In this article we will discuss about Cell Cycle:- 1. Introduction to Cell 2. Phases of Cell Cycle 3. Regulatory Activities 4. Enzymes that Control 5. Mode of Action of M Phase Kinase 6. The Transition from G1 to S Phase and G2 to M Phase.
- Introduction to Cell
- Phases of Cell Cycle
- Regulatory Activities of the Cell Cycle
- Enzymes that Control Cell Cycle
- Mode of Action of M Phase Kinase
- The Transition from G1 to S Phase and G2 to M Phase
1. Introduction to Cell:
All living organisms of the biological world start life as one cell, i.e., unicellular zygote, the product of the union of gametes—a sperm and an egg. Of course, unicellular organisms live their entire lives as one cell. But in a multicellular organism, the unicellular zygote undergoes countless divisions and produces many cells.
These cells ultimately build the organism to a level of cellular complexity and organisation. The process by which any cell produces its own replica is known as cell division. Thus, simply by cell division a zygote enables an organism to grow. During this period of growth, many cells undergo a course of specialisation that commits them to perform specific functions.
Some cells function in cell division—either they divide to produce gametes for sexual reproduction or they divide to make new cells for growth or to replace old and damaged cells. Thus cell division is at the core of life itself. It helps organisms to grow, reproduce and repair damaged and worn tissue—three fundamental activities of life.
New cells originate only from other living cells. In 1850, Virchow enunciated that every cell originates through division of pre-existing cells. The cell that undergoes division is termed a mother cell, while the cells derived from the division of a mother cell are known as daughter cells.
There is no reason for using these familial terms. The mother cell transmits copies of its hereditary material in the form of DNA or chromosome to its daughter cells, the next cell generation of cells.
For hereditary information to be transmitted from generation to generation, DNA must be replicated before the cells divide so that each new daughter cell receives a complete copy of hereditary instruction. Since DNA is a part of an eukaryotic cell’s chromosome, the chromosomes duplicate as well.
After chromosomal duplication, the rest of the division activities proceed in a way that ensures each daughter cell receives the same share of genetic information as well as almost equal proportion of the cell’s cytoplasm and organelles. Therefore, in order to divide, a cell must double its mass and increase in shape and size. Cells generally divide when they attain the maximum size.
2. Phases of Cell Cycle:
Most cells divide one or more times during their life time. When they do, they pass through an ordered sequence of events that collectively forms the cell cycle. The duration of the cell cycle varies greatly from one cell to another.
The shortest cell cycle occurs in early embryo and can last as little as 8 minutes. The cell cycle of growing eukaryotic cell lasts from 90 minutes to more than 24 hours, its duration varying considerably within a population of cells.
The cell cycle of the eukaryotic cell is divided into two fundamental parts:
i. Interphase, and
ii. Mitosis (including Cytokinesis)
Interphase is the period of non-apparent division whereas mitosis is the period of division. Actually, for many years cell biologists were concerned with the period of division in which changes visible under the compound microscope could be observed—whereas during interphase no visible changes under compound microscope were seen.
Even chromosomes were not visible in the interphase because the refractive index of the nuclear sap and that of the chromosome present in re-condensed, hydrated and dispraised state become identical. The whole nucleus appears as idle. So interphase was mistakenly considered as resting stage.
During interphase of nucleus several changes take place at the molecular level that are not visible microscopically. Interphase is a period of intense biosynthetic activity in which the cell doubles in size and duplicates precisely its chromosome complement. So this phase is also known as metabolic phase and the nucleus is known as the metabolic nucleus.
The time from the end of one mitosis to the start of the next mitosis is called interphase. It is the phase between the end of last telophase and subsequent prophase. This is the longest period in cell division. Feulgen staining of metabolic nucleus followed by a cytophotometric quantitative assay first suggested that doubling of DNA takes place during interphase.
Auto-radiographic studies with labelled thymine demonstrated that doubling of DNA—i.e., replication or synthesis of DNA— did not take place throughout the entire interphase. It occurs only in a restricted portion of the interphase—the so called S phase, i.e., synthetic period. This period is preceded and followed by two gap periods of interphase (G1 and G2) in which there is no DNA synthesis. Thus the interphase can be subdivided into three successive sub-phases G1,S and G2 and it normally comprises 90% or more of the total cell cycle (Fig. 14.1).
