Intracellular Control:

Cyclin-dependent Kinases

1
 The Cell Cycle
• Since this is review, we will not discuss at length the cell cycle itself.
• Focus on mechanisms of control that occur at specific stages.
• What allows a cell to stay within a phase?
“mitosis”

“growth”

“growth”
“synthesis”

2
 Why Control the Cell Cycle?
• Well orchestrated – sequence of events that have to occur and if they
don’t occur in the sequence, then there can be catastrophic results

• Biochemical switches: regulatory proteins that through binding can

control a cell’s progression through cell cycle, so by interacting
together they allow a cell to progress or pause (wait until the cell fixes
itself and then continue)

• The cell cycle is a complex system of coordinated processes that must

occur in a specific sequence.

• If performed incorrectly or out of sequence, results may be catastrophic.

• Regulatory proteins and biochemical switches control progression through

the cell cycle.

• This system monitors intracellular and extracellular environments.

3
 Cdks Control the Cell Cycle
• Cdk = “cyclin-dependent kinase”:
- depend on protein called cyclin
- cyclin’s concentration is cyclical (in a cycle – rises and falls)
- conc of active Cdk changes at diff phases of the cell cycle and
rises and falls
• Activity of cyclin (and thus Cdks) rises and falls with cell cycle.
• Molecular switches that regulate important events:
– control synthesis or DNA replication (s-phase)
– mitosis
– chromosome segregation: prep for mitosis
– cell proliferation

4
Classification of Cyclins and Cdks
Cyclins in all eukaryotes (4 major classes):
• This helps better map the rising and falling
• Many diff types of cyclins in eukaryotes
• G1/S cyclins: bind Cdk near end of G1 and lead cell into
DNA replication. (G1 at both ends = shows the cycle)
- G1/s cyclin goes up and then goes down and stays
down for the rest of the cycle
- as cells are coming out of G1 and entering S1, this
is where DNA is replicated
- So, important for getting cell out of G1 and entering
DNA replication
• S-cyclins: bind Cdk during S phase and are required for
DNA replication, control early mitotic events.
- stay high until early phases of mitosis
- For S phase to take place, S-cyclins need to be on
the rise
• M-cyclins: promote the events of mitosis.
- steadily rise during second growth phase
- Important in getting cell into mitosis
- have to decrease for getting cell out of mitosis
• G1-cyclins: (in most cells) promote passage through
restriction point in late G1.
- important for getting cell past restriction point

5
Cdks are Protein Kinases
 Protein kinase is a protein that phosphorylates another
protein
 Takes phosphate from ADP and turns it into ATP and
transfers the phosphate to a protein, affecting the protein by
either activating/deactivating it
 Phosphate can be added to one or multiple binding sites;
changing the confirmation of the protein and affecting the
function
 Protein kinase can deactivate a protein if that is the end
result of placing the phosphate in a specific area
 Protein phosphate: another enzyme that instead removes a
phosphate group
6
Cdk Activity is Regulated
 Cyclin is going up and down and this is controlled by protein
ligases which can lead to the destruction of cyclins
 There are cyclins that are present in the cytoplasm
 Ligase that cause the activity to drop and once they are
deactivated, then the cyclin activity increases again – this
directly results in activation of Cdk
 M cyclins increasing activity and bind to Cdk and this can
trigger mitosis
 M Cdk’s are destroyed, and this deactivates M Cdk’s
 Same with S cyclins (cyclins rise in activity bind to S Cdk and
then S cyclin is destroyed, S cdk’s are then deactivated)
7
 Activation of Cdk-cyclin
 When cyclin comes in and binds to Cdk, it changes the
conformation of T-loop (it opens the loop up and exposes a
site of phosphorylation)
 T-loop is now joined to the protein complex
 A cyclin dependent kinase which activates kinase comes in
and donates a phosphate, now there is phosphorylation of
Cdk
 For Cdk to be fully active: has to be bound to cyclin and
phosphorylated
 ATP is a source of phosphate
 Inhibition of Cdk
 CDK can be activated and another kinase can come long (such as Wee1)

 CDK is held in limbo by inhibiting it temporarily

 Wee1 is another protein kinase and puts the inhibitory phosphate which binds to site to
prevent cdk from performing phosphorylating activity (affects site of phosphorylation)

e.g. M-Cdk
 CDK can also be inhibited with presence of cyclin dependent kinase inhibitor (CKI)
 p27 is the protein: slides in and interacts with entire protein complex and prevents it from
undergoing conformational changes or interaction with other proteins (prevents cdk from
phosphorylating)
 p27 blocks part of active sit needed for phosphorylation and function

p27 is a CKI

(Cyclin-dependent
Kinase
Inhibitor)

9
 Intracellular Control: Role of
Ubiquitin Ligases
 when cyclins are broken down and then control activation of cdk (control is
caused by ubiquitin ligases)

1
Ubiquitin and Protein Degradation
 Ubi is attached to target protein through enzymatic reaction and to a chain
 Many molecules of ubiquitin attached in a chain to the protein
 Polyubiquitin chain: composed of many copies of the protein
 Proteasome: structure in cells that takes care of crunching up degrading proteins and
spitting out amino acids
 Protein will enter into vestibule of proteasome and identified by presence of polyubiquitin
chain
 ubiquitin is then let go and protein moves through proteasome and is reduced to amino
acids and protein is destroyed and amino acids are reused in cell to form other proteins
Ubiquitin

The proteasome

A ubiquitin ligase is required to catalyze addition of a polyubiquitin chain to a protein

SCF are Ubiquitin Ligases
• SCF is needed to break down G1/s cyclins to prevent activation of
G1/s CDKs and this moves the cell forward
• SCF can lead to destruction of G1/S cyclins (see slide 5 from lecture
video 14).
• SCF can lead to destruction of CKI.

