Supplementary,Information,,

Frequency and Amplitude Control of Cortical Oscillations by Phosphoinositide Waves

Ding Xiong1, 2, 3, Shengping Xiao1, 2, Su Guo1, 2, 3, Qinsong Lin1, Fubito Nakatsu4,

Min Wu1, 2, 3, †

1 Department of Biological Sciences, 2 Centre for Bioimaging Sciences,

3 Mechanobiology Institute, National University of Singapore, Singapore

4 Department of Neurochemistry and Molecular Cell Biology, Graduate School of

Medical and Dental Sciences, Niigata University, Japan

†To whom correspondence should be addressed.

Email: dbswum@nus.edu.sg

Nature Chemical Biology: doi:10.1038/nchembio.2000

CIP4

PTEN

CIP4

SHIP2

CIP4

SYNJ1

CIP4

FBP17

a b

c d

TIRF Kymograph

TIRF Kymograph

TIRF Kymograph

TIRF Kymograph

Supplementary Figure 1. Patterns of other F-BAR proteins and lipid enzymes.(a) Image and kymograph of cells expressing mCherry-CIP4 and FBP17-EGFP (n=2 cells, 2

experiment). (b) Image and kymograph of cells expressing 5-phosphatase GFP-SHIP2 and

mCherry-CIP4 (n=20 cells, 4 experiments). (c) Image and kymograph showing that GFP-Syn-

aptojanin1 is punctate but did not display apparent correlation with mCherry-CIP4 waves

(n=10 cells, 2 experiments). (d) Image and kymograph showing uniform GFP-PTEN distribu-

tion in cells with mCherry-CIP4 waves (n=53 cells, 5 experiments). For TIRF images, scale

bar 5 μm; for kymographs, scale bar= 1 min (horizontal bar), 5 μm (vertical bar).

Supplementary Results

Nature Chemical Biology: doi:10.1038/nchembio.2000

p110-α/ PIK3CAp110-β/ PIK3CBp110-δ/ PIK3CD

class IACatalytic

Regulatoryp85-α/ PIK3R1

p55-γ/ PIK3R3p85-β/ PIK3R2

class IB p110-γ/ PIK3CGCatalytic

Regulatoryp87/ PIK3R6

p101/ PIK3R5

class II CatalyticPI3K-C2β/ PIK3C2BPI3K-C2α/ PIK3C2A

PI3K-C2γ/ PIK3C2G

class III CatalyticRegulatory

Vps34/ PIK3C3p150/ PIK3R4

Synaptojanin2/ Synj2Synaptojanin1/ Synj1

SHIP1/ Inpp5d

SHIP2/ Inppl1

PTEN/ PTEN

Phosphoinositide Phosphatases

RPKM

(N.D)

0 20 40 60 80 100

Supplementary Figure 2. RNA-Seq analysis to assess expression of lipid phosphatase and

PI3K genes in RBL-2H3.

Gene expression levels are interpreted in read per kilobase per million mapped expression

(RPKM) (n=2 total RNA samples; error bar: s.e.m. N.D: not detected).

Nature Chemical Biology: doi:10.1038/nchembio.2000

0 2 4 6 8 102500

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Laser On Time x Number of Repeating Cycle/ Cycle Time/ Power

114msec x6/ 40sec/ 0.1mW 228msec x6/ 40sec/ 0.1mW

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PHPLCδ Cry2-5ptaseINPP5E

114msec x6/ 40sec/ 0.1mWPHAkt Cry2-5ptaseINPP5E

PHPLCδ Cry2-5ptaseINPP5E

228msec x6/ 40sec/ 0.1mWPHAktCry2-5ptaseINPP5E

228msec x12/ 20sec/ 0.1mWPHPLCδ Cry2-5ptaseINPP5E

228msec x10/ 20sec/ 0.1mWPHAkt Cry2-5ptaseINPP5E

Inte

nsity

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nsity

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nsity

0 1 2 3 4 52500

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228msec x6/ 40sec/ 0.1mWFBP17Cry2-5ptaseINPP5E

Supplementary Figure 3. Effect of the rapid recruitment of 5-phosphatase domain of

INPP5E.

(a) Intensity profiles of mCherry-CRY2-5ptaseINPP5E and iRFP-PHPLCδ, monitoring levels of

PtdIns(4,5)P2. Sustained decrease of PtdIns(4,5)P2 could be induced by a train of laser pulses

with indicated blue light power and pulse intervals (n=6 cells from 2 experiments). (b) Intensi-

ty profiles of mCherry-CRY2-5ptaseINPP5E and iRFP-PHAkt, monitoring levels of

PtdIns(3,4)P2 and PtdIns(3,4,5)P3 levels (n=8 cells from 2 experiments). (c) Intensity profile

of mCherry-CRY2-5ptaseINPP5E and FBP17-iRFP shows reduced amplitude of FBP17 waves

with membrane recruitment of CRY2-5ptaseINPP5E ( n=3 cells from 2 experiments).

