Transcript Document

Multicellularity: From cells to tissues to organisms
ECB 16-1
shmoos
Mating dance of a budding yeast (S.cerevisiae)…
Haploid a- and a-cells form shmoos in response to chemical signals
Shmoos mate to form diploid a/a cell
Examples of:
- “differentiated” cell types (a-, a-, and a/a-cells) cell-cell adhesion
-cell-cell signaling
Human body consists of trillions of cells, 200+ specialized cell types that must
differentiate (next time) and communicate (today) with one another
Cell-cell communication required to coordinate:
- physiology and metabolism
- behavior
-growth, proliferation, and differentiation
Basic categories of cell-cell signaling in animals
“Contact-mediated” (short range)
“Paracrine” (local)
ex. - nerve cell production
ex. inflammation
Signaling
cell
Signaling
cell
Target
cell
Target
cells
“Autocrine”
“Endocrine” (long distance)
“Neuronal”
(ex.-hormones)
Endocrine
(signaling) cell
Post-synaptic
target (muscle,
neuron, etc)
Target cells
Axon
hormone
Action potential
Cell body
of neuron
Bloodstream
Synapse
ECB 16-3
Cellular response depends on specific combination of signals
ECB 16-6
No signal often results in activation of apoptosis
Common features of cell-cell signaling pathways
Other
signals
ECB 16-7
Receptors for diffusible signals can be intracellular or surface
Cell surface receptors
Intracellular receptors
Small non-polar molecules
cross plasma membrane by
simple diffusion
Large polar molecules
…cannot cross
membrane
They bind
cell surface
receptors
ECB 16-9
And bind to intracellular receptors
Intracellular signals
Plasma
membrane
Transcription
Transcription
Most receptors for hydrophobic
signaling molecules act in nucleus to
regulate gene transcription
Membrane receptors for hydrophilic
signaling molecules activate a wide
variety of intracellular “signal
transduction” pathways, including gene
regulation
A few examples of hydrophobic hormones
ECB 16-11
CH2OH
OH
C=O
HO
O
OH
OH
O
HO
Cortisol
Estradiol
Testosterone
I
HO
HO
O
I
Cholesterol
(not hormone)
I
H
CH2
I
C
COO-
NH3+
Thyroid hormone
Responses mediated by a conserved family of “steroid” receptors
Responses to hydrophobic hormones are
mediated by intracellular receptors
Plasma
membrane
Target
cell
Cytoplasm
Intracellular
receptor
Translation
Nuclear
envelope
Lipophilic hormone carried in
blood
Hormone binds intracellular
receptor inducing receptor
dimerization and activation
Complex is imported into
nucleus
Binds to “hormone response
element” to regulate gene
expression
Transcription
“Hormone
response
element”
Promoter
Target gene
Nucleus
ECB 16-12
Cell-surface receptors - three classes
Ion channel-linked receptor
Signaling
ligand
Ions
G-protein linked receptor
Target
Signaling
ligand
Target
(inactive)
Receptor
G-protein
(inactive)
Target
(inactive)
Receptor
G-protein
(active)
(active)
(active)
Receptor
G-protein
(active)
(active)
Enzyme-linked receptor
Signaling
ligand
Catalytic
domain
(active)
Catalytic
domain
(active)
ECB 16-14
Activation of surface receptor can cause fast
(cytoplasmic) or slow (transciptional) changes
Review: phosphorylation and GTPases as molecular switches
Signaling with phosphorylation
Signal in
Signaling with GTPases
Signaling
protein
Signal in
Off
Off
Pi
ATP
Signaling
GTPase
GDP
Pi
GDP
Kinase
Signal activates
protein kinase
Phosphatase
GEF
Signal activates GEF
GTP
ADP
On
On
P
Signal out
GTP
ECB 16-15
Signal out
Energy (in the form of ATP or GTP hydrolysis) used to activate (or
inactivate) signaling molecules
Energy use allows transient, high affinity/specificity interactions
GAP
“Heterotrimeric G-proteins” mediate many cell signals
b g
Ga, Gbg subunits
a
Ga binds guanine nucleotide
GDP
GDP
Gabg
(inactive GDP form)
Heterotrimeric
G-proteins
Pi
Receptor acts as GEF, activating
G-protein
Activated Ga- and Gbg regulate
targets
Ga inactivated by GTP
hydrolysis, subunits reassociate
GTP
Active Ga and Gbg
(GTP form)
b g
See ECB 16-17
+
a
GTP
Downstream targets
Multiple G-proteins with distinct
a-, b-, and g-subunits (>20 known)
“Gs” stimulates or activates
effectors
“Gi” inhibits effectors
“Gq” mediates Ca2+ signaling
“Heterotrimeric G-proteins” are activated by a
