Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
Polarized cytoskeletal networks organize cells
and control interactions with their environment
Which of the structures below would you predict
to organize intracellular trafficking routes?
a) Green
b) Red
c) Blue
Which of the structures below would you predict
to organize intracellular trafficking routes?
a) Green (Microtubules)
b) Red (Actin)
c) Blue (Nucleus)
Microtubules are inherently polarized
(from subunit, to protofilament, to network)
Each protofilament is made of
heterodimers of the monomeric
proteins a-tubulin and b-tubulin
Tubulin monomers bind and
hydrolyze GTP (shown in red)
Each heterodimer is asymmetric
The heterodimers assemble
head to tail forming
polarized filaments
g-Tubulin complexes nucleate microtubules
• g-tubulin binds tubulin heterodimers assembling protofilaments into tubes
• g-tubulin nucleates microtubules at their minus ends
• Plus ends grow away from nucleation sites
• g-tubulin often associates with large microtubule organizing centres (MTOCs)
e.g. the centrosomes (above)
Contain 2 centrioles surrounded by hundreds of proteins
with g-tubulin nucleation sites on the surface
Microtubule movement by dynamic instability
• Single microtubules switch between growing & shrinking
•termed dynamic instability
This behaviour allows microtubules to search the full 3-D space of the cytoplasm
Microtubule movement by dynamic instability
• Single microtubules switch between growing & shrinking
•termed dynamic instability
• Growing microtubules have a protective cap of GTP-bound tubulin
• If GTP hydrolysis is faster
than subunit addition
the cap is lost and
catastrophe occurs
*depolymerization is
~100x faster at an
exposed GDP end*
•Regaining a GTP cap
rescues growth
Microtubule networks can form a coordinate system on their own
A purified centrosome was mixed
with purified tubulin subunits
in an artificial membrane-bound container.
It moves to the centre of the container
as microtubule plus ends push on
the outer membrane.
àminus ends central/plus ends outwards
Microtubule networks can form a coordinate system on their own
This may contribute to microtubule
organization in cells
(but many regulatory proteins
are also involved)
How could a microtubule coordinate system be used?
How is cargo transported along microtubules?
Sun Peaks Trail Map
Wikimedia
Motors move cargo through the microtubule networks
The motor activity is polarized.
– Dynein moves to microtubule minus ends
– Kinesin moves to microtubule plus ends
Motors move cargo through the microtubule networks
The motor activity is polarized.
– Dynein moves to microtubule minus ends
– Kinesin moves to microtubule plus ends
The role for microtubules in positioning the Golgi can be seen
after the addition of a microtubule inhibitor
Control cells
Nocodazole-treated cells
What motor is likely key for Golgi positioning?
a) Dynein
b) Kinesin
The microtubule network is very dynamic and can be reorganized
How could microtubules be used to control the colour of this fish?
The African cichlid fish, Tilapia mosssambica
Male dominant behavior
àBlack color change
Kinesin and dynein
compete
for melanosomes
Haimo and Thaler, Bioessays 16:727
Camouflage behavior
àWhite colour change
Kinesin inhibited
Dynein moves
melanosomes
to centre
Melanosomes are
pigment-containing
vesicles.
When they are
throughout cell,
the cell is black.
When at the centre,
the cell is white
The actin cytoskeleton also plays a critical role
in organizing cell structure and controlling cell behaviour
The actin cytoskeleton is inherently polarized
(from subunit, to filaments, to networks)
Actin monomers are
asymmetric
Actin monomers bind
and hydrolyze ATP
Actin monomers assemble
head to tail forming
polarized filaments
The actin cytoskeleton is inherently polarized
(ATP-ADP polarity along actin filaments)
• After polymerization Actin-ATP is hydrolyzed to Actin-ADP
• Hydrolysis reduces binding affinities to neighbouring subunits increasing dissociation
• However, if the rate of addition of Actin-ATP is faster than rate of removal of ActinADP, a relatively stable “cap” of Actin-ATP subunits can be formed
ATP
ADP
ADP ADP ATP ATP ATP
ADP ADP ADP ATP ATP
ADP
cap
plus end
ATP
Which way will this filament grow?
The actin cytoskeleton is inherently polarized
(Polarized assembly and disassembly leads to treadmilling)
Because of the ATP-ADP polarity along the filament,
subunits can undergo net assembly at plus end
equal to (or greater than) the net disassembly at the minus end
Thus, the polymer can maintain a constant length (or grow)
with a flux of subunits through the filament (treadmilling)
Net direction of treadmilling
The actin cytoskeleton is inherently polarized
(from subunit, to filaments, to networks)
The ARP complex nucleates actin filaments…
..and branches
actin filaments
to form polarized
2-D networks
Polarized treadmilling of large actin networks
can produce significant protrusive power
These networks
drive polarized
cell movement
Treadmilling actin networks need traction
to drive cells forward
Net direction of treadmilling
Without traction actin filaments can treadmill
but maintain their overall position
Treadmilling microfilaments can engage
stationary anchors to create ‘protrusive machines’
Cell
Protrusion
A stationary anchor binds one part of the filament
The treadmilling filament extends from that point
This extension pushes against the cell membrane driving cell protrusion
Large regions of actin networks are anchored
to create ‘protrusive machines’
ANCHORED
Large regions of actin networks are anchored
to create ‘protrusive machines’
Top View
Side View
What is the network
anchored to?
In animals, cells migrate on (and through) the extracellular matrix,
a non-cellular material made of proteins and polysaccharides
Integrins connect the actin cytoskeleton to extracellular matrix
molecules
Main receptors that bind
extracellular molecules
Transmembrane heterodimers
of non-covalently associated
a and b subunits
Linked to the actin cytoskeleton
via adaptor proteins
How is the actin cytoskeleton directed to follow a target?
Video
Chemoattractant receptors orient the actin networks
Chemoattractant receptors orienting actin networks
is analogous to food sources orienting ant trails
Actin networks and ant trails are each made of polarized subunits
Each are dynamic and can rapidly reorient to changes in the target position
mtkilimanjarologue.com
Actin networks can undergo
other large scale rearrangements
e.g. localized assembly of the contractile ring
that divides daughter cells after mitosis
Actin and microtubule networks are also integrated in cells
but we are just beginning to understand the crosstalk
Advice on required e-text reading
Supplement your lecture notes by writing out the following as you read.
à forces you to think and organize as you read,
and thus increases your understanding
• The steps of processes and the key molecules of the complexes involved
– for those discussed in class, add information from the e-textbook
– for examples not discussed, write short summaries or diagrams
How do I choose which processes/complexes to take notes on?
Emphasize those with…
– relevance to a key function of a cell, organ or organism
– relevance to disease
– an organization that is similar to other
processes/complexes/concepts discussed
…and make short notes about these points
Do this within 24h of each lecture (preferably the same day),
and review your notes frequently.
à cements the material in your longer term memory
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
4. Tissue morphogenesis
5. Tissue patterning
6. Stem cells
9-10: Mitosis and Cell Division & Cancer
11-12: Development of Multicellular Organisms
All structures need maintenance
Once cells and tissues are formed they continually regenerate
This can occur at the level of molecular turnover or cellular turnover
1. Some tissues contain the same cells for the life of the organism,
but the molecular components of these cells do turn over
-typically cells with very specialized architecture
e.g. auditory hair cells or photoreceptor cells
2. Other tissues renew their cells rapidly
-typically cells exposed to harsh environments or activities
e.g. skin cells, gut cells or blood cells
3. Other tissues are between these extremes
The specialized architecture of auditory hair cells in the organ of Corti
This architecture functions to convert sound waves into nerve impulses
(it allows us to hear)
Sound waves cause stereocilia
atop hair cells to tilt
With tilting, tethers pull open
ion channels on neighbouring stereocilia
(this initiates a nerve impulse)
This architecture functions to convert sound waves into nerve impulses
(it allows us to hear)
Sound waves cause stereocilia
atop hair cells to tilt
With tilting, tethers pull open
ion channels on neighbouring stereocilia
(this initiates a nerve impulse)
In mammals, these cells do not re-grow when lost
anatomybox.com
Image by By F. Kalinec and B. Kachar, NIH
Their loss from disease, toxins or extreme noise leads to permanent
hearing loss
However, the molecules that make up these cells are continually made
and destroyed
Human photoreceptor cells are another
permanent cell type with a specific architecture.
Their architecture
converts light waves
into nerve impulses
The overall photoreceptor cells are permanent,
but do they turn over at the molecular level?
Pulse-chase experiment
-cells exposed to radiolabeled
leucine for a short time
-they take up the labeled amino
acid and incorporate it into newly
synthesized proteins for a short
period of time
What will happen to the labeled
Leucine?
a) It will be detected in the cells
for their entire lifetime
b) It’s detection will gradually
be lost
The overall photoreceptor cells are permanent,
but do they turn over at the molecular level?
Degrades the protein
+ photoreceptive
discs
Pulse-chase experiment
-cells exposed to radiolabeled
leucine for a short time
-they take up the labeled amino
acid and incorporate it into newly
synthesized proteins for a short
period of time
What will happen to the labeled
Leucine?
a) It will be detected in the cells
for their entire lifetime
b) It’s detection will gradually
be lost
1. Some tissues contain the same cells for the life of the organism,
but the molecular components of these cells do turnover
-typically cells with very specialized architecture
e.g. auditory hair cells or photoreceptor cells
2. Other tissues renew their cells rapidly
-typically cells exposed to harsh environments or activities
e.g. skin cells, gut cells or blood cells
3. Other tissues are between these extremes
Cell turnover can be stem cell dependent or independent
Stem cell definition
1. It is not terminally differentiated
2. It can divide without limit
3. Its daughters can remain a stem cell
or differentiate
Cell turnover can be stem cell dependent or independent
Stem cell definition
1. It is not terminally differentiated
2. It can divide without limit
3. Its daughters can remain a stem cell
or differentiate
Cell renewal can occur from division
of differentiated cells
(e.g. liver cells and insulin-secreting cells
of the pancreas)
The use of stem cells requires specific
mechanisms
Mechanism 1
The fates of stem cell daughters must be controlled
1. Divisional asymmetry
One daughter receives factors promoting
‘stemness’, and the other receives
factors promoting differentiation
A drawback:
If stem cells are lost, their original numbers
can’t be restored
Mechanism 1
The fates of stem cell daughters must be controlled
2. Environmental asymmetry
The cell division is symmetric and
and the daughters’ fates are determined
by the environment they are born in to
If stem cells are lost, then their numbers
can be increased by having both
daughters of divisions enter the
environment promoting “stemness”
Mechanism 2
Stem cells divide slowly for their long-term preservation
This protects the stem cell from:
1. Mutations associated with
cell division
2. Telomere depletion associated
with cell division
However, large numbers of cells
are needed to renew differentiated
cell populations
Mechanism 2
Stem cells divide slowly for their long-term preservation
Transit amplifying cells
expand cell numbers
before final differentiation
Mechanism 3
Stem cells are supported by a local environment, their niche
e.g. Skin stem cells and their progeny
The stem cells reside in the
basal layer and require
basal lamina attachment
to remain as stem cells
àthe basal lamina provides
a niche for the stem cells
After detaching from the
basal lamina, the cells
differentiate through
a linear sequence
of cell types and are
finally shed from the animal
Mechanism 3
Stem cells are supported by a local environment, their niche
e.g. Skin stem cells and their progeny
The stem cells reside in the
basal layer and require
basal lamina attachment
to remain as stem cells
àthe basal lamina provides
a niche for the stem cells
After detaching from the
basal lamina, the cells
differentiate through
a linear sequence
of cell types and are
finally shed from the animal
Without renewal from stem cells,
our skin would be lost in a month
Blood stem cells and their progeny
Blood stem cells
differentiate into
various populations
creating
a branched pathway
to final differentiation
Signals can promote
specific branches
depending on the
need for cell types
Blood stem cells and their progeny reside in bone marrow
An electron micrograph of bone marrow
Blood cell precursors are in bone marrow
but are the stem cells there?
If all blood stem cells were killed off
would ‘new’ bone marrow restore them?
Blood stem cells and their progeny reside in bone marrow
An electron micrograph of bone marrow
Blood cell precursors are in bone marrow
but are the stem cells there?
If all blood stem cells were killed off
would ‘new’ bone marrow restore them?
Identifying blood stem cells and their progeny
Separate cells based on arbitrary differences
………
How would you test each separated group for stem cell activity?
Identifying blood stem cells and their progeny
Homogenize mouse bone
marrow to release single cells
Expose cells to fluorescent
antibodies recognizing specific
cell surface molecules
Isolate labeled cells by
Fluorescence-Activated
Cell Sorting (FACS)
Identifying blood stem cells and their progeny
Homogenize mouse bone
marrow to release single cells
Expose cells to fluorescent
antibodies recognizing specific
cell surface molecules
Isolate labeled cells by
Fluorescence-Activated
Cell Sorting (FACS)
Test ability of isolated cells
to restore all blood cells
of an irradiated mouse
~1/10 000 bone marrow cells can
~5 of such cells are sufficient for
the restoration
Blood stem cells are maintained
through interactions with stromal
cells in the bone marrow
Once detached cells differentiate
Stromal cells provides a niche for
for the blood stem cells
NOTE:
“stromal cell”,
“mesenchymal cell”,
“connective tissue cell”
…are all close synonyms
Stem cells and tissue renewal affect how we age…
…and can treat diseases and disabilities
Stem cells and tissue renewal affect how we age…
…and can treat diseases and disabilities
Medical uses for stem cells
Using blood stem cells to treat leukemia
Problems of immune rejection
à careful tissue matching
and immunosuppressive drugs
à if the cancer arises from a mutation
in one of the progenitor populations
then the patient’s own stems cells
can be used after sorting
!!
Remove from
marrow sample
Inject the rest
or a patient
Medical uses for stem cells
What if the tissue to be replaced doesn’t have a readily
available supply of its own stem cells?
(e.g. spinal cord injuries or neurodegenerative diseases)
Can cells of a different tissue be used to make stem cells
for treatment?
This occurs naturally during
limb regeneration in newts
•Muscle cells de-differentiate,
and start dividing
•They form a bud similar to the
embryonic limb bud
•Their progeny form all cell types
needed to re-grow the limb
Medical uses for stem cells
What if the tissue to be replaced doesn’t have a readily
available supply of its own stem cells?
(e.g. spinal cord injuries or neurodegenerative diseases)
Can cells of a different tissue be used to make stem cells
for treatment?
Current technology cannot do this from adult human cells
at the scale or reliability needed for medical purposes,
but is has been done in experiments
This technique could avoid immune rejection by using
the patient’s own cells, but cancer development is a
potential problem if cell differentiation isn’t properly controlled
Embryonic stem (ES) cells can proliferate indefinitely in culture
and have full developmental potential
This can increase the yield of cells needed for treatments, but
ethical issues, immune rejection and the potential of cancer
are still concerns
Two potential ways to avoid immune rejection of ES cells
1. Somatic cell nuclear transfer—use a nucleus from one of the patient’s own cells
and transfer it into an unfertilized egg to develop an embryo from which ES cells
can be harvested
Two potential ways to avoid immune rejection of ES cells
1. Somatic cell nuclear transfer—use a nucleus from one of the patient’s own cells
and transfer it into an unfertilized egg to develop an embryo from which ES cells
can be harvested
2. Treat some of the patient’s own cells with factors that generate ES cells
à a combination of Oct3/4, Sox2, Myc and Klf4 (all transcription factors) can
convert differentiated cells into cells with ES cell characteristics
+ protein
expression
Continual renewal is important for maintaining all structures
Tissues often renew themselves naturally,
but when they can’t medical treatments can be used
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
Polarized cytoskeletal networks organize cells
and control interactions with their environment
Which of the structures below would you predict
to organize intracellular trafficking routes?
a) Green
b) Red
c) Blue
Which of the structures below would you predict
to organize intracellular trafficking routes?
a) Green (Microtubules)
b) Red (Actin)
c) Blue (Nucleus)
Microtubules are inherently polarized
(from subunit, to protofilament, to network)
Each protofilament is made of
heterodimers of the monomeric
proteins a-tubulin and b-tubulin
Tubulin monomers bind and
hydrolyze GTP (shown in red)
Each heterodimer is asymmetric
The heterodimers assemble
head to tail forming
polarized filaments
g-Tubulin complexes nucleate microtubules
• g-tubulin binds tubulin heterodimers assembling protofilaments into tubes
• g-tubulin nucleates microtubules at their minus ends
• Plus ends grow away from nucleation sites
• g-tubulin often associates with large microtubule organizing centres (MTOCs)
e.g. the centrosomes (above)
Contain 2 centrioles surrounded by hundreds of proteins
with g-tubulin nucleation sites on the surface
Microtubule movement by dynamic instability
• Single microtubules switch between growing & shrinking
•termed dynamic instability
This behaviour allows microtubules to search the full 3-D space of the cytoplasm
Microtubule movement by dynamic instability
• Single microtubules switch between growing & shrinking
•termed dynamic instability
• Growing microtubules have a protective cap of GTP-bound tubulin
• If GTP hydrolysis is faster
than subunit addition
the cap is lost and
catastrophe occurs
*depolymerization is
~100x faster at an
exposed GDP end*
•Regaining a GTP cap
rescues growth
Microtubule networks can form a coordinate system on their own
A purified centrosome was mixed
with purified tubulin subunits
in an artificial membrane-bound container.
It moves to the centre of the container
as microtubule plus ends push on
the outer membrane.
àminus ends central/plus ends outwards
Microtubule networks can form a coordinate system on their own
This may contribute to microtubule
organization in cells
(but many regulatory proteins
are also involved)
How could a microtubule coordinate system be used?
How is cargo transported along microtubules?
Sun Peaks Trail Map
Wikimedia
Motors move cargo through the microtubule networks
The motor activity is polarized.
– Dynein moves to microtubule minus ends
– Kinesin moves to microtubule plus ends
Motors move cargo through the microtubule networks
The motor activity is polarized.
– Dynein moves to microtubule minus ends
– Kinesin moves to microtubule plus ends
The role for microtubules in positioning the Golgi can be seen
after the addition of a microtubule inhibitor
Control cells
Nocodazole-treated cells
What motor is likely key for Golgi positioning?
a) Dynein
b) Kinesin
The microtubule network is very dynamic and can be reorganized
How could microtubules be used to control the colour of this fish?
The African cichlid fish, Tilapia mosssambica
Male dominant behavior
àBlack color change
Kinesin and dynein
compete
for melanosomes
Haimo and Thaler, Bioessays 16:727
Camouflage behavior
àWhite colour change
Kinesin inhibited
Dynein moves
melanosomes
to centre
Melanosomes are
pigment-containing
vesicles.
When they are
throughout cell,
the cell is black.
When at the centre,
the cell is white
The actin cytoskeleton also plays a critical role
in organizing cell structure and controlling cell behaviour
The actin cytoskeleton is inherently polarized
(from subunit, to filaments, to networks)
Actin monomers are
asymmetric
Actin monomers bind
and hydrolyze ATP
Actin monomers assemble
head to tail forming
polarized filaments
The actin cytoskeleton is inherently polarized
(ATP-ADP polarity along actin filaments)
• After polymerization Actin-ATP is hydrolyzed to Actin-ADP
• Hydrolysis reduces binding affinities to neighbouring subunits increasing dissociation
• However, if the rate of addition of Actin-ATP is faster than rate of removal of ActinADP, a relatively stable “cap” of Actin-ATP subunits can be formed
ATP
ADP
ADP ADP ATP ATP ATP
ADP ADP ADP ATP ATP
ADP
cap
plus end
ATP
Which way will this filament grow?
The actin cytoskeleton is inherently polarized
(Polarized assembly and disassembly leads to treadmilling)
Because of the ATP-ADP polarity along the filament,
subunits can undergo net assembly at plus end
equal to (or greater than) the net disassembly at the minus end
Thus, the polymer can maintain a constant length (or grow)
with a flux of subunits through the filament (treadmilling)
Net direction of treadmilling
The actin cytoskeleton is inherently polarized
(from subunit, to filaments, to networks)
The ARP complex nucleates actin filaments…
..and branches
actin filaments
to form polarized
2-D networks
Polarized treadmilling of large actin networks
can produce significant protrusive power
These networks
drive polarized
cell movement
Treadmilling actin networks need traction
to drive cells forward
Net direction of treadmilling
Without traction actin filaments can treadmill
but maintain their overall position
Treadmilling microfilaments can engage
stationary anchors to create ‘protrusive machines’
Cell
Protrusion
A stationary anchor binds one part of the filament
The treadmilling filament extends from that point
This extension pushes against the cell membrane driving cell protrusion
Large regions of actin networks are anchored
to create ‘protrusive machines’
ANCHORED
Large regions of actin networks are anchored
to create ‘protrusive machines’
Top View
Side View
What is the network
anchored to?
