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Imaging signal transduction in single dendritic spines during synaptic plasticity. Ryohei Yasuda (HHMI, Duke). Spine. Spine: Biochemical compartment. Small ~0.1 fL. Narrow neck (~100nm Φ ) : Diffusional barrier Ca 2+ signaling in spines Synaptic plasticity Memory. - PowerPoint PPT Presentation
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Imaging signal transduction in single dendritic spines during
synaptic plasticity
Ryohei Yasuda
(HHMI, Duke)
Spine
Transmitter Receptors Calcium Channels
Dendrite
AxonTransmitter Vesicles
Post SynapticDensity (PSD)
Ca2+
Signaling
~ 0.5 µm
Spine
Spine: Biochemical compartment
• Small ~0.1 fL.• Narrow neck (~100nmΦ) : Diffusional barrier• Ca2+ signaling in spines Synaptic plasticity Memory
Signaling networks
R. Iyenger
80S1
Ran-GAP
Rap-GAP
Raf-1
Microtubule
a-actinin
L1
Axina-catenin
SoSFAK
CRK
CAS Grb2
SMC
Talin Vinculin
b-catenin
ILK
Cofilin
integrin
ankyrin
N cadherin
eEF2K
Grb2SoS
RTK
aq PLC
Ca++
cAMP
Ras
AC5,6AA
GPCR(ai)CRAC(Ca++) GPCR(aq
)
bgai/o
cSrc
DSH
E1
E2
PHAS
E
2BIR
GPCR(as) AC2 AC1/8
Ca++ Ch
NMDARglutamate
IRS-1
PI3K 1,4,5PIP3
PKB/AKT
PDK
S6K
MTOR
MAPKAP
Rho
MEKK
MEK4 MEK6
MLK3
p38
DAG
PKC
IP3
IP3R
RhoK
LIMK
MNK
PAK
IQGAP
MAPK1,2
MEK1,2
Raccdc42
syntaxinSNAP25
RM
Synaptotagmin
Rabphilin
SynaptobrevinCa++
Synaptophysin
Rab3
Ca++
Ca++
CaM
CaMKK
CaMK2
I1 PP1PKA
PDE
mRNA
CITRON
PSD95 cSrc
Na+Ch
Ank-4bg
as
B-Raf
EPACRap1
Ras-GAP
80S2
48S
43S
40s60S
60S
Ran
Plectin
cd2
FOS Elk
SHP2 SIRP
FzWnt
YOTIAO
Neurofacin
RSK
jun
AMPAR
GPCR(aq)
mGLU-R
aq
HOMER
cPLA2
SRE
CAMK4 ZIF268ATF2
TAL PAX
FYM
profilin
a-actinin
PIP2
Ca++
Actin
Gelsolin
CREM
CBP
Ran-GEF
GSK-3 MKP
JNK/SAPK
myosin
SHC
MARCK
SHP2
NSKMUNC18
AK
AP-7
9
Can
S6
S6
WAVEJIP
GRIP
STAT3
Jak
CK2
Abl
CATCool/PIX
GAP
MP1
LamininA ,B
MyosinPPase
PP2A
STAT3
BAD
MEK5
BMK
CREBAP1 MEF2
NFAT
NFATTcf/Lef
b-cateninmSin3
CoRestNRSF
H202
NO
cGMP
IF's
80S1
Ran-GAP
Rap-GAP
Raf-1
Microtubule
a-actinin
L1
Axina-catenin
SoSFAK
CRK
CAS Grb2
SMC
Talin Vinculin
b-catenin
ILK
Cofilin
integrin
ankyrin
N cadherin
eEF2K
Grb2SoS
RTK
aq PLC
Ca++
cAMP
Ras
80S1
Ran-GAP
Rap-GAP
Raf-1
Microtubule
a-actinin
L1
Axina-catenin
SoSFAK
CRK
CAS Grb2
SMC
