Biophysical Journal Volume 94 February 2008 1483–1496 1483

Investigating Interactions Mediated by the Presynaptic Protein Bassoon

in Living Cells by Foerster’s Resonance Energy Transfer and
Fluorescence Lifetime Imaging Microscopy

Mini Jose,* Deepak K. Nair,* Wilko D. Altrock,* Thomas Dresbach,y Eckart D. Gundelfinger,*
and Werner Zuschratter*
*Leibniz Institute for Neurobiology, Magdeburg, Germany; and yInstitute for Anatomy and Cell Biology, University of Heidelberg,
Heidelberg, Germany

ABSTRACT Neuronal synapses are highly specialized structures for communication between nerve cells. Knowledge about
their molecular organization and dynamics is still incomplete. The large multidomain protein Bassoon plays a major role in scaf-
folding and organizing the cytomatrix at the active zone of neurotransmitter release in presynaptic boutons. Utilizing immuno-
fluorescence techniques, we show that Bassoon is essential for corecruitment of its synaptic interaction partners, C-terminal
binding protein 1/brefeldin A-dependent ADP-ribosylation substrate and CAZ-associated structural protein, into protein complexes
upon heterologous expression in COS-7 cells. A combination of Foerster’s resonance energy transfer and fluorescence lifetime
imaging microscopy in the time domain was adopted to investigate the potential for the association of these proteins in the same
complexes. A direct physical association between Bassoon and CtBP1 could also be observed at synapses of living hippocampal
neurons. Simultaneous analysis of fluorescence decays of the donor and the acceptor probes along with their decay-associated
spectra allowed a clear discrimination of energy transfer.

INTRODUCTION
Making use of the current advances in fluorescence micros- trafficking events underlying neurotransmitter release (7,8).
copy and the development of variants of green fluorescent Bassoon and its sister molecule Piccolo are major scaffolding
proteins (GFPs), Foerster’s resonance energy transfer (FRET) components of the CAZ (9,10). The CAZ-associated struc-
is becoming a powerful tool to map protein-protein interac- tural protein CAST/ELKS2 (11–13) and the C-terminal bind-
tions in living specimens. In contrast to in vitro methods, ing protein 1/brefeldin A (BFA)-dependent ADP-ribosylation
which are useful to assess the potential of proteins for physical substrate (CtBP1/BARS) (14) have been suggested to directly
interactions, FRET allows direct access to these interactions interact with Bassoon-containing complexes, though the clear
within a macromolecular complex inside a living cell at nano- functional relevance of these interactions is yet to be ad-
meter resolution (1,2). FRET combined with fluorescence dressed. CtBP1/BARS plays a key role as a transcription
lifetime imaging microscopy (FLIM) is gaining importance as corepressor during embryonic development and in oncogen-
a powerful technique in probing these interactions in biolog- esis, and as a promoter of Golgi membrane fission in intra-
ical systems at the molecular level due to its independence cellular trafficking (15).
from excitation intensities and fluorophore concentrations It has been shown biochemically that a central fragment of
(3,4). The advantage of using the contributions of lifetimes Bassoon of ;1500 amino acids (Bsn1692-3263) (16) has the
or decay-associated spectra (DAS) by simultaneous donor- potential to interact with CtBP1 (14), while it also harbors
acceptor analysis to discriminate FRET in biological samples the binding site for CAST/ELKS2 (11). Upon blockade of
has been shown in previous works (5,6). Thus, a combination the Golgi exit in neurons or upon expressing heterologously
of these techniques provides high spatial (nanometer) and in nonneuronal cells, Bassoon forms protein clusters in the
temporal (picosecond) resolution in monitoring interactions cell soma (17). It would be interesting to know whether these
within protein complexes in their natural environment. macromolecular complexes are functional and can recruit
Active zones are specialized regions of the presynaptic binding partners of Bassoon. A deeper understanding of the
plasma membrane designed for the regulated exocytosis of nature of the association of CAZ proteins with these com-
neurotransmitters. A highly organized cytomatrix of proteins plexes is also crucial in providing insight into their basic
assembled at the active zone, i.e., the cytomatrix at the active molecular network. In neurons, though Bassoon and CtBP1
zone (CAZ), is thought to mediate and regulate membrane are colocalized in synapses (14), a direct physical interaction
between them at these sites in living cells has not yet been
proven. A direct confirmation of such an in vivo interaction
Submitted April 27, 2007, and accepted for publication September 26, 2007.
would provide deeper insight into the molecular organization
Mini Jose and Deepak K. Nair contributed equally to this work.
of the CAZ.
Address reprint requests to Mini Jose, Leibniz Institute for Neurobiology,
Neuronal development is characterized by high ionic
Brenneckestrasse 6, 39118 Magdeburg, Germany. Tel.: 49-391-626-3704;
Fax: 49-391-626-3328; E-mail: mini.jose@ifn-magdeburg.de. changes that significantly affect the properties of fluorescent
Editor: David W. Piston.
Ó 2008 by the Biophysical Society
0006-3495/08/02/1483/14 $2.00 doi: 10.1529/biophysj.107.111674
1484 Jose et al.

