thereby yielding the two daughter nuclei. Subsequently, each cell divides into two daughter cells (cytokinesis).
Cytokinesis:
In animal cells, electron dense material condenses in the equatorial region of spindle at mid-anaphase, and forms layer called “mid body”. During telophase, the microtubules embedded in the mid-body remain intact. There occurs the in-folding of plasma membrane which causes a slight depression of a “cleavage furrow” in the surface around the cell (Fig. 9.10).
Then a band of microfilaments called “contractile ring” appears below the cleavage furrow in the cytoplasm. The microfilaments are responsible for creating the tension that cleaves the cell into two daughter cells. As the anaphase ends in plant cells, a layer of membrane vesicles is formed across the equatorial region of the spindle.
This structure is known as “phragmoplast”; it continues to grow outward and extends across the wall. The phragmoplast vesicles fuse to form a double layered membrane sheet or “cell plate” dividing the cell into two parts (Fig. 9.10).
Then the cell wall is formed by the accumulation of cell wall material between the two membranes of the cell plate. After sufficient thickness, the cell wall splits down in the middle to produce two cells that have intact plasma membranes and walls.
Thus the mechanism of cytokinesis in animal cells is different from that in plant cells. In animal cells, in-folding of plasma membrane extends from outside of the cell towards the inner side, while in plant cells, the cytokinesis starts from inside the cell and extends towards the periphery.
The distribution of cell organelles other than chromosomes, e.g., mitochondria, chloroplasts, endoplasmic reticulum, ribosomes and microtubules etc. to the two daughter cells not necessarily equal. So long as the two cells receive the minimum number of each of the granules, the shortage is made up by the production of additional organelles during the next cell cycle.
