Recombinant Probes for Visualizing Endogenous
Synaptic Proteins in Living Neurons
Garrett G. Gross,1,7Jason A. Junge,2,7Rudy J. Mora,2,7Hyung-Bae Kwon,3C. Anders Olson,4Terry T. Takahashi,1
Emily R. Liman,2Graham C.R. Ellis-Davies,5Aaron W. McGee,6Bernardo L. Sabatini,3Richard W. Roberts,1,*
and Don B. Arnold2,*
1Department of Chemistry
2Department of Biology
University of Southern California, Los Angeles, CA 90089, USA
3Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA
4Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90090; USA
5Department of Neuroscience, Mount Sinai School of Medicine, New York, NY 10029, USA
6Saban Research Institute, Childrens Hopsital Los Angeles, Los Angeles, CA 90027, USA
7These authors contributed equally to this work
*Correspondence: email@example.com (R.W.R.), firstname.lastname@example.org (D.B.A.)
The ability to visualize endogenous proteins in living
neurons provides a powerful means to interrogate
neuronal structure and function. Here we gene-
rate recombinant antibody-like proteins, termed
display (FingRs), that bind endogenous neuronal
proteins PSD-95 and Gephyrin with high affinity and
that, when fused to GFP, allow excitatory and inhib-
itory synapses to be visualized in living neurons.
Design of the FingR incorporates a transcriptional
regulation system that ties FingR expression to the
level of the target and reduces background fluores-
cence. In dissociated neurons and brain slices,
FingRs generated against PSD-95 and Gephyrin did
not affect the expression patterns of their endoge-
nous target proteins or the number or strength of
synapses. Together, our data indicate that PSD-95
and Gephyrin FingRs can report the localization
and amount of endogenous synaptic proteins in
living neurons and thus may be used to study
changes in synaptic strength in vivo.
Immunocytochemistry, a technique invented almost 70 years
ago, has made it possible to visualize the spatial distribution
of specific molecules in cells and tissues (Coons et al., 1942).
Despite its utility, however, a number of properties of immuno-
cytochemistry drastically limit the range of experimental ques-
tions to which it can be applied. For instance, staining of
cytoplasmic proteins requires that cells first be fixed and per-
meabilized, which precludes its use in labeling live cells. Also,
application of antibodies to tissue results in the labeling of all
molecules within the tissue. Thus, it is often difficult to extract
information about the localization of the molecule within an indi-
vidual cell. Some of these limitations were overcome with the
cloning of the gene encoding the green fluorescent protein
(GFP) (Chalfie et al., 1994). GFP can be genetically fused to a
protein of interest, making it possible to visualize that protein
within living cells (Marshall et al., 1995). If GFP-tagged proteins
are introduced into sparsely distributed cells, the subcellular
localization of the protein can be easily interpreted, even in
complex tissue preparations such as brain slices (Arnold and
Clapham, 1999). However, introduced GFP-fusion proteins
may fail to localize properly, due to saturation of targeting
machinery, and overexpression of proteins can have dramatic
morphological and/or functional effects on cells (El-Husseini
et al., 2000). For instance, when the potassium channel Kv4.2
is exogenously expressed in neurons in culture or slices, it local-
izes diffusely to the somatodendritic region (Chu et al., 2006;
Rivera et al., 2003), whereas endogenous Kv4.2 localizes in a
conspicuously punctate manner (Burkhalter et al., 2006; Jinno
et al., 2005). These problems may be circumvented by intro-
ducing tagged proteins into a knockout background (Lu et al.,
2010) or by knocking GFP into the locus of the endogenous
gene (Chiu et al., 2002). However, the former method may fail
if the expression of the introduced transgene is not regulated
at precisely the same level and with the same temporal pattern
as the endogenous protein and the latter method is time
consuming and costly. Moreover, both methods have three
serious limitations that restrict their applicability: (1) they do
not readily allow labeling of two or more proteins in the same
cell, (2) it is difficult to confine the expression of the tagged pro-
teins to a genetically defined subset of cells, and (3) they do not
allow any analysis of either posttranslational modifications or
specific protein conformations.
Recently, a novel strategy was used to label endogenous pro-
teins in a manner that avoids the drawbacks associated with
traditional approaches (Nizak et al., 2003). Recombinant anti-
body-like proteins (termed intrabodies) that bind to endogenous
target proteins were selected from a library of single-chain anti-
bodies, scFvs (Huston et al., 1988), using phage display. The
genes encoding intrabodies were then fused to GFP genes and
Neuron 78, 971–985, June 19, 2013 ª2013 Elsevier Inc. 971
transfected into cells in culture allowing an activated form of
Rab6 to be visualized in real time. Phage display selection of
scFvlibraries hasalsobeenusedtogenerateintrabodies against
neuronal proteins such as Gephyrin and Huntingtin (Southwell
et al., 2008; Varley et al., 2011). Nonetheless, this method has
a serious drawback: the scFv scaffold requires disulfide bonds
for stable folding, but the reducing environment of the cell pre-
cludes the formation of disulfide bonds. Thus, the scFv scaffold
is prone to misfolding and/or aggregation (Goto and Hamaguchi,
1979; Goto et al., 1987; Proba et al., 1998). This problem was
subsequently solved by using the 10thfibronectin type III domain
fromhumanfibronectin (10FnIII)asascaffold (Koide etal.,1998).
This domain has an overall beta-sandwich topology and loop
structure similar to the VH domain of IgG but folds stably with
no disulfide bonds (Dickinson et al., 1994; Koide et al., 1998;
Main et al., 1992). Libraries composed of 10FnIII domains have
been combined with phage display selection to create binders
to targets, such as one against the Src SH3 domain (Karatan
et al., 2004), that work in reducing environments. Another inno-
vation has been the use of mRNA display, an entirely in vitro
selection method that uses libraries with > 1012sequences,
103- to 104-times higher diversity than phage display. This
method has been used to create protein aptamers that bind to
targets such as the SARS virus N-protein and phospho-iKappa-
Balpha with very high target binding affinity and selectivity
(Ishikawa et al., 2009; Olson et al., 2008; Olson and Roberts,
2007; Xu et al., 2002).
