Supplemental Figure Legends

1

Drosophila Cdc42-interacting protein 4 (dCIP4) restrains synaptic growth by inhibiting the secretion of the retrograde Gbb signal Minyeop Nahm, Sungdae Kim, Sang Kyoo Paik, Mihye Lee, Seongsoo Lee, Zang Hee Lee, Jaesang Kim, Daekee Lee, Yong Chul Bae, and Seungbok Lee

Supplemental Figure Legends

Supplemental Figure 1. Localization of dCIP4 in the embryonic CNS. Stage 16 wild-

type embryos were double stained for dCIP4 (green) and the presynaptic marker

Synaptotagmin (Syt) (red; A) or the postsynaptic marker Dlg (red; B). White arrows

indicate salivary glands. Ventral view, anterior to the left. Scale bar, 100 μm.

Supplemental Figure 2. Evolutionarily conserved properties of dCIP4. A, B,

Representative images of COS-7 cells transfected with wild-type Myc-dCIP4 (dCIP4;

A) and Myc-dCIP4-L145D (dCIP4[LD]; B). Myc immunoreactivity was examined by

confocal microscopy. Note that the L145D mutation in dCIP4 completely abolishes its

membrane-tubulating activity. Scale bar, 20 μm. C, dCIP4 interacts with the

constitutively active Cdc42 (Cdc42V12) via the HR1 domain and Wsp via the SH3

domain. In pull-down assays, cell lysates from HEK293T cells expressing Myc-

Cdc42V12, Myc-Cdc42N17, or Wsp-GFP were incubated with immobilized GST,

GST-dCIP4, GST-dCIP4-L436S (GST-dCIP4[LS]), or GST-dCIP4-W603K (GST-

dCIP4[WK]). The pull-downs were analyzed by western blotting with either anti-Myc

(two upper panels) or anti-GFP (middle panel). The lower panel shows the Coomassie

Blue staining following SDS-PAGE of GST and GST-dCIP4 preparations. The arrow

indicates the position of the GST-dCIP4 fusion and its mutants. D, dCIP4 can bind

2

simultaneously to active Cdc42 and Wsp. Cell lysates prepared from HEK293T cells

expressing HA-Cdc42V12 along with Myc-dCIP4 (left panels) or Myc-dCIP4-W603K

(right panels) were incubated with immobilized GST or GST-Wsp-PRD (amino acids

301-378). The pull-downs were analyzed by western blot analysis with both anti-Myc

(upper panels) and anti-HA (middle panels) antibodies. The lower panels show the

Coomassie Blue staining following SDS-PAGE of GST and GST-Wsp-PRD

preparations. The arrows indicate the position of GST-Wsp-PRD. Numbers on the left

are molecular masses in kDa.

Supplemental Figure 3. Western blot of body-wall muscle extracts from wild-type

(WT), BG57-GAL4/+, dcip41/Df(3L)ED4342 (dcip41/Df), BG57-GAL4,dcip41/UAS-HA-

dcip4,Df(3L)ED4342 (dCIP4 rescue-post), BG57-GAL4,dcip41/UAS-HA-dcip4-

L145D,Df(3L)ED4342 (dCIP4[LD] rescue-post), BG57-GAL4,dcip41/UAS-HA-dcip4-

L436S,Df(3L)ED4342 (dCIP4[LS] rescue-post), and BG57-GAL4,dcip41/UAS-HA-

dcip4-W603K,Df(3L)ED4342 (dCIP4[WK] rescue-post) larvae. The blot was probed

with anti-HA and reprobed with anti-β-actin.

Supplemental Figure 4. Cdc42 controls the postsynaptic localization of dCIP4, which

in turn controls the postsynaptic localization of Wsp. A, Quantification of the ratio of

average dCIP4 or Wsp to HRP signal intensities for experiments in Figure 4B-E. Values

are percentages of wild-type. ***p < 0.001. B, C, Western blots of total extracts of third

instar cdc422/cdc422 larvae probed with anti-dCIP4 (B) and dcip41/Df(3L)ED4342

larvae probed with anti-Wsp (C). Each blot was stripped and reprobed with anti-β-

actin.

