Cell Biology III(The Cell Cycle)

hQp://25.media.tumblr.com/tumblr_lcn6XAey11qezvqko1_500.jpg

Oct 29, Nov.5, Nov.1, Dec.3
Shin SUGIYAMA
Oﬃce: E207 Ext. 5039
ssugiya@bio.nagoya-u.ac.jp

図の出典
Essen\al 細胞生物学 原書第3版（Bruce Alberts他著、
中村桂子・松原謙一監訳、南江堂、2011年）
細胞の分子生物学 第5版（Bruce Alberts他著、中村桂子・松原謙一監訳、Newton Press、2010年）
The Cell Cycle an Introduc\on. Andrew Murray and Tim
Hunt. Oxford Univ. Press 1993
ECB

Wikipedia

教科書

MBoC

高価だけど、レフェランス
本としては必要。

What is the cell cycle？

hQp://25.media.tumblr.com/tumblr_lcn6XAey11qezvqko1_500.jpg

During the cell cycle the chromosomes are replicated andthen equally divided to sister cells
hQp://www.nature.com/
EssGen3-4_HumanChromosomesFi
g3_MID_0.jpg

The cell cycle can be considered as being the replica\on andprecise distribu\on of gene\c material
Today’s Concept IMitosis: Walther Flemming ofGermany ﬁrst descibed it in 1882

hQps://
upload.wikimedia.org/
wikipedia/commons/f/fe/
Walther_ﬂemming.gifDNAtubulin

hQp://25.media.tumblr.com/tumblr_lcn6XAey11qezvqko1_500.jpg

hQps://upload.wikimedia.org/wikipedia/commons/6/6d/
Zellsubstanz-Kern-Kerntheilung.jpg

Mendel’s theories were not widely known so he didin’t appreciate the behavior of the chromosomes 。

Why is an understanding of the cell
cycle important?

What happens when the cell cycle goes out of control？

hQp://www.nature.com/principles/ebooks/principles-of-biology-104015/9523064

example:Breast Cancer
Today’s Concept IIDiﬀeren\a\on and the Cell Cycle

Undiﬀeren\ated：
Prolifera\onDiﬀeren\a\on：Less（No）Cell Division

Today’s Concept III

What will be taught•Control of the cell cycle•Mechanisms of mitosis•Excep\ons to the rule

What won’t be taught• The prokaryo\c cell cycle• Meiosis• DNA replica\onHow is the cell cycle controlled？

The experiment that got things started
Cells at diﬀerent phases of the cell cycle werefused together and sometmes the cell phases
were shimed or delayed.

Cell fusion

hQp://www.cosmobio.co.jp/
export_e/products/cells/
products_ISK_20070518/
CF_02.gif

Viruses can be used to make cells fuse.
Johnson, R.T. & Rao, P.N., Mammalian Cell Fusion: Induc;on ofPremature Chromosome Condensa;on in Interphase Nuclei.
Nature 226, 717–722 (1970)

How do viruses induce cellfusion?

MBoC p.1506
MBoC p.1497
Figure 24-24 Molecular Biology of the Cell (© Garland Science 2008)

The experiment that got things started
Usually: G1 → S → G２ → M → G1
1.

2.

When S phase cells andG1phase cells were fused, the G1
cells replicated DNA prematurely.

What happened…
hQp://ars.sciencedirect.com/content/image/1-s2.0-S0304419X97000334-gr1.jpg

However, when S phase cells
and G2phase cells were fused, G2
cells would NOT replicate DNA.

3. When S phase cells and G2phasecells were fused, G2 cells did notproceed to M phase.

M
Johnson, R.T. & Rao, P.N., Mammalian Cell Fusion: Induc;on ofPremature Chromosome Condensa;on in Interphase Nuclei.
Nature 226, 717–722 (1970)

The experiment that got things started(i) Existance of factor in S phasecells that induces DNA
replica\on.
Licensing Factor(ii) G1 phase cells can replicate DNA
but G2 phase cells can not.
Re-replica;on block(iii) Existance of factor in S phasecells that blocks progression to
M phase.
Check Point(iv) Once cells have gone through Mphase they can replicate DNA
again.
Re-licensing

What does all this suggest?