Despite these advances, intrabodies have not been widely
used for imaging protein localization and expression. A central
problem in the application of intrabodies to cellular imaging is
that they are only expected to colocalize with the target protein
if the expression level of the intrabody is the same as or lower
than that of the cognate protein; otherwise, the unbound intra-
body that is freely diffusible in the cytoplasm will overwhelm
the image. Here we describe a method that overcomes these
obstacles and allows endogenous protein to be visualized in
real time in living cells. Our method is based on the generation
of disulfide-free intrabodies, known as FingRs, that are tran-
scriptionally regulated by the target protein. Specifically, we
used a 10FnIII-based library in combination with mRNA display
to identify FingRs that bind two synaptic proteins, Gephyrin
and PSD95. After the initial selection, we screened binders using
a cellular localization assay to identify potential FingRs that bind
at high affinity in an intracellular environment. We also created a
intrabody to that of the target protein regardless of the target’s
expression level. This system virtually eliminates unbound
visualization of thetarget proteins. Thus,the FingRspresented in
this study allow excitatory and inhibitory synapses to be visual-
ized in living neurons in real time, with high fidelity, and without
affecting neuronal function.
Generating FingRs that bind to PSD-95 or Gephyrin
Our goal in this work was to create reagents that could be used
to label excitatory and inhibitory synapses in live neurons. To do
this, we chose two well-established protein targets that serve as
immunocytochemical markers for these structures: PSD-95, a
marker of excitatory postsynaptic sites (Cho et al., 1992), and
Gephyrin, a marker of inhibitory postsynaptic regions (Craig
et al., 1996; Langosch et al., 1992; Prior et al., 1992; Takagi
et al., 1992). Within each protein, we targeted well-structured
regions where binding to FingRs would be unlikely to disturb
function. For PSD-95 we chose the SH3-GK domain, which
mediates intra- and intermolecular interactions (McGee et al.,
2001), while for Gephyrin, we chose the G domain, which medi-
ates trimerization (Sola et al., 2001). In the case of Gephyrin we
used protein in a trimerized state as a target in order to generate
binders to the external surface.
To isolate FingRs, we generated recombinant disulfide-free
antibody-like proteins based on the Fibronectin 10FnIII scaffold
using mRNA display (Roberts and Szostak, 1997). The naive
FingR library was constructed as described (Olson and Roberts,
2007), with the addition of point mutations that enhance expres-
sion and folding (Olson et al., 2008). The resulting library was
predominantly full-length, in-frame clonesandhad anexpressed
FG loops (Figure 1A).
Using this library, two selections were performed—one target-
the target protein was immobilized on a solid support and used
to purify functional library members via affinity chromatography.
The purified mRNA-protein fusions were then amplified to
provide a new library enriched for binders to the targets, which
was used for the next round of selection. After six rounds, the
number of PCR cycles needed to generate the enriched pool
decreased markedly, indicating that both selections had
converged to predominantly functional clones. A radioactive
pull-down assay confirmed this observation (Figures 1C and
1D), demonstrating that 42% of the Gephyrin FingR pool
(round 7) and 45% of the PSD-95 FingR pool (round 6) bound
to target with very low background binding. Importantly, cloning
and sequencing of each pool indicated that both contained
numerous, independent, functional FingRs.
Since numerous independent FingRs bound to target, we
wished to choose proteins that gave the best intracellular label-
ing. To do this, we devised a stringent COS cell screen, wherein
the target (e.g., Gephyrin) was localized to the cytoplasmic face
of the Golgi apparatus by appending a short Golgi-targeting
sequence (GTS) (Andersson et al., 1997) (Figure 1E). Functional
FingRs (‘‘winners’’) were defined as those that showed tight sub-
cellular colocalization between the rhodamine-labeled target
and the GFP-labeled FingR (Figures 1F–1H). Suboptimal
sequences (Figure 1I, ‘‘losers’’) result in diffuse staining (Fig-
ure 1K), poor expression, and/or poor colocalization (Figures
1J and 1L). This experiment allowed us to choose FingR proteins
that satisfied three essential criteria: (1) good expression and
folding inside a mammalian cell, (2) lack of aggregation, and (3)
high-affinity binding to the intended target under cellular condi-
tions and despite the high levels of other proteins present. Our
results confirm the importance and stringency of the screen,
as only 10%–20% of FingR clones (4/30 PSD-95 FingRs and
3/14 Gephyrin FingRs) that bind to the target in vitro colocalized
with target intracellularly.
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PSD95 and Gephyrin FingRs Label Endogenous Targets
in Neurons in Culture
Gephyrin or PSD-95 in native cells, GFP-tagged FingR cDNAs
can label endogenous
that were positive in the COS cell assay were expressed in
dissociated cortical neurons in culture. After incubation for
14 hr, the cultures were fixed and immunostained for both
GFP and the endogenous target proteins. In each selection,
Figure 1. Selection of Fibronectin Binders of PSD-95 and Gephyrin by mRNA Display and by a Cellular Localization Assay
(A) A library consisting of 10FnIII domains with 17 random residues in the BC and FG loops was used to select binders to PSD-95 and Gephyrin.
(B) The selection protocol is as follows: (1) DNA encoding the randomized Fibronectins was transcribed and a puromycin molecule attached to linker DNA was
fused to the 30end of the transcript. (2) The mRNA-puromycin fusion was translated to give an mRNA-puromycin-peptide molecule. An anti-sense cDNA strand
hybridized to the mRNA was synthesized that allows individual library members to be amplified by PCR. (3) The library was exposed to target molecules con-
sisting of either the G domain of Gephyrin or the SH3-GK domains of PSD-95. Binders were purified by precipitation. (4) Library members that bound were
amplified by PCR to reconstitute a library that is enriched for binders to target.
(C and D) Percentage of the library that bound to the beads after each round of selection.
specificity, it becomes localized to the Golgi apparatus and colocalized with GTS-GPHN:G.
(F and G) Expression pattern of GTS-GPHN:G tagged with Rhodamine (red, F) colocalizes with that of GPHN.FingR.W-GFP (green, G).
(H) Yellow indicates colocalization of GTS-GPHN:G and GPHN.FingR.W-GFP.
(I) Schematic of COS cell expressing GTS-GPHN:G and a FingR that does not bind to target with high affinity and specificity, GPHN.FingR.L-GFP.
