Assigned Reading:

Murray and Kirschner, 1989. Cyclin Synthesis drives the early embryonic
cell cycle. Nature 339, 275-280

Murray, Solomon, and Kirschner, 1989. The role of cyclin synthesis and
degradation in the control of maturation promoting factor activity. Nature
339, 280-286

Complementation used to clone a human homologue of the fission yeast

cell cycle control gene cdc2. (1987) Lee and Nurse, Nature 327, 31-35.

Optional Reading:

Building a cell cycle oscillator: hysteresis and bistability in the activation of

Cdc2. (2003) Pomerening, Sontag, and Ferrell, Nature Cell Biology 5,346-
351.

Charvin et al. 2010. Origin of Irreversibility of Cell Cycle Start in Budding

Yeast. PLoS Biology 8(1) e1000284. A very beautiful paper, but very tough
to read--requires cell cycle expertise.
Optional Reading

Andrew W Murray. Recycling the Cell Cycle: Cyclins Revisited. Cell, vol.
116, 2004.

Modeling the cell cycle: why do certain circuits oscillate?

Ferrell JE Jr, Tsai TY, Yang Q.
Cell. 2011 Mar 18;144(6):874-85.

Johnson and Skotheim, Current Opinion in Cell Biology 2013, 25:717-723.

“Start and the Restriction Point”

Coudreuse and Nurse, Nature 2010, “Driving the cell cycle with a minimal
CDK network”, Nature 468, 1074-9 (very difficult paper)

Background: YouTube: David O. Morgan (UCSF) Controlling the Cell

Cycle, parts 1, 2, 3.
 The Cell Cycle

G1 = Gap 1
S = DNA Synthesis

G1 S

M G2
G2 = Gap 2
M = Mitosis

The contents of the cell have to be duplicated.

Then, the contents have to be divided between the two new cells.
G1: Gap 1. Normal business of cell. Growth, if a dividing cell.
“Post-mitotic”, mature cells (liver, kidney, muscle, brain) are in G1.

S: DNA Synthesis. Duplicate all DNA.

G2: Gap 2. Prepare for cell division (mitosis)

M: Mitosis. Divide into two cells.

Cytokinesis is the end of mitosis, when the two cells separate.

Return to G1. Repeat.

We will be interested in:

1. What exactly is happening in these four cell division phases?

2. How is cell division controlled?
 The Cell Cycle is driven by a protein kinase
activity called “Cyclin Dependent Kinase”, or
“CDK”

Kinase activity

G1 S G2 M G1 S G2 M G1

Cyclin Dependent Kinase phosphorylates other proteins (substrates) and changes

their behavior. The phosphorylated substrates carry out the individual events of
the cell cycle. In many cases, we know fairly exactly how some phosphorylations
bring about a cell cycle change (e.g., initiation of DNA replication).

A consensus site for phosphorylation by CDK is (S/T)-P-x-(K/R)

Many important CDK substrates have many such consensus sites (e.g., 10 or so).
Part 1: Events of the Cell Cycle
Movies
• https://www.youtube.com/watch?v=N97cgUqV0Cg
1. Growth to a sufficiently large size allows Commitment to cell division.

CDK: Very low.

 2. S-phase (DNA Synthesis)
1. Pre-replication complexes are set up at origins in G1, when
CDK (Cyclin Dependent Kinase) activity is low or zero.
2. In S, CDK activity rises, and causes the origins to “fire”, and
DNA replication begins at multiple origins (hundreds or
thousands).
3. Replication forks proceed bi-directionally from origins.
4. Cohesins are put in place, gluing sister chromatids together.
5. Forks meet and merge, and replication finishes.

Pre-Replication Complex Cohesin

CDK: Low, to set up origins, then rising, to fire origins.

3. Duplicated chromosomes are Kim Nasmyth lab
held together inside cohesin
rings.

Cohesin ring
G2 Phase

The cohesin ring will get cut at a critical

moment in mitosis, releasing the two sister
chromatids, so that they can partition into
daughter cells.