S-Cdk not deactivated;

e.g. cell proceeds to S phase
CKI must first be phosphorylated
before it is recognized by SCF/F-box
Positive effect on cell cycle
• CKI is an inhibitor that inhibits Cdk’s
• This mechanism is degrading an inhibitor and removing the
inhibition of Cdk
• Proteolysis: destruction of a protein
• SCF is constituently active through diff phases of the cell cycle
• SCF is present in high activity, so a CKI has to be
phosphorylated
• For SCF to recognize a CKI: F-box protein NEEDS TO BE
present, and the target protein needs to be phosphorylated
• If these conditions are met, the polyubiquitin chain is applied and
binding occurs
• Polyubiquitin is applied to the CKI which is phosphorylated
• CKI goes to the proteasome and is destroyed
• S-Cdk is not deactivated and thus the cell proceeds to the S-
phase
• Important to have high levels of S-Cdk (S-cyclins are elevated)
• This mechanism prevents destruction of active S-Cdk, thus
positive effect on cell cycle allowing it to progress
 APC are Ubiquitin Ligases
• APC can lead to destruction of securin, leading to chromatid
separation (next lecture video).
• APC can lead to destruction of M-cyclin.

Cell permitted to
leave M-phase

Positive effect on cell cycle

4
Skp/Cullin/F box family of proteins; Anaphase Promoting Complex
 APC leads to destruction of M cyclin
 For APC to be active, another subunit Cdc20 needs to present
 Cdc20 binds to APC and becomes active, polyubiquitin is then
applied to cyclin and cyclin is then destroyed
 Cyclin leaves the cdk, and cdk is left inactive, thus degrading m
cyclin in a proteosome
 Polyubiquitin chain is needed for proteasome to recognize the
protein that needs to be degraded.
 M cyclin is needed for cells to get into M phase (when M cyclin is
high, Mcdk’s are active)
 Cell also has to get out of M phase, and so Mcyclin is degraded and
Mcdk is deactivated and this causes cell to leave M phase.
SCF and APC are Active During Different Stages
(SCF here keeps G1/S cyclins low and S cyclins high)

SCF

APC

(-)
êM-cyclin êM-Cdk éAPC-Cdh1 Keeps M-cyclin
APC-Cdc20 (above) levels low and cell
activation leaves M-phase

Separase activation 5
and anaphase
 Puts everything in a linear scale
 SCF and APC changes cause changes in cyclin
 SCF is high and so it keeps G1/S cyclins low and does this by degrading the
S cyclins and deactivating G1/S Cdk’s
 SCF will break G1/S cyclins, leaving G1/S cdks low, allowing cell to progess
into S phase
 S cyclins are high because SCF will mark CKIs for destruction, not allowing
it to inhibit S-CDKs

 APC:
o Important for getting a cell through M phase and out of Mphase
o Anaphase promoting complex leads to chromatid separation
o APC is activated by CDC-20 leads to destruction of the M cycle, so the
active conc of M cycle is reduced
o To get out of M cycle the Mcdk activation has to crash, reduction in
active mcdk
o M cyclin is degraded by ubiquitin ligase and is reduced and so, mcdk
won’t be active
 Reduction mcdk is going to feedback and prevent further activation
of cdc20 (this is why there is a spike and reduction)
 Mcdk is the first thing to activate cdc20 and goes up and up.
Reaches a crest and deactivates enough mcdk (feedbacking
negatively on cdc20) – thus, rise and fall
o Once active mcdk is low it leads to activation of other apc (activated by
Cdh1)
 Cdh1 stays at higher levels to maintain low M cycle
 Reduced m cycle levels but keeps going all the way until the end of
G1, this is to prevent mitosis from happening during growth phase
 So, cdh1 levels are high to ensure m cyclin levels are low, or
else mcdk will be triggered again
Intracellular Control:
Examples

1
Activation of M-Cdk Triggers Mitosis
 This allows for immediate transition into mitosis
 The cell is on the brink of mitosis and waits until the conditions are right and then thrusts in
mitosis
 This mechanism exists to allow for checks and balances to occur

 M cyclin binds to Active Cdk1, conformational change of the t-loop and gets phosphorylated
 Wee1 (inhibitory protein kinase) that phosphorylates an active mCDK complex
o It places 2 phosphates
o Its phosphorylating a part of the complex which causes a conformational change which
leads to inactivation of the complex
o Its putting in more phosphates than Cdk would and phosphorylating a site
o Mcdk is held in limbo state (bring of entering mitosis)
 The trigger is the protein phosphatase (cdc25) – steals phosphate and leads to active M-cdk
o CDC25 Removes the phosphate that was added by Wee1, allowing to move into mitosis
 When we get active M-cdk, there is a positive feedback loop
o So, active M-cdk will continue to activate more cdc25 which continues to remove more
phosphates from other inactive mdck’s, and active mcdk will feedback negatively on
Wee1
 So, if m-CDK is active, it will inhibit the activity
 Since S-Cdk is high, it provides a basal level of activation for Cdc-25
Here, we are in the latter stages of G2 on the brink of mitosis…

(Phosphorylation by Wee1
blocks activity)

Enhances Feedback

S-Cdk phosphorylates (activates) Cdc25

during M-phase
2
 “Checkpoints”
2
1

1. DNA replication checkpoint

o End of G2
o Important that a cell verifies whether all
DNA has been replicated and if the cell
should enter mitosis
o If DNA hasn’t replicated (in S-phase),
then the cell should not undergo mitosis
o This is to ensure that either the cell
undergoes apoptosis or waits until all the
DNA has been replicated
2. Spindle attachment
checkpoint
o Important to determine
if all chromosomes are
attached to spindle
and
whether to trigger
anaphase
 o If all chromosomes are
not attached at mitotic
spindle at metaphase
plate, it should not
divide into sister
chromatids (won’t
evenly be divided into
daughter cells)
3. DNA damage checkpoints (several)
 Before cell enters S-phase and
so if the DNA is damaged, then it
should not be replicating,
because then replicating DNA will
be damaged too
DNA Replication Checkpoint
Detection of unreplicated DNA
If there is unreplicated dna, CDC-25 is not activated, M-CDK Is then
blocked and cell doesn’t proceed into Mitosis

Cdc 25 not activated; M-Cdk activation is

blocked (See previous slide)