(J and K) Expression pattern of GTS-GPHN:G tagged with Rhodamine (red, J) does not colocalize with that of GPHN.FingR.L-GFP, but instead localizes diffusely
(L) A relative lack of yellow staining in merge of GTS-GPHN:G and GPHN.FingR.L-GFP indicates a lack of colocalization. Scale bar represents 5 mm.
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at least one FingR (PSD95.FingR for PSD-95, GPHN.FingR for
Gephyrin) localized in a punctate manner characteristic of
both target proteins (Figures 2A and 2D). The expression pat-
terns of each FingR showed striking colocalization with its
cognate endogenous protein (Figures 2B, 2C, 2E, and 2F). In
addition, when GPHN.FingR-GFP was expressed in either
excitatory or inhibitory neurons it appeared in puncta adjacent
to or overlapping presynaptic terminals labeled for GAD-65
(Figure S1 available online). These results are consistent with
FingRs binding at high affinity to PSD-95 or Gephyrin. To
corroborate the results from colocalization experiments we
used biochemical means to test for interaction between each
FingR and its endogenous target protein in cortical neurons in
culture. cDNAs encoding each FingR were incorporated into
a lentivirus, which was used to infect the cultures. After expres-
sion of either PSD95.FingR-GFP or GPHN.FingR-GFP for 96 hr,
we collected cell lysate, which was exposed to immobilized
anti-GFP antibody. The immunoprecipitated protein complexes
were blotted and stained for the presence of the endogenous
target proteins. In cells infected with PSD95.FingR-GFP, the
anti-GFP antibody coimmunoprecipitated a band at 95 kD
that was labeled by the anti-PSD-95 antibody (Figure 2G), but
the precipitate was not labeled with anti-Gephyrin antibodies.
In cells where GPHN.FingR-GFP was expressed, the precipi-
tate pulled down by the anti-GFP antibody contained a band
at 80 kD that was labeled with the anti-Gephyrin antibody,
but the precipitate was not labeled with the anti-PSD-95 anti-
body (Figure 2G).
The GK domain of PSD95, which is contained within the target
of the PSD95.FingR selection, interacts with guanylate kinase-
associated protein (GKAP), a protein that links PSD-95 to
Shank-Homer complexes (Naisbitt et al., 1999; Tu et al., 1999)
and has been implicated in synaptic remodeling (Shin et al.,
2012). To determine whether binding of PSD95.FingR with
PSD-95 interferes with the interaction between PSD-95 and
GKAP, we asked whether GKAP could pull down both PSD-95
and PSD95.FingR-GFP when all three proteins were expressed
in COS cells. We found that, indeed, immunoprecipitation
of GKAP resulted in coprecipitation of both PSD-95 and
PSD95.FingR-GFP (Figure S1). Furthermore, in COS cells
expressing only GKAP and PSD95.FingR-GFP, immunoprecipi-
tation of GKAP did not cause coprecipitation of PSD95.FingR-
GFP. Thus, in the GKAP/PSD95/PSD95.FingR-GFP complex,
GKAP and PSD95.FingR must both bind to PSD95, indicating
that binding of PSD95.FingR to PSD-95 does not disrupt binding
of PSD-95 to GKAP.
Figure 2. FingRs Recognizing Gephyrin or
PSD-95 Bind to Endogenous Targets after
Expression in Neurons
(A) A FingR against Gephyrin (GPHN.FingR-GFP,
green) localizes in a punctate fashion after
expression in a dissociated cortical neuron.
(B) Endogenous Gephyrin (red).
(C) Merge of GPHN.FingR-GFP and endogenous
Gephyrin shows colocalization (yellow).
(D) A FingR against PSD-95 (PSD95.FingR-GFP,
green) localizes in a punctate fashion after
expression in a cortical neuron.
(E) Endogenous PSD-95 (red).
(F) Merge of PSD95.FingR-GFP and endogenous
PSD-95 shows colocalization (yellow). Scale bar
represents 5 mm.
(G) Coimmunoprecipitation of endogenous target
proteins with FingRs after expression in cortical
neurons in culture. Neurons were infected with
lentivirus expressing either PSD95.FingR(FLAG)-
GFP (lanes 1, 3) or GPHN.FingR(FLAG)-GFP (lanes
2, 4), both of which contained a FLAG-tag that
enabled immunolabeling. After 96 hr of expres-
sion, cells were lysed and a portion of the lysates
The resulting precipitates were run on an SDS
PAGE gel (lanes 1, 2) along with the lysates
(lanes 3, 4), blotted, and probed with anti-
FLAG antibodies (top), anti-Gephyrin antibodies
(middle), and anti-PSD-95 antibodies (bottom).
with endogenous PSD95, but not with endoge-
nous Gephyrin, whereas GPHN.FingR(FLAG)-GFP
coimmunoprecipitated with Gephyrin, but not with
See also Figure S1.
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A Transcriptional Control System to Regulate the
Expression Level of FingRs
In order for FingRs to accurately report the localization and traf-
ficking of their endogenous targets, it is necessary to minimize
the excess, unbound FingR. To demonstrate the effect of over-
expressing FingRs, we expressed either GPHN.FingR-GFP (Fig-
to 6 days and found that each appears in a diffuse pattern
consistent with the signal from the excess, unbound FingR over-
whelming the signal from the FingR bound to its target (Figures
3A–3C, Figure S2). In order to minimize this background signal,
we developed a transcriptional control system that was
designed to closely match the expression level of a FingR with
that of its endogenous target (Figure 3G). This transcriptional
control system uses the transcription repressor KRAB(A) fused
to a ZINC-finger (ZF) binding domain (Margolin et al., 1994; Witz-
gall et al., 1994). In our system ZF fused to KRAB(A) is fused to
the FingR itself and ZF binding sites are inserted into the DNA
upstream of the promoter that controls FingR expression (Fig-
ure 3G). When bound to target proteins in the dendrites, via
FingRs, ZF-KRAB(A) is physically prevented from moving to
the nucleus and turning off transcription (Figure 3G). Thus, as
long as there is unbound target present the ZF-KRAB(A) tran-
scription factor will be prevented from turning off transcription.
Figure 3. A Transcriptional Control System
Causes FingRs to Be Expressed at the
Same Level as the Endogenous Target
(A) GPHN.FingR-GFP(green) expressed for7days
localizes in a nonspecific pattern, probably due to
high background from unbound FingR.