Talin Vinculin
b-catenin
ILK
Cofilin
integrin
ankyrin
N cadherin
eEF2K
Grb2SoS
RTK
aq PLC
Ca++
cAMP
Ras
AC5,6AA
GPCR(ai)CRAC(Ca++) GPCR(aq
)
bgai/o
cSrc
DSH
E1
E2
PHAS
E
2BIR
GPCR(as) AC2 AC1/8
Ca++ Ch
NMDARglutamate
IRS-1
PI3K 1,4,5PIP3
AC5,6AA
GPCR(ai)CRAC(Ca++) GPCR(aq
)
bgai/o
cSrc
DSH
E1
E2
PHAS
E
2BIR
GPCR(as) AC2 AC1/8
Ca++ Ch
NMDARglutamate
IRS-1
PI3K 1,4,5PIP3
PKB/AKT
PDK
S6K
MTOR
MAPKAP
Rho
MEKK
MEK4 MEK6
MLK3
p38
DAG
PKC
IP3
IP3R
RhoK
LIMK
MNK
PAK
IQGAP
MAPK1,2
MEK1,2
Raccdc42
syntaxinSNAP25
RM
Synaptotagmin
Rabphilin
SynaptobrevinCa++
Synaptophysin
Rab3
Ca++
Ca++
CaM
CaMKK
CaMK2
I1 PP1PKA
PDE
mRNA
CITRON
PSD95 cSrc
Na+Ch
PKB/AKT
PDK
S6K
MTOR
MAPKAP
Rho
MEKK
MEK4 MEK6
MLK3
p38
DAG
PKC
IP3
IP3R
RhoK
LIMK
MNK
PAK
IQGAP
MAPK1,2
MEK1,2
Raccdc42
syntaxinSNAP25
RM
Synaptotagmin
Rabphilin
SynaptobrevinCa++
Synaptophysin
Rab3
Ca++
Ca++
CaM
CaMKK
CaMK2
I1 PP1PKA
PDE
mRNA
CITRON
PSD95 cSrc
Na+Ch
Ank-4bg
as
B-Raf
EPACRap1
Ras-GAP
80S2
48S
43S
40s60S
60S
Ran
Plectin
cd2
FOS Elk
SHP2 SIRP
FzWnt
YOTIAO
Neurofacin
RSK
jun
AMPAR
GPCR(aq)
mGLU-R
aq
HOMER
cPLA2
SRE
CAMK4 ZIF268
Ank-4bg
as
B-Raf
EPACRap1
Ras-GAP
80S2
48S
43S
40s60S
60S
Ran
Plectin
cd2
FOS Elk
SHP2 SIRP
FzWnt
YOTIAO
Neurofacin
RSK
jun
AMPAR
GPCR(aq)
mGLU-R
aq
HOMER
cPLA2
SRE
CAMK4 ZIF268ATF2
TAL PAX
FYM
profilin
a-actinin
PIP2
Ca++
Actin
Gelsolin
ATF2
TAL PAX
FYM
profilin
a-actinin
PIP2
Ca++
Actin
Gelsolin
CREM
CBP
Ran-GEF
GSK-3 MKP
JNK/SAPK
myosin
SHC
CREM
CBP
Ran-GEF
GSK-3 MKP
JNK/SAPK
myosin
SHC
MARCK
SHP2
NSKMUNC18
AK
AP-7
9
Can
S6
S6
WAVEJIP
GRIP
STAT3
Jak
CK2
Abl
CATCool/PIX
GAP
MP1
LamininA ,B
MyosinPPase
PP2A
STAT3
BAD
MEK5
BMK
CREBAP1 MEF2
NFAT
NFAT
MARCK
SHP2
NSKMUNC18
AK
AP-7
9
Can
S6
S6
WAVEJIP
GRIP
STAT3
Jak
CK2
Abl
CATCool/PIX
GAP
MP1
LamininA ,B
MyosinPPase
PP2A
STAT3
BAD
MEK5
BMK
CREBAP1 MEF2
NFAT
NFATTcf/Lef
b-
Imaging signaling in single spines
• Measure FRET with 2-photon fluorescence lifetime imaging (2-photon FLIM)
• Develop and use FRET sensors optimized for 2-photon FLIM
• Image signal transduction, while inducing plasticity in single spine with 2-photon glutamate uncaging
FRET and fluorescence lifetime
GFP RFP
Excitation Emission
Donor Acceptor
FRET
KFRET
• FRET decreases fluorescence lifetime.