Cell does not progress into mitosis

4
 Chromatid Separation
 CDC20 binds with APC and provides activation, also activated by M-CDK
(has to be at high concentration)
 APC provides ubiquitylation of securin (chain gets added) which is marked for
proteasome destruction, securing is separated, and enzyme separate is active
 Protein between sister chromatids holding it together, cohesin complex holds it
together, but separase comes in and breaks down proteins holding chromatids
together, separating at anaphase
 Spindle Attachment Checkpoint

Unattached
kinetochore

Mad2 binds

Cdc20-APC
activation blocked
 In green (mitotic spindle)
 In blue (sister chromosomes aligned along metaphase plate)
 Pair of chromatids not aligned at plate

No chromatid separation 

Mad 2 protein in red
Kinetochore is the centre of the chromosomes where the
sister chromatids are held together before separating in
anaphase
 Kinetochore binds to the mitotic spindle
 If the kinetochore is unattached, Mad2 binds to it which
causes inhibition of CDC20-APC complex
 If Mad2 is present, it will lead to inactivation and this is
meant to prevent chromatid separation in anaphase

Nond
Non-disjunction:

 Chromosomes can split even if pair isn’t attached

 You get an extra pair of chromosomes in one daughter cell
 Happens when there is a defect in the way the cell is monitoring these checkpoints
 Genetic component
DNA Damage Checkpoint (G1)
 Can occur through radiation – when a checkpoint is doing what is should and preventing
cell from going through cell cycle
 Signal that leads to activation of P53 (GENE REGULATORY PROTEIN – protein that
translocates into nucleus and has effect on transcription of mRNA and translation of a
new protein)
 Protein that can be translocated into nucleus and modify the transcription of mRNA from
a gene (mRNA once combined with ribosomes will produce proteins)
 P53 is degraded when bound to another regulatory protein called Mdm2, not kept in high
levels in the cell
 In damage, protein kinase leads to phosphorylation of p53, dissociating from Mdm2,
leading to its activation and can go on to bind with a regulatory region (P21) of the gene
 P21 gene transcribes P21 mRNA and P21 protein (Cdk inhibitor)
 Since it’s a cdk inhibitor, it takes G1/S and S CDKs and inhibits them – further interfering
with regions that would be phosphorylated, preventing the cell from entering S-phase
because DNA is damaged.
 By inhibiting S-CDK, this prevents the cell from getting into the S-phase
p53 is a gene regulatory protein, GRP

Negative effect on cell cycle

Cell does not enter S-phase

7
Extracellular Control:
Growth Factors

Fig. 22-103. Alberts et al.

1
 Total Cell Mass
 What determines the size of organ?
 Total cell mass is affected by:
o total cell # and the cell growth (how big the cells are)
 Depends on the difference in cell division vs cell death
 Cell death refers to apoptosis
 Cell division – Cell death: this difference determines total cell number
 Collectively, cell # and cell growth determines cell mass
 Ex: Mouse vs Human
o Difference in total cell mass of the organisms
o Fully developed organism (different masses)
o But same order of magnitude
 Total # of cells are controlled by mitogens (important for proliferation)

Total cell mass (size of organ / organism)

Total cell number Cell Growth

Cell division – Cell death

2
 Total Cell Mass
Dependent on Extracellular Factors:

1. Growth Factors: increase synthesis of proteins needed inside

the cell and decrease degradation of proteins

2. Mitogens: these proliferative factors are released and cell will

depend on it to move through cell division/cycle

3. Survival Factors: decrease apoptosis (programmed cell death);

a cell that is deprived of these factors cannot survive, cell needs
to be continuously exposed

Note: It’s confusing, but some “growth factors” also have mitogenic and
survival effects. Therefore, please remember processes and not
specific factors.
3
 Growth Factors
• Needed for growth of animal cells.
• Increase synthesis and decrease degradation.
• e.g. platelet-derived growth factor (PDGF), epidermal growth factor (EGF),
nerve growth factor (NGF).

Extracellular signalling proteins (GFs) from outside the cell which bind
to cell surface receptors

Cell surface receptors (binding initiates intracellular pathways)

 signal transduction (way a signal is transformed from one form to
another
 protein binds to receptor and a conformational change occurs
which activates intracellular pathways
 signal crosses the membrane via this receptor and activity

Intracellular pathways
 Growth Factors
• Act through receptor tyrosine kinase (“enzyme-linked receptors”): kinase activity embedded
in the structure of the large protein complex, allows for cross/auto-phosphorylation
- signalling molecule (dimer) binds and it brings together two inactive parts of the kinase,
- the bilayer is fluid, allows for conformational change and brings together two parts,
activating them
- tyrosine kinase domains phosphorylate each other and this adds more phosphates
- When TK is activated, it recruits many proteins and phosphorylates them, sending signals
• Extracellular binding, transmembrane domain, and intrinsic enzyme acitivity
(“autophosphorylation”).
• Ligand (dimer) binding ® receptor dimerization and transfer of Pi from ATP by tyrosine
kinase (TK domain).
• “Autophosphorylation” initiates intracellular pathways.

A receptor tyrosine kinase

with bound ligand (dimer)
 Growth Factors
• Increase synthesis of new proteins
• Phosphorylated receptor activates
PI-3 kinase…
• Phosphorylation of ribosomal protein
S6 and translation of many important
proteins.
• e.g. NGF and TrkA receptors (binds to nerve
growth factors) in neurons.
NGF dimer (a protein kinase)

(a protein kinase)

(a ribosomal protein)
TrkA receptor
Modified from Fig. 15-5 0a.
Alberts et al., 4th ed.

Modified from Fig. 17-65, Alberts, 5th ed. 6

phosphatidylinositol 3-kinase; phosphoinositol di/triphosphate
 Nerve growth factor binds, and cross-phosphorylation occurs
 PI-3 kinase is recruited and activated, will grab phosphate and phosphorylate PIP2,
now it is PIP3
 PIP3 will carry the signal forward and activate TOR (a protein kinase), which will
then activate S6K, which will phosphorylate a ribosomal protein (S6) = this
stimulates synthesis of new proteins which will initiate or increase the growth of cells
 Nerve Growth Factor (NGF)
 Rita isolated dorsal root ganglion cells from mice
 Presence in high concentration of NGF, in vitro – growing out in all directions
 Cell cannot determine what direction it should produce dendrites or extensions
of membrane processes
 Rita studied to identify the effects of NGF
 In tissue:
o Neuron in tissue growing, there is a growth cone which eventually
reached termination point and flourished into terminus of neuron,
producing nerve terminals
o Extended the long axon because in tissue in development, NGF secreted
by sites meant to attract axons from growing nerve cell (can occur in brain
or spinal cord)
o Detect a chemical gradient being released, so it would grow in that
direction (then differentiate and grow complex terminals)
 Nobel Prize, 1986

Rita Levi-Montalcini
The Nobel Foundation

Mouse sarcoma tissue released

“neurotrophins”.

