Supporting Information for

Fluorescent biosensors for multiplexed imaging of phosphoinositide dynamics

Fabian Hertel1,5,6, Simin Li1,6, Mingyuan Chen2, Lutz Pott4, Sohum Mehta1, Jin Zhang1,2,3*

1
Department of Pharmacology, 2Department of Bioengineering, and 3Department of

Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA.
4
Institute of Physiology, Ruhr-University Bochum, Bochum, Germany.
5
Current address: Department of Nuclear Medicine, University Hospital RWTH Aachen,

Aachen, Germany.
6
Equal contribution.

*Correspondence: jzhang32@ucsd.edu (J.Z.)

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Supplementary Figures and Legends

Figure S1. Phosphoinositide binding to PH domains. Schematic overview of PIP2 and PIP3

binding to the PH domains of phospholipase Cδ1 (aa 22-139) and Akt1 (aa 4-111),

respectively, of Rattus norvegicus. The residues involved in binding are highlighted according

to the NCBI reference sequences NP_058731.1 (PLCδ1) and NP_150233.1 (Akt1). Many of

these residues are positively charged, illustrating the importance of electrostatic interactions

for the binding of the negatively charged phosphate groups of phosphoinositides.

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Figure S2. Lipid-binding mutant of FRET-based PIP2 reporter PlcR does not respond to

Ach stimulation in Cos7 cells expressing M3 receptor. Single-cell traces of Lyn-PlcRK30/32L

imaged in M3-expressing Cos7 cells (n = 15). Thick line indicates the mean, and thin lines

represent individual cells. Traces were taken from a single experiment and are representative

of 3 independent experiments.

3
 PlcR
RCaMP

Figure S3. Characterization of FRET-based PIP2 reporter PlcR in Cos7 cells expressing

M3 receptor. Single-cell traces of Lyn-PlcR (purple) co-imaged with Ca2+ indicator RCaMP

(red) in M3-expressing Cos7 cells. These traces were from 3 independent experiments.

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 PlcR
RCaMP

Figure S4. Validation of FRET-based PIP2 reporter PlcR in Cos7 cells with no M3

receptor. Single-cell traces of Lyn-PlcR (purple) co-imaged with Ca2+ indicator RCaMP (red)
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in Cos7 cells with no M3. These traces were from 2 independent experiments.

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Figure S5. Lipid-binding mutant of single-color PIP2 reporter dPlcR does not respond to

Ach stimulation in Cos7 cells expressing M3 receptor. Single-cell traces of Lyn-

dPlcRK30/32L imaged in M3-expressing Cos7 cells (n = 13). Thick line indicates the mean, and

thin lines represent individual cells. Traces were taken from a single experiment and are

representative of 3 independent experiments.

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 dPlcR
RCaMP

Figure S6. Characterization of single-color PIP2 reporter dPlcR in Cos7 cells expressing

M3 receptor. Single-cell traces of Lyn-dPlcR (green) co-monitored with Ca2+ indicator

RCaMP (red) in M3-exressing Cos7 cells. These traces were from 3 independent experiments.

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 dPlcR
RCaMP

Figure S7. Validation of single-color PIP2 reporter dPlcR in Cos7 cells with no M3

receptor. Single-cell traces of Lyn-dPlcR (green) co-monitored with Ca2+ indicator RCaMP

(red) in Cos7 cells with no M3. These traces were from 2 independent experiments.

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 dPlcR
RCaMP
sapphireCKAR

Figure S8. Simultaneous real-time monitoring of PIP2, Ca2+ and PKC activity in single

cells. Single-cell traces of PIP2 depletion time course monitored by Lyn-dPlcR in green,

intracellular Ca2+ influx monitored by RCaMP in red and PKC activation monitored by

sapphireCKAR in blue in Cos7 cells expressing M3 receptor when acetylcholine (Ach, 10

µM) was added at t = 0 min, collected from 3 independent experiments.

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 dPlcR
RCaMP
sapphireCKAR

Figure S9. Validation of dPlcR, RCaMP and sapphireCKAR co-imaging experiments in

Cos7 cells with no M3 receptor. Single-cell traces of PIP2 depletion time course monitored

by Lyn-dPlcR in green, intracellular Ca2+ influx monitored by RCaMP in red and PKC

activation monitored by sapphireCKAR in blue in Cos7 cells with no M3 receptor when

acetylcholine (Ach, 10 µM) was added at t = 0 min. Data were collected from 3 independent

experiments.

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Figure S10. Validation of dPlcR response to endogenous Gq-coupled receptor

stimulation. Time course of Lyn-dPlcR in response to histamine stimulation (200 μM) in

HeLa cells (n = 9). Shaded area represents SEM. Data are representative of 2 independent

experiments.

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 dPlcR
dInPAkt

Figure S11. Simultaneous real-time monitoring of PIP2 and 3-phosphoinositide signaling

dynamics in single cells. Single-cell traces of Lyn-dInPAkt and Lyn-dPlcR co-imaging in

Cos7 cells in response to EGF (100 ng/mL) collected from 3 independent experiments.

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 EGF
EGF + PI3K Inh.

Figure S12. Characterization of PI3K inhibition conditions in Cos7 cells. Time course of

InPAkt in response to EGF stimulation (100 ng/mL) in Cos7 cells with no pre-treatment

(circles, n = 5) or pre-incubation with PI3K inhibitors (triangles, n = 6) prior to EGF. Results

indicate 60 min pre-incubation with the PI3K inhibitors LY294002 (10 µM) and wortmannin

(10 µM) sufficiently inhibited PI3K activity and abolished EGF-induced 3-phosphoinositide

formation. Shaded areas represent SEM.

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Figure S13. Lipid-binding mutant of single-color PIP3 reporter dInPAkt does not

respond to EGF stimulation in Cos7 cells. Single-cell traces of Lyn-dInPAktR23/25A imaged

in Cos7 cells (n =7). Thick line indicates the mean, and thin lines represent individual cells.

Traces were taken from a single experiment and are representative of 2 independent

experiments.

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 dPlcR
dInPAkt

Figure S14. Validation of dInPAkt and dPlcR co-imaging experiments in Cos7 cells with

PI3K pre-inhibition. Single-cell traces of Lyn-dInPAkt and Lyn-dPlcR co-imaging in Cos7

cells in response to EGF (100 ng/mL), collected from 3 independent experiments.

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