2009 Group 5 Project

From CellBiology

The Cell Cycle

Image of Rudolf Ludwig Karl Virchow.

Introduction

German Pathologist, Rudolf Ludwig Karl Virchow’s realization in 1885 that "cells only arise from pre-existing cells" sparked the beginning of an intense investigation into the cell cycle, which is still as active and dynamic today. [1] Over the years, a great number of discoveries and advances have been made in bettering our understanding of this cycle.

Early scientists described the cytology of the cell division [2] and more recently, scientists from an array of disciplines, spanning embryologists to microbiologists have collaborated and begun to understand the underlining mechanisms and workings involved in the cell cycle. [3][4]


Historic Background

  • Since 1953, when James Watson and Francis Crick described the double-helical base-paired structure of DNA, [5] a lot of studies on cell cycle has been focused on DNA synthesis and replication.
  • In the 1970s, general control mechanisms that regulate the onset of each phases of the cell cycle were established. [6] Also, the Gap 0 phase (G0) was identified, and Pardee (1974) studied the regulatory mechanisms and how cells switch between proliferative and quiescent (G0) states in the cell cycle. [7]
The structure of DNA as first described by Watson and Crick. It shows the double-helical base-paired structure.
  • During the 1980s, the function of centromeres in segragating chromosomes were studied and a functional centromere was isolated from a budding yeast. [8] Also, the precise control mechanisms between the phases, and the changes in levels of 'cyclins' - "proteins that showed cell-cycle-oscillatory behaviour" [9] were discovered and studied in more details. [10][11]
  • During the late 1980s, replication origins in cells were identified and the importance of 'checkpoint controls' that arrest cells following DNA damage was discovered. [12] These help to avoid improper transitions between phases, and provided important clues to the nature of cell cycle regulation. Furthermore, the importance of tumour suppressor genes such as retinoblastoma and p53 were also discovered. [13]
  • In the 1990s, a new class of proteins, known as cyclin-dependent kinase (CDK) was identified. Their interaction with cyclins were studied in detail and has lead to the discovery of many more new classes of cyclins such as cyclins C, D and E. [14][15] In 1993, CDK inhibitors (CKIs) were identified and their interaction with CDKs was studied in detail. [16][17][18][19]


Current Research

Throughout the history, five Nobel prizes for Physiology or Medicine have been awarded in the development and understanding of the cell cycle. A lot of information have been collaborated to present, but there still exists areas that are not fully understood. Current research is now centred on the use of cell-cycle-specific treatments in cancer and other cell cycle related diseases. Cancer therapy may greatly benefit from a better understanding of the connenctions between cell cycle and apoptosis. Major advances have been recently made in the discovery of `cell cycle drugs', and their use as anticancer drugs is being extensively investigated. [20] The discovery of cell cycle regulators in the brain of Alzheimer's disease patients leaves us with the idea that cell cycle studies, initially supported by the antitumour purpose, may have applications in quite unexpected fields.


The Cell Cycle

Figure 1. Approximate 24 hour cell cycle of a mammalian cell.

The cell cycle is a series of changes a cell undergoes from the time it forms until it reproduces itself. [1] Depending on what text you read, this cycle can be divided into two or four major phases. We will be discussing the division as four phases, as the prescribed text; Essentials in Cell Biology: 2nd Ed. [1] defines.

These are:

  • Gap 1 phase (G1)
  • Synthetic phase (S phase)
  • Gap 2 phase (G2) and
  • Mitotic phase (M phase) (see Figure 1).

It is important to understand that the timing, or length, of the cell cycle differs from cell to cell and from organism to organism. For instance, a small single celled organism can quickly reproduce, whereas a more complex multicelluar organism takes longer. [21] From here on in, we will be discussing the phases of the cycle for that of a typical fast dividing mammalian cell, with an approximate cell cycle of twenty-four hours. [22]


Figure 2. Different phases of the cell cycle.

Gap 1 Phase (G1)

G1 is the interval between the completion of the M phase and the beginning of the S phase. [1] It is typically the longest and most variable phase of the cell cycle (see Figure 2).