Fig.17-45. Alberts et al.

7
 Extracellular Control:
Mitogens and Survival Factors

Fig. 22-103. Alberts et al.

1
 Mitogens
• Extracellular signals necessary for cell proliferation (cell to divide and
undergo mitosis)
• e.g. PDGF (platelet derived GF), EGF (epithelial GF), erythropoietin
(EPO).
• Act to increase cyclin synthesis, and thus Cdk activation.
- mitogens are there to increase cyclin synthesis to make sure there are
adequate number of cyclins (further regulated by ubiquitin ligase) –
important for cdk activation
• Promotes entry into S-phase from G1.
- Mitogen can have an impact on intracellular pathways (synthesis of
cyclins) and this can promote entry into S-phase from G1
 G1 Restriction Point
• For a cell to divide, it must progress out of
growth phase and into S-phase
• Type of checkpoint where the environment of
cell is taken into consideration to determine
whether the cell should move through cycle
• Also a “checkpoint”, but requires an
extracellular signal (mitogen), thus called a
restriction point
• Most vertebrate cells aren’t in a differentiated state
And are in G0
• With mitogens, cell renters G1 (out of G0),
pass the restriction point and cycle through
the cell cycle (ex: wound healing, process
more cells)
No mitogens Mitogens

Cell enters G0 Cell re-enters G1 and

continues

e.g. neurons, e.g. fibroblasts, PDGF,

muscle wound healing
Mitogens Stimulate Cell Division
(translocates from the cytosol
• These are receptors that to nucleus) and then binds to
bind to platelet derived specific site and increases
GFs expression of MYC gene
- growth factor may have • Gene regulatory protein
mitogenic properties modifies transcription of mrna
• Mechanisms of activation: and that translocates back to
PDGF would bind to both cytosol, combines with
sides and bring the ribosomes and produces the
together, thus permitting myc protein
cross-phosphorylation • Myc gene leads to increased
• PDGF binds to its translation of myc protein
receptors (alpha and beta • Transduction of signal across
subunits), activates RAS membrane and activation of
protein (G-protein pathway led by G-protein
• This activates MAP kinase
and this activates gene
regulator protein
Mitogens Stimulate Cell Division
 MYC protein can modify many other genes (cyclin, scf) and increase translation
 MYC increase Cyclin in the cytosol, cyclin gene will be transcribed (more cyclin in
the cytosol) and this will get activation of G1-CDK
 MYC also produces SCF subunit in the cytosol and SCF (protein ligase) will
mediate the destruction of P27 (CKI)
o By producing more SCF, we get more degradation of P27 and less inhibition
of S-CDK
 Both of these activities have an effect on Rb phosphorylation, eventually leading to
inactivation and leading cell into S-phase.
Mitogens Stimulate Cell Division
a grp
• Myc is a gene-regulatory
protein.
• G1-Cdk and S-Cdk
activity via transcription
of cyclin or SCF.
• Rb proteins (which inh. (a CKI)

cell cycle) are S-Cdk activation

inactivated.
• Production of S-cyclin
and S-Cdk activation. (inactivation)

• Progression to S-phase.

5
Rb: retinoblastoma protein, an inhibitor of cell cycle progression
 Abnormal Mitogenic Stimulation
• Mutations can sometimes lead to abnormal mitogenic stimulation.
• Some mutations that can lead to abnormal mitosis
- If a cell has increase in expression of RAS or Myc protein, cell will
produce more proteins, where the cell will increasingly divide
(lead to tumor production)
• Excessive Ras or Myc mimics stimulation.
• e.g. cancer, inappropriate entry into S-phase.
• Normal cells detect these changes and prevent further cell division.

6
 Prevention of Abnormal Mitogenic Stimulation
(in normal cells)
• Recall, p53 is normally degraded, and cell cycle normal.
• But if excessive Myc production, signal that allows p19ARF will bind to Mdm2,
dissociating Mdm2 from p53 and stabilizes (releases) p53.
• This can cause cell cycle arrest or apoptosis.
• In cancer, this mechanism may not
work because of p19 and p53
mutation
• Some cases where there is tumor
production, where this mechanism
doesn’t sense excessive myc
production, not allowing cell to
undergo apoptosis or arrest.

7
 Survival Factors
• Extracellular signals required for cells to survive.
• Secreted by cells in surrounding tissue.
• Without survival factors, cells will undergo apoptosis (next lecture).
• For a cell to continue to divide, survival factors must be present.
• Some growth factors are survival factors.
• e.g. nervous system development.

8
 Survival Factors
• Extracellular signal.
• Protein kinase B (PKB) activation.
• Phosphorylation of BAD and GRP inhibit
apoptosis.
• This example:
- Mechanism where SF bind to
transmembrane complex, activating
PKB and phosphorylating it
- This changes activity of BCL2 family of
proteins
- Bcl2 family of proteins promote or
inhibit apoptosis by regulating
cytochrome C release from the
mitochondrion
- Bcl2 itself inhibits apoptosis
- When Bcl 2 dissociates from
phosphorylated BAD, bcl2 is activated
- If BAD isn’t phosphorylated, then it
remains bound to Bcl2
- If BAD is phosphorylated, it is now
inhibited and wont produce proteins for
apoptosis
 Things to Consider...
1. There are many details that you’ve learned in the
lectures on the cell cycle. Try to piece everything
together and think about where in the cell cycle
everything fits, i.e. in ‘chronological’ order.
2. What roles do feedback mechanisms play in cell
cycle control?
3. Remember the differences between extracellular
and intracellular mechanisms of cell cycle control.
10
 Review of Intracellular Control of the Cell Cycle

1 3 7
2 6

Modified from Figure 17-34. Alberts et al.