probes (6,18). Therefore, in this study, heterologous expression were transfected with Cerulean, Citrine, or fusion constructs of full-length
in COS-7 cells was initially used to assess the formation of Bsn, Bsn95-3938, Bsn1692-3263 (16), CtBP1, or CAST cDNAs using
Polyfect Transfection Reagent (Qiagen, Hilden, Germany) and Lipofectamine
macromolecular complexes involving CtBP1 and CAST (Invitrogen), respectively. The cells were fixed with 4% paraformaldehyde
recruited by Bassoon. This system also provided an environ- (PFA) in phosphate buffer saline and immunostained with primary
ment free of Piccolo, the sister molecule of Bassoon, another antibodies against CtBP1 (mouse, 1:1000, BD Transduction Laboratories,
potential binding partner of both CtBP1 (14) and CAST (11). Lexingon, CA), Bassoon (mouse/rabbit, 1:1000) (9), CAST (rabbit, 1:200,
Here, we assessed the role of Bassoon in organizing macro- Synaptic Systems, Goettingen, Germany), or SAP90/PSD-95 (rabbit, 1:200,
Synaptic Systems). Alexa 594 (anti-mouse/-rabbit, 1:200, Molecular Probes,
molecular complexes linking its different synaptic interaction Eugene, OR), Alexa 488 (anti-rabbit, 1:200, Molecular Probes), and Cy3
partners. Bassoon-dependent recruitment of CtBP1 was stud- (anti-mouse, 1:100, Dianova, Hamburg, Germany) were used as secondary
ied using different functional fragments of Bassoon, including antibodies. Primary antibody incubation was done overnight at 4°C.
the short central fragment (Bsn1692-3263). A combination of
FRET and time domain FLIM was utilized for monitoring a
direct physical association of these proteins in living COS-7 Quantification
cells. Similar approaches were adopted for a deeper under- Fluorescence quantification experiments were performed using Leica-TCS-
standing of the association of Bassoon and CtBP1 in presynaptic SP2-AOBS (633 oil immersion objective, Leica Microsystems, Mannheim,
complexes of living hippocampal neurons. A combination of Germany). To quantify the nuclear expression of CtBP1 (Supplementary
recently generated photostable variants of cyan fluorescent Materials Fig. 1), COS 7 cells were transfected with Cerulean-Bsn1692-
3263, fixed, and immunostained with anti-CtBP1 (mouse, 1:1000) with the
protein (CFP) and yellow fluorescent protein (YFP), namely protocols mentioned previously. Alexa 594 (anti-mouse, 1:200) was used as
Cerulean and Citrine (19,20), was used as a suitable donor- the secondary antibody. Confocal stacks of 18 sections with a section size of
acceptor pair for the purpose. Unlike the conventional way of 0.60 mm were collected. The maximum projection images of the stacks were
monitoring donor mean lifetimes alone, simultaneous multi- taken, and the fluorescence quantified using Image J 1.38. The expressions
exponential analysis of donor and acceptor fluorescence of CtBP1 in transfected and untransfected cells of the same stacks were
compared. To quantify the Bassoon fluorescence at synaptic sites (Supple-
decays along with DAS allowed the discrimination of excited mentary Material Fig. 2), mature neurons (after 15 days in vitro (DIV 15))
state reactions, such as energy transfer. were transfected with Cerulean full-length Bsn. The cells were fixed 14–15 h
after transfection and immunostained with anti-Bassoon (mouse, 1:1000,
Stressgen, North Yorkshire, UK) and anti-SAP90/PSD-95 (rabbit, 1:200);
the latter served as a postsynaptic marker. Cy3 (anti-mouse, 1:100) and Alexa
MATERIALS AND METHODS
594 (anti-rabbit, 1:200) were used as secondary antibodies. Untransfected
Cloning cultures subjected to the same treatment, which were measured under similar
excitation and detection conditions, were used as control. Confocal stacks of
The central fragment of Bassoon complementary DNA (cDNA) encoding 24 sections with a section size of 0.28 mm were collected. Image processing
amino acid residues 1692-3263 (Bsn1692-3263, see Fig. 7) (16) was cloned and fluorescence quantification were performed as for COS-7 cells.
into a frame-shifted variant of p-Cerulean or pCMV-Tag2A expression vec- For quantitative Western blot analysis (Supplementary Material Fig. 3),
tors using BamHI/BamHI restriction sites, and CtBP1 cDNA (14) was cloned Citrine-CtBP1 transfected COS-7 cells were harvested in lysis buffer (150
into frame-shifted variants of p-Cerulean (19) and p-Citrine (20) using EcoRI/ mM NaCl, 1% TritonX-100, complete protease inhibitor (Roche Applied
XhoI restriction sites introduced via linkers. Full-length Bsn cDNA (16) was Science, Mannheim, Germany), 10 mM TrisHCl pH 7.4). Proteins were
subcloned from a cloning vector into p-Cerulean or pCMV-Tag2A using NotI precipitated, each sample was loaded as a triplet on a Tris-acetate buffered gel,
sites. Bsn1692-3263 and full-length Bsn lacking the fluorescent tag are and after separation proteins were transferred to an Immobilon fluoropolymer
referred to as the untagged Bsn1692-3263 and untagged full-length Bsn, polyvinylidene difluoride membrane (Millipore, Schwalbach, Germany) as
respectively. CAST/ELKS2 (11,13) was cloned into p-Cerulean and p-Citrine previously described (14). Blots were incubated with anti-CtBP1 (mouse,
expression vectors using BamHI/SacI restriction sites. The frame-shifted 1:5000) as primary and anti-mouse immunoglobulin G-Alexa 680 (Invitrogen)
variants of p-Cerulean and p-Citrine were generated using standard molecular as secondary antibodies and scanned on an Odyssey Imager (LI-COR, Bad
biological techniques. Bsn or bsn is the official abbreviation in databases for Homburg, Germany) according to the manufacturer’s protocols. Fluores-
the Bassoon protein and gene, respectively. In this article, we have used the cence intensities of bands for endogenous CtBP1 and Citrine-CtBP1 were
abbreviation to name all cDNA constructs. The numbers indicate the amino determined with Odyssey Application Software 2.1 (LI-COR).
acids covered by the cDNA.