Nature Chemical Biology: doi:10.1038/nchembio.2000

FBP17

FBP17

FBP17

PI(3,4,5)P3

PI(4,5)P2

PI(3,4)P2(RFP-PHTapp1)

(mCherry-PHGrp1)

(iRFP-PHPLCδ)

10

10

10

Supplementary Figure 4. Effects of PI3K inhibitor wortmannin on lipid levels.

The concurrent decrease of PtdIns(3,4)P2 (monitored by RFP-PHTapp1, n=5 cells from 4 exper-

iments) and PtdIns(3,4,5)P3 levels (monitored by mCherry-PHGrp1, n=12 cells from 5 experi-

ments) are shown. PtdIns(4,5)P2 (iRFP-PHPLCδ) level on the plasma membrane was not affect-

ed (n=6 cells from 3 experiments). The gray/pseudocolor scale kymographs are made from a

Nature Chemical Biology: doi:10.1038/nchembio.2000

a

1 0 1 2 3 4 5 6 7 8 9 100

0.10.20.30.40.50.60.70.80.9

1

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0.60.010.050.10.18

0.010.050.10.18

0.010.050.10.18

FBP17

Cry2-PI3K

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Cry2-iSH2PI3K PHG p1

T ( )

T ( ) T ( ) T ( )

T ( ) T ( )

T ( ) T ( ) T ( )

II

I

2P

I3K (

..)

I

P

I

P

Gp1

(.

.)

Tpp

1 (.

.)

11 / / 0.01

Cry2-iSH2PI3K PHG p111 / / 0.1

Cry2-iSH2PI3K PHG p111 5/ 1 / 0.01

Cry2-iSH2PI3K PHG p111 5/ 1 / 0.1

Cry2-iSH2PI3K PHG p111 10/ 30 / 0.01

Cry2-iSH2PI3K PHG p111 10/ 30 / 0.1

N R p C / C / P

Supplementary Figure 5. Characterization of the optogenetic method employed for

acute PI3K activation.

(a) Quantification of mCherry-CRY2-iSH2PI3K, PtdIns(3,4,5)P3 (monitored by iRFP-PHGrp1)

and PtdIns(3,4)P2 (monitored by iRFP-PHTapp1) recruitment to the plasma membrane by

different laser power (n=9 cells from 6 experiments). (b) Effect of activation conditions on the

levels of PtdIns(3,4,5)P3 (n=28 cells from 6 experiments).

Nature Chemical Biology: doi:10.1038/nchembio.2000

a bTIRF Kymograph TIRF Kymograph

FBP17

PI3Kδ

FBP17

PI3Kδ

Supplementary Figure 6. Patterns of FBP17 and PI3Kδ.P δ C P P

P δ CP P

Nature Chemical Biology: doi:10.1038/nchembio.2000

PXTks5FBP17

PXTks5FBP17

Time (sec)

FBP17

-10 -5 0 5 10 15 20

PXTks5

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Sample

Cell with waveCell without wave

Fluo

resc

ence

inte

nsity

o

f Tan

dem

-PH T

app1

a b

Inte

nsity

(a.u

.)In

tens

ity (a

.u.)

Supplementary Figure 7. Additional PtdIns(3,4)P2 sensor and effect of PtdIns(3,4)P2

sequestering on waves.

(a) Kymographs and intensity profile of FBP17-EGFP and PXTks5-mCherry ( n= 5 cells from

2 experiments). (b) Quantification shows GFP-PHTAPP1-PHTAPP1 overexpression could inhibit

C C Pm (vertical bar).

Nature Chemical Biology: doi:10.1038/nchembio.2000

140kd

100kd

40kd

35kd

260kd

70kdWestern

Blot

RBL cell lysate

140kd100kd70kd

50kd

40kd35kd

25kd

CoomassieStaining

FBP17GST-SH3

CIP4GST-SH3

PACSIN1GST-SH3GST

+-+-+-+-

RBL cell lysateFBP17GST-SH3

CIP4GST-SH3

PACSIN1GST-SH3GST

+-+-+-+-

Supplementary Figure 8. Full gel image of Figure 1b.

Dashed box indicates the cropped portion.

Nature Chemical Biology: doi:10.1038/nchembio.2000