11
1. G1 Restriction Point. Cell cycle proceeds only if mitogen present. Ras,
MAP kinase, myc… This brings cell out of G0.

2. DNA Damage Checkpoint. Stabilization of p53 leads to inactivation of S-

Cdk via p21 (a CKI).

3. Excess Mitogenic Stimulation. Excess Myc production removes Mdm2

by p19 and stabilizes p53. Cell cycle arrests.

4. S-phase and DNA Replication. Increased S-cyclin (and active S-Cdk),

and levels of M-cyclin are kept low because of APC.

5. Entry into Mitosis. S-Cdk activates Cdc25, which will in turn remove
inhibitory phosphate from M-Cdk and trigger entry into mitosis. Note that
M-Cdk feeds back positively on Cdc25.

6. Unreplicated DNA Checkpoint. If unreplicated DNA present, Cdc25

inhibited and M-Cdk not activated. Entry into mitosis does not occur.

7. Spindle Attachment Checkpoint. Unattached kinetochore leads to

binding of Mad2 and prevents activation of APC by Cdc20. Chromatids not
separated. (Recall that M-Cdk activates APC.) 12
The Mitotic Spindle: General Structure

Cell from Newt lung in prometaphase; nuclear envelope broken down,

chromosomes attaching to spindle (in green), centrosome of microtubules, foci
points of microtubule generation to allow for mitotic spindle generation 1
 Some Major Events of Mitosis
The Centrosome
Cycle

 Centrosome duplication (G2).

o Centrosome is home to the centrioles used to
organize the microtubules
o centrosome is duplicated (S-G2)
 Mitotic spindle formation (prophase).
 Chromosome condensation (prophase).
o Chromatin becomes discrete chromosome when
cell is ready to divide
 Breakdown of nuclear envelope
(prometaphase).
o Early metaphase
 Chromosome attachment to MTs begins
(prometaphase).
 The metaphase plate seen in metaphase,
chromosomes pairs attached to mitotic spindle
and nicely aligned.
 Anaphase (separation)
 Increased Dynamic Changes
 Levels of M-Cyclin are increasing as the cell approaches M phase.
 If MCyclin is going up, active MCDK is going up too
 MCDK as it becomes active in high concentrations, it phosphorylates MAP proteins,
leading to a decrease in their activity - at the same time, phosphorylating Kinesins
 Molecular motors: once phosphorylated, they co-opt in another time of way, used to
increase dynamic changes of microtubules + decreasing their stability
 Will not cause catastrophe, dynamic instability = swings between these 2 states
o By having phosphorylation, providing conditions for catastrophe
o Once catastrophe occurs, whole process occurs again
 Result is = we have shorter more dynamic microtubules, don’t need to extend to entire
length of the cell
 Increased Dynamic Changes
(by phosphorylation)

¯MAP activity
M-Cdk Dynamic changes of MTs
Kinesin activity

Recall from previous lecture:

(Kinesins)

3
Components of the Mitotic Spindle
1. Microtubules (Based on the function)
• Astral (star-shaped): in all directions,
- plus end growing, minus end in centrosome
• Kinetochore
- ejected from centrosome
- interact with kinetochores of the chromosome
- allow for attachment with MS
• Overlap
- other MT never make contact with
chromosomes and cross-link with each other,
stabilize structure of MS

2. Motor proteins
• Kinesin-related (+): at the centre
• Once the MS is formed, + is removed from
centrosome and in the cytoplasm
• Dynein (-):

3. Chromosomes (chromatids): important for the structure

And become a part of mitotic spindle

4. Centrosome: needed to nucleate initial MTs

• Centrioles
• PCM (regulating gamma tubulin and nucleating growth at – end)
 Motor Proteins Contribute to Spindle Assembly
• Multimeric motor proteins “cross-link” antiparallel MTs.
• (+) end-directed kinesin-related proteins (KRPs, e.g. BimC or
kinesin 5):
 Motor proteins specialized for formation/cross-linking of Mitotic spindle

• KRPs self-associate with each other at their tail domains, allow for cross-
linking at the MT tails
• This can promote elongation of mitotic spindle (MS forms and cross-linking
helps because of overlap of MTs)
• Kinesins go towards + end (lengthen the mitotic spindle), MT will go to - end
• Distance between catalytic end and + will decrease
• Slide overlap MTs past each other, pushed apart by kinesins.
• Promotes elongation of mitotic spindle.

5
 Motor Proteins Contribute to Spindle Assembly
• Dynein’s walk towards the minus end
• Distance between catalytic heads and minus end will decrease
• Don’t cause expansion but create minus ends of stability and foci
• Dynein motor proteins cross-link adjacent MTs.
• Multimeric (-) end-directed dyneins and kinesin-14 arrange minus
ends.
• Creation of foci at spindle poles and stability at the minus end

6
 Spindle Pole Separation
• Prophase:
• Before nuclear envelope breaks down
mitotic spindle is formed to cast
a net over the nucleus
• Mitotic spindle is formed, nuclear
envelope breaks down and chromosomes
come down and are captured by
the spindle
• Kinesin Related Proteins push on
the overlap MTs,
 dynein’s create foci
• dynein’s pull astral MTs.
 The tails of the dynein are attached to cortex (beneath membrane)
 Catalytic heads attached to MTs
 dynein’s are stabilized, distance between heads and minus end
decreases
 So, dynein’s pull on astral MTs, to bring them closer to the minus end
 Centrosomes are pulled closer to the cell membrane, expanding the
mitotic spindle
The Mitotic Spindle: Balance