(B and C) Endogenous Gephyrin (red) does not
colocalize with GPHN.FingR-GFP.
(D–F) GPHN.FingR-GFP (green, D) with transcrip-
tional regulation expressed for 7 days appears in a
punctate pattern that precisely colocalizes with
that of endogenous Gephyrin (red, E; yellow, F).
Note that red staining that does not colocalize in
(F) is from untransfected cells. Scale bar repre-
sents 5 mm.
(G) To control its transcription the FingR (blue) is
fused to a transcription factor consisting of a zinc
finger DNA binding domain (ZF, purple) fused with
a KRAB(A) transcriptional repressor domain (pink).
In addition, a ZF DNA binding site (light blue) was
inserted upstream of the CMV promoter. When
less than 100% of the target is bound by FingR (i),
then 100% of newly made FingR binds to endog-
enous target and is prevented from moving to the
nucleus. When 100% of the target is bound (ii),
then newly made FingR can no longer bind to the
target and, instead, moves to the nucleus as a
result of the nuclear localization signal within the
ZF. Once in the nucleus the transcription factor
binds to the ZF binding site and represses tran-
scription. Thus, the level of FingR is matched to
the level of the endogenous target protein.
(H) Live cortical neuron coexpressing regulated
mKate2 (red). Note that the regulation systems for
PSD95.FingR-GFP and GPHN.FingR-mKate2 are
based on DNA binding domains from CCR5 and
IL2RG, respectively. Each FingR is expressed in a
punctate, nonoverlapping fashion consistent with
labeling of PSD-95 and Gephyrin.
(I) Neuron in cortical culture expressing transcrip-
tionally controlled GPHN.FingR-GFP after lenti-
viral infection. Images taken at 1 s intervals show a
vesicle moving with a velocity of ?7 mm.s?1in the
the axon. Scale bar represents 5 mm.
See also Figure S2 and Movie S1.
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Figure 4. Knockdown ofGephyrin or PSD-95 in Dissociated Cortical Neurons Leads to Elimination ofGPHN.FingR and PSD95.FingR Staining
(A–D) Cortical neuron in dissociated culture coexpressing siRNA against Gephyrin and the transcriptionally controlled GPHN.FingR (A). Note that virtually no
staining against the GPHN.FingR is visible in the dendrites or axon except for a single punctum (arrowhead), which colocalizes with a punctum of endogenous
Gephyrin (B and C).
HA-mCherry staining of the transfected cell (D). (E–H) In contrast, when the transcriptionally regulated GPHN.FingR was coexpressed with scrambled siRNA it
expressed abundantly in a punctate pattern (E) that colocalized with endogenous Gephyrin (F and G).
(H) HA-mCherry staining of the transfected cell.
(legend continued on next page)
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However, if all of the target is bound, the unbound ZF-KRAB(A)
transcription factor moves to the nucleus and turns off transcrip-
tion. In this manner the expression level of the FingR should be
closely matched to that of its target.
To test whether this transcriptional control system can effec-
tively regulate the expression level of FingRs, we expressed
transcriptionally controlled versions of GPHN.FingR-GFP or
PSD95.FingR-GFP in cortical neurons in culture for 7 days.
Both transcriptionally controlled FingRs localized in a punctate
manner, precisely colocalizing with their target proteins (Figures
3D–3F; Figure S2), in contrast to the nonspecific localization of
the uncontrolled FingRs (Figures 3A–3C; Figure S2). To quanti-
tate the degree to which transcriptionally controlled and uncon-
trolled FingRs localized to postsynaptic sites, we calculated
the ratio of the amount of FingR at nonsynaptic sites on den-
drites versus at postsynaptic sites (Rn/s). Rn/sfor uncontrolled
GPHN.FingR-GFP was 0.96 ± 0.16 (n = 100 synapses) as
compared with 0.033 ± 0.005 (n = 100) for the same construct
with transcriptional control and 0.002 ± 0.006 for endoge-
nous Gephyrin (Figure S2). Similarly, Rn/s for unregulated
PSD95.FingR-GFP was 0.90 ± 0.02, 0.16 ± 0.01 for regulated
PSD95.FingR-GFP, and 0.16 ± 0.008 for endogenous PSD-95
(Figure S2). Thus, our results are consistent with the transcrip-
tional control drastically reducing the amount of unbound FingR
that contributes to background signal. Note that the transcrip-
tional control system causes the accumulation of some FingR
in the nucleus (Figure S2).
To further test the transcriptional control system, we asked
whether regulated FingRs could maintain high-fidelity labeling
in response to a sudden increase in target protein. To simulate
such an increase, we first transfected cortical neurons in cul-
ture with an inducible construct containing Gephyrin-mKate2
along with a second construct containing transcriptionally
regulated GPHN.FingR-GFP, but without inducing transcription
of Gephyrin-mKate2. After 1 week in culture, expression of
Gephyrin-mKate2 was induced by adding an Ecdysone analog
for 24 hr. This induction of Gephyrin-mKate2 produced an
uninduced cells as measured by immunocytochemistry (Fig-
between induced versus uninduced, consistent with a coordi-
nated upregulation of FingR expression. To quantify the relative
fidelity with which GPHN.FingR-GFP-labeled uninduced versus
induced cells, we calculated the ratio of total Gephyrin staining
that the ratio of total Gephyrin staining versus GPHN.FingR-GFP
staining was 1.40 ± 0.03 (n = 200 puncta, 10 cells) for uninduced
two ratios are not significantly different (p = 0.15, Wilcoxon),
indicating that GPHN.FingR-GFP labeled Gephyrin with similar
fidelity in the uninduced versus induced cells, a result that is
consistent with the transcriptional regulation system responding
to the increase in target with an appropriately graded increase in
To test whether the transcriptional regulation system could
work fortwo FingRs simultaneously,
PSD95.FingR-GFP and GPHN.FingR-mKate2 for 7 days. Both
had independently regulated transcriptional control systems.
PSD95.FingR-GFP was fused to the CCR5L zinc finger (Mani
et al., 2005), and GPHN.FingR-mKate2was fused to the IL2RG2L
zinc finger (Gabriel et al., 2011). PSD95.FingR-GFP and
GPHN.FingR-mKate2 each expressed in a distinctly punctate
manner with very little background or overlap between the two
(Figure 3H), indicating that each transcriptional feedback system
worked efficiently and independently.
