Lesson: Cell Cycle: Regulation Lesson Developer: …...Cell Cycle - Regulation Institute of Lifelong Learning, University of Delhi 2Introduction An understanding of the mechanisms

Cell Cycle - Regulation

Institute of Lifelong Learning, University of Delhi 0

Lesson: Cell Cycle: Regulation

Lesson Developer: Rama Sisodia

College/ Department: Department of Botany, Maitreyi College, University

of Delhi



Table of Contents

Chapter: Cell Cycle - Regulation

Introduction

Cell cycle regulation by internal check points

G1 check point

G2 check point

M check point

Regulator molecules of the cell cycle

Cyclin dependent kinases

Identification

Regulation

Classes of Cdks and Cyclins

Cdk regulation of M phase

Extracellular signals

Checkpoint control

DNA damage induced check points

Spindle assembly check point

Regulation of meiosis

Fertilization

Summary

Exercise/ Practice

Glossary

References/ Bibliography/ Further Readings



Introduction

An understanding of the mechanisms that control cell cycle is important not only for basic

biology but also since it underlies the basis of studying cancer- the uncontrolled proliferation

of cells. The transition from one phase to other of the cell cycle is very well coordinated and

is under the control of extracellular signals as well as certain internal signals.

Cell cycle regulation by internal checkpoints

The progression of the cell cycle is regulated by check points in response to specific

signals.

Figure: Cell cycle check points

Source: http://setarosite.org/images/biobook_images/Checkpoints1.jpg

G1 check point

o Start a regulatory check point characterized in budding yeast Saccharomyces

cerevisiae that occurs in G1 and controls the transition from G1 to S in

response to signals like presence of nutrients, cell size and mating factors.

http://setarosite.org/images/biobook_images/Checkpoints1.jpg



Figure: Cell cycle regulation in Saccharomyces cerevisiae

Source: http://commons.wikimedia.org/wiki/File:Ch14f5a.jpg

Similarly in animal cells a restriction point regulates the progression of cell

cycle from G1 to S phase is controlled by extracellular signals like growth

regulators. When the required growth factors are present the cell cycle

progresses beyond the restriction point into the S phase. However in the

absence of appropriate growth factors the cells enter a quiescent stage- Go and

remain metabolically active but stop dividing and have reduced size. On

availability of suitable growth factor these regain their ability to divide and

reenter the cell cycle.

Figure: Regulation of cell cycle in animal cells in response to growth factors

Source: http://upload.wikimedia.org/wikipedia/commons/c/c2/Ch14f6.jpg

http://commons.wikimedia.org/wiki/File:Ch14f5a.jpg

http://upload.wikimedia.org/wikipedia/commons/c/c2/Ch14f6.jpg

http://commons.wikimedia.org/wiki/File:Ch14f5a.jpg



G2 check point

In contrast to these examples cell cycle is regulated by a check point that exists

in the G2 phase and controls the progression into the M phase as seen in fission

yeast Schizosaccharomyces pombe monitored by appropriate cell size and

availability of nutrients. Similarly in vertebrate oocytes the control of cell cycle in

G2 phase is seen in response to hormones and the cells remain arrested in G2

phase until triggered to divide.

M check point

A checkpoint exists at the end of the mitosis – the spindle assembly checkpoint

that monitors the assembly and alignment of the spindle and therefore ensures

that a complete set of chromosomes are distributed between the two newly

formed daughter cells.

In addition to these, several checkpoints in the G1, S, and G2 exist to ensure integrity of

genome i.e. detect the presence of damaged and unreplicated DNA and coordinate the

recruitment of repair machinery to these sites.

Figure: The three main cell cycle checkpoints

Source: ILLL Inhouse

Animation: http://outreach.mcb.harvard.edu/animations/checkpoints.swf

http://sites.sinauer.com/cooper5e/animation1603.html

Regulator molecules of the cell cycle

http://outreach.mcb.harvard.edu/animations/checkpoints.swf



Cyclin dependent kinases

Studies in numerous organisms like yeasts, frogs and mammals etc. have revealed that the

progression of cell cycle is controlled by a conserved set of proteins called kinases. These

regulate the progression of cell cycle in association with another group of proteins called as

cyclins. The concentration of cyclins fluctuates throughout the cell cycle while that of Cdk

remain constant.