• Use donor fluorescence only.– Independent of fluorophore
concentrations.– Independent of wavelength-dependent
light scattering• Multiple populations can be
deconvolved.• Easy to combine with 2-photon
microscopy2 4 6 8 10 12
Log
(flu
ores
cenc
e)
Time (ns)
Laser pulse
FRET
Mixture
Donor
2-photon fluorescence lifetime imaging microscopy
• High resolution and sensitivity in deep tissue.• Quantitative measurements of FRET
Pixel clock
Stop
Start
PMT
Scanmirrors
TiSa Laser200 fs, 76 MHz
Photodiode
Photon
Laser pulse
PC
Mirror control
Timer
Memory
Stimulate single spines using 2-photon glutamate uncaging
Synapse-specific Ca2+ elevation
4 ms, ~5 mW720nm Laser
Spontaneous
Ca2+
Glutamate
Caged-glutamate
20 ms10 pA
Uncaging
Matsuzaki, Ellis-Davies, Kasai
2p-uncaging to produce long lasting synaptic potentiation and spine growth
30-60 times, 0.5-1 Hz in Zero extracellular Mg2+
Matsuzaki 2001, 2004
Imaging activity of
CaMKIICa2+/Calmodulin-dependent kinase II
Ca2+/Calmodulin-dependent kinase II: biochemical memory?
Kinase domain
Low Ca2+
Ca2+Activation
Memory?
Ca2+/CaM
CaM
CaM
PCa2+
P
InactiveGFP
FRETd-YFP
Takao et al, 2004Lisman 2002
Autoinhibitory domain
Kinase domain
Fluorescence lifetime change in lysates
Time (min)
0
0.1
0.2
0.3
0.4
0.5
-5 0 5 10 15
CaM+, ATP+
CaM+, ATP+ (T286A)
CaM+, ATP–CaM–, ATP+
CaM–, ATP–
Flu
ores
cenc
e lif
etim
e ch
ange
(ns
)
Ca2+ EGTA
Lee et al., Nature 2009
CaMKII activation during structural plasticity of spines
Lee et al., Nature 2009
Transient and spine-specific activation of CaMKII
Lee et al., Nature 2009
1.6
Glutamate uncaging (0.5 Hz, 45 stim)
4sec 12sec 20sec-4sec-12sec
2 μm
28sec 36sec 44sec
68sec 74sec 84sec60sec52sec 92sec 100sec 108sec
2.0Lifetime (ns)
Transient and spine-specific activation of CaMKII
-5 0 5 10 15 20 25 30 35
?0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
Ca
MK
II A
ctiv
ity C
ha
ng
e
Stimulated SpineAdjacent SpineDendrite
Uncaging
35Time (min)
-5 0 5 10 15 20 25 30
0
100
200
300
400
Vo
lum
e c
ha
ng
e (
%)
2 min
CaMKII
Uncaging
Volume
Lee et al., Nature 2009
Ca2+/Calmodulin-dependent kinase II: biochemical memory?
Kinase domain
Low Ca2+
Ca2+Activation
Memory?
Ca2+/CaM
CaM
CaM
PCa2+
P
Inactive
Not for 1 hour
What is the role of phosphorylation?
T286A
Wild-type
Fast fluorescence lifetime imaging
Lee et al., Unpublished
-2 0 2 4 6 8 10 12 14-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0.7 s
5.8 s
Time (s)
Flu
ore
sce
nce
life
time
(ns) 1 μM
Δ[Ca2+]
~0.1 s
Glutamate uncaging (4 ms)
Ca2+/Calmodulin-dependent kinase II: biochemical memory?
Kinase domain
Low Ca2+
Ca2+Activation
Memory?
Ca2+/CaM
CaM
CaM
PCa2+
J. Lisman
P
Inactive
Yes, but only for 6 s
~ 6 s
Ca2+ CaMKIILong-termplasticity
0.1 s 10 s 10 min 1 hour1 min
Previous view of LTP
Ca2+ CaMKIILong-termplasticity
0.1 s 10 s 10 min 1 hour1 min
Now ….