To this point our experiments have concentrated on using
FingRs to visualize the localization of endogenous proteins at
single points in time. However, because FingRs can be visual-
ized in living cells, it should be possible to use them to observe
trafficking of their endogenous target proteins. To visualize
trafficking of Gephyrin we used lentivirus to express transcip-
tionally controlled GPHN.FingR-GFP in neurons in culture for
7 days. Time-lapse imaging of these cultures revealed numerous
vesicles moving in both directions in the cell body, axons, and
dendrites (Figures 3I and 3J). Interestingly, axonal vesicles
appeared more elongated and moved at higher velocity than
dendritic vesicles, hitting speeds of ?7 mm.s?1(Movie S1).
Thus, GPHN.FingR-GFP can be used to visualize trafficking of
endogenous Gephyrin in addition to its localization.
Testing FingRs for Binding Specificity and Off-Target
To test whether FingRs label their endogenous targets spe-
cifically in cortical neurons in culture, we expressed either
transcriptionally controlled PSD95.FingR-GFP or GPHN.FingR-
GFP along with siRNA against either PSD-95 or Gephyrin. Cells
in which either endogenous PSD-95 or endogenous Gephyrin
was knocked down expressed extremely low levels of the corre-
spondingFingR (Figures 4A–4D, 4I–4L,andS3). Incontrast, cells
expressing either PSD95.FingR or GPHN.FingR along with
scrambled siRNA expressed considerably higher levels of each
FingR. In addition, each FingR was expressed in a punctate
(I–L) Neuron expressing transcriptionally controlled PSD95.FingR and siRNA (I) shows a very low level of FingR staining that is comparable to the level of
endogenous PSD-95 staining (J and K).
HA-mCherry staining of transfected cell (L). M-P In contrast, staining of the transcriptionally regulated PSD95.FingR is robust in a cell coexpressing scrambled
siRNA (M) and colocalizes with staining of endogenous PSD-95.
(P) HA-mCherry staining of transfected cell. Scale bar represents 5 mm.
(Q) Quantitative comparison of the total amount of staining by anti-Gephyrin antibody (total Gephyrin) and by GPHN.FingR-GFP in the processes of neurons
transfected with siRNA against Gephyrin versus with scrambled siRNA. Note that staining by anti-Gephyrin antibody and by GPHN.FingR-GFP are reduced by
comparable amounts in cells expressing Gephyrin siRNA versus scrambled siRNA.
(R) Quantitative comparison of the total amount of staining by anti-PSD-95 antibody (total PSD95) and by PSD95.FingR-GFP in the processes of neurons
transfected with siRNA against PSD-95 versus with scrambled siRNA. Note that staining by anti-PSD-95 antibody and by PSD95.FingR-GFP are reduced by
comparable amounts in cells expressing PSD-95 siRNA versus scrambled siRNA.
All error bars represent SEM. See also Figure S3.
Probes for Visualizing Synaptic Proteins
Neuron 78, 971–985, June 19, 2013 ª2013 Elsevier Inc. 977
pattern that localized with the corresponding endogenous pro-
tein (Figures 4E–4H, 4M–4P, and S3). In the presence of siRNA
against Gephyrin, the total Gephyrin expressed in processes
per cell was reduced by 96% ± 1% (Figure 4Q; n = 10 cells)
compared with scrambled siRNA, whereas the staining of
GPHN.FingR-GFP was reduced by 98.1% ± 1% (n = 10 cells)
under the same circumstances, a difference that is not signifi-
cant (p > 0.13, t test). Similarly, in cells where the amount of total
PSD-95 was reduced by 96% ± 1% (Figure 4R; n = 10 cells), the
amount of PSD95.FingR staining in processes was reduced by
99% ± 1% (n = 10 cells) compared with cells expressing scram-
bled siRNA, a difference that is not significant (p > 0.1, t test).
These results are consistent with the majority of PSD95.FingR
and GPHN.FingR labelingtheir target proteins within dissociated
In the CNS there are three close homologs of PSD-95 that are
also found at postsynaptic sites: PSD-93, SAP-97, and SAP-102
(Brenman et al., 1998). To determine whether PSD95.FingR
could distinguish between different MAGUK proteins, we inde-
pendently tested whether PSD95.FingR-GFP bound to PSD-
93, SAP-102, or SAP-97 in our COS cell assay. We found that
PSD-93 did not colocalize with PSD95.FingR-GFP, whereas
SAP-102 and SAP-97 did (Figures 5A–5C). To determine
whether SAP-102 and SAP-97 interact with PSD95.FingR-GFP
in a more stringent assay, we coexpressed HA-tagged versions
of these proteins in cultured cortical neurons where PSD-95
expression had been knocked down with siRNA. We found
that when PSD95.FingR is coexpressed with either SAP-97
or SAP-102, the coexpressed proteins colocalize (Figures
5D–5K). Thus, PSD95.FingR probably binds to heterologous
SAP-97 and SAP-102 with relatively high affinity, but not to het-
erologous PSD-93. Additional testing will be required to deter-
mine the exact specificity of binding of PSD95.FingR-GFP with
endogenous MAGUK proteins in vivo. However, even in the
Figure 5. Interaction of PSD95.FingR with MAGUK Proteins
(A–C) In a COS cell coexpressing Golgi-targeted PSD-93 (GTS-PSD-93) and PSD95.FingR (A), the two proteins do not colocalize, indicating the PSD95.FingR
does not interact with PSD-93. In contrast, PSD95.FingR colocalizes with GTS-SAP-102 (B) or GTS-SAP-97 (C) when it is coexpressed with either MAGUK
(D–G) PSD95.FingR (D) colocalizes with exogenous HA-SAP-102 (E and G) after expression in dissociated cortical neurons where PSD-95 (F) has been knocked
down with siRNA.
(H–K) PSD95.FingR (H) colocalizes with exogenous HA-SAP-97 (I and K) after expression in dissociated cortical neurons where PSD-95 (J) has been knocked
down with siRNA. Scale bar represents 5 mm.