Steady-state imaging
Cell cultures, transfection,
and immunocytochemistry The cells were imaged in an extracellular buffer (pH 7.4, 129 mM NaCl,
5 mM KCl, 2 mM CaCl2 2H2O, 1 mM MgCl2 6H2O, 20 mM Hepes, 30 mM
COS-7 cells were grown in DMEM (Dulbecco’s modified Eagle’s medium, Glucose). To perform steady-state imaging, the microscope was equipped
Invitrogen, Karlsruhe, Germany) that included 10% fetal calf serum (FCS), with a charge-coupled device (CCD) camera (F View, SIS Imaging Systems,
antibiotics (100 units/ml penicillin, 100 mg/ml streptomycin), and 2 mM Duesseldorf, Germany). The filter settings (AHF Analysentechnik, Tuebingen,
glutamine at a density of 105 cells per well (0.5 ml) at 37°C, 5% CO2 Germany) used were D436/20 excitation filter, 455 DCLP dichroic beam
environment. Hippocampal neurons from rat brains of embryonic day 19 were splitter, and D480/40 emission filter for Cerulean and HQ 500/20 excitation
cultured in DMEM that included 10% FCS, antibiotics (100 units/ml filter, Q515 LP dichroic beam splitter, and HQ 535/30 emission filter for
penicillin, 100 mg/ml streptomycin) and 2 mM glutamine. The neurons were Citrine. Only the cells showing moderate expression levels of transfected
grown at a density of 60,000 cells/0.5 ml in Neurobasal medium (without constructs were used for imaging. Confocal fluorescence images as well as
phenol red, Invitrogen) containing B27, antibiotics (100 units/ml penicillin, differential interference contrast (DIC) images were captured using Leica-
100 mg/ml streptomycin), and 2 mM glutamine. COS-7 cells and neurons TCS-SP2-AOBS (Leica Microsystems).