Imaging the activity of
Ras superfamily proteins
Small GTPase signaling
GDP GTP
Membrane
Effector
GEF
GAP
Signaling
Inactive Active
•Several major subgroups: Ras, Rho, Rab, Rap, Arf, Ran etc…•Acts as signaling switch.•Regulate organization of actin cytoskeleton, membrane trafficking etc.•Important for morphogenesis of dendritic spines and plasticity•Mutations in the pathway are associated with mental retardation
Imaging binding between Ras and Ras binding domain (RBD) of Raf1
Yasuda et al., Nat.Neurosci. 2006Harvey et al., Science 2008
51-131 a.a. R59A
mGFP HRAS
RAF1mRFP mRFP
CaMKII
Ras
Ca2+ CaMKIILong-termplasticity
0.1 s 10 s 10 min 1 hour1 min
Ras ERK
AMPAR exocytosis
General approach to make sensors for Ras superfamily
Cdc42 / RhoA: Important for regulation of actin cytoskeleton and dendritic spine morphology.
65-118 a.a.S79A, F89A
mGFP Cdc42
Pak3mRFP mRFP8-89 a.a..
WT
mGFP RhoA
RTKNmRFP mRFP
Cdc42 sensor RhoA sensor
Making small GTPase sensors
1. Screen RXX Binding Domain in cuvetteKd ~ 1 – 5 uM for GTP form
(RBD inhibits Rho inactivation)
Kd > 50 uM for GDP form for low background
2. Test sensitivity & specificity in cell line
3. Test sensitivity & kinetics in neurons
Step1: Screen RBD and mutants
0 2 4 6 8 100.01
0.1
1
No
rma
lize
d n
um
be
r o
f ph
oto
ns
Time (ns)
GDPGMPPNP
0
5
10
15
20
25
Bin
din
gfr
act
ion
(%)
[PAK2] (µM)
GMPPNP: KD
= 0.47 µM
GDP: KD
= 63.6 µM
2 4 6 8 100
A
FRET between GFP-CDC42 and PAK2-mCherry
GFP-Ras Cdc42 Cdc42 Rac1 Rac1 RhoA RhoA
mCherry-RBD GDP GMPPNP GDP GMPPNP GDP GMPPNP
PAK1 6.9 0.1 17 0.4
PAK1 (F89A) 74 0.6 78 4.6
PAK1 (F89A, F96A) 91 3.4 > 300 41
PAK2 10 0.2 22 0.5
PAK3 57 0.3 69 1.8
PAK3 (F89A) 139 1.0 143 12.5
PAK3 (S79A, F89A) 150 1.8 77 22
WASP 43 2.9
RTKN 47 3.9
Step 2: Test sensitivity & specificity in HeLa cells
0
10
20
30
40
50
Bin
din
g f
ract
ion
(%)
Cdc42 T17N Q61L WT WTWTPBD3m + + + ++
- - Dbl p50RhoGAP
-GEF or GAP
RhoA T17N Q61L WT WTWTRTKN + + + ++
- - Dbl p50RhoGAP
-GEF or GAP
Bin
din
g f
ract
ion
(%)
Cdc42(T17N) Cdc42(Q61L) Cdc42(wt)Cdc42(wt) +Dbl
Cdc42(wt)+p50RhoGAP
50 mm
2.0
2.7
ns
1.9
2.6
RhoA(T19N) RhoA(Q63L) RhoA(wt)RhoA(wt) +Dbl
RhoA(wt)+p50RhoGAP
ns
b
c d
a
0
10
20
30
40
Step 3: Test sensitivity & reversibility in neurons
-10 0 10 20 30 40
0
10
20
Cdc42 n=18RhoA n=27
Bin
din
g f
ract
ion
cha
ng
e (%
)
Time (min)