Sister Chromatids

CDK: Medium
 4. M-phase (Mitosis)

1. A spindle forms from microtubules.

2. Chromosomes condense.
3. Kinetochores attach to microtubules.
4. When all kinetochores are attached to microtubules,
cohesins are destroyed, and chromosomes separate.
(5) In some species, the nuclear membrane breaks down before M.
(6) These changes require higher CDK activity.

Microtubule

Kinetochore

Spindle Pole Body

a.k.a. Microtubule
Organizing Center
Cohesin
Chromatid CDK: High to Very High
At Mitosis, the two sets of chromosomes are partitioned to the two
daughter cells.

The chromosomes move because they are attached to

microtubules.

There are two kinds of movement:

First, the chromosomes move along the microtubules
towards the spindle poles (Anaphase A).
Second, the spindle poles anchoring the microtubules move
away from each other (Anaphase B).

Anaphase B movement is caused by a family of “motor proteins”

called kinesins. The kinesins have one end that is a molecular
motor, and binds to microtubules, and another end that can carry
some kind of “cargo”.
Kinesin motor protein
Video: The Inner Life of a Cell, Xvivo, BioVisions,
3:15 to 5:30
 How Spindles Elongate (Anaphase B)

SPB

+ SPB

BimC-like kinesin

microtubule
SPB = Spindle Pole Body.
 Metaphase Spindle

SPB SPB

kinetochore/centromere

cohesin
chromosome

microtubule CDK: Very High,

BimC-like kinesin as high as it gets
 Activation of the E3 Cdc20 (part of Anaphase Spindle
the Anaphase Promoting Complex,
APC) results in ubiquitin-dependent
destruction of cohesion and cyclin.
Anaphase A

Anaphase B

SPB +
+ SPB

kinetochore

chromosome

BimC-like kinesin, microtubule Cdk: Collapsing from

drives Anaphase B High to Low
Anaphase (chromosome separation) is triggered
when Cdc20, an E3 for ubiquitin-mediated
proteolysis, is activated and indirectly destroys
cohesin, freeing sister chromosomes from each
other.

At the same time, this activation of Cdc20 causes

ubiquitylation and destruction of cyclin (see later).

Cdc20 is part of an E3 called the APC (Anaphase

Promoting Complex), and it recognizes Destruction
boxes and KEN boxes.

Cdc20 was found as a mutant in a “Cell Division

Cycle” screen, hence “CDC”.
The activation of Cdc20 (and so the destruction of cyclin and cohesion) requires
two things:

1. CDK phosphorylation of proteins of the Anaphase Promoting Complex (APC)

(i.e., the E3). To get the high levels of Cyclin Dependent Kinase activity
required to phosphorylate this E3, a lot of cyclin is needed. Thus, high levels
of cyclin are required for the (slightly later) destruction of cyclin. This is called
a “negative feedback loop”. Cyclin accumulation leads to its own destruction.

2. All chromosomal kinetochores must be occupied by microtubules. This

silences an inhibitor of Cdc20 called Mad2.

Once these two conditions are met (phosphorylation of APC, all chromosome
kinetochores attached to microtubules), Cdc20 is activated, cohesion and cyclin
are destroyed, and anaphase occurs.
Cdc20 is activated by high CDK kinase

CDK off: Cdc20 off At the metaphase-anaphase

transition, CDK kinase is
high, so Cdc20 is in the
active form. At the
transition, Mad2 turns off,
releasing active Cdc20. The
active Cdc2 destroys
cohesin and cyclin,
allowing mitosis to finish,
CDK on: Cdc20 on and the cell cycle to return
to the ground state with no
cyclin or CDK activity.

Destroys Cohesin, Cyclin

The Spindle Checkpoint: Letting Go at Mitosis

Mad2
Cdc20

Wait, wait Cohesin

 Cdc20 and Cohesin

Mad2 Critical protein in Spindle Assembly Checkpoint;

Tension on centromeres relieves inhibition by Mad2.