(a) G1 Phase:
The period between the end of telophase and just before the entry S phase is called G1 phase. The period of G1 is usually greater and is subjected to greater variation. Generally speaking, the S and G1 and mitotic periods are relatively constant in the cells of the same organism. But the G1 period is the most variable in length.
It may constitute 25-50% of the total interphase duration. In some cells G1 may be very short or absent. Depending on the physiological condition of the cell it may retain in G1 phase for days, months or years. Cells that have stopped growing also become arrested at a specific point of G1 (e.g., liver cell, lymphocytes etc.) and the cells contain the amount of DNA present in G1 period.
Arrested cells can be induced to divide again. For example, liver cells normally neither grow nor divide but liver damage rapidly induces them to divide. Intensive cellular synthesis takes’ place during G1 phase. Mitochondria, chloroplasts, endoplasmic reticulum’s, lysosomes, Golgi apparatus, vacuoles and vesicles are produced.
In cells preparing for cell division there is a marked synthesis of mRNA, tRNA and proteins during G1 but there is no DNA synthesis (Fig. 14.2). The enzymes and substrates necessary for DNA synthesis during S phase are also synthesised during this phase.
Nucleolus produces rRNA and ribosomes are synthesised. This is Tiecessary for the entry of cells into mitosis as inhibition of their production delays the entry of the cell into mitosis. As a whole, cellular metabolic rate is very high in G1. As a result, cell growth occurs.
Commitment to chromosome or DNA replication in S phase occurs in G1 phase. If conditions to pass the commitment point are satisfied, after a lag a cell will enter S phase. The conditions mean the nutritional state of the medium, the mass of the cell etc. The commitment point is clearly observed in yeast cell where it is called start. The comparable feature of the animal cell is called the restriction point.
(b) G0 State:
Some cells do not divide at all. These cells are often considered to have indefinitely withdrawn from the cell cycle into another state, resembling G1 but distinct from it because they are not able to go to S phase, i.e., cells are arrested to non-cycling state.
This non-cycling state is called Go state or resting state and the cells are called resting cells. Some cells such as neurons have left the cell cycle irreversibly and can never divide again. But certain types of cell can be stimulated to leave Go and reenter a cell cycle. For example, liver cells normally neither grow nor divide, but liver damage rapidly induces them to divide.
Indefinite withdrawal from or reactivation into the cell cycle takes place effectively at an early part G1 phase. The absence of nutrients or growth factors cause cells to enter a resting state. Yeast cells starved of nutrients or mammalian cells deprived of growth factors arrest early in G1 in the stage G0.
G0 cells usually contain fewer ribosomes and less RNA than the corresponding cycling G1 cells and they synthesise protein less than half the G1rate. When a G0 cell is stimulated to grow by growth factor or by providing nutrients, changes in the rate of protein synthesis generally go hand in hand with effect on the chromosome cycle.
The coupling between protein synthesis and the chromosome cycle is not always rigid. With suitable combination of protein synthesis inhibitors and growth factors, it is possible to depress protein synthesis in cultured cells without delaying progress through the cell cycle or conversely stimulate protein synthesis without stimulating cell division.
Comparison of the size of a mammalian neuron and a lymphocyte reveal that both contain the same amount of DNA, but a neuron grows progressively larger during its development while remaining in a Go state. During this time the ratio of cytoplasm to DNA increases enormously. On the other hand, lymphocyte maintains its constant cell size and the definite cytoplasmic ratio by means of cell division.
(c) S Phase:
S phase is the intermediate phase between G1 and G2 phases. When G1 phase ends, S phase starts. It is a highly specialised phase of interphase and the word S stands for synthesis. Actually, DNA synthesis takes place in this phase. Before a cell can divide, it must produce a new copy of its chromosomes.
For making a new copy of chromosome it needs both the replication of the long DNA molecule in each chromosome and the assembly of a new set of chromosomal proteins onto the DNA to form chromatin or chromatid.
By its end each chromosome has been copied to two complete chromatids which remain joined together at their centromeres until the M phase that soon follows.
