Multiplex,
Quantitative,
High-Resolution
Imaging
of Protein:Protein
Complexes
via Hybridization
Chain
Reaction
Samuel
J. Schulte,
Boyoung
Shin,
Ellen
V. Rothenberg,
and Niles
A. Pierce
*
Cite
This:
ACS Chem.
Biol.
2024,
19, 280−288
Read
Online
ACCESS
Metrics
& More
Article
Recommendations
*
sı
Supporting
Information
ABSTRACT:
Signal
amplification
based
on the mechanism
of
hybridization
chain
reaction
(HCR)
facilitates
spatial
exploration
of
gene
regulatory
networks
by enabling
multiplex,
quantitative,
high-
resolution
imaging
of RNA
and protein
targets.
Here,
we extend
these
capabilities
to the imaging
of protein:protein
complexes,
using
proximity-dependent
cooperative
probes
to conditionally
generate
a
single
amplified
signal
if and
only
if two
target
proteins
are
colocalized
within
the sample.
HCR
probes
and amplifiers
combine
to provide
automatic
background
suppression
throughout
the
protocol,
ensuring
that even
if reagents
bind
nonspecifically
in the
sample,
they
will
not
generate
amplified
background.
We
demonstrate
protein:protein
imaging
with
a high
signal-to-back-
ground
ratio
in human
cells,
mouse
proT
cells,
and
highly
autofluorescent
formalin-fixed
paraffin-embedded
(FFPE)
human
breast
tissue
sections.
Further,
we demonstrate
multiplex
imaging
of three
different
protein:protein
complexes
simultaneously
and validate
that HCR
enables
accurate
and precise
relative
quantitation
of protein:protein
complexes
with
subcellular
resolution
in an anatomical
context.
Moreover,
we establish
a unified
framework
for
simultaneous
multiplex,
quantitative,
high-resolution
imaging
of RNA,
protein,
and protein:protein
targets,
with one-step,
isothermal,
enzyme-free
HCR
signal
amplification
performed
for all target
classes
simultaneously.
■
INTRODUCTION
Methods
for imaging
molecular
complexes
1,2
have
been
comparatively
less explored
than
methods
for imaging
RNA
and protein
targets
3
−
8
yet represent
an important
frontier
for
spatial
exploration
of the interactome.
Generating
one signal
conditional
on the proximity
of two molecules
provides
a
subdiffraction-limit
readout,
in contrast
to independent
imaging
of the
same
two
molecules
with
two
signals.
Protein:protein
complexes
play central
roles
in diverse
cellular
processes
including
transcription,
translation,
signaling,
development,
and
disease.
9
−
12
To
date,
imaging
of
protein:protein
complexes
has predominantly
been
performed
using
proximity
ligation
assays
(PLA)
that
exploit
enzyme-
mediated
ligation
and rolling
circle
amplification,
13
−
17
leading
to challenges
with
both
false-negatives
(formation
of
noncircular
ligation
products
17,18
) and false-positives
(back-
ground
evident
in technical
controls
that omit
one reaction
component
17
), as well
as issues
with
cost
and
variable
enzyme
activity.
13,17
Alternatively,
to avoid
the
use
of
enzymes,
a proximity-based
HCR
approach
has
been
developed
that
uses
a kinetic
trigger
mechanism
to
desequester
an HCR
initiator
if two probes
are bound
to
proximal
target
proteins;
18,19
this approach
has so far been
limited
to 1-plex
applications.
Over
the course
of nearly
two decades,
we have
developed
simple
and robust
HCR
RNA
fluorescence
in situ hybrid-
ization
(RNA-FISH)
and immunofluorescence
(IF)
methods
that
enable
biologists,
drug
developers,
and pathologists
to
perform
multiplex,
quantitative,
high-resolution
imaging
of
RNA
and
protein
targets
in highly
autofluorescent
sam-
ples.
20
−
26
Here,
we sought
to use HCR
principles
to extend
these
benefits
to the imaging
of protein:protein
complexes.