3

Supplemental Figure 5. Postsynaptic Cdc42 and Wsp regulate synaptic growth at the

NMJ. A1-C2, Confocal images of third instar larval NMJs 6/7 (A1, B1, C1) and muscle

6 (A2, B2, C2) labeled with anti-HRP (green) and phalloidin (red), respectively. The

genotypes analyzed include MHC-GS-GAL4/+ (control; A1, A2), MHC-GS-GAL4/UAS-

cdc42V12 (UAS-cdc42V12; B1, B2), and MHC-GS-GAL4/UAS-cdc42N17 (UAS-

cdc42N17; C1, C2). Cdc42V12 larvae display an NMJ undergrowth phenotype, while

Cdc42N17 larvae have the opposite phenotype. Actin filaments are organized regularly

into striated myofibrils in control (A2) and Cdc42N17 (C2) larvae, while Cdc42V12

(B2) larvae display abnormal actin structures (arrow). Scale bar, 25 μm. D-F,

Quantification of total bouton number (D), satellite bouton number (E), and total NMJ

length (F) at NMJ 6/7 in MHC-GS-GAL4/+ (control), MHC-GS-GAL4/UAS-cdc42V12

(UAS-cdc42V12), and MHC-GS-GAL4/UAS-cdc42N17 (UAS-cdc42N17) larvae. All

animals were raised on RU486-free medium. The numbers of NMJs analyzed are

indicated inside bars. Statistically significant differences versus the MHC-GS-GAL4/+

control are indicated on top of bars (**p < 0.01; ***p < 0.001). G-I, Quantification of

bouton number (G), satellite bouton number (H), and NMJ length (I) at NMJ 6/7 in

wild-type (WT), wsp1/Df(3R)3450 (wsp1/Df), BG57-GAL4,wsp1/UAS-wsp,Df(3R)3450

(Wsp rescue-post), and C155-GAL4; wsp1/UAS-wsp,Df(3R)3450 (Wsp rescue-pre).

Statistically significant differences versus wsp1/Df(3R)3450 are indicated on top of bars

(***p < 0.001). Raw data are shown in supplemental Table 3.

Supplemental Figure 6. Levels of postsynaptic F-actin are significantly reduced in

dcip4, cdc42, wsp, and arp3 mutants. Single confocal slices of third instar NMJ

4

branches at muscles 12 and 13 double stained with anti-HRP (green) and rhodamine-

conjugated phalloidin (red). The genotypes analyzed include wild-type (A1, A2),

dcip41/Df(3L)ED4342 (B1, B2), cdc422/cdc422 (C1, C2), wsp1/Df(3R)3450 (D1, D2),

and arp3EP3640/arp3EP3640 (E1, E2). Scale bar, 5 μm. F, Quantification of the ratio of

average postsynaptic F-actin to HRP levels in the indicated genotypes. Values are the

percentage of wild-type. Statistically significant differences versus wild-type are

indicated on top of bars (**p < 0.01; ***p < 0.001).

Supplemental Figure 7. Postsynaptic dCIP4, Cdc42, Wsp, and Arp2/3 complex are

required for the normal accumulation of P-Mad in the nuclei of motor neurons. A1-F2,

Cell body nuclei in third instar larval ventral nerve cords stained with anti-P-Mad (red)

and anti-Elav (green) antibodies in control (BG57-GAL4/+, A1, A2), BG57-GAL4/UAS-

dcip4RNAi (B1, B2), UAS-cdc42RNAi/+; BG57-GAL4/+ (C1, C2), BG57-GAL4/UAS-

wspRNAi (D1, D2), UAS-arp2RNAi/+; BG57-GAL4/+ (E1, E2), and BG57-GAL4/UAS-

arp3RNAi (F1, F2). The numbers of nuclei analyzed are indicated inside bars. Scale bar,

20 μm. G, Quantification of the ratio of average P-Mad to HRP staining intensities.

Values are percentages of the BG57-GAL4/+ control. Statistically significant differences

versus the control are indicated on top of bars (***p < 0.001).