In animals, cells migrate on (and through) the extracellular matrix,
a non-cellular material made of proteins and polysaccharides
Integrins connect the actin cytoskeleton to extracellular matrix
molecules
Main receptors that bind
extracellular molecules
Transmembrane heterodimers
of non-covalently associated
a and b subunits
Linked to the actin cytoskeleton
via adaptor proteins
How is the actin cytoskeleton directed to follow a target?
Video
Chemoattractant receptors orient the actin networks
Chemoattractant receptors orienting actin networks
is analogous to food sources orienting ant trails
Actin networks and ant trails are each made of polarized subunits
Each are dynamic and can rapidly reorient to changes in the target position
mtkilimanjarologue.com
Actin networks can undergo
other large scale rearrangements
e.g. localized assembly of the contractile ring
that divides daughter cells after mitosis
Actin and microtubule networks are also integrated in cells
but we are just beginning to understand the crosstalk
Advice on required e-text reading
Supplement your lecture notes by writing out the following as you read.
à forces you to think and organize as you read,
and thus increases your understanding
• The steps of processes and the key molecules of the complexes involved
– for those discussed in class, add information from the e-textbook
– for examples not discussed, write short summaries or diagrams
How do I choose which processes/complexes to take notes on?
Emphasize those with…
– relevance to a key function of a cell, organ or organism
– relevance to disease
– an organization that is similar to other
processes/complexes/concepts discussed
…and make short notes about these points
Do this within 24h of each lecture (preferably the same day),
and review your notes frequently.
à cements the material in your longer term memory
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
BIO 230
Lecture 3 :
Prokaryotic Transcriptional Regulation
Continued…
1) Recap of prokaryotic gene regulation
2) Bacteriophage Lamba
3) Synthetic Biology
4) Transcription Attenuation
Readings (Alberts et al. custom text)
Pages 400-405, 413-416, 876-878
2
3
Catabolite Activator Protein
Trp Repressor
Lac Repressor
Recap: Prokaryotic Gene Regulation
Example 1: The Tryptophan Operon
Tryptophan repressor contains a
Helix-Turn-Helix
DNA binding motif (most common DNA-binding motif)
Helix-Turn-Helix
Binds in major
groove of DNA
double helix
Tryptophan Repressor
Tryptophan binding induces
Conformational change
Fits in major groove 4
Recap: Prokaryotic Gene Regulation
To summarize:
Negative regulation:
Competition between
RNA polymerase and
repressor protein for promoter binding
Positive regulation:
activator protein recruits RNA polymerase
to the promoter to activate transcription
5
Recap: Prokaryotic Gene Regulation
Gene regulatory elements are typically close to the
transcriptional start site of prokaryotic genes
BUT regulatory elements can also be found
Far upstream of gene
Downstream of gene (eukaryotes)
Within gene (introns; eukaryotes)
6
Recap: Prokaryotic Gene Regulation
Some regulatory elements are distant from the
transcriptional start site and influence transcription – How?
DNA looping
(Euk. Video)
NtrC protein is a transcriptional activator
DNA looping allows NtrC to directly interact with
RNA polymerase to activate transcription from a distance
Bacteriophage Lambda
Virus that infects bacterial cells
Positive and negative regulatory mechanisms work
together to regulate the lifestyles of bacteriophage lamba
Two proteins repress each others synthesis
Bacteriophage Lambda
Bacteriophage lambda can exist as
one of two states in bacteria
Under favorable
bacterial growth
conditions
When host cell
is damaged
Two gene regulatory proteins are responsible for initiating this switch
Bacteriophage Lambda
Two gene regulatory proteins are responsible for initiating the switch
between prophage and lytic pathways
lambda repressor protein (cI) and
Cro protein
Repress each other’s synthesis, giving rise to the two states.
Bacteriophage Lambda
Bacteriophage lamba: a genetic switch
State 1: Prophage
Lambda repressor
occupies the operator.
blocks synthesis
of Cro
activates its own
synthesis
most bacteriophage
DNA not transcribed
Bacteriophage Lambda
eg. bacteriophage lamba: a genetic switch
State 2: Lytic
Cro occupies the operator
blocks synthesis
of repressor
allows its own
synthesis
most bacteriophage
DNA is extensively
transcribed
What triggers switch?
DNA is replicated, packaged,
new bacteriophage released
by host cell lysis
Bacteriophage Lambda
eg. bacteriophage lamba: a genetic switch
What triggers switch between prophage and lytic states?
Host response to DNA damage
-switch to lytic state
inactivates repressor
Under good growth conditions repressor protein turns off
Cro and activates itself
positive feedback loop
-maintains prophage state
Example of a transcriptional circuit.
Different types exist, control various biological processes
Transcriptional Circuits
Transcriptional Circuits
eg. repressor
protein
eg. Cro / Repressor
switch
Transcriptional Circuits
Transcriptional Circuits
Positive Feedback loops can be used to create cell memory
Transcriptional Circuits
Transcriptional Circuits
Feed-forward loops can measure the duration of a signal
– both A and B required for transcription of Z
Brief input
B does not
accumulate
Z not
transcribed
Prolonged
input B
accumulates
Z is
transcribed
Transcriptional Circuits
Transcriptional Circuits
Combinations of
regulatory circuits
combine in eukaryotic
cells to create
exceedingly complex
regulatory networks
Scientists can construct
artificial circuits and
examine their behavior in
cells synthetic biology
Gene circuit of developing sea urchin embryo
Synthetic Biology
Synthetic Biology
eg. creating a simple gene oscillator using a delayed
negative feedback circuit – “the repressillator”
A: Lac repressor
B: Tet repressor (response to antibiotic)
C: Lambda repressor
Predicted: delayed negative feedback
gives rise to oscillations
Introduced this circuit into bacterial cells
and observed expression of the repressor
genes
Synthetic Biology
Synthetic Biology
Synthetic Biology: “the repressillator”, how does it work?
1)
A expressed
A expression
4)
2)
B repressed
3)
C expressed
4)
C represses A expression
Synthetic Biology
Synthetic Biology: “the repressillator”, how does it work?
5)
A repressed
A expression
6)
B expressed
7)
C repressed
8) Repeat 1-4
Did it work?
Synthetic Biology
Synthetic Biology
eg. creating a simple gene oscillator using a negative
feedback circuit
Looking at 1 Protein
(Fluorescently tagged)
Observed
Predicted
Increasing amplitude due to
bacterial growth
Transcriptional Circuits
Feedback loops also circadian gene regulation
~ 24-hour cycle: eg. Drosophila
http://www.hhmi.org/biointeractive/drosophila-molecular-clock-model
Delayed Negative Feedback Loop
Transcription Attenuation
-In both prokaryotes and eukaryotes there can be a
premature termination of transcription called
Transcription attenuation
-RNA adopts a structure that interferes with RNA
polymerase
-Regulatory proteins can bind to RNA and interfere with
attentuation
-Prokaryotes, plants and some fungi also use
Riboswitches to regulate gene expression
Transcription Attenuation
Riboswitches
Short RNA sequences that change conformation when
bound by a small molecule
eg. prokaryotic riboswitch that regulates purine biosynthesis
Recall that bases making up DNA/RNA include:
pyrimidines (C,T,U)
purines (A,G)
Transcription Attenuation
Riboswitches
eg. prokaryotic riboswitch that regulates purine biosynthesis
Low guanine levels
-Transcription of purine biosynthetic genes is
on
Transcription Attenuation
Riboswitches
eg. prokaryotic riboswitch that regulates purine biosynthesis
High guanine levels
-Guanine binds riboswitch
-Riboswitch undergoes
conformational change
-Causes RNA polymerase
to terminate transcription
-Transcription of purine biosynthetic genes is
off
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
BIO 230
Lecture 4:
Eukaryotic Gene Regulation
1) Eukaryotic transcriptional activation
2) Eukaryotic transcriptional repression
Readings (Alberts et al. custom text)
Pages 310-314, 187-193, 196-197, 198-201
2
Reminder from a couple of
lectures ago…
Transcriptional Regulation
Gene expression in both prokaryotes and
eukaryotes is regulated by:
Gene Regulatory Proteins (transcription factors)
Which bind specifically to:
Regulatory regions of DNA (cis elements)
Gene regulatory proteins can turn genes:
-ON; Positive regulators; activators
-OFF; Negative regulators;
(eg. Trp operon)
repressors
4
Transcriptional Regulation
Recall that DNA is transcribed into RNA by the
enzyme
RNA polymerase
5
Transcriptional Regulation
Cells produce several
types of RNA:
Different RNAs transcribed
by different RNA polymerases
in eukaryotes
Prokaryotes have a single type of RNA polymerase
Transcriptional Regulation
Transcription initiation in eukaryotes requires many proteins:
general transcription factors
Help position RNA polymerase at
eukaryotic promoters contain TATA box
Required by nearly all promoters used by
RNA polymerase II
Eukaryotic Gene Regulation
Eukaryotic transcription
– RNA polymerase II transcribes protein coding genes
– Requires five general transcription factors; TFIID, TFIIB, TFIIF,
TFIIE, and TFIIH (prokaryotes only need one; σ factor)
– Eukaryotic genomes lack operons
– Eukaryotic DNA is packaged into chromatin which provides
an additional mode of regulation
– Eukaryotic transcriptional activation requires many gene
regulatory proteins
Eukaryotic Gene Regulation
Eukaryotic transcription
– Mediator acts an intermediate between regulatory
proteins and RNA polymerase
RNA Polymerase
Eukaryotic Gene Regulation
-Eukaryotic gene expression controlled by many regulatory
proteins (~2000 encoded by the human genome)
both activators and repressors
-Gene regulatory proteins can act over very large distances,
sometimes >10000 base pairs away
– One mechanism is DNA looping
Eukaryotic Gene Regulation
Eukaryotic gene regulatory proteins often function
as protein complexes on DNA
Coactivators and corepressors assemble on DNA-bound
gene regulatory proteins do not directly bind DNA
Eukaryotic Gene Regulation
Eukaryotic Activator Proteins
Modular design:
1) DNA binding domain (DB)
– recognizes specific
DNA sequence
2)
Activation domain (AD)
– accelerates rate of
transcription
Can mix-and-match DBs and ADs
Eukaryotic Gene Regulation
How do Activator Proteins activate transcription?
Attract, position and modify:
General transcription factors
Mediator
RNA polymerase II
They can do this either:
1)
Directly by acting on these components
2) Indirectly modifying chromatin structure
Eukaryotic Gene Regulation
1) Activator proteins can bind directly to transcriptional
machinery or mediator and attract them to promoters (like
prokaryotic activators)
Eukaryotic Gene Regulation
2) Activator proteins can alter
chromatin structure
Nucleosomes are the basic structure of Eukaryotic chromatin
– DNA wound around a histone octamer
(H2A, H2B, H3, and H4 x 2)
Eukaryotic Gene Regulation
Nucleosomes pack as compact chromatin fibers
Zigzag model
Solenoid Model
Transcriptional machinery cannot assemble on
promoters tightly packaged in chromatin
Activator proteins can alter chromatin structure and
increase promoter accessibility
How?
Eukaryotic Gene Regulation
4 major ways activator proteins can alter chromatin
1.
2.
3.
4.
Eukaryotic Gene Regulation
Nucleosome structure can be altered by
chromatin remodeling complexes in an
manner to increase promoter accessibility
1) Nucleosome sliding
ATP-dependent
Eukaryotic Gene Regulation
2, 3) Nucleosome removal and histone exchange
Requires cooperation with histone chaperones
Eukaryotic Gene Regulation
4 major ways activator proteins can alter chromatin
1.
2.
3.
4.
Signal for
chromatin
remodeling
Eukaryotic Gene Regulation
4)
Histone modifying enzymes produce specific patterns of
histone modifications histone code
phosphorylation
Enzyme: kinase
acetylation
Enzyme: acetyltransferase
methylation
Enzyme: methyltransferase
Addition of phosphate group:
Addition of acetyl group:
Addition of methyl group:
Histone modifications occur on specific amino acids of
histone tails
Eukaryotic Gene Regulation
The histone code:
Specific modifications to histone tails by histone
modifying enzymes “writers”
Histone H3
Eukaryotic Gene Regulation
The histone code:
Code- “reader” proteins can recognize specific
modifications and provide meaning to the code
Histone H3
Eukaryotic Gene Regulation
Transcriptional regulation using the histone code
eg. human interferon gene promoter
Step 1: Activator protein
binds to chromatin DNA
and attracts a
histone
acetyltransferase (HAT)
Step 2: HA acetylates lysine 9
of histone H3 and lysine 8 of
histone H4.
Eukaryotic Gene Regulation
Transcriptional regulation using the histone code
eg. human interferon gene promoter
Step 3: Activator protein
attracts a
histone kinase (HK)
Step 4: HK phosphorylates
serine 10 of histone H3. Can only
occur after acetylation of lysine 9
Eukaryotic Gene Regulation
Transcriptional regulation using the histone code
eg. human interferon gene promoter
Step 5: Serine modification signals
the acetyltransferase to acetylate
lysine 14 of histone H3
Histone code for transcription
Initiation is written
Eukaryotic Gene Regulation
Transcriptional regulation using the histone code
eg. human interferon gene promoter
Step 6:
TFIID and a chromatin
remodelling complex
bind to acetylated histone tails and initiate
transcription
Eukaryotic Gene Regulation
Transcriptional Repression
– Unlike prokaryotes, eukaryotic repressor proteins rarely
compete with RNA polymerase for access to DNA
– Instead use a variety of mechanisms to inihibit transcription
1) Interfering with activator function
Eukaryotic Gene Regulation
Transcriptional Repression
Interfering with activator function
2)
3)
Eukaryotic Gene Regulation
Transcriptional Repression by altering chromatin
structure
4)
Eukaryotic Gene Regulation
Transcriptional Repression by altering the histone code
5)
6)
Eukaryotic Gene Regulation
Guided by gene regulatory proteins histone “reader” and “writer”
proteins can establish a repressive form of chromatin
histone
code can
spread
This chromatin can be stabilized
Eukaryotic Gene Regulation
Spreading the histone code along chromatin carried out by
Reader-writer complexes
Eukaryotic Gene Regulation
DNA methylase
enzyme is attracted by
Reader and methylates
nearby cytosines in
DNA
DNA methyl-binding
proteins bind methyl
groups and stabilize
structure
-Methylation and therefore gene expression patterns can be inherited
A process called epigenetic inheritance
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
How can small and fragile cells form large and stable organisms?
?
Cells are organized into one of two main tissue categories
(these support the animal body plan)
Epithelial tissues à cells directly connected with minimal extracellular matrix beneath
Connective tissues à cells dispersed with extracellular matrix providing overall structure
Cells are organized into one of two main tissue categories
(these support the animal body plan)
Epithelial tissues à cells directly connected with minimal extracellular matrix beneath
Connective tissues à cells dispersed with extracellular matrix providing overall structure
>60% of the cell types in our bodies are epithelial, forming our skin
and coating our organs.
Connective tissue cells include muscle cells, neurons and immune cells.
Moving through your body, what tissues would you pass by?
Schematic Cross Section
of the Human Abdomen
(inds.co.uk)
MBoC 4th Ed.
Epithelial structure and function requires junctional complexes
Adherens junction structure
Adherens junctions can form strong continuous adhesion belts critical for
adhering cells to form epithelia
Adherens junction structure
Adherens junctions can form strong continuous adhesion belts critical for
adhering cells to form epithelia
Cadherin clusters mediate the adhesion
Cadherin cluster
Adherens junction structure
Cadherin clusters mediate the adhesion through:
1. Homophilic interactions between cadherin receptors
Adherens junction structure
Cadherin clusters mediate the adhesion through:
1. Homophilic interactions between cadherin receptors
2. Links to the actin cytoskeleton
Adherens junction function:
Tissue maintenance during development
Movement of the outer epithelium of the Drosophila embryo
Adherens junction function:
Tissue maintenance during development
Tissue structure is lost
in adherens junction
mutants
Adherens junction function:
Tumour suppression
• The loss of epithelial structure
is a hallmark of cancer
• Cadherins are
tumour suppressors
Epithelia have distinct ‘apical’ and ‘basal’ sides
-the apical surface faces the organ lumen or the animal surface
-the basal surface faces underlying tissue
This epithelial polarity is critical for organ function
[email protected]
Epithelial polarity controls solute diffusion
between our body compartments
e.g. Controlling glucose
transport into the blood
The glucose is blocked from
diffusing between cells by tight junctions
Instead, it must be actively transported
through cells by plasma membrane
channels allowing for precise regulation.
How are the channels positioned?
What would happen without tight junctions?
Epithelial polarity controls solute diffusion
between our body compartments
e.g. Controlling glucose
transport into the blood
The glucose is blocked from
diffusing between cells by tight junctions
Instead, it must be actively transported
through cells by plasma membrane
channels allowing for precise regulation.
How are the channels positioned?
What would happen without tight junctions?
Epithelial polarity controls solute diffusion
between our body compartments
e.g. Controlling glucose
transport into the blood
The glucose is blocked from
diffusing between cells by tight junctions
Instead, it must be actively transported
through cells by plasma membrane
channels allowing for precise regulation.
How are the channels positioned?
What would happen without tight junctions?
Testing the permeability barrier across an epithelia
Testing the permeability barrier across an epithelia
(a)
(b)
If a dye was added below an epithelium
with tight junctions how far could it diffuse?
(c)
Testing the permeability barrier across an epithelia
added apically
or basally?
added apically
or basally?
Tracer molecule
(black) added to
apical or basal side
of the epithelium
What would happen without tight junctions?
Tight junctions encircle the apical end of each cell in an epithelial sheet
Apical
Basal
Tight junctions are formed from
strands of interacting transmembrane proteins
Apical
Tight junctions are formed from
strands of interacting transmembrane proteins
Core tight junction proteins:
Claudin
• 4-pass transmembrane protein
Occludin
• 4-pass transmembrane protein
Tight junctions block two movements.
Movement of aqueous molecules
through the extracellular space between cells
Movement of membrane molecules
between the apical and basolateral domains of
each cell’s plasma membrane
Cell Polarity is Fundamental
to Cell and Developmental Biology
How is cell polarity established?
How does asymmetry arise
to make one end of the cell
different from the other?
(and what does this polarity do
for cells and tissues?)
In general, cells use ‘landmarks’
to establish and elaborate polarity
Primary
Landmark
Subsequent
Elaboration
Chemoattractants polarize cells and the cells chase prey
Adherens junctions (AJs) are important
landmarks for epithelial polarity
Lumen or Exterior Space
Apical
Domain
AJs
Basolateral
Domain
Underlying Tissue
Adherens junctions (AJs) are important
landmarks for epithelial polarity
Cadherin
Cadherin
Arm
Cadherin
a-cat
F -a
n
c ti
Apical
Domain
Basolateral
Domain
Adherens junctions (AJs) are important
landmarks for epithelial polarity
Apical
Cues
Cadherin
Arm
a-cat
F
ti
c
-a
n
Basal
Cues
Conserved apical and basal cues controlling epithelial polarity
The integration of polarity complexes, adhesion complexes,
cytoskeletal networks and trafficking routes is critical for the
structure and function of epithelia that form our organs
Complexes required for epithelial structure
A functional
epithelium
Body compartments constructed
from functional epithelia
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
4. Tissue morphogenesis
5. Tissue patterning
6. Stem cells
9-10: Mitosis and Cell Division & Cancer
11-12: Development of Multicellular Organisms
All structures need maintenance
Once cells and tissues are formed they continually regenerate
This can occur at the level of molecular turnover or cellular turnover
1. Some tissues contain the same cells for the life of the organism,
but the molecular components of these cells do turn over
-typically cells with very specialized architecture
e.g. auditory hair cells or photoreceptor cells
2. Other tissues renew their cells rapidly
-typically cells exposed to harsh environments or activities
e.g. skin cells, gut cells or blood cells
3. Other tissues are between these extremes
The specialized architecture of auditory hair cells in the organ of Corti
This architecture functions to convert sound waves into nerve impulses
(it allows us to hear)
Sound waves cause stereocilia
atop hair cells to tilt
With tilting, tethers pull open
ion channels on neighbouring stereocilia
(this initiates a nerve impulse)
This architecture functions to convert sound waves into nerve impulses
(it allows us to hear)
Sound waves cause stereocilia
atop hair cells to tilt
With tilting, tethers pull open
ion channels on neighbouring stereocilia
(this initiates a nerve impulse)
In mammals, these cells do not re-grow when lost
anatomybox.com
Image by By F. Kalinec and B. Kachar, NIH
Their loss from disease, toxins or extreme noise leads to permanent
hearing loss
However, the molecules that make up these cells are continually made
and destroyed
Human photoreceptor cells are another
permanent cell type with a specific architecture.