This experiment showedthat the cell cycle iscontrolled by an intricate
mechanism.
And gave researchers
speciﬁc ques\ons to beanswered.

Johnson, R.T. & Rao, P.N., Mammalian Cell Fusion: Induc;on ofPremature Chromosome Condensa;on in Interphase Nuclei.
Nature 226, 717–722 (1970)

MMPF, Cyclin, Cdk

Discovery of MPF activity�(maturation/mitosis promoting factor)�

Rana pipiens
ヒョウガエル/Leopard Frog

hQps://upload.wikimedia.org/wikipedia/
commons/d/d8/Rana_sphenocephala.jpg

ECB p.616

ECB p.616

hQp://www.brh.co.jp/s_library/j_site/scien\stweb/no25/img/
face.jpg

So what kind ofmolecule was MPF?

hQps://upload.wikimedia.org/wikipedia/commons/2/2d/
Echinometra_mathaei_MHNT_Philippines.jpg

ウニ！

Synchronized cell divisionKCltreatment

hQp://www2s.biglobe.ne.jp/~nkazu/jugyou/image/uni_jusei/Resized800/sDSCN0920.jpg

hQps://
upload.wikimedia.org/
wikipedia/commons/
4/48/
Sea_urchin_eggs.jpg

hQps://embryology.med.unsw.edu.au/embryology/images/b/b6/Sea_Urchin_early_embryo_cleavage_paQern.jpg

Tim HunthQps://upload.wikimedia.org/wikipedia/
commons/a/a3/Tim_Hunt_at_UCSF_05_2009_%284%29.jpg

Wood’s Hole
海洋学研究所hQps://upload.wikimedia.org/wikipedia/
commons/c/c6/
Marine_Biological_Laboratory%2C_Woods_Hole_by_Pam_Wilmot.jpg

The Cell Cycle an Introduc\on p.25

2001

hQp://www.cell.com/cell/abstract/
S0092-8674(08)00888-X

Experiment focusing onproteins newly synthesized in
fer\lized sea urchin eggs.

hQps://
upload.wikimedia.org/
wikipedia/en/e/ed/
Nobel_Prize.png

Tim Hunt
Proof of the func\on of cyclin•lem： control•center： de novo protein synthesis is inhibited by RNase•right： same, with subsequent blocking of RNase and addi\on of cyclin mRNA
The Cell Cycle an Introduc\on p.54

Why is it called “Cyclin”？
hQp://visit-hungary.com/site/upload/2009/12/Cycling.jpg"By the way the name cyclin,
which I coined, was really ajoke, it's because I like
cycling so much at the \mebut they did come and go in
the cell..."ECB 614頁

Cyclin binds to Cdk which was
discovered in yeast

ECB 614頁

So how was Cdk discovered?

MBoC 1056頁

The size and shape of yeast indicate thephase of cell cycle(possible with live cells)

Figure 17-5 Molecular Biology of the Cell (© Garland Science 2008)

A very useful type of muta\on

MBoC 1057頁

At diﬀerent phases

All at G1
When cells are
returned topermissive
temperature they all
resume cell cycle
progression.

Why are certain mutant allelestemperature-sensi\ve?

The cell cycle can be synchronized with temperature shims.

• In this case amino acidchanges of Ile207 caused
temperature- sensi\vity.
• TS mutants usually aﬀect
higher structure and notcataly\c domains.

SHOWN HERE AS
AN EXAMPLE

Wildtypecon\nues togrow whenshimed from
30℃ to 36℃.

Many of the cell cycle
genes were iden\ﬁed as
Cdc ts-mutants
hQp://images.the-scien\st.com/content/
ﬁgures/images/yr2003/apr21/hartwell.jpg

In this example, allcells of the cdc28
temperaturesensi\ve mutant hQps://en.wikipedia.org/wiki/Cell_cycle
stop at G1 phase.

