2013 Group 1 Project
- 1 Regulation of Cell Division
- 1.1 Introduction
- 1.2 History
- 1.3 Mitogens and Cell Division
- 1.4 Entry into M-phase
- 1.5 Metaphase to Anaphase Transition
- 1.6 Disease
- 1.7 Current Research
- 1.8 Future Research
- 1.9 References
Regulation of Cell Division
The cell cycle is broken down into 4 main phases: G1 phase, S phase, G2 phase and M phase. The ultimate goal is cell division. M phase is where the chromosomes segregate and the cell physically divides into two daughter cells .
Cell division (M-phase) at first glance appears to be a simple and common process. However, as each step is broken down it becomes evident how many processes there are and how many things could go wrong, illustrating that cell division is anything but simple. During cell division there are various steps that need tight regulation, for instance the entry into M-phase, the spindle checkpoint, the reorganization of the actin cytoskeleton, distribution of organelles and fragmentation of the nuclear envelope .
The regulation of cell division is essential and even the breakdown of one seemingly small component of this regulation could be catastrophic . Due to the immense nature of the regulation of cell division, for the purpose of this page, only a couple of key regulatory checkpoints and their components will be discussed.
Mitogens will be addressed as they play a role in the commencement of G1/S phase. Although mitogens act before M-phase, they are an extremely important regulatory component of cell division .
The first checkpoint addressed will be the entry checkpoint to mitosis, where the regulation ensures that DNA replication is complete and cell division commences as a result of the activation of the Mitosis Promoting Factor (MPF).
The second checkpoint being addressed is the Spindle formation checkpoint, where the regulation ensures that all chromosomes are attached to the appropriate kinetochores before activation of the Anaphase Promoting Complex (APC) and hence the separation of chromosomes in anaphase .
Cells dividing movie- need to check for copyright
|1950||The discovery of growth factors were established. Nerve growth factors were one of the first growth-regulating signal substances that was discovered by Rita Levi Montalcinigrowth. |
|1970||The dependence of S-phase on M-phase initially came from cell fusion experiments who were performed by Potu Rao and Robert Johnson.|
|1970||Zirkle was one of the first to realise the existance of the spindle checkpoint in the transition from metaphase to anaphase by holding back single chromosomes from reaching the metaphase plate. He observed that anaphase was postponed until the chromosomes all arrived. |
|1971||Maturation Promoting Factor (MPF) was described as a cytoplasmic activity appearing during meiosis in amphibian oocytes that could cause maturation when injected into resting oocyte.|
|1980||The first mitogen-activated protein kinase that is called ERK1 (MAPK3) in mammals was discovered. |
|1983||Timothy Hunt discovered cyclins by studying the fertilisation of sea urchin eggs. |
|1992||Reider and Palazzo further researched the “checkpoint mechanism” and discovered a checkpoint that paused the cell division in the middle of metaphase to anaphase transition if these mitotic checkpoints were not reached. |
Mitogens and Cell Division
A mitogen is an extracellular substance, such as a growth factor, that stimulates cell proliferation. The rate of cell proliferation in unicellular organisms is highly dependant on the availability of nutrients in its environment. However, the cells in multicellular organisms only divide when new cells are necessary. Therefore nutrients alone is not sufficient for the process of cell proliferation . In order to aid this process, cells receive stimulatory extracellular signals via specific mitogens of other cells. One such mitogen is Platelet-derived growth factor (PDGF). Mitogens effect cell division by overcoming the intracellular braking mechanisms that block progression through the cell cycle, therefore increasing the rate of cell division.
Mitogens stimulate cell division by triggering the cells to enter Start in the G1 phase at which point the cells begin to divide. Entry into the S phase of the cell cycle is regulated by the G1 cyclins (cyclin D) by initiating expression of G1/S and S cyclins. This is done by removing inhibitory proteins from G1/S cyclin-CDK (cyclin dependant kinase) complexes . Throughout the G1 phase of the cell cycle there are several mechanisms that act to suppress Cdk activity therefore inhibiting entry in to S phase. Mitogens act in a way to stimulate Cdk activity in order to allow S phase to begin. This occurs by mitogens binding to cell-surface receptors to initiate a complex array of intracellular signals that penetrate deep into the cytoplasm and nucleus . The activation of G1-Cdk and G1/S-Cdk complexes by mitogens will overcome inhibitory barriers that usually block the cell cycle from progressing in to the S phase.
Mitogens are responsible for signalling cells to proliferate and therefore activate Cdk in order to initiate the S phase. Therefore in their absence the G1 phase is maintained and the cell cycle will arrest. A specialised non-dividing state, G0, can be achieved if cells partly disassemble from their cell- cycle control system . Many cells in our body exist in G0 phase therefore their cell-cycle control system is completely dismantled and the expression of specific Cdk and cyclin encoding genes being permanently turned off. Therefore resulting in the arrest of cell division.The presence of mitogens is imperative for the process of cell division due to the presence of pRB proteins preventing expression of the G1/S cyclins, therefore keeping cells in the G0 phase. Mitogens are responsible for the initiation of phosphorylation of pRB proteins and dissociate them from E2F proteins. The complexes responsible for the phosphorylation of the pRB proteins are cyclinD-CDK4 and cyclinD-CDK6. Mitogens stimulate cell division by increasing the expression of cyclin D in the G1 phase.