Their architecture
converts light waves
into nerve impulses
The overall photoreceptor cells are permanent,
but do they turn over at the molecular level?
Pulse-chase experiment
-cells exposed to radiolabeled
leucine for a short time
-they take up the labeled amino
acid and incorporate it into newly
synthesized proteins for a short
period of time
What will happen to the labeled
Leucine?
a) It will be detected in the cells
for their entire lifetime
b) It’s detection will gradually
be lost
The overall photoreceptor cells are permanent,
but do they turn over at the molecular level?
Degrades the protein
+ photoreceptive
discs
Pulse-chase experiment
-cells exposed to radiolabeled
leucine for a short time
-they take up the labeled amino
acid and incorporate it into newly
synthesized proteins for a short
period of time
What will happen to the labeled
Leucine?
a) It will be detected in the cells
for their entire lifetime
b) It’s detection will gradually
be lost
1. Some tissues contain the same cells for the life of the organism,
but the molecular components of these cells do turnover
-typically cells with very specialized architecture
e.g. auditory hair cells or photoreceptor cells
2. Other tissues renew their cells rapidly
-typically cells exposed to harsh environments or activities
e.g. skin cells, gut cells or blood cells
3. Other tissues are between these extremes
Cell turnover can be stem cell dependent or independent
Stem cell definition
1. It is not terminally differentiated
2. It can divide without limit
3. Its daughters can remain a stem cell
or differentiate
Cell turnover can be stem cell dependent or independent
Stem cell definition
1. It is not terminally differentiated
2. It can divide without limit
3. Its daughters can remain a stem cell
or differentiate
Cell renewal can occur from division
of differentiated cells
(e.g. liver cells and insulin-secreting cells
of the pancreas)
The use of stem cells requires specific
mechanisms
Mechanism 1
The fates of stem cell daughters must be controlled
1. Divisional asymmetry
One daughter receives factors promoting
‘stemness’, and the other receives
factors promoting differentiation
A drawback:
If stem cells are lost, their original numbers
can’t be restored
Mechanism 1
The fates of stem cell daughters must be controlled
2. Environmental asymmetry
The cell division is symmetric and
and the daughters’ fates are determined
by the environment they are born in to
If stem cells are lost, then their numbers
can be increased by having both
daughters of divisions enter the
environment promoting “stemness”
Mechanism 2
Stem cells divide slowly for their long-term preservation
This protects the stem cell from:
1. Mutations associated with
cell division
2. Telomere depletion associated
with cell division
However, large numbers of cells
are needed to renew differentiated
cell populations
Mechanism 2
Stem cells divide slowly for their long-term preservation
Transit amplifying cells
expand cell numbers
before final differentiation
Mechanism 3
Stem cells are supported by a local environment, their niche
e.g. Skin stem cells and their progeny
The stem cells reside in the
basal layer and require
basal lamina attachment
to remain as stem cells
àthe basal lamina provides
a niche for the stem cells
After detaching from the
basal lamina, the cells
differentiate through
a linear sequence
of cell types and are
finally shed from the animal
Mechanism 3
Stem cells are supported by a local environment, their niche
e.g. Skin stem cells and their progeny
The stem cells reside in the
basal layer and require
basal lamina attachment
to remain as stem cells
àthe basal lamina provides
a niche for the stem cells
After detaching from the
basal lamina, the cells
differentiate through
a linear sequence
of cell types and are
finally shed from the animal
Without renewal from stem cells,
our skin would be lost in a month
Blood stem cells and their progeny
Blood stem cells
differentiate into
various populations
creating
a branched pathway
to final differentiation
Signals can promote
specific branches
depending on the
need for cell types
Blood stem cells and their progeny reside in bone marrow
An electron micrograph of bone marrow
Blood cell precursors are in bone marrow
but are the stem cells there?
If all blood stem cells were killed off
would ‘new’ bone marrow restore them?
Blood stem cells and their progeny reside in bone marrow
An electron micrograph of bone marrow
Blood cell precursors are in bone marrow
but are the stem cells there?
If all blood stem cells were killed off
would ‘new’ bone marrow restore them?
Identifying blood stem cells and their progeny
Separate cells based on arbitrary differences
………
How would you test each separated group for stem cell activity?
Identifying blood stem cells and their progeny
Homogenize mouse bone
marrow to release single cells
Expose cells to fluorescent
antibodies recognizing specific
cell surface molecules
Isolate labeled cells by
Fluorescence-Activated
Cell Sorting (FACS)
Identifying blood stem cells and their progeny
Homogenize mouse bone
marrow to release single cells
Expose cells to fluorescent
antibodies recognizing specific
cell surface molecules
Isolate labeled cells by
Fluorescence-Activated
Cell Sorting (FACS)
Test ability of isolated cells
to restore all blood cells
of an irradiated mouse
~1/10 000 bone marrow cells can
~5 of such cells are sufficient for
the restoration
Blood stem cells are maintained
through interactions with stromal
cells in the bone marrow
Once detached cells differentiate
Stromal cells provides a niche for
for the blood stem cells
NOTE:
“stromal cell”,
“mesenchymal cell”,
“connective tissue cell”
…are all close synonyms
Stem cells and tissue renewal affect how we age…
…and can treat diseases and disabilities
Stem cells and tissue renewal affect how we age…
…and can treat diseases and disabilities
Medical uses for stem cells
Using blood stem cells to treat leukemia
Problems of immune rejection
à careful tissue matching
and immunosuppressive drugs
à if the cancer arises from a mutation
in one of the progenitor populations
then the patient’s own stems cells
can be used after sorting
!!
Remove from
marrow sample
Inject the rest
or a patient
Medical uses for stem cells
What if the tissue to be replaced doesn’t have a readily
available supply of its own stem cells?
(e.g. spinal cord injuries or neurodegenerative diseases)
Can cells of a different tissue be used to make stem cells
for treatment?
This occurs naturally during
limb regeneration in newts
•Muscle cells de-differentiate,
and start dividing
•They form a bud similar to the
embryonic limb bud
•Their progeny form all cell types
needed to re-grow the limb
Medical uses for stem cells
What if the tissue to be replaced doesn’t have a readily
available supply of its own stem cells?
(e.g. spinal cord injuries or neurodegenerative diseases)
Can cells of a different tissue be used to make stem cells
for treatment?
Current technology cannot do this from adult human cells
at the scale or reliability needed for medical purposes,
but is has been done in experiments
This technique could avoid immune rejection by using
the patient’s own cells, but cancer development is a
potential problem if cell differentiation isn’t properly controlled
Embryonic stem (ES) cells can proliferate indefinitely in culture
and have full developmental potential
This can increase the yield of cells needed for treatments, but
ethical issues, immune rejection and the potential of cancer
are still concerns
Two potential ways to avoid immune rejection of ES cells
1. Somatic cell nuclear transfer—use a nucleus from one of the patient’s own cells
and transfer it into an unfertilized egg to develop an embryo from which ES cells
can be harvested
Two potential ways to avoid immune rejection of ES cells
1. Somatic cell nuclear transfer—use a nucleus from one of the patient’s own cells
and transfer it into an unfertilized egg to develop an embryo from which ES cells
can be harvested
2. Treat some of the patient’s own cells with factors that generate ES cells
à a combination of Oct3/4, Sox2, Myc and Klf4 (all transcription factors) can
convert differentiated cells into cells with ES cell characteristics
+ protein
expression
Continual renewal is important for maintaining all structures
Tissues often renew themselves naturally,
but when they can’t medical treatments can be used
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
7-9: How do cells communicate with each other?
7. Principles of cellular signalling
8. Signalling via small molecules
9. Signalling via protein modifications
10-12: How are cell numbers controlled?
Cellular signaling
Cells must communicate to develop and maintain multicellular organisms
Unicellular bacterial-like organisms existed on earth for ~2.5 billion years
before complex multicellular organisms arose
In part, this delay may have been due to the time needed to evolve
complex signaling systems
However, unicellular organisms do communicate…
Examples of unicellular communication
Quorum sensing in bacteria:
Many bacteria release and respond to chemical signals.
This signaling coordinates motility, antibiotic production, spore formation and
sexual conjugation in bacterial populations.
Mating in budding yeast:
Signaling between yeast cells
prepares them to mate.
Aggregation of ameboid cells:
Signaling between Dictyostelium
cells draws them together
to form a fruiting body.
http://www.biocircle.fu-berlin.de/mikrobio2/signaltransduktion_en.php?id=mb2&p=9&lang=en
How do cells communicate to develop and maintain
complex multicellular organisms?
?
Mammals, flies and worms use similar
communication pathways.
Many core pathway components were
first discovered in mutants affecting cell
communication in Drosophila,
C. elegans and yeast.
The basics of sending and receiving signals
Cells can send out hundreds of different
types of signaling molecules
(e.g. proteins, small peptides, amino acids,
nucleotides, other small molecules and
dissolved gases)
They are exocytosed, emitted by diffusion,
or displayed on the cell surface
Cells receive signals in two main ways
Signaling occurs over short or long distances
Contact-dependent signaling
-signals are retained on the cell surface
Paracrine signaling
-signals are released from the cell but act locally
-signal movement is restricted by:
1. internalization by neighbouring cells
2. signal instability or destruction by extracellular enzymes
3. binding to extracellular matrix molecules
Signaling occurs over short or long distances
Synaptic signaling
-neurons extend axons to contact distant target cells
-the released signaling molecules act locally at target
Endocrine signaling
-endocrine cells secrete hormones into the bloodstream
for long-range distribution
Cells use signal transduction pathways
to respond to extracellular signals
Definitions:
Signal transduction
The conversion of extracellular
signals into intracellular signals
Effector
A downstream molecule in a
signal transduction pathway
(upstream molecules have their
effects on them)
Cells use signal transduction pathways
to respond to extracellular signals
Small intracellular signaling
molecules are called second
messengers
-made in large numbers and
diffuse through the cytoplasm
(e.g. cyclic AMP) or plasma
membrane (e.g. diacylglycerol)
-bind and alter effector molecules
Large intracellular signaling
molecules are typically proteins
-organized into pathways and
networks
Many components of signaling pathways act like switches
Enzymatic versus non-enzymatic conformation
Protein-binding versus non-binding conformation
Gain or loss of phosphate groups
Small molecule binding or unbinding
Switching “off” is just as important
as switching “on”
-if the pathway isn’t turned off,
it won’t be able to send another signal
Addition and removal of phosphate groups on proteins
~30% of human proteins
are phosphorylated
The human genome encodes
~520 protein kinases and
~150 protein phosphatases
There are serine/threonine
kinases and tyrosine kinases
GTP or GDP binding
GTP binding proteins
-Large trimeric and small monomeric types
-Have low GTPase activity
GTP or GDP binding
GTPase activating proteins (GAPs) increase the GTP hydrolysis
Guanine nucleotide exchange factors (GEFs) promote the exchange
of GDP for GTP
Many components of signaling pathways interact like Lego
Proteins often contain one or more interaction domains
These bind to structural motifs in other molecules
(short peptide sequences, other domains,
covalent modifications)
During evolution, domains can be added or removed from
proteins to alter interactions and re-wire signaling pathways
Some examples:
SH2 and PTB domains bind
phosphotyrosine containing
sequences
PH domains bind
phosphoinositides
SH3 domains bind prolinerich sequences
If this portion of “Sos” was replaced by a PTB domain,
what do you predict would happen?
Some examples:
SH2 and PTB domains bind
phosphotyrosine containing
sequences
PH domains bind
phosphoinositides
SH3 domains bind prolinerich sequences
How are complex signaling events orchestrated?
How is signaling specificity achieved?
How are signaling pathways coordinated?
How are signaling pathways organized in space and time?
Targeting specific cells
Synaptic signaling specificity:
Neurons make connections with
specific target cells (the same
signaling molecules can be used
at all connections)
Endocrine signaling specificity:
Different molecules are released and
target cells express specific receptors
to respond to specific molecules
Different cells can also have different responses to the same molecule
by changing the signal receptor or downstream components of the pathway
Within a cell, a signal transduction molecule will often function
in many different pathways
What prevents an upstream signal from activating
all of the pathways?
PNAS 104:12890
Within a cell, a signal transduction molecule will often function
in many different pathways
What prevents an upstream signal from activating
all of the pathways?
The formation of local complexes helps insulate pathways
from each other
Within a cell, a signal transduction molecule will often function
in many different pathways
What prevents an upstream signal from activating
all of the pathways?
The formation of local complexes helps insulate pathways
from each other
How are complex signaling events orchestrated?
How is signaling specificity achieved?
How are signaling pathways coordinated?
How are signaling pathways organized in space and time?
Cellular outcomes often depend on multiple signaling inputs
Coincidence detectors only activate downstream signals
when two upstream signals are both detected
This ensures two conditions are met before the cell responds
How are complex signaling events orchestrated?
How is signaling specificity achieved?
How are signaling pathways coordinated?
How are signaling pathways organized in space and time?
How are complex signaling events orchestrated?
How is signaling specificity achieved?
How are signaling pathways coordinated?
How are signaling pathways organized in space and time?
In addition to forming local complexes, signaling pathways can be
organized at subcellular compartments such as…
…primary cilia…
…or synapses
biochemistry.ucsf.edu/labs/reiter/index-home.shtml
cumc.columbia.edu/publications/press_releases/STVimaging.html
Signaling also occurs over different time frames
Synaptic signaling is very fast
(electrical impulses travel at 100 m/s)
Endocrine signaling is relatively slow
(limited by blood flow)
The response speed to a signal can vary
depending on the cellular machinery involved
Cellular outcomes also depend on feedback mechanisms
Output
Output
Effects of positive feedback
Output
stimulus
Output
stimulus
The feedback will enhance the response.
If the feedback is strong enough, it can be self-sustaining.
System kept at high activation even with loss of the original signal.
àa ‘bistable’ system that can stably exist in ‘off’ or ‘on’ states
Effects of positive feedback
Output
stimulus
The feedback will enhance the response.
stimulus
lost
If the feedback is strong enough, it can be self-sustaining.
System kept at high activation even with loss of the original signal.
àa ‘bistable’ system that can stably exist in ‘off’ or ‘on’ states
Effects of positive feedback
Biphasic
switches
promote
differentiation
stimuli
lost
Output
stimulus
The feedback will enhance the response.
stimulus
lost
If the feedback is strong enough, it can be self-sustaining.
System kept at high activation even with loss of the original signal.
àa ‘bistable’ system that can stably exist in ‘off’ or ‘on’ states
Effects of negative feedback
Output
stimulus
Output
stimulus
If feedback occurs quickly, the signaling is suppressed.
System adapts to the stimulus.
Needs boosted stimulus for output.
Effects of negative feedback
Output
stimulus
Output
stimulus
If feedback occurs quickly, the signaling is suppressed.
System adapts to the stimulus.
Needs boosted stimulus for output.
Allows cells to respond to changes in upstream signals
rather than their absolute amounts.
(cells can respond to a wider range of signal strengths.)
Effects of negative feedback
Output
stimulus
If feedback occurs slowly,
then the system can oscillate
(e.g. for circadian rhythms)
Delay
Recover
Run every 2 days as long as
the driving stimulus is there
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
7-9: How do cells communicate with each other?
7. Principles of cellular signalling
8. Signalling via small molecules
9. Signalling via protein modifications
10-12: How are cell numbers controlled?
Signaling via small molecules
1. Independently of plasma membrane proteins
2. Through plasma membrane channels
3. Downstream of plasma membrane G-protein coupled receptors
Signaling via small molecules
1. Independently of plasma membrane proteins
2. Through plasma membrane channels
3. Downstream of plasma membrane G-protein coupled receptors
Which class of molecules could signal
from outside to inside the cell
without a channel or receptor?
A
B
C
D
A very small signaling molecule: Nitric oxide (NO)
Made by the deamination of arginine by NO synthases
Acts locally because of a 5-10 second half life
Affects smooth muscle and other target cells
Nitroglycerine is used to treat heart pain
It is converted to NO and relaxes blood vessels, reducing workload on the heart
Small hydrophobic signaling molecules
Steroid hormones
-made from cholesterol
-affect sexual characteristics and metabolism
Thyroid hormones
-made from tyrosine
-increase metabolic rate
Retinoids
-made from Vitamin A
-regulate development
Vitamin D
-affects metabolism
Small hydrophobic signaling molecules
Steroid hormones
-made from cholesterol
-affect sexual characteristics and metabolism
Thyroid hormones
-made from tyrosine
-increase metabolic rate
Retinoids
-made from Vitamin A
-regulate development
Vitamin D
-affects metabolism
Transported in extracellular fluids by
carrier proteins
Dissociate from carriers upon cell entry
In the cell they bind a member of the
nuclear receptor superfamily
The nuclear receptor superfamily
Contain binding sites for a small
hydrophobic molecule and for DNA
48 identified in the human genome
More than half only identified based
on sequence analyses
àtheir ligands are unknown
àtermed orphan nuclear receptors
Ligand binding alters receptor conformation, and releases inhibitors,
to promote DNA binding and downstream transcription
Signaling via small molecules
1. Independently of plasma membrane proteins
2. Through plasma membrane channels
3. Downstream of plasma membrane G-protein coupled receptors
Ion channels are a major class
of signaling molecules
Electrochemical gradients across the plasma membrane
Ion channel properties
• Have narrow selective pores
• Open and close rapidly
• Up to 100 million ions can pass though an open channel per second
• Transport is passive
à based on electrochemical gradients across the plasma
membrane and ion diffusion down these gradients
Ion channels can be activated in a number of ways
(e.g. hair cells of
the Organ of Corti)
Ion channel functions
-electrical excitability of muscle cells
-electrical signaling in the nervous system
-leaf-closure responses in plants
-signal the single-celled Paramecium to reverse its movement upon collision
-others (these channels are present in all animal cells)
A nerve impulse releases neurotransmitters
that open channels of a postsynaptic target cell
With exocytososis, synaptic vesicles
release neurotransmitters which bind
and open ligand-gated ion channels
These open channels allow ion passage
into the target cell to create another
nerve impulse or a different effect
Neurons also activate other cell types via synaptic connections
(e.g. muscle contraction)
The connections between neurons can be very complex
Signaling via small molecules
1. Independently of plasma membrane proteins
2. Through plasma membrane channels
3. Downstream of plasma membrane G-protein coupled receptors
Signaling by G-protein coupled receptors
G-protein coupled receptors
– 7 pass transmembrane proteins
– activated by proteins, small molecules and light
– more than 700 in humans
Signaling by G-protein coupled receptors
All G-protein coupled receptors signal into the
cytoplasm via a membrane associated
trimeric GTP-binding protein
(a G protein)
The activated receptor functions
as a guanine nucleotide exchange
factor to exchange GDP for GTP
on the α-subunit
The α-subunit undergoes a
conformational change which
alters the conformation of the
other subunits or induces their
release
The altered subunits bind
downstream effectors
The activity is turned off by a
regulator of G protein signaling
(RGS) which acts as a GTPase
activating protein
G-proteins have a number of downstream effects
G proteins can signal rapidly via cyclic AMP (cAMP)
cAMP is synthesized from ATP by adenylyl
cyclase and is destroyed by cAMP
phosphodiesterase
The canonical cAMP pathway
The signal is transduced by increasing
adenylyl cyclase activity above a constant
background of phosphodiesterase activity
The accumulated cAMP activates
Protein Kinase A (PKA) (a serinethreonine kinase)
[other targets are activated as well]
Note the release of separate
molecules from a tetramer
The canonical cAMP pathway
The signal is transduced by increasing
adenylyl cyclase activity above a constant
background of phosphodiesterase activity
The accumulated cAMP activates
Protein Kinase A (PKA) (a serinethreonine kinase)
[other targets are activated as well]
PKA phosphorylates CREB to activate
transcription
[PKA has other targets as well]
A sense of smell with GPCRs and cAMP
~350 G-protein coupled receptors
allow us to smell
Each receptor recognizes
a different set of odourants and
then produces cAMP which opens
cAMP-gated cation channels
which induce an action potential
Each olfactory neuron expresses
just one of these receptors
A smell is a compilation of
different odourants
Signals from different combinations
of neurons allows us to distinguish
>10 000 different smells
The receptors localize to
specialized cilia emanating
from olfactory neurons
in the lining of the nose
GPCR signaling via calcium
IP3 is a water soluble molecule that diffuses through the cytoplasm
DAG is a hydrophobic molecule that diffuses along the plasma membrane
GPCR signaling via calcium
GPCR signaling via calcium
PKC is a serine/threonine kinase with a range of targets
Calcium also has different targets (e.g. calmodulin)
DAG also has different signaling mechanims
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
7-9: How do cells communicate with each other?