Biophysical Journal 94(4) 1483–1496

Synaptic Interactions by FRET-FLIM 1485

FLIM system lifetime. A set of intervals was defined for each analyzed lifetime in the
model. The values of the upper and lower limits for the interval were set to a
A femtosecond mode-locked titanium sapphire laser (Tsunami Model 3955, 20% variation for the long lifetimes (t1 and t2) in all cases. The variation
690–1080 nm, 80 MHz, Spectra Physics, Mountain View, CA) pumped by a was increased to 60% for t3 to obtain a realistic spread of the x2 minimum
continuous diode laser (Millennia Vs, 5W, TEM00 532 nm, Spectra Physics) surface. The number of points between the upper and lower limits was set to
was tuned and frequency doubled using a frequency doubler/pulse picker 10. The examined parameter was fixed at the current trial value, but all other
(Model 3986, Spectra Physics) to the desired wavelength of 420 nm with a parameters were allowed to vary to minimize the value of x 2. A plot of the
pulse repetition rate of 8 MHz. The excitation probability at this wavelength change of x2 with the change in lifetimes was obtained (Supplementary
was .80% for Cerulean, whereas for Citrine it was ,5% (21). The laser beam Material Fig. 4 c). The maximum error on the lifetimes corresponding to
was coupled alternatively via two optical fibers to different ports of an inverted 67% x 2 confidence was deduced from this plot. Comparison of these results
microscope (IX81, Olympus, Hamburg, Germany). The beam was directed with the obtained multiexponential model was used to judge the quality of
to the sample after passing a beam splitter, 450 DCLP (AHF Analysentechnik), lifetimes in the fit. At 67% x 2 confidence, the maximum error for the in-
and an oil immersion 1003 objective (Plan Apo 1003/1.45 oil, total internal dividual lifetimes obtained was in the range 0.07–0.11 ns for all samples.
reflection fluorescence microscopy, Olympus). The system allowed illumi- Data obtained from the point detector were fit with linked lifetimes for the
nation of the sample for the two types of detectors. The delay line (DL) and decays corresponding to the different emission wavelength bands. The in-
quadrant anode (QA) detectors (Europhoton, Berlin, Germany) (22), based dividual decays were obtained by binning data over a fixed number of con-
on time- and space-correlated single photon counting, were used to study the tinuous wavelength channels, resulting in a net resolution of 7.14 nm per
interaction between tagged fluorophores by simultaneous acquisition of time decay. The contributions of the lifetimes in each of these intensity decays
and space information on a picosecond timescale. More detailed discussions were obtained as preexponential factors ai. ai was plotted along the wave-
of the FLIM system are presented in Jose et al. (6) and Nair et al. (5). length to obtain the DAS. The preexponential factor ai, corresponding to a
A focused beam in combination with an iris was used to limit the area of lifetime in an intensity decay, was positive except in the case of excited state
excitation for recording using the DL detector. The fluorescence emissions reactions, where it changes to negative (25). The comparison of plots of DAS
from the tiny selected area passed an emission filter, HQ 460 ALP (AHF of different multiexponential components allowed us to discriminate among
Analysentechnik), and the slit (11 mm 3 0.10 mm) of the polychromater fixed the fluorescent species involved in different excited state processes. The
in front of the sensitive area of the point detector to translate the spectrally fractional contributions were calculated as jaij/+jaij for the different ex-
resolved intensity decays on the detector. Whole field illumination was ponentials in the model. The mean lifetime t mean of each intensity decay was
provided for the QA detector, which imaged the fluorescence decays simul- calculated as tmean ¼ +jaitij/+jaij, where ti is the lifetime and ai is the
taneously within the whole illuminated region. The fluorescence emission corresponding preexponential factor in the decay. There was no averaging
was split by a beam splitter (dichroic 505 DCXR) and passed into two specific over the entire spectra, since mean lifetimes as well as the contributions of
