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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: [email protected]
Nature Chemical Biology: doi:10.1038/nchembio.2000
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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
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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
Page 4
0 2 4 6 8 102500
3000
3500
4000
4500
5000
5500
1.05
1.1
1.15
1.2
1.25
x 104
0 2 4 6 8 10
2500
3000
3500
4000
6600
6800
7000
7200
7400
7600
Laser On Time x Number of Repeating Cycle/ Cycle Time/ Power
114msec x6/ 40sec/ 0.1mW 228msec x6/ 40sec/ 0.1mW
0 2 4 6 8 101500
2000
2500
3000
3500
7200
7400
7600
7800
8000
8200
a
b
c
Time(min) Time(min) Time(min)
0 2 4 6 8 10
1400
1600
1800
2000
2200
2400
1650
1700
1750
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1850
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1950
Time(min)
Time(min)
Time(min) Time(min)0 2 4 6 8 10
2500
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1850
0 2 4 6 8 10
1400
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1450
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
Inte
nsity
Inte
nsity
0 1 2 3 4 52500
3000
3500
4000
4500
5000
5500
6000
900
1000
1100
1200
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
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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
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a
1 0 1 2 3 4 5 6 7 8 9 100
0.10.20.30.40.50.60.70.80.9
1
1 0 1 2 3 4 5 6 7 8 9 100
0.1
0.2
0.3
0.4
0.5
0.6
1 0 1 2 3 4 5 6 7 8 9 100
0.1
0.2
0.3
0.4
0.5
0.60.010.050.10.18
0.010.050.10.18
0.010.050.10.18
FBP17
Cry2-PI3K
0 2 4 6 8 10 12 1 1 18 20
1800
2000
2200
2400
1350
1 00
1 50
1500
1550
1 00
0 2 4 6 8 101 00
1 00
1800
2000
2200
1 00
2000
2100
2200
2300
0 2 4 6 8 101200
1 00
1 00
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2000
0 2 4 6 8 10
2000
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0 2 4 6 8 10 12 1 1 18 20
2600
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2100
2200
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2600
b
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
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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
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PXTks5FBP17
PXTks5FBP17
Time (sec)
FBP17
-10 -5 0 5 10 15 20
PXTks5
0 20 40 60 800
1000
2000
3000
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
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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