of 11
Proteins, Air and Water: Reporter Genes for Ultrasound and
Magnetic Resonance Imaging
George J. Lu
1,#
,
Arash Farhadi
2,#
,
Arnab Mukherjee
3
, and
Mikhail G. Shapiro
1,*
1
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
CA 91125, USA
2
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA
91125, USA
3
Department of Chemical Engineering, University of California, Santa Barbara, Santa Barbara,
CA 93106, USA
Abstract
A long-standing goal of molecular imaging is to visualize cellular function within the context of
living animals, necessitating the development of reporter genes compatible with deeply penetrant
imaging modalities such as ultrasound and magnetic resonance imaging (MRI). Until recently, no
reporter genes for ultrasound were available, and most genetically encoded reporters for MRI were
limited by metal availability or relatively low sensitivity. Here we review how these limitations are
being addressed by recently introduced reporter genes based on air-filled and water-transporting
biomolecules. We focus on gas-filled protein nanostructures adapted from buoyant microbes,
which scatter sound waves, perturb magnetic fields and interact with hyperpolarized nuclei, as
well as transmembrane water channels that alter the effective diffusivity of water in tissue.
Introduction
Molecular imaging seeks to visualize the location and function of cells and molecules within
a variety of biological settings, including deep inside intact animals. Within this context,
much of the powerful repertoire of genetically encoded reporters and sensors based on green
fluorescent protein (GFP) and its analogues has limited utility due to the strong scattering
and absorption of light by tissue. In his influential 2003 perspective titled “Imagining
Imaging’s Future” Roger Tsien recognized this limitation and predicted that “the prevalence
*
Correspondence should be addressed to MGS: mikhail@caltech.edu, Phone: 626-395-8588, 1200 E. California Blvd, MC 210-41,
Pasadena, CA 91125.
#
Equal contribution
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AUTHOR CONTRIBUTIONS
All authors wrote the manuscript.
COMPETING INTERESTS
The authors declare no competing financial interests.
HHS Public Access
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Curr Opin Chem Biol
. 2018 August ; 45: 57–63. doi:10.1016/j.cbpa.2018.02.011.
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and success of GFP indicate that comparable revolutions might result from genetic
sequences that robustly encode image contrast for other methods.” [
1
] Indeed, reporter genes
for noninvasive deep-tissue imaging modalities such as ultrasound and magnetic resonance
imaging (MRI) could have great value in both basic biomedical research and the
development of cellular diagnostics and therapeutics. However, no such reporter genes have
so far achieved the prevalence of GFP, and new ideas are therefore needed to spark the
envisioned revolutions.
This review summarizes progress on two recently introduced classes of genetically encoded
contrast agents for ultrasound and MRI that operate via new biophysical principles. One
class is based on gas-filled proteins derived from buoyant microbes, which serve as the first
reporter genes for ultrasound and produce contrast in susceptibility-based MRI and
hyperpolarized xenon MRI. The second class is based on water channels such as aquaporin,
whose overexpression in mammalian cells is brightly detectable with diffusion-based MRI.
These reporter genes use fundamental properties of air and water to introduce new forms of
contrast to the field of molecular imaging, providing unique capabilities for visualizing
cellular function
in vivo
.
Proteins with air: gas vesicles as acoustic reporter genes
Until very recently, no reporter genes were available for ultrasound, a versatile modality
capable of imaging centimeters-deep into soft tissue with spatial and temporal resolution on
the order of 100 μm and 1 ms. In addition to being one of the most widely used modalities in
medicine, ultrasound scales to smaller model organisms to enable basic and translational
research. Recent advances in equipment and signal processing have provided ultrasound
with the ability to image faster (down to tens of μs) and more precisely (below 10 μm with
super-localization techniques) [
2
4
] (ref
2*
). The classic contrast agents used in ultrasound
are micron-sized bubbles of gas stabilized by a lipid or protein shell, which scatter sound
waves due to their differential density and compressibility relative to water [
5
]. Could
similar physical principles be embodied in a genetic sequence?
In 2014, it was discovered that a unique class of gas-filled protein nanostructures known as
gas vesicles (GVs), which evolved in certain photosynthetic microbes as a means to achieve
buoyancy in water, could produce ultrasound contrast *[
6
]. GVs are cylindrical or spindle-
shaped gas-filled compartments with dimensions on the order of 200 nm, surrounded by a 2-
nm thick protein shell [
7
] (Fig. 1A, B). This shell allows gases dissolved in the surrounding
media to exchange freely in and out, while preventing water from forming a liquid inside the
GV due to the strong hydrophobicity of the shell’s interior face. GVs are encoded in diverse
organisms by operons of 8–14 genes, comprising a mixture of structural proteins and
assembly factors [
8
]. Because they contain gas, it was hypothesized that GVs could scatter
sound waves and produce ultrasound contrast. Indeed, this ability was demonstrated using
GVs isolated from cyanobacteria and haloarchaea [
6
]. Building on this initial discovery,
several other studies have been undertaken to understand the acoustic properties of GVs [
9
],
to engineer them through genetic and biochemical modifications [
10
,
11
] (*ref.