Supplemental Figure 8. The expression and processing of Gbb-GFP is not altered in

dcip4 and wsp mutants. A1-B2, High magnification images of Ib boutons at NMJ 6/7

labeled with anti-P-Mad (red) and anti-HRP (blue) in BG57-GAL4/+ (A1, A2) and

BG57-GAL4/UAS-gbb-GFP (B1, B2) third instar larvae. Note that presynaptic P-Mad

levels and satellite bouton formation (indicated by arrowheads) are significantly

5

increased by muscle-specific expression of Gbb-GFP. Scale bar, 5 μm. C,

Quantification of the ratio of average P-Mad to HRP levels in Ib boutons. The

difference between BG57-GAL4/+ (control) and BG57-GAL4/UAS-gbb-GFP is

statistically significant (***p < 0.001). D, Western blot of body-wall muscles dissected

from BG57-GAL4/+, BG57-GAL4/UAS-gbb-GFP, BG57-GAL4,Df(3L)ED4342/UAS-

gbb-GFP,dcip41, and BG57-GAL4/UAS-gbb-GFP,UAS-wspRNAi third instar larvae

probed with anti-GFP. The same blot was reprobed with anti-β-actin. Note that the ratio

of processed Gbb-GFP to unprocessed Gbb-GFP is not significantly changed by dcip4

mutations or wsp RNAi. E-H, Confocal images of NMJs 6/7 stained for total Gbb-GFP

(green) and HRP (red) in BG57-GAL4/+ (E), BG57-GAL4/UAS-gbb-GFP (F), BG57-

GAL4,Df(3L)ED4342/UAS-gbb-GFP,dcip41 (G), and BG57-GAL4/UAS-gbb-GFP,UAS-

wspRNAi (H) third instar larvae. Note that levels of total Gbb-GFP in muscle-cell

cytoplasm and at the NMJ are not significantly different according to genotype. Scale

bar, 50 μm.

Supplemental Figure 9. Increased glutamate receptor abundance at the third-instar

NMJ in larvae expressing ShiK44A postsynaptically. A1-D2, Confocal images of NMJs

6/7 stained with anti-HRP (green) and either anti-GluRIIA (red; A1-B2) or anti-

GluRIIB (red; C1-D2) antibodies in MHC-GS--GAL4/+ and MHC-GS--GAL4/UAS-

shiK44A larvae. Scale bar, 10 μm. E, Quantification of the ratio of GluRIIA (black bars)

or GluRIIB (gray bars) versus HRP staining intensities in the indicated genotypes.

Wild-type and shits mutant larvae were pulsed at restrictive temperature for 3 h before

staining. The numbers of NMJ 6/7 branches analyzed are indicated inside bars. Values

are percentages of the corresponding control. ***p < 0.001.

Bou

ton

num

ber

p va

lue

vs W

TM

uscl

e ar

ea( μ

m2 X1

03 )p

valu

evs

WT

Bou

ton

num

ber

/Mus

cle

area

(X 1

0-3 μ

m-2

)

p va

lue

vs W

TN

MJ

leng

th( μ

m)

p va

lue

vs W

T

NM

J le

ngth

/Mus

cle

area

(X 1

0-3 μ

m-1

)

p va

lue

vs W

TSa

telli

tebo

uton

num

ber

p va

lue

vs W

T

w11

18 (W

T)11

8.2

± 4

91.7

± 1

.71.

29 ±

0.0

454

3 ±

215.

9 ±

0.2

13.1

± 1

dcip

41/D

f(3L)

ED

4342

147.

1 ±

5.6

<0.0

0187

± 2

.40.

670

1.7

± 0.

07<0

.001

624

± 26

0.00

57.

3 ±

0.3

<0.0

0121

.9 ±

1.2

<0.0

01B

G57

-GA

L4/+

112

± 4.

20.

992

87.9

± 1

.90.

855

1.27

± 0

.04

1.00

051

3 ±

210.

280

5.8

± 0.

20.

745

13.6

± 1

.31.

000

BG

57-G

AL4

,dci

p41

/UA

S-H

A-d

cip4

,Df(3

L)E

D43

4211

3.7

± 3.

90.

999

90 ±

1.9

0.99

91.