wavelength bands defined by the band-pass emission filters, D 480/40 M for multiple lifetimes were wavelength dependent. The individual counts at the
Cerulean and 540/40 ALP for Citrine. This allowed the simultaneous moni- peak for each analysis were maintained at 6000–7000. Data were acquired
toring of donor and acceptor dynamics. continuously for 10–15 min using the point detector to get the required pho-
The pulse width of the instrument response function (IRF) in the point tons for analysis.
detector (DL) was reduced to a minimum of 150 6 25 ps, measured at full Data obtained by the imaging detector were analyzed by selecting cor-
width half-maximum. The wavelength sensitivity of the system was calcu- responding regions of interest (ROIs) for the Cerulean and Citrine channels,
lated to be 1.02 nm/channel. The time channel resolution of the point detector as defined by the filter settings. The data sets of individual channels were
was calculated to be 24.81 ps/channel. The pulse width of the IRF of the exported to the Globals Unlimited software format. The donor and acceptor
imaging detector (QA) was reduced to a minimum of 200 6 20 ps at full width decays were analyzed with linked lifetimes. The peak counts were always
half-maximum. The time channel resolution of the imaging detector was ensured to be .10,000. Continuous measurements of 15–20 min were per-
calculated to be 9.72 ps/channel. Independent control measurements of the formed using the imaging detector.
monoexponential dye coumarin6 in ethylene glycol at magic angle, excited The FRET efficiency was calculated as E ¼ 1  tDA/t D, where tD is the
at 420 nm, and observed in a band of 515 6 15 nm (HQ 515/30, AHF unperturbed mean lifetime of the donor and tDA is the mean lifetime of the
Analysentechnik) were performed to ensure the absence of any systemic donor in the presence of an acceptor or the FRET lifetime. The lifetime maps
effects. Lifetimes of 2.30 ns and 2.29 ns were obtained for the point and were generated using the QA Analysis software (Europhoton), fitting from
imaging detectors, respectively; these were similar to the published value of the peak of the intensity decay. An estimate of the background was per-
2.30 ns (23). formed by obtaining mean counts per pixel of areas of background and then
subtracting this level from the whole field image. A lifetime map was then
Data analysis produced by assigning a color on a 16-bit pseudocolor scale to a fitted single-
exponential decay time. The resulting pseudocolor lifetime image was dis-
The data analysis has been discussed in previous works (5,6). To obtain played over 1.1–3.1 ns; this was used to analyze the morphological variation
lifetimes from fluorescence decays, the data were modeled by the convo- of lifetimes in cells in detail.
lution product of a multiexponential theoretical model with the IRF: i(t) ¼
IRF(t)5Saiet/ti, where ai is the relative contribution of the fluorescent
species characterized by the fluorescence lifetime, ti, and IRF is the mea- RESULTS
surement of the pulsed excitation. Data were analyzed by the Levenberg-
Marquardt nonlinear least-squares algorithm using the Globals Unlimited Colocalization studies of Bassoon and its
software package (version 1.20) (24). The quality criterion for global fit was interaction partners in COS-7 cells and neurons
defined as x 2 , 1.3 for all analyzed decays. The criteria for the improvement
of x2 upon addition of multiexponential components were set to a value of It has been proposed that the presynaptic protein Bassoon
Dx 2—the ratio between the x 2 of the previous model and the current model interacts with both CtBP1 (14) and CAST (11) and that these
with the addition of a single lifetime component, where Dx2 . 1.05. x 2 was interactions contribute to the molecular organization of the
checked using the linked multiexponential model and the unlinked model,
active zone of neurotransmitter release. To characterize these
and the data set was discarded if the ratio of x 2 was .1.05, indicating a
random error originating from the data acquisition. interactions in living cells, we used a heterologous expres-
Rigorous error analysis using global analysis software was performed to sion of these binding partners in COS-7 cells. As Bassoon
obtain a realistic estimation of the variation of x 2 associated with each forms macromolecular complexes in the cytoplasm when