7. Principles of cellular signalling
8. Signalling via small molecules
9. Signalling via protein modifications
10-12: How are cell numbers controlled?
Signaling via protein modifications
1. Enzyme-coupled receptors (and protein phosphorylation)
2. Use of proteolysis in signaling
Signaling via protein modifications
1. Enzyme-coupled receptors (and protein phosphorylation)
There are six main classes of enzyme-coupled receptors
i)
ii)
iii)
iv)
v)
vi)
Receptor tyrosine kinases
Tyrosine-kinase-associated receptors
Receptor serine/threonine kinases
Histidine-kinase-associated receptors
Receptor guanylyl cyclases
Receptorlike tyrosine phosphatases
Receptor tyrosine kinases
~60 encoded in the human genome
Some bind secreted proteins and others bind cell surface proteins
Receptor tyrosine kinases mediate essential functions
Signaling molecules induce the transautophosphorylation
of receptor tyrosine kinases
Ligand binding dimerizes the receptor
Their kinase domains are brought together and phosphorylate each other
Phosphorylation of the kinase domains enhances their activity
Phosphorylation of other regions creates docking sites to assemble a signaling complex
If a receptor tyrosine kinases with a defective kinase domain was
expressed together with the normal receptor what would happen?
A) The cell would respond
normally to the signal
B) The cell would have no
response to the signal
C) The cell would have a
reduced response to the
signal
A more complicated structure-activity relationship…
(not on the exam)
EGFR as a RTK example
Andy Schulte
Phosphorylated receptor tyrosine kinases recruit proteins
which mediate downstream signaling
The recruited proteins contain domains (SH2 or PTB domains)
that bind to phosphotyrosine and neighbouring sequences
3D structure of an SH2 domain
The basic functionality of an SH2 domain
Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway
The Drosophila compound eye consists of ~800 ommatidia
The ommatidia are composed of 8 photoreceptor cells
and 12 support cells
They arise from a simple epithelial sheet through the
sequential differentiation of the photoreceptor cells
The R7 photoreceptor cell is needed to detect UV light
A screen was performed to identify mutants that failed
to specify R7 (based on insensitivity to UV light)
Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway
The first mutant identified was called Sevenless (Sev)
àthe normal Sevenless protein was shown to be a receptor tyrosine
kinase expressed in R7 cells
Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway
The second mutant identified was called Bride-of-sevenless (Boss)
àthe normal Bride-of-sevenless protein was shown to be the
ligand for Sevenless expressed on R8 cells
Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway
Drk and Son-of-sevenless (Sos) were identified in subsequent screens
àDrk links Sevenless to Son-of-sevenless
àSon-of-sevenless is a GEF for Ras
Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway
This basic mechanism is used in different contexts in all animals
What is Ras?
Ras is a molecular switch downstream of receptor tyrosine kinases
•monomeric GTPase
•attached to the cytoplasmic face of the plasma membrane by a lipid anchor
•activated by Ras-GEFs and inactivated by Ras-GAPs
•its activity leads to cell proliferation or differentiation
•30% of human tumours have hyperactive mutant forms of Ras
Ras activates a mitogen-activated protein kinase module
(MAP kinase module) to change protein activity and gene expression
At least 5 parallel MAP kinase modules can operate in mammalian cells
(there are at least 12 MAPKs, 7 MAPKKs, and 7 MAPKKKs)
àHow is non-specific cross-talk controlled?
àWhat stops a MAPKKK from phosphorylating multiple MAPKKs?
At least 5 parallel MAP kinase modules can operate in a mammalian cells
(there are at least 12 MAPKs, 7 MAPKKs, and 7 MAPKKKs)
àHow is non-specific cross-talk controlled?
In yeast, scaffolds bind specific MAP kinase modules, insulating them from other
modules and increasing response specificity
What might happen if kinase D exchanged its double-lobed domain
with the square domain of kinase C?
Signaling via protein modifications
1. Enzyme-coupled receptors (and protein phosphorylation)
There are six main classes of enzyme-coupled receptors
i)
ii)
iii)
iv)
v)
vi)
Receptor tyrosine kinases
Tyrosine-kinase-associated receptors
Receptor serine/threonine kinases
Histidine-kinase-associated receptors
Receptor guanylyl cyclases
Receptorlike tyrosine phosphatases
Receptor serine/threonine kinases
-the largest class of cell surface receptors in plants (also function in animals)
~6 major families in plants
àthe largest is the leucine-rich repeat receptor kinase family
(175 members in Arabidopsis [e.g. the Clavatal1/Clavatal2 complex])
Clv1/Clv2 signaling stimulates
the development of
stems, leaves and flowers
Gene regulation
Histidine-kinase-associated receptors
-activate a “two-component” signaling pathway
-used by bacteria, yeast and plants, but not animals
e.g. regulation of bacterial chemotaxis
attractant
Flagella rotating for propulsion
repellent
Rotation reverses for re-direction
Histidine-kinase-associated receptors
-activate a “two-component” signaling pathway
-used by bacteria, yeast and plants, but not animals
e.g. regulation of bacterial chemotaxis
attractant
repellent
CheA is the histidine kinase
It phosphorylates itself and then
transfers the phosphate to an
aspartic acid on CheY
Signaling via protein modifications
1. Enzyme coupled receptors and protein phosphorylation
2. Use of proteolysis in signaling
Notch signaling
Hedgehog signaling
Wingless signaling
TNFα / NFκB signaling
Lateral inhibition by Notch signaling
Clathrin-mediated
Delta binding
leads to Notch
cleavage…
…and movement
of a Notch fragment
to the nucleus
Note the single cleavage
on the cytoplasmic tail
Organizer function in vertebrate limb development
Source of the morphogen sonic hedgehog (Shh)
Shh spreads from this source
The Shh gradient controls
the formation of distinct digits
POSTERIOR
Without hedgehog signaling
the transcriptional activator
Ci is sequestered in the cytoplasm
by a microtubule-associated complex
Plus, this complex promotes the
proteolysis of Ci to create a
transcriptional repressor
At the same time, the Smoothened protein
is sequestered in intracellular vesicles
by Patched
The binding of Hedgehog to Patched
allows Smoothened to transfer to the plasma membrane
where it releases Ci from its inhibitory complex
Signaling via protein modifications
1. Enzyme coupled receptors and protein phosphorylation
2. Use of proteolysis in signaling
Notch signaling
Hedgehog signaling
Wingless signaling
TNFα / NFκB signaling
The release of a transcriptional activator from an inhibitory complex
is also at the core of Wingless and TNFα / NFκB signaling
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
7-9: How do cells communicate with each other?
7. Principles of cellular signalling
8. Signalling via small molecules
9. Signalling via protein modifications
10-12: How are cell numbers controlled?
How can we understand all of the signaling occurring in a cell?
PNAS 104:12890
e.g a network controlling human pancreatic cancer
How can we understand all of the signaling occurring in a cell?
How can we understand all of the signaling occurring in a cell?
How can we understand all of the signaling occurring in a cell?
Mapping Brain Circuits: The Connectome
http://www.sfn.org/index.aspx?pagename=brainBriefings_09_mapping
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
7-9: How do cells communicate with each other?
10-12: How are cell numbers controlled?
10. The cell cycle
11. Programmed cell death
12. Cancer
9-10: Mitosis and Cell Division & Cancer
11-12: Development of Multicellular Organisms
How do cells change
their shapes, interactions
and numbers
to build tissues, organs
and organisms?
What goes wrong
when cells lose control of
their shapes, interactions and numbers
during cancer progression
or with degenerative disease?
Eukaryotic cell cycle
• Life requires cell growth
and division.
• The cell cycle is the
duplication of cellular
contents and division of
these contents by two.
• A critical aspect of cell
division is the fidelity with
which the cell duplicates
and segregates its
genome
Four main phases of the eukaryotic cell cycle
(Cell growth and remaining doubling
of proteins and organelles)
(Cell growth and partial doubling
of proteins and organelles)
The cell must check if the process is occurring properly
The cell must check if the process is occurring properly
What are the molecular bases for these
checkpoints?
How are they regulated?
Cyclin-Dependent Kinases (Cdks):
traffic lights of the cell cycle control system
• Cdks are protein kinases
with targets that control the
cell cycle
• Their activity allows passage
through a check point
• Their activity depends on
Cyclin binding and other
modifications.
Different cyclin-Cdk checkpoints
act at different stages
of the cell cycle
Cdk activation by cyclin binding
and Cdk-activating kinase activity
Cdks regulate the machinery
that directly replicate the cell
S-Cdk activity
promotes DNA replication
M-Cdk phosphorylates multiple
targets required to start mitosis
Examples:
M-Cdk phosphorylation of lamin
leads to nuclear envelope breakdown
M-Cdk regultes proteins required
for chromosome condensation
M-Cdk phosphoryates microtubule regulators
important for making the mitotic spindle
Cdks regulate the machinery
that directly replicate the cell
S-Cdk activity
promotes DNA replication
What turns Cdks OFF?
M-Cdk phosphorylates multiple
targets required to start mitosis
Examples:
M-Cdk phosphorylation of lamin
leads to nuclear envelope breakdown
M-Cdk regultes proteins required
for chromosome condensation
M-Cdk phosphoryates microtubule regulators
important for making the mitotic spindle
Targeted degradation of cyclins turns Cdks off
Targeted degradation of cyclins turns Cdks off
e.g. anaphase promoting complex (APC) targets M-cyclin
to the proteasome allowing the completion of mitosis
The sequential synthesis and degradation of
different cyclins can drive the cell cycle
… but more control is needed
Regulating Cdk activity by
Cdk inhibitor proteins (CKIs)
e.g. p27
Regulating Cdk activity by phosphorylation
A summary of cell cycle checkpoint control
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Proteins
Cyclins
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
A summary of cell cycle checkpoint control
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Cyclins
Proteins
What acts upstream of these signals?
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
Rb blocks G1 progression and S phase
by inhibiting cyclin synthesis
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Proteins
Cyclins
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
Rb blocks G1 progression and S phase
by inhibiting cyclin synthesis
E2F is a transcription factor
Rb blocks G1 progression and S phase
by inhibiting cyclin synthesis
Rb blocks G1 progression and S phase
by inhibiting cyclin synthesis
What would happen to cells
without any Rb protein?
Rb blocks G1 progression and S phase
by inhibiting cyclin synthesis
Rb is short for “Retinoblastoma protein”
Loss of both copies of the Rb gene leads to
eye cancer in children due to excess cell
proliferation
Rb is a tumour suppressor
Mitogen activation of the cell cycle
Mitogen signaling
induces Myc transcription
via a Ras-MAP kinase
signaling cascade
Myc increases cyclin synthesis and
CKI degradation by regulating transcription
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Proteins
Cyclins
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
Myc increases cyclin synthesis and
CKI degradation by regulating transcription
Myc increases cyclin synthesis and
CKI degradation by regulating transcription
Myc over-activity leads to cancer due to
excess cell proliferation
Myc is an oncogene
Temporal feedback promoting M phase
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Proteins
Cyclins
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
Temporal feedback promoting M phase
What initially activiates Cdc25?
Polo Kinase
Temporal feedback promoting M phase
What protects against a sudden problem?
(Cell growth and remaining doubling
of proteins and organelles)
(Cell growth and partial doubling
of proteins and organelles)
p53 stops the cell cycle
in response to DNA damage
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Proteins
Cyclins
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
p53 stops the cell cycle
in response to DNA damage
p53 stops the cell cycle
in response to DNA damage
p53 stops the cell cycle
in response to DNA damage
p53 protects cells
Loss of p53 leads to cancer
p53 is a tumour suppressor
Intricate regulation of these signals is critical for
proper cell cycle progression and
and the prevention of cancer
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Proteins
Cyclins
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
7-9: How do cells communicate with each other?
10-12: How are cell numbers controlled?
10. The cell cycle
11. Programmed cell death
12. Cancer
9-10: Mitosis and Cell Division & Cancer
11-12: Development of Multicellular Organisms
Programmed cell death (PCD) (Apoptosis)
PCD sculpts
fingers and
toes
PCD removes
juvenile
body parts
Programmed cell death (PCD) (Apoptosis)
PCD kills dangerous cells
Apoptosis is highly regulated and stereotyped
(Necrosis is not)
Necrosis: Accidental cell death.
e.g. with acute injury cells
can swell and burst into the
surrounding tissue
This can lead to a damaging
inflammatory reaction
Apoptosis avoids this by neatly..
-shrinking the cell
-collapsing the cytoskeleton
-fragmenting the DNA
-signaling to macrophages for
cell removal by engulfment
Necrosis
Apoptosis
Apoptosis is highly regulated and stereotyped
(Necrosis is not)
Necrosis: Accidental cell death.
e.g. with acute injury cells
can swell and burst into the
surrounding tissue
Necrosis
This can lead to a How
damaging
is apoptosis triggered?
inflammatory reaction
Apoptosis
Apoptosis avoids this by neatly..
-shrinking the cell
-collapsing the cytoskeleton
-fragmenting the DNA
-signaling to macrophages for
cell removal by engulfment
Caspases trigger apoptosis
•
Cysteine proteases that cleave target
proteins at specific aspartic acid residues
•
Synthesized as procaspase precursor
molecules
•
Procaspases are cleaved and activated by
other caspases
•
This leads to an amplified cascade of
caspase activity
Individual caspase activation
Two polypeptides
One multi-protein
complex
The caspase
amplification cascade
Detecting fragmented DNA
in a gel after induction
of apotosis
Detecting fragmented DNA
in cells by TUNEL labeling
after induction of apotosis
Terminal deoxynucleotidyl transferasemediated dUTP Nick End Labeling
Apoptotic cells also change their
cell surface properties
The phospholipid phosphatidylserine
is normally restricted to the inner leaflet
of the plasma membrane
In apoptotic cells it flips to the outer leaflet
In vivo, macrophages recognize the
exposed lipid and phagocytose the cell
In experiments, probes for phosphatidylserine can detect apoptotic cells
What activates
caspase cascades?
Caspase cascades can be activated
by extrinsic or intrinsic signals
Triggering apoptosis from outside the cell via death
receptors (e.g. Killer lymphocyte activation of Fas receptors)
Need death domain activation to activate DED
Caspase cascades can be activated
by extrinsic or intrinsic signals
Triggering apoptosis from outside the cell via death
receptors (e.g. Killer lymphocyte activation of Fas receptors)
Caspase cascades can be activated
by extrinsic or intrinsic signals
Triggering apoptosis from outside the cell via death
receptors (e.g. Killer lymphocyte activation of Fas receptors)
Caspase cascades can be activated
by extrinsic or intrinsic signals
Triggering apoptosis from within (e.g. release of electron
carrier protein cytochrome c from damaged mitochondria
after cell stress)
Caspase cascades can be activated
by extrinsic or intrinsic signals
Triggering apoptosis from within (e.g. release of electron
carrier protein cytochrome c from damaged mitochondria
after cell stress)
Caspase cascades can be activated
by extrinsic or intrinsic signals
A common mechanism:
Signals lead to the aggregation of the “top” caspase
promoting self cleavage.
Many cells make inhibitors of apoptosis
(this increases the threshold for activating the apoptotic program)
Extracellular inhibitors:
Decoy receptors act by competitive inhibition
-have a ligand-binding domain but not a death domain
-out-compete functional Fas death receptors for ligands
Why isn’t the apoptotic signal sent?
Decoy receptor
Fas death receptor
Many cells make inhibitors of apoptosis
(this increases the threshold for activating the apoptotic program)
Extracellular inhibitors:
Decoy receptors act by competitive inhibition
-have a ligand-binding domain but not a death domain
-out-compete functional Fas death receptors for ligands
Intracellular inhibitors:
Examples of competitive inhibition
e.g. mimic of an initiator caspase that lacks a
proteolytic domain
Other inhibitors simply block apoptotic machinery
Bcl2 inhibits
channel formation
in the outer
mitochondrial
membrane
Inhibitors of apoptosis (IAPs)
block caspase activity in the cytoplasm
Inhibitors of apoptosis (IAPs)
block caspase activity in the cytoplasm
Signaling can either induce or prevent apoptosis
In populations of dividing or stable cells, signals can induce apoptosis.
After cell populations fully grow,
apoptosis often becomes the default state
and signals are required to prevent apoptosis
à The default state kills off cells if they leave their protective environment
à Survival factors are used to control proper cell numbers in body tissues
Survival factors act through apoptotic inhibitors
to protect cells from apoptosis
Determining neuronal cell numbers
via competition for survival factors
secreted by target cells
Signaling between cells in a tissue
allows the tissue to regulate its size
Cell “A”
Cell “C”
protected from
apoptosis
apoptosis
Cell “B”
Cell “D”
told to
divide
Signaling between cells in a tissue
allows the tissue to regulate it’s size
Cell “A”
Cell “B”
Billions of cells die in our bone marrow and intestine each hour
The system is regulated so that cell division equals cell death
Cell “C”
protected from
apoptosis
apoptosis
Cell “D”
told to
divide
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
BIO230 FINAL EXAM 2011 ANSWER KEY
1D
2A
3C
4B
5A
6B
7C
8D
9A
10B
11C
12ABCD
13A
14B
15D
16C
17A
18B
19C
20B
21A
22B
23C
24D
25A
26B
27C
28D
29D
30C
31B
32B
33A
34C
35A
36D
37A
38D
39A
40CD
41B
42C
43C
44B
45C
46A
47D
48C
49B
50B
51D
52A
53D
1. If a chemical that inhibits PI 3‐kinase were added to cells, which of the following would occur?
a.
b.
c.
d.
Specific proteins would dissociate from the cytosolic face of cell membranes.
The concentrations of PIP2 species would be affected.
Rab activities would be affected.
All of the above.
2. In a cell with a clathrin mutation, which of the following would most likely be affected?
a.
b.
c.
d.
Endocytosis.
Transport of molecules from the endoplasmic reticulum to the golgi apparatus.
Transport of molecules from the golgi apparatus to the endoplasmic reticulum.
Transport of molecules between golgi cisternae.
3. Which of the following statements concerning vesicle regulation is incorrect?
a.
b.
c.
d.
Inositol phospholipids are phosphorylated.
Rab5‐GDP is converted to Rab5‐GTP by Rab5‐GEF.
Rab5A is localized on late endosomes.
The presence of Rab5‐GTP and PI(3)P forms a coincidence detector.