2001

Lee
HartwellNon-cdc
temperaturesensi\ve mutants
stop growth atvarious phases.

The Cell Cycle an Introduc\on 32頁

Searched for temperaturesensi\ve cell cycle mutants
They are known as
Cdc (Cell division cycle) mutantsand 32 loci were iden\ﬁed.

Budding yeast（Normally cultured at 30℃）

Screening of Cdc (Cell division cycle)
Mutants

Mutagenesis（EMS or UV)
Grown fromsingle cells at
25℃A) Grow ◎B)Don’t grow X“Replica” made ofcolonies
Replica grown at
36℃A-A) Grow X
A-B) Don’t grow this \me ◎（temperature sensi;ve）

Lee Hartwell (USA)A-B-A) non-cdc mutant（stop at any phase in
the cell cycle）X

Study cell cycle phase A-B-B) （cdc mutant）◎
Growth blocked at a par\cular cell cycle phaseat 36℃

Iden\ﬁed Cdk1
(cdc2) in Fission
Yeast

The Cell Cycle an Introduc\on 49頁

Iden\ﬁca\on of the gene bymutant rescue

2001 Paul Nurse

hQps://upload.wikimedia.org/wikipedia/commons/
a/ac/Paul_Nurse_portrait.jpg• When the cdc2ts mutant is shimed to 35℃ it is arrested at G2 phase. Because cell growth
con\nues it becomes longer.
• The mutant is randomly transformed with aof genomic DNA.
• Most plasmids will not change (rescue) the mutant phenotype.
• However if the plasmid contains the full-length cdc2 gene it will complement (rescue) the
mutant. The plasmid is then, recovered and sequenced.
• Fission yeast was later found to be homologous to budding yeast Cdc28 and human Cdk1.

Fission yeast Cdc2 temperaturesensi\ve mutant（permissive temperature 25℃）

酵母の温度感受性変異を
Iden\ﬁed Cdk1
使って一番重要な細胞周期(cdc2)
制御遺伝子を同定
in Fission

Yeast

Transformed with plasmids carrying
random fragments of genomic DNA
Grown at 35℃
A)
B)
C)
D)

Non-transformed yeast do not grow X
Yeast transformed with genes other than cdc2 do not grow X
Yeast transformed with par\al fragments of cdc2 do not grow X
Yeast transformed with the full length cdc2 gene are “rescued” ◎Plasmid isolated from yeast that grow
Sequence of plasmid that “rescued”
the mutant is determined

Sequence of same gene in the original
mutant strain is also sequenced（protein coded bycdc2 gene is iden\ﬁed）

Paul Nurse (UK)The gene was ﬁrst iden\ﬁed as
the mutant (cdc2) and the DNA
sequence was not known“Mutant Rescue”（”Plasmid Rescue” method）

PNAS 85, 3009 (1988)

Summary (for part1)

Frog Egg
MPF(Experimental
Biology)

Yeast
Cdk(gene\cs)

Cell fusion
experiments indicatedexistance of a control
mechanism

Sea Urchin
Cyclin（Biochemistry）

Activation of M-Cdk�

You learned this last week

Regula\on of Cdk-Cyclin by Wee1 & Cdc25
Both were iden\ﬁed as mutants in ﬁssion yeast (S. pombe)

Figure17-18 Molecular Biology of the Cell S/e (©Garland science 2008)
hQp://eishinoguchi.com/NoguchiLab.html

cdc2: Cdk (Cyclin-dependent kinase)wee1: Cdk inhibitory kinase
cdc25: Cdk ac\va\ng phosphatase

Paul Nurse
The Cell Cycle an Introduc\on p.48

Wee1

(his ﬁrst mutant)wee (adj.) deﬁni;on;
1. Very small; ;ny.
2. Very early: the wee hours of the morning.

Paul Nursecdc2: Cdk (Cyclin-dependent kinase)wee1: Cdk inhibitory kinase
cdc25: Cdk ac\va\ng phosphatase
Normal mitosis(ﬁssion yeast)

Mitosis is inhibited but cell growth con\nues

Mitosis is inhibited but cell growth con\nues
Mitois induced before G2
growth is ﬁnished.

