0

200

400

600

800

470 550530510490wavelength (nm)

norm

aliz

ed fl

uore

scen

ce in

tens

ity (A

DU

)

B

FED

A C

emis

sion

ratio

(525

nm/4

90nm

)

wt Q63L T19N0.8

1.0

1.4

1.2

1.6IHG

GDPPAK

462nm492nm

462nm

528nm

venus mTFPRac1

venu

sm

TFP

GTP

GEF GAP

GDP

mTFPvenusRhoA PKN

GTP

462nm492nm

462nm

528nm

venu

sm

TFP

GEF GAP

GDP

mTFPvenuscdc42 PAK

GTP

462nm492nm

462nm

528nm

venu

sm

TFP

GEF GAP

470

RhoA-wtRhoA-Q63LRhoA-T19N

Cdc42-wtCdc42-G12VCdc42-T17N

norm

aliz

ed fl

uore

scen

ce in

tens

ity (A

DU

)

5505305104900

200

400

600

800

wavelength (nm)

emis

sion

ratio

(525

nm/4

90nm

)

0.8

1.0

1.4

1.2

1.6

wt G12V T17N0

200

400

600

800

470 550530510490wavelength (nm)

norm

aliz

ed fl

uore

scen

ce in

tens

ity (A

DU

)

emis

sion

ratio

(525

nm/4

90nm

)wt G12V T17N

0.8

1.0

1.4

1.2

1.6

Rac1-wtRac1-G12VRac1-T17N

Rac

1C

dc42

Rho

A

A B

E F

I J

highlowhighlow

C

G

K

P.V.

1.1

1.0

0.9

0.8

0.7

1.2 P < 0.0001

Rac

1 ac

tivity

(FR

ET/T

FP)

I.P.

Cdc

42 a

ctiv

ity (F

RET

/TFP

)

P.V. I.P.

1.1

1.0

0.9

0.8

0.7

1.2P < 0.0001

Rho

A ac

tivity

(FR

ET/T

FP)

P < 0.005

P.V. I.P.

1.1

1.0

0.9

0.8

0.7

1.2

D

H

L

perivascular migrationintraparenchymal migration

Cdc

42

0.7

0.8

0.9

1.0

1.1

seg.

1

seg.

12

seg.

14

seg.

7

seg.

5

seg.

6

seg.

13

seg.

8

seg.

9

seg.

10

seg.

11

seg.

4

seg.

3

seg.

2

outer region (O)medial region (M)inner region (I)

Cdc

42 a

ctiv

ity (F

RE

T/TF

P)

TFP

K

G JIH

P = 0.004

O

I

M

0.7 0.8 0.9Cdc42 activity (FRET/TFP)

L

seg.

17

seg.

16

seg.

151

1312111098

765432

14

171615

Rac

1

inner region outer regionmedial region

0.7

0.8

0.9

1.0

1.1

1.2

seg.

1

seg.

12

seg.

14

seg.

7

seg.

5

seg.

6

seg.

13

seg.

8

seg.

9

seg.

10

seg.

11

seg.

4

seg.

3

seg.

2outer region (O)medial region (M)inner region (I)

Rac

1 ac

tivity

(FR

ET/

TFP

)

TFP

E

A1

DCB

F

P < 0.0001

O

I

M

0.8 0.9 1.0 1.1Rac1 activity (FRET/TFP)

1312

111098765432

14

Rho

A

0.7

0.8

0.9

1.0

1.1

1.2

seg.

1

seg.

12

seg.

7

seg.

5

seg.

6

seg.

13

seg.

8

seg.

9

seg.

10

seg.

11

seg.

4

seg.

3

seg.

2

outer region (O)medial region (M)inner region (I)

Rho

A a

ctiv

ity (F

RE

T/TF

P)

TFP

Q

M PON

R

P = 0.03

O

I

M

0.8 0.9 1.0RhoA activity (FRET/TFP)

1

1312111098765432

inner region outer regionmedial region

inner region outer regionmedial region

Rac

1

0.65

1.10

Cdc

42

0.65

1.15

Rho

A

0.65

1.10

Figure S5 Hirata et al.

A

B

Rac1-GTP

total Rac1

total Cdc42

total RhoA

Cdc42-GTP

RhoA-GTP

DMSO NSC23766 Y27632

0.65

1.10

Rac

1

NSC23766 Y27632

Cdc

42R

hoA

0.65

1.15

0.65

1.10

cont

rol

Zizi

min

-KD

-AZi

zim

in-K

D-B

Zizi

min

-KD

-C

Zizi

min

-KD

-Cco

ntro

lZi

zim

in-K

D-A

Zizi

min

-KD

-B

Figure S8 Hirata et al.

AEGFP

Cherry merge

PH

10 20 30 40transfection effecacy (%)

50 6030 40H1-mCherry positive leading cells / all leading cells (%)

70 80

F

D

EG

FP

E

B

C

250

50

Zizimin1/DOCK9

-tubulin

KD-AKD-A

+ hDOCK9m

erge

Figure S9 Hirata et al.

0.65

1.10

FRET/TFP (IMD image)

Rac

1C

dc42

Rho

A

TFP + Cherry

0.65

1.15

0.65

1.10

A B

C D

E F