Cell from Newt lung in prometaphase 1

 Chromosome Condensation
o Condensation occurs at prophase.
o When cell prepares for division, discrete
chromosomes are produced
o Cohesin (green circles in pic) and
condensin (Red circles) have similar
ATP and DNA-binding domains.
 Cohesin over time is replaced with
condensin
2
 Cohesin exists between sister G2 Prophase Metaphase
chromatids
 Cohesin is integrated within
chromosomes and compresses it
 Both dimer proteins, similar coiled COHESIN
domains that allow for cross-linking
o Cross-linking with DNA and cause CENTROMERE
CONDENSIN
attachment or compression
o Most cohesin is replaced by
condensin during prophase
o Condensation is dependent on ATP
hydrolysis and phosphorylation of
condensin by M-Cdk.
o Later stages of G2, mCDK is high –
phosphorylates condensin
MT – Kinetochore Attachment
• Prometaphase
• Astral MTs “capture” kinetochore
(highly dynamic astral MTs
o High levels of MCDK, MAP
proteins and kinesins
phosphorylated
o Chromosome pairs attach via
kinetochore to astral MTs –
refer to it as kinetochore MT
o Kinetochore is attached
alongside; dynamic changes
occurring with MTs until the
kinetochore interact end-on
o No longer dynamic instability
once it is attached
• Increases MT stability.
• Astral MTs become kinetochore
MTs.
• Bipolar attachment: attachment at both ends of mitotic spindle to both ends of kinetochores
o Bipolar attachment allows for attachment of MTs originating from each centrosome to make
attachment with kinetochore of the chromosome pair
 MT – Kinetochore Attachment
• Bundle of kinetochore MTs on either side
• (+) end attached “end-on” to kinetochore.
• Metaphase
• Formation of metaphase plate
• (+) and (-) end-directed motor proteins
provide stability of chromosomes along
equator of spindle.
o Motor proteins help achieve balance
and center chromosomes along the
equator of the mitotic spindle
.

4
 MT – Kinetochore Attachment
Motor proteins: balance at the MT-kinetochore interface.
• KRP (i.e. depolymerase): walk along microtubule towards + end, destabilizing it
• CENP-E (centromere-associated protein E): walk towards + end
• Dynein: walk towards – end
• MTs are not directly attached to kinetochores – has to remain unattached, + end has to be
dynamic

Note: Ndc80 not shown

See next lecture

(KRP/catastrophin)

(Karp 2008) 5
 MT – Kinetochore Attachment
• Motor proteins: balance by astral ejection force.
• Instead of having motor proteins working at the kinetochore, they’re also found near the
telomeres that interact with other microtubules (astral MTs)
• Pulling/pushing force by molecular motors at kinetochores and plus end of MT
• Force pushes the chromosome out to the metaphase plate

6
 Consequence of Missing a Motor Protein
Upper
• Normal plate formation.
Lower
• Chromosomes trapped near (-) end of
MT (poles).
• Kinesin protein (Kid) is lacking
- prevents astral ejection force
- not carrying chromosomes to
the metaphase plate

7
 Poleward flux
• Contributes to balance at metaphase plate
• Poleward flux
o Addition of subunits to plus end and lose of subunits at the minus end
o As motor proteins are pushing/pulling, the length of MTs is accommodating (getting
longer on one side and shorter on the other)
o Signals that allow for shorter MTs to gain subunits at + end and slowing
polymerization at the – end
o Rapid loss of subunits at longer MTs, and slow depolymerization at – end
o MTs have to remain dynamic (minus and plus ends exposed to allow for length
changes)
o Poleward flux allows for BALANCE at metaphase plate and movement of
kinetochores and chromosome pairs

Balance
Summary: balance at metaphase plate

1. Motor proteins:
- exist at + end, get push/pull
force at – end
- produce astral ejection force
2. Poleward flux:
- treadmilling
- balancing at metaphase plate
The Mitotic Spindle: Separation

Cell from Newt lung in prometaphase 1

 Anaphase A Separation
• Poleward (minus pole where the centrosomes are)
movement of chromatids towards – end by
shortening of kinetochore MTs.
o Anaphase A involves shortening of the
kinetochore MTs, bringing with them a sister
chromatid

1.MT depolymerization at (+) ends by KRPs.

o MT at + are degrading because of Kinesin-
Related Proteins, when they interact with
ends, they cause de-stability and bonds to
break
2.Continual loss of tubulin at (-) ends (i.e. as in
poleward flux) without addition at (+) ends.

o Always a loss of subunits at – end

o At equilibrium, what’s lost at – end is gained
at the + end
o During anaphase A, lose contribution of new
subunits at + end, continual loss
(destabilization); tubulin is in D-form
o Subunits are at end, so they fall off (little
subunits interacting)
o Subunits in middle don’t fall off, stable –
held by other things
 2 Separation Forces
1. MT disassembly drives chromatid movement (below)
 Ndc80 is a series of coiled protein that integrate with a part of MT
 The + end of MT remain free and can fall apart
 Falling apart of protofilaments that pulls back NDC80, with it comes the kinetochores
2. Poleward (or microtubule) flux at onset of anaphase.
 Because of destabilization at + end and poleward flux at – end, we get an overall shortening
 Loss of subunits at – end, dynein’s still remain focus, getting shorter and shorter
 No additional energy required: energy was initially stored in the formation of the microtubules

No additional energy required!

3
 Combined Model for Anaphase A
• Movement of chromatids is unknown
• Kinesin-13 depolymerizes at both ends.
• Dam1 ring (circular structure of protein wrapped
Dam1 ring surrounding a MT
around MT) maintains attachment between MT and
chromatid in yeast.
• When Kinesin-13 depolymerizes the MT, Dam1 ring
Maintains attachment of MT and kinetochore despite
depolymerization at + end
 Protofilaments expand from center of MT it
forces the Dam1 ring down and this pulls the
entire kinetochore
• Depolymerization powers movement.
 Experimental Evidence of Anaphase A separation
• Enough energy caused by depol of MTs to pull the sister chromatids
• MT attached to the pole body to the kinetochore of the chromosome pair; chromosome has
travelled all the way to the basal pole body
• MT depolymerization may drive chromatid movement.
• Nucleated MTs deprived of tubulin: cause depolymerization of MTs
• Depolymerization of MTs pulls chromosome pair towards the pole body
• Therefore, MT depolymerization provides sufficient energy.

5 μm
 Anaphase B Separation
• It occurs at the same time as anaphase A
• Expansion of the mitotic spindle at
anaphase; position of centrosome is
changed, now further apart
• Pulling by motor proteins at poles
• Pushing by motor proteins by overlap MTs at
central spindle (i.e. at overlap MTs) – expand
the mitotic spindle
• Thus, further elongation of spindle.