Probes for Visualizing Synaptic Proteins
978 Neuron 78, 971–985, June 19, 2013 ª2013 Elsevier Inc.
case where PSD95.FingR does identify other synaptic MAGUK
proteins, it is still suitable for marking synapses.
In addition to testing the specificity of binding, we asked
whether expression of the FingR had a morphological effect on
cells. In light of the dramatic increase in spine size and density
caused by overexpression of PSD-95 (El-Husseini et al., 2000;
Kanaani et al., 2002) and the large aggregates seen with overex-
pression of Gephyrin (Yu et al.,2007), wetested whether expres-
sion of PSD95.FingR-GFP or GPHN.FingR-GFP had an effect on
the size of PSD-95 or Gephyrin puncta, respectively. We found
that the amounts of total Gephyrin associated with individual
puncta (stained with an anti-Gephyrin antibody) were nearly
identically distributed in cells expressing GPHN.FingR-GFP
(mGPHN= 18.1 ± 0.7 a.u., n = 200 puncta) versus with untrans-
fected cells (mGPHN= 18.4 ± 0.8 a.u., n = 200, p > 0.8; Figures
6A, 6B, and S4). Similar results were also obtained for PSD-95
puncta in PSD95.FingR-GFP expressing cells (mPSD-95= 20 ±
2 a.u., n = 200) and in untransfected cells (mPSD-95 = 20 ±
2 a.u., n = 200, p > 0.5; Figures 6E, 6F, and S4). In contrast,
puncta from cells expressing Gephyrin-GFP contained signifi-
cantly more total Gephyrin (mGPHN= 55 ± 3 a.u., n = 200) than
puncta from comparable untransfected cells (mGPHN = 21 ±
1 a.u., n = 200, p < 0.001; Figures 6C, 6D, and S4). Similar mea-
surements in cells expressing PSD95-GFP (mPSD-95= 41 ± 2 a.u.,
n = 200) were also higher than in untransfected cells (mPSD-95=
18 ± 1 a.u., n = 200, p < 0.001; Figures 6G, 6H, and S4). In addi-
gates of protein, as was observed previously (Yu et al., 2007).
Such aggregates were never seen in cells expressing transcrip-
tionally controlled GPHN.FingR-GFP. Thus, expressing GFP-
tagged FingRs does not affect the size of Gephyrin or PSD-95
puncta, in contrast to overexpressed, tagged PSD-95 and
PSD95.FingR and GPHN.FingR Do Not Affect
the Electrophysiological Properties of Neurons
To furthertest PSD95.FingR and GPHN.FingR in acontext that is
closer to in vivo, we expressed them in organotypic rat hippo-
campal slices using biolistic transfection. Slices cut from rats
at 8 days postnatal, transfected 2–3 days later, and then incu-
bated for 7–8 days were imaged live using two-photon micro-
scopy. Both transcriptionally controlled PSD95.FingR-GFP and
GPHN.FingR-GFP expressed in a punctate pattern that was
similar to their respective localization patterns after expression
in dissociated neurons (Figures 7A and 7F). Furthermore,
PSD95.FingR-GFP was clearly concentrated in dendritic spines,
while GPHN.FingR-GFP was found in puncta on the dendritic
shaft, consistent with the former being localized to postsynaptic
excitatory sites and the latter being localized to postsynaptic
inhibitory sites. The morphology of neurons transfected with
PSD95.FingR-GFP was not different from untransfected cells,
and, in particular, spine density did not differ significantly
between cells expressing PSD95.FingR-GFP (Figure 7B; spine
density = 0.94 ± 0.08 spines.mm?1; n = 1,064 spines, 8 cells)
and control cells, (spine density = 0.97 ± 0.06 spines.mm?1;
n = 1,396 spines, 9 cells; p > 0.5, t test). In order to determine
whether expressing FingRs had a physiological effect on cells
we measured spontaneous miniature excitatory postsynaptic
currents (mEPSCs) in neurons expressing PSD95.FingR-GFP
and spontaneous miniature inhibitory postsynaptic currents
(mIPSCs) in neurons expressing GPHN.FingR-GFP. We found
that neither mEPSCs nor mIPSCs from cells expressing the cor-
responding FingR differed qualitatively from untransfected con-
trol cells (Figures 7C and 7G). In addition, in cells expressing
PSD95.FingR-GFP mEPSC frequency (f) and amplitude (A) mea-
surements (f = 1.59 ± 0.1 s?1, A = 9.7 ± 0.6 pA, n = 8 cells) did not
differ significantly from that in control cells (Figures 7D and 7E;
f = 1.66 ± 0.2 s?1, A = 10.8 ± 0.7 pA, n = 8 cells; p > 0.5
t test). Similarly, measurements of frequencies and amplitudes
of mIPSCs in cells expressing GPHN.FingR-GFP (Figures 7H
and 7I; f = 4.2 ± 0.5 s?1, A = 14.1 ± 1.0 pA, n = 8 cells) were
not significantly different from comparable measurements in
control cells (f = 4.3 ± 0.5 s?1, A = 13.5 ± 1.1 pA, n = 9 cells;
p > 0.1 t test). Thus, our results indicate that expressing
PSD95.FingR-GFP and GPHN.FingR-GFP does not cause
changes in synaptic physiology and has no effect on the number
or neurotransmitter receptor content of individual synapses.
To determine whether GPHN.FingR-GFP signals represent
functional inhibitory synapses, we measured IPSCs evoked by
GABA photolysis at individual green punta. Hippocampal CA1
pyramidal neurons were transfected with GPHN.FingR-GFP
and TdTomato to visualize inhibitory synapses and neuronal
structure (Figure 8A). Two-photon GABA uncaging 0.5 mm
away from puncta of GPHN.FingR-GFP triggered IPSCs. IPSC
amplitude diminished when uncaging occurred further away
from the dendrite, demonstrating that IPSCs originated from
the activation of receptors localized in dendrites of the recorded
neuron (Figures 8B and 8C). When GABA was photoreleased on
the dendritic shaft at locations where GPHN.FingR-GFP was
present, robust IPSCs were evoked. However, GABA photore-
lease at two locations, one in a dendritic spine and a second
on a dendritic shaft, where there was no GPHN.FingR-GFP
signal elicited small or negligible IPSCs (Figures 8D and 8E).