Cancel each otherAnalysis of thephosphoryla\on sites

Tyrosine15 of Cdc2 exchanged with
Phenylalanine

human Cdk(Cdc2) phosphoryla\on
Tyrosine15: inhibi\on
Threonine161: ac\va\on

The Cell Cycle an Introduc\onp.58•Cdc2 regular ts muta\on: inhibi\on of mitosis•Cdc25 regular ts muta\on: inhibi\on of mitosis•Cdc2 Y15→F mutant : induc\on of mitosis• Cdc2Y15F and Cdc25 mutants do not cancel each other out,
and the Cdc2Y15F phenotype is dominant.
• Indicates Cdc25 func\ons through Cdk Tyrosine 15

1. Binding of Cyclin
2. Phosphoryla\on ofac\va\ng site
3. Dephosphoryla\on of
inhibitory site
4. Release from Cdk
inhibitor protein

Cdk inhibitor proteins (CKIs)

MBoC17-63（ECB 18−16）•P53: tumor suppressor protein•Name from apparent molecularmass (53kD)• Omen mutated in tumors

hQps://en.wikipedia.org/wiki/P53

hQps://en.wikipedia.org/wiki/P53

先週！
Carl Zimmer

像は癌になりにくい！

Peto’s Paradox
推定変異率• 変異率は年齢で変わらない？（マウス）• 年齢による癌の上昇は変異の蓄積で説明される• 細胞数から推定するとヒトの発ガン率はもっと高
いはず

大腸の幹細胞数
hQp://rstb.royalsocietypublishing.org/content/370/1673/20140222

死後解剖での腫瘍発見率• 癌によるヒトの死亡率： 11〜25％• 動物園での癌によるゾウの死亡率： 5％• 体重〜100x像

体重（g）x寿命（年）p53• ゾウはチェックポント因子p53のコ
ピー数を増やすことで発癌率を抑えるよう進化してきた？
• ヒトはどうして同じように進化しなかった？

How many \mes can a cell divide？

Hayﬂick limit (1965)

hQp://images.the-scien\st.com/content/ﬁgures/images/yr1997/may/may_art/hayﬂick.jpg

Human culture cells cease growth amer about 50 divisions.
Individual cells have a life span too!
aging= stop in prolifera\on？
Why do they stop amer 50 divisions?

The telomere theory

hQps://upload.wikimedia.org/wikipedia/commons/4/4a/Telomere_caps.gif

hQp://bioenv.gu.se/digitalAssets/1311/1311740_bpop_telomeres_picture3.jpg

What are telomeres?
Specialized structures at the end ofchromosomes
Chromosomes become
shorter each \me
DNA is replicated.
hQp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/telomere3.gifTelomeres(: end structures of chromosomes)

and DNA replica\on

Replica\on of both
DNA strands is not
the same.

hQps://upload.wikimedia.org/wikipedia/
commons/8/8f/DNA_replica\on_en.svg

Chromosomes inmost cells get
shorter witheach division

Reiji and Tsuneko Okazaki

DNA replica\on at the telomeres

How many cells in the human body？
37,200,000,000,000( =about 246）
Average number ofdivisions: 47
If you believe Hayﬂick,
how many divisions do you
have lem？

Ann Hum Biol. 2013 An
es;ma;on of the number ofcells in the humanbody.Bianconi E, Piovesan,
Facchin F, Beraudi A, Casadei R,
FrabeT F, Vitale L, Pelleri MC,
Tassani S, Piva F, Perez-Amodio
S, Strippoli P, Canaider S.

So how dotheyactually
func\on?

From yourtext book

INTERPRETATION
The assembly of the pre-RC is the“licensing”.
Phosphoryla\on of Cdc6 by Cdk induces
its destruc\on and the start of DNA
replica\on
Phosphoryla\on of ORC by S-Cdk is the“re-replica\on block”.