Cdc20 Adaptor protein for E3 (APC); allows ubiquitylation.

Requires high CDK activity and absence of Mad2 activity.
Destroys Securin and Cyclin (and other stuff).
Securin Binding partner and inhibitor of Separase
(What protein motifs might Securin have?

Separase Very specific protease for Cohesin

Cohesin Holds chromatids together

Pictures of Newt lung cells, from Conley Reider

Newt cells are good for microscopy. Newts

have a lot of DNA, so big, easy-to-see
chromosomes. The cells are large. The lung
cells are flat, so it is easy to get all the
chromosomes into focus.

In the next few pictures, DNA is stained

blue, and microtubules are stained green. I
think the red is vimentin.
https://www.youtube.com/watch?v=L61Gp_d7evo

https://www.youtube.com/watch?v=CLk6nUmPRdA
 Other Cell Division movies:

https://youtu.be/N97cgUqV0Cg Kidney cells

https://youtu.be/hapQ9swZIa4 Apoptosis

https://youtu.be/Wz4igVjNGq4 Xenopus

https://youtu.be/RG9TMn1FJzc Bonus video: baby ibex

https://www.youtube.com/watch?v=tCJ5pfiL7aQ
Bonus: Alphonse Davies’ goal
Part 2: Mechanisms and Control of the Cell Cycle
 Cell Division Cycle (cdc) Mutants
To help study the cell cycle, Hartwell (S. cerevisiae) and Nurse (S. pombe)
isolated “cdc”mutants, specifically defective in various steps in the cell cycle.

There were two key ideas:

1. The mutants had to be temperature sensitive (ts) mutants, since cell cycle
processes are essential for life.
2. Mutants were recognized by their homogenous “terminal phenotype.

Petri dishes

mutagenize

23 degrees Replicate 37 degrees

Obtain large collection of ts lethals.

 Microscope
Views !
ts33

tsNS

ts20

ts28

23 37
 24

1N 2N

19, 33 28 4, 7, 2, 8, 21 16, 17, 20, 23, 28 5, 14, 15

no spindle

31

Phenotypes of cdc mutants (Hartwell)

 cdc2 in S. pombe (Paul Nurse)

Earlier, an equivalent gene, CDC28, had been

discovered in S. cerevisiae by Lee Hartwell.

Cdc2 and Cdc28 encode Protein Kinases.

“CDC” mutants are “Cell Division Cycle” mutants
Upon sequencing, the cloned human protein was largely identical (~ 63%) to
the S. pombe cdc2 protein, and also to the Cdc28 protein of S. cerevisiae !!!
 The Cell Cycle is driven by a protein kinase
activity called “Cyclin Dependent Kinase”, or
“CDK”

Kinase activity

CDK Protein Amount

G1 S G2 M G1

Cyclin Dependent Kinase phosphorylates other proteins (substrates) and changes

their behavior. The phosphorylated substrates carry out the individual events of
the cell cycle. In many cases, we know fairly exactly how some phosphorylations
bring about a cell cycle change (e.g., initiation of DNA replication).

A consensus site for phosphorylation by CDK is (S/T)-P-x-(K/R)

Many important CDK substrates have many such consensus sites (e.g., 10 or so).
 Protein Kinase: Adds phosphates to other proteins

Protein
Kinase

The phosphate will change the behavior of the substrate, e.g., turning its activity on or off.

The protein kinase will typically have a surface for specific binding to the substrate.
The protein kinase will have a pocket for binding ATP.
The phosphate can be added to an –OH group, as found on Ser, Thr, and Tyr.

The activity of the protein kinase can typically be regulated by various means.
From T. Hunt Sea Urchin
 Cdc2 is a Cyclin Dependent Kinase

Kinase activity

Cyclin
Cdc2 protein

G1 S G2 M G1 S G2 M

Ruderman and Hunt

 Cyclin Dependent Kinases (CDKs)
Cdc28 and Cdc2 are the protypical protein kinases of a family called the cyclin-dependent
kinases. The monomeric catalytic subunits have no protein kinase activity, but gain protein
kinase activity when a cyclin is bound. Protein kinase activity is often regulated by many
mechanisms: availability of cyclin; inhibitory tyrosine phosphorylation of the CDK; presence
or absence of specific inhibitors of CDKs (CKIs, Cyclin Kinase Inhibitors).