10
), to devise
ultrasound imaging techniques tailored to distinguish their signal from background [
12
], and
to characterize their
in vivo
biodistribution as purified, injectable agents [
13
]. These studies
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revealed remarkable non-linear acoustic properties and engineering versatility, enabling
selective detection, multiplexed imaging and molecular targeting.
In parallel, a major effort was undertaken to express GVs heterologously as acoustic reporter
genes, initially in commensal and pathogenic bacteria being developed as microbial
diagnostic and therapeutic agents **[
14
]. This was challenging because nearly a dozen
genes need to be co-expressed at correct stoichiometry, fold into functional proteins and self-
assemble into correctly shaped nanostructures. Initial attempts to transplant GV-forming
genes from cyanobacteria to
Escherichia coli
were unsuccessful, while the expression of a
more
E. coli
-compatible gene cluster from
Bacillus megaterium
[
15
] yielded small GVs
producing little ultrasound contrast. The breakthrough was to combine structural genes from
cyanobacteria with assembly factor genes from
B. megaterium
, resulting in correctly
assembled GVs with size, shape and acoustic properties producing strong ultrasound
contrast (Fig. 1, C–D).
E. coli
and
Salmonella typhimurium
expressing the resulting acoustic
reporter genes could be imaged at concentrations on the order of 10
8
cells ml
−1
, representing
a volume fraction of approximately 0.01%, and could be visualized
in vivo
in the
gastrointestinal (GI) tract (Fig. 1E) and inside tumor xenografts. Furthermore, it was
possible to distinguish two versions of the gene cluster from each other based on their
acoustic properties, enabling multiplexed imaging (Fig. 1F).
While this initial development of acoustic reporter genes opens exciting new possibilities in
the fields of ultrasound and molecular imaging, additional work must be done to maximize
the acoustic contrast obtained from GV expression and apply this technology to imaging
microbes
in vivo
in real basic biology, diagnostic and therapeutic scenarios. In addition,
another breakthrough is needed to express GVs in mammalian cells, which poses additional
challenges due to the differences in gene expression machinery between eukaryotes and
prokaryotes.
Air in a magnet: gas vesicles as reporter genes for susceptibility-based and
hyperpolarized MRI
Relative to ultrasound, much more work has been done to develop reporter genes for MRI,
starting with pioneering work on enzymes that convert synthetic T
1
contrast agents to forms
with higher relaxivity[
16
], overexpression of the iron storage protein ferritin [
17
,
18
] and the
detection of proteins with large numbers of exchangeable protons using chemical exchange
saturation transfer (CEST) [
19
]. However, while these technologies continue to improve,
they face intrinsic limitations due to their reliance on metal co-factors, and difficulty in
being distinguished from background tissue contrast at low concentrations [
20
]. New
contrast mechanisms are therefore needed to help overcome these limitations.
Air is well known as a source of contrast in T
2
-based MRI, with air-filled tissues such as
lungs and nasal cavities generating unwanted image “blackouts” due to the differential
magnetic susceptibility of air and water (set apart by ~ 9 ppm). Could air be turned into a
source of genetically encodable MRI contrast? This concept was recently demonstrated
using GVs, whose air contents produce microscale magnetic field gradients in their vicinity
(Fig. 2A), efficiently dephasing aqueous protons and resulting in contrast in T
2
-weighted
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and quantitative susceptibility images (Fig. 2B) **[
21
]. However, what really distinguishes
this contrast from that produced by metal-based T
2
reporters is that it can be made to
disappear: when ultrasound is applied above a threshold level, the GVs collapse, the air
inside them dissolves, and their MRI contrast vanishes (Fig. 2C). This acoustically
modulated MR imaging paradigm allows the contrast produced by the GVs to be
distinguished from background, solving a major problem with existing contrast agents. In
addition, it allows the acoustic properties of GVs to be utilized in MRI, for example
enabling multiplexed imaging via serial acoustic collapse of GVs with engineered collapse
thresholds (Fig. 2, D–E).