(d) G2 Phase:
The period from the end of S phase until mitosis is called G2 phase. G2 phase is usually the shortest part of interphase. In this phase intensive cellular synthesis occurs. Mitochondria and chloroplasts divide. Energy stores increase.
Mitotic spindle begins to form. In the interphase there are two control points such as G1/S and G2/M at which the cell takes a decision on whether to proceed or not to the next step. Two control points are also called check points.
This provides an opportunity for the cells to ensure:
a. Whether all conditions are favourable for DNA replication or not?
b. Whether the cytoplasmic mass has increased to a level adequate for division or not.
c. Whether replication has been completed and thus DNA is undamaged.
If the check points did not give any green signal, the cells may halt in G1/S or G2/M. Some embryonic cycles bypass some of these controls at some stages of embryogenesis. Thus, the control of the cell cycle can be coupled as required to time, growth rate, mass and the completion of replication.
3. Regulatory Activities of Cell Cycle:
The presence of different regulators at different stages of the cell cycle can be determined by means of cell fusion in different stages of the cycle. Cell fusion makes a hybrid cell. Hybrid cells are heterokaryons which contain two different nuclei in a common cytoplasm. Cell fusion can be induced in presence of chemical agents or using inactivated sendai virus.
Interphase nuclei during G1,S and G2 and mitotic cell (M phase) can be fused by various combinations:
(i) When S phase cell is fused with a cell in G1, it reveals that both nuclei in the heterokaryon replicate DNA. This suggested that the cytoplasm of the S phase cell contains an activator or regulator of DNA replication.
The regulator identified by this fusion is called S phase activator (Fig. 14.3). The nature of the S phase activator is unknown. It could be regulator whose activation is decided when cells in G1 are ready to enter to a cycle of replication.
(ii) When a cell in S phase is fused with G2 cell, the S phase nucleus continues to replicate but the G2 nuclei does not replicate. This suggests that DNA that has been replicated once becomes recalcitrant to the effects of the S phase activator.
It means that S phase activator fails to induce the replication of DNA of the G2 cell where DNA replication has already been completed once. But in this fusion experiment, the S phase nucleus enters M phase sooner than it would have in its former cytoplasm, but the G2 cell does not enter mitosis. This can be explained that some regulators in the S phase cell—possibly the S phase activator itself inhibits the start of mitosis.
(iii) When a cell in mitotic phase or M phase is fused with a cell at either G1 or G2 stage of interphase, it brings about the interphase nucleus to enter a pseudo mitosis which is characterised by premature chromosome condensation in the interphase nuclei. This suggests that a mitotic phase inducer is present in dividing cells. The existence of inducer can be proved by the fact that the fusion between G1 and G2 cells do not induce replication or mitosis in either nucleus of the heterokaryon.
(iv) When S phase nuclei are fused to mitotic cells a more complex pattern is found in which the S phase chromosomes have a fragmented appearance or pulverized configuration.
It has also been noticed that blocking of DNA replication with inhibitors such as hydroxy urea prevents somatic cells from processing through S phase into G2 and M phase. Thus a common feature in the cell cycle of all somatic eukaryotic cell is that completion of DNA replication is a prerequisite for cell division.
4. Enzymes that Control Cell Cycle:
It has been observed from the experiments on frog eggs that when an arrested immature oocyte (equivalent to G2 somatic cell) is injected with cytoplasm extracted from arrested eggs (equivalent to M phase somatic cells), the oocyte starts to divide. This experiment suggests that the extract contains an active component that induces the immature oocyte to enter M phase.
The active component of the extract is, therefore, called maturation promoting factor (MPF) because MPF causes the cells to enter M phase. MPF is now understood to stand for M phase promoting factor. In fact the MPF is seen to have an enzymatic activity and it has the ability to phosphorylate target protein and so it is also known as M phase kinase.
It consists of two protein sub-units, P34 and P45 (the numbers indicating molecular weight; P34 = 34,000 Dalton’s).
The two sub-units have different function:
i. The sequence of P34 is a catalytic sub-unit which phosphorylates serine and threonine residues of target protein.
ii. The other sub-unit, i.e., P54 is a regulatory sub-unit which has kinase activity with appropriate substrate. This sub-unit is also named as cyclin.