Transcription
Cyclin
Proteolysis
Inhibitory Tyr Phosphorylation
CDK
Inhibitor proteins

Only the complex (the Cyclin-CDK complex) has protein kinase activity

Many labs: Nurse, Beach, labs working on marine invertebrates

Cyclin Proteolysis Activated By:
1. CDK-dependent spindle (loss of Mad2)
Embryonic Cell Cycle Cyclin Stabilized By:
1. Loss of spindle (gain of Mad2)
2. High Cyclin Dependent Kinase Activity 2. Low Cyclin Dependent Kinase Activity

Cyclin Proteolysis Cyclin Proteolysis

P P
P P
P P
P P

P P

Mitosis Mitosis
Cyclin Negative
Feedback loop

Cyclin Cyclin Cyclin

Stabilized Stabilized Stabilized

Cyclin protein ( ) made continuously

Maternal Cyclin mRNA present continuously (no transcription)
 Cyclin Negative
Feedback loop

The Core Cell Cycle

(runs in embryos)
https://www.youtube.com/watch?v=ERnzwtj4YPs
Cdk activity drives the cell cycle

The cell cycle is driven by changes

(oscillations) in the activity of Cyclin
Dependent Kinase (CDK).

The oscillations in CDK activity are driven

largely by oscillations in the abundance of
Cyclin, the activating subunit.
Cyclin is necessary for mitosis

Minshull, Blow, and Hunt (Cell, 1989) made cycling extracts from
activated frog oocytes. When cyclin mRNAs in these extracts
were specifically destroyed using cloned cyclin DNA and Rnase H,
cycling no longer occurred. Histone H1 kinase activity (a.k.a CDK
activity, which is assayed using histone H1 as a substrate) did not
rise in the extracts that lacked cyclin.
 Histone H1 kinase activity in
embryonic clam (?) cells

Effect of protein synthesis inhibitor

Dabauvalle et al. 1988

Cyclin is the translated protein
sufficient for mitosis

Murray and Kirschner (Nature, 1989) made cycling frog oocyte extracts, and
destroyed all mRNAs using RNase A. They then added back cyclin RNA, and
this, without any other RNA, induced multiple cycles of mitosis. Histone H1
kinase activity (a.k.a. CDK activity) went up and down with cyclin protein.

Read this paper.

Cyclin Destruction is necessary for
cells to exit mitosis
Murray, Solomon and Kirschner (Nature 1989) deleted the sequence
encoding 90 amino acids at the N-terminus of cyclin (which, it was later
found, contains the “destruction box” allowing ubiquitination and
proteolysis). The mRNA encoding this truncated cyclin allowed cells to
go into mitosis, but the cells could not degrade the cyclin, and the cells
were not able to exit mitosis. Histone H1 kinase activity (a.k.a. CDK
activity) stayed high.

Read this paper.

 Why does Cyclin collapse at the end
of mitosis?

• It is destroyed by ubiquitylation and the proteasome.

• It is targeted for ubiquitylation by Cdc20, which is an E3.

• Cdc20 activity requires both high CDK activity, and low (zero)
Mad2 activity.

• So, high kinase activity leads to its own destruction: it activates

mitosis (and so inactivates Mad2), and it activates Cdc20, which
causes degradation of cyclin.
Cycling of Kinase activity in a simple embryonic system
Cdc20 Activated
(by lack of
Mad2 Mad2, high kinase)
inactivated

Cdc25/Wee1
Pos. feedback
loop

Cdc20 Inactivated
(by lack of CDK)

Cyclin made continuously

Constant cyclin mRNA, constant translation
 Cyclin Negative
Feedback loop

The Core Cell Cycle

(runs in embryos)
CDK off: Cdc20 off At the metaphase-anaphase
transition, CDK kinase is
high, so Cdc20 is in the
active form. At the
transition, Mad2 turns off,
releasing active Cdc20. The
active Cdc2 destroys
cohesin and cyclin,
allowing mitosis to finish,
CDK on: Cdc20 on and the cell cycle to return
to the ground state with no
cyclin or CDK activity.

