Review of Cell Cycle Regulation in Physiology

An introduction to the cell cycle

The cell is the fundamental unit of fundamental unit of living creatures and life processes. One of the basic tenants states that all cells arise from pre-existing cells by cell division. Cell division or reproduction is when cell increases in the number and cell differentiation explains the process when one cell type changes to another or increases the type of cell. A cell reproduces by carrying out an orderly sequence of events in which its duplicates its content and then divides in two. This cycle of duplication and division is known as the cell cycle. The detail of the cell cycle varies throughout eukaryotes from organisms such as yeast to humans, each, at times, in the cycle produces a new organism. The components that regulate cell growth and division also play key roles in the termination of cell division that accompanies cell differentiation. The goal or basic function of the cell cycle most times is to produce two daughter cells that are accurate copies of the parent cell. Therefore the cell cycle regulation has to be carried out in ordered and controlled way as it is very crucial to human health because any disturbance the regulation of the cell cycle can lead to many forms of diseases, notable, cancer.

Principles behind the cell cycle regulation

In understanding the principle behind the regulation of the cell cycle, it is important to know the different stages that eukaryotic cells go through leading to their division. The cell cycle combines a continuous growth cycle (increase in cell mass) with a discontinuous division or chromosome cycle (the replication and partitioning of the genome into two daughter cells). As was mentioned before the main goal of the cell cycle is to accurately produce two identical copies. The chromosome cycle is driven by a sequence of enzymatic cascades that produce a sequence of discrete biochemical “states” of the cytoplasm. Each state arises by the destruction or inactivation of key enzymatic activities characteristic of the preceding state and the expression or activation of a new cohort of activities. This means that each phase or stage of the cell cycle is highly regulated in an orderly manner so to ensure that each stage is completed before going to the next. Before we can understand these mechanisms of regulation, we have to know the different stages of the cell cycle.

An overview of the cell cycle phases

Interphase and its phases

The cell cycle can be divided into two major phases: Firstly: an M phase which includes mitosis and cytokinesis and secondly, the interphase which describes all the stages outside the M phase consisting of G1 phase, S phase ad the G2 phase. Each cell is born at the end of M phase through mitosis, where the components and other cellular components are partitioned and Cytokinesis, where the cytoplasm is divided. The chromosomal DNA is replicated in the S phase (synthetic phase). G1 (first gap phase) and the G2 (second gap phase) are gaps between mitosis and the S phase. In G1 phase, metabolic changes prepare the cell for division; at a certain point known as the restriction point, this is where a crucial decision is made to move to the next stage. If the supply of nutrients is poor or if cells receive an anti-proliferative stimulus such as a signal to embark on terminal differentiation, they delay their progress through the cell cycle in G1 or exit the cycle to enter G0. The G0 phase is a quiescent cellular state outside the replicative cell cycle where the cells are neither dividing nor preparing to divide. G0 is not necessarily permanent so in some cases some cells may be permitted to re- enter the cell cycle in response to variety of stimuli. If however, the appropriate stimuli are received then the cells will trigger a program of gene expression that commits them to a new cycle of cell division. S phase is where DNA synthesis takes place. DNAs are initiated at many different sites called origins of replication. G2 phase, finally, is a very brief period where key enzymatic activities will trigger the entry into mitosis. Here the chromatin and cytoskeleton are prepared for the structural changes that will occur in the M phase.

Mitosis and its phases

The M phase as previously mentioned consists of mitosis and cytokinesis. Mitosis can be explained as a process of nuclear division in which the replicated DNA molecules of each chromosome are segregated into two nuclei. Mitosis is normally divided into 5 stages: 

