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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