6
 Action of Motor Proteins in Anaphase B
• KRPs cross-link and push overlap MTs apart.
• Here, the Dyneins anchored to cell cortex pull themselves toward (-) end of astral MTs.
• Dyneins pull on the astral MTs as they become closer to the minus end, this will bring poles to the
minus end

7
 Things to Consider...
1. Are you familiar with the roles of each different type
of MT during mitosis?

2. Motor proteins?

3. Think about how chromosomes are balanced at

metaphase, and how chromatids separate
during anaphase.

8
Apoptosis: Caspases

1
 Apoptosis
 “Programmed cell death”: cells dispose of themselves following a program
 Total # of cells: proliferation of cells – cells that are apoptotic
 When organisms are developing, there are more cells that are proliferated, less cells that are
apoptotic
 Number of cells is, in part, controlled by regulation of cell death.
 Necrosis: death of cells due to injury to tissue or tissue/cell poisoning
o Can happen from reduction in ATP
o Lead to decrease in sodium, potassium ATPase and this can result in lysis of the cell
+ +
 Differs from necrosis: trauma or cytotoxicity leading to ¯ATP and ¯Na /K -ATPase activity,
followed by lysis.
o Calcium toxicity: should be at low concentrations but some conditions lead to overload
of calcium which leads to cell lysis
 Why does apoptosis occur?
o (Ex: neatly disposing a structure to make room for a new one, not make mess, i.e. building
destruction
Apoptosis Necrosis
 Apoptosis
 Apoptosis in development.
o Ex: Mouse paw
 During development of mouse embryo
 Bright Green (indicating cells undergoing apoptosis)
 Missing tissues in between digits (Wedding present)
 Through apoptosis, we get more mature looking digits (wedding disappears)
 BALANCE between proliferation of cells and loss of cells to create the structure
 Balance of cell division with death in adult tissues.

Mouse paw
Cells undergoing apoptosis Loss of cells between digits
 General Characteristics
• In order for apoptosis to occur, a cell must undergo significant
biochemical and morphological change – orchestrated during
development
• Part of the control is to make specific cell disappear through apoptosis: Initiation
by intracellular or extracellular signals
• Proteins called caspases that go off and destroy everything and cell is eaten up
by phagocyte
• Activation of a series of proteins involved in promoting apoptosis (or
inhibition of those that prevent it).
• Important intracellular proteins and signals necessary for survival are
cleaved/destroyed during apoptosis.
• Orderly disposal of dead cell.
Structural Changes During Apoptosis
o Normal cells of epithelial layer (stained and fixed): membranes joined together with a
little space
o During apoptosis, these normal cells contract and look separated – stop
communicating and form structures called “blebs”: smaller bits of cells that may
contain parts of mitochondria, nucleus, cytoplasm, some DNA
o Earliest phases of apoptosis (after destruction), we see shrinkage of cytoplasm and
chromatin condensation
o Second phase: Get DNA laddering, fragmenting of DNA all over the place, blebbing
occurs
o Final phase: phagocytosis – entire cell picked up by phagocyte; phagocyte will clean
up mess and debris in cell and destroy bits of apoptotic cells
 Structural Changes During Apoptosis
2 3
1

Normal cell

Normal Loss of adhesion “Blebbing”

5
 Phagocytosis
 In image: phagocyte on outside and apoptotic cell in the centre (in inside)
 Asymmetric distribution of plasma membrane is lost: many diff phospholipids delivered to
plasma membrane oriented asymmetrically (outer layer is different than inner layer)
o During apoptosis, loss of this asymmetry because things are getting broken down
 Signal that attracts phagocytes to the apoptotic cells at end of apoptosis
 This signal is a negatively charged phospholipid: phosphatidylserine which
then becomes exposed on the outside of cell.
 Phagocytes detect negatively charged phospholipids in the outer
layer
 The cell is then marked for phagocytosis by a macrophage.

6
 Cell-death Mutants
Normal
• Caenorhabditis elegans is a nematode
(important model)
• At end of development, they have:
• 959 somatic cells.
• 131 apoptose every time.
• Discovery of ced-3 gene that encodes
proteins similar to mammalian protease.
• Ced-3 gene produced proteins that were
enzymes which led to destruction of cells
• These cells refract light in a way where they can be
observed with apoptosis
• Ced-3 gene in mutants are knocked down,
won’t produce proteins it usually does,
preventing apoptosis of the 131 cells
• Cell is developing but not undergoing
apoptosis
• Lead to caspases (found in mammals) and
ced-4 encodes Apaf-1; ced-9 encodes Bcl-2.
ced-3 mutant
ced-3 mutation prevents apoptosis in all 131 cells 7
Ellis and Horvitz 1986. Cell 44:817-829.
 Caspases
 These are important for destruction of proteins inside a cell
needed for survival
 Called caspases:
- proteases
- mediate changes in apoptosis
- cysteine residue at catalytic site which cleaves other proteins at
aspartate sites
- caspases can also cleave each other; can lead to amplification
of more caspase activation
 Identification of ced-3 gene led to the discovery of homologous
family of proteins, called “caspases”, in mammals.
 They are proteases that cleave essential proteins.
 Involved in most changes observed during cell death.
 This enzyme has a cysteine residue at its catalytic site and cleaves
other protiens at an aspartate site.
 Caspases may also cleave each other, leading to their activation.
 Caspases Cleave Essential Proteins
 Protein Kinases:
o Focal adhesion Kinase: important in allowing cells attach to each other
in ECM; these are cleaved by caspases (distribution in cell adhesion,
BLEB)
 Lamins:
o Important for construction of nuclear lamina
o Caspases break down Lamins and this is how the nuclear envelope is
broken down and DNA is destroyed
 Cytoskeleton:
o Changes in cell shape, can no longer move (disintegrates)
o Breaks down tubulin, actin, intermediate filaments
 CAD
o Caspases cleave Caspase-activate DNase (CAD)
 This activates CAD and allows it to translocate into nucleus and
break down DNA and its components
 Caspases Cleave Essential Proteins

= cleavage
Caspases
( = activation

(+)

Protein Kinases Lamins Cytoskeleton CAD

e.g. FAK, disrupt Disassembly of Changes in cell DNA

cell adhesion nuclear lamina shape fragmentation

From Karp 2008. Cell and Molecular Biology.