Destroys Cohesin, Cyclin

Cdk works through multiple
pathways
Consensus phosphorylation site: (S/T) P x (K/R)
Roughly 300 substrates in the cell.

• Cdk activity changes transcription of cell cycle protein genes by

changing activity of cell cycle transcription factors (SBF, MBF,
Forkhead, Swi5 factors)
• Cdk activity changes stability of cell cycle proteins via
proteasomal degradation (Sic1, Clb2, Cdc20)
• Cdk activity changes activity of some cell cycle protein through
direct phosphorylation (Wee1, Cdc25, Cdc20, Cdh1)
• Cdk activity changes cellular localization of some cell cycle
proteins through phosphorylation (Swi5, Ace2 transcription
factors; Cdc14)
However, the embryonic cell cycle is insufficient, because it has no
coupling to growth. In the embryonic cycle, a large egg is
subdivided into smaller and smaller cells over a finite number of
divisions. E.g., in Drosophila, there are 13 embryonic divisions,
dividing the original cell into ~ 8,192 cells. This happens without
growth. Necessarily, cells get smaller. Xenopus video.

In mature organisms, cells stay within a narrow, constant size

range. This requires some kind of coupling between cell growth in
size and mass, and cell division.

A key insight of Lee Hartwell was that Cell Growth, on the one
hand, and Cell Division, on the other, are separate, and separable,
processes.
Drosophila again, then Xenopus
• https://www.youtube.com/watch?v=XSKh-GLQn4E

• https://www.youtube.com/watch?v=Wz4igVjNGq4
Division is coupled to cell size and cell growth through “G1 cyclins”.

These are expressed in G1 phase, cause an increase in a G1-CDK

activity, and activate a transcription factor (SBF in yeast, E2F in
mammals).

The transcription factor ultimately and indirectly causes

transcription of the mitotic cyclins, which increases the mitotic CDK
activity.

Meanwhile, the mitotic CDK activity shuts off the G1 cyclins,

completing the transition from G1 into G2/M.
fission
Yeast cyclins Cdc2
Cdc13
Puc1 Cig2

G1 S G2 M
budding
Yeast cyclins Cdc28
Clb3 Clb1
Cln1 Cln2 Clb5 Clb2
Clb4
Clb6
Cln3

G1 S G2 M
Human cyclins Cdk3= GO->G1?
B + Cdc2 Cdk5= neuronal
Ibrance, Kisqali, Cdk7=CAK
A + Cdc2 (Cdk1)
Verzenio
D + Cdk4 E + Cdk2
(Cdk1) Cdk8=txn.
D + Cdk6 A + Cdk2
Cdk9=txn.
+splicing

G1 S G2 M
In yeast, about 700 genes of 6000 are regulated in their transcription
through the cell cycle. A key G1 transcription factor is activated by the G1
cyclins, and it allows transcription of the mitotic cyclins. The mitotic cyclins
then repress the G1 cyclins.

++

G1 Cyclin
Mitotic Cyclin
 Regulatory Motifs in the Mature Cell Cycle

Size ++

G1 Cyclin
Mitotic Cyclin
Cancer is the result of abnormal, persistent cell division.

Most anti-cancer drugs attack specific steps of the cycle.

For example:
1. DNA damaging agents. Alkylate or otherwise modify or crosslink DNA. Kill
cells in S-phase (DNA replication). Chlorambucil, Carboplatin, Cisplatin,
Cyclophosphamide, Paraplatin, Ifosfamide, many others.
2. Prevents DNA replication. Fluorouracil, fludarabine.
3. Topoisomerase 2 inhibitors. Kill cells in S-phase (DNA replication).
Etoposide, Doxorubicin, Etopophos, Toposar, many others.
4. Microtubule function. Kills cells in Mitosis. Vincristine, Vinblastin,
Paclitaxel, many others.

