supplementary information coverpage 1350 1400 1450 cry2-5ptaseinpp5e phplc δ 114msec x6/ 40sec/...
TRANSCRIPT
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
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
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Laser On Time x Number of Repeating Cycle/ Cycle Time/ Power
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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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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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0.010.050.10.18
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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1000
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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
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