4. How many of the following complexes/proteins draw two lipid bilayers into direct contact?
a. 1
b. 2
Version 11
Clathrin
Dynamin
SNARE proteins
Rab small GTPases
Page 1 of 13
c. 3
d. 4
5. An artificial membrane‐bound container is filled with purified tubulin subunits, a purfied
centrosome, purified Dynein complexes and the necessary small molecules for p…
Purchase answer to see full
attachment
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
Polarized cytoskeletal networks organize cells
and control interactions with their environment
Which of the structures below would you predict
to organize intracellular trafficking routes?
a) Green
b) Red
c) Blue
Which of the structures below would you predict
to organize intracellular trafficking routes?
a) Green (Microtubules)
b) Red (Actin)
c) Blue (Nucleus)
Microtubules are inherently polarized
(from subunit, to protofilament, to network)
Each protofilament is made of
heterodimers of the monomeric
proteins a-tubulin and b-tubulin
Tubulin monomers bind and
hydrolyze GTP (shown in red)
Each heterodimer is asymmetric
The heterodimers assemble
head to tail forming
polarized filaments
g-Tubulin complexes nucleate microtubules
• g-tubulin binds tubulin heterodimers assembling protofilaments into tubes
• g-tubulin nucleates microtubules at their minus ends
• Plus ends grow away from nucleation sites
• g-tubulin often associates with large microtubule organizing centres (MTOCs)
e.g. the centrosomes (above)
Contain 2 centrioles surrounded by hundreds of proteins
with g-tubulin nucleation sites on the surface
Microtubule movement by dynamic instability
• Single microtubules switch between growing & shrinking
•termed dynamic instability
This behaviour allows microtubules to search the full 3-D space of the cytoplasm
Microtubule movement by dynamic instability
• Single microtubules switch between growing & shrinking
•termed dynamic instability
• Growing microtubules have a protective cap of GTP-bound tubulin
• If GTP hydrolysis is faster
than subunit addition
the cap is lost and
catastrophe occurs
*depolymerization is
~100x faster at an
exposed GDP end*
•Regaining a GTP cap
rescues growth
Microtubule networks can form a coordinate system on their own
A purified centrosome was mixed
with purified tubulin subunits
in an artificial membrane-bound container.
It moves to the centre of the container
as microtubule plus ends push on
the outer membrane.
àminus ends central/plus ends outwards
Microtubule networks can form a coordinate system on their own
This may contribute to microtubule
organization in cells
(but many regulatory proteins
are also involved)
How could a microtubule coordinate system be used?
How is cargo transported along microtubules?
Sun Peaks Trail Map
Wikimedia
Motors move cargo through the microtubule networks
The motor activity is polarized.
– Dynein moves to microtubule minus ends
– Kinesin moves to microtubule plus ends
Motors move cargo through the microtubule networks
The motor activity is polarized.
– Dynein moves to microtubule minus ends
– Kinesin moves to microtubule plus ends
The role for microtubules in positioning the Golgi can be seen
after the addition of a microtubule inhibitor
Control cells
Nocodazole-treated cells
What motor is likely key for Golgi positioning?
a) Dynein
b) Kinesin
The microtubule network is very dynamic and can be reorganized
How could microtubules be used to control the colour of this fish?
The African cichlid fish, Tilapia mosssambica
Male dominant behavior
àBlack color change
Kinesin and dynein
compete
for melanosomes
Haimo and Thaler, Bioessays 16:727
Camouflage behavior
àWhite colour change
Kinesin inhibited
Dynein moves
melanosomes
to centre
Melanosomes are
pigment-containing
vesicles.
When they are
throughout cell,
the cell is black.
When at the centre,
the cell is white
The actin cytoskeleton also plays a critical role
in organizing cell structure and controlling cell behaviour
The actin cytoskeleton is inherently polarized
(from subunit, to filaments, to networks)
Actin monomers are
asymmetric
Actin monomers bind
and hydrolyze ATP
Actin monomers assemble
head to tail forming
polarized filaments
The actin cytoskeleton is inherently polarized
(ATP-ADP polarity along actin filaments)
• After polymerization Actin-ATP is hydrolyzed to Actin-ADP
• Hydrolysis reduces binding affinities to neighbouring subunits increasing dissociation
• However, if the rate of addition of Actin-ATP is faster than rate of removal of ActinADP, a relatively stable “cap” of Actin-ATP subunits can be formed
ATP
ADP
ADP ADP ATP ATP ATP
ADP ADP ADP ATP ATP
ADP
cap
plus end
ATP
Which way will this filament grow?
The actin cytoskeleton is inherently polarized
(Polarized assembly and disassembly leads to treadmilling)
Because of the ATP-ADP polarity along the filament,
subunits can undergo net assembly at plus end
equal to (or greater than) the net disassembly at the minus end
Thus, the polymer can maintain a constant length (or grow)
with a flux of subunits through the filament (treadmilling)
Net direction of treadmilling
The actin cytoskeleton is inherently polarized
(from subunit, to filaments, to networks)
The ARP complex nucleates actin filaments…
..and branches
actin filaments
to form polarized
2-D networks
Polarized treadmilling of large actin networks
can produce significant protrusive power
These networks
drive polarized
cell movement
Treadmilling actin networks need traction
to drive cells forward
Net direction of treadmilling
Without traction actin filaments can treadmill
but maintain their overall position
Treadmilling microfilaments can engage
stationary anchors to create ‘protrusive machines’
Cell
Protrusion
A stationary anchor binds one part of the filament
The treadmilling filament extends from that point
This extension pushes against the cell membrane driving cell protrusion
Large regions of actin networks are anchored
to create ‘protrusive machines’
ANCHORED
Large regions of actin networks are anchored
to create ‘protrusive machines’
Top View
Side View
What is the network
anchored to?
In animals, cells migrate on (and through) the extracellular matrix,
a non-cellular material made of proteins and polysaccharides
Integrins connect the actin cytoskeleton to extracellular matrix
molecules
Main receptors that bind
extracellular molecules
Transmembrane heterodimers
of non-covalently associated
a and b subunits
Linked to the actin cytoskeleton
via adaptor proteins
How is the actin cytoskeleton directed to follow a target?
Video
Chemoattractant receptors orient the actin networks
Chemoattractant receptors orienting actin networks
is analogous to food sources orienting ant trails
Actin networks and ant trails are each made of polarized subunits
Each are dynamic and can rapidly reorient to changes in the target position
mtkilimanjarologue.com
Actin networks can undergo
other large scale rearrangements
e.g. localized assembly of the contractile ring
that divides daughter cells after mitosis
Actin and microtubule networks are also integrated in cells
but we are just beginning to understand the crosstalk
Advice on required e-text reading
Supplement your lecture notes by writing out the following as you read.
à forces you to think and organize as you read,
and thus increases your understanding
• The steps of processes and the key molecules of the complexes involved
– for those discussed in class, add information from the e-textbook
– for examples not discussed, write short summaries or diagrams
How do I choose which processes/complexes to take notes on?
Emphasize those with…
– relevance to a key function of a cell, organ or organism
– relevance to disease
– an organization that is similar to other
processes/complexes/concepts discussed
…and make short notes about these points
Do this within 24h of each lecture (preferably the same day),
and review your notes frequently.
à cements the material in your longer term memory
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
4. Tissue morphogenesis
5. Tissue patterning
6. Stem cells
9-10: Mitosis and Cell Division & Cancer
11-12: Development of Multicellular Organisms
All structures need maintenance
Once cells and tissues are formed they continually regenerate
This can occur at the level of molecular turnover or cellular turnover
1. Some tissues contain the same cells for the life of the organism,
but the molecular components of these cells do turn over
-typically cells with very specialized architecture
e.g. auditory hair cells or photoreceptor cells
2. Other tissues renew their cells rapidly
-typically cells exposed to harsh environments or activities
e.g. skin cells, gut cells or blood cells
3. Other tissues are between these extremes
The specialized architecture of auditory hair cells in the organ of Corti
This architecture functions to convert sound waves into nerve impulses
(it allows us to hear)
Sound waves cause stereocilia
atop hair cells to tilt
With tilting, tethers pull open
ion channels on neighbouring stereocilia
(this initiates a nerve impulse)
This architecture functions to convert sound waves into nerve impulses
(it allows us to hear)
Sound waves cause stereocilia
atop hair cells to tilt
With tilting, tethers pull open
ion channels on neighbouring stereocilia
(this initiates a nerve impulse)
In mammals, these cells do not re-grow when lost
anatomybox.com
Image by By F. Kalinec and B. Kachar, NIH
Their loss from disease, toxins or extreme noise leads to permanent
hearing loss
However, the molecules that make up these cells are continually made
and destroyed
Human photoreceptor cells are another
permanent cell type with a specific architecture.
Their architecture
converts light waves
into nerve impulses
The overall photoreceptor cells are permanent,
but do they turn over at the molecular level?
Pulse-chase experiment
-cells exposed to radiolabeled
leucine for a short time
-they take up the labeled amino
acid and incorporate it into newly
synthesized proteins for a short
period of time
What will happen to the labeled
Leucine?
a) It will be detected in the cells
for their entire lifetime
b) It’s detection will gradually
be lost
The overall photoreceptor cells are permanent,
but do they turn over at the molecular level?
Degrades the protein
+ photoreceptive
discs
Pulse-chase experiment
-cells exposed to radiolabeled
leucine for a short time
-they take up the labeled amino
acid and incorporate it into newly
synthesized proteins for a short
period of time
What will happen to the labeled
Leucine?
a) It will be detected in the cells
for their entire lifetime
b) It’s detection will gradually
be lost
1. Some tissues contain the same cells for the life of the organism,
but the molecular components of these cells do turnover
-typically cells with very specialized architecture
e.g. auditory hair cells or photoreceptor cells
2. Other tissues renew their cells rapidly
-typically cells exposed to harsh environments or activities
e.g. skin cells, gut cells or blood cells
3. Other tissues are between these extremes
Cell turnover can be stem cell dependent or independent
Stem cell definition
1. It is not terminally differentiated
2. It can divide without limit
3. Its daughters can remain a stem cell
or differentiate
Cell turnover can be stem cell dependent or independent
Stem cell definition
1. It is not terminally differentiated
2. It can divide without limit
3. Its daughters can remain a stem cell
or differentiate
Cell renewal can occur from division
of differentiated cells
(e.g. liver cells and insulin-secreting cells
of the pancreas)
The use of stem cells requires specific
mechanisms
Mechanism 1
The fates of stem cell daughters must be controlled
1. Divisional asymmetry
One daughter receives factors promoting
‘stemness’, and the other receives
factors promoting differentiation
A drawback:
If stem cells are lost, their original numbers
can’t be restored
Mechanism 1
The fates of stem cell daughters must be controlled
2. Environmental asymmetry
The cell division is symmetric and
and the daughters’ fates are determined
by the environment they are born in to
If stem cells are lost, then their numbers
can be increased by having both
daughters of divisions enter the
environment promoting “stemness”
Mechanism 2
Stem cells divide slowly for their long-term preservation
This protects the stem cell from:
1. Mutations associated with
cell division
2. Telomere depletion associated
with cell division
However, large numbers of cells
are needed to renew differentiated
cell populations
Mechanism 2
Stem cells divide slowly for their long-term preservation
Transit amplifying cells
expand cell numbers
before final differentiation
Mechanism 3
Stem cells are supported by a local environment, their niche
e.g. Skin stem cells and their progeny
The stem cells reside in the
basal layer and require
basal lamina attachment
to remain as stem cells
àthe basal lamina provides
a niche for the stem cells
After detaching from the
basal lamina, the cells
differentiate through
a linear sequence
of cell types and are
finally shed from the animal
Mechanism 3
Stem cells are supported by a local environment, their niche
e.g. Skin stem cells and their progeny
The stem cells reside in the
basal layer and require
basal lamina attachment
to remain as stem cells
àthe basal lamina provides
a niche for the stem cells
After detaching from the
basal lamina, the cells
differentiate through
a linear sequence
of cell types and are
finally shed from the animal
Without renewal from stem cells,
our skin would be lost in a month
Blood stem cells and their progeny
Blood stem cells
differentiate into
various populations
creating
a branched pathway
to final differentiation
Signals can promote
specific branches
depending on the
need for cell types
Blood stem cells and their progeny reside in bone marrow
An electron micrograph of bone marrow
Blood cell precursors are in bone marrow
but are the stem cells there?
If all blood stem cells were killed off
would ‘new’ bone marrow restore them?
Blood stem cells and their progeny reside in bone marrow
An electron micrograph of bone marrow
Blood cell precursors are in bone marrow
but are the stem cells there?
If all blood stem cells were killed off
would ‘new’ bone marrow restore them?
Identifying blood stem cells and their progeny
Separate cells based on arbitrary differences
………
How would you test each separated group for stem cell activity?
Identifying blood stem cells and their progeny
Homogenize mouse bone
marrow to release single cells
Expose cells to fluorescent
antibodies recognizing specific
cell surface molecules
Isolate labeled cells by
Fluorescence-Activated
Cell Sorting (FACS)
Identifying blood stem cells and their progeny
Homogenize mouse bone
marrow to release single cells
Expose cells to fluorescent
antibodies recognizing specific
cell surface molecules
Isolate labeled cells by
Fluorescence-Activated
Cell Sorting (FACS)
Test ability of isolated cells
to restore all blood cells
of an irradiated mouse
~1/10 000 bone marrow cells can
~5 of such cells are sufficient for
the restoration
Blood stem cells are maintained
through interactions with stromal
cells in the bone marrow
Once detached cells differentiate
Stromal cells provides a niche for
for the blood stem cells
NOTE:
“stromal cell”,
“mesenchymal cell”,
“connective tissue cell”
…are all close synonyms
Stem cells and tissue renewal affect how we age…
…and can treat diseases and disabilities
Stem cells and tissue renewal affect how we age…
…and can treat diseases and disabilities
Medical uses for stem cells
Using blood stem cells to treat leukemia
Problems of immune rejection
à careful tissue matching
and immunosuppressive drugs
à if the cancer arises from a mutation
in one of the progenitor populations
then the patient’s own stems cells
can be used after sorting
!!
Remove from
marrow sample
Inject the rest
or a patient
Medical uses for stem cells
What if the tissue to be replaced doesn’t have a readily
available supply of its own stem cells?
(e.g. spinal cord injuries or neurodegenerative diseases)
Can cells of a different tissue be used to make stem cells
for treatment?
This occurs naturally during
limb regeneration in newts
•Muscle cells de-differentiate,
and start dividing
•They form a bud similar to the
embryonic limb bud
•Their progeny form all cell types
needed to re-grow the limb
Medical uses for stem cells
What if the tissue to be replaced doesn’t have a readily
available supply of its own stem cells?
(e.g. spinal cord injuries or neurodegenerative diseases)
Can cells of a different tissue be used to make stem cells
for treatment?
Current technology cannot do this from adult human cells
at the scale or reliability needed for medical purposes,
but is has been done in experiments
This technique could avoid immune rejection by using
the patient’s own cells, but cancer development is a
potential problem if cell differentiation isn’t properly controlled
Embryonic stem (ES) cells can proliferate indefinitely in culture
and have full developmental potential
This can increase the yield of cells needed for treatments, but
ethical issues, immune rejection and the potential of cancer
are still concerns
Two potential ways to avoid immune rejection of ES cells
1. Somatic cell nuclear transfer—use a nucleus from one of the patient’s own cells
and transfer it into an unfertilized egg to develop an embryo from which ES cells
can be harvested
Two potential ways to avoid immune rejection of ES cells
1. Somatic cell nuclear transfer—use a nucleus from one of the patient’s own cells
and transfer it into an unfertilized egg to develop an embryo from which ES cells
can be harvested
2. Treat some of the patient’s own cells with factors that generate ES cells
à a combination of Oct3/4, Sox2, Myc and Klf4 (all transcription factors) can
convert differentiated cells into cells with ES cell characteristics
+ protein
expression
Continual renewal is important for maintaining all structures
Tissues often renew themselves naturally,
but when they can’t medical treatments can be used
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
Polarized cytoskeletal networks organize cells
and control interactions with their environment
Which of the structures below would you predict
to organize intracellular trafficking routes?
a) Green
b) Red
c) Blue
Which of the structures below would you predict
to organize intracellular trafficking routes?
a) Green (Microtubules)
b) Red (Actin)
c) Blue (Nucleus)
Microtubules are inherently polarized
(from subunit, to protofilament, to network)
Each protofilament is made of
heterodimers of the monomeric
proteins a-tubulin and b-tubulin
Tubulin monomers bind and
hydrolyze GTP (shown in red)
Each heterodimer is asymmetric
The heterodimers assemble
head to tail forming
polarized filaments
g-Tubulin complexes nucleate microtubules
• g-tubulin binds tubulin heterodimers assembling protofilaments into tubes
• g-tubulin nucleates microtubules at their minus ends
• Plus ends grow away from nucleation sites
• g-tubulin often associates with large microtubule organizing centres (MTOCs)
e.g. the centrosomes (above)
Contain 2 centrioles surrounded by hundreds of proteins
with g-tubulin nucleation sites on the surface
Microtubule movement by dynamic instability
• Single microtubules switch between growing & shrinking
•termed dynamic instability
This behaviour allows microtubules to search the full 3-D space of the cytoplasm
Microtubule movement by dynamic instability
• Single microtubules switch between growing & shrinking
•termed dynamic instability
• Growing microtubules have a protective cap of GTP-bound tubulin
• If GTP hydrolysis is faster
than subunit addition
the cap is lost and
catastrophe occurs
*depolymerization is
~100x faster at an
exposed GDP end*
•Regaining a GTP cap
rescues growth
Microtubule networks can form a coordinate system on their own
A purified centrosome was mixed
with purified tubulin subunits
in an artificial membrane-bound container.
It moves to the centre of the container
as microtubule plus ends push on
the outer membrane.
àminus ends central/plus ends outwards
Microtubule networks can form a coordinate system on their own
This may contribute to microtubule
organization in cells
(but many regulatory proteins
are also involved)
How could a microtubule coordinate system be used?
How is cargo transported along microtubules?
Sun Peaks Trail Map
Wikimedia
Motors move cargo through the microtubule networks
The motor activity is polarized.
– Dynein moves to microtubule minus ends
– Kinesin moves to microtubule plus ends
Motors move cargo through the microtubule networks
The motor activity is polarized.
– Dynein moves to microtubule minus ends
– Kinesin moves to microtubule plus ends
The role for microtubules in positioning the Golgi can be seen
after the addition of a microtubule inhibitor
Control cells
Nocodazole-treated cells
What motor is likely key for Golgi positioning?
a) Dynein
b) Kinesin
The microtubule network is very dynamic and can be reorganized
How could microtubules be used to control the colour of this fish?
The African cichlid fish, Tilapia mosssambica
Male dominant behavior
àBlack color change
Kinesin and dynein
compete
for melanosomes
Haimo and Thaler, Bioessays 16:727
Camouflage behavior
àWhite colour change
Kinesin inhibited
Dynein moves
melanosomes
to centre
Melanosomes are
pigment-containing
vesicles.
When they are
throughout cell,
the cell is black.
When at the centre,
the cell is white
The actin cytoskeleton also plays a critical role
in organizing cell structure and controlling cell behaviour
The actin cytoskeleton is inherently polarized
(from subunit, to filaments, to networks)
Actin monomers are
asymmetric
Actin monomers bind
and hydrolyze ATP
Actin monomers assemble
head to tail forming
polarized filaments
The actin cytoskeleton is inherently polarized
(ATP-ADP polarity along actin filaments)
• After polymerization Actin-ATP is hydrolyzed to Actin-ADP
• Hydrolysis reduces binding affinities to neighbouring subunits increasing dissociation
• However, if the rate of addition of Actin-ATP is faster than rate of removal of ActinADP, a relatively stable “cap” of Actin-ATP subunits can be formed
ATP
ADP
ADP ADP ATP ATP ATP
ADP ADP ADP ATP ATP
ADP
cap
plus end
ATP
Which way will this filament grow?
The actin cytoskeleton is inherently polarized
(Polarized assembly and disassembly leads to treadmilling)
Because of the ATP-ADP polarity along the filament,
subunits can undergo net assembly at plus end
equal to (or greater than) the net disassembly at the minus end
Thus, the polymer can maintain a constant length (or grow)
with a flux of subunits through the filament (treadmilling)
Net direction of treadmilling
The actin cytoskeleton is inherently polarized
(from subunit, to filaments, to networks)
The ARP complex nucleates actin filaments…
..and branches
actin filaments
to form polarized
2-D networks
Polarized treadmilling of large actin networks
can produce significant protrusive power
These networks
drive polarized
cell movement
Treadmilling actin networks need traction
to drive cells forward
Net direction of treadmilling
Without traction actin filaments can treadmill
but maintain their overall position
Treadmilling microfilaments can engage
stationary anchors to create ‘protrusive machines’
Cell
Protrusion
A stationary anchor binds one part of the filament
The treadmilling filament extends from that point
This extension pushes against the cell membrane driving cell protrusion
Large regions of actin networks are anchored
to create ‘protrusive machines’
ANCHORED
Large regions of actin networks are anchored
to create ‘protrusive machines’
Top View
Side View
What is the network
anchored to?