15 mM NMDA for 2 min
Before 2 min 4 min 10 minBefore 2 min 4 min 10 min
5 mm
RhoA Cdc42 15 mM for 2 min15 mM for 2 min
0 100 200 3000
2
4
6
8
[mCherry-RBD-mCherry] (mM)
Cdc42RhoA
5 mm2.6 1.9ns 2.6 1.9ns
Cdc42RhoA
0
2
4
6
8
De
cay
time
con
stan
t(m
in)
De
cay
time
con
stan
t(m
in)
[mEGFP-Rho GTPase] (mM)0 21 3 4 5
Cdc42/RhoA activation during LTP
Cdc42 activation is compartmentalized and sustained
Hideji Murakoshi
0 10 20 30
0
5
10
Time (min)
Cd
c42
act
iva
tion
(%
)
40
Uncaging
200
400
0Vol
ume
chan
ge (
%)
0 10 20 30Time (min)
40
Uncaging
Stimulated spine
Adjacent spine
0 10 20 30 40
0.00
0.05
0.10
0.15
Time (min)0 10 20 30 40
0
100
200
300
400
500
%V
olum
ech
ange
Rho
A a
ctiv
atio
n
Time (min)
Stim. spineAdj. spine
RhoA activation spreads and sustained
Hideji Murakoshi
Stimulated spine
Adjacent spine
AP5Rho
A a
ctiv
atio
n (%
)
0 10 20 30
Time (min)
0
5
10
Uncaging
Cdc42
0
5
10
Bin
din
g fr
actio
n c
han
ge (
%) Cdc42
8-56 s 1-5 min 5-10 min 10-20 min
0 5 10Distance (µm)
0 5 10 0 5 10 0 5 10
Before
24 s
2.15 ns 2.65 5 µm
Spine
Dendrite
Spatial profile of Cdc42
Spatial spreading of RhoARhoA
24 s
Before
2.0 ns 2.60 5 10
Distance (µm)
RhoA
0
5
10
15
0 5 10 0 55 µm
10 0 5 10
8-56 s 1-5 min 5-10 min 10-20 min
Bin
din
g fr
actio
n c
han
ge (
%)
H-Ras
Distance (µm)
0
5
10
15
Bin
din
g fr
actio
n c
han
ge (
%)
20
0 10 20
2 min
0 10 20
6 min
5 15 155
2 min
H-Ras
5 µm
2.2 ns 2.9
Spine
Dendrite
Effects of overexpression on length constant
Len
gth
con
sta
nt (
µm
)
Len
gth
con
sta
nt (
µm
)
0 50 100
0
2
4
6
8
[mCherry-RBD-mCherry] (µM)0 2 4 6 8
0
2
4
6
8
[mEGFP-RhoA] (µM)
RhoA RhoA n = 20/18r = 0.05
n = 20/18r = 0.16
Diffusion coupling at the spine neckC
aMK
II
Tim
e co
nsta
nt (
s)
0 20 40
150
100
50
0
Time (s)
Gre
en in
tens
ity (
A.U
.)
8.192 s
2.048 s
0.512 s
-0.512 s
1μm
103
102
101
100
10-1
Cam
ui
Cdc42
H-R
as
MA
RC
KS
paGF
P
Rac1
Rho
A
Lee, Harvey, Murakoshi
Ras proteins: ~5 sCaMKII: ~60 s
PA-GFP tagged Ras
*Constitutively active mutants
* * **
Ca2+ CaMKIILong-termplasticity
0.1 s 10 s 10 min 1 hour
Cdc42
1 min
RhoA
RhoA activation is CaMKII dependent
(NMDA receptor inhibitor)(CaMKII inhibitor)
Late phase is CaMKII dependent
Partial inhibition at early phase
0 10 20 30Time (min)
0
5
10
Rho
A a
ctiv
atio
n (%
)Ctrl (stim)KN62AP5
Uncaging
Hideji Murakoshi
Control
Hideji Murakoshi