These data confirm that GPHN.FingR-GFP does indeed label
functional inhibitory synapses.
PSD95.FingR and GPHN.FingR label their endogenous target
proteins in dissociated neurons, as well as in neurons in slices.
To determine whether FingRs can be used to label endogenous
proteins in vivo, we transfected PSD95.FingR-GFP into neurons
in mouse embryos in utero using electroporation and then
assessed expression at approximately 7 weeks of age. Images
of dendrites of layer V cortical pyramidal neurons coexpressing
HA-mCherry and taken from unstained sections cut from
perfused, fixed brains clearly show punctate patterns of GFP
expression consistent with labeling of PSD-95 (Figures 9A
and 9B). In addition, lower-magnification images show labeling
of layer V and layer II/III pyramidal neurons that is also consistent
PSD-95 labeling (Figures 9C and 9D). Finally, an image obtained
from a living animal of PSD95.FingR-GFP expressed in an apical
tuft from a cortical pyramidal neuron (Figure 9E) demonstrates
that PSD95.FingR-GFP can be imaged in vivo.
In this paper we demonstrate that Fibronectin intrabodies
generated with mRNA display (FingRs) can be used to visualize
Probes for Visualizing Synaptic Proteins
Neuron 78, 971–985, June 19, 2013 ª2013 Elsevier Inc. 979
Figure 6. Expression of FingRs Does Not Change the Size of Gephyrin or PSD-95 Puncta
(A) Immunostained Gephyrin puncta from untransfected cells and cells transfected with GPHN.FingR-GFP are of similar size and brightness.
(B) The average intensity associated with Gephyrin puncta is not significantly different in neurons expressing GPHN.FingR-GFP versus untransfected neurons
(p > 0.5). The amounts of Gephyrin/puncta are distributed similarly in neurons expressing GPHN.FingR-GFP versus untransfected neurons.
(C) Puncta stained with an anti-Gephyrin antibody from cells transfected with Gephyrin-GFP tend to be brighter and larger than those from untransfected cells.
(D) The average intensity associated with Gephyrin puncta is significantly larger in neurons expressing Gephyrin-GFP versus untransfected neurons (p < 0.0001).
In cells expressing Gephyrin-GFP the amount of Gephyrin/puncta is distributed over a larger range encompassing higher values as compared with similar
measurements in untransfected cells. Note that vertical axes of the histograms in (B) and (D) have the same scale.
(E) Puncta stained with an anti-PSD-95 antibody from untransfected cells and cells transfected with PSD95.FingR-GFP are of similar size and brightness.
(F) The average intensity associated with PSD-95 puncta is not significantly different in neurons expressing PSD95.FingR-GFP versus untransfected neurons
(p > 0.5). The amounts of PSD-95/puncta are distributed similarly in neurons expressing PSD95.FingR-GFP versus untransfected neurons.
(legend continued on next page)
Probes for Visualizing Synaptic Proteins
980 Neuron 78, 971–985, June 19, 2013 ª2013 Elsevier Inc.
the localization of the endogenous postsynaptic proteins
Gephyrin and PSD-95 in living neurons without affecting
neuronal structure and function. FingRs represent a substantial
improvement over traditional antibody approaches that, in
general, require that cells be fixed and permeabilized prior to
staining. In addition, FingRs are genetically encoded so that
they can be expressed in neurons using methods of transfection
and transgenesis that can produce cell-specific expression pat-
terns that are easily interpretable. Expressing FingRs is superior
to expressing tagged, exogenous neuronal proteins that tend
not to localize in the same manner as their endogenous counter-
parts and that can cause morphological and functional pheno-
types (El-Husseini et al., 2000). Finally, FingR expression is
controlled by a transcriptional feedback system, based on
a zinc-finger DNA binding domain that closely matches the
amount of FingR expressed to that of its endogenous target.
Thus, transcriptional regulation allows FingRs to accurately
report not just the localization of a protein, but also its expres-
The innovations in methodology that we have devised here
could also be useful for generating FingRs against proteins other
than PSD-95 and Gephyrin. We used as targets multimerization
domains of cytoskeletal proteins because they provide a rigid
surface that FingRs can bind to without disrupting protein func-
tion. We screened FingRs produced by mRNA display using an
intracellular assay to identify binders that work efficiently in the
cytoplasm. Finally, we regulated the FingR transcription using
a zinc finger-based negative feedback system. When using
this system to produce novel FingRs it should be noted that,
as with antibodies, the utility of each FingR is limited by its spe-
cific characteristics. Each new FingR must be optimized to bind
its target specifically and with high affinity over long periods of
time. In addition, each FingR must be thoroughly tested to deter-
mine whether binding to its target disrupts the target’s functional
properties or the ability of the target to interact with other
molecules. Regulation must also be tested to ensure that
FingR expression can respond dynamically to changes in target.
Finally, these properties need to be assessed in the contexts in
which the FingRs will be applied.
The properties of PSD-95 and Gephyrin suggest that the
FingRs discussed in this paper will be useful for many applica-
tions. PSD-95 as well as its homologs PSD-93, SAP-102, and
SAP-97 interact either directly or indirectly with AMPA receptors
be markers for the size and location of postsynaptic densities
(Brenman et al., 1996; Cho et al., 1992; Mu ¨ller et al., 1996; Valt-
schanoff et al., 2000). Although dendritic spines have been used
as morphological markers of excitatory postsynaptic sites, there
is only a rough correlation between synapse size and spine size
(Harris and Stevens, 1989). With PSD95.FingR it will now be
possible to precisely map the sizes and locations of excitatory
postsynaptic sites. The potential applications of GPHN.FingR
are even greater, as there is no morphological structure compa-
rable to a dendritic spine that marks inhibitory synapses. For
instance, we showed that GPHN.FingR-GFP could be used to
ses, enabling a paradigm for probing inhibitory circuitry. In
addition, the amount of Gephyrin at postsynaptic inhibitory sites
is precisely correlated with the number of GABA or Glycine
receptors (Essrich et al., 1998) and thus with the strength of
the corresponding inhibitory synaptic connection. Gephyrin
and PSD-95 FingRs, therefore, provide a map of the location
and strengths of synaptic connections onto specific neurons.