family of “Seven-pass” transmembrane receptors
G-protein
–GDP
Ligand
binding domain
(inactive)
Extracellular space
b g
1 2 3 4 5 a6 7 Plasma membrane
Inactive
receptor
GDP
Cytoplasm
Effector domain
See ECB 16-16
Seven transmembrane domains (a-helices)
Extracellular ligand-binding domain (N-terminal)
Cytoplasmic “effector” domain
Activated receptor acts as GEF to activate “heterotrimeric G-protein”
“Heterotrimeric G-proteins” are activated by a
family of “Seven-pass” transmembrane receptors
G-protein –GDP
(inactive)
b g
Seven-pass
receptor
a
GDP
Inactive
target
Binding of ligand activates receptor
ECB 16-18 thru 16-18
“Heterotrimeric G-proteins” are activated by a
family of “Seven-pass” transmembrane receptors
b g
a
Active
receptor
GTP
GTP
GDP
Binding of ligand activates receptor
Heterotrimeric G-protein binds activated receptor
Activated
target
ECB 16-18 thru 16-18
Activated receptor acts as GEF for heterotrimeric G-protein
Activated components (a- and b/g-) regulate downstream targets
GTP hydrolysis inactivates G-protein, subunits reassociate (switches off)
Activated target can be enzyme that makes
“intracellular messenger”
ECB 16-20
Ephinephrine (adrenaline) acts via heterotrimeric G
protein and cAMP (intracellular messenger)
Activated adenylate cyclase
forms cAMP
cAMP activates protein
kinase A (PKA)
PKA enters nucleus
and phosphorylates
a gene regulatory
protein
ECB 16-24
Result: altered
transcription (slow)
Adenylate cyclase converts ATP to 5’,3’ cAMP
O
A
O
O
5’
-
O P O P O P O CH2
-
O
-
O
-
O
O
4’
3’
2’
OH
OH
“Adenylate cyclase”
PPi
A
5’
CH2
O
O
P
-
O
3’
2’
O
OH
O
O
4’
Adenosine 3’,5’ cyclic
monophosphate
(cAMP)
A
5’
-
2Pi
Methylated xanthines (caffiene,
theophylline, and theobromine )
inhibit cAMP PDE
O P O CH2
O
1’
O
cAMP
phosphodiesterase
-
ATP
1’
3’
OH
1’
2’
OH
AMP
ECB 16-21
cAMP levels rise rapidly in response to
extracellular signal
Assay fluorescence of protein that binds cAMP
5 X 10
ECB 16-22
-8
M cAMP
10 -6 M cAMP
Serotonin is a neurotransmitter
G-protein coupled receptors also activate IP3 and
Ca2+-mediated signaling pathways
Activate receptor acts as GEF
Activated Ga activates
phospholipase C (PLC)
Active PLC cleaves PIP2 to IP3
and diacylglycerol (DAG)
IP3 opens Ca2+ channels in ER
releasing Ca2+ to cytoplasm
DAG and Ca2+ activate protein
kinase C (PKC)
Active PKC phosphorylates
target proteins…
Other Ca2+-dependent responses are regulated by
“Calmodulin” (CaM) and “CaM kinases”
Ca2+
CaM contains 4 Ca2+ binding
domains
Inhibitory domain
Catalytic domain
Inactive
CaM kinase
Ca2+
Ca2+-CaM binds to
regulatory domains of
effector proteins (e.g.
CaM kinases)
ADP ATP
P
Autophosphorylation
Ca2+
Calmodulin (CaM)
Active CaM
kinase
Phosphorylates target
proteins in cytoplasm
see ECB 16-27
Ca2+-calmodulin activates CaM kinases, which phosphorylate
and regulate target proteins
Cells carefully regulate “free” Ca2+ levels in their cytoplasm
Ca2+
[Ca2+] >1 mM
ATP
ADP + Pi
Ca2+
2Na+
In a resting cell, intracellular
[Ca2+]free is low relative to external
Ca2+…
Ca2+ is pumped into the ER (plant
vacuole)
[Ca2+ ]free ~0.2 mM
ATP
ADP + Pi
Ca2+
ER
Ca2+ is pumped out of the cell by a
Ca2+ ATPase and antiport with Na+
(antiport with H+ in plants/fungi)
Intracellular [Ca2+]free may increase
10-30-fold during signaling…
Moves in through channels and is
released from internal stores
(mostly from the ER, vacuole)
Last class of cell surface receptors
Signaling
ligand
1. Ligand gated ion channel
Ions
2. G-protein coupled receptor
Target
Signaling
ligand
Target
(inactive)
Receptor
G-protein
(inactive)
Receptor
G-protein
(active)
(active)
3. Enzyme-linked receptor
Signaling
ligand
Catalytic
domain
(active)
Target
(inactive)
Catalytic
domain
(active)
(active)
Receptor
G-protein
(active)
(active)
Many growth factors bind to receptor tyrosine kinases
(enzyme-linked receptor)
Receptor binds growth factor and dimerizes
Kinase activity activated and receptor autophosphorylates
Signaling proteins bind phosphotyrosine, activating signaling cascades
EGF and other growth factors activate Ras signaling
Ras found to be mutated in ~30% of human tumors!