3 ±

0.05

1.00

051

7 ±

140.

368

5.8

± 0.

20.

663

12.4

± 1

1.00

0

BG

57-G

AL4

,dci

p41

/UA

S-H

A-d

cip4

-L14

5D,D

f(3L)

ED

4342

120.

7 ±

4.5

1.00

090

.3 ±

11.

000

1.34

± 0

.05

1.00

054

2 ±

200.

968

6 ±

0.2

0.82

710

.6 ±

0.7

0.87

1

BG

57-G

AL4

,dci

p41

/UA

S-H

A-d

cip4

-L43

6S,D

f(3L)

ED

4342

140.

7 ±

3.8

0.02

088

.9 ±

1.5

0.97

71.

59 ±

0.0

60.

004

595

± 22

0.07

26.

7 ±

0.3

0.01

724

± 1

.3<0

.001

BG

57-G

AL4

,dci

p41

/UA

S-H

A-d

cip4

-14

6.1

± 5.

60.

002

91.7

± 1

.31.

000

1.6

± 0.

070.

007

610

± 22

0.03

06.

7 ±

0.3

0.04

124

± 1

.5<0

.001

C15

5-G

AL4

/+13

1.8

± 3.

80.

468

96.9

± 1

.70.

468

1.4

± 0.

040.

992

555

± 21

0.67

75.

7 ±

0.2

0.56

913

.3 ±

1.1

1.00

0C

155-

GA

L4/+

; dci

p41

/UA

S-H

A-d

cip4

,Df(3

L)E

D43

4214

9.4

± 5.

3<0

.001

91.5

± 1

.61.

000

1.64

± 0

.07

<0.0

0166

1 ±

21<0

.001

7.3

± 0.

3<0

.001

21.7

± 1

.1<0

.001

Supp

lem

enta

l Tab

le 1

. Q

uant

ifica

tion

of N

MJ

para

met

ers

for e

xper

imen

ts in

Fig

ure

2

Bou

ton

num

ber

p va

lue

vsB

G57

-GA

L4/+

Mus

cle

area

( μm

2 X103 )

p va

lue

vsB

G57

-GA

L4/+

Bou

ton

num

ber

/Mus

cle

area

(X 1

0-3 μ

m-2

)

p va

lue

vsB

G57

-GA

L4/+

NM

J le

ngth

( μm

)

p va

lue

vsB

G57

-GA

L4/+

NM

J le

ngth

/Mus

cle

area

(X 1

0-3 μ

m-1

)

p va

lue

vsB

G57

-GA

L4/+

Sate

llite

bout

on n

umbe

r

p va

lue

vsB

G57

-GA

L4/+

BG

57-G

AL4

/+11

5.4

± 2.

192

.3 ±

1.5

1.25

± 0

.02

549

± 13

6 ±

0.1

13 ±

0.7

UA

S-c

dc42

RN

Ai /+

; BG

57-G

AL4

/+16

1.4

± 5.

4<0

.001

91.3

± 1

.80.

999

1.77

± 0

.05

<0.0

0162

6 ±

200.

002

6.9

± 0.

2<0

.001

24.2

± 1

.5<0

.001

BG

57-G

AL4

/UA

S-w

spR

NA

i15

2.1

± 3.

5<0

.001

89 ±

1.3

0.67

51.

71 ±

0.0

4<0

.001

631

± 20

0.00

17.

1 ±

0.2

<0.0

0123

± 1

.2<0

.001

UA

S-a

rp2

RN

Ai /+

; BG

57-G

AL4

/+15

4.8

± 3.

9<0

.001

92.1

± 1

.81.

000

1.69

± 0

.05

<0.0

0159

0 ±

160.

073

6.4

± 0.

20.

044

20 ±

1.1

<0.0

01

BG

57-G

AL4

/UA

S-a

rp3

RN

Ai

161

± 5

<0.0

0190

± 1

.40.

859

1.79

± 0

.05

<0.0

0159

3 ±

160.

066

6.6

± 0.

20.

