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Cell cycle and checkpoint control
2-16-2016
How do we follow the cellular processes during cell division?
Under microscopy!
Mitosis: chromosome condensation and segregation.
Time to complete one cell division cycle.
How about DNA replication?
Drawing of chromosomes during mitosis by Walther Flemming, circa 1880
When DNA is replicated?
How to measure DNA
replication during cell
division?
Pulse label H3-thymidine in nonsynchronized cell population, then
measure the kinetics of
appearance of labeled mitotic cell!
Labeled nuclei
Labeled
mitotic cell
1-hour radioactive thymidine ( ) pulse (DNA synthesis).
4% of cell nucleus are labeled. So, interphase has at least 2
phases (?). Can we see labeled mitotic cell in this experiments?
After pulsing, no
labeled mitotic cell was
observed.
When will you see the
first labeled mitotic cell?
Why then the number of
labeled mitotic cell
increase with time?
Why the number of
labeled mitotic cell
decrease then?
G2
Tcell cycle
Ts
G2
TG1 = Tcell cycle – Ts – TG2 - Tmitosis
How to identify control genes of cell cycle in
eukaryotes?
Experimental approaches to identify control
genes of cell cycle in eukaryotes:
— Genetic approach
Yeast CDC (cell division control) genes
— Biochemical approach
Xenopus oocytes
Brief history of career development of
Dr. Leland H. Hartwell
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High school – sports, girls and car
Make up at a two years junior college
BS in physics from Caltech in 1961
Ph.D. in biology from MIT in 1964
Post doctor of Dr. Renato Dulbecco (Nobel
Laureate in 1975 with Temin and Baltimore)
• Faculty of UC Irvine 1965-1968: from cancer to
yeast
• Faculty of U of Washington, Seattle 1968-
Genetic approach to study the regulation of macromolecular
biosynthesis (protein, RNA and DNA)
1, to pick up a good model system.
2, to isolate conditional mutant such as temperature sensitive
mutant (ts) which grows normally in low (permissive)
temperature but no longer grow at high (non permissive)
temperature.
3, to examine defect occurred in the mutant: DNA, RNA or
protein synthesis
4, to identify mutated gene which is responsible for
the phenotype of the mutant.
5, to understand how the gene works (biochemical approach).
Why budding yeast?
• Budding yeast is a
good model to study
cell cycle control!
Because..
• It’s has defined cell
cycle events.
• It’s easy to follow cell
cycle progression by
monitoring the ratio of
bud size to parent cell
size.
• Yeast has both
haploid and diploid
life cycle
• It’s easy to isolate
recessive mutant in
haploids and to
perform
complementation test
in diploids.
How to isolated Temperature sensitive mutants of yeast
Mutagenize Cells
(i.e. EMS)
Plate Yeast Cells
Make
Replica
Plates
Grow Cells at
2 Temperatures
Permissive
temperature
Restrictive
temperature
Permissive
Restrictive
Most of ts mutant defect in macromolecular
biosynthesis stop growing at non-permissive
temperature with normal cell morphology
What’s wrong about this?
Too many mutants to be examined in detail.
Most mutated genes are “bore” (house keeping) genes!
Until a undergraduate student Brain Reid came…
The Behavior of a Temperature Sensitive cdc Mutant
cdc mutant growing
at permissive temp (23C)
cdc mutant growth arrested after
6 hrs at non-permissive temp (36C)
All mutant cell has similar staining pattern of their chromosome
indicates they may be arrested at the same stage of cell cycle!
Those mutants may be cell cycle control mutants!
Cdc Mutants Arrest at the Same Cell Cycle Phase
Permissive (low) temperature
Restrictive (high) temperature
Isolation of temperature sensitive mutants of cell
cycle control in budding yeast
• Total 150 ts mutants with unique morphology were
isolated.
• Do they carry mutations on the same or different genes?
– Complementation test to define how many mutated genes
could be find!
• Total 32 complementation groups (gene) were identified!
How to determine the phenotype of the cdc mutants?
• To shift temperature from 23C to 36C when cells just
separated after mitosis.
• The initial defect is defined as the first cell cycle event that
fails to occur at 36C.
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Initiation of DNA synthesis (iDS)
Bud emergence (BE)
DNA synthesis (DS)
Medial nuclear division (mND)
Late nuclear migration (lND)
Cytokinesis (CK)
Cell separation (CS)
How are the events coordinated in the yeast
cell cycle so that their sequence is fixed?
Two models to account for the ordering of cell
cycle events
• Dependent pathway: product vs substrate.
• Independent pathway: signals from one single timer.
Two models to account for the ordering of cell
cycle events
• Dependent pathway: product vs substrate.
• Independent pathway: signals from one single timer.
How to distinguish two cell cycle events
are dependent or independent?
To block one gene by ts mutation and to see
whether the following event will continue
(independent) or stop (dependent)?