An HCR
amplifier
consists
of two
species
of kinetically
trapped
DNA
hairpins
(h1 and h2) that coexist
metastably
in
solution,
storing
the energy
to drive
conditional
self-assembly
of an HCR
amplification
polymer
upon
exposure
to a cognate
initiator
sequence
(i1; Figure
1A).
27
Using
HCR
RNA-FISH,
an RNA
target
is detected
using
one or more
pairs
of split-
initiator
DNA
probes,
each
carrying
a fraction
of HCR
initiator
i1 (Figure
1B).
24
Probe
pairs
that
hybridize
specifically
to proximal
binding
sites
on the target
RNA
colocalize
a full HCR
initiator
i1 capable
of triggering
HCR
signal
amplification.
Meanwhile,
any individual
probes
that
bind
nonspecifically
in the sample
do not colocalize
full HCR
Received:
July 22, 2023
Revised:
November
18, 2023
Accepted:
December
11, 2023
Published:
January
17,
2024
Articles
pubs.acs.org/acschemicalbiology
© 2024
The Authors.
Published
by
American
Chemical
Society
280
https://doi.org/10.1021/acschembio.3c00431
ACS Chem.
Biol.
2024,
19, 280
−
288
This article is licensed under CC-BY 4.0
initiator
i1 and do not trigger
HCR.
Using
HCR
IF, a protein
target
is detected
using
an unlabeled
primary
antibody
probe,
which
in turn
is detected
by an initiator-labeled
secondary
antibody
probe
that carries
an HCR
initiator
i1 capable
of
triggering
HCR
signal
amplification
(Figure
1C).
26
We hypothesized
that the split-initiator
concept
from
HCR
RNA-FISH
(Figure
1B)
could
be generalized
using
the
antibody
probes
of HCR
IF (Figure
1C)
to enable
simple
and robust
HCR
imaging
of protein:protein
complexes
using
a split-initiator
antibody
probe
pair in conjunction
with
a new
proximity
probe
(Figure
1D).
Here,
we demonstrate
that this
Figure
1.
Applying
HCR
principles
to enable
simple
and robust
imaging
of protein:protein
complexes.
(A) HCR
mechanism.
Stars
denote
fluorophores.
Arrowhead
indicates
the 3
′
end of each
strand.
(B) HCR
RNA-FISH:
an RNA
target
is detected
using
a pair of split-initiator
DNA
probes,
each
carrying
a fraction
of HCR
initiator
i1. (C) HCR
IF: a protein
target
is detected
using
an unlabeled
primary
antibody
probe
and an initiator-labeled
secondary
antibody
probe
carrying
HCR
initiator
i1. (D)
HCR
protein:protein
imaging:
a protein:protein
target
complex
is detected
with
a pair of unlabeled
primary
antibodies,
a pair of split-initiator
secondary
antibodies
each
carrying
a fraction
of HCR
initiator
i1, and a proximity
probe.
Figure
2.
Imaging
protein:protein
complexes
using
HCR.
(A) Three-stage
protocol.
Detection
stage:
unlabeled
primary
antibody
probes
bind
to
protein
targets
1 and 2; wash;
split-initiator
secondary
antibody
probes
p1 and p2 bind
to primary
antibody
probes;
wash.
Proximity
stage:
if p1
and p2 are proximal,
a proximity
probe
hybridizes
to the proximity
domains
of p1 and p2 to colocalize
full HCR
initiator
i1. Amplification
stage:
colocalized
full HCR
initiator
i1 triggers
self-assembly
of fluorophore-labeled
HCR
hairpins
into
a tethered
fluorescent
amplification
polymer;
wash.
(B) Multiplexing
timeline.
The
same
three-stage
protocol
is used
independent
of the number
of protein:protein
target
complexes.
ACS
Chemical
Biology
pubs.acs.org/acschemicalbiology
Articles
https://doi.org/10.1021/acschembio.3c00431
ACS Chem.
Biol.
2024,
19, 280
−
288
281
combination
of proximity-dependent
cooperative
probes
and
metastable
HCR
amplifiers
enables
multiplex,
quantitative,
high-resolution
imaging
of protein:protein
complexes,
includ-
ing full compatibility
with
HCR
RNA-FISH
and HCR
IF.