010

23.3

± 1

<0.0

01

Bou

ton

num

ber

p va

lue

vsC

155-

GA

L4/+

Mus

cle

area

( μm

2 X103 )

p va

lue

vsC

155-

GA

L4/+

Bou

ton

num

ber

/Mus

cle

area

(X 1

0-3 μ

m-2

)

p va

lue

vsC

155-

GA

L4/+

NM

J le

ngth

( μm

)

p va

lue

vsC

155-

GA

L4/+

NM

J le

ngth

/Mus

cle

area

(X 1

0-3 μ

m-1

)

p va

lue

vsC

155-

GA

L4/+

Sate

llite

bout

on n

umbe

r

p va

lue

vsC

155-

GA

L4/+

C15

5-G

AL4

/+13

1.8

± 3.

896

.9 ±

1.7

1.36

± 0

.04

555

± 21

5.7

± 0.

213

.3 ±

1.1

C15

5-G

AL4

/+; U

AS

-cdc

42R

NA

i /+14

1.4

± 6

0.29

389

± 1

.90.

010

1.58

± 0

.05

0.00

158

9 ±

200.

192

6.6

± 0.

1<0

.001

19.7

± 1

.5<0

.001

C15

5-G

AL4

/+; U

AS

-wsp

RN

Ai /+

131.

6 ±

3.1

0.99

995

.5 ±

1.6

0.83

31.

38 ±

0.0

30.

937

545

± 13

0.69

15.

7 ±

0.1

0.94

413

.4 ±

0.7

0.99

7

Supp

lem

enta

l Tab

le 2

. Q

uant

ifica

tion

of N

MJ

para

met

ers

for e

xper

imen

ts in

Fig

ure

5

Bou

ton

num

ber

p va

lue

vsM

HC

-GS

-GA

L4/+

Mus

cle

area

( μm

2 X103 )

p va

lue

vsM

HC

-GS-

GAL

4/+

Bou

ton

num

ber

/Mus

cle

area

(X 1

0-3 μ

m-2

)

p va

lue

vsM

HC

-GS-

GAL

4/+

NM

Jle

ngth

( μm

)

p va

lue

vsM

HC

-GS

-GAL

4/+

NM

J le

ngth

/Mus

cle

area

(X 1

0-3 μ

m-1

)

p va

lue

vsM

HC

-GS-

GAL

4/+

Sate

llite

bout

onnu

mbe

r

p va

lue

vsM

HC

-GS

-GA

L4/+

MH

C-G

S-G

AL4/

+ 10

8.5

± 1.

581

.9 ±

21.

33 ±

0.0

357

8 ±

197.

1 ±

0.3

10.7

± 0

.8

MH

C-G

S-G

AL4/

UA

S-c

dc42

V12

74.9

± 1

.8<0

.001

80.8

± 1

.70.

913

0.93

± 0

.03

<0.0

0143

6 ±

11<0

.001

5.4

± 0.

1<0

.001

7 ±

0.4

0.00

2M

HC

-GS

-GAL

4/U

AS

-cdc

42N

1713

0 ±

3.1

<0.0

0182

.6 ±

2.4

0.97

21.

59 ±

0.0

5<0

.001

663

± 23

0.00

58.

1 ±

0.2

0.00

520

.4 ±

0.9

<0.0

01

Bou

ton

num

ber

p va

lue

vs w

sp1/D

fM

uscl

e ar

ea( μ

m2 X1

03 )p

valu

evs

wsp

1/D

f

Bou

ton

num

ber

/Mus

cle

area

(X 1

0-3 μ

m-2

)

p va

lue

vs w

sp1/D

f

NM

Jle

ngth

( μm

)

p va

lue

vs w

sp1/D

f

NM

J le

ngth

/Mus

cle

area

(X 1

0-3 μ

m-1

)

p va

lue

vs w

sp1/D

f

Sate

llite

bout

onnu

mbe

r

p va

lue

vs w

sp1/D

f

w11

18 (W

T)11

7.8

± 2.

8<0

.001

89.6

± 1

.30.

949

1.32

± 0

.04

<0.0

0152

9 ±

20<0

.001

5.9

± 0.

3<0

.001

13.6

± 0

.8<0

.001

wsp

1/D

f(3R

)345

016

9.3

± 2.