Biophysical Journal 94(4) 1483–1496

1486 Jose et al.

expressed in nonneuronal cells (17), we tested whether and CAST to the Bassoon-containing molecular complexes
Bassoon is functional within these complexes, i.e., whether it (Fig. 2, a–d). No such extranuclear recruitment of endoge-
can recruit its binding partners. nous CtBP1 was observed in the presence of CAST alone
Immunofluorescence studies with antibodies against CtBP1 (Fig. 2, e–g).
showed a nuclear localization (Fig. 1, a–c), which changed When expressed in young neurons at DIV 7, a similar
significantly in the presence of a Bassoon construct similar to recruitment of CtBP1 and CAST to the Bassoon-containing
the full-length, namely, Bsn95-3938 (16) (Fig. 1, d–f). In complexes (Fig. 3, a–d) was also observed with Bsn1692-
COS-7 cells, which endogenously do not express Bassoon, 3263. In mature neurons at DIV 16, CtBP1 colocalized with
the protein showed the expected punctate pattern when Bassoon-containing complexes along axons, most likely
expressed heterologously (Fig. 1 d). In this case, the nuclear representing presynapses (Fig. 3, e–g, (14)). Quantification
staining of CtBP1 was significantly reduced with increased of overexpression levels of Bassoon indicated a fivefold in-
immunoreactivity in Bassoon-containing macromolecular crease at synapses (n ¼ 5000 from 10 independent experi-
complexes in the cytosol. Using a fragment of Bassoon ments, Supplementary Material Fig. 2). These findings suggest
(Bsn1692-3263, see Fig. 7), which in neurons is still recruited that Bassoon has a role in recruiting its binding partners to
to the presynapse, a similar change in the distribution of protein complexes at presynaptic active zones.
CtBP1 immunoreactivity was observed (Fig. 1, g–i). Quan-
titative analysis revealed a 1.4-fold reduction of CtBP1
Photophysics of Cerulean and Citrine in
immunoreactivity in the nucleus of COS-7 cells in the
living cells
presence of Cerulean-Bsn1692-3263 (n ¼ 100, Supplemen-
tary Material Fig. 1). Thus, clear interference with the nuclear The photophysical properties of the common fluorescent probes
recruitment of CtBP1 was observed in the presence of both CFP and YFP have been observed to be perturbed by cellular
Bassoon constructs. Coexpression of Bassoon (Bsn95-3938 environments (6). Therefore, it was crucial to understand the
or Bsn1692-3263) with CAST in COS-7 cells followed by behavior of photostable variants of them, namely Cerulean
CtBP1 immunostaining revealed a corecruitment of CtBP1 and Citrine, used for the FRET-FLIM studies in living cells.

FIGURE 1 Immunofluorescence of CtBP1 in

COS-7 cells in the absence and presence of
Bassoon. (a–c) DIC images (a) together with
immunostainings against CtBP1 (c) of the same
cells showed clear nuclear localization of CtBP1.
(d–i) Disruption of the nuclear localization of CtBP1
(f and i) in the presence of a Bassoon construct
similar to the full length, namely, Bsn95-3938 (d)
as well as in the presence of Bsn1692-3263 (g). In
the overlay images (e and h), GFP-Bsn95-3938 and
Cerulean-Bsn1692-3263 are shown in green, whereas
endogenous CtBP1 stained with Alexa 594 is shown
in red. Yellow denotes the degree of colocalization
of these proteins. In both cases, CtBP1 was en-
riched outside the nucleus in contrast to its original
localization pattern.

Biophysical Journal 94(4) 1483–1496

Synaptic Interactions by FRET-FLIM 1487

FIGURE 2 Corecruitment of Bassoon, CtBP1,

and CAST to the same intracellular complexes in
COS-7 cells (a–d). Cells were transfected with
Cerulean-CAST (a) and Citrine-Bsn1692-3263 (b)
and immunostained with antibodies against CtBP1
(c). The overlay of the trimeric complex is given in
(d), where Cerulean-CAST, Citrine Bsn1692-3263,
and CtBP1 are shown in green, red, and blue,
respectively. (e–g) In the absence of Bassoon, the
presence of CAST did not affect the nuclear lo-
calization of endogenous CtBP1. Cerulean-CAST
(g) was observed to form cytosolic complexes, but
no extranuclear recruitment of CtBP1 to these com-
plexes was observed as visualized by Alexa 594
immunostainings (e).

COS-7 cells expressing Cerulean alone showed emission ns. The latter arose most likely from cell autofluorescence, as
maxima at 486 nm (Fig. 4 a). In contrast to the monoex- observed from the DAS (Supplementary Material Fig. 4 d),
ponential fluorescence decay previously reported in solu- which showed a completely different distribution compared
tions (19), a biexponential decay was observed for Cerulean to the 3.20 ns component.
in living COS-7 cells with lifetimes of 3.37 6 0.03 ns (t 1)
and 1.32 6 0.05 ns (t 2) (Table 1, Fig. 4 b). DAS revealed
Interaction studies of the trimeric complex
similar distributions for the lifetimes, confirming the source
Bassoon, CtBP1, and CAST by FRET-FLIM in
of both lifetimes to be the Cerulean chromophore (Fig. 4 c).
living COS-7 cells
The quality of decay fits is presented in Supplementary Ma-
terial Fig. 4, a and b. The rigorous error analysis of multiple To achieve deeper insight into the interactions and complex
lifetimes was performed to ensure the reliability of the values formation of the different CAZ proteins including Bassoon,
(Supplementary Material Fig. 4 c). The mean lifetime at the CtBP1, and CAST, the colocalization studies of these pro-
donor emission maxima was calculated to be 2.46 6 0.04 ns, teins were extended to live cell interaction studies by FRET
which did not show any deviation along the wavelength axis and FLIM. Different binding strategies with Cerulean- and
(Fig. 4 d). The advantage of FRET determination by donor Citrine-fused constructs as donor-acceptor pair were adopted
excitation and simultaneous analysis of donor and acceptor for this purpose, as discussed below.
emission was signified by coexpressing Cerulean with a stable
variant of YFP called Citrine. The fluorescence emission spec-
Interaction studies of Bassoon and CtBP1
tra or decay of this mixture were not different from those of
Cerulean, except for a small emission near the YFP peak Though the possibility for binding between Bassoon and
(Fig. 4, a and b) arising due to the high molar extinction CtBP1 has been indicated by biochemical techniques (14), a
coefficient of Citrine (20). Direct excitation of Citrine alone confirmation of this interaction under real cell conditions was
at 420 nm resulted in a single lifetime of 3.20 6 0.03 ns crucial to appreciate its physiological relevance. Since the
contaminated by a short lifetime contribution of 0.22 6 0.14 Cerulean full-length Bsn was too dim for FRET studies with