In part, this lengthy time frame can be explained by the fact that G1 ensues the M phase, a phase in which cell division takes place. [23] The first opportunity for newly formed cells from the M phase to grow and develop is in G1, a phase of rest and growth. In addition to this function, G1 (along with the other gap phase G2) provides a time for cells to monitor their surrounding environment, both inside and outside of the cell, to ensure that conditions are appropriate and preparations are complete before committing to the next phase, the S phase. [22]

There is another sub phase within G1, known as the Gap zero phase (G0) (see Figure 3). When conditions are unfavorable, cells delay progress through G1 and enters G0. In this specialized phase, cells can remain in a rest form of periods from days to even years, [22] before returning to a proliferating state.

Some cells, such as cardiac muscle cells and nerves cells are unable to divide. That is, they have lost their ability to replicate themselves and as a consequence never leave G0 [1]. Other cells like the red blood cells are continually dividing throughout our life. Some cells will only divide under certain circumstance, for instance if part of the cell population is damaged or dies.


Figure 3. Different phases of the cell cycle with the addition of Gap 0 phase.

Synthesis

The synthetic phase or S phase of the cell cycle occurs between the gap phases of the cycle. It is a short phase consisting of few hours in which the entire nuclear content of the cell is replicated. [24] Replication of DNA molecule is undertaken in 3 steps; where a bidierectional mechanism is initiated to facilitate multiple replication sites along a chromosome. [25] This is subsequent to a preparation period where all hereditary material of the parent DNA molecule is portioned prior to cellular division. [12] A parent chromosome is composed of 2 complementary strands of DNA, where every base on one strand is paired with a specific partner on the other strand. That is, Adenine (A) pairs with Thymine (T); Guanine (G) pairs with Cytosine (C). [1]


The process of DNA Synthesis:

• The initial step of DNA replication in vertebrate cells involves the separation of the double stranded parent molecule of DNA, where hydrogen bonds linking base pairs are broken. [26] Separation of the DNA molecule is facilitated via an unwinding mechanism and leads to the formation of a 'v' by the two single stranded molecules.

Figure 4. Process of DNA replication
Figure 5. Semi-conservative nature of DNA

• As the molecule unwinds, DNA synthesis occurs at this location of the 'v' which is also referred to as the replication fork. [27] The process of DNA synthesis in eukaryotes is semi-conservative and occurs in a bi-directional manner. Consequently, there is a 'leading strand' and a 'lagging strand' formed. The leading strand replicates in the same direction as the proceeding replication fork, and is therefore faster. The lagging strand copies the molecule in the opposite direction, and therefore produces copied fragments of DNA known as Okazaki fragments, which will be joined together to form a single strand. [27] This is illustrated in Figure 4, which shows the process of replication in a double stranded molecule.

• As a result of separation of the double-stranded molecule and the synthesis of the single strands of DNA, new nucleotides are able to bind to the exposed bases with the assistance of a DNA polymerase enzyme. [26] Each strand is then used as a template for determining the order of base pairs in the new complementary strand. This results in the synthesis of a new strand that is complementary to the pre-existing template strand. [27] This then leads to the formation of duplicate daughter strands in an anti-parallel orientation, as shown in Figure 5.

• The final step of replication and synthesis produces one parent and one new strand of DNA. At this point, the cycle progresses into a phase of rapid protein synthesis to ensure cellular growth. Different cyclin-dependent kinase complexes trigger the process of DNA synthesis. [25] There are 3 main trigger complexes of the S phase, which are classified into a group known as the S phase promoting factors (SPF). [28] There is firstly an accumulation of cyclin E/cdk2 during late G1 phase, initiating the S phase. [29] Cyclin B/cdk2 is then synthesised, but phosphorylation of the complex occurs at Thr14 -Tyr15 and renders it inactive. [30] Lastly, cyclin A/cdk2, which is considered the most prominant factor in the group, enters the cell nucleus and prepares for cellular duplication. [28] It accumulates during S phase and its activation triggers the transition to G2 of the cycle.


A newt lung cell stained with fluorescent dye, undergoing mitosis. G2 prepares the cell for entry into mitosis.