Cyclins can be classified into two general types, viz., A and B. About 30% overall identity is found between A and B. In mammals and frogs, the B cyclins can be divided into the sub-types B1 and B2.
The sub-unit P34 is activated by modification at the start of M phase. The other sub-unit, i.e., P45, or cyclin, is destroyed gradually during mitosis. Its destruction is responsible for inactivating M phase kinase (P34) and releasing the daughter cells to leave mitosis.
The cell in G2 phase does not enter mitosis (M phase) until and unless M phase kinase is activated. During G2 phase two sub-units, i.e., P34 and cyclin, bind with each other to form an inactive P34-cyclin dimer. Thereafter P34 undergoes phosphorylation at three sites by two steps. P34 is a long chain protein molecule containing several amino acids.
In the first step threonine at 14 position (Thr 14) and Tyrosine at 15 position (Tyr 15) of the amino acid chain of P34 are phosphorylated. In the second step, another phosphorylation occurs on Threonine 167 (Thr 161) of P34.
After phosphorylation, Thr 14 and Tyr 15 are dephosphorylated, and at the same time, cyclin is phosphorylated. The phosphorylation and dephosphorylation of P34 and cyclin respectively, are major activities that induce the cell to enter the mitosis (M phase). The phosphate at Thr 167 of P34 is required for its activity during M phase.
The phosphorylated cyclin is destroyed by proteolysis during mitosis. Destruction of cyclin sub-unit causes dephosphorylation of P34 and P34 becomes inactive, Indeed, this process is necessary for cell to exit mitosis and the cell returns to an interphase where further synthesis of cyclin takes place to initiate a new cell cycle. Fig. 14.4 summarizes the whole process.
5. Mode of Action of M Phase Kinase:
M phase kinase acts directly or indirectly upon the various potential substrates and provides a means to control the passage of mitosis.
Two general models have been proposed to explain the mode of action of M phase kinase:
i. M phase kinase phosphorylates target proteins which, in turn, act to regulate other necessary functions. So this is an indirect action.
ii. M phase kinase directly phosphorylates the crucial substrates that are needed to regulate M phase.
The action of M phase kinase is always reversible. The M phase kinase directly or indirectly triggers several activities that causes the onset of M phase.
The activities are:
i. Condensation of chromatin;
ii. Dissolution of the nuclear lamina and breakdown of nuclear envelope (except yeast where the nuclear envelope does not breakdown).
iii. Reconstruction of microtubules into a spindle.
iv. Reconstruction of actin filaments for cytokinesis.
The list of potential action of M phase kinase is given in the Table 14.1.:
One of the most important functions of M phase kinase is the phosphorylation of H1 protein of nucleosome—a major protein constituent of chromatin. It might be connected with the chromosome condensation at M phase. Nuclear integrity is lost when underlying lamina of the nuclear membrane dissociates. As a result, nuclear envelop breaks down and laminas are actually phosphorylated during mitosis.
This phosphorylation takes place due to direct action of M phase kinase.
The gene responsible for encoding M phase kinase have been identified in yeast cell. In fission yeast Schizosaccharomyces prombe, P34 is encoded by the cell division cycle gene cdc 2; in budding yeast Saccharomyces cerevisiae, P34 is encoded by the homologous gene CDC 28. Homologous sub-units can be recognised in (probably) all eukaryotic cells.
The mitotic cycle of fission yeast, S pombe and baker’s yeast (S cerevisiae) is given in Fig. 14.5.:
S pombe has a conventional cell cycle. The cell grows and lengthens double in size and then divides. The cell cycle of S cerevisiae is unusual cells proceed almost directly from S phase into division. S cerevisiae buds during a cell in which G2 is absent or very brief and M phase comprises the greatest part.
Fission yeast Schizosaccharomyces pombe are rod shaped cells that grow by elongation and divide by laying down a cell wall across the middle of rod. Budding yeast Saccharomyces cerevisiae are round-shaped that grows by budding. Buds enlarge continually and, ultimately, separates from the mother cell.
