The Cell Growth and Division Cycle

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The Cell Growth and Division Cycle

• How our current understanding of the cell cycle
  emerged, based on descriptive and analytic cell
  biology

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The Cell Cycle
The durations of G1 and G2 are variable, even
within an organism or cell type. The
durations of S and M are usually quite
consistent.
Mitosis
Cytokinesis
Cell Division
Lodish et al., Molecular Cell Biology Figure 13-1
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SEM of cultured mammalian cells
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Structural differences in cells with time in cell
cycle allows selection

• Mitotic shakeoff for mammalian cell synchrony
• Synchrony by centrifugal elutriation
• Synchrony by arrest with drugs also works
  mitotic blockers, S-phase retarders
• Synchrony by use of TS mutants

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Different experimental systems have contributed
distinct kinds of information to our
understanding of the cell division cycle

• Mammalian cells showed us the subdivision of
  interphase into G1, S, and G2 and have helped
  with the description of cell growth cycles
• Yeast cells have provided insight into the
  pathways that control the decision to move from
  one stage to the next
• Embryos (amphibian and echinoderm) have provided
  large numbers of synchronized cells for useful
  biochemistry, which has in turn led to the
  identification of important control proteins

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Yeast Size and Morphology Through the Cell Cycle
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Both yeasts have been useful for the
identification of cell division cycle (CDC) genes
through conditionally lethal mutation

• Review in your minds the processes of mutagenesis
  and identification of TS lethals
• CDC-ts mutants fail to cycle at restrictive
  temperature, but they DO grow. This allows one
  to distinguish them from simple lethals
• Like any other ts mutation, the wild-type allele
  at this locus can be cloned by complementation
  with a plasmid library

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Problem With cdc mutant collections
Cant distinguish major regulators from minor
players
Loss of function at the top of pathway can look
just like loss of function at the bottom of the
pathway
wild type
cdc2, cdc13, cdc25, etc
- Cant distinguish which among, for example,
cdc2, cdc13 and cdc25 is a regulator, and
which is regulated
-Loss of Function of any prevents initiation of
mitosis
-All these mutants have the same phenotype, but
they map to different loci
From Nurse (2002) ChemBioChem 3596
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However, continued screening identified CDC
mutants with different phenotypes, e.g., those
that initiated mitosis early
From Nurse (2002) ChemBioChem 3596
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The first wee Mutants Identified a Single Gene
WEE1, identified by several LOF alleles
From Nurse (2002) ChemBioChem 3596
- Wee1 acts in G2 to inhibit mitosis - loss of
Wee1 function causes mitosis to begin prematurely
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Further work identified both gain of function
alleles at the Wee1 locus (big cells that delayed
division) and a gain of function in another gene
(Cdc2) that produced the Wee phenotype

• The real power of yeast genetics emerged not from
  the identification of a mutant here and there,
  but from the possibility of getting multiple of
  alleles of any one gene
• Even then, CDC genetics was mostly an
  intellectual exercise until molecular biology
  made it possible to clone the genes, obtain their
  sequences, and purify the corresponding gene
  products

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A Dominant, GOF allele of CDC2 revealed a Wee
phenotype
From Susan Forsburg, Salk Institute
These phenotypes imply that CDC2 is an important
gene
- LOF mutations are cdc (big, fail to enter
mitosis) - Cdc2 function is required to initiate
mitosis - GOF mutations are wee, implying that
Cdc2 is a regulator
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Cdc2 was cloned and sequenced, revealing a
Protein Kinase
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Limited biochemistry confirmed that Cdc2p was a
Protein Kinase
Functional characterization - Cdc2
immunoprecipitates have kinase activity - a good
kinase for histone phosphorylation - this kinase
activity peaks at G2/M - the kinase activity
from cdc2ts lysates disappears at high
temperature
Studies of genetic interactions suggested,
however, that Cdc2 did not function alone Cdc13
LOF mutations had just the same phenotype as Cdc2
LOF, and mild alleles of the two genes were
additive, an example of a synthetic genetic
interaction.
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Cloning and sequencing of Cdc13 showed that it
was a cyclin, a kind of protein recently
identified though biochemistry on sea urchin eggs
Cdc13p functions at the same time in mitosis as
Cdc2p - Cdc13p required for Cdc2p function CDC13
Cloning reveals that Cdc13 is a Cyclin -
Homology to Starfish Cyclin B (Tim Hunt) - Cdc13
protein levels cycle - correspond with Cdc2
activity - Cdc13 is required for Cdc2 function
in-vitro and in-vivo
Nurse and collaborators propose that Cdc2 is
Cyclin-Dependent Kinase, but clarifying this with
authority required biochemistry. Meanwhile
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Oocyte cytoplasm can be obtained in quantity and
manipulated, allowing both experimental and
biochemical studies
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Maturation of frog eggs is induced by
progesterone, induction of embryogenesis is
induced by a sperm
Lodish et al., Molecular Cell Biology Figure 13-5
Maturation
Activation
Yoshio Masui Use Oocyte maturation to study a
simple, synchronized cell cycle Hormone can
drive G2 - M Activation can drive M-G1
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Once they are activated, oocytes produce a
cytoplasmic Maturation Promoting Factor (MPF),
which is Transferable and Autocatalytically
produced
100x dil. (106 x dil. In toto)
100x dil.
100x dil.
Yoshio Masui, Differentiation (2001) 691-17
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MPF turns out to regulate the embryonic Cell
Cycle as well
MPF Regulates Mitosis as well as Meiosis
Yoshio Masui, Differentiation (2001) 691-17
Lodish et al., Molecular Cell Biology Figure 13-5
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How to pursue the purification of MFP? Take
advantage of synchrony