Gap 2 Phase (G2)

G2 is the third stage of the cell cycle, preparing the cell for entry into mitosis (M phase). It is the second shortest phase of the cell cycle after mitosis, lasting approximately four to five hours in a 24-hour cell cycle. [31] It is a period of rapid cell growth and production of new proteins, similar to G1 and includes key mechanisms that control the completion of DNA and chromosomal replication following DNA synthesis during the S phase. [7]

In G2, the nucleus bound by a nuclear envelope is well defined, and includes at least one nucleolus. However, the replicated chromosomes occur as loosely packed chromatin fibres and cannot be distinguished individually. [32] One key process that occurs during G2 is the duplication of the centrosomes resulting with two centrosomes by the end of G2. [7] This centrosome replication is necessary in order to guide the chromosomes during the M phase that follows.

Cyclins and cyclin dependent kinases (CDKs) are a family of proteins which regulate the distinct stages of the cell cycle. [28] During the G2 phase, cyclins A and B coupled to CDK-1 drives the cell up to the M phase. [33]

The successful transition from G2 to the M phase is controlled by a key checkpoint at the end of this gap phase. This ‘DNA structure checkpoint’ has the important role of providing a quality check ahead of mitosis. [12] If DNA replication is incomplete or if there are mistakes in the copies of DNA, the G2 checkpoint prevents the transition to M phase and cells will undergo repair or apoptosis (programmed cell death). [34]

Diagramatic representation of the different stages of mitosis.

p53 is a protein which can block the cell cycle if the DNA is damaged. By blocking the cell cycle, it provides more time for DNA repair and increased p53 levels are present in damaged cells. [35] More information on p53 can be found here.


Mitosis

Mitosis is the division of a mother cell into two identical daughter cells, in which a eukaryotic cell separates the chromosomes in its cell nucleus. [36] Eukaryotic mitosis can be divided into five key stages. These are;

  • * prophase
  • * prometaphase
  • * metaphase
  • * anaphase and
  • * telophase

The prophase stage is signified when the DNA has been replicated and the resulting chromosomes become condensed. Each chromosome is made up of two chromatids, these pairs of chromatids are linked by a structure called centromere. [37]. Near the nucleus are structures called centrosomes, made from pairs of centrioles, which coordinate the cell's microtubules. The centrioles also start moving to opposite poles of the cell, allowing the mitotic spindle to develop in-between. Microtubules disassemble into their tubulin components which will be used to form the mitotic spindle. [38]

As the nuclear envelope deteriorates, the cell enters the prometaphase stage. In this stage, the centrioles move to opposite poles of the cell and the chromosomes attach to the spindle fibres via the kinetochores located at the centromere. [39] Prometaphase is sometimes considered part of prophase.

Once the chromosomes have completely bound to the spindle fibres, the chromosomes align to the metaphase plate located at the centre of the cell, signalling that the cell is at metaphase. [40]

Following metaphase, anaphase is characterised by the shortening kinetochore microtubules and the separation of the chromatid pairs. The chromosomes move towards opposite poles of the cell as they follow along the mitotic spindle. [38] This chromosomal movement and separation is referred to as anaphase A, while the separation of the spindle poles is called anaphase B. [37]

The final stage of mitosis is telophase whereby new nuclear envelopes develop on both sides of the cell. [36] The chromosomes beginning to decondense into chromatin, and the breakdown of the mitotic spindle also begins. [41] Also during this time the mother cell is completely divided creating two daughter cells by the process of cytokinesis. These daughter cells then enter interphase at G1, restarting the cell cycle.


A series of cells showing the mitotic division of a eukaryotic cell (above), and images of a cell in the corresponding stages of mitosis, stained with fluorescent dyes (below).

Checkpoints

Progression through the cell cycle is monitored at various checkpoints, where inhibition or activation of the cell cycle is possible depending on the formation of cyclin/CDK complexes and their binding activity. [12] In the cell cycle, 3 major checkpoints are elicited and these are:

  1. G1/S checkpoint (restriction point)
  2. G2/M checkpoint and
  3. M-phase checkpoint.


1. The first checkpoint is the G1 checkpoint, also known as the restriction point, which occurs midway through the cell cycle's G1 phase. At this point, the cell can decide whether it should divide, delay division, or enter a resting period (G0). [42] This restriction point is mainly controlled by the actions of Cdk inhibitors. [7] Activation at the restriction point is a result of appropriate cyclin-cdk complex forming and will trigger the phosphorylation of proteins, which in tern activates M-phase cyclins, thereby leading to the transition into G2/M checkpoint. [7] Conversely should an error occur during DNA replication, G1/S cyclin-cdk complexes (at the restriction point) will not bind and hence inhibition of cycle progression to G2/M checkpoint will occur. [12]

