My humble pranaams to the lotus feet……

The Cell Cycle

•Complex system
•Components are identified
•Highly regulated
•Defined parameters
Cell Cycle Characteristics

•Temporally ordered events

•Irreversibility
•Oscillations
•Checkpoints
•Positive and negative feedback loops
Cell Cycle
 Proliferating Cells-Brdu

+ Retinoic acid

DAPI / BrdU / Nestin / Merge

 Cell Cycle Times

• Many human cell types have cell cycle times of ~1 day

• Mitosis is usually the shortest phase (usually ~1 hour)
• S phase is usually 1/3 to 2/3 of cell cycle (can't be too short)
                                  

Figure 14.4. Determination of cellular DNA content A population of

cells is labeled with a fluorescent dye that binds DNA. The cells are then
passed through a flow cytometer, which measures the fluorescence
intensity of individual cells. The data are plotted as cell number versus
fluorescence intensity, which is proportional to DNA content. The
distribution shows two peaks, corresponding to cells with DNA contents
of 2n and 4n; these cells are in the G1 and G2/M phases of the cycle,
respectively. Cells in S phase have DNA contents between 2n and 4n and
are distributed between these two peaks.
 Cell Cycle Analysis By Flow Cytometer
G0-G1- 63.7%

A G2-M-15.4%
S-20.9%
G0-G1- 80.5%
G2-M-9.5%
B
S-20.9%

G0-G1- 80.0%
G2-M-9 %

C S-11%
G0-G1- 80.4%
G2-M-8.4% D
S-11.2%
 Regulatory Mechanisms
Restriction point or G1/S Transition
Regulated by protein phosphorylation,
protein degradation, and inhibitory proteins

G2/M Transition - Entry into M phase

Regulated by protein phosphorylation

Anaphase onset - Exit from M phase

Regulated by protein degradation
S. cerevisiae (budding yeast)
Cell Cycle - Regulation by Extra cellular signals S.cerevisiae.

G1: Gap 1, Varying time

S: DNA synthesis
G2: Gap 2 Nutrients

M: Mitosis
Mating
factors

Cell size
 Cell Cycle Regulation

2 3

Key cell cycle transitions:

1. Restriction point (in late G1; regulates S phase onset)
2. G2 - M transition (in late G2, regulates M phase onset)
3. Metaphase-anaphase transition (regulates exit from M)
 Cell cycle checkpoints
• Cell cycle checkpoints prevent entry in to the next phase of the cell
cycle until the events of the preceding phase have been completed.
• These checkpoints sense unreplicated or damaged DNA and
coordinate further cell cycle progression with the completion of DNA
replication or repair.
• For example, the checkpoint in G2 prevents the initiation of mitosis
until DNA replication is completed.
• This G2 checkpoint senses unreplicated DNA, generates a signal that
lead to cell cycle arrest.
• Operation of the G2 checkpoint therefore prevents the initiation of M
phase before completion of S phase, so cell remain in G2 until the
genome has been completely replicated.
 Cell cycle checkpoints
• Arrest at the G1 checkpoint allows repair of the
damage to take place before the cell enters S
phase.
• The S phase checkpoint provides continual
monitoring of the integrity of DNA to ensure that
damaged DNA is repaired before it is replicated.
• A complex of sensor proteins binds to damaged
DNA and activates the ATM and ATR protein
kinases. These inturn phosphorylate and activate
the checkpoint 2 and checkpoint 1 kinases leading
to cell cycle arrest.
 Role of P53 in G1 arrest
• In mammalian cells arrest at the G1 check point is
mediated by additional protein known as P53 which is
phsophorylated by ATM and ChkP2.
• Phsophoyrlation stabilizes p53, p53 is a trascriptional
factor and – an increased expression lead to induction of
target genes that induces, cell cycle arrest.
• Loss of p53 function is usually found in human cancers
due to mutation.
• Spindle check point monitors the alignment of
chromosomes on the mitotic spindle to ensure that a
complete set of chromosomes are distributed accurately to
the daughter cells.
 Restriction of DNA Replication
• DNA replication is restricted to once per cell cycle
by MCM protein that bind to origins of replication
together with ORC (origin replication complex)
protein and are required for the initiation of DNA
replication.
• MCM proteins are only able to bind to DNA in
G1, allowing DNA replication to initiate in S
phase.
• Once initiation has occurred, the MCM proteins
are displaced, so that replication cannot initiate
again until after mitosis.
 Role of P53 in G1 arrest
induced by DNA Damage
X-rays
P-tyr15

cdk2
strand
ATM p53 p21
break
cyclin E

p21 = CKI class (cyclin dependent kinase inhibitors)