Figure: Cyclin concentration varies with

Source: http://upload.wikimedia.org/wikipedia/commons/0/08/Ch14f14.jpg

Infact there is a direct correlation between the concentration of cyclin and the three main

checkpoints of the cell cycle.

Figure: Variation in the concentration of cyclin with reference to the stage of the cell cycle

http://upload.wikimedia.org/wikipedia/commons/0/08/Ch14f14.jpg



Source: OpenStax College, http://cnx.org/content/m44466/1.3/

Source: http://sgugenetics.pbworks.com/f/1269908653/cyclinCDK.jpg

Identification

Molecules that control the progression of cell cycle were initially identified from cell fusion

experiments. When cultured mammalian cells arrested in different stages of cell cycle were

fused to form heterokaryons (a single cell with two nuclei) the progression to the next stage

was possible indicating the presence of molecules controlling the progression to the next

stage( Rao and Johnson, 1970). For example a fusion of a cell in S phase with a cell in G1

phase resulted in a heterokaryon which showed transition to S phase and the inititation of

DNA replication indicating the S phase cells contained the molecules that triggers the

progression from G1 to S phase.

In 1971, investigations involving frog oocytes ( Masui,Y. and Markert, C.; Smith,D and

http://cnx.org/content/m44466/1.3/

http://sgugenetics.pbworks.com/f/1269908653/cyclinCDK.jpg



Ecker,R) arrested in G2 showed that these cells showed transition to M phase upon either

hormonal stimulation or even on injecting cytoplasm from hormonally stimulated oocytes.

This cytoplasmic factor was called as maturation promoting factor (MPF) since it

stimulated the maturity of oocytes. In somatic cells MPF induces the dividing cells to enter

into the mitotic phase.

Figure: Experiment demonstrating the role of cytoplasmic factor (MPF) in inducing cell

cycle progression

Source: http://commons.wikimedia.org/wiki/File:Ch14f12.jpg

In budding yeast Saccharomyces cerevisiae, the role of MPF was highlighted in studies

involving temperature sensitive mutants that were unable to proceed forward in cell cycle

http://commons.wikimedia.org/wiki/File:Ch14f12.jpg



and were arrested at particular

r points of cell cycle. For example the cell divison cycle mutants called cdc28 were arrested

at the check point START and only in the presence of the protein Cdc28 the progression to

the next phase was achieved. Similarly in fission yeast Schizosaccaromyces pombe the

product of cdc2 gene was required for the transition of cell cycle through the G1 START and

the G2 to M transition. It was later revealed that the genes cdc 28 and cdc2 encode a

protein kinase Cdk1. Related genes were identified in higher eukaryotes including human

beings.

Cell divisions in early embryo development in sea urchin were found to be

accompanied with new protein synthesis

Periodic variation in accumulation of proteins-cyclins.

Microinjection of cyclin in frog oocytes was found to trigger G2 to M transition.

MPF purified from frog eggs in 1988, James Maller

Molecular characterization of MPF revealed that it is composes of two important

subunits-Cyclin dependent kinases (Cdk) and cyclin.

Figure: The components of MPF


Cdks are enzymes-protein kinases that phosphorylate their targets.The cyclin acts as a

catalytic subunit regulating the activity of Cdk (therefore the name- cyclin dependent

kinases). Cyclin undergoes a periodic variation in its content with an increase in

concentration coinciding with onset of M phase ( named cyclin since these undergo

cyclic variation on concentration) and rapid decrease with the termination of M phase.

Cdk is active only when it is associated with cyclin.

– Low cyclin CdK Inactive Cell cycle blocked Interphase




- Cyclin concentration increases and it associates with Cdk Cdk active

Progression of cell cycle

Regulation of Cdk

The Cdk acitivity is regulated by four molecular mechanisms

Figure: Regulation of Cdk activity

Source: http://upload.wikimedia.org/wikipedia/commons/c/c4/Ch14f18.jpg

1. The association of cyclin with Cdk

2. Phosphorylation of Cdk1 on threonine-161 that triggers its kinase activity

3. Phosphorylation of tyrosine 15 (threonine-14 in vertebrates) inhibits Cdk1 acitivity. A

protein kinase Wee1 inhibits the Cdk1 activity by phosphorylating the tyrosine 15

residue. A protein phosphatase Cdc25C deposphorylates the threonine-14 and

tyrosine -15 residue and activates Cdk1.