2. What takes place at the G2 checkpoint?

  • The cell is checked to make sure that DNA is replicated successfully and that two identical sets of the genome are now present and intact. [42]
  • Proof reading of both sets of the genome takes place and a check for molecular damage decides on whether the genome will be maintained, repaired or rejected. [24]
  • If DNA repair is required, the cell cycle is delayed for the duration of the DNA repair. [43]
  • If DNA is badly damaged and rejected, apoptosis (programmed cell death) will be triggered.
  • Overall competence of the cell to enter mitosis is determined. If the cell is ready, mitosis promoting factor (MPF) promotes the entrance into mitosis. [34]

3. Also known as the mitotic spindle checkpoint, it controls the entry into anaphase from metaphase. At this checkpoint, the cell senses the tension of the bipolar attachment that is created by the correct alignment of chromosomes at the mitotic plate. [12] If this checkpoint is satisified, it results in the degradation of cyclin B, which usually blocks the entry to anaphase by inhibiting the anaphase promoting complex. [34] The cell can now progress and continue the cell cycle.

Presence of checkpoints in the cell cycle ensure duplication of genomic material occurs without error such that abnormalities resulting from genomic instability can be avoided. [42]


Abnormalities

When a cell starts dividing uncontrollably producing more and more cells, it will eventually produce a mass of cell known as a tumor. This tumor might them begin to interfere with normal body function causing complications and leading to other diseases. Although not fully understood, it has been identified that the formation of a tumor is the direct result of an abnormal regulation of the cell cycle.

Cellular abnormalities may also be a result of errors incurred during DNA replication wherein discrepancies in nucleotide sequences of the chromosome may be found. Such errors within a genome have the potential to cause cellular malfunctioning and mutation of the gene. Accumulation of mutated genetic materials will lead to the other complications, including the development of cancer.


Glossary

  • Centriole: Cylindrical structures composed of groupings of microtubules. Assist in the assembly of microtubules during cell division.
  • Centromere: A condensed and constricted region of a chromosome, to which the spindle fiber is attached during mitosis.
  • Chromatid: One of the two strands of the chromosome pair undergoing cell division.
  • Chromosome: A Strand of DNA associated with proteins in the nucleus of eukaryotic cells. It contains the hereditary material of the organism required for cellular function and life.
  • Cyclin: A class of proteins that vary in abundance during specific points of the cell cyclin and are responsible for its regulation by binding to kinases.
  • Cytokinesis: The phase of cell division in which the cytoplasm divides.
  • Cytology: Division of biology that is associated with the formation, structure, and function of cells.
  • Kinetochore: A large multi-protein complex which binds to the centromeres of the microtubules of the mitotic spindle in the cell cycle during metaphase.
  • Proliferation: Term used to describe the growth or rapid multiplication of cellular material and tissues.
  • Replication fork: The point at which the two strands of DNA are separated to allow replication of each strand.
  • Retinoblastoma: A malignant tumor of the retina that occurs predominantly in young children.


Links


References

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Timeline

Weeks 2-3

  • Allocate sections to group members (See above)
  • Research topic and headings

Weeks 3-6

  • Write and format Wiki
  • Citation and reference
  • Use 'edit' and 'discussion' to make comments/ explanations

Week 7

  • Review and edit project page as allocated

Week 9-10

  • Monitor and review all the edits
  • Write group reflection
  • Download all the edits made during the 'edit' period

Week 11

  • Submit final wiki version by WEDNESDAY


2009 Group Projects

Group 1 Meiosis | Group 2 Cell Death - Apoptosis | Group 3 Cell Division | Group 4 Trk Receptors | Group 5 The Cell Cycle | Group 6 Golgi Apparatus | Group 7 Mitochondria | Group 8 Cell Death - Necrosis | Group 9 Nucleus | Group 10 Cell Shape