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