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)
ABSTRACT
Calcium imaging has enabled major biological discoveries. However, the scattering of light by tissue limits the use of standa
rd
fluorescent calcium indicators in living animals. To address this limitation, we introduce the first genetically encoded ultr
aso
nic
reporter of calcium (URoC). Based on a unique class of air
-
filled protein nanostructures called gas vesicles, we engineered URoC
to produce elevated nonlinear ultrasound signal upon binding to calcium ions. With URoC expressed in mammalian cells, we
de
monstrate noninvasive ultrasound imaging of calcium signaling
in vivo
during drug
-
induced receptor activation. URoC brings
the depth and resolution advantages of ultrasound to the
in vivo
imaging of dynamic cellular function and paves the way for
acoustic biosensing of a broader variety of biological signals.
INTRODUCTION
Calcium is a ubiquitous
signaling molecule
involved in
essential cellular functions in various tissues and organisms,
including synaptic transmission in neurons
1,2
, insulin
regulation
in islet cells
3,4
, immune activation in T
-
cells
5
–
7
and
cytotoxicity in tumors
8,9
. Due to the importance of this
signaling molecule, calcium imaging with state
-
of
-
the
-
art
synthetic and genetically encoded calcium indicators (GECIs)
has proven to be of immense value in biological research
7,10
–
15
.
However, light scattering in tissue makes it challenging to use
fluorescent calcium sensors at scale in intact organisms
16
. In
most cases, it is limited to single
-
cell imaging in small, optically
clear, or surgically accessed tissues, or lower
-
sensitivity and
lower
-
resolution imaging in larger and deeper areas in non
-
transparent organisms
17
. The ability to image calcium at
sufficient resolution and depth in the context of intact living
organisms would promote our understanding of fundamental
physiology and facilitate the development of novel diagnostic
and therapeutic agents.
Compared to other prevalent non
-
invasive imaging modalities
such
as
magnetic
resonance
imaging
18,19
and
photoacoustics
15,20,21
, ultrasound provides an unparalleled
combination of penetration depth (centimeters), imaging
volume (multiple cm
3
), spatiotemporal resolution (~100 μm
and ~1 ms
22,23
), accessibility, and compatibility with freely
moving experimental subjects
23
. Recently, the first genetically
encoded ultrasound contrast agents were introduced based on
gas vesicles (GVs), a unique class of air
-
filled protein
nanostructures derived from buoyant microbes
24
. GVs
produce ultrasound contrast due to the low density and high
compressibility of their gaseous core relative to aqueous
tissues
24
, and their heterologous
expression in bacteria
25,26
or
mammalian cells
26,27
allows GVs to serve as acoustic reporter
genes (ARGs).
GVs’ ultrasound contrast depends on the composition and
mechanics of their protein shell
28,29
. For example, an alpha
-
helical protein called GvpC sits on the outside of the shell and
stiffens the GV against deformation under
acoustic pressure;
removal of this protein results in reversible GV buckling and
enhanced nonlinear contrast
28
–
32
.
The first GV
-
based acoustic
biosensors
–
of protease activity
–
were developed by
engineering GvpC to contain specific protease recognition sites,
such that cleavage by the cognate protease makes the GV shell
more flexible, leading to stronger nonlinear co
ntrast
33
. While
these protease sensors herald an important advance in
biomolecular ultrasound
22,23,34
, they produce only a one
-
time,
irreversible change in acoustic contrast due to the permanent
covalent modification of the sensor and were only shown to
function in bacteria.
Here, we set out to develop the first dynamic, reversible,
allosteric acoustic biosensor
–
an ultrasonic reporter of calcium
(URoC)
–
to enable noninvasive, deep
-
tissue calcium imaging
in mammalian cells (
Fig. 1a
). Inspired by fluorescent GECIs
35
,
we hypothesized that we could engineer GvpC to incorporate
calmodulin (CaM) and a calmodulin
-
binding peptide (CBP),
such that calcium binding would result in a reversible
conformational change that weakens
GvpC binding to the GV
shell. This would make the GV more flexible and increase its
nonlinear acoustic response
(
Fig. 1a
-
b)
. After engineering and
systematically characterizing
a range of URoC designs, we
obtained an acoustic biosensor that produces a 4.7
-
fold
increase in nonlinear contrast in response
to calcium
in vitro
,
with a
sensitivity midpoint of 113 nM. We showed that this
construct can be fully genetically encoded and functional in
mammalian cells, producing a contrast enhancement of more
than 170% in response to elevated intracellular calcium.
To
validate the performance of the URoC
in vivo
, we engineer
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted November 12, 2023.
;
https://doi.org/10.1101/2023.11.09.566364
doi:
bioRxiv preprint