590

.8 ±

1.3

1.87

± 0

.03

706

± 17

7.8

± 0.

226

.5 ±

1.3

BG

57-G

AL4,

wsp

1

/UA

S-w

sp,D

f(3R

)345

013

1.3

± 3.

8<0

.001

96.6

± 1

.70.

187

1.52

± 0

.03

<0.0

0156

5 ±

15<0

.001

6.5

± 0.

1<0

.001

16.3

± 0

.8<0

.001

C15

5-G

AL4

; wsp

1

/UA

S-w

sp,D

f(3R

)345

015

4.7

± 3

0.00

488

.1 ±

1.4

0.54

61.

76 ±

0.0

40.

161

690

± 17

0.90

37.

9 ±

0.2

0.99

118

± 1

.1<0

.001

Supp

lem

enta

l Tab

le 3

. Q

uant

ifica

tion

of N

MJ

para

met

ers

for e

xper

imen

ts in

Sup

plem

enta

l Fig

ure

5

Supplemental Table 4. Quantification of NMJ parameters for experiments in Figure 6

Bouton number WT dcip4 1 /+ cdc42 2 /+ wsp 1 /+ arp3 EP3640/+ cdc42 2 /+; dcip4 1 /+ dcip4 1 , +/+,wsp 1

w 1118 (WT) 117.9 ± 3.3 1.000 0.904 1.000 1.000 <0.001 <0.001

dcip4 1 /+ 118.7 ± 3.3 1.000 0.360 0.999 1.000 <0.001 <0.001

cdc42 2 /+ 125.3 ± 2.1 0.904 0.360 <0.001

wsp 1 /+ 119.4 ± 3 1.000 0.999 <0.001

arp3 EP3640/+ 118.1 ± 3.4 1.000 1.000

cdc42 2 /+; dcip4 1 /+ 145.2 ± 2.8 <0.001 <0.001 <0.001 0.957

dcip4 1 , +/+,wsp 1 147.9 ± 4.1 <0.001 <0.001 <0.001 0.957

cdc42 2 /+; dcip4 1 ,+/+,wsp 1 178.8 ± 4.2 <0.001 <0.001 <0.001

dcip4 1 ,+/ +,arp3 EP3640 155.9 ± 4.5 <0.001 <0.001 <0.001

Muscle area(μm2X103)

WT dcip4 1 /+ cdc42 2 /+ wsp 1 /+ arp3 EP3640/+ cdc42 2 /+; dcip4 1 /+ dcip4 1 , +/+,wsp 1

w 1118 (WT) 89.7 ± 1.8 0.983 1.000 1.000 1.000 0.952 1.000

dcip4 1 /+ 92 ± 1.3 0.983 0.844 0.942 0.850 0.996 0.842

cdc42 2 /+ 90.5 ± 1.2 1.000 0.844 0.718

wsp 1 /+ 90.8 ± 1.7 1.000 0.942 0.993

arp3 EP3640/+ 90 ± 1.8 1.000 0.850

cdc42 2 /+; dcip4 1 /+ 92.4 ± 1.1 0.952 0.996 0.718 0.564

dcip4 1 , +/+,wsp 1 90.2 ± 1.4 1.000 0.842 0.993 0.564

cdc42 2 /+; dcip4 1 ,+/+,wsp 1 96.9 ± 0.7 0.027 0.043 <0.001

dcip4 1 ,+/ +,arp3 EP3640 97.4 ± 2 0.017 0.112 0.017

Bouton number/Muscle area(X 10-3 μm-2)


w 1118 (WT) 1.32 ± 0.05 1.000 0.983 1.000 1.000 0.006 <0.001

dcip4 1 /+ 1.3 ± 0.03 1.000 0.210 0.956 0.975 <0.001 <0.001

cdc42 2 /+ 1.4 ± 0.03 0.983 0.210 0.003

wsp 1 /+ 1.33 ± 0.04 1.000 0.956 <0.001

arp3 EP3640/+ 1.32 ± 0.04 1.000 0.975

cdc42 2 /+; dcip4 1 /+ 1.57 ± 0.03 0.006 <0.001 0.003 0.629

dcip4 1 , +/+,wsp 1 1.65 ± 0.06 <0.001 <0.001 <0.001 0.629

cdc42 2 /+; dcip4 1 ,+/+,wsp 1 1.85 ± 0.04 <0.001 <0.001 0.020

dcip4 1 ,+/ +,arp3 EP3640 1.61 ± 0.05 <0.001 <0.001 <0.001

NMJ length(μm)