1. Prophase is mainly defined by chromosomal condensation. Chromosomes are seen to be composed of two chromatids attached to the centrosome. The centrosome duplicate and forms two poles of the mitotic spindle. 
2. Prometaphase commences with the breakdown of the nuclear envelope and the chromosome begins to attach randomly to microtubules emerging from two poles of the forming mitotic spindle. Once both kinetochores on a pair of sister chromatids are attached to opposite spindle poles, the chromosome slowly moves to a point midway between the poles. 
3. Metaphase is when all chromosomes are properly attached at the metaphase plate. The exit from mitosis begins at Anaphase. 
4. Anaphase is defined as the stage where the sister chromatids separate from each other. The metaphase-anaphase transition is triggered by the proteolytic degradation of molecules that regulate sister chromatid cohesion. During anaphase, the separated sister chromatids move to the two spindle poles (anaphase A), which themselves move apart (anaphase B). As the chromatids approach the spindle poles, the nuclear envelope reforms on the surface of the chromatin. At this point, the cell is said to be in telophase. 
5. Telophase concludes the stages and the initiation of cytokinesis. A contractile ring of actin and myosin is formed in a circumferential belt in the cortex between the spindle poles and constricts the equator of the cell. Cytokinesis is the separation of two daughter cells from one another.

Regulation of the cell cycle

The timing of these different events of the cell cycle are highly regulated and checkpoints control the changes and transition between cell cycle stages. There are two systems to regulate the cell cycle: 

1. a power system consisting of CDK which uses the expression of cyclin proteins and 
2. The other is checkpoint in eukaryotes which can be defined as the biochemical circuits that detect external or internal problems and send inhibitory signals to the cell cycle system. The checkpoint ensures proper division of the cell.

The first discovery of regulatory factors in yeast cells: Cyclin-dependent kinases.

The discovery and genetic analysis of cell cycle regulatory factors were carried out in a series of experiments in budding and fission yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe) cell cycles. These are considered quite important for the study of the cell cycle for several reasons: one is that the proteins that control the cell cycle are conserved between yeasts and mammals and both yeasts grow as haploids and incorporate cloned DNA into their chromosomes by homologous recombination thus genetic analysis is facilitated. Therefore the yeast cell cycle is a dependent pathway through which events in the cycle occur normally only after earlier processes has been completed making them suitable for genetic study. In the genetic analysis of the fission yeast, a gene was identified called the cell division cycle 2 (cdc2) gene which is essential for the progression of the cell cycle during both the G1 to S phase and G2 to M phase transitions. The protein kinase called pk34cdc2 (now called cdk1) a product of the cdc2 gene is the framework for a family of protein kinases necessary for cell cycle progression in all eukaryotes. Each stage in the cell cycle corresponds to the action of a gene product that is essential for progression so as each stage progresses the gene product tied to that stage gets broken down so a gene product of the same family follows. According to this type of order, mutations in genes that are essential for cell cycle progression cause the entire culture of yeast to accumulate at a single point referred as the arrest point. This is the point where defective gene products become essential. These are called cell division cycle mutants or CDC mutants. When compared to the normal CDC genes, it is impossible to make known the strains of yeast cells carrying the CDC mutant genes unless the mutant has a conditional lethal phenotype. Temperature-sensitive (ts) is the most commonly used lethal mutation. Temperature-sensitive is where yeast mutants' permissive temperature (23 degrees operable) is lower than restrictive temperature (ceases at 36 degrees). Temperature-sensitive mutants often display altered sequence and lack of gene product causing the temperature-sensitive (ts) phenotype.

Fission yeasts with CDC mutants affecting the entry into mitosis have distinctive morphologies. Mutant cells in Wee1, the kinase that keeps Cdk1 inactive prior to mitosis, enter mitosis prematurely and are shorter than normal. In contrast, the cells lacking Cdc25, a phosphatase that counteracts Wee1 and activates Cdk1, are unable to enter mitosis but continue their growth cycle and hence become enormously large. This distinctive classification in phenotype of the yeast CDC genes compares a Cdc mutant and a normal cdc gene in which one delays entry pass into mitosis and the other stimulates progression through mitosis.