Biophysical Journal 94(4) 1483–1496

1488 Jose et al.

FIGURE 3 Colocalization of Bassoon, CtBP1,

and CAST to the same intracellular complex in
hippocampal neurons (a–d). Cells were trans-
fected with Cerulean-CAST (a) and Citrine-
Bsn1692-3263 (b) and immunostained with
anti-CtBP1-Alexa 594 (c). The overlay of the
trimeric complex is given in d, where Cerulean-
CAST, Citrine-Bsn1692-3263, and CtBP1 are
shown in green, red, and blue, respectively. (e–g)
In the presence of Bassoon (green), CtBP1 (red)
colocalized with Bassoon-containing complexes
at DIV 16, most of which are likely to represent
synapses.

high contamination from cell autofluorescence (;5- to 10-fold) in the contributions of individual lifetimes were observed
at the excitation wavelength used, discrimination of energy (Table 1). For the FRET sample, the contribution of
transfer from cellular artifacts was difficult. Therefore, a t 1 decreased from 44% to 24%, whereas contributions of t 2
brighter, shorter fragment of it, namely Bsn1692-3263 tagged and t 3 increased from 38% and 19% to 45% and 31%,
with Cerulean, was used for the purpose (see Fig. 7). This respectively. This resulted in a reduction of the mean lifetime at
construct showed a presynaptic recruitment similar to the the donor emission maxima from 2.09 6 0.17 ns to 1.51 6
full-length construct (16). 0.10 ns (Table 1), which increased along the spectra to 2.87 6
Bsn1692-3263 showed high aggregating properties when 0.15 ns at the acceptor maxima (Fig. 5 f). DAS displayed
expressed in COS-7 cells. As a result, though the fluorescence negative preexponential factors for t 2 and reduced contribu-
emission spectrum was unaffected, the fluorescence decay tion for t 3 at the acceptor emission maxima (Fig. 5 e).
of Cerulean Bsn1692-3263 was deviated, resulting in a third For a detailed analysis of the interaction, COS-7 cells
short lifetime component of 0.18 6 0.05 ns (t 3) (Table 1, Fig. expressing Cerulean-Bsn1692-3263 and Citrine-CtBP1 were
5, a and b). A cellular variability of this lifetime component imaged using the QA detector. Small ROIs were defined, and
was observed (with contributions ,20%) due to the aggre- the corresponding fluorescence decays were analyzed for the
gating nature of Bsn1692-3263. DAS showed similar distri- donor and the acceptor probes simultaneously (Fig. 6, a and
butions for all three lifetimes with positive preexponential b). Multiexponential analysis revealed similar lifetimes as
factors (Fig. 5 c). The contribution of the short lifetime observed with the point detector (Table 2), and the quality of
component resulted in a reduction of the mean lifetime of this decay fits of both donor and acceptor probes were confirmed
construct to 2.09 6 0.17 ns at its emission peak (Table 1), (Fig. 6 c). The acceptor fluorescence decay displayed nega-
which displayed no deviation along the wavelength axis tive preexponential factors (,10%) for the 1.4 ns (t 2) com-
similar to Cerulean alone (Fig. 5 d). ponent. The lifetime distributions of the puncta were mapped
Coexpression of Cerulean-Bsn1692-3263 and Citrine- for a deeper understanding of the complex formation (Fig. 6 d).
CtBP1 resulted in significant differences in the fluorescence Significant reduction in the donor mean lifetimes with a
emission spectra as well as the decay of both probes (Fig. 5, a simultaneous increase in the mean lifetimes of the acceptor
and b). A Citrine enhancement at 529 nm in the spectra arose were observed, as indicated by the transition from blue to red
due to energy transfer (Fig. 5 a). The absolute values of dif- in the lifetime scale. Small deviations in the fluorescence
ferent lifetimes of Cerulean-Bsn1692-3263 remained un- lifetimes from the two detectors were expected due to the
changed in the presence of FRET, though significant changes differential illumination methods used.