■
RESULTS
AND
DISCUSSION
HCR
Imaging
of Protein:Protein
Complexes
Using
a
Three-Stage
Protocol.
HCR
imaging
of protein:protein
complexes
is performed
using
the
three-stage
protocol
summarized
in Figure
2A. In the detection
stage,
two protein
targets
are detected
with
unlabeled
primary
antibody
probes
that are in turn detected
by a pair of split-initiator
secondary
antibody
probes
(p1 and p2) each
carrying
a fraction
of HCR
initiator
i1 and a proximity
domain.
In the proximity
stage,
if
the two protein
targets
are colocalized
in the sample,
then
the
proximity
probe
is able
to hybridize
to p1 and
p2 to
colocalize
a full HCR
initiator
i1 capable
of triggering
HCR
signal
amplification.
Note
that the proximity
probe
creates
a
cooperative
probe
junction
(Figure
1D)
inspired
by the
cooperative
probe
junction
created
in HCR
RNA-FISH
(Figure
1B),
with
the DNA
proximity
probe
taking
the place
of the RNA
target.
Any
split-initiator
probes
that
bind
nonspecifically
or to isolated
protein
targets
in the sample
can
hybridize
to the proximity
probe
but will not colocalize
a full
HCR
initiator
i1 and
will
not
trigger
HCR.
In the
amplification
stage,
each
colocalized
full HCR
initiator
i1
triggers
self-assembly
of metastable
fluorophore-labeled
HCR
hairpins
(h1 and h2) into a tethered
fluorescent
amplification
polymer
to generate
an amplified
signal
at the site of the
protein:protein
target
complex.
Imaging
Protein:Protein
Complexes
in Human
Cells,
Mouse
proT
Cells,
and
FFPE
Human
Breast
Tissue
Sections.
To evaluate
the performance
of our split-initiator
approach
for imaging
protein:protein
complexes,
we com-
pared
the fluorescence
intensity
between
three
pairs
of
biological
sample
types
using
the same
imaging
settings
for
both
sample
types.
Positive
samples
are expected
to form
the
Figure
3.
Imaging
protein:protein
complexes
in human
cells,
mouse
proT
cells,
and FFPE
human
breast
tissue
sections.
(A,B)
Imaging
β
-
catenin:E-cadherin
target
complex
in A-431
cells
expressing
β
-catenin
and E-cadherin
(panel
A) or HeLa
cells
expressing
N-cadherin
instead
of
E-cadherin
(panel
B). (C,D)
Imaging
RUNX1:PU.1
target
complex
in Scid.adh.2C2
mouse
proT
cells
retrovirally
transduced
with
a PU.1-
expressing
vector
(panel
C) or an empty
vector
(panel
D). (E,F)
Imaging
β
-catenin:E-cadherin
target
complex
in 5
μ
m FFPE
human
breast
tissue
sections
from
the same
patient:
normal
(panel
E) or invasive
lobular
carcinoma
(panel
F). All panels:
confocal
image;
single
optical
section;
0.18
×
0.18
×
0.8
μ
m pixels
(panels
A
−
D)
or 0.57
×
0.57
×
3.3
μ
m pixels
(panels
E,F).
Signal-to-backround
ratio
for each
row (mean
±
SEM
for representative
regions
of
N
= 3 replicate
samples).
See sections
S2.2
−
S2.4
for additional
data.
ACS
Chemical
Biology
pubs.acs.org/acschemicalbiology
Articles
https://doi.org/10.1021/acschembio.3c00431
ACS Chem.
Biol.
2024,
19, 280
−
288
282
protein:protein
complex
of interest;
negative
samples
are
expected
to have
minimal
or no formation
of the
protein:protein
complex
of interest.
For each
pair of sample
types,
we calculate
an estimated
signal-to-background
ratio
using
the positive
sample
type
to estimate
signal
plus
background
and
the
negative
sample
type
to estimate
background.
This
approach
yields
a conservative
estimate
of
performance,
as characterizing
background
in a sample
containing
little
or no protein:protein
target
complex
places
an upper
bound
on background
and hence
a lower
bound
on
the signal-to-background
ratio.