N-terminal of p21 forms complex with cyclin / cdk - inhibit
kinase
 DNA Damage - Cell Cycle Arrest
damage dependent checkpoints
G1 - S - G2 G1 - S - G2

CELL
No. wild-type

DNA content DNA content

X-ray treated
asynchronous G1/S block loss of G1/S in
p53 deficient
G2/M block cells
(6-9 hours)
 Cell Cycle Checkpoints
• Checkpoints are “quality control” steps in the cell cycle
that determine if the cell is prepared to progress to the
next stage of the cell cycle and ensure that one cell cycle
phase has been completed before moving to the next

• Questions asked at checkpoints:

Environment good? (G1)
Growth factors present? (G1)
DNA damage? (G1, S, G2)
DNA replicated? (S)
DNA replication errors? (G2-M)
Mitotic spindle properly formed? (G2-M)
Chromosomes attached to spindle? (anaphase transition)

• Checkpoints are enforced by regulation of CDK activities

MPF: M-phase Promoting Factor

 MPF was first isolated from the cytoplasm of

maturing xenopus oocytes.
 The concentration of MPF was found to vary
dramatically during oocyte development.
 Injection of cytoplasm or purified MPF into
oocytes arrested in G2 caused the oocytes to
proceed through mitosis.
MPF: M-phase Promoting Factor

 Identification of MPF

1971- Yoshio M. and Clement M., Dennis S. Robert E.,

 Mitotic CDK (MPF) Activation
Cyclin-Cdk kinase activity

Cyclin protein level

Cyclin-dependent kinase (CDK) protein level

G2 M

1. CDK protein level changes little during the cell cycle

2. Mitotic cyclin levels rise in G2 and reach a critical threshold
3. Active CDK/cyclin complex yields a peak of kinase activity
4. CDK complex phosphorylates proteins and drives G2 to M
5. Cyclin is degraded to inactivate CDK and permit exit from M
 Second Approach understanding Cell Cycle Regulation

Schizo saccharomyces pombe- Paul Nurse characterized this

mutants
 Regulators of Cell Cycle Progression

FROM YEAST TO MAN: cdks

conservation of sequence across evolution
• cdk1 = G2/M transition
• fission yeast (S pombe) = cdc2
• budding yeast (S cerevisiae) = cdc28
• man = cdc2 homolog; p34
• cdk2 = G1/S transition
• fission yeast (S pombe) = cdc2 (S form)
• budding yeast (S cerevisiae) = cdc28
• man = cdk2; p33 - PSALRE motif
 Third line of investigation- In sea Urchin embryo

MPF activity is dependent upon

Cyclin B

 Accumulation and degradation of cyclins

MPF: M-phase Promoting Factor

 MPF is composed of two key subunits:

Cdc2 and Cyclin B.
– Cdc2 is the protein that encoded by genes
which are required for passage through
START as well as for entry into mitosis.
– Cyclin B is a regulatory subunit required for
catalytic activity of the Cdc2 protein kinase.
MPF: M-phase Promoting Factor

 Structure of MPF

Molecular Characterization of MPF from frog egg-

James Maller
MPF activity is dependent upon
Cyclin B

 The cyclins were identified as proteins that

accumulate throughout interphase and are
rapidly degraded toward the end of mitosis.
 It is suggested that they might function to
induce mitosis, with their periodic
accumulation and destruction controlling
entry and exit from M phase.
MPF regulation

 Cdc2 forms complexes with cyclin B during

S and G2.
 Cdc2 is then phosphorylated on threonine-
161, which is required for Cdc2 activity, as
well as on tyrosine-15 (and threonine-14 in
vertebrate cells), which inhibits Cdc2
activity. Dephosphorylation of Thr14 and
Tyr15 activates MPF at the G2 to M
transition.
 MPF activity is then terminated toward the
end of mitosis by proteolytic degradation of
cyclin B.
MPF regulation
 Demonstration
of regulation of
MPF
Families of Cyclins and Cyclin-
Dependent Kinases