Xenopus oocyte and MPF

The Xenopus oocyte assay and cell fusion are two notable experiments that lead to the discovery of the maturation-promoting factor of the cell cycle. Amphibian’s oocytes and eggs are storehouses of most components necessary for cell cycle progression. The oocytes are arrested in meiotic G2 where they mature to eggs by a surge of the hormone progesterone. The eggs now are naturally arrested in the metaphase of the second meiotic division. They, therefore, provide abundant sources of cytoplasm from these defined stages of the cell cycle. So when an M phase cytoplasm from a mature unfertilized egg is injected into a G phase oocyte, the G2 phase oocyte will ‘mature’ into M phase and completes its maturation. This activity of the cell cytoplasm was initially called maturation promoting factor (MPF).

Cell fusion and MPF

Another early evidence of the existence of positive inducers of cell cycle transitions was obtained in cell fusion experiments. It revealed that when cells cultured in S phase were fused with cells in G1, the G1 nuclei initiated DNA replication thereafter. In contrast, if S phase cells are fused with G2 cells, the G2 nuclei did not re-replicate their DNA after passing through mitosis.

The most dramatic results were obtained when mitotic cells were fused with interphase cells. This caused the interphase cells to enter mitosis abruptly. This was termed premature chromosome condensation (PCC). If mitotic cells (M cells) were fused with G1 phase, the G1 nucleus undergoes premature chromosome condensation into long, single filaments. If the mitotic cells were fused with an S-phase cell, the partially replicated chromosomes condensed into a complex pattern of single and double condensed regions separated by regions of de-condensed chromatin corresponding to the sites where DNA was actively replicating at the time of fusion.

Finally, when mitotic cells are fused with G2 cells, the G2 cells undergo PCC also but because DNA replication had already occurred, the compacted G2 chromosomes were visibly doubled.

Cyclins, Cdks and MPF

The stimulation to enter M phase was termed maturation and the unknown factor in the egg that induced oocyte maturation was termed maturation promoting factor (MPC) or M phase promoting factor. This concept was coined early on through the various experiments explained above and was thought to be the inducer of mitosis. Through biological studies later revealed that a protein might be involved with MPF, which is called cyclin. It was noted that cyclin first accumulated was greatly reduced at the metaphase-anaphase transitions. Another component relating to MPF is a subunit with kinase activity that transfers a phosphate group from ATP to specific serine or threonine residues of specific protein substrates. Only a few protein kinases are involved in the cell cycle control. For them to be active, these enzymes must each associate with the regulatory subunit, cyclin thus termed cyclin-dependent kinases (Cdks). Mammalian cells expressed Cdks that function in different stages of the cell cycle. Cdk1, formally called p34cdc2, functions primarily in the regulation of the G2 to M transition. A second family member, Cdk2, is involved in the regulation of G1 to S and G2 to M transitions whereas two other family members, Cdk4 and Cdk 6 are involved in the passage of the restriction point (G1). Cdk 7 is involved in the activation of other Cdks and also seen in RNA transcription and repair of damaged DNA. Other Cdks participate in diverse processes from transcriptional regulation to neuronal differentiation which may not be linked to the cell cycle.

Cyclins

As was previously explained the defining feature of Cdks is that they require the binding of cyclins. Cyclins are a group of diverse proteins with a similar core structure based on two symmetrical domains of five alpha-helices. The term cyclin however was derived because of the process of cyclic accumulation and destruction. The level of cyclin during each stage of the cell cycle is regulated through gene expression and proteolysis thus its concentration rises and falls in a predictable pattern during each stage of the cell cycle. This regulation is carried largely through phosphorylation and dephosphorylation of proteins involved. Although there are a large of cyclins only a small amount is required in the cell cycle. Of those that are, some function during G1the  phase that binds Cdks at the end of G1 and commits the cell to DNA replication, others during G2 phase, and still others during M phase which promotes the event of mitosis. S-cyclins bind Cdks during S phase and are required for DNA replication. The four classes of cyclins expressed are: Cyclin D, Cyclin A, Cyclin B, and Cyclin E. 

Cyclin D induced growth factors or mitogens early in the G1phase, cyclin A is required for the initiation of DNA replication, cyclin B is necessary for the entry to mitosis, and cyclin E is required for the passage through G1/ restriction point

• The regulation and activity of Cdks during cell cycle progression.
• The activity of Cdks during cell cycle progression is regulated by four molecular mechanisms.
• The first level of regulation involves association with cyclin as they are needed for activation.