First,
we compared
the fluorescence
intensity
for the
β
-
catenin:E-cadherin
complex
in A-431
and
HeLa
adherent
human
cell lines.
While
A-431
cells
form
the
β
-catenin:E-
cadherin
complex
at the cell
membrane
of intercellular
junctions,
28
HeLa
cells
express
N-cadherin
rather
than
E-
cadherin
29,30
and
therefore
lack
the
β
-catenin:E-cadherin
complex.
As expected,
A-431
cells
(Figure
3A)
display
a
strong
signal
at intercellular
junctions
and HeLa
cells
display
no visible
staining
(Figure
3B),
with
a signal-to-background
ratio
of 26
±
4 between
the two cell lines
(mean
±
SEM
for
representative
regions
of
N
= 3 replicate
wells
on a slide).
Next,
we imaged
Scid.adh.2C2
31
mouse
proT
cells
in
search
of the RUNX1:PU.1
target
complex.
The Scid.adh.2C2
cell line has emerged
as a useful
proT
cell line for studying
T
cell
development,
with
exogenous
introduction
of PU.1
protein
capable
of reverting
the cell
line
to an earlier
developmental
time
point,
in part
via direct
or indirect
interactions
between
PU.1
and
other
proteins
such
as
RUNX1.
32
−
34
Because
the Scid.adh.2C2
cell line does
not
endogenously
express
the PU.1
protein,
31
Scid.adh.2C2
cells
cannot
natively
form
the RUNX1:PU.1
complex.
When
the
Scid.adh.2C2
cell line is retrovirally
transduced
with
PU.1,
it
is unknown
whether
PU.1
forms
a complex
with
RUNX1
or
interacts
less
directly.
33
Here,
imaging
the RUNX1:PU.1
target
complex,
we observe
signal
in cells
retrovirally
transduced
with
a PU.1-containing
vector
(Figure
3C)
and
no visible
staining
for cells
retrovirally
transduced
with
an
empty
vector
(Figure
3D),
with
a signal-to-background
ratio
of 15
±
3 between
the two experiment
types
(mean
±
SEM
for representative
regions
of
N
= 3 replicate
wells
on a slide).
These
results
provide
evidence
that
RUNX1
and PU.1
are
spatially
colocalized
in PU.1-transduced
Scid.adh.2C2
cells
and are not merely
logically
linked.
To test performance
in highly
autofluorescent
samples,
we
detected
the
β
-catenin:E-cadherin
complex
in normal
and
pathological
FFPE
human
breast
tissue
sections.
The
β
-
catenin:E-cadherin
complex
is robustly
formed
in normal
breast
epithelial
cells,
but the expression
of and interaction
between
the
β
-catenin
and E-cadherin
proteins
is interrupted
when
breast
epithelial
cells
become
cancerous
in the invasive
lobular
carcinoma
disease
process.
35,36
We obtained
paired
normal
and invasive
lobular
carcinoma
FFPE
breast
tissue
sections
from
the same
patient
and evaluated
them
for the
β
-
catenin:E-cadherin
complex,
observing
a strong
signal
in
normal
breast
tissue
(Figure
3E) and no visible
staining
in
cancerous
tissue
(Figure
3F),
with
a signal-to-background
ratio
of 30
±
3 between
the two tissue
types
(mean
±
SEM
for representative
regions
of
N
= 3 replicate
sections).
In summary,
protein:protein
complexes
are imaged
with
high
signal-to-background
across
three
different
paired
sample
types,
including
highly
autofluorescent
FFPE
tissues.
Multiplex
Protein:Protein
Imaging.
HCR
RNA-FISH
and HCR
IF enable
straightforward
multiplexing
for RNA
and
protein
targets
to allow
multidimensional
analyses
of
gene
expression
in an anatomical
context.
20
−
22,24
−
26
To
likewise
enable
multiplex
imaging
of protein:protein
com-
plexes,
we used
NUPACK
37,38
to design
proximity
probes
for
three
orthogonal
HCR
amplifiers.