Mitosis

http://upload.wikimedia.org/wikipedia/commons/c/c4/Ch14f18.jpg



Figure: Regulation of mitosis by wee1 and Cdc25C

Source : ILLL Inhouse

4. Cdk activity is also controlled by their binding with inhibitory proteins- Cdk

inhibitors or CKIs.

Wee1 Cdc 25

Mitosis

Cyclin Cdk



Figure: Regulation of Cyclin-Cdk activity


cdk cyclin.swf

Animation: Regulation of Cyclin-Cdk activity.

Source:ILLL Inhouse

Classes of cyclins and cyclin-dependent kinases

There are several classes of cyclins and cyclins dependent kinases. These are the key

molecules that control transition from one phase of cell cycle to the next through the check

points:

I. Cyclins-




G1/S Cyclins – trigger the transition through the START into the S phase. Their

levels falls in the S phase.

S phase cyclins- stimulate chromosome duplication and also early mitotic events.

There levels remain high even during M phase.

M phase cyclins- regulate the G2/M checkpoint and entry into the M phase.

The concentration of these three cyclins varies at different stages of the cell cycle.

II. Cyclin dependent kinases (Cdks). There is a single cyclin identified in yeast (Cdk1)

while four are known in vertebrates:

G1 phase Cdks – Cdk4,6

S phase Cdks- Cdk2, Cdk1

M phase Cdks-Cdk1

Table : Types of Cyclins and their associated Cdks in vertebrates and yeast

Cell cycle stage Vertebrates Yeast

Cyclin Cdk Cyclin Cdk1

G1 Cyclin D Cdk 4, Cdk6 Cln3 Cdk1

G1/S Cyclin E Cdk2 Cln1,2 Cdk1

S Cyclin A Cdk2,Cdk1 Clb5,6 Cdk1

M Cyclin B Cdk1 Clb 1,2,3,4 Cdk1



Figure: Different classes of cyclins and Cdks regulate different phases of cell cycle

Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817888/figure/F2/

The target proteins of the activated cyclin-Cdk complex include a variety of proteins each

specific to the stage of cell cycle. Proper phosphorylation of these proteins is important for

the cell cycle to proceed.

Cdk regulation of M phase

Cdk1/cyclin B complex regulate several important activities in the M phase including:

Chromatin condensation – the duplicated DNA strands of the two sister

chromatids are held close by proteins called cohesins. As mitosis

proceeds another group of proteins called condensins get activated by

Cdk1/ cyclin B phosphorylation and replace cohesins. As their name

indicates these proteins are involved in the condensation of the chromatin

into metaphase chromosomes.

Nuclear envelope breakdown- the nuclear lamina, the network of

protein filaments underlying the nuclear envelope get phosphorylated by

Cdk1/cyclin B complex and depolymerize resulting in the breakdown of

the nuclear envelope.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817888/figure/F2/



Breakdown of the golgi apparatus- The phosphorylation of the golgi

matrix proteins by the Cdk1/cyclin B complex results in the fragmentation

of the golgi apparatus into small vesicles.

Spindle formation – As mitosis progresses Cdk1 activation leads to

centrosome separation and movement towards the poles. The activation

of protein kinases result in phosphorylation and depolymerisation of

microtubule proteins that make up the spindle. The rate of microtubule

turnover therefore increases and the short microtubules come to

surround the centrosomes. The nuclear envelope breaks down and the

microtubules attach to the chromosomes at their kinetochores. As the

microtubules attach to the kinetochore, the chromosomes align in the

centre.

Animation: http://sites.sinauer.com/cooper5e/animation1604.html

Extracellular signals

Extracellular signals like hormones trigger the progression of the cell cycle beyond the

restriction point that exists in the late G1 phase. In the absence of such signals the cell

enters into a quiescent phase (G0) until the presence of the signals triggers their entry into

the next phase. In response to the growth factors cyclin D are synthesized through the

signaling pathways involving Ras/Raf/MEK/ERK pathways. The cyclin D1 associates with and

activates the Cdk4/6. The concentration of cycin D1 is sensitive to the presence of the

growth factors and falls in their absence.