The formation of Cdk/cyclin complexes, therefore, is controlled by cyclin synthesis and degradation. Secondly, the activation of the Cdk/cyclin complexes requires phosphorylation of conserved Cdk threonine residue. This is catalyzed by an enzyme called CAK (Cdk activating kinase) which itself contains a Cdk (Cdk7) complex with cyclin H. The third mechanism, in contrast, involves inhibitory phosphorylation of tyrosine near the Cdk amino terminus. It is catalyzed by Wee1 protein kinases. Both Cdk1 and Cdk 2 are inhibited by the phosphorylation of tyrosine -15 and threonine 14 in vertebrates. In particular, these Cdks are then activated by the dephosphorylation of the residues by members of the theCdc25 family of phosphatase. In addition, a second strategy for inactivating Cdks involves the binding of small inhibitory subunits of the cyclin-dependent kinase inhibitor (CKI) and inhibitor of Cdk4 (INK4) families.

Checkpoints

Cell cycle checkpoints as already discussed ensure that all the events that make up the cell cycle occur accurately and in proper order. They are termed ‘surveillance mechanisms’. In general, they block cell-cycle progression if any DNA is damaged or critical processes have not been accurately completed. They are three main types of checkpoints: 1. the G1 checkpoint also known as the restriction point in G1 which we explained as the point where the decision to proceed to replication is made. The restriction point is sensitive to the physiological state of the cell and interactions with their surrounding extracellular matrix, cells that do not receive the appropriate growth stimuli from their environment do not progress from this point in the G1phase and may commit suicide by apoptosis. 2. The G2 /M checkpoint and 3. The metaphase checkpoint is known as the spindle checkpoint.

A brief look at the G2/M checkpoint

At the transition from G2 to M, the checkpoint system confirms that the DNA is undamaged and fully replicated ensuring that the cell doesn’t enter mitosis unless its DNA is fully intact. If DNA is damaged after it is replicated the cell can use the information present in sister chromatids to guide the repair process. This, however, have to happen before the separation of sister chromatids or it could be a potential danger. If a cell enters mitosis before completing the replication of chromosomes, the attempt to separate sister chromatids causes extensive chromosomal damage. This is the need for the checkpoint in G2 to block DNA entry if there’s some form of DNA damage or the replication is incomplete. The way G2 checkpoint works is through sensors, transducers, and effectors such as ATR and ATM. Defects in G2 checkpoint are normally associated with cancer.

Spindle checkpoint and mitotic transitions

Since we already reviewed the restriction point and the G2/M checkpoint, the spindle checkpoint is also of great importance during the cell cycle. During the early stages of mitosis, starting at prophase the cytoplasm is dominated by highly active mitotic Cdks (Cdk1 and Cdk2 in combination with cyclins A and B). Phosphorylation of key components by these kinases leads to dramatic reorganization of the cell. The Cdk1/cyclin B complex induces multiple nuclear and cytoplasmic changes such as condensins, components of the nuclear envelope, Golgi matrix proteins, and proteins associated with centrosomes and microtubules. At prometaphase, unattached kinetochores catalyze the mitotic checkpoint complex (MCC) consisting of CDC20 and other proteins. The Anaphase promoting complex /cyclome (APC/C) is a key factor regulating the proteolysis of cyclins. The APC/C is inactive during S and G2 phases. APCcdc20 degrades cyclin A but the spindle checkpoint inhibits the destruction of other substrates. The spindle checkpoint role is to ensure that APC does not tag cyclin and cohesion until all chromosomes are attached to microtubules. Kinetochores on the chromosomes that are not attached to microtubules keep cdc20 inactive state. At metaphase, once all the chromosomes are aligned with their kinetochores the generation of MCC ceases. When the last chromosome has attached correctly to both spindle poles, the checkpoint block is released, and APC/CCdc20 starts to gradually degrade cyclin B and securin, an inhibitor of a key protease called separase. This degradation continues throughout the metaphase. At anaphase leading to the mitotic exit, when the securin levels fall below a critical threshold, separase is activated which in turn cleaves a key substrate (scc1 kleisin subunit) in the cohesion complex. This opens the ring triggering the separation of sister chromatids. Cyclin destruction continues throughout anaphase and telophase, and falling Cdk1 activity allows the formation of APC/CCdh1, which destroys Cdc20 and completes the destruction of the B-type cyclins. This leads to the mitotic exit. In fact, low Cdk activity is required for cytokinesis, spindle disassembly, chromosome decondensation, nuclear envelope reassembly, reactivation of transcription, reassembly of the Golgi apparatus, and assembly of pre-replication complexes on the chromosomes.