In animals, cells migrate on (and through) the extracellular matrix,
a non-cellular material made of proteins and polysaccharides
Integrins connect the actin cytoskeleton to extracellular matrix
molecules
Main receptors that bind
extracellular molecules
Transmembrane heterodimers
of non-covalently associated
a and b subunits
Linked to the actin cytoskeleton
via adaptor proteins
How is the actin cytoskeleton directed to follow a target?
Video
Chemoattractant receptors orient the actin networks
Chemoattractant receptors orienting actin networks
is analogous to food sources orienting ant trails
Actin networks and ant trails are each made of polarized subunits
Each are dynamic and can rapidly reorient to changes in the target position
mtkilimanjarologue.com
Actin networks can undergo
other large scale rearrangements
e.g. localized assembly of the contractile ring
that divides daughter cells after mitosis
Actin and microtubule networks are also integrated in cells
but we are just beginning to understand the crosstalk
Advice on required e-text reading
Supplement your lecture notes by writing out the following as you read.
à forces you to think and organize as you read,
and thus increases your understanding
• The steps of processes and the key molecules of the complexes involved
– for those discussed in class, add information from the e-textbook
– for examples not discussed, write short summaries or diagrams
How do I choose which processes/complexes to take notes on?
Emphasize those with…
– relevance to a key function of a cell, organ or organism
– relevance to disease
– an organization that is similar to other
processes/complexes/concepts discussed
…and make short notes about these points
Do this within 24h of each lecture (preferably the same day),
and review your notes frequently.
à cements the material in your longer term memory
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
BIO 230
Lecture 3 :
Prokaryotic Transcriptional Regulation
Continued…
1) Recap of prokaryotic gene regulation
2) Bacteriophage Lamba
3) Synthetic Biology
4) Transcription Attenuation
Readings (Alberts et al. custom text)
Pages 400-405, 413-416, 876-878
2
3
Catabolite Activator Protein
Trp Repressor
Lac Repressor
Recap: Prokaryotic Gene Regulation
Example 1: The Tryptophan Operon
Tryptophan repressor contains a
Helix-Turn-Helix
DNA binding motif (most common DNA-binding motif)
Helix-Turn-Helix
Binds in major
groove of DNA
double helix
Tryptophan Repressor
Tryptophan binding induces
Conformational change
Fits in major groove 4
Recap: Prokaryotic Gene Regulation
To summarize:
Negative regulation:
Competition between
RNA polymerase and
repressor protein for promoter binding
Positive regulation:
activator protein recruits RNA polymerase
to the promoter to activate transcription
5
Recap: Prokaryotic Gene Regulation
Gene regulatory elements are typically close to the
transcriptional start site of prokaryotic genes
BUT regulatory elements can also be found
Far upstream of gene
Downstream of gene (eukaryotes)
Within gene (introns; eukaryotes)
6
Recap: Prokaryotic Gene Regulation
Some regulatory elements are distant from the
transcriptional start site and influence transcription – How?
DNA looping
(Euk. Video)
NtrC protein is a transcriptional activator
DNA looping allows NtrC to directly interact with
RNA polymerase to activate transcription from a distance
Bacteriophage Lambda
Virus that infects bacterial cells
Positive and negative regulatory mechanisms work
together to regulate the lifestyles of bacteriophage lamba
Two proteins repress each others synthesis
Bacteriophage Lambda
Bacteriophage lambda can exist as
one of two states in bacteria
Under favorable
bacterial growth
conditions
When host cell
is damaged
Two gene regulatory proteins are responsible for initiating this switch
Bacteriophage Lambda
Two gene regulatory proteins are responsible for initiating the switch
between prophage and lytic pathways
lambda repressor protein (cI) and
Cro protein
Repress each other’s synthesis, giving rise to the two states.
Bacteriophage Lambda
Bacteriophage lamba: a genetic switch
State 1: Prophage
Lambda repressor
occupies the operator.
blocks synthesis
of Cro
activates its own
synthesis
most bacteriophage
DNA not transcribed
Bacteriophage Lambda
eg. bacteriophage lamba: a genetic switch
State 2: Lytic
Cro occupies the operator
blocks synthesis
of repressor
allows its own
synthesis
most bacteriophage
DNA is extensively
transcribed
What triggers switch?
DNA is replicated, packaged,
new bacteriophage released
by host cell lysis
Bacteriophage Lambda
eg. bacteriophage lamba: a genetic switch
What triggers switch between prophage and lytic states?
Host response to DNA damage
-switch to lytic state
inactivates repressor
Under good growth conditions repressor protein turns off
Cro and activates itself
positive feedback loop
-maintains prophage state
Example of a transcriptional circuit.
Different types exist, control various biological processes
Transcriptional Circuits
Transcriptional Circuits
eg. repressor
protein
eg. Cro / Repressor
switch
Transcriptional Circuits
Transcriptional Circuits
Positive Feedback loops can be used to create cell memory
Transcriptional Circuits
Transcriptional Circuits
Feed-forward loops can measure the duration of a signal
– both A and B required for transcription of Z
Brief input
B does not
accumulate
Z not
transcribed
Prolonged
input B
accumulates
Z is
transcribed
Transcriptional Circuits
Transcriptional Circuits
Combinations of
regulatory circuits
combine in eukaryotic
cells to create
exceedingly complex
regulatory networks
Scientists can construct
artificial circuits and
examine their behavior in
cells synthetic biology
Gene circuit of developing sea urchin embryo
Synthetic Biology
Synthetic Biology
eg. creating a simple gene oscillator using a delayed
negative feedback circuit – “the repressillator”
A: Lac repressor
B: Tet repressor (response to antibiotic)
C: Lambda repressor
Predicted: delayed negative feedback
gives rise to oscillations
Introduced this circuit into bacterial cells
and observed expression of the repressor
genes
Synthetic Biology
Synthetic Biology
Synthetic Biology: “the repressillator”, how does it work?
1)
A expressed
A expression
4)
2)
B repressed
3)
C expressed
4)
C represses A expression
Synthetic Biology
Synthetic Biology: “the repressillator”, how does it work?
5)
A repressed
A expression
6)
B expressed
7)
C repressed
8) Repeat 1-4
Did it work?
Synthetic Biology
Synthetic Biology
eg. creating a simple gene oscillator using a negative
feedback circuit
Looking at 1 Protein
(Fluorescently tagged)
Observed
Predicted
Increasing amplitude due to
bacterial growth
Transcriptional Circuits
Feedback loops also circadian gene regulation
~ 24-hour cycle: eg. Drosophila
http://www.hhmi.org/biointeractive/drosophila-molecular-clock-model
Delayed Negative Feedback Loop
Transcription Attenuation
-In both prokaryotes and eukaryotes there can be a
premature termination of transcription called
Transcription attenuation
-RNA adopts a structure that interferes with RNA
polymerase
-Regulatory proteins can bind to RNA and interfere with
attentuation
-Prokaryotes, plants and some fungi also use
Riboswitches to regulate gene expression
Transcription Attenuation
Riboswitches
Short RNA sequences that change conformation when
bound by a small molecule
eg. prokaryotic riboswitch that regulates purine biosynthesis
Recall that bases making up DNA/RNA include:
pyrimidines (C,T,U)
purines (A,G)
Transcription Attenuation
Riboswitches
eg. prokaryotic riboswitch that regulates purine biosynthesis
Low guanine levels
-Transcription of purine biosynthetic genes is
on
Transcription Attenuation
Riboswitches
eg. prokaryotic riboswitch that regulates purine biosynthesis
High guanine levels
-Guanine binds riboswitch
-Riboswitch undergoes
conformational change
-Causes RNA polymerase
to terminate transcription
-Transcription of purine biosynthetic genes is
off
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
BIO 230
Lecture 4:
Eukaryotic Gene Regulation
1) Eukaryotic transcriptional activation
2) Eukaryotic transcriptional repression
Readings (Alberts et al. custom text)
Pages 310-314, 187-193, 196-197, 198-201
2
Reminder from a couple of
lectures ago…
Transcriptional Regulation
Gene expression in both prokaryotes and
eukaryotes is regulated by:
Gene Regulatory Proteins (transcription factors)
Which bind specifically to:
Regulatory regions of DNA (cis elements)
Gene regulatory proteins can turn genes:
-ON; Positive regulators; activators
-OFF; Negative regulators;
(eg. Trp operon)
repressors
4
Transcriptional Regulation
Recall that DNA is transcribed into RNA by the
enzyme
RNA polymerase
5
Transcriptional Regulation
Cells produce several
types of RNA:
Different RNAs transcribed
by different RNA polymerases
in eukaryotes
Prokaryotes have a single type of RNA polymerase
Transcriptional Regulation
Transcription initiation in eukaryotes requires many proteins:
general transcription factors
Help position RNA polymerase at
eukaryotic promoters contain TATA box
Required by nearly all promoters used by
RNA polymerase II
Eukaryotic Gene Regulation
Eukaryotic transcription
– RNA polymerase II transcribes protein coding genes
– Requires five general transcription factors; TFIID, TFIIB, TFIIF,
TFIIE, and TFIIH (prokaryotes only need one; σ factor)
– Eukaryotic genomes lack operons
– Eukaryotic DNA is packaged into chromatin which provides
an additional mode of regulation
– Eukaryotic transcriptional activation requires many gene
regulatory proteins
Eukaryotic Gene Regulation
Eukaryotic transcription
– Mediator acts an intermediate between regulatory
proteins and RNA polymerase
RNA Polymerase
Eukaryotic Gene Regulation
-Eukaryotic gene expression controlled by many regulatory
proteins (~2000 encoded by the human genome)
both activators and repressors
-Gene regulatory proteins can act over very large distances,
sometimes >10000 base pairs away
– One mechanism is DNA looping
Eukaryotic Gene Regulation
Eukaryotic gene regulatory proteins often function
as protein complexes on DNA
Coactivators and corepressors assemble on DNA-bound
gene regulatory proteins do not directly bind DNA
Eukaryotic Gene Regulation
Eukaryotic Activator Proteins
Modular design:
1) DNA binding domain (DB)
– recognizes specific
DNA sequence
2)
Activation domain (AD)
– accelerates rate of
transcription
Can mix-and-match DBs and ADs
Eukaryotic Gene Regulation
How do Activator Proteins activate transcription?
Attract, position and modify:
General transcription factors
Mediator
RNA polymerase II
They can do this either:
1)
Directly by acting on these components
2) Indirectly modifying chromatin structure
Eukaryotic Gene Regulation
1) Activator proteins can bind directly to transcriptional
machinery or mediator and attract them to promoters (like
prokaryotic activators)
Eukaryotic Gene Regulation
2) Activator proteins can alter
chromatin structure
Nucleosomes are the basic structure of Eukaryotic chromatin
– DNA wound around a histone octamer
(H2A, H2B, H3, and H4 x 2)
Eukaryotic Gene Regulation
Nucleosomes pack as compact chromatin fibers
Zigzag model
Solenoid Model
Transcriptional machinery cannot assemble on
promoters tightly packaged in chromatin
Activator proteins can alter chromatin structure and
increase promoter accessibility
How?
Eukaryotic Gene Regulation
4 major ways activator proteins can alter chromatin
1.
2.
3.
4.
Eukaryotic Gene Regulation
Nucleosome structure can be altered by
chromatin remodeling complexes in an
manner to increase promoter accessibility
1) Nucleosome sliding
ATP-dependent
Eukaryotic Gene Regulation
2, 3) Nucleosome removal and histone exchange
Requires cooperation with histone chaperones
Eukaryotic Gene Regulation
4 major ways activator proteins can alter chromatin
1.
2.
3.
4.
Signal for
chromatin
remodeling
Eukaryotic Gene Regulation
4)
Histone modifying enzymes produce specific patterns of
histone modifications histone code
phosphorylation
Enzyme: kinase
acetylation
Enzyme: acetyltransferase
methylation
Enzyme: methyltransferase
Addition of phosphate group:
Addition of acetyl group:
Addition of methyl group:
Histone modifications occur on specific amino acids of
histone tails
Eukaryotic Gene Regulation
The histone code:
Specific modifications to histone tails by histone
modifying enzymes “writers”
Histone H3
Eukaryotic Gene Regulation
The histone code:
Code- “reader” proteins can recognize specific
modifications and provide meaning to the code
Histone H3
Eukaryotic Gene Regulation
Transcriptional regulation using the histone code
eg. human interferon gene promoter
Step 1: Activator protein
binds to chromatin DNA
and attracts a
histone
acetyltransferase (HAT)
Step 2: HA acetylates lysine 9
of histone H3 and lysine 8 of
histone H4.
Eukaryotic Gene Regulation
Transcriptional regulation using the histone code
eg. human interferon gene promoter
Step 3: Activator protein
attracts a
histone kinase (HK)
Step 4: HK phosphorylates
serine 10 of histone H3. Can only
occur after acetylation of lysine 9
Eukaryotic Gene Regulation
Transcriptional regulation using the histone code
eg. human interferon gene promoter
Step 5: Serine modification signals
the acetyltransferase to acetylate
lysine 14 of histone H3
Histone code for transcription
Initiation is written
Eukaryotic Gene Regulation
Transcriptional regulation using the histone code
eg. human interferon gene promoter
Step 6:
TFIID and a chromatin
remodelling complex
bind to acetylated histone tails and initiate
transcription
Eukaryotic Gene Regulation
Transcriptional Repression
– Unlike prokaryotes, eukaryotic repressor proteins rarely
compete with RNA polymerase for access to DNA
– Instead use a variety of mechanisms to inihibit transcription
1) Interfering with activator function
Eukaryotic Gene Regulation
Transcriptional Repression
Interfering with activator function
2)
3)
Eukaryotic Gene Regulation
Transcriptional Repression by altering chromatin
structure
4)
Eukaryotic Gene Regulation
Transcriptional Repression by altering the histone code
5)
6)
Eukaryotic Gene Regulation
Guided by gene regulatory proteins histone “reader” and “writer”
proteins can establish a repressive form of chromatin
histone
code can
spread
This chromatin can be stabilized
Eukaryotic Gene Regulation
Spreading the histone code along chromatin carried out by
Reader-writer complexes
Eukaryotic Gene Regulation
DNA methylase
enzyme is attracted by
Reader and methylates
nearby cytosines in
DNA
DNA methyl-binding
proteins bind methyl
groups and stabilize
structure
-Methylation and therefore gene expression patterns can be inherited
A process called epigenetic inheritance
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
How can small and fragile cells form large and stable organisms?
?
Cells are organized into one of two main tissue categories
(these support the animal body plan)
Epithelial tissues à cells directly connected with minimal extracellular matrix beneath
Connective tissues à cells dispersed with extracellular matrix providing overall structure
Cells are organized into one of two main tissue categories
(these support the animal body plan)
Epithelial tissues à cells directly connected with minimal extracellular matrix beneath
Connective tissues à cells dispersed with extracellular matrix providing overall structure
>60% of the cell types in our bodies are epithelial, forming our skin
and coating our organs.
Connective tissue cells include muscle cells, neurons and immune cells.
Moving through your body, what tissues would you pass by?
Schematic Cross Section
of the Human Abdomen
(inds.co.uk)
MBoC 4th Ed.
Epithelial structure and function requires junctional complexes
Adherens junction structure
Adherens junctions can form strong continuous adhesion belts critical for
adhering cells to form epithelia
Adherens junction structure
Adherens junctions can form strong continuous adhesion belts critical for
adhering cells to form epithelia
Cadherin clusters mediate the adhesion
Cadherin cluster
Adherens junction structure
Cadherin clusters mediate the adhesion through:
1. Homophilic interactions between cadherin receptors
Adherens junction structure
Cadherin clusters mediate the adhesion through:
1. Homophilic interactions between cadherin receptors
2. Links to the actin cytoskeleton
Adherens junction function:
Tissue maintenance during development
Movement of the outer epithelium of the Drosophila embryo
Adherens junction function:
Tissue maintenance during development
Tissue structure is lost
in adherens junction
mutants
Adherens junction function:
Tumour suppression
• The loss of epithelial structure
is a hallmark of cancer
• Cadherins are
tumour suppressors
Epithelia have distinct ‘apical’ and ‘basal’ sides
-the apical surface faces the organ lumen or the animal surface
-the basal surface faces underlying tissue
This epithelial polarity is critical for organ function
[email protected]
Epithelial polarity controls solute diffusion
between our body compartments
e.g. Controlling glucose
transport into the blood
The glucose is blocked from
diffusing between cells by tight junctions
Instead, it must be actively transported
through cells by plasma membrane
channels allowing for precise regulation.
How are the channels positioned?
What would happen without tight junctions?
Epithelial polarity controls solute diffusion
between our body compartments
e.g. Controlling glucose
transport into the blood
The glucose is blocked from
diffusing between cells by tight junctions
Instead, it must be actively transported
through cells by plasma membrane
channels allowing for precise regulation.
How are the channels positioned?
What would happen without tight junctions?
Epithelial polarity controls solute diffusion
between our body compartments
e.g. Controlling glucose
transport into the blood
The glucose is blocked from
diffusing between cells by tight junctions
Instead, it must be actively transported
through cells by plasma membrane
channels allowing for precise regulation.
How are the channels positioned?
What would happen without tight junctions?
Testing the permeability barrier across an epithelia
Testing the permeability barrier across an epithelia
(a)
(b)
If a dye was added below an epithelium
with tight junctions how far could it diffuse?
(c)
Testing the permeability barrier across an epithelia
added apically
or basally?
added apically
or basally?
Tracer molecule
(black) added to
apical or basal side
of the epithelium
What would happen without tight junctions?
Tight junctions encircle the apical end of each cell in an epithelial sheet
Apical
Basal
Tight junctions are formed from
strands of interacting transmembrane proteins
Apical
Tight junctions are formed from
strands of interacting transmembrane proteins
Core tight junction proteins:
Claudin
• 4-pass transmembrane protein
Occludin
• 4-pass transmembrane protein
Tight junctions block two movements.
Movement of aqueous molecules
through the extracellular space between cells
Movement of membrane molecules
between the apical and basolateral domains of
each cell’s plasma membrane
Cell Polarity is Fundamental
to Cell and Developmental Biology
How is cell polarity established?
How does asymmetry arise
to make one end of the cell
different from the other?
(and what does this polarity do
for cells and tissues?)
In general, cells use ‘landmarks’
to establish and elaborate polarity
Primary
Landmark
Subsequent
Elaboration
Chemoattractants polarize cells and the cells chase prey
Adherens junctions (AJs) are important
landmarks for epithelial polarity
Lumen or Exterior Space
Apical
Domain
AJs
Basolateral
Domain
Underlying Tissue
Adherens junctions (AJs) are important
landmarks for epithelial polarity
Cadherin
Cadherin
Arm
Cadherin
a-cat
F -a
n
c ti
Apical
Domain
Basolateral
Domain
Adherens junctions (AJs) are important
landmarks for epithelial polarity
Apical
Cues
Cadherin
Arm
a-cat
F
ti
c
-a
n
Basal
Cues
Conserved apical and basal cues controlling epithelial polarity
The integration of polarity complexes, adhesion complexes,
cytoskeletal networks and trafficking routes is critical for the
structure and function of epithelia that form our organs
Complexes required for epithelial structure
A functional
epithelium
Body compartments constructed
from functional epithelia
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
4. Tissue morphogenesis
5. Tissue patterning
6. Stem cells
9-10: Mitosis and Cell Division & Cancer
11-12: Development of Multicellular Organisms
All structures need maintenance
Once cells and tissues are formed they continually regenerate
This can occur at the level of molecular turnover or cellular turnover
1. Some tissues contain the same cells for the life of the organism,
but the molecular components of these cells do turn over
-typically cells with very specialized architecture
e.g. auditory hair cells or photoreceptor cells
2. Other tissues renew their cells rapidly
-typically cells exposed to harsh environments or activities
e.g. skin cells, gut cells or blood cells
3. Other tissues are between these extremes
The specialized architecture of auditory hair cells in the organ of Corti
This architecture functions to convert sound waves into nerve impulses
(it allows us to hear)
Sound waves cause stereocilia
atop hair cells to tilt
With tilting, tethers pull open
ion channels on neighbouring stereocilia
(this initiates a nerve impulse)
This architecture functions to convert sound waves into nerve impulses
(it allows us to hear)
Sound waves cause stereocilia
atop hair cells to tilt
With tilting, tethers pull open
ion channels on neighbouring stereocilia
(this initiates a nerve impulse)
In mammals, these cells do not re-grow when lost
anatomybox.com
Image by By F. Kalinec and B. Kachar, NIH
Their loss from disease, toxins or extreme noise leads to permanent
hearing loss
However, the molecules that make up these cells are continually made
and destroyed
Human photoreceptor cells are another
permanent cell type with a specific architecture.