• Collect many eggs that contain active MPF
• Crush them and make cytoplasm or egg extract
• Fractionate by standard biochemistry, but use a
  bioassay for MPF activity
• Achieve several thousand-fold purification

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MPF
Manfred Lohka and Jim Maller (UCHSC) - used cell
free assay to purify MPF - tested various
cytosolic fractions for MPF activity
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Manfred Lohka and Jim Maller (UCHSC) - used cell
free assay to purify MPF - tested various
cytosolic fractions for MPF activity
Purified MPF 3000-fold MPF is a 2 subunit
protein kinase - 32 kDa and 45 kDa -
Autophosphorylates 45 kDa component
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MPF regulation was elucidated by Tim Hunts
discovery of Cyclins
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MPF is a cyclin-dependent kinase (CDK)
Lodish et al., Molecular Cell Biology Figure 13-7
1) MPF Activity Requires Cyclin B Production by
Oocyte - Cyclin B ONLY Protein Synthesis
Required to Initiate Development 2) Suggests that
45 kDa MPF subunit is Cyclin B
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The Gautier from the Nurse lab and the Maller lab
got together to work this out and determine for
sure that MPF is the cyclin-dependent kinase that
had been identified as Cdc2 in complex with Cdc13
in fission yeast. Now we can try to understand
how this complex works to regulate cell cycle
progression
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Structure of a CDK (Human CDK2)
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These steps in CDK regulation are, however, only
a beginning

• The fully active CDK (cyclin bound and CAK
  phosphorylated) can be down regulated by a
  different phosphorylation
• The extent of this down regulation by the Wee1
  kinase is controlled by an antagonistic
  phosphatase, called Cdc25

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Wee1 and Cdc25 Regulate Cdc2 Phosphorylation
Tyrosine 15 is a major site of Cdc2
phosphorylation
Tyrosine 15 Phosphorylation - correlates
negatively with Cdc2 activity - added by Wee1
in-vitro, kinase activity goes down - removed by
Cdc25 in-vitro, kinase goes up Phosphorylation
at Tyrosine 15 inhibits Cdc2 activity - Wee1
inhibits Cdc2 in-vitro - Cdc25 activates Cdc2
in-vitro - Tyrosine 15 to Phenylalanine
mutation makes cells wee!! (cant be
phosphorylated)
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Tyr 15 Inhibitory Added byWee1 Removed by Cdc25
Thr 160 Activating CAK adds after Cyclin binding
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Cdc2 Activates Cdc25 and Inhibits Wee1 Positive
Feedback Loop A little Cdc2 goes a long way
Tyr 15
Thr 160
This means Cdc25 Must also be regulated
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How Universal is CDK Control of Mitosis?
Steve Reeds lab told a similar story for Cdc28
in Budding yeast - Cdc28 is a CDK, controls
G2/M transition - Budding yeast CDC28 fully
complements fission yeast cdc2ts -Suggests CDK
control of mitosis may be Universal
Melanie Lee found a Human cDNA that COMPLETELY
complements fission yeast cdc2ts
Human CDC2 gene product is 63 identical to
fission yeast Cdc2
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cdc2ts Cells Human CDC2 plasmid
Wild-Type Cells
cdc2ts Cells
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CDK Control of Mitosis is Universal
Fission Yeast Cdc2-Cdc13 heterodimer is
equivalent to Xenopus MPF
PSTAIRE antibody - cross reacts with ALL
CDKs - cross reacts with MPF - removes MPF
activity from Xenopus Oocyte extracts
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This CDK drives cells into mitosis, but how does
it do it?

• This CDK (known in mammals as CDK1) is the most
  important lamin kinase and thereby promotes the
  solubilization of the nuclear envelope
• It also phosphorylates MT-related proteins and
  promotes mitotic behavior in MTs, though just how
  is not clear
• It is also an active histone kinase, though the
  significance of this activity is again unclear
• No one has wanted yet to take the time to figure
  out just how MPF regulates the onset of M-phase,
  but its activity does push a cell into mitosis,
  even at inappropriate times in the cell cycle,
  like S phase

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So MPF gets us into mitosis, how do we get out?
One can imagine many possibilities

• Activate Wee kinase and turn off MPF
• Inactivate Cdc25 phosphatase
• Activate a phosphatase that undoes the work of
  CAK
• Synthesize an inhibitor of MPF, a CKI
• It turns out all these possibilities are wrong.
  The solution lies in the behavior of CYCLIN

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Wee1
Cdc25
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Cyclin Destruction Drives Mitotic Exit
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Cyclin B degradation in Live Cells (HeLa)
Jonathan Pines lab
http//www.welc.cam.ac.uk/jplab/Movies/b1-degrada
tionmovie.htm
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Mitotic Cyclins are Destroyed by
Ubiquitin-Dependent Proteolysis
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Ubiquitin
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Fortuitous Discovery of Cyclin B Ubiquitination
IP of 35S-Labeled Cyclin B
Cyclin B-Ub Ladder
Cyclin B
Over Exposure
Normal Exposure
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Ubiquitin is Transferred via Thioester Bonds
High Energy Thioester Bond, can be transferred
from E1 to E2, E3, or substrate
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Substrate Ubiquitination is Processive
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Substrate Ubiquitination is Processive
Processivity
Isopeptide Bond
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Ubiquitinated Proteins are Degraded by the
Proteosome
Binds to Ubiquitinated Proteins, Cleaves and
Recycles Ubiquitin
(Isopeptidase)
Destroys Target Proteins
(Peptidase)
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Ubiquitination of Protein Substrates
E3 Enzymes responsible for specificity and
diversity of Ub system