A review of Meiosis

Meiosis is the specialized program of two coupled cell divisions used by eukaryotes to maintain the proper chromosome number for the species during sexual reproduction. Its importance is that it ensures the production of a haploid phase in the life cycle and fertilization ensures a diploid phase.

Therefore without meiosis, the chromosome number would double with each generation and sexual reproduction wouldn’t be possible. It has two parts: 

1. Meiosis I. The first division of meiosis, in which homologous chromosomes separate, is also known as the reductional division because the

number of chromosomes is halved and 

2. Meiosis II. The second division of meiosis (also called the equational division); resembles mitosis as sister chromatids segregate from each other and the number of chromosomes remains the same Like mitosis, it can be divided into phases. 

It also has premeiotic interphase diving into G1, S, and G2 phases. I won’t go into details of all the stages only prophase 1 which is the most complex stage of the cycle. The first stage of prophase1 is leptotene, during which the chromosomes are revealed to be composed of paired chromatids, and start to condense. Lateral elements of the synaptonemal complex assemble. During the second stage, called Zygotene, is marked by the visible pairing of homologous with one another. The process of chromosomes pairing is called synapsis. The chromosomes synapsis is accompanied by the synaptonemal complex. The synaptonemal complex is a ladder-like protein structure that forms between homologous chromosomes. Pairing is highly specific. The complex formed by a pair of synapsed homologous chromosomes is called a bivalent or a tetrad. During the third stage, pachytene, which is characterized by a fully formed synaptonemal complex, the homologous are held closely together along their length by the SC. The DNA of the sister chromatids is extended in parallel loops. The beginning of the diplotene stage, the fourth stage, is recognized by the dissolution of the SC, which leaves chromosomes attached to each other at specific points by X- shaped structure called chiasmata. During the final stage of prophase 1, called diakinesis, the meiotic spindle is assembled and the chromosomes are prepared for separation. It also marks the breakdown of the nuclear envelope and the movement of the tetrads to the metaphase plate.

After prophase 1, similar stages to that of mitosis follows prometaphase 1, in which spindle apparatus is formed and chromosomes are attached to spindle fibers by kinetochores. Metaphase 1 is where the two homologous chromosomes of each bivalent are connected to the spindle fibers from the opposite poles, at anaphase 1, each pole receives a random assortment of maternal and paternal chromosomes and finally, telophase 1 is where chromosomes undergo some dispersion and the nuclear envelope may or may not develop. The stage between the two meiotic divisions is called interkinesis and is very short-lived followed by the stages of meiotic II. Prophase II is much simpler than prophase 1 and the other events of meiosis II are analogous to those of mitotic division, although the number of chromosomes has been halved. Meiosis II ends with the chromosomes enclosed by a nuclear envelope at telophase.

Conclusion

Cell division is mediated by a number of biochemical events and must be highly regulated. The mechanics and the timing of these events are described in terms of the cell cycle. The cell cycle is divided into two main stages: the interphase and the M phase which can be further divided. The regulation of these events is mediated by a protein called cyclin which activates Cdk triggering different cell cycle events and checkpoints which monitor the progress of cell cycle events and prevent entry to stages until the current stage is completed. The cell cycle is therefore important for proper cellular homeostasis to control the rate of cell growth and division. Without these mechanisms, cells would grow uncontrollably resulting in major medical problems, notably, cancer.