Their architecture
converts light waves
into nerve impulses
The overall photoreceptor cells are permanent,
but do they turn over at the molecular level?
Pulse-chase experiment
-cells exposed to radiolabeled
leucine for a short time
-they take up the labeled amino
acid and incorporate it into newly
synthesized proteins for a short
period of time
What will happen to the labeled
Leucine?
a) It will be detected in the cells
for their entire lifetime
b) It’s detection will gradually
be lost
The overall photoreceptor cells are permanent,
but do they turn over at the molecular level?
Degrades the protein
+ photoreceptive
discs
Pulse-chase experiment
-cells exposed to radiolabeled
leucine for a short time
-they take up the labeled amino
acid and incorporate it into newly
synthesized proteins for a short
period of time
What will happen to the labeled
Leucine?
a) It will be detected in the cells
for their entire lifetime
b) It’s detection will gradually
be lost
1. Some tissues contain the same cells for the life of the organism,
but the molecular components of these cells do turnover
-typically cells with very specialized architecture
e.g. auditory hair cells or photoreceptor cells
2. Other tissues renew their cells rapidly
-typically cells exposed to harsh environments or activities
e.g. skin cells, gut cells or blood cells
3. Other tissues are between these extremes
Cell turnover can be stem cell dependent or independent
Stem cell definition
1. It is not terminally differentiated
2. It can divide without limit
3. Its daughters can remain a stem cell
or differentiate
Cell turnover can be stem cell dependent or independent
Stem cell definition
1. It is not terminally differentiated
2. It can divide without limit
3. Its daughters can remain a stem cell
or differentiate
Cell renewal can occur from division
of differentiated cells
(e.g. liver cells and insulin-secreting cells
of the pancreas)
The use of stem cells requires specific
mechanisms
Mechanism 1
The fates of stem cell daughters must be controlled
1. Divisional asymmetry
One daughter receives factors promoting
‘stemness’, and the other receives
factors promoting differentiation
A drawback:
If stem cells are lost, their original numbers
can’t be restored
Mechanism 1
The fates of stem cell daughters must be controlled
2. Environmental asymmetry
The cell division is symmetric and
and the daughters’ fates are determined
by the environment they are born in to
If stem cells are lost, then their numbers
can be increased by having both
daughters of divisions enter the
environment promoting “stemness”
Mechanism 2
Stem cells divide slowly for their long-term preservation
This protects the stem cell from:
1. Mutations associated with
cell division
2. Telomere depletion associated
with cell division
However, large numbers of cells
are needed to renew differentiated
cell populations
Mechanism 2
Stem cells divide slowly for their long-term preservation
Transit amplifying cells
expand cell numbers
before final differentiation
Mechanism 3
Stem cells are supported by a local environment, their niche
e.g. Skin stem cells and their progeny
The stem cells reside in the
basal layer and require
basal lamina attachment
to remain as stem cells
àthe basal lamina provides
a niche for the stem cells
After detaching from the
basal lamina, the cells
differentiate through
a linear sequence
of cell types and are
finally shed from the animal
Mechanism 3
Stem cells are supported by a local environment, their niche
e.g. Skin stem cells and their progeny
The stem cells reside in the
basal layer and require
basal lamina attachment
to remain as stem cells
àthe basal lamina provides
a niche for the stem cells
After detaching from the
basal lamina, the cells
differentiate through
a linear sequence
of cell types and are
finally shed from the animal
Without renewal from stem cells,
our skin would be lost in a month
Blood stem cells and their progeny
Blood stem cells
differentiate into
various populations
creating
a branched pathway
to final differentiation
Signals can promote
specific branches
depending on the
need for cell types
Blood stem cells and their progeny reside in bone marrow
An electron micrograph of bone marrow
Blood cell precursors are in bone marrow
but are the stem cells there?
If all blood stem cells were killed off
would ‘new’ bone marrow restore them?
Blood stem cells and their progeny reside in bone marrow
An electron micrograph of bone marrow
Blood cell precursors are in bone marrow
but are the stem cells there?
If all blood stem cells were killed off
would ‘new’ bone marrow restore them?
Identifying blood stem cells and their progeny
Separate cells based on arbitrary differences
………
How would you test each separated group for stem cell activity?
Identifying blood stem cells and their progeny
Homogenize mouse bone
marrow to release single cells
Expose cells to fluorescent
antibodies recognizing specific
cell surface molecules
Isolate labeled cells by
Fluorescence-Activated
Cell Sorting (FACS)
Identifying blood stem cells and their progeny
Homogenize mouse bone
marrow to release single cells
Expose cells to fluorescent
antibodies recognizing specific
cell surface molecules
Isolate labeled cells by
Fluorescence-Activated
Cell Sorting (FACS)
Test ability of isolated cells
to restore all blood cells
of an irradiated mouse
~1/10 000 bone marrow cells can
~5 of such cells are sufficient for
the restoration
Blood stem cells are maintained
through interactions with stromal
cells in the bone marrow
Once detached cells differentiate
Stromal cells provides a niche for
for the blood stem cells
NOTE:
“stromal cell”,
“mesenchymal cell”,
“connective tissue cell”
…are all close synonyms
Stem cells and tissue renewal affect how we age…
…and can treat diseases and disabilities
Stem cells and tissue renewal affect how we age…
…and can treat diseases and disabilities
Medical uses for stem cells
Using blood stem cells to treat leukemia
Problems of immune rejection
à careful tissue matching
and immunosuppressive drugs
à if the cancer arises from a mutation
in one of the progenitor populations
then the patient’s own stems cells
can be used after sorting
!!
Remove from
marrow sample
Inject the rest
or a patient
Medical uses for stem cells
What if the tissue to be replaced doesn’t have a readily
available supply of its own stem cells?
(e.g. spinal cord injuries or neurodegenerative diseases)
Can cells of a different tissue be used to make stem cells
for treatment?
This occurs naturally during
limb regeneration in newts
•Muscle cells de-differentiate,
and start dividing
•They form a bud similar to the
embryonic limb bud
•Their progeny form all cell types
needed to re-grow the limb
Medical uses for stem cells
What if the tissue to be replaced doesn’t have a readily
available supply of its own stem cells?
(e.g. spinal cord injuries or neurodegenerative diseases)
Can cells of a different tissue be used to make stem cells
for treatment?
Current technology cannot do this from adult human cells
at the scale or reliability needed for medical purposes,
but is has been done in experiments
This technique could avoid immune rejection by using
the patient’s own cells, but cancer development is a
potential problem if cell differentiation isn’t properly controlled
Embryonic stem (ES) cells can proliferate indefinitely in culture
and have full developmental potential
This can increase the yield of cells needed for treatments, but
ethical issues, immune rejection and the potential of cancer
are still concerns
Two potential ways to avoid immune rejection of ES cells
1. Somatic cell nuclear transfer—use a nucleus from one of the patient’s own cells
and transfer it into an unfertilized egg to develop an embryo from which ES cells
can be harvested
Two potential ways to avoid immune rejection of ES cells
1. Somatic cell nuclear transfer—use a nucleus from one of the patient’s own cells
and transfer it into an unfertilized egg to develop an embryo from which ES cells
can be harvested
2. Treat some of the patient’s own cells with factors that generate ES cells
à a combination of Oct3/4, Sox2, Myc and Klf4 (all transcription factors) can
convert differentiated cells into cells with ES cell characteristics
+ protein
expression
Continual renewal is important for maintaining all structures
Tissues often renew themselves naturally,
but when they can’t medical treatments can be used
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
7-9: How do cells communicate with each other?
7. Principles of cellular signalling
8. Signalling via small molecules
9. Signalling via protein modifications
10-12: How are cell numbers controlled?
Cellular signaling
Cells must communicate to develop and maintain multicellular organisms
Unicellular bacterial-like organisms existed on earth for ~2.5 billion years
before complex multicellular organisms arose
In part, this delay may have been due to the time needed to evolve
complex signaling systems
However, unicellular organisms do communicate…
Examples of unicellular communication
Quorum sensing in bacteria:
Many bacteria release and respond to chemical signals.
This signaling coordinates motility, antibiotic production, spore formation and
sexual conjugation in bacterial populations.
Mating in budding yeast:
Signaling between yeast cells
prepares them to mate.
Aggregation of ameboid cells:
Signaling between Dictyostelium
cells draws them together
to form a fruiting body.
http://www.biocircle.fu-berlin.de/mikrobio2/signaltransduktion_en.php?id=mb2&p=9&lang=en
How do cells communicate to develop and maintain
complex multicellular organisms?
?
Mammals, flies and worms use similar
communication pathways.
Many core pathway components were
first discovered in mutants affecting cell
communication in Drosophila,
C. elegans and yeast.
The basics of sending and receiving signals
Cells can send out hundreds of different
types of signaling molecules
(e.g. proteins, small peptides, amino acids,
nucleotides, other small molecules and
dissolved gases)
They are exocytosed, emitted by diffusion,
or displayed on the cell surface
Cells receive signals in two main ways
Signaling occurs over short or long distances
Contact-dependent signaling
-signals are retained on the cell surface
Paracrine signaling
-signals are released from the cell but act locally
-signal movement is restricted by:
1. internalization by neighbouring cells
2. signal instability or destruction by extracellular enzymes
3. binding to extracellular matrix molecules
Signaling occurs over short or long distances
Synaptic signaling
-neurons extend axons to contact distant target cells
-the released signaling molecules act locally at target
Endocrine signaling
-endocrine cells secrete hormones into the bloodstream
for long-range distribution
Cells use signal transduction pathways
to respond to extracellular signals
Definitions:
Signal transduction
The conversion of extracellular
signals into intracellular signals
Effector
A downstream molecule in a
signal transduction pathway
(upstream molecules have their
effects on them)
Cells use signal transduction pathways
to respond to extracellular signals
Small intracellular signaling
molecules are called second
messengers
-made in large numbers and
diffuse through the cytoplasm
(e.g. cyclic AMP) or plasma
membrane (e.g. diacylglycerol)
-bind and alter effector molecules
Large intracellular signaling
molecules are typically proteins
-organized into pathways and
networks
Many components of signaling pathways act like switches
Enzymatic versus non-enzymatic conformation
Protein-binding versus non-binding conformation
Gain or loss of phosphate groups
Small molecule binding or unbinding
Switching “off” is just as important
as switching “on”
-if the pathway isn’t turned off,
it won’t be able to send another signal
Addition and removal of phosphate groups on proteins
~30% of human proteins
are phosphorylated
The human genome encodes
~520 protein kinases and
~150 protein phosphatases
There are serine/threonine
kinases and tyrosine kinases
GTP or GDP binding
GTP binding proteins
-Large trimeric and small monomeric types
-Have low GTPase activity
GTP or GDP binding
GTPase activating proteins (GAPs) increase the GTP hydrolysis
Guanine nucleotide exchange factors (GEFs) promote the exchange
of GDP for GTP
Many components of signaling pathways interact like Lego
Proteins often contain one or more interaction domains
These bind to structural motifs in other molecules
(short peptide sequences, other domains,
covalent modifications)
During evolution, domains can be added or removed from
proteins to alter interactions and re-wire signaling pathways
Some examples:
SH2 and PTB domains bind
phosphotyrosine containing
sequences
PH domains bind
phosphoinositides
SH3 domains bind prolinerich sequences
If this portion of “Sos” was replaced by a PTB domain,
what do you predict would happen?
Some examples:
SH2 and PTB domains bind
phosphotyrosine containing
sequences
PH domains bind
phosphoinositides
SH3 domains bind prolinerich sequences
How are complex signaling events orchestrated?
How is signaling specificity achieved?
How are signaling pathways coordinated?
How are signaling pathways organized in space and time?
Targeting specific cells
Synaptic signaling specificity:
Neurons make connections with
specific target cells (the same
signaling molecules can be used
at all connections)
Endocrine signaling specificity:
Different molecules are released and
target cells express specific receptors
to respond to specific molecules
Different cells can also have different responses to the same molecule
by changing the signal receptor or downstream components of the pathway
Within a cell, a signal transduction molecule will often function
in many different pathways
What prevents an upstream signal from activating
all of the pathways?
PNAS 104:12890
Within a cell, a signal transduction molecule will often function
in many different pathways
What prevents an upstream signal from activating
all of the pathways?
The formation of local complexes helps insulate pathways
from each other
Within a cell, a signal transduction molecule will often function
in many different pathways
What prevents an upstream signal from activating
all of the pathways?
The formation of local complexes helps insulate pathways
from each other
How are complex signaling events orchestrated?
How is signaling specificity achieved?
How are signaling pathways coordinated?
How are signaling pathways organized in space and time?
Cellular outcomes often depend on multiple signaling inputs
Coincidence detectors only activate downstream signals
when two upstream signals are both detected
This ensures two conditions are met before the cell responds
How are complex signaling events orchestrated?
How is signaling specificity achieved?
How are signaling pathways coordinated?
How are signaling pathways organized in space and time?
How are complex signaling events orchestrated?
How is signaling specificity achieved?
How are signaling pathways coordinated?
How are signaling pathways organized in space and time?
In addition to forming local complexes, signaling pathways can be
organized at subcellular compartments such as…
…primary cilia…
…or synapses
biochemistry.ucsf.edu/labs/reiter/index-home.shtml
cumc.columbia.edu/publications/press_releases/STVimaging.html
Signaling also occurs over different time frames
Synaptic signaling is very fast
(electrical impulses travel at 100 m/s)
Endocrine signaling is relatively slow
(limited by blood flow)
The response speed to a signal can vary
depending on the cellular machinery involved
Cellular outcomes also depend on feedback mechanisms
Output
Output
Effects of positive feedback
Output
stimulus
Output
stimulus
The feedback will enhance the response.
If the feedback is strong enough, it can be self-sustaining.
System kept at high activation even with loss of the original signal.
àa ‘bistable’ system that can stably exist in ‘off’ or ‘on’ states
Effects of positive feedback
Output
stimulus
The feedback will enhance the response.
stimulus
lost
If the feedback is strong enough, it can be self-sustaining.
System kept at high activation even with loss of the original signal.
àa ‘bistable’ system that can stably exist in ‘off’ or ‘on’ states
Effects of positive feedback
Biphasic
switches
promote
differentiation
stimuli
lost
Output
stimulus
The feedback will enhance the response.
stimulus
lost
If the feedback is strong enough, it can be self-sustaining.
System kept at high activation even with loss of the original signal.
àa ‘bistable’ system that can stably exist in ‘off’ or ‘on’ states
Effects of negative feedback
Output
stimulus
Output
stimulus
If feedback occurs quickly, the signaling is suppressed.
System adapts to the stimulus.
Needs boosted stimulus for output.
Effects of negative feedback
Output
stimulus
Output
stimulus
If feedback occurs quickly, the signaling is suppressed.
System adapts to the stimulus.
Needs boosted stimulus for output.
Allows cells to respond to changes in upstream signals
rather than their absolute amounts.
(cells can respond to a wider range of signal strengths.)
Effects of negative feedback
Output
stimulus
If feedback occurs slowly,
then the system can oscillate
(e.g. for circadian rhythms)
Delay
Recover
Run every 2 days as long as
the driving stimulus is there
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
7-9: How do cells communicate with each other?
7. Principles of cellular signalling
8. Signalling via small molecules
9. Signalling via protein modifications
10-12: How are cell numbers controlled?
Signaling via small molecules
1. Independently of plasma membrane proteins
2. Through plasma membrane channels
3. Downstream of plasma membrane G-protein coupled receptors
Signaling via small molecules
1. Independently of plasma membrane proteins
2. Through plasma membrane channels
3. Downstream of plasma membrane G-protein coupled receptors
Which class of molecules could signal
from outside to inside the cell
without a channel or receptor?
A
B
C
D
A very small signaling molecule: Nitric oxide (NO)
Made by the deamination of arginine by NO synthases
Acts locally because of a 5-10 second half life
Affects smooth muscle and other target cells
Nitroglycerine is used to treat heart pain
It is converted to NO and relaxes blood vessels, reducing workload on the heart
Small hydrophobic signaling molecules
Steroid hormones
-made from cholesterol
-affect sexual characteristics and metabolism
Thyroid hormones
-made from tyrosine
-increase metabolic rate
Retinoids
-made from Vitamin A
-regulate development
Vitamin D
-affects metabolism
Small hydrophobic signaling molecules
Steroid hormones
-made from cholesterol
-affect sexual characteristics and metabolism
Thyroid hormones
-made from tyrosine
-increase metabolic rate
Retinoids
-made from Vitamin A
-regulate development
Vitamin D
-affects metabolism
Transported in extracellular fluids by
carrier proteins
Dissociate from carriers upon cell entry
In the cell they bind a member of the
nuclear receptor superfamily
The nuclear receptor superfamily
Contain binding sites for a small
hydrophobic molecule and for DNA
48 identified in the human genome
More than half only identified based
on sequence analyses
àtheir ligands are unknown
àtermed orphan nuclear receptors
Ligand binding alters receptor conformation, and releases inhibitors,
to promote DNA binding and downstream transcription
Signaling via small molecules
1. Independently of plasma membrane proteins
2. Through plasma membrane channels
3. Downstream of plasma membrane G-protein coupled receptors
Ion channels are a major class
of signaling molecules
Electrochemical gradients across the plasma membrane
Ion channel properties
• Have narrow selective pores
• Open and close rapidly
• Up to 100 million ions can pass though an open channel per second
• Transport is passive
à based on electrochemical gradients across the plasma
membrane and ion diffusion down these gradients
Ion channels can be activated in a number of ways
(e.g. hair cells of
the Organ of Corti)
Ion channel functions
-electrical excitability of muscle cells
-electrical signaling in the nervous system
-leaf-closure responses in plants
-signal the single-celled Paramecium to reverse its movement upon collision
-others (these channels are present in all animal cells)
A nerve impulse releases neurotransmitters
that open channels of a postsynaptic target cell
With exocytososis, synaptic vesicles
release neurotransmitters which bind
and open ligand-gated ion channels
These open channels allow ion passage
into the target cell to create another
nerve impulse or a different effect
Neurons also activate other cell types via synaptic connections
(e.g. muscle contraction)
The connections between neurons can be very complex
Signaling via small molecules
1. Independently of plasma membrane proteins
2. Through plasma membrane channels
3. Downstream of plasma membrane G-protein coupled receptors
Signaling by G-protein coupled receptors
G-protein coupled receptors
– 7 pass transmembrane proteins
– activated by proteins, small molecules and light
– more than 700 in humans
Signaling by G-protein coupled receptors
All G-protein coupled receptors signal into the
cytoplasm via a membrane associated
trimeric GTP-binding protein
(a G protein)
The activated receptor functions
as a guanine nucleotide exchange
factor to exchange GDP for GTP
on the α-subunit
The α-subunit undergoes a
conformational change which
alters the conformation of the
other subunits or induces their
release
The altered subunits bind
downstream effectors
The activity is turned off by a
regulator of G protein signaling
(RGS) which acts as a GTPase
activating protein
G-proteins have a number of downstream effects
G proteins can signal rapidly via cyclic AMP (cAMP)
cAMP is synthesized from ATP by adenylyl
cyclase and is destroyed by cAMP
phosphodiesterase
The canonical cAMP pathway
The signal is transduced by increasing
adenylyl cyclase activity above a constant
background of phosphodiesterase activity
The accumulated cAMP activates
Protein Kinase A (PKA) (a serinethreonine kinase)
[other targets are activated as well]
Note the release of separate
molecules from a tetramer
The canonical cAMP pathway
The signal is transduced by increasing
adenylyl cyclase activity above a constant
background of phosphodiesterase activity
The accumulated cAMP activates
Protein Kinase A (PKA) (a serinethreonine kinase)
[other targets are activated as well]
PKA phosphorylates CREB to activate
transcription
[PKA has other targets as well]
A sense of smell with GPCRs and cAMP
~350 G-protein coupled receptors
allow us to smell
Each receptor recognizes
a different set of odourants and
then produces cAMP which opens
cAMP-gated cation channels
which induce an action potential
Each olfactory neuron expresses
just one of these receptors
A smell is a compilation of
different odourants
Signals from different combinations
of neurons allows us to distinguish
>10 000 different smells
The receptors localize to
specialized cilia emanating
from olfactory neurons
in the lining of the nose
GPCR signaling via calcium
IP3 is a water soluble molecule that diffuses through the cytoplasm
DAG is a hydrophobic molecule that diffuses along the plasma membrane
GPCR signaling via calcium
GPCR signaling via calcium
PKC is a serine/threonine kinase with a range of targets
Calcium also has different targets (e.g. calmodulin)
DAG also has different signaling mechanims
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
7-9: How do cells communicate with each other?