w 1118 (WT) 524 ± 10 0.769 0.745 0.697 0.850 0.004 0.004

dcip4 1 /+ 531 ± 15 0.769 0.970 0.903 0.925 0.002 <0.001

cdc42 2 /+ 532 ± 18 0.745 0.970 0.002

wsp 1 /+ 533 ± 11 0.697 0.903 <0.001

arp3 EP3640/+ 529 ± 19 0.850 0.925

cdc42 2 /+; dcip4 1 /+ 598 ± 14 0.004 0.002 0.002 0.950

dcip4 1 , +/+,wsp 1 597 ± 13 0.004 <0.001 <0.001 0.950

cdc42 2 /+; dcip4 1 ,+/+,wsp 1 692 ± 19 <0.001 <0.001 <0.001

dcip4 1 ,+/ +,arp3 EP3640 656 ± 25 <0.001 <0.001 <0.001

NMJ length/Muscle area(X 10-3 μm-1)


WT 5.9 ± 0.1 0.796 0.919 0.877 0.877 0.030 0.004

dcip4 1 /+ 5.8 ± 0.2 0.796 0.658 0.606 0.690 0.003 <0.001

cdc42 2 /+ 5.9 ± 0.2 0.919 0.658 0.012

wsp 1 /+ 5.9 ± 0.1 0.877 0.606 <0.001

arp3 EP3640/+ 5.9 ± 0.2 0.877 0.690

cdc42 2 /+; dcip4 1 /+ 6.5 ± 0.1 0.030 0.003 0.012 0.445

dcip4 1 , +/+,wsp 1 6.7 ± 0.2 0.004 <0.001 <0.001 0.445

cdc42 2 /+; dcip4 1 ,+/+,wsp 1 7.1 ± 0.2 <0.001 0.006 0.037

dcip4 1 ,+/ +,arp3 EP3640 6.7 ± 0.2 0.002 0.001 0.005

Satellitebouton number WT dcip4 1 /+ cdc42 2 /+ wsp 1 /+ arp3 EP3640/+ cdc42 2 /+; dcip4 1 /+ dcip4 1 , +/+,wsp 1

w 1118 (WT) 13.8 ± 0.9 0.997 0.139 0.989 0.989 <0.001 <0.001

dcip4 1 /+ 15 ± 0.9 0.997 0.098 0.188 0.181 <0.001 <0.001

cdc42 2 /+ 17.9 ± 0.8 0.139 0.098 <0.001

wsp 1 /+ 12.3 ± 0.8 0.989 0.188 <0.001

arp3 EP3640/+ 12.3 ± 0.6 0.989 0.181

cdc42 2 /+; dcip4 1 /+ 25.8 ± 1 <0.001 <0.001 <0.001 0.080

dcip4 1 , +/+,wsp 1 22 ± 1.1 <0.001 <0.001 <0.001 0.080

cdc42 2 /+; dcip4 1 ,+/+,wsp 1 32.2 ± 1.3 <0.001 <0.001 <0.001

dcip4 1 ,+/ +,arp3 EP3640 21.3 ± 1.1 <0.001 <0.001 <0.001

p value vs

p value vs

p value vs

p value vs

p value vs

p value vs

Supplemental Table 5. Quantification of NMJ parameters for experiments in Figure 7