Figure
4 demonstrates
multiplex
protein:protein
imaging
for three
target
complexes
that
localize
to different
compartments
of A-431
adherent
human
cells:
cytoskeletal
α
-tubulin:
β
-tubulin
complex,
mem-
branous
β
-catenin:E-cadherin
complex,
and nuclear
speckle
SC35:SON
complex.
High
signal-to-background
is observed
for all three
protein:protein
target
complexes,
with
back-
ground
estimated
based
on technical
control
experiments
that
omit
the primary
and secondary
antibody
probes
for one
protein
or the other
within
a given
complex
(see
Table
S13
for details).
Multiplexing
is straightforward
using
a three-stage
protocol
independent
of the number
of protein:protein
target
complexes
(Figure
2B):
all protein
targets
are detected
in
parallel,
proximity
is verified
for all protein
target
pairs
in
parallel,
and amplification
is performed
for all colocalized
full
HCR
initiators
in parallel.
qHCR
Imaging:
Relative
Quantitation
of Protein:-
Protein
Complexes
with
Subcellular
Resolution.
We
have
previously
demonstrated
that
HCR
imaging
enables
accurate
and precise
relative
quantitation
of both
RNA
and
protein
targets
with
subcellular
resolution
in an anatomical
context,
generating
an amplified
signal
that
scales
approx-
imately
linearly
with
the number
of target
molecules
per
imaging
voxel.
24
−
26
Here,
we validate
that
the proximity
Figure
4.
Multiplex
imaging
of protein:protein
complexes.
Three-channel
confocal
image
in A-431
cells;
single
optical
section;
0.18
×
0.18
×
0.8
μ
m pixels.
Ch1:
cytoskeletal
α
-tubulin:
β
-tubulin
complex
(Alexa488).
Ch2:
membranous
β
-catenin:E-cadherin
complex
(Alexa546).
Ch3:
nuclear
speckle
SC35:SON
complex
(Alexa647).
Signal-to-background
ratio
for each
channel
(mean
±
SEM
for representative
regions
of
N
= 3
replicate
wells
on a slide).
See section
S2.5
for additional
data.
ACS
Chemical
Biology
pubs.acs.org/acschemicalbiology
Articles
https://doi.org/10.1021/acschembio.3c00431
ACS Chem.
Biol.
2024,
19, 280
−
288
283
probe
and
split-initiator
antibody
probe
pair
preserve
the
quantitative
nature
of HCR
imaging
for protein:protein
target
complexes.
To test
relative
quantitation,
we detect
each
protein
in the complex
with
an unlabeled
primary
antibody
probe
as usual
and then
redundantly
detect
each
primary
antibody
probe
with
two batches
of split-initiator
secondary
antibody
probes,
where
each
batch
interacts
with
a different
proximity
probe
and
triggers
a different
spectrally
distinct
HCR
amplifier
(Figure
5A),
yielding
a two-channel
image
(Figure
5B).
If HCR
signal
scales
approximately
linearly
with
the number
of target
protein:protein
complexes
per voxel,
a
two-channel
scatter
plot
of normalized
voxel
intensities
will
yield
a tight
linear
distribution
with
zero
intercept.
25
Consistent
with
expectation,
we observe
high
accuracy
(linearity
with
zero
intercept)
and precision
(scatter
around
the line)
for subcellular
voxels
in both
cultured
human
cells
(Figure
5C; top)
and highly
autofluorescent
FFPE
human
breast
tissue
(Figure
5C; bottom).
Simultaneous
Multiplex
Imaging
of Protein,
Pro-
tein:Protein,
and
RNA
Targets.
We have
previously
shown
that
HCR
RNA-FISH
and
HCR
IF enable
multiplex,
quantitative,
high-resolution
RNA
and
protein
imaging
in
highly
autofluorescent
samples.
26
Here,
we demonstrate
compatible
multiplex
imaging
of protein,
protein:protein,
and RNA
targets
using
initiator-labeled
antibody
probes
for
protein
targets,
proximity
probes
and split-initiator
antibody
probe
pairs
for protein:protein
targets,
and
split-initiator
DNA
probe
pairs
for RNA
targets
with
simultaneous
HCR
signal
amplification
for all target
classes
(Figure
6A).