The target of cyclin D /Cdk4,6 is a protein Rb (Retinoblastoma). The Rb protein was

identified as a product of a gene responsible for a rare inherited eye tumour -

retinoblastoma. The Rb genes are a type of tumour suppressor genes and their

malfunctioning results in uncontrolled cell divisions.

In their unphosphorylated state the Rb proteins are associated with E2F family of

transcription factors acting as transcriptional repressors. However when Rb proteins become

phosphorylated by Cdk4,6/cyclin D complex it disassociates from E2f and thus allows for the

transcription of E2F target genes. These target genes encode proteins required for the G1 to

S phase transition . For example the synthesis of cyclin E.

http://sites.sinauer.com/cooper5e/animation1604.html



Figure: Regulation of cell cycle by Rb

Source: OpenStax College, http://cnx.org/content/m44466/1.3/

Figure: Regulation of G1 to S phase transition by Rb :E2F

http://cnx.org/content/m44466/1.3/



Check point control

DNA damage induced checkpoints

The integrity of the genetic material is monitored through the G1, S and G2 phases. The cell

cycle gets arrested in response to damaged, unreplicated DNA. This is mediated through

two protein kinases- ATM and ATR. ATM is activated in response to double strand break

while ATR recognizes single strand breaks. These activated kinases further phosphorylate

and activate CHK1 and CHK2 protein kinases respectively. CHCK1 and CHCK2 then

phosphorylate and inhibit the protein phosphatases Cdc25A and Cdc25C. The latter two are

required to activate Cdk2 and Cdk1 the important kinases required for the progression of

cell cycle. Their inhibition therefore arrests the cell cycle at the DNA damage checkpoints in

G1, S and G2.

Figure: The regulation of DNA damage checkpoints in response to DNA damage.

Source: ILLL inhouse

Spindle assembly checkpoint

The progression to anaphase is mediated by a protein complex- Anaphase Promoting



Complex (APC). The APC is a ubiquitin ligase that ubiquitinates its target leading to its

degradation.

The APC remains inactivated till the cell passes through the spindle assembly checkpoint.

It requires a protein called Cdc20 for its activation. A complex of proteins called as the

Mad/ Bub proteins attach to unattached kinetochores and cdc20 inactivating the latter. As

the cell cycle progresses beyond the spindle assembly check point the chromosomes align

on the spindle the Mad/Bub complex dissociates from the Cdc20 leading to the activation

of APC which then:

Ubiquitinates cyclin B targeting it for destruction and thereby inactivating Cdk1

allowing the cell to then exit mitosis and enter interphase.

Ubiquitinates a protein securin leading to activation of associated protein

separase. The separase degrades the protein cohesion that holds the two

sister chromatids together thereby allowing the two to separate and move to

opposite poles.

Figure: Progression through anaphase controlled by APC

http://www.nature.com/nrm/journal/v7/n9/images/nrm1988-f2.jpg

Regulation of Meiosis

The vertebrates oocytes because of their large size and ease of manipulation have been

http://www.nature.com/nrm/journal/v7/n9/images/nrm1988-f2.jpg



used as model systems to investigate meiosis.

The oocytes remain arrested for a long period in the diplotene stage where active DNA

synthesis takes place. In response to hormonal stimulation the cells proceed through and

enter meiosis I. This division is asymmetric resulting in a small polar body and a large cell-

oocyte. This involves the activation of Cdk1 and similar accompanying cellular changes as

seen in mitosis. The oocyte then arrests again in meiosis II at the metaphase II stage and

proceeds further upon hormonal stimulation again.

Interestingly the levels of Cdk1 gets reduced but not completely as the oocyte proceeds

from meiosis I to meiosis II with the chromatin remaining condensed and the nuclear

envelope remaining disintegrated. In 1971 in a experiment carried out by Yoshio Masui

and Clement Markert it was seen that the upon injection of cytoplasm of an oocyte

arrested in meiosis II into a early embryonic cell undergoing mitosis, arrested the

divisions in the embryonic cell. This indicated the presence of a cytostatic factor in the

cytoplasm since it could arrest a dividing cell. Recent experiments have shown that a

component of the cytostatic factor to be a protein serine/threonine kinase Mos.

