Jin et al
.
, Ultrasonic reporters of
calcium for deep tissue imaging of cellular signals (2023)
1
Ultrasonic reporters of calcium for deep tissue imaging of
cellular
signals
Zhiyang Jin
1
, Anupama Lakshmanan
2,
†
, Ruby Zhang
2,
†
, Teresa A. Tran
2
, Claire Rabut
2
, Przemys
ł
aw Dutka
2,3
,
Mengtong Duan
3
, Robert C. Hurt
3
, Dina Malounda
2
, Yuxing Yao
2
,
Mikhail G. Shapiro
1,2,4,*
1
Division of Engineering and Applied Sciences
, California Institute of Technology; Pasadena, CA 91125, USA
2
Division of Chemistry and Chemical Engineering
, California Institute of Technology; Pasadena, CA 91125, USA
3
Division of Biology and Biological Engineering
, California Institute of Technology; Pasadena, CA 91125, USA
4
Howard Hughes Medical Institute
; Pasadena, CA 91125, USA
†
These authors contributed equally
.
*
Corresponding authors:
MGS
(mikhail@caltech.edu)
Supplementary Materials
Supplementary Figure 1. URoC screening and URoC1a characterization
. (
a
) Nonlinear signal after background subtraction and
GV concentration normalization of GVs with 3
-
repeat Ana GvpC (3R GvpC) or no
GvpC after incubation with EGTA, calcium or first with
calcium and then with EGTA. (
b
,
c
) The nonlinear signal mean fold change of URoC variants with different CBP insertion sites as a function
the p
-
value, comparing conditions with calcium and with EGTA (
b
) or first with calcium and then reversed with EGTA and only with EGTA
(
c
). The vertical dash lines represent the significance threshold of p = 0.05 and the horizontal lines represent no calcium
-
dependent change (
b
)
or fully reversible (
c
). (
d
) Nonlinear
SBR in dB scale as a function of applied acoustic pressure for control dURoC1a GVs (with all EF hands
knocked out) after incubation with 200 μM CaCl
2
, 5 mM EGTA or first with 200 μM CaCl
2
and then with 5 mM EGTA. Solid curves represent
the mean of all biological replicates. (
e
) Nonlinear SBR in dB scale as a function of magnesium concentration for URoC1a after incubation
Ca
2+
dURoC1a -
dURoC1a +
Ca
2+
dURoC1a reverse
200
400
600
800
1000
0
5
10
15
20
25
Acoustic Pressure (kPa)
Nonlinear US SBR (dB)
e
d
0
1
2
3
4
0
5
10
15
20
25
[Mg
2+
] (mM)
Nonlinear US SBR (dB)
Ca
2+
URoC1a -
URoC1a +
Ca
2+
6.5
7.0
7.5
8.0
0
5
10
15
20
25
pH
*
ns
Nonlinear US SBR (dB)
Ca
2+
URoC1a -
URoC1a +
Ca
2+
Ca
2+
No GvpC -
No GvpC +
Ca
2+
Ca
2+
dURoC1a -
dURoC1a +
Ca
2+
g
f
3R GvpC
No GvpC
0.0
0.2
0.4
0.6
0.8
Nonlinear US Signal (a.u.)
Ca
2+
-
+
Ca
2+
Reverse
-3
-2
-1
0
log
10
(P-value)
0.2
0.4
0.6
0.8
1
1.2
1.4
Mean Fold Change (EGTA/Rev)
-3
-2
-1
0
log
10
(P-value)
0
2
4
6
8
10
12
14
Mean Fold Change (Ca
2+
/EGTA)
2
2
3
4
4
5
6
6
7
8
8
9
10
10
11
11
12
12
13
13
14
14
15
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
CBP Position
CBP Position
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
b
c
CBP Position
P = 0.05
P = 0.05
Fold change = 1
Fold change = 1
a
0
2
4
6
8
10
Time (s)
0
50
100
150
200
250
Raw xAM (mV)
Pump injecting
(1 second)
Estimated
deadtime
Use as t = 0 s for analysis
URoC1a + Ca
2+
(pre-incubated with Ca
2+
)
1
1
3
5
7
9
15
Jin et al
.
, Ultrasonic reporters of
calcium for deep tissue imaging of cellular signals (2023)
2
with 1 mM CaCl
2
(solid lines) or 5 mM EGTA (dash lines). Curves represent the mean of all biological replicates. (
f
) Nonlinear SBR in dB scale
as a function of pH for URoC1a, control GVs or GVs without GvpC after incubation with 200 μM CaCl
2
(solid lines) or 5 mM EGTA (dash
lines). (
g
) Nonlinear ultrasound signal of URoC1a preincubated with 200 μM CaCl
2
as a function of time after being mixed into 200 μM CaCl
2
.
The time needed for the signal to become stable was used to estimate the dead time for the kineti
cs measurement. Ultrasound data were
acquired with xAM at 547 kPa for (
a
-
c
,
e
-
f
) and at 472 kPa for (
g
). Curves represent the mean of all biological replicates. N = 3 biological
replicates for all panels and each biological replicate has 2 technical replicates for
d, e, f
. Dots represent individual measurement of each
replicate for
a
and
g
, or the mean of two technical replicates for
d, e, f
.