In order to increase the expression of cyclin D, mitogens act via a “classic tyrosine kinase pathway” . When bound to mitogens, their receptors become active and therefore phosphorylate one another on the cytoplasmic domains. A guanine nucleotide exchange factor for Ras recognises these phosphorylated domains resulting in an increase in the amount of Ras-GTP. Activation of the small GTPase Ras is one of the early steps of mitogen activation. The activation of Ras leads to MAP kinase pathways being activated with the final kinase phosphorylating transcription factors. This induces the expression of early response genes such as FOS and MYS. Separate complexes are formed by these proteins, which causes activation of G1 cyclin (cyclin D) expression .
Platelet-derived growth factor (PDGF) is one of many mitogens that are involved in extracellular regulation of cell division. It has been suggested that PDGF is a fundamental component involved in scar formation by functioning as a wound hormone. It is thought to do so by increasing the number of fibroblast population in the wound tissue . It also effects the types and total amount of matrix components that are being synthesised in early wound healing thereby effecting scar formation. The results of research done by Savage et al. (1987) confirmed that the mitogenic effects of PDGF have been shown to have more of an effect on normal skin fibroblasts than scar-derived fibroblasts.
History of Platelet-Derived Growth Factor (PDGF)
PDGF was first isolated while the cellular and molecular mechanisms that underly the lesions of atherosclerosis were being investigated. It was observed that cultured fibroblasts do not proliferate when given plasma but in fact do so when they are given serum. Plasma and serum are prepared using very different methods. In order to prepare plasma, cells from blood are removed without allowing clotting to occur whereas serum is prepared by removing the remaining cell-free liquid from the blood after the blood has clotted. Platelets are miniature cells that are found in blood that are imperative to the process of blood clotting at sites of tissue damage. They are necessary in order to stop excessive bleeding and also release other factors that stimulate healing.
When the process of blood clotting occurs it triggers the involved platelets in the blood clot to release the contents of their secretory vesicles. “The superior ability of serum to support cell proliferation suggested that platelets contain one or more mitogens”. Further research showed that extracts of platelets were able to substitute for serum in order to stimulate fibroblast proliferation, therefore supporting this hypothesis. It was shown that the critical factor in the platelet extracts that allowed this to occur was a protein that was later purified and confirmed as PDGF. As mentioned earlier, it has also been shown that PDGF in the body has a particularly crucial role in increasing the rate of cell division of fibroblasts during wound healing . However, it is not isolated to stimulation of fibroblast cell division. PDGF is also involved in the stimulation of proliferation of other cells such as smooth muscle cells, and neuroglial cells.
Entry into M-phase
During different stages of the cell cycle, there are certain events that take place such as DNA replication, division of cells (M-phase) and the spindle checkpoint. These events need to be operated with one another so that they occur in the appropriate order. For example a cell cannot divide unless its genome has already been replicated. Therefore to assure these types of processes occur correctly, cell cycle presents specific checkpoints such as G1 (Restriction) checkpoint, G2 checkpoint and the spindle check point. Entry to M-phase requires the activation of G2 checkpoint. Nevertheless before the role of G2 phase is established, it is important to describe briefly the M-phase.
What is M-phase?
M-phase also known as mitotic phase, is a period of the eukaryotic cell cycle during which the nucleus and the cytoplasm divide. M-phase involves a series of dramatic procedures that begin with nuclear division, consists of chromosome segregation and ends with cytokinesis. In mammalian cell this occurs in less than an hour, therefore require less time.
At the end of G2 phase, regulation ensures that replicated DNA is not damaged and the cell is ready to enter the M-phase. Therefore G2/M phase involves activation of key regulators such as cyclins, cyclin-dependent kinases (CDKs) and Mitosis-Promoting-Factor (MPF).
Cyclins are from the family of proteins that are involved in regulation of cell division. They control the progression of cells through the cell cycle by activating cyclin-dependent kinases enzymes. Timothy Hunt discovered cyclins by studying the fertilization of sea urchin eggs. In his experiment, while he was measuring the level of proteins in newly fertilized eggs, he found one protein that shortly disappeared at the end of cell division and then gradually appeared again as eggs began the next round of division. He named this protein as “cyclin” and concluded that this protein was driving the cell cycle. Furthermore, Hunt and other scientists deducted that making and destroying cyclins were essential for cell division. There are many different types of cyclins (Cyclin: A, B, C, D, E, F, G & H), but cyclin A, B, D and E are particularly important in cell cycle.