Is it possible to go beyond air? A major limitation on the sensitivity of conventional MRI
arises from the low equilibrium polarization of nuclear spins (10
−5
–10
−6
), meaning that only
a few of the available spins in a biological sample contribute to the MRI signal. This
limitation can be overcome with hyperpolarization – the preparation of certain nuclear spins
in a non-equilibrium state of near-unity polarization with the applied field[
22
24
]. One such
nucleus used in biological imaging is the noble gas
129
Xe, which is biocompatible and can
be delivered to tissues via inhalation. In 2006, it was discovered that synthetic contrast
agents acting on
129
Xe can be detected at nanomolar concentrations using hyperpolarized
CEST (HyperCEST)[
25
], inspiring the search for reporter genes that could serve a similar
purpose. As a protein compartment allowing its gas contents to be in dynamic exchange with
the surrounding media, GVs made a natural candidate (Fig. 2E). Indeed, it was shown in
2014 that GVs can be detected with HyperCEST at picomolar concentrations – a level
unprecedented for MRI contrast agents (Fig. 2F) *[
26
]. Although other xenon-binding
proteins capable of HyperCEST have since been identified [
27
,
28
] (* for ref
27
), GVs
continue to provide unique capabilities, including elastic contrast via their ability to scale
129
Xe binding capacity according to the ideal gas law to match the concentration of xenon in
solution – an important property for
in vivo
applications with variable xenon delivery. In
addition to
129
Xe, other hyperpolarized nuclei such as
13
C may also be substrates for
reporter gene detection [
29
,
30
].
Calling nature’s plumber: water channels as reporter genes for diffusion-
weighted MRI
In addition to the advances described above, one of the newest forms of MRI contrast arises
from a far more ubiquitous class of proteins: aquaporins. These transmembrane channels,
present in all domains of life, enable the selective diffusion of water across lipid bilayers,
providing a way for cells to transport water, deform and respond to changes in osmolarity
[
31
]. In a 2016 study, it was hypothesized that the overexpression of aquaporins in
mammalian tissues would significantly increase the effective diffusivity of water in such
tissues by minimizing the impact of the primary diffusion barrier – cell membranes (Fig.
3A) **[
32
]. This was expected to produce contrast in diffusion-weighted MRI. In fact,
overexpressing aquaporin 1 (AQP1) in several mammalian cell lines produced increases of
up to 200% in the apparent diffusion coefficient of water when these cells were imaged as
pellets (Fig. 3B). Concentrations of the protein below 500 nM were sufficient for detection.
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Even when maximally expressed, AQP1 was found to have no effect on cell viability or
morphology, as expected for a passive water channel under osmotically balanced conditions.
A key insight to making aquaporin work as a reporter involved setting the diffusion
timescale in the MRI sequence to longer than 100 ms, allowing water to effectively sample
the cell membrane as a diffusion barrier. Another important aspect of this contrast
mechanism is that it involves water transport among cells and therefore depends on the
fraction of cells in a given voxel expressing the reporter gene. Simulation and experiments
showed that this fraction can be as small at 10%. The functionality of AQP1 as a reporter
gene
in vivo
was demonstrated with its chemically induced expression in a brain tumor
model (Fig. 3C). Together with another water channel published shortly after this work
*[
33
], aquaporin represents a new category of MRI reporter genes that is sensitive, compact,
autologous to its host, nonmetallic and non-toxic.
Outlook
As described in this article, the armory of reporter genes available for non-invasive
molecular imaging has expanded to include proteins with air and water as their primary
sources of contrast. By leveraging the unique properties of these materials, reporter genes
are for the first time available for ultrasound imaging, acoustically modulated susceptibility-
based MRI, hyperpolarized xenon MRI, and diffusion-weighted imaging. Whether any of
these constructs achieves the impact of GFP remains to be seen. Since the initial
demonstration of GFP as a fluorescent reporter, the engineering of its genetic sequence and
mining of additional proteins with similar properties produced the toolkit that today is
indispensable in biological imaging. Analogously, advances in the molecular engineering of
GVs are needed to maximize the impact of these genetic constructs in deep-tissue imaging.
The native biodiversity of GVs sets the stage for such engineering: for example, the particle
volume of GVs varies by an order of magnitude between those encoded in haloarchaea and
B. megaterium
[
11
], and could potentially be reduced or expanded further through
mutagenesis [
34
]. Exploiting this phenotypic diversity could yield GVs with material
properties optimized for each imaging modality: the ability of the protein shell to
harmonically scatter sound waves for ultrasound imaging, the optimal particle size and shape
for susceptibility-based MRI, and the optimal diffusivity of gas across the shell for
hyperpolarized xenon MRI. Additionally, a mechanistic understanding of the GV assembly
process, especially in the context of heterologous expression in target cell types, will be
important for their utility as imaging agents in challenging
in vivo
settings. Likewise,
tapping into the evolutionary heritage and diversity of aquaporin genes [
35
] could yield
proteins with enhanced or conditional water transport for improved sensitivity or functional
sensing. Revolutions in biomolecular ultrasound and several forms of MRI await the results
of this further research.