 Both Cdc2 and cyclin B are members of large

families of related proteins, with different
members of these families controlling progression
through distinct phases of the cell cycle.
 The cell cycles of higher eukaryotes are controlled
not only by multiple cyclins, but also by multiple
Cdc2-related protein kinases. These Cdc2-related
kinases are known as Cdk's (for cyclin-dependent
kinases). Cdc2 is also known as Cdk1, with others
being designated Cdk2 – Cdk8.
 CDKs and Cell Cycle

Diagram shows
CDK activity
levels during the
human cell cycle

• Human have multiple cyclins and multiple CDKs

• Specific cyclin-CDK complexes are active during specific
cell cycle stages (CDK complexes are not active in G0)
Families of Cyclins and Cyclin-
Dependent Kinases

 In yeast, passage through

START is controlled by
Cdc2 in association with
G1 cyclins (Cln1, Cln2,
and Cln3). Complexes of
Cdc2 with distinct B-type
cyclins (Clb's) then
regulate progression
through S phase and
entry into mitosis.
 Families of Cyclins and Cyclin-
Dependent Kinases
 In animal cells, progression through the G1
restriction point is controlled by complexes of
Cdk4 and Cdk6 with D-type cyclins. Cdk2/cyclin E
complexes function later in G1 and are required
for the G1 to S transition.
Cdk2/cyclin A complexes are
then required for progression
through S phase, and
Cdc2/cyclin B complexes
drive the G2 to M transition.
 Temperature-sensitive mutations
These are mutations that give rise to mutant proteins that
exhibit an increased (or decreased) sensitivity to temperature.

For example, a change from one amino acid to another at a

critical location in the protein can result in a protein that
is unstable at 37oC, the “nonpermissive temperature”.
Thus if that protein is essential for normal cell growth,
then the cell will not grow. Reducing the temperature to 30oC,
the “permissive temperature”, may allow the cell to grow.

Cdc mutants fail to progress through the cell cycle;

wee mutants are defective in proteins that prevent
cells from dividing prematurely (check points).
 Loss of the kinase activity

The wee cell

20-12: Recessive and dominant cdc2 mutants have opposite phenotypes.

Recessive cdc2- mutants cannot enter mitosis at the non-permissive
temperature and thus appear as elongated cells with a single nucleus.
The dominant cdc2D mutant enters mitosis prematurely, and is thus “wee”.
Four molecular mechanisms that
regulate Activities of Cdk's

 Mechanisms of Cdk regulation

CAK-cdk-activating
kinase
Cdc25 family-
phosphatases
Regulation By
 CDK Inhibitors (CKIs)
p27
Inhibitory
Domain

Cyclin A

CKIs bind to and inactivate CDK/cyclin complexes

 Function of Tumour Suppressor
gene
3. Regulate cell cycle :
– Rb gene: that inhibits the cell cycle in the G1 phase
decrease cell proliferation.
– INK-4 gene: that produces P16 that inhibits
cdk4/cyclin D action ( to phosphorylate Rb gene to
inactivate it’s action)
– P53: that produces P21 that has the same action of
P16 in inhibiting the action of cdk4/cyclin D
Cell cycle regulation of Rb and E2F In its underphosphorylated form,
Rb binds to members of the E2F family, repressing transcription of E2F-
regulated genes. Phosphorylation of Rb by Cdk4, 6/cyclin D complexes
results in its dissociation from E2F in late G1. E2F then stimulates
expression of its target genes, which encode proteins required for cell
cycle progression.
 Regulate cell cycle
P16
P
Cdk4/cyclin D
Rb Rb
Rb inactive

G1
G1 S
S

M G2 M G2

Cell Cycle Blocked Cell Cycle Proceeds

 E2F: Transcriptional Factor

• E2F refers to a family of at least five

distinct transcriptional factors. It is
noteworthy that only three members of E2F
interact with Rb. The other two E2F’s may
instead be regulated by other Rb-related
proteins. However the interaction of E2F by
Rb represent a key mechanism responsible
for controlling cell cycle progression.
Inhibitor of Cell Cycle Progression