Supplementary Figure 2. Molecular mechanism of URoC.
(
a
) Cryo
-
EM 2D density map of the side view of the dURoC1a GV shell
incubated with 200 μM CaCl
2
or 5 mM EGTA prior to freezing and an integrative model of the Ana GvpA:GvpC (PDB: 8GBS)
31
complex
was overlaid on the GV shell density in the 2D class averages. (
b
) Coomassie
-
stained SDS
-
PAGE gel of OD
500nm
-
matched URoC1a GVs and
dURoC1a with unbound GvpC molecules removed through buoyancy purification after incubation with calcium or EGTA at 37 ̊C. (
c
)
Structure prediction of calcium
-
saturated CaM bound to the URoC GvpC without the linker. The GvpC was colored by the pLDDT
confidence from AlphaFold prediction. The CaM was colored in rainbow from blue to red (N
-
terminus to C
-
terminus) and the CB
P in the
CaM
-
CBP complex was colored orange. The dash line indicates the distance between the center carbon of the C
-
terminal glycine of GvpC and
the that of the N
-
terminal aspartic acid of CaM.
Supplementary Figure 3. Ultrasound imaging of control non
-
calcium
-
sensing GVs in mammalian cells and viability assay.
(
a
) Nonlinear SBR in dB scale as a function of applied acoustic pressure for cells expressing control dURoC1b after incubation
with ionomycin
and EGTA, ionomycin and calcium or calcium only. (
b
) Viability assays of cells transiently expressing URoC1b or jRCaMP1b. The data were
normalized to the mean of each measurement done with jRCaMp1b. The difference in viability measured by Trypan Blue is statist
ically
different but both are above 93% (93.5%
for URoC1b and 99% for jRCaMP1b).
50
37
25
20
75
100
kDa
Ladder
URoC1a+EGTA
dURoC1a
+EGTA
URoC1a+Ca
2+
dURoC1a
+Ca
2+
~ 92 A
dURoC1a - Ca
2+
dURoC1a + Ca
2+
a
b
c
CBP
CaM
N-terminus (GvpC)
C-terminus (GvpC)
0
50
70
90
100
pLDDT
Trypan Blue
ATP Content
Resazurin Reduction
URoC1b
jRCaMP1b
0.0
0.5
1.0
1.5
2.0
Relative Viability
p = 0.07
p = 0.06
p = 2e-4
b
+ion/+Ca
2+
-
ion/+Ca
2+
+ion/+EGTA
400
600
800
0
10
20
30
Acoustic Pressure (kPa)
SBR (dB)
a
dURoC1b
Jin et al
.
, Ultrasonic reporters of
calcium for deep tissue imaging of cellular signals (2023)
3
Supplementary Figure 4. Ultrasound imaging of ligand
-
induced GPCR
-
driven calcium dynamics
in vivo
.
(
a
-
c
) Representative
nonlinear US images of DRUM and DRUM
mut
implants in the brain of mouse 2R1 (
a
), 3N (
b
) and 4R1 (
c
). The relative signal change at t =
-
100/100/200/300/450/600 s compared to t = 0 s was overlaid on a Doppler image. The Scale bar = 1 mm. Color bars represent ult
rasound
signal intensity in the dB scale.
The intensity of the Doppler image represents the blood volume that circulated in each vox
el within an
integration time of 400 ms (see Methods).
Imaging was conducted through polymer cranial windows.
Supplementary Figure 5. Immunofluorescence characterization of DRUM and DRUM
mut
implants
in vivo
.
(
a
) Representative
immunofluorescence micrograph of a 100
-
μ
m
-
thin brain section. Red color shows mCherry fluorescence from the hM3D(Gq) receptor (direct
fusion); green color shows GFP fluorescence from the
gvpNV
genes (P2A chained); blue shows DAPI nuclear stain. (
b
) Top: the nonlinear
ultrasound image of the baseline signals from the implants overlaid on a Doppler image for the same mouse before DCZ injectio
n
(t = 0 s). The
dashed line indicates the effective field of view of xAM due to the side aperture partially blocked by the edge of the
s
kull
/cranial window
.
Bottom: The ultrasound image overlaid with the fluorescence micrograph. All the scale bars = 1 mm. Color bars represent ultra
sound signal
intensity in the dB scale. The nonlinear ultrasound signal was acquired with xAM at 426 kPa (calibrated in water)
and through polymer cranial
windows.
t = -100 s
Mouse 4R1
t = -100 s
t = 100 s
t = 200 s
t = 300 s
t = 450 s
t = 600 s
Nonlinear US ΔS/S
0.2
2
Mouse 2R1
t = -100 s
t = 100 s
t = 200 s
t = 300 s
t = 450 s
t = 600 s
Nonlinear US ΔS/S
0.2
1.2
Mouse 3N
Nonlinear US ΔS/S
0.2
1.8
t = -100 s
t = 100 s
t = 200 s
t = 300 s
t = 450 s
t = 600 s
a
b
c