AP5 (NMDA receptor inhibitor)KN62 (CaMKII inhibitor)
0 20 40
0
5
10
Cdc
42 a
ctiv
atio
n (%
)
Time (min)
Uncaging
Cdc42 activation is CaMKII dependent
Ca2+ CaMKII Long-termplasticity
0.1 s 10 s 10 min 1 hour
Cdc42
1 min
RhoA
Cdc42 is required for long-term structural maintenance
0 10 20 30
0
100
200
300
400
Time (min) Time (min)
Vo
lum
e C
ha
ng
e (
%)
sh-Cdc42
Ctrlsh-Cdc42
0 10 20 30
0
100
200
300
400
Rescue
CtrlmEGFP-Cdc42 + sh-Cdc42
Uncaging
Hideji MurakoshiCdc42 Sustained growth
Cdc42 is required for long-term structural maintenance
Hideji MurakoshiCdc42 Sustained growth
0
0
20 40
100
200
300
400ControlWASP
Vol
ume
chan
ge (
%)
Time (min)
Cdc42 binding domain(24 hours)
RhoA is required for transient phase
0 10 20 30
0
100
200
300
400
sh-RhoA and B
Ctrl
sh-Rho
0 10 20 30
0
100
200
300
400
Rescue
Ctrl
sh-Rho + mEGFP-RhoA
Vo
lum
e C
ha
ng
e (
%) Uncaging Uncaging
Time (min)
Hideji MurakoshiRhoA Transient growth
Stronger inhibition of RhoA inhibits both transient and sustained phases
0 10 20 30
0
100
200
300
400
C3 transferase(Rho inhibitor)
CtrlV
olum
e ch
ange
(%
)
Time (min)
RhoA Transient/Sustained growth
RhoA/Cdc42 does not alter Ca2+-CaMKII
Cdc42RhoA
CaMKII
Ca2+CaMKII activation
Structural plasticity
ControlC3: Rho inhibitorWASP: Cdc42 inhibitor
-5 0 5 10
0
0.05
0.1
0.15
Time (min)
Life
time
chan
ge (
ns)
0 1 2
0
0.05
0.1
0.15
-5 0 5 100
100
200
300
400
Time (min)
Vol
ume
chan
ge (
%)
0 1 20
100
200
300
400
Time (min)
Time (min)
Spinegrowth
Regulation of spine volume by Rho GTPases
Cdc42/RhoA
Spine growth
CaMKII
Hideji Murakoshi
Cdc42Volume
CaMKII
2 min
Cdc42Volume
CaMKII
10 min
Uncaging
RhoA
RhoA
Ca2+ CaMKII Long-termplasticity
0.1 s 10 s 10 min 1 hour
Cdc42
Actin
1 min
RhoA
ActinTransientplasticity
Downstream of RhoA/Cdc42
0 20 40
0
100
200
300
400
Time (min)
Vo
lum
e C
ha
ng
e (
%)
IPA3(Pak inhibitor)
0 20 40
Glycyl-H1152(ROCK inhibitor)
0
100
200
300
Vol
ume
Cha
nge
(%)
0
50
100
150
Vol
ume
Cha
nge
(%)
*
ControlIPA3
ControlG-H1152
0
100
200
300
400
Vo
lum
e C
ha
ng
e (
%)
Time (min)
* *
Con
trolIP
A3
Con
trolG
-H1152
Con
trolIP
A3
Con
trolG
-H1152
Sustained phaseTransient phase
Uncaging
NMDAR
Ca2+
CaMKII
Cdc42RhoA
Transientvol. change
Sustainedvol. change
ROCK Pak
Ca2+ CaMKII Long-termplasticity
0.1 s 10 s 10 min 1 hour
Cdc42 PAK
Actin
1 min
RhoA ROCK
ActinTransientplasticity
Ras ERK