We have expressed FingRs in cultured neurons, in slices, and
in intact mice using in utero electroporation, suggesting that
FingRs will be useful for mapping synaptic connections in
many different contexts. Note that because PSD95.FingR-GFP
labels the MAGUK proteins SAP-102 and SAP-97 in cultured
cells, caution must be used when interpreting its expression
pattern in tissue where MAGUK proteins other than PSD-95
are present. However, an advantage of this nonspecific labeling
is that PSD95.FingR-GFP can be used to mark synapses in
neurons in which PSD-95 is either absent or present at a low
One possible application of PSD95.FingR and GPHN.FingR is
in the study of how neurons respond to changes in firing rate by
tuning the strengths of synaptic inputs (Watt et al., 2000). Previ-
ously it has not been possible to monitor strengths of individual
excitatory or inhibitory synapses during this tuning process.
With the FingRs described in this paper it will now be possible
to measure synaptic strengths, providing temporal and spatial
information about homeostatic responses in individual neurons.
FingRs could also be used in other paradigms where synaptic
strength changes are induced, such as LTP and LTD. These
experiments could probe how synaptic inputs are controlled
with a temporal and/or spatial precision that surpasses current
methods. Finally, PSD95.FingR and GPHN.FingR could be
used to monitor the changes in synaptic strength in the brains
of living mice that occur during behavioral paradigms, for
instance during sleep and wake cycles or before and after
learning a cognitive task. Thus, with the FingRs generated in
this study it may be possible to correlate changes in synaptic
structure with events at the cell, circuit, and behavioral levels.
Target Preparation, mRNA Display, and Intracellular Screening
Targets for the mRNA screens consisted of the G domain of Gephyrin
(GPHN[1-113]) or the SH3-GK domains of PSD-95 (PSD-95[417-724]) fused to
a biotin acceptor tag (AviTag, Avidity). mRNA display was carried out essen-
tially as described (Olson et al., 2008). For screening for FingRs that were
well-behaved in vivo, GFP-tagged candidates were coexpressed in COS
cells with fusion proteins consisting of their respective target (Gephyrin or
PSD-95) fused to a Golgi localization signal from the G1 protein of Uukuniemi
virus (Andersson et al., 1997). After 14 hr of expression cells were fixed and
(G) Puncta stained with an anti-PSD-95 antibody from cells transfected with PSD95-GFP tend to be brighter and larger than those from untransfected cells.
(H) The average intensity associated with PSD-95 puncta is significantly larger in neurons expressing PSD95-GFP versus untransfected neurons (p < 0.0001). In
cells expressing PSD95-GFP the amount of PSD-95/puncta is distributed over a larger range encompassing higher values as compared with similar mea-
surements in untransfected cells. Note that vertical axes of the histograms in (F) and (H) have the same scale.
All error bars represent SEM. See also Figure S4.
Probes for Visualizing Synaptic Proteins
Neuron 78, 971–985, June 19, 2013 ª2013 Elsevier Inc. 981
stained, and FingRs were selected on the basis of colocalization with Golgi-
Immunocytochemistry, Imaging, and Data Analysis
Immunocytochemistry of dissociated cultures was performed as in (Lewis
et al., 2009). Fixed dissociated cortical neurons were imaged on an Olympus
FV1000 confocal microscope. Pixel intensity levels were measured with
ImageJ (U.S. National Institutes of Health). All analyses were performed
blinded. Live cells were imaged on an Olympus IX81 microscope. All analysis
was performed by blinded observers.
In Utero Electroporation and In Vivo Imaging
Electroporations were performed as described in (Saito, 2006). Cranial win-
dows were inserted as described in (Holtmaat et al., 2005). Live images
Figure 7. The Presence of FingRs Does Not
Change the Morphology or Electrophysio-
logical Properties of Neurons in Hippocam-
(A) PSD95.FingR-GFP (green) expressed in CA1
neurons of the hippocampus shows punctate
staining of spine heads, consistent with staining at
postsynaptic sites, which is distinct from the
pattern of coexpressed TdTomato (red).
(B) The morphology of dendrites in cells express-
different from control cells. Similarly, spine den-
sity was not significantly different between cells
expressing PSD95.FingR-GFP and control cells.
PSD95.FingR-GFP (C) were not qualitatively dif-
ferent from those recorded from control cells. In
addition, the frequencies (D) and amplitudes (E) of
mEPSCs did not differ between cells expressing
PSD95.FingR-GFP and control cells.
(F) In a cell expressing GPHN.FingR-GFP (green)
and TdTomato (red), the FingR expresses in
puncta on the dendritic shaft in a manner similar to
inhibitory postsynaptic sites.
(G) mIPSCs recorded from cells expressing
GPHN.FingR-GFP do not differ qualitatively from
mIPSCs from untransfected control neurons (H).
(I) Similarly, neither the frequency nor the ampli-
tude of mIPSCs recorded from cells expressing
recorded from control cells.
All error bars represent SEM.
Probes for Visualizing Synaptic Proteins
982 Neuron 78, 971–985, June 19, 2013 ª2013 Elsevier Inc.
were acquired with a Movable Objective Microscope (MOM) (Sutter Instru-
ments). Experimental protocols were conducted according to the U.S.
National Institutes of Health guidelines for animal research and were approved
by the Institutional Animal Care and Use Committee at the University of South-
Supplemental Information includes Supplemental Experimental Procedures,
four figures, and one movie and can be found with this article online at
We thank Liana Asatryan (USC, Lentivirus Core Facility) for producing lenti-
virus, Aaron Nichols for help in producing the naive FingR library, Samantha
Ancona-Esselmann for technical assistance and help in data analysis, Jerardo
Viramontes Garcia for help in data analysis, and Ryan Kast for technical help
with in vivo two-photon imaging. We thank Matthew Pratt, David McKemy,
Samantha Butler, and members of the Arnold and Roberts laboratories for
helpful suggestions on the manuscript. D.B.A. was supported by grants
GM-083898 and MH-086381. R.W.R. was supported by GM-083898, GM
060416, and OD 006117. G.C.R.E.-D. was supported by GM53395 and
NS69720. B.L.S. was supported by NS-046579. G.C.R.E.-D. has filed a
preliminary patent declaration on the synthesis of dinitroindolinyl-caged
Accepted: April 10, 2013
Published: June 19, 2013
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