Mos maintains the Cdk1/cyclin B activity in two ways:

Stimulating cyclin B synthesis

Inhibiting the degradation of cyclin B by APC

The levels of Cdk1/cyclin B therefore remains high arresting the progression of the cell

cycle. The arrested oocyte would then remain so till fertilization.

Fertilization

Upon fertilization of the oocyte several important changes are seen:

The Ca+2 level increase in the cytoplasm due to the hydrolysis of

phosphatidylinositol4,5-bisphosphate (PIP2).

The raised Ca+2 levels prevent other sperms from entering the egg due to

alterations in the wall.

The arrested oocyte resumes the cell cycle and proceeds beyond metaphase II.

This involves the Ca+2 dependent activation of the APC.

The oocyte meiosis completes and the fertilized egg now contains two haploid

nuclei. Each of these nuclei replicate their DNA and the zygote enters into the M

phase producing two diploid cells that divide further to give rise to the



multicelled embryo.

Summary

The cell cycle is divided into interphase (G1, S, G2) and the divisional M phase. There are

several checkpoints that regulate the transition from one phase to the other. The key

component of the control system that coordinates the progress of the cell cycle are the

cyclin dependent protein kinases. The activity of Cdks is regulated primarily by their

association with proteins-cyclins. The concentration of cyclin fluctuates during the various

phases of cell cycle while that of Cdks remains constant. When the concentration of cyclin is

low, it is not associated with Cdk and therefore the latter is inactive. Phosphorylation and

association with inhibitors (CkIs) are some of the mechanisms that control the activity of

Cdks.

Extracellular signals like hormones stimulate the synthesis of D type cyclins and the

activation of Cdk4/6 that control the movement of the cell cycle beyond the G1/S

checkpoint. The transition to metaphase is mediated by Cdk1/cyclin B complex that

phosphorylates and activates proteins involved in the M phase. These include the proteins

involved in breakdown of the nuclear membrane, condensation of chromatin, spindle

assembly etc.

The progression into anaphase is regulated by the spindle assembly checkpoint. The

assembly and the alignment of the chromosomes triggers the anaphase promoting complex.

This promotes the ubiqutination and degradation of proteins like cohesions that results in

separation of the sister chromatids and also the degradation of cyclinB promoting to

transition to anaphase.

Glossary

Anaphase: Stage during mitosis during which the sister chromatids separate and move

towards the poles. Composed of Anaphase A – chromosomes move towards the spindle

poles and Anaphase B- Spindle poles move apart.

APC: Anaphase promoting complex which is a ubiquitin ligase that catalyzes the

ubiquitylation and destruction of proteins –securin and M and S cyclins resulting in the

separation of chromatids and transition from metaphase to anaphase.

Cdk: Cyclin dependent protein kinases. These trigger the progression of cel cycle by



phosphorylating their target molecules.

Cell cycle: Reproductive cycle of a cell involving the orderly sequence of events that

includes the duplication of the genetic material and division into two cells.

Checkpoints: the mechanism that controls the progression of the cell cycle in response to

(1) availability of growth hormones/ appropriate size (2) DNA damage (3) proper spindle

assembly etc.

Chiasma (plural Chiasmata) : The X shaped connections between the paired homologous

chromosomes during the prophase stage of meiosis. These represent the sites where

crossing over (genetic recombination has occurred).

Cdk: Cyclin dependent protein kinases which are active when associated with cyclins. These

trigger the different steps of the cell cycle by phosphorylating their targets.

CKI: Cdk inhibitor proteins that bind to and inhibit the Cyclin-Cdk complexes involved in the

control of G1 and S phase.

Condensins: Complex of proteins involved in condensation of chromosomes on the onset

of mitosis.

Cyclin: a family of enzymes which vary in their concentrations in particular phase of cell

cycle. Cyclins activate important protein kinases called as cyclin dependent protein kinases

that trigger the progression of the cell cycle.

E2F protein: Gene regulatory protein that switches on many genes that encode proteins

required for entry into the S phase of the cell cycle.