Six events comprise a dependent pathway of cell cycle progression
Six events comprise a dependent pathway of cell cycle progression
CS CK lND mND DS IDS
A common step (cdc28) controls both pathways
Properties of S. cerevisiae cdc28 (START) Mutants
What Start is doing?
Very short
period for
mitosis
Regulation of Animal Cell Cycles by Growth Factors
Mating factor
This model system allows scientist to work out biochemical
function of each gene product involved in cell cycle regulation for
the following 30 years!
Genetic Control of the Cell Division Cycle in Yeast
A model to account for the order of cell cycle events is deduced
from the phenotypes of yeast mutants
LH Hartwell, J Culotti, JR Pringle, BJ Reid
Science 183: 46-51; 1974
Schizosaccharomyces pombe
(Fission Yeast)
S. pombe (the fission yeast) has a well defined process for mitosis.
Cell Cycle of Fission Yeast
Two kinds of yeast
cdc2ts
Cdc2wee2
LOF mutations in CDC2 fail to enter mitosis and become large
GOF mutations are wee-initiate mitosis prematurely, get small
From Susan Forsburg, Salk Institute
A dominant wee mutant turned out
to be an allele of CDC2
Genetic model in fission yeast
G1
S
G2
CDC28
Genetic model in budding yeast
What is function of cdc2 and CDC28 in cell cycle control?
Evans et al (1983) Cyclin: a protein specified by maternal mRNA in sea
urchin eggs that is destroyed at each cleavage division. Cell 33: 389-396.
System: the sea urchin Arbacia punctulata. After fertilization, it undergoes 8
very rapid cell divisions, which require continual protein synthesis.
Very simple experiment: add 35-S-methionine to eggs. Then extract proteins
and examine the synthesis of "new" (radiolabeled) protein.
Cleavage index
“Protein A” rises and
falls in advance of
each cycle of cell
division.
Hunt names the
protein “cyclin.”
Evidence for cytoplasmic signals in
cell cycle regulation
South African clawed frog (Xenopus laevis)
and its big eggs
Evidence for the existence of soluble factors
in the Xenopus oocyte to trigger meiosis
“Maturation Promoting Factor” (MPF) in the
cytosol of mature egg!
The oscillation of MPF activity
during the cell cycle
interphase
mitosis
interphase
MPF
activity
Seen in all eukaryotic cells
mitosis
interphase
Further purification work showed that MPF consisted of a kinase
subunit and a regulatory subunit – cyclin.
Cdk: Cyclin-dependent protein kinase
Targets of MPF
A simplified view of the
core of the cell cycle
control system: dynamic
level of cyclin proteins
determine whether the
Cdk is active or not!
Synthesis and
degradation of specific
cyclins become another
key control point!
How to make sure the cell cycle
progression is in a right process?
What happen if some key events of
cell cycle is going wrong?
DNA damage will delay mitosis: why and how?
Two possible models
Some control genes to check!
A few mutants of yeast have been
identified to relief dependence of
ordered events of cell cycle
Hartwell proposed “checkpoint” for
these control mechanisms
Dependence of mitosis on DNA synthesis
• cdc9: DNA ligase
– Cell cycle arrest at G2/M at non-permissive temperature
• RAD9 was isolated as mutant that permit cell division of cells with DNA
damaged induced by x-irradiation
– Should be viable at high temperature.
– Has higher ( x21) rate of chromosome lost than the wild type cells
• Rad9 mutant can relief cdc9 induced mitotic arrest
RAD9 is a checkpoint of cell cycle progression
What happen when RAD59 mutation relief dependence of mitosis on DNA synthesis?
In the absence of RAD9, DNA ligase deficient cells die much more
rapidly at the non-permissive temperature.
Mitotic catastrophe!
Is checkpoint essential?
• For somatic cells
• No, but it is very important for normal cells!
• No checkpoint makes low fidelity in DNA
replication and separation during cell
division.
• Accumulation of somatic mutation and
aneuploidy causes cancer formation!
Is checkpoint essential?
• For embryonic cell cycle
• Absolute no! because….
Why embryonic cell cycle don’t need
checkpoing?
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What’s wrong to have checkpoint?
Synchrony and speed of cell division is the priority!
How to handle “damaged product-defective cells”?
When cell divisions slow down (mid-blastula transition in
Xenopus and division 14 of Drosophla), abnormal nuclei may
fall into the interior of the egg and do not contribute to the
larval cells.
• The Drosophila embryo has capacity to replace lost nuclei
Cell Cycle Checkpoints
Why always negative regulation?
A Mechanism Involving Negative Regulation is More Robust
Very different
situations
Anaphase:
Not very
different
situations
Anaphase:
GO x 5
GO x 6
How do Cell Cycle Checkpoints Work?
Zhou and Elledge Nature 408, 433 - 439 (2000)