Typically have dose-limiting toxicities, because they also affect normal dividing
cells. Typically kill by apoptosis, so cancer cells defective in apoptosis (e.g. p53
mutants) survive.

But, more specific anti-cancer cell cycle drugs are possible.

 Top 10 anti-cancer drugs by sales, 2017
1. Revlimid (analog of thalidomide, famous for causing terrible birth defects). Multiple mechanisms, but
prevents tumor angiogenesis (development of blood vessels to feed tumor). $8 billion.
2. Rituxan. Antibody against CD20. Kills CD20+ B-cells. Treats B-cell malignancies. High sales because
also used against rheumatoid arthritis. $7 billion.
3. Herceptin. Antibody against HER2, a receptor tyrosine protein kinase, involved in signaling for
mitogenesis. Breast cancer, gastric cancer. $7 billion.
4. Avastin. Antibody against vascular endothelial growth factor A (VEGF-A). Prevents angiogenesis (i.e.,
prevent blood vessel growth in tumors. Similar idea as Revlimid.
5. Neulasta/Neupogen. Recombinant granulocyte colony stimulating factor. Increases production of
neutrophils, other blood cells. By promoting recovery of hematopoietic system, allows larger, longer
doses of chemotherapeutic agents to be used (i.e., affects the dose-limiting toxicity). $4.5 billion.
6. Opdivo. Antibody against Programmed death receptor-1 (PD-1). PD-1 allows natural cancer-killing T-
cells to be down-regulated by PD-L1 or PD-L2. Opdivo blocks this interaction, maintaining the cancer-
killing effect of these T-cells. One of a group of new agents that work in this way. ~ $5 billion.
7. Imbruvica. Small molecule inhibitor of Bruton’s tyrosine kinase, which is needed for mitogenesis in B-
cells. $4 billion.
8. Keytruda. Antibody against PD-1. Like Opdivo, but from Merck. $4 billion.
9. Ibrance. Small molecule (palbociclib) that inhibits the CDK4 and CDK6 protein kinases. $3 billion.
10. Velcade. Small molecule inhibitor of proteasome. Interferes with cell cycle (?). $ 2 billion.

These drugs are still under patent, so the high sales reflect high prices more than wide-spread usage.
Patient cost of Lipitor (statin, anti-cholesterol) before and after
patent expiration
$1,980
Lipitor sales 2006, ~ 13 billion.
Patent expired 2011.

But keep in mind, recent estimate to

develop one new drug and get FDA
approval is:
Lipitor
Prescription $ 2.6 billion
Cost Per year
Typically, the cost to manufacture a drug is
negligible. Almost all the cost is in
development/approval. The
pharmaceutical industry resembles the
video game industry or the software
industry or the movie industry in this
respect. Very high prices are charged until
$6 patent expiration (and sometimes after),
which may or may not be sufficient to pay
2006 2017 for development/approval.
 Specific Cell Cycle Events are Targeted
by some highly specific anti-cancer drugs.

1. Palbociclib (Ibrance), abemaciclib (Verzenio), ribociclib

(Kisqali) are inhibitors of the CDK4 and CDK6 G1 cell
cycle kinases. Inhibit cell cycle entry. May cause Size-
Induced Senescence (SIS). FDA approved.

2. ICEC0942. Inhibitor of Cdk7. Causes Size-Induced

Senescence specific to cancer cells. In trials.

3. RP-6306. Repare Therapeutics. Inhibitor of human Myt1

(Wee1 homolog). Causes mitotic catastrophe specific to
cancer cells. In trials. Repare Therapeutics uses
synthetic lethal CRISPR screens to find specific targets.
Verzenio

Ibrance

Inhibitors of Cyclin D/CDK4,6

Kisqali