Genetic analysis of both yeasts during cell cycle can be studied by the isolation of mutants. The first cell cycle conditional mutants came from a large collection of temperature sensitive (ts) mutants that could grow at 23°C but not at 36°C. Temperature-sensitive mutants mean that the gene product can function at one temperature, called the permissive temperature but not at a higher, restrictive temperature.
At the elevated temperature (36° C) the cell cycle is arrested at specific point but the cell cycle continues at 23° C. The mutant phenotype allows cell to continue growing while the cycle is arrested causing an obvious aberration; in S pombe the cells become highly elongated and S cerevisiae they fail to bud.
A number of temperature-sensitive cdc mutants have been isolated and characterised. These mutations define at least thirty unlined genes which are involved in DNA synthesis, nuclear division or cell plate formation.
A crucial point in the cell cycle is defined by the behaviour of the yeast cell. A haploid yeast cell (Schizosaccharomyces pombe) decides at a point early in Gi whether to proceed through a mitotic cycle or to mate with another haploid cell to form an diploid which may undergo meiosis to generate haploid spores (Fig. 14.6).
Start is the point in G1at which cells become committed to the mitotic cell cycle. Cells which have completed this point are committed to the mitotic cycle in progress and are unable to undergo an alternative developmental pathway such as mating and meiosis.
In fission yeast, two genes cdc 2 and cdc 10 have been identified whose functions are required for start, these two mutants have the ability to mate after arrest at the restrictive temperature.
The cdc 2 gene also participates prior to M phase of the cell cycle. Thus the cdc 2 gene product appears to have a central role in the regulation of the cell cycle. The other two genes cdc 20 and cdc 22 have their function in G1 after start since these mutants cannot conjugate from their arrest points. They are likely to be required for the initiation of DNA replication.
The control regulating the initiation of mitosis at late G2 phase has been investigated using mutants. The wee mutants initiate mitosis and cell division at reduced cell size. Genetic evidence indicates the wee 1 gene usually inhibits cells from initiating mitosis until their size is adequate. On the other hand, cdc 2 gene product may also act as an inducer of mitosis (Fig. 14.7).
The cdc 25 gene has been identified and characterised by experimental means. The result suggests that the cdc 25 and wee 1 gene functions are antagonistic and independent in their regulation of the initiation of mitosis. The product of cdc 25 is needed for the activation of cdc 2.
Another gene controlling the cell cycle is Sue 1. The Sue 1 gene product acts as a regulatory component of cdc 2.
The cdc 13 gene is one of the cdc genes required for the progression of mitosis. In S pombe, cycline of M phase kinase is encoded by cdc 13.
The nim 1 gene was identified by its ability to supress a cdc 25 mutation. This gene encodes a protein kinase that phosphorylates and inactivates wee 1.
The gene mik 1 encodes a homologue of wee 1 that also regulates the phosphorylation of cdc 2.
The analysis of cdc mutant in budding yeast shows that the cell cycle in fusion yeast consists of three cycles that separate after start and join before cytokinesis cells may be diverted into the mating pathway before the point of start.
The cell cycles of S cerevisiae are:
i. Chromosomal Cycle;
ii. Centrosome Cycle; and
iii. Cytoplasmic Cycle.
The chromosomal cycle consists of events needed to duplicate and separate the chromosomes. The mutations in the chromosomal cycle do not stop the cytoplasmic cycle. The cytoplasmic cycle consists of bud emergence and nuclear migration into the bud, the centrosome cycle consists of the events associated with duplication and then separation of the spindle pole body.
Completion of an entire cell cycle requires all three constituent cycles to be functioned. Nuclear division needs both the chromosome and centrosome cycle and cytokinesis requires the cytoplasmic cycle.
The crucial point of cell cycle is the start point. The decision on whether to initiate a division cycle is made before the point start.
If the cell does not decide to go to divisional cycle, the cell may enter into the mating type pathway by mating factors and cdc 36, cdc 39, fus 3 and far 1 which appear to block the cell cycle before start.
If the cell has to move the divisional cycle, it should pass through the start point. The crucial gene in passing start is cdc 2 in S pombe. Most mutants in cdc 28 are blocked at start. Therefore, cdc 28 is required to enter mitosis.
6. The Transition from G1 to S Phase and G2 to M Phase:
Growing cells have two important transition points in their cell cycle.