9
CAD = caspase-activated DNAse; FAK = focal adhesion kinase
 Procaspase Activation
 Caspases are not always active until they are activated

 Initiator (or “pro”) caspases are inactive.

 An apoptotic signal triggers assembly of adaptor proteins that activate

procaspases.

 These caspases go on to activate downstream executioner caspases by

proteolytic interactions (causing cleavage and break down of proteins).

 Adapter proteins cause dimerization and bring 2 procaspases together and

activate them (threshold concentration = widespread activation) – procaspases
will cleave aspartate sites at the executioner caspase and this will cause a
caspase cascade
11
 The Caspase Cascade
 Amplified signal which will lead to apoptosis
 An active caspase activates another caspase…
 procaspases will cleave aspartate sites at the executioner caspase, and this will cause a caspase
cascade
 Irreversible, “all-or-none” cascade – no turning back
 Amplification: one caspase will activate several others
 Result: intracellular proteins are
cleaved.

12
Figure 18-5b Molecular Biology of the Cell (© Garland Science 2008)
Apoptosis: Extrinsic and Intrinsic
Pathways

1
 2 Major Apoptotic Pathways
Extrinsic pathway
• Extracellular signal that initiates the pathway
• Procaspase activation triggered from outside the cell.
• “Cell death” receptors.

Intrinsic pathway (intracellular signal)

• Eupoptism
• Procaspase activation triggered from inside the cell.
• e.g. intracellular damage.
• e.g. via Bcl-2 family of proteins (note: this may involve an extracellular
survival factor).

2
 Extrinsic Pathway
• Cell undergoes apoptosis
• Ex: T-cells of immune system
• Cell-to-cell interaction (transmembrane protein that acts as a ligand)
• Lymphocyte: expresses large protein complex, and when it comes in contact with target cell,
it binds FAS death receptor
• Extracellular signal (Fas ligand) activates death receptor (Fas protein).
• Recruitment of adaptor proteins and procaspase activation.
- FAD ligand binds to FAS receptor and that recruits FAD adaptor protein and this recruits
procaspase-8 = producing death inducing signal complex (DISC)
- DISC complex brings procaspases in close proximity with each other, activating executioner
• When we have produced the DISC, we have a means of activating the Caspase cascade
• Binding of extracellular domains causes change in confirmation in intracellular domains

FADD = Fas-associated death domain; DISC = death-inducing signalling complex

 Intrinsic Pathway
 An intracellular death signal initiates caspase Cytochrome c in mitochondria DNA in nucleus
cascade and cell death.
o Initiated by e.g. DNA damage or loss of
survival factor
o Intrinsic: formation of apoptosome that
initiates cascase
 Bcl-2 protein family
o Bcl-2 inhibits apoptosis
o Bax and Bak (part of family) act to initiate
apoptosis on mitochondria and release
cytochrome c.
 Balance of these proteins determines fate (life or
death) of cell
 DNA in nucleus
 Mitochondria contain cytochrome c
 Below: cell in the process of undergoing apoptosis,
the cytochrome c is released from mitochondria
and DNA is fragmented
o This initiates the process of recruitment of
apoptosome (important for this pathway)

Cytochrome c released DNA fragmented

(Karp, 2008) 4
 Intrinsic Pathway
 Early phases of apoptosis:
o Activation of Bcl2 family (most importantly, Bax)
o Apoptotic signal activates Bcl2 family proteins (e.g. Bax) to form aggregates in
outer mitochondrial membrane.
 Bax proteins are dispersed as transmembrane proteins but when there is a
signal, there is an aggregation of Bax proteins into complex, they get together
and form a large pore (allow the passage of proteins)
 Proteins are spit out into the cytosol (one is cytochrome C)
 Induces cytochrome c release from intermembrane space
 Intrinsic Pathway
• Cytochrome C is out and will bind to the other proteins (Apaf1): conformational change of Apaf1, allows
for increased binding affinity of CAR domain to interact with other domains (forms pinwheel)

• CAR domains form this complex that has increased binding affinity for procaspases
• Formation of “apoptosome” to recruit procaspases close to each other (aligned now)
• Alignment of procaspases leads to activation of executioner caspase which leads to the Caspase
cascade.
• Survival factors can cause inhibition activation of Bax (inhibiton of apoptosis)

6
Apaf-1 = apoptotic protease activating factor-1; CARD = caspase recruitment domain
Some Mechanisms of the Intrinsic Pathway
survival factor

DNA damage

(synthesis)
(-)
(-)
Cytochrome c release
Bax
from mitochondria

(irreversible)

Caspase cascade Cleavage of

intracellular proteins
7
 Apoptosis in the Developing Nervous System
• Recall NGF is important to leading to external outgrowth
• A growth factor, but also a survival factor: nerve cells that are deprived of survival factor
will enter into apoptosis
• Half of cells die in vertebrates because of apoptosis – needs to happen!
- important to produce more cells to grow and form synapses with target cells
- cells that don’t reach target die (not needed)
- axons extending to meet targets
• Survival factors are released by “target” cells: so the further away from target cells, the
weaker the gradient
• From synapses at target cells, reduction in release of NGF from the target cells
• Cells that reach cell and have access to growth factors will survive (those that don’t will
die)
• Bax is an important mediator* in apoptosis
 Apoptosis in the Developing Nervous System
• Ex: Mouse embryos:
• mutation preventing synthesis casp3, casp9 and apaf1
• Mutations in genes encoding important proteins in the apoptotic pathway
have drastic consequences.
• In developing mouse embryo, forebrain protrusion/malformations because of
excessive mitosis and differentiation result, apoptosis was not occurring at
the required level
• having apoptosis is important!

9
Nijhawan et al. 2000. Annu Rev Neurosci 23:73-87
 Things to Consider...

How do extrinsic and intrinsic pathways of

apoptosis differ? For example, think about how
the caspase cascade is initiated in each case.

10