RAS
(inactive)
GTP Exchange Factors
(GEFs) promote
GDP/GTP exchange
GDP
Pi
“Off”
GDP
GAP
GEF
GTP
“On”
“GTPase Activating
Protein” (Ras-GAPs)
promote GTP hydrolysis
by intrinsic GTPase
RAS
Active Ras activates
downstream signaling
proteins…
GTP
Downstream
effectors
Receptor tyrosine kinases activate
intracellular Ras signaling cascades
Growth
Factors
P
RAS
P
RAS
MAPKKK
(inactive)
GTP
P
P
P
P
DRK
GDP
inactive
active
MAP kinase kinase kinase
(MAPKKK)
Ras GEF
Receptor kinase
(active)
ATP
ADP
P
MAP kinase kinase
(MAPKK)
MAPKK
MAPKK
active
inactive
ATP
ADP
Downstream of Receptor Kinase
activates Ras GEF
P
“Mitogen-activ. protein kinase”
MAP kinase
P
MAP kinase
inactive
active
ATP
ADP
P
ECB 16-31, 16-32
Transcription
factors
P
Other
proteins
Regulate gene expression
and protein activity
Mutations in Ras signaling pathway cause
uncontrolled cell proliferation: cancer
P
RAS
P
RAS
MAPKKK
(inactive)
GDP
P
P
P
P
DRK
active
GTP
ATP
ADP
Ras GEF
P
GTP
MAPKK
active
Receptor kinase
(active)
ATP
ADP
Downstream of Receptor Kinase
The Ras pathway activates
expression of G1 cyclins that
stimulate cell proliferation
P
P
MAP kinase
active
ATP
Constituitive activation of pathway
components results in uncontrolled cell
proliferation = “cancer”
Cancer causing genes = “Oncogenes”
Predict effects of Ras mutations?
ADP
P
Transcription
factors
P
Other
proteins
Regulate gene expression
and protein activity…
Signal transduction cascades are complex and interconnected
G-protein coupled receptors
G-protein
G-protein
P
P
P
P
P
P
Phospholipase C
IP3
Adenylate cyclase
Diacylglycerol
Receptor
tyrosine
kinases
• Integration
Adapter
Ras activator
Ras
Ca2+
cAMP
Calmodulin
Protein kinase A
CaM kinase
Gene regulatory proteins
Protein kinase C
Why?
Multiple inputs to a
single response…
• Divergence
Single input to
multiple responses
Kinase I
• Amplification
Kinase II
• Regulation
Kinase III
Cytoplasmic target proteins
ECB 16-38
Communication by direct cytoplasmic continuity
between cells
Cytoplasmic bridges and cell junctions
Communication via cell junctions: some embryonic
cells and/or tissues are “dye-coupled”
100 Da
1,000 Da
10,000 Da
Membrane-impermeant dye injected into on cell passes into neighbors
Cytoplasmic coupling is limited to small molecules (<1000 Da)
“Gap junctions” are responsible for cytoplasmic coupling of
animal cells
Membranes of coupled cells
closely apposed, separated
by 2-4 nm “gap”
Large
“gap jnctn”
TEM/Freeze fracture of gap
junctions reveals “plaques” of
intra-membrane particles
ECB figure 19-28
MBoC figure 19-16
Common in developing embryo, cardiac muscle, liver, and lens
Gap junctions are composed of “connexons”
made of “connexin” hexamers
Cytoplasm of cell #1
Channel is ~ 1.5
nm (~1000 Da
cutoff)
Plasma membrane
of cell #1
“Connexon”
(2 per channel)
= “connexin” x 6
Extracellular “gap”
(2-4 nm)
Plasma membrane
of cell #2
Cytoplasm of cell #2
Two connexons in register form channel
coupling cytoplasm of adjacent cells
ECB 21-28
The cytoplasm of plant cells is coupled by “plasmadesmata”
Cytoplasm
Desmotubule
Cell wall
Cytoplasm
Vacuole
Nucleus
Plasma membrane
of adjacent cells
Cell wall
Nucleus
Cytoplasm
Nucleus
Plasmadesmata
Endoplasmic
reticulum
100 nm
ECB 21-30
Membranes continuous from cell to cell
ER continuous from cell to cell thru “desmotubule”
Limited to small molecules (<800 Da), but can open to let through 20,000 Da
Primarily (but not exclusively) formed during cell division