Acknowledgments
We thank members of the Shapiro Laboratory for helpful discussions. Related work in the Shapiro laboratory is also
supported by the Heritage Medical Research Institute, the National Institutes of Health, the Defense Advanced
Research Projects Agency, the Jacobs Institute for Molecular Engineering in Medicine, the Caltech Center for
Environmental Microbial Interactions, the Human Frontiers Science Program, the Burroughs Wellcome Fund, the
Lu et al.
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Pew Scholarship in the Biomedical Sciences, the Sontag Foundation, the Packard Fellowship for Science and
Engineering. A.F. is supported by the Natural Sciences and Engineering Research Council of Canada PGSD.
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Figure 1. Acoustic Reporter Genes
(
a
) Transmission electron micrograph (TEM) of an individual gas vesicle from
A. flos-
aquae.
(
b
) Diagram of the structure and composition of a gas vesicle. (
c
) Engineered genetic
construct, ARG1, comprising genes from
A. flos aquae
(green) and genes from
B.
megaterium
(gray) to produce ultrasound-detectable gas vesicles in heterologous bacteria.
(Bottom) Ultrasound images of
E. coli
and
S. typhimurium
expressing ARG1 or the
luminescent LUX operon. (
d
) TEM of a an
E. coli
Nissle 1917 cell expressing ARG1. (
e
)
Ultrasound image of live mouse with ARG1-expressing
E. coli
arranged in the colon as
indicated in diagram. Color map represents collapse-subtracted contrast within the colon
region of interest (outlined in blue), overlaid on grayscale anatomical image. (
f
) Ultrasound
images of
E. coli
expressing ARG1 or ARG2 before and after the application of two
different collapse pressures. (Bottom) Unmixed contrast maps corresponding to each type of
bacterium. Scale bars represent 150 nm (a), 2 mm (c), 500 nm (d), 2.5 mm (e) and 2 mm (f).
Data adapted with permission from Ref.
14
.
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Figure 2. Gas-based MRI reporters
(
a
) Finite element model of the magnetic field gradient produced by a single air-filled GV in
water exposed to a horizontal magnetic field (B
0
= 7 Tesla). (
b
) Quantitative susceptibility
map (QSM), T2*-weighted (T2*w) and T2-weighted (T2w) images of wells containing GVs
at concentrations ranging from 0 to 1.1 nM. The QSM color scale ranges linearly from −2 to
+50 parts per billion (ppb), and T2*w and T2w images have linear scales adjusted for
optimal contrast. (
c
) Diagram and representative images of acoustically modulated MRI
imaging of GVs injected in mouse brain. A T2*-weighted image is acquired followed by a
second one after the application of ultrasound, and the difference between these images
reveals contrast specific to the GVs. This difference image is overlaid on an anatomical
image. (
d
) Schematic of the pressure-based multiplexing paradigm, wherein sequential
ultrasound pulses are applied between MR images. The low-pressure ultrasound (Low US)
selectively collapses Ana
ΔC
GVs and eliminates their MRI contrast; subsequently, high-
pressure ultrasound (High US) collapses Ana
WT
GVs. (
e
) Representative QSM images taken
before ultrasound application in wells containing Ana
WT
, Ana
ΔC
or a 1:1 mixture of the two,
followed by an unmixed image indicating the quantity of each of the two populations in each
well. (
f
) Diagram of
129
Xe exchanging between the gas phase inside the GV and the
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dissolved phase in surrounding media. A radiofrequency (RF) pulse at the chemical shift of
129
Xe in the GV saturates its signal, which is transferred by exchange to the bulk. (
g
)
Saturation contrast image of a three-compartment phantom containing 400pM GVs, 100pM
GVs and buffer. Panels (a)–(e) adapted with permission from ref.
21
. Panel (g) adapted with
permission from ref.
26
.
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Figure 3. Diffusion-based MRI reporters
(
a
) Illustration of diffusion-based reporter gene mechanism. Cells overexpressing aquaporin
have facilitated transport of water in and out of the cell, thereby increasing the effective
diffusivity. (
b
) Diffusion-weighted MR image of pellets of CHO, U87 and N2a cells
genetically modified to express AQP1 or GFP. Darker voxels indicate faster diffusion. (
c
)
Illustration of
in vivo
injection of CHO cells to form brain tumors comprising cells with
doxycycline (dox)-dependent expression of GFP or AQP1; after tumor growth, and
preliminary imaging, expression is induced and the mice are imaged again. (
d
) Diffusion-
weighted MR images of mouse before and after dox administration. The tumor expressing
AQP1 appears darker after gene expression is induced. Data adapted with permission from
ref.
32
.
Lu et al.
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