Bouton number WTBG57-GAL4/UAS-cip4 RNAi

BG57-GAL4/UAS-wsp RNAi wit A12 /wit B11

w 1118 (WT) 119.4 ± 2.7 <0.001 <0.001 <0.001BG57-GAL4/+ 114.8 ± 1.7 0.978 <0.001 <0.001 <0.001

BG57-GAL4/UAS-cip4 RNAi 142.1 ± 3 <0.001 0.720 <0.001

BG57-GAL4/UAS-wsp RNAi 149.6 ± 3.5 <0.001 0.720 <0.001

wit A12 /wit B11 61.6 ± 3.3 <0.001 <0.001 <0.001BG57-GAL4,wit B11 /UAS- 65 ± 1.7 <0.001 <0.001 <0.001 0.998BG57-GAL4,wit B11 /UAS- 61.2 ± 2.8 <0.001 <0.001 <0.001 1.000

wit A12 /+ 121.1 ± 2 1.000 <0.001 <0.001 <0.001

BG57-GAL4/UAS-cip4 RNAi ,wit A12 120.1 ± 3.6 1.000 <0.001 <0.001 <0.001

BG57-GAL4/UAS-wsp RNAi ,wit A12 119.1 ± 3.1 1.000 <0.001 <0.001 <0.001

Muscle area(μm2X103)

WTBG57-GAL4/UAS-cip4 RNAi


w 1118 (WT) 89.4 ± 1.2 <0.001 0.999 <0.001BG57-GAL4/+ 91.6 ± 1.3 0.985 <0.001 1.000 <0.001

BG57-GAL4/UAS-cip4 RNAi 79.2 ± 1.2 <0.001 <0.001 0.001

BG57-GAL4/UAS-wsp RNAi 90.8 ± 1.2 0.999 <0.001 <0.001

wit A12 /wit B11 70.3 ± 1.1 <0.001 0.001 <0.001BG57-GAL4,wit B11 /UAS- 68 ± 0.7 <0.001 <0.001 <0.001 0.980BG57-GAL4,wit B11 /UAS- 71.1 ± 1.2 <0.001 0.005 <0.001 1.000

wit A12 /+ 89.8 ± 1.2 1.000 <0.001 1.000 <0.001

BG57-GAL4/UAS-cip4 RNAi ,wit A12 79.4 ± 2.1 <0.001 1.000 <0.001 0.001

BG57-GAL4/UAS-wsp RNAi ,wit A12 91.8 ± 1.7 0.961 <0.001 1.000 <0.001

Bouton number/Muscle area(X 10-3 μm-2)

WTBG57-GAL4/UAS-cip4 RNAi


w 1118 (WT) 1.34 ± 0.04 <0.001 <0.001 <0.001BG57-GAL4/+ 1.26 ± 0.02 0.869 <0.001 <0.001 <0.001

BG57-GAL4/UAS-cip4 RNAi 1.8 ± 0.03 <0.001 0.238 <0.001


wit A12 /wit B11 0.88 ± 0.05 <0.001 <0.001 <0.001BG57-GAL4,wit B11 /UAS- 0.96 ± 0.03 <0.001 <0.001 <0.001 0.918BG57-GAL4,wit B11 /UAS- 0.86 ± 0.04 <0.001 <0.001 <0.001 1.000

wit A12 /+ 1.36 ± 0.03 1.000 <0.001 <0.001 <0.001

BG57-GAL4/UAS-cip4 RNAi ,wit A12 1.52 ± 0.05 0.052 <0.001 0.418 <0.001


Satellitebouton number WT

BG57-GAL4/UAS-cip4 RNAi


w 1118 (WT) 13.8 ± 0.8 <0.001 <0.001 <0.001BG57-GAL4/+ 13.6 ± 0.6 1.000 <0.001 <0.001 <0.001

BG57-GAL4/UAS-cip4 RNAi 19 ± 0.6 <0.001 0.486 <0.001


wit A12 /wit B11 3.7 ± 0.5 <0.001 <0.001 <0.001BG57-GAL4,wit B11 /UAS- 4.6 ± 0.3 <0.001 <0.001 <0.001 0.996BG57-GAL4,wit B11 /UAS- 2.8 ± 0.3 <0.001 <0.001 <0.001 0.998

wit A12 /+ 12 ± 0.5 0.722 <0.001 <0.001 <0.001

BG57-GAL4/UAS-cip4 RNAi ,wit A12 11.8 ± 0.5 0.677 <0.001 <0.001 <0.001


p value vs

p value vs

p value vs

p value vs

Documents

Supplemental Figure Legends