The transition points are as follows:
i. G1 to S phase that regulates the initiation of DNA replication. The transition called start is unicellular eukaryotes and the restriction point in animal cells.
ii. G2 to M phase—when mitotic spindle begins to form or the duplication of the microtubule organising centre starts.
It has been noted that in both fission and baker’s yeast the same catalytic sub unit of M phase kinase, i.e., P34 is required for the G1/S and G2/M transitions. It has also been noted that the same catalytic sub-unit uses different regulatory partners at each transition. The regulatory sub-units used at two stages of the cycle Eire sometimes called G1 cyclines and G2 cyclines, respectively.
A single regulatory partner for P34 is used at mitosis (G2/M transition) in S pombe, the product of cdc 13 gene. In case of S cerevisiae there are multiple patterns. These are encoded by the CLB 1-4 genes which produces B-like cyclin.
The genes can survive with only CLB 2. Mutants that lack all four of this group of cyclin-like products of the CLB genes proceed through start, bud and synthesise DNA but fails to assemble a spindle, i.e., G2/M transition.
But blocking of the cell cycle in G1 was not previously detected by mutation. The absence of such mutants has been later explained by the discovery of three independent genes—CLN1, CLN2 and CLN3. All such genes must be inactivated to block passage through start is S cerevisiae.
Therefore, triple mutation of CLN genes cannot pass G1. But mutation in any one or even any two of these genes fail to block the G1; thus the CLN genes are functionally redundant. Therefore, activation or inactivation of all three CLN genes is the main step for controlling the G1/S transition.
When all three CLN genes are inactivated, a cell leaves the cell cycle and may enter the mating type pathway. When all three CLN genes are activated the cell enters the cell cycle and pass G1– Although the CLN and CLB genes are identified as cyclin-like by their functions and by their sequences, the proteins do not appear to have the features of cyclic proteolysis by which the cyclins were originally identified.
In case of S pombe the same catalytic sub-unit, i.e., P34 plus regulatory partner, the product of PUC 1 gene, encodes a G1 cyclin. Over- expression of such gene causes a delay in G2 and is lethal in CDC 13 mutant.
In case of higher eukaryotes, it is plausible that the catalytic sub-unit P34 is needed only for G2/M transition or mitosis. P34 sub-unit is probably encoded by the genes which are homologous to cdc 2 genes of yeast. However, G1/S transition in higher eukaryotic cell the same catalytic sub-unit P34 is not required.
The catalytic sub-unit that has the potential for the transition of G1/S phase is the product of some genes called cyclin-dependent kinases (CDKs).
Two of the CDK genes, CDK 2 and CKD 4, code for proteins that form pairwise combinations with potential G1 cyclins. The first and best characterised gene is CDK 2. It codes for a protein that was initially called P33 which is different from the activity of P34.
The product of CDK genes is required for DNA replication but is not needed for the entry into mitosis. During the transition of G2/S phase, P34 associates with several alternative partners of cyclin-like cyclin A, B1 and B2. Three new types of G1 cyclin have been identified.
These are cyclin C, D and E. These are distantly related to one another and to other cyclins. Cyclin E can bind to the product CDK 2 constituting a dimer. There are 3D-type cyclins such as D1;D2 and D3. They form dimers with the product of CDK2 and/or CDK4.
Cyclin A also plays a regulatory sub- unit with the catalytic sub-unit during G1/S transition. Thus cyclin A could be involved at both stages of transitions. The potential catalytic sub-units and their regulatory sub- units are summarised in Fig 14.9.
Besides mutation, inhibitors of DNA replication can arrest the cell cycle directly and the un-replicated DNA can provide an impediment to passage into G2. On the basis of in vitro experimental data, it suggests that non-replicated DNA at concentrations equivalent to percent of the genome can inhibit progress from S phase to mitosis.
Another interesting relationship between DNA and the cell cycle has been noted. Wild type yeast cell cannot pass from G2 into M if they are damaged by X-irradiation or due to mutation of cdc 9 gene (responsible for the synthesis of DNA ligase). Yeast cells contain RAD 9 gene that prevents nuclear division in cells that contains damaged DNA.