In A-
431
adherent
human
cells,
mitochondrial
HSP60
protein
targets,
cytoskeletal
α
-tubulin:
β
-tubulin
protein:protein
target
complexes,
and
nuclear
U6 RNA
targets
are all imaged
simultaneously
(Figure
6B)
with
high
signal-to-background
(see
Table
S18 for additional
details).
Unified
Framework
for
Multiplex,
Quantitative,
High-Resolution
Imaging.
We
have
shown
that
HCR
imaging
provides
a unified
framework
for multiplex,
quantitative,
high-resolution
imaging
of RNA
targets,
protein
targets,
and protein:protein
target
complexes
simultaneously.
A high
signal-to-background
ratio
is achieved
even
in highly
autofluorescent
samples.
As a natural
property
of this
method,
the amplified
signal
scales
approximately
linearly
with
target
abundance,
enabling
accurate
and precise
relative
quantitation
of each
target
with
subcellular
resolution
in an
anatomical
context.
By contrast,
the amplified
signal
using
PLA
methods
does
not scale
linearly
with
target
abundance.
16
Using
the validated
proximity
probes
presented
here,
up to
three
protein:protein
target
complexes
can
be imaged
simultaneously,
in combination
with
RNA
or protein
targets
of choice.
Multiplex
HCR
protein:protein
imaging
is achieved
using
a three-stage
protocol
(detection
stage,
proximity
stage,
and amplification
stage)
involving
two overnight
incubations.
Simultaneous
multiplex
imaging
of protein,
protein:protein,
and
RNA
targets
is achieved
using
a four-stage
protocol
(protein
detection
stage,
proximity
stage,
RNA
detection
stage,
and
amplification
stage)
involving
three
overnight
incubations.
The
use of overnight
incubations
reflects
our
longstanding
focus
on developing
versatile
protocols
that are
suitable
for diverse
sample
types
including
whole-mount
vertebrate
embryos
while
allowing
researchers
to maintain
a
normal
sleep
schedule.
20,21,24,26
If desired,
HCR
imaging
protocols
can be optimized
to use shorter
incubation
times
in
sample
types
of interest.
39
−
41
Automatic
Background
Suppression
Throughout
the
Protocol.
As is the case
for HCR
RNA-FISH,
the use of
split-initiator
probes
during
the
detection
stage
and
metastable
HCR
hairpins
during
the amplification
stage
provides
automatic
background
suppression
throughout
the
protocol,
ensuring
that even
if reagents
bind
nonspecifically
Figure
5.
qHCR
imaging:
relative
quantitation
of protein:protein
complexes
with
subcellular
resolution
in an anatomical
context.
(A) Two-
channel
redundant
detection
of a protein:protein
complex:
each
target
protein
is detected
by an unlabeled
primary
antibody
probe
and two
batches
of secondary
antibody
probes
that interact
with
orthogonal
proximity
probes
to colocalize
full HCR
initiators
that trigger
orthogonal
spectrally
distinct
HCR
amplifiers
(Ch1,
Alexa546;
Ch2,
Alexa647).
(B) Two-channel
confocal
images;
single
optical
sections.
Top:
β
-catenin:E-
cadherin
complex
in A-431
cells
(0.18
×
0.18
×
0.8
μ
m pixels).
Bottom:
β
-catenin:E-cadherin
complex
in a 5
μ
m FFPE
normal
human
breast
tissue
section
(0.57
×
0.57
×
3.3
μ
m pixels).
(C) High
accuracy
and precision
for protein:protein
relative
quantitation
in an anatomical
context.
Highly
correlated
normalized
signal
(Pearson
correlation
coefficient,
r
) for subcellular
voxels
in the indicated
regions
in panel
B. Top:
2.0
×
2.0
×
0.8
μ
m voxels.
Bottom:
2.0
×
2.0
×
3.3
μ
m voxels.
Accuracy:
linearity
with
zero intercept.
Precision:
scatter
around
the line. See section
S2.6
for additional
data.
ACS
Chemical
Biology
pubs.acs.org/acschemicalbiology
Articles
https://doi.org/10.1021/acschembio.3c00431
ACS Chem.
Biol.
2024,
19, 280
−
288
284