7. Principles of cellular signalling
8. Signalling via small molecules
9. Signalling via protein modifications
10-12: How are cell numbers controlled?
Signaling via protein modifications
1. Enzyme-coupled receptors (and protein phosphorylation)
2. Use of proteolysis in signaling
Signaling via protein modifications
1. Enzyme-coupled receptors (and protein phosphorylation)
There are six main classes of enzyme-coupled receptors
i)
ii)
iii)
iv)
v)
vi)
Receptor tyrosine kinases
Tyrosine-kinase-associated receptors
Receptor serine/threonine kinases
Histidine-kinase-associated receptors
Receptor guanylyl cyclases
Receptorlike tyrosine phosphatases
Receptor tyrosine kinases
~60 encoded in the human genome
Some bind secreted proteins and others bind cell surface proteins
Receptor tyrosine kinases mediate essential functions
Signaling molecules induce the transautophosphorylation
of receptor tyrosine kinases
Ligand binding dimerizes the receptor
Their kinase domains are brought together and phosphorylate each other
Phosphorylation of the kinase domains enhances their activity
Phosphorylation of other regions creates docking sites to assemble a signaling complex
If a receptor tyrosine kinases with a defective kinase domain was
expressed together with the normal receptor what would happen?
A) The cell would respond
normally to the signal
B) The cell would have no
response to the signal
C) The cell would have a
reduced response to the
signal
A more complicated structure-activity relationship…
(not on the exam)
EGFR as a RTK example
Andy Schulte
Phosphorylated receptor tyrosine kinases recruit proteins
which mediate downstream signaling
The recruited proteins contain domains (SH2 or PTB domains)
that bind to phosphotyrosine and neighbouring sequences
3D structure of an SH2 domain
The basic functionality of an SH2 domain
Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway
The Drosophila compound eye consists of ~800 ommatidia
The ommatidia are composed of 8 photoreceptor cells
and 12 support cells
They arise from a simple epithelial sheet through the
sequential differentiation of the photoreceptor cells
The R7 photoreceptor cell is needed to detect UV light
A screen was performed to identify mutants that failed
to specify R7 (based on insensitivity to UV light)
Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway
The first mutant identified was called Sevenless (Sev)
àthe normal Sevenless protein was shown to be a receptor tyrosine
kinase expressed in R7 cells
Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway
The second mutant identified was called Bride-of-sevenless (Boss)
àthe normal Bride-of-sevenless protein was shown to be the
ligand for Sevenless expressed on R8 cells
Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway
Drk and Son-of-sevenless (Sos) were identified in subsequent screens
àDrk links Sevenless to Son-of-sevenless
àSon-of-sevenless is a GEF for Ras
Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway
This basic mechanism is used in different contexts in all animals
What is Ras?
Ras is a molecular switch downstream of receptor tyrosine kinases
•monomeric GTPase
•attached to the cytoplasmic face of the plasma membrane by a lipid anchor
•activated by Ras-GEFs and inactivated by Ras-GAPs
•its activity leads to cell proliferation or differentiation
•30% of human tumours have hyperactive mutant forms of Ras
Ras activates a mitogen-activated protein kinase module
(MAP kinase module) to change protein activity and gene expression
At least 5 parallel MAP kinase modules can operate in mammalian cells
(there are at least 12 MAPKs, 7 MAPKKs, and 7 MAPKKKs)
àHow is non-specific cross-talk controlled?
àWhat stops a MAPKKK from phosphorylating multiple MAPKKs?
At least 5 parallel MAP kinase modules can operate in a mammalian cells
(there are at least 12 MAPKs, 7 MAPKKs, and 7 MAPKKKs)
àHow is non-specific cross-talk controlled?
In yeast, scaffolds bind specific MAP kinase modules, insulating them from other
modules and increasing response specificity
What might happen if kinase D exchanged its double-lobed domain
with the square domain of kinase C?
Signaling via protein modifications
1. Enzyme-coupled receptors (and protein phosphorylation)
There are six main classes of enzyme-coupled receptors
i)
ii)
iii)
iv)
v)
vi)
Receptor tyrosine kinases
Tyrosine-kinase-associated receptors
Receptor serine/threonine kinases
Histidine-kinase-associated receptors
Receptor guanylyl cyclases
Receptorlike tyrosine phosphatases
Receptor serine/threonine kinases
-the largest class of cell surface receptors in plants (also function in animals)
~6 major families in plants
àthe largest is the leucine-rich repeat receptor kinase family
(175 members in Arabidopsis [e.g. the Clavatal1/Clavatal2 complex])
Clv1/Clv2 signaling stimulates
the development of
stems, leaves and flowers
Gene regulation
Histidine-kinase-associated receptors
-activate a “two-component” signaling pathway
-used by bacteria, yeast and plants, but not animals
e.g. regulation of bacterial chemotaxis
attractant
Flagella rotating for propulsion
repellent
Rotation reverses for re-direction
Histidine-kinase-associated receptors
-activate a “two-component” signaling pathway
-used by bacteria, yeast and plants, but not animals
e.g. regulation of bacterial chemotaxis
attractant
repellent
CheA is the histidine kinase
It phosphorylates itself and then
transfers the phosphate to an
aspartic acid on CheY
Signaling via protein modifications
1. Enzyme coupled receptors and protein phosphorylation
2. Use of proteolysis in signaling
Notch signaling
Hedgehog signaling
Wingless signaling
TNFα / NFκB signaling
Lateral inhibition by Notch signaling
Clathrin-mediated
Delta binding
leads to Notch
cleavage…
…and movement
of a Notch fragment
to the nucleus
Note the single cleavage
on the cytoplasmic tail
Organizer function in vertebrate limb development
Source of the morphogen sonic hedgehog (Shh)
Shh spreads from this source
The Shh gradient controls
the formation of distinct digits
POSTERIOR
Without hedgehog signaling
the transcriptional activator
Ci is sequestered in the cytoplasm
by a microtubule-associated complex
Plus, this complex promotes the
proteolysis of Ci to create a
transcriptional repressor
At the same time, the Smoothened protein
is sequestered in intracellular vesicles
by Patched
The binding of Hedgehog to Patched
allows Smoothened to transfer to the plasma membrane
where it releases Ci from its inhibitory complex
Signaling via protein modifications
1. Enzyme coupled receptors and protein phosphorylation
2. Use of proteolysis in signaling
Notch signaling
Hedgehog signaling
Wingless signaling
TNFα / NFκB signaling
The release of a transcriptional activator from an inhibitory complex
is also at the core of Wingless and TNFα / NFκB signaling
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
7-9: How do cells communicate with each other?
7. Principles of cellular signalling
8. Signalling via small molecules
9. Signalling via protein modifications
10-12: How are cell numbers controlled?
How can we understand all of the signaling occurring in a cell?
PNAS 104:12890
e.g a network controlling human pancreatic cancer
How can we understand all of the signaling occurring in a cell?
How can we understand all of the signaling occurring in a cell?
How can we understand all of the signaling occurring in a cell?
Mapping Brain Circuits: The Connectome
http://www.sfn.org/index.aspx?pagename=brainBriefings_09_mapping
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
7-9: How do cells communicate with each other?
10-12: How are cell numbers controlled?
10. The cell cycle
11. Programmed cell death
12. Cancer
9-10: Mitosis and Cell Division & Cancer
11-12: Development of Multicellular Organisms
How do cells change
their shapes, interactions
and numbers
to build tissues, organs
and organisms?
What goes wrong
when cells lose control of
their shapes, interactions and numbers
during cancer progression
or with degenerative disease?
Eukaryotic cell cycle
• Life requires cell growth
and division.
• The cell cycle is the
duplication of cellular
contents and division of
these contents by two.
• A critical aspect of cell
division is the fidelity with
which the cell duplicates
and segregates its
genome
Four main phases of the eukaryotic cell cycle
(Cell growth and remaining doubling
of proteins and organelles)
(Cell growth and partial doubling
of proteins and organelles)
The cell must check if the process is occurring properly
The cell must check if the process is occurring properly
What are the molecular bases for these
checkpoints?
How are they regulated?
Cyclin-Dependent Kinases (Cdks):
traffic lights of the cell cycle control system
• Cdks are protein kinases
with targets that control the
cell cycle
• Their activity allows passage
through a check point
• Their activity depends on
Cyclin binding and other
modifications.
Different cyclin-Cdk checkpoints
act at different stages
of the cell cycle
Cdk activation by cyclin binding
and Cdk-activating kinase activity
Cdks regulate the machinery
that directly replicate the cell
S-Cdk activity
promotes DNA replication
M-Cdk phosphorylates multiple
targets required to start mitosis
Examples:
M-Cdk phosphorylation of lamin
leads to nuclear envelope breakdown
M-Cdk regultes proteins required
for chromosome condensation
M-Cdk phosphoryates microtubule regulators
important for making the mitotic spindle
Cdks regulate the machinery
that directly replicate the cell
S-Cdk activity
promotes DNA replication
What turns Cdks OFF?
M-Cdk phosphorylates multiple
targets required to start mitosis
Examples:
M-Cdk phosphorylation of lamin
leads to nuclear envelope breakdown
M-Cdk regultes proteins required
for chromosome condensation
M-Cdk phosphoryates microtubule regulators
important for making the mitotic spindle
Targeted degradation of cyclins turns Cdks off
Targeted degradation of cyclins turns Cdks off
e.g. anaphase promoting complex (APC) targets M-cyclin
to the proteasome allowing the completion of mitosis
The sequential synthesis and degradation of
different cyclins can drive the cell cycle
… but more control is needed
Regulating Cdk activity by
Cdk inhibitor proteins (CKIs)
e.g. p27
Regulating Cdk activity by phosphorylation
A summary of cell cycle checkpoint control
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Proteins
Cyclins
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
A summary of cell cycle checkpoint control
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Cyclins
Proteins
What acts upstream of these signals?
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
Rb blocks G1 progression and S phase
by inhibiting cyclin synthesis
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Proteins
Cyclins
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
Rb blocks G1 progression and S phase
by inhibiting cyclin synthesis
E2F is a transcription factor
Rb blocks G1 progression and S phase
by inhibiting cyclin synthesis
Rb blocks G1 progression and S phase
by inhibiting cyclin synthesis
What would happen to cells
without any Rb protein?
Rb blocks G1 progression and S phase
by inhibiting cyclin synthesis
Rb is short for “Retinoblastoma protein”
Loss of both copies of the Rb gene leads to
eye cancer in children due to excess cell
proliferation
Rb is a tumour suppressor
Mitogen activation of the cell cycle
Mitogen signaling
induces Myc transcription
via a Ras-MAP kinase
signaling cascade
Myc increases cyclin synthesis and
CKI degradation by regulating transcription
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Proteins
Cyclins
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
Myc increases cyclin synthesis and
CKI degradation by regulating transcription
Myc increases cyclin synthesis and
CKI degradation by regulating transcription
Myc over-activity leads to cancer due to
excess cell proliferation
Myc is an oncogene
Temporal feedback promoting M phase
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Proteins
Cyclins
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
Temporal feedback promoting M phase
What initially activiates Cdc25?
Polo Kinase
Temporal feedback promoting M phase
What protects against a sudden problem?
(Cell growth and remaining doubling
of proteins and organelles)
(Cell growth and partial doubling
of proteins and organelles)
p53 stops the cell cycle
in response to DNA damage
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Proteins
Cyclins
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
p53 stops the cell cycle
in response to DNA damage
p53 stops the cell cycle
in response to DNA damage
p53 stops the cell cycle
in response to DNA damage
p53 protects cells
Loss of p53 leads to cancer
p53 is a tumour suppressor
Intricate regulation of these signals is critical for
proper cell cycle progression and
and the prevention of cancer
Inhibitory
Phosphorylation
Kinases
Phosphatases
Inhibitory
Proteins
Cyclins
Degradation
Synthesis
Cdk
“Green light”
Degradation
Synthesis
Cell allowed to pass checkpoint
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
Reminders
Watch the video lectures
• Add to your notes so that you can understand
the material
• Replay/re-watch sections as necessary
• Take breaks
• Change the video speed as necessary; use
shortcut keys
Read the textbook. Check the textbook for
answers to your questions before posting on the
Discussion Board.
To understand normal cell biology and disease
we must understand the molecular machinery
that functions inside cells to control their
shapes, functions, interactions and numbers.
Lectures
1-3: How do cells and tissues organize themselves spatially?
4-6: How do multicellular organisms develop?
7-9: How do cells communicate with each other?
10-12: How are cell numbers controlled?
10. The cell cycle
11. Programmed cell death
12. Cancer
9-10: Mitosis and Cell Division & Cancer
11-12: Development of Multicellular Organisms
Programmed cell death (PCD) (Apoptosis)
PCD sculpts
fingers and
toes
PCD removes
juvenile
body parts
Programmed cell death (PCD) (Apoptosis)
PCD kills dangerous cells
Apoptosis is highly regulated and stereotyped
(Necrosis is not)
Necrosis: Accidental cell death.
e.g. with acute injury cells
can swell and burst into the
surrounding tissue
This can lead to a damaging
inflammatory reaction
Apoptosis avoids this by neatly..
-shrinking the cell
-collapsing the cytoskeleton
-fragmenting the DNA
-signaling to macrophages for
cell removal by engulfment
Necrosis
Apoptosis
Apoptosis is highly regulated and stereotyped
(Necrosis is not)
Necrosis: Accidental cell death.
e.g. with acute injury cells
can swell and burst into the
surrounding tissue
Necrosis
This can lead to a How
damaging
is apoptosis triggered?
inflammatory reaction
Apoptosis
Apoptosis avoids this by neatly..
-shrinking the cell
-collapsing the cytoskeleton
-fragmenting the DNA
-signaling to macrophages for
cell removal by engulfment
Caspases trigger apoptosis
•
Cysteine proteases that cleave target
proteins at specific aspartic acid residues
•
Synthesized as procaspase precursor
molecules
•
Procaspases are cleaved and activated by
other caspases
•
This leads to an amplified cascade of
caspase activity
Individual caspase activation
Two polypeptides
One multi-protein
complex
The caspase
amplification cascade
Detecting fragmented DNA
in a gel after induction
of apotosis
Detecting fragmented DNA
in cells by TUNEL labeling
after induction of apotosis
Terminal deoxynucleotidyl transferasemediated dUTP Nick End Labeling
Apoptotic cells also change their
cell surface properties
The phospholipid phosphatidylserine
is normally restricted to the inner leaflet
of the plasma membrane
In apoptotic cells it flips to the outer leaflet
In vivo, macrophages recognize the
exposed lipid and phagocytose the cell
In experiments, probes for phosphatidylserine can detect apoptotic cells
What activates
caspase cascades?
Caspase cascades can be activated
by extrinsic or intrinsic signals
Triggering apoptosis from outside the cell via death
receptors (e.g. Killer lymphocyte activation of Fas receptors)
Need death domain activation to activate DED
Caspase cascades can be activated
by extrinsic or intrinsic signals
Triggering apoptosis from outside the cell via death
receptors (e.g. Killer lymphocyte activation of Fas receptors)
Caspase cascades can be activated
by extrinsic or intrinsic signals
Triggering apoptosis from outside the cell via death
receptors (e.g. Killer lymphocyte activation of Fas receptors)
Caspase cascades can be activated
by extrinsic or intrinsic signals
Triggering apoptosis from within (e.g. release of electron
carrier protein cytochrome c from damaged mitochondria
after cell stress)
Caspase cascades can be activated
by extrinsic or intrinsic signals
Triggering apoptosis from within (e.g. release of electron
carrier protein cytochrome c from damaged mitochondria
after cell stress)
Caspase cascades can be activated
by extrinsic or intrinsic signals
A common mechanism:
Signals lead to the aggregation of the “top” caspase
promoting self cleavage.
Many cells make inhibitors of apoptosis
(this increases the threshold for activating the apoptotic program)
Extracellular inhibitors:
Decoy receptors act by competitive inhibition
-have a ligand-binding domain but not a death domain
-out-compete functional Fas death receptors for ligands
Why isn’t the apoptotic signal sent?
Decoy receptor
Fas death receptor
Many cells make inhibitors of apoptosis
(this increases the threshold for activating the apoptotic program)
Extracellular inhibitors:
Decoy receptors act by competitive inhibition
-have a ligand-binding domain but not a death domain
-out-compete functional Fas death receptors for ligands
Intracellular inhibitors:
Examples of competitive inhibition
e.g. mimic of an initiator caspase that lacks a
proteolytic domain
Other inhibitors simply block apoptotic machinery
Bcl2 inhibits
channel formation
in the outer
mitochondrial
membrane
Inhibitors of apoptosis (IAPs)
block caspase activity in the cytoplasm
Inhibitors of apoptosis (IAPs)
block caspase activity in the cytoplasm
Signaling can either induce or prevent apoptosis
In populations of dividing or stable cells, signals can induce apoptosis.
After cell populations fully grow,
apoptosis often becomes the default state
and signals are required to prevent apoptosis
à The default state kills off cells if they leave their protective environment
à Survival factors are used to control proper cell numbers in body tissues
Survival factors act through apoptotic inhibitors
to protect cells from apoptosis
Determining neuronal cell numbers
via competition for survival factors
secreted by target cells
Signaling between cells in a tissue
allows the tissue to regulate its size
Cell “A”
Cell “C”
protected from
apoptosis
apoptosis
Cell “B”
Cell “D”
told to
divide
Signaling between cells in a tissue
allows the tissue to regulate it’s size
Cell “A”
Cell “B”
Billions of cells die in our bone marrow and intestine each hour
The system is regulated so that cell division equals cell death
Cell “C”
protected from
apoptosis
apoptosis
Cell “D”
told to
divide
Remember to read the textbook. Check the
textbook for answers to your questions.
After reading the textbook, questions are
welcome… please ask on the Discussion
Board, and/or after classes.
Help one another on the Discussion Board.
BIO230 FINAL EXAM 2011 ANSWER KEY
1D
2A
3C
4B
5A
6B
7C
8D
9A
10B
11C
12ABCD
13A
14B
15D
16C
17A
18B
19C
20B
21A
22B
23C
24D
25A
26B
27C
28D
29D
30C
31B
32B
33A
34C
35A
36D
37A
38D
39A
40CD
41B
42C
43C
44B
45C
46A
47D
48C
49B
50B
51D
52A
53D
1. If a chemical that inhibits PI 3‐kinase were added to cells, which of the following would occur?
a.
b.
c.
d.
Specific proteins would dissociate from the cytosolic face of cell membranes.
The concentrations of PIP2 species would be affected.
Rab activities would be affected.
All of the above.
2. In a cell with a clathrin mutation, which of the following would most likely be affected?
a.
b.
c.
d.
Endocytosis.
Transport of molecules from the endoplasmic reticulum to the golgi apparatus.
Transport of molecules from the golgi apparatus to the endoplasmic reticulum.
Transport of molecules between golgi cisternae.
3. Which of the following statements concerning vesicle regulation is incorrect?
a.
b.
c.
d.
Inositol phospholipids are phosphorylated.
Rab5‐GDP is converted to Rab5‐GTP by Rab5‐GEF.
Rab5A is localized on late endosomes.
The presence of Rab5‐GTP and PI(3)P forms a coincidence detector.
4. How many of the following complexes/proteins draw two lipid bilayers into direct contact?
a. 1
b. 2
Version 11
Clathrin
Dynamin
SNARE proteins
Rab small GTPases
Page 1 of 13
c. 3
d. 4
5. An artificial membrane‐bound container is filled with purified tubulin subunits, a purfied
centrosome, purified Dynein complexes and the necessary small molecules for p…
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