AMPAR exocytosis
There are 100 more small GTPase proteins…
Rac1 sensor
-20
0
20
40
60
80
100
No
rma
lize
d R
ac1
act
iva
tion
(%
)
0 20 3010Time (min)
40
PAK1PAK1
F89A
Rac1GTP
Pulldown
0 0 2 6 104 20 30 40Time after EGF stimulation (min)
8 15
Imaging
A B
C
0 20 3010Time (min)
40
Pull-down assayD1.9 2.7
Lifetime (ns)
-2min 8min 42min
Swess-3T3 cell
Total Rac1Whole lysate
mGFP Rac1
PAK1mRFP mRFP
Rac1activityBefore Stimulation
30 s
High
Low
RhoA/Cdc42/Rac1 activation time courses
0 10 20 30 40
0.00
0.05
0.10
0.15
Time (min)
Rho
act
ivat
ion
0 10 20 30 400
100
200
300
400
500
%V
olum
ech
ange
Time (min)
Stim. spineAdj. spine
0 100
100
200
300
400
500
%V
olum
ech
ange
Time (min)
0 10
0.00
0.05
0.10
0.15
Time (min)
Rho
act
ivat
ion
RhoACdc42Rac1
RhoACdc42Rac1
Rap1 sensor
0 60
Binding fraction (%)
-2 min 6 min
ControlForskolin
IBMX
0
20
40
WT G12V S17N
ControlForskolin
IBMX
10 um
Bin
ding
fra
ctio
n (%
)
mGFP Rap1A
RalGDSmRFP mRFP
Neuroblastoma cell
Signaling networks
R. Iyenger
80S1
Ran-GAP
Rap-GAP
Raf-1
Microtubule
a-actinin
L1
Axina-catenin
SoSFAK
CRK
CAS Grb2
SMC
Talin Vinculin
b-catenin
ILK
Cofilin
integrin
ankyrin
N cadherin
eEF2K
Grb2SoS
RTK
aq PLC
Ca++
cAMP
Ras
AC5,6AA
GPCR(ai)CRAC(Ca++) GPCR(aq
)
bgai/o
cSrc
DSH
E1
E2
PHAS
E
2BIR
GPCR(as) AC2 AC1/8
Ca++ Ch
NMDARglutamate
IRS-1
PI3K 1,4,5PIP3
PKB/AKT
PDK
S6K
MTOR
MAPKAP
Rho
MEKK
MEK4 MEK6
MLK3
p38
DAG
PKC
IP3
IP3R
RhoK
LIMK
MNK
PAK
IQGAP
MAPK1,2
MEK1,2
Raccdc42
syntaxinSNAP25
RM
Synaptotagmin
Rabphilin
SynaptobrevinCa++
Synaptophysin
Rab3
Ca++
Ca++
CaM
CaMKK
CaMK2
I1 PP1PKA
PDE
mRNA
CITRON
PSD95 cSrc
Na+Ch
Ank-4bg
as
B-Raf
EPACRap1
Ras-GAP
80S2
48S
43S
40s60S
60S
Ran
Plectin
cd2
FOS Elk
SHP2 SIRP
FzWnt
YOTIAO
Neurofacin
RSK
jun
AMPAR
GPCR(aq)
mGLU-R
aq
HOMER
cPLA2
SRE
CAMK4 ZIF268ATF2
TAL PAX
FYM
profilin
a-actinin
PIP2
Ca++
Actin
Gelsolin
CREM
CBP
Ran-GEF
GSK-3 MKP
JNK/SAPK
myosin
SHC
MARCK
SHP2
NSKMUNC18
AK
AP-7
9
Can
S6
S6
WAVEJIP
GRIP
STAT3
Jak
CK2
Abl
CATCool/PIX
GAP
MP1
LamininA ,B
MyosinPPase
PP2A
STAT3
BAD
MEK5
BMK
CREBAP1 MEF2
NFAT
NFATTcf/Lef
b-cateninmSin3
CoRestNRSF
H202
NO
cGMP
IF's
80S1
Ran-GAP
Rap-GAP
Raf-1
Microtubule
a-actinin
L1
Axina-catenin
SoSFAK
CRK
CAS Grb2
SMC
Talin Vinculin
b-catenin
ILK
Cofilin
integrin
ankyrin
N cadherin
eEF2K
Grb2SoS
RTK
aq PLC
Ca++
cAMP
Ras
80S1
Ran-GAP
Rap-GAP
Raf-1
Microtubule
a-actinin