Kinases: Enzymes that phosphorylate their targets.

MPF: Maturation promoting complex. Has two components Cdk1/Cyclin B and controls the

G2 and M checkpoint.

PIP2: Phosphatidyl inositol phosphate.

Protein Kinase: Enzyme that transfers the terminal phosphate group of ATP to one or

more specific amino acids for e.g. serine, threonine or tyrosine of a target protein.

Retinoblastoma protein (Rb): Tumour suppressor protein involved in the regulation of

cell cycle. Its main function is to bind and inhibit the activity of E2F genes thus binding

blocking the progression of DNA replication and cell cycle. Rb protein is mutated in the

retinoblastoma cancer.

Ubiquitin: Small conserved eukaryotic protein that becomes covalently attached to lysines

of other proteins and acts as a tag for intracellular proteolytic destruction.



Ubiquitin ligase: Enzymes that catalyze the addition of ubiquitin molecules to a protein

targeting them for destructution.

Exercises

1. Identify the different stages of cell cycle.

2. What is the difference between the cells in G0 and G1 phases?

3. What is the importance of DNA replication in cell cycle.

4. What is the effect on fusing cells :

In G1 phase with one in S phase

In G2 phase with one in M phase.

5. Briefly mention the four mechanisms which regulate Cdk activity.

6. Discuss the role of Anaphase Promoting Complex in separation of sister chromatids

during anaphase.

References

1. Campbell, N.A. and Reece, J. B. (2008) Biology 8th edition, Pearson Benjamin

Cummings, San Francisco.

2. Raven, P.H et al (2006) Biology 7th edition Tata McGrawHill Publications, New Delhi

3. Karp, G. 2010 Cell and Molecular Biology: Concepts and Experiments. 6th edition. John

Wiley & Sons. Inc.

4. De Robertis, E.D.P. and De Robertis, E.M.F. 2006 Cell and Molecular Biology. 8th edition.

5. Cooper, G.M. and Hausman, R.E. 2009 The Cell: A Molecular Approach. 5th edition. ASM

Lippincott Williams and Wilkins, Philadelphia.Press & Sunderland, Washington, D.C.; Sinauer

Associates, MA.

6. Becker, W.M., Kleinsmith, L.J., Hardin. J. and Bertoni, G. P. 2009 The World of the Cell.

7th edition. Pearson Benjamin Cummings Publishing, San Francisco.

Suggested Readings

1. Enders,G.H. (2010) Cell Division 5:12.Gauchos and ochos: a Wee1-Cdk tango regulating

mitotic entry.

2. Yang et al. 2006. Cell Division 1: 32 Variations in cyclin D1 levels through the cell cycle

determine the proliferative fate of a cell.

Web Links

1. http://www.nature.com/scitable/ebooks/essentials-of-cell-biology-

14749010/14759908

http://www.nature.com/scitable/ebooks/essentials-of-cell-biology-14749010/14759908

http://www.nature.com/scitable/ebooks/essentials-of-cell-biology-14749010/14759908



2. http://www.sciencedirect.com/science/article/pii/S1357272596001781#

3. http://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=9&cad=rja&ved=0

CG8QFjAI&url=http%3A%2F%2Fbiology.buffalo.edu%2Fcourses%2Fbio404%2Fberezn

ey%2Farticles%2FLodish_Chapter_13_p495_533.pdf&ei=PXVnUp7YBsWRiQeo5oD4B

g&usg=AFQjCNGP_YL30vp98ksnPCzdvKwe_yLK1A

http://www.sciencedirect.com/science/article/pii/S1357272596001781

http://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=9&cad=rja&ved=0CG8QFjAI&url=http%3A%2F%2Fbiology.buffalo.edu%2Fcourses%2Fbio404%2Fberezney%2Farticles%2FLodish_Chapter_13_p495_533.pdf&ei=PXVnUp7YBsWRiQeo5oD4Bg&usg=AFQjCNGP_YL30vp98ksnPCzdvKwe_yLK1A




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Lesson: Cell Cycle: Regulation Lesson Developer: …...Cell Cycle - Regulation Institute of Lifelong Learning, University of Delhi 2Introduction An understanding of the mechanisms