CDKs are enzymes that present throughout the cell cycle, but they are inactive at the absence of cyclins. In other words cyclins activate CDKs and need to bind to them in order to reach a maximum at a certain point, particularly when moving from one cell phase to another. CDKs are consecutively expressed in cells whereas cyclins are synthesized and degraded at specific stages of the cell cycle. Animal cells contain at least nine CDKs, however only four of them (CDK 1, 2, 4 & 6) are directly involved in cell cycle control. Cyclin B/CDK1 complex is one of the main key regulators that is involved in transition of G2 checkpoint to M-phase.
Metaphase to Anaphase Transition
Anaphase Promoting Complex
The main function of APC is to transition the cell from metaphase to anaphase by degrading proteins that inhibit anaphase.
APC triggers the transition of metaphase to anaphase by “tagging” protiens to be degraded (usually securin and S and M cyclins).  Degraded securin releases separase, which triggers the separation of the sister chromatids together in the cleavage of cohesin, which normally keeps the sister chromatids together. 
Activator subunits, cdc20 and cdh 1, drives the APC. 
When the spindle-checkpoint is activated, it inhibits the activation of APC, thus keeping the cell from transitioning between metaphase and anaphase. The spindle-checkpoint is only inactivated when all kinetochores of sister chromatids are attached to microtubules stemming from opposite chromatids. Even a single unconnected kinetochore can activate the spindle checkpoint .
The Spindle Assemble Checkpoint (SAC) functions to make sure the kinetochores and spindle microtubules are properly connected. CDC20 ans SAC proteins are concentrated at the kinetochores during prometaphase until every kinetochore is attached. 
The Spindle Checkpoint blocks anaphase by inhibiting the APC. The checkpoint complex also activates a protein,BUBR1, through CENP-E (a centromete protein) to block anaphase. 
|Chronic Myeloid Leukaemia (CML)||A somatic translocation of chromosomes 9 and 22 called the "Philadelphia chromosome" results in an BCR-ABL chimeric gene that acts as an oncogene and impacts various signalling pathways. As a result, patients with CML can exhibit enlargement of the spleen due to the accumulation of neoplastic leukocytes and splenic infarctions due to hyperviscosity of the blood .|
|Ovarian Clear Cell Carcinoma||A study found that over expresseion of Early Mitosis Inhibitor 1 (Emi1) was present in 82% of clear cell carcinomas. Clear cell carcinomas are more aggressive than other forms, and it is thought that the upregulation of Emi1 results in the upregualtion of different cyclins and disregulation of APC .|
Some cancers can stimulate their own growth by producing their own growth factors. Glioblastomas can secrete PDGF and has receptors that recognize it (autocrine).
Things to search in current research
- Sensor mechanisms that check DNA replication and activate M-Cdk
- How M-Cdk initiates the morphological changes that occur at the start of cell division, ie actin cytoskeleton rearrangement
- What triggers APC? It’s known that a Cdc20-APC complex forms and that M-Cdk activity is required, but still unclear what kinases phosphorylate the complex.
- Why do cells with a defective spindle-attachment checkpoint still undergo anaphase with normal timing? Useful accessory or essential component.
- Stopping mechanisms for number of divisions- replicative cell senescence.
- How is the actin cytoskeleton regulated in cell division?
Due to the impact that CDK's have on the regulation of cell division, some current research is exploring the importance of associated protein kinases such as Polo-like kinases .
 <pubmed>11389834</pubmed> The article presents the discovery of a new early mitotic inhibitor, Emi1. Emi1 inhibits the Anaphase Promoting Complex (APC) by binding to Cdc20, a component which is essential for the activation of APC . APC is an important regulatory complex in cell division as it triggers the transition between metaphase and anaphase, but there has been some unknown components surrounding the mechanism of its activation. The discovery of this inhibitor answers some of the questions about how APC is regulated and activated but there are questions left unanswered. Firstly, a question that may be asked with regards to destruction of the protein is what happens if the protein isn't destroyed after its interaction with Cdc20? The presence of nondestructable isoforms of the protein results in somatic cells not being able to commence through mitosis. The protein, after removal, is destroyed by proteolysis, mediated by the attachment of ubiquitin. Further mechanisms surrounding Emi1's destruction are being pursued by the authors. Another question that presents itself is are there any other inhibitory mechanisms or proteins interacting with Emi1?
Impact of tropomyosin in the regulation of the actin cytoskeleton in cell division is a field for future research. We know a lot about how different isoforms can impact cellular activity but don't know a lot about it's impact on cell division.
Tropomyosin is a coiled-coil actin binding protein that has over 40 different isoforms. Tropomyosin regulates the formation of different actin filament populations as discussed in the Microfilaments lecture .
Actin cytoskeleton rearrangement occurs upon entry into cell division as well as the exit, cytokinesis. So the following questions could be asked:
- How does tropomyosin regulate cell division? Does it affect the rearrangement of the cytoskeleton upon entry into M-phase or does it have an impact on cytokinesis?
- What affect do the different isoforms have on cell division?
- As the Tm5NM1 isoform is upregulated in cancer, does it have an impact on the replicative potential typical of cancer cells?
- Dr Mark Hill 2013, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G