L1
Axina-catenin
SoSFAK
CRK
CAS Grb2
SMC
Talin Vinculin
b-catenin
ILK
Cofilin
integrin
ankyrin
N cadherin
eEF2K
Grb2SoS
RTK
aq PLC
Ca++
cAMP
Ras
AC5,6AA
GPCR(ai)CRAC(Ca++) GPCR(aq
)
bgai/o
cSrc
DSH
E1
E2
PHAS
E
2BIR
GPCR(as) AC2 AC1/8
Ca++ Ch
NMDARglutamate
IRS-1
PI3K 1,4,5PIP3
AC5,6AA
GPCR(ai)CRAC(Ca++) GPCR(aq
)
bgai/o
cSrc
DSH
E1
E2
PHAS
E
2BIR
GPCR(as) AC2 AC1/8
Ca++ Ch
NMDARglutamate
IRS-1
PI3K 1,4,5PIP3
PKB/AKT
PDK
S6K
MTOR
MAPKAP
Rho
MEKK
MEK4 MEK6
MLK3
p38
DAG
PKC
IP3
IP3R
RhoK
LIMK
MNK
PAK
IQGAP
MAPK1,2
MEK1,2
Raccdc42
syntaxinSNAP25
RM
Synaptotagmin
Rabphilin
SynaptobrevinCa++
Synaptophysin
Rab3
Ca++
Ca++
CaM
CaMKK
CaMK2
I1 PP1PKA
PDE
mRNA
CITRON
PSD95 cSrc
Na+Ch
PKB/AKT
PDK
S6K
MTOR
MAPKAP
Rho
MEKK
MEK4 MEK6
MLK3
p38
DAG
PKC
IP3
IP3R
RhoK
LIMK
MNK
PAK
IQGAP
MAPK1,2
MEK1,2
Raccdc42
syntaxinSNAP25
RM
Synaptotagmin
Rabphilin
SynaptobrevinCa++
Synaptophysin
Rab3
Ca++
Ca++
CaM
CaMKK
CaMK2
I1 PP1PKA
PDE
mRNA
CITRON
PSD95 cSrc
Na+Ch
Ank-4bg
as
B-Raf
EPACRap1
Ras-GAP
80S2
48S
43S
40s60S
60S
Ran
Plectin
cd2
FOS Elk
SHP2 SIRP
FzWnt
YOTIAO
Neurofacin
RSK
jun
AMPAR
GPCR(aq)
mGLU-R
aq
HOMER
cPLA2
SRE
CAMK4 ZIF268
Ank-4bg
as
B-Raf
EPACRap1
Ras-GAP
80S2
48S
43S
40s60S
60S
Ran
Plectin
cd2
FOS Elk
SHP2 SIRP
FzWnt
YOTIAO
Neurofacin
RSK
jun
AMPAR
GPCR(aq)
mGLU-R
aq
HOMER
cPLA2
SRE
CAMK4 ZIF268ATF2
TAL PAX
FYM
profilin
a-actinin
PIP2
Ca++
Actin
Gelsolin
ATF2
TAL PAX
FYM
profilin
a-actinin
PIP2
Ca++
Actin
Gelsolin
CREM
CBP
Ran-GEF
GSK-3 MKP
JNK/SAPK
myosin
SHC
CREM
CBP
Ran-GEF
GSK-3 MKP
JNK/SAPK
myosin
SHC
MARCK
SHP2
NSKMUNC18
AK
AP-7
9
Can
S6
S6
WAVEJIP
GRIP
STAT3
Jak
CK2
Abl
CATCool/PIX
GAP
MP1
LamininA ,B
MyosinPPase
PP2A
STAT3
BAD
MEK5
BMK
CREBAP1 MEF2
NFAT
NFAT
MARCK
SHP2
NSKMUNC18
AK
AP-7
9
Can
S6
S6
WAVEJIP
GRIP
STAT3
Jak
CK2
Abl
CATCool/PIX
GAP
MP1
LamininA ,B
MyosinPPase
PP2A
STAT3
BAD
MEK5
BMK
CREBAP1 MEF2
NFAT
NFATTcf/Lef
b-
Ca2+
Hong WangCaMKII Seok-Jin LeeRas/ERK/Rap Ana Oliveira Erzsebet Szatmari
Shenyu ZhaiRho Hideji Murakoshi
Nathan HedrickAMPAR Michael PattersonTechnical assistance Airong Wan
David KloetzerFunding•NIH/NIMH•NIH/NINDS•NIH/NIDA•NSF•HHMI•Alzheimer’s Association
Brandeis Univ.J. Lisman
HHMI/Janelia FarmChristopher HarveyKarel Svoboda
Yasuda labYasuda lab
Duke Sridhar Raghavachari Michael Ehlers Scott Soderling
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