You may not believe it but by the end of the semester
This will make sense!
Hanahan and Weinberg, Cell 100:57-70 (2000)
Cell cycle and its control
Cells must be able to proliferate
- during development
- wound healing
- stem cells in blood, small intestine, immune system
For cells to copy themselves they need to:
- Grow; make more stuff; e.g. proteins, lipids
- Copy their genetic material
- Segregate contents to daughter cells, especially…
- Segregate replicated chromosomes to daughter cells
The Cell Cycle
Interphase
cells duplicate chromosomes
Mitosis
cells segregate duplicated chromosomes into
two daughter cells
Many of the images in the cell cycle part of the course are taken from The Cell Cycle , by David O Morgan (New Science Press)
Restriction Point /
START
Interphase has 3 periods: G1, S, G2
G1: cells decide whether to divide or not:
- Have I grown big enough to enter the cell cycle?
- Am I OK?
Execution of these decisions commits a cell to
complete a full division cycle
S: chromosomes are duplicated
G2: cell prepare to enter mitosis by asking:
- Have I completed DNA synthesis properly?
- Am I OK?
The main jobs of the cell cycle:
1. To accurately transmit the genetic information!
2. To maintain normal ploidy; i.e. diploidy!
Regulatory mechanisms:
- Accuracy in the “assembly line” (e.g. DNA
polymerase)
- Extrinsic regulatory mechanisms (all processes
follow a correct order)
Let’s remind ourselves some basic stuff
Starting with the S phase
Helicase
Early G1
Pre-replicative complex
(origin licensing)
Early S
Activation of
helicase;
Assembly of
pre-initiation
complex
DNA does not come naked
It is packed into chromatin
Mainly, histone proteins
Thus, duplicating chromosome
= duplicating DNA and
duplicating histones
In addition, we need to repack
the duplicated DNA
Histone synthesis increases sharply during the S phase
Increase in transcription, in processing, and in stability
Chromatin Inheritance
Reproducing chromatin organization during
the S phase
- Telomeres
Cis-elements: sequences recruiting proteins that
modify histones
- Centromere
Epigenetic mechanisms, not clearly understood
Mitosis
During the S phase, the
duplicated DNA is rearranged
through cohesion to form
two sister-chromatids
attached to each other by
cohesins
Gradually, the cohesins will
be removed to allow sisterchromatid separation
Prophase
- Sister-chromatids condense
- Centrosomes move to
opposite poles of the cell,
nucleating microtubules
(MTs)
- Nuclear envelope breakdown
Prometaphase
- Nuclear envelope
breakdown is completed
- The centrosomes nucleate
MTs towards each other,
forming the spindle MTs
- The growing (+) ends of the MTs capture
the chromosomes at the site of the
centromere through a protein complex
called the kinteochore
Centromere
Kinetochore
Microtubule
Kerry Bloom
Ted Salmon
Microtubule
Kinetochore
Prometaphase
- Nuclear envelope
breakdown is completed
- The centrosomes nucleate
MTs towards each other,
forming the spindle MTs
- The growing (+) ends of the MTs capture
the chromosomes at the site of the
centromere through a protein complex
called the kinteochore
At the end of the day: Metaphase
Now, we are ready for Anaphase
Anaphase (A+ B)
Salmon lab
Prophase
Mitosis
Chromatid condensation
Prometaphase
Kinetochore-MTs binding
Spindle assembly
Metaphase
Chromosomes align at the midline
Anaphase
Segregation of sister-chromatids
Telophase and Cytokinesis
Birth of two daughter cells
Silverman-Gavrila lab
Cell cycle is controlled
Cells can be fused
Rao and Johnson (1970)
Cell fusion experiments
- Fuse S phase cell with G1
cell: The G1 nucleus enters
S phase
- Fuse M phase cell with
interphase cell: Interphase