 A variety of signals act to inhibit cell cycle

progression.
 The effects of inhibitory signals are also
mediated by regulators of the cell cycle
machinery, frequently via the induction of
Cdk inhibitors.
Inhibitor of Cell Cycle Progression

 DNA damage results in the elevation of

intracellular levels of p53, which activates
transcription of the gene encoding the Cdk
inhibitor p21. In addition to inhibiting cell
cycle progression by binding to Cdk/cyclin
complexes, p21 may directly inhibit DNA
synthesis by interacting with PCNA (a
subunit of DNA polymerase d).
Inhibitor of Cell Cycle Progression

 Induction of p21 by
DNA damage
Inhibitor of Cell Cycle Progression

 A complex of checkpoint proteins recognizes

unreplicated or damaged DNA and activates
the protein kinase Chk1, which phosphorylates
and inhibits the Cdc25 protein phosphatase.
Inhibition of Cdc25 prevents dephosphorylation
and activation of Cdc2.
Inhibitor of Cell Cycle Progression

 Control of the G2 checkpoint

 Tumor Suppressor p53
p53 gene encodes a 53 kDa protein

p53 protein functions as a transcription factor

p53 responds to DNA damage - “guardian of the genome”

p53 can arrest cell cycle progression or promote apoptosis

About 50% of human tumors contain mutations in p53 gene

Most point mutants map to DNA-binding regions in p53

(inability to bind DNA causes loss of tumor suppressor activity)

Some DNA tumor virus oncoproteins bind to and inactivate p53

 p53 Function
• Mdm2 binds to p53,
causing its degradation
• DNA damage triggers
phosphorylation of p53
(stable and active)
• p53 induces expression
of p21, a CDK inhibitor
• p21 arrests the cell
cycle, which allows
repair of DNA damage
• p53 can also trigger
apoptosis rather than
cell cycle arrest
Stages in Carcinogenesis

p16 - a CDK inhibitor

p53 - tumor suppressor
 MPF and progression to Meta phase

Targets of MPF MPF induces multiple nuclear and cytoplasmic changes

at the onset of M phase, both by activating other protein kinases and by
phosphorylating proteins such as condensins and the nuclear lamins.
Dissolution of the nuclear lamina The nuclear lamina consists of a
meshwork of lamin filaments. At mitosis, Cdc2 and other protein kinases
phosphorylate the lamins, causing the filaments to dissociate into free
lamin dimers.
Meta Phase Spindle
Model for control of entry into anaphase by APC-regulated
degradation of the cohesin link betweensister chromatids.
Fig. 3-29:
a) The ubiquitin-mediated
proteolytic pathway.

Ub is a small peptide
that is activated and
then attached to a
target protein, or to itself
on a protein.

b) A model of the proteasome.

It recognizes ubiquitinated
proteins and degrades them
to constituent peptides.
MCB Fig. 13-9
 Cytokinesis in animal cells

During cytokinesis in animal cells the cell membrane squeezes together

around the middle of the cell.
• The cytoplasm forms into two cells mediated by contractile ring of actin and
myosin two filaments that forms beneath the plasma membrane.
• Each daughter cell gets about 1/2 of the organelles.

Cytokinesis in plant cells

• Because the plant cell has a cell wall unlike the animal cell, it can’t squeeze
together like the cell membrane can.
• Instead a structure called the cell plate forms across the middle of the cell and
the cell gradually splits into two cells.
• Then two new cell wall forms.
Overview of eukaryotic cell cycle regulation
 Cell Cycle Analysis By Flow Cytometer
G0-G1- 63.7%

A G2-M-15.4%
S-20.9%
G0-G1- 80.5%
G2-M-9.5%
B
S-20.9%

0-G1- 80.0%
G2-M-9 %

C S-11%
G0-G1- 80.4%
G2-M-8.4% D
S-11.2%
 Checkpoint and Core Layers

Cytokine sis in animal cells

Checkpoint layer - collects information about environment,

cell (e.g, DNA) damage, and completion of cellular processes
Core layer - CDK and APC activities drive progression of cell
cycle (thick arrows) and are regulated by checkpoints
 Acknowledgement
I thank Mr. Nawaz S.S. for all the help
towards this presentation
I thank Dr. Manjula for the academic help
towards this lecture.
Thanking you