MBoC 289頁

• Origin Recogni\on Complex:
Orc1-Orc6 subunits•Cdc6： Iden\ﬁed as one of theyeast cdc mutants. Upregulated inmany tumors.
•Cdt1：cdc10-dependent transcript 1

Figure 17-23 (part 1 of 3) Molecular Biology of the Cell (© Garland Science 2008)

MCM
designer “genes”•Mcm：minichromosome
maintenance (6 subunits) Isolated as genes
required to maintain ar\ﬁcial chromosomes inyeast.

Bik-Kwoon Tye
hQp://bmcb.cornell.edu/faculty/tye.html

p.356-359

Meier-Gorlin syndrome:mutants of subunits in white

Growth of speciﬁc\ssues is inhibited

hQps://en.wikipedia.org/wiki/Lini\s_plas\ca

Scirrhous gastriccarcinomas arecaused by cdc6
upregula\on

Cdc6 RNAitreatment

The experiment that got things started(i) Existance of factor in S phasecells that induces DNA
replica\on.
Licensing Factor(ii) G1 phase cells can replicate DNA
but G2 phase cells can not.
Re-replica;on block(iii) Existance of factor in S phasecells that blocks progression to
M phase.
Check Point(iv) Once cells have gone through Mphase they can replicate DNA
again.
Re-licensing
Johnson, R.T. & Rao, P.N., Mammalian Cell Fusion: Induc;on ofPremature Chromosome Condensa;on in Interphase Nuclei.
Nature 226, 717–722 (1970)

M

Trigger
Mechanism
for Mitosis�

Figure 18-18 Essential Cell Biology (© Garland Science 2010)

M cyclin-Cdk ac\va\on

1. Rise in M cyclin transcrip\on during G2 phase.
2. Cdk-Ac\va\ng Kinase (CAK) phosphorylates
Cyclin-Cdk on its ac\va\on site.

5. Ac\vated Cdk-Cyclin induces
inhibi\on of Wee1 (double-nega\vefeedback).
3. At the same \me Wee1 phosphorylates Cyclin6. Ac\vated Cdk-Cyclin induces
Cdk on its inhibitory site.
ac\va\ng phosphoryla\on of Cdc25
4. Dephosphoryla\on of the inhibitory site byforming a posi\ve feedback loop.
Cdc25 \ps the balance towards ac\va\on. The
mechanism that ini\ally induces Cdc25
MBoC17-25
ac\va\on is unknown.
（ECB18−17）

Cell-cycle times�

typically

Only 20 minutes!
hQps://end\messigns.ﬁles.wordpress.com/2011/08/ecoli-bacteria.jpg

The paradox of E. coli cell division•E. coli can divide once every 20 minutes.
•Their genome is 4,600,000 base pairs•Their DNA replica\on rate is 1000bases ／sec.
• 4.6 million/1000/2=2300sec.=38min（Replica\on goes in both direc\ons）

There are many
•Not enough ;me！
exceptions to rules inthe•Single replica\on origin！（OriC) cell cycle�

•Replica\on starts before the previous cycle isﬁnished and division occurs even during
replica\on•They cheat！

hQp://sandwalk.blogspot.jp/
2008/05/dna-replica\on-in-ecoli-solu\on.html

Summary• A cell fusion experiment provided insight on the mechanisms
regula\ng the cell cycle.
• Cyclin was discovered by biochemistry, and Cdk was discovered by
gene\cs.
• Many cell cycle factors were iden\ﬁed as temperature-sensi\ve
Cdc (Cell division cycle) mutants.
• Par\al destruc\on of the pre-replica\ve complex (Orc, MCM
proteins etc.) triggered by Cdk enables ini\a\on of S phase(Replica\on).
• Phosphoryla\on of ORC by Cdk blocks re-replica\on within thesame cell cycle.
• Ac\va\on of Cdk-Cyclin by Cdc25 \ps the balance of ac\va\oninhibi\on toward ac\va\on and in reinforced by posi\ve feedback.

Next week
• The Mito\c Spindle• The Cell Cycle Checkpoint• Anaphase Progression