nucleus enters M
Cell cycle has a clock, regulated by
promoting factors and checkpoints
For example, anaphasemetaphase transition will take
place only if ALL the
kinetochores are attached to
MTs
If the checkpoint regulators are
compromised, unattached
chromosome might be lagging
behind, resulting in aneuploidy
G1
Cyclin Dependent Kinases Regulate the Cell Cycle
Experimental Systems Important for Cell Cycle Studies
Saccharomyces cerevisiae
Schizosaccharomyces pombe
Arbacia punctulata
Xenopus laevis
Budding Yeast: Saccharomyces cerevisiae
Budding Yeast: a genetic eukaryotic model organism
Hartwell was interested in the
protein synthesis machinery
Let’s look for mutants that cannot
synthesize proteins
Lee Hartwell
Isolating temperature sensitive mutants in
haploid yeast
Budding Yeast: Saccharomyces cerevisiae
Lee Hartwell
Brian Reid
Serendipity, our old friend
Brian Reid, an undergrad, needs to look at a
microscope to follow a mutant. They realize that bud
size stores information about the cell cycle
An assay for isolating cdc mutants
cdc: cell division cycle mutants
Permissive (low) temperature Restrictive (high) temperature
(mixed population of cells in different
stages of the cell cycle)
Temperature sensitive cdc mutant
cdc mutant growing
at permissive temp
cdc mutant growth arrested
after 6 hrs at restrictive temp
Genetic and descriptive analysis discover the
interactions between the mutants
How to clone cdc genes in yeast?
Let’s say you have a candidate sequence
DNA
cdc28 (-)
WT
If the candidate sequence complements (rescues)
the mutated phenotype: that’s your gene!
How to Clone cdc Genes in Yeast
Gene Z
Many of the cdc genes encode proteins needed for
DNA replication
cdc28 gene encodes a kinase
Fission yeast: Schizosaccharomyces pombe
Sir Paul Nurse
cdc genes encode proteins needed for the
G2-M transition: studies in s. pombe
cdc2D = gain of function mutant
Cloning cdc2
The same approach used in budding yeasts:
complementation by a library
Only using a budding yeast library
Cdc2 (fission)
START/Restriction Point
Cdc2 (fission)
Cdc28 (budding)
This is all great
Yeast are really cute and interesting
Can we really learn something from that
about humans?
Schizosaccharomyces
pombe
Crazy idea
It worked for us with budding yeast
genes. Why not try human genes?
Sir Paul Nurse
Let’s try to complement (rescue)
the cdc2 (-) mutant of pombe with
a human cDNA library
Human cdc2 rescues cdc2 mutants
Melanie Lee
Elongated cdc2 mutants,
failing to undergo mitosis
cdc2 mutants,
complemented by a
human cdc2 gene
Summary
- A genetic approach in fission and budding
yeasts reveal genes that are essential in
promoting the cells through the cell cycle
- These genes encode kinases proteins and are
called CDKs for Cyclin-Dependent Kinases
Cdk1 = the protein encoded by cdc2/CDC28
Woods Hole Marine Biological Laboratory
Tim Hunt
Sea urchins
can be stimulated to
lay lots of eggs
The summer project: to
follow protein synthesis
upon fertilization by
following incorporation
of S35 - Met and getting
samples every 10’
Proteins X,Y,Z are synthesized only in unfertilized eggs
Proteins A,B,C are synthesized upon fertilization
mitosis mitosis mitosis
Protein A disappears 10’
before completion of mitosis
In clams two proteins, A and
B, express this cyclic behavior
Cyclins are synthesized and degraded in a cyclic
manner and with correlation to the cell cycle
Protein
Level
cyclin A
cyclin B
Time
M
M
M
Something needs to go away in order for the
cell cycle to proceed
CDK
Yeast genetics
Needed for promoting cells through the cell cycle
Cyclin
Biochemistry in sea urchin
Appear in correlation with the cell cycle
Time to bring them together