Biophysical Journal 94(4) 1483–1496

Synaptic Interactions by FRET-FLIM 1489

FIGURE 4 Fluorescence emission of

Cerulean at 420 nm excitation in living
COS-7 cells. (a) Comparison of fluo-
rescence emission spectra of Cerulean
alone (black) with coexpression of
Cerulean and Citrine (shading). The
fluorescence emission maximum for
Cerulean was observed at 486 nm. A
small Citrine component due to its high
extinction coefficient was observed for
the latter. (b) Fluorescence decay of
Cerulean in the absence (black) and in
the presence of Citrine (shading). IRF
is shown in light shading. (c) DAS of
Cerulean alone, which was unchanged
in the presence of Citrine. Intensity
decays were analyzed in 17 emission
bands from 470 nm to 590 nm, and the
preexponential factors of lifetimes
t1 (solid) and t2 (shading) were plotted
along the wavelength (Table 1). (d)
Mean lifetimes of each emission band
of Cerulean were plotted along the
wavelength. Error bars are not shown
due to their very small variability
(,0.05 ns).

The FRET efficiencies were directly calculated from the FLIM studies of Bassoon and CAST
differences in the donor mean lifetimes in the absence and
Cerulean-Bsn1692-3263 and Citrine-CAST expressing COS-7
presence of FRET. Using mean lifetimes of Cerulean-
cells were imaged to study interactions between these pro-
Bsn1692-3263 alone as t D (Table 1) and in the presence of
Citrine-CtBP1 as t DA (Table 1), the FRET efficiency of the teins. The coexpressed cells revealed fluorescence dynamics
Bassoon-CtBP1 complex in COS-7 cells was 28%. The orig- (Table 1) similar to the donor control alone (i.e., Cerulean-
inal energy transfer efficiency may be higher than this value Bsn1692-3263), except for a slight Citrine enhancement in
due to the inclusion of the contribution of free conformers of the fluorescence emission spectra (Supplementary Material
donor not participating in FRET (6). But this influence of Fig. 5, a and b). A slight reduction in the mean lifetime of the
free donors on t DA could not be determined because of the coexpressed sample at the donor emission maxima was ob-
aggregation of Bsn1692-3263 in COS-7 cells, which causes served (1.93 6 0.14 ns), which increased to 2.17 6 0.24 ns at
variability in control properties. the acceptor emission maxima. Similar to the previous case,

TABLE 1 The decay kinetics of Cerulean and its fusion constructs in COS-7 cells in the absence and presence of FRET
Construct n t1% t1 (ns) t 2% t2 (ns) t 3% t 3 (ns) t mean (ns)
Cerulean 5 56 6 1 3.37 6 0.03 45 6 1 1.32 6 0.05 - - 2.46 6 0.04
Cerulean1Citrine 5 57 3.32 6 0.02 43 1.23 6 0.06 - - 2.41 6 0.04
Cerulean-Bsn1692-3263 5 44 6 5 3.35 6 0.12 38 6 2 1.56 6 0.12 19 6 5 0.23 6 0.07 2.09 6 0.17
Cerulean-Bsn1692-32631Citrine-CtBP1 5 24 6 2 3.39 6 0.06 45 6 2 1.42 6 0.11 31 6 2 0.20 6 0.10 1.51 6 0.10
Cerulean-Bsn1692-32631Citrine-CAST 9 34 6 6 3.41 6 0.23 44 6 4 1.60 6 0.21 22 6 4 0.31 6 0.12 1.93 6 0.14
Cerulean-CtBP1 7 58 6 1 3.44 6 0.04 42 6 1 1.39 6 0.08 - - 2.57 6 0.07
Cerulean-CtBP1 1 Citrine-CAST 8 60 6 1 3.41 6 0.05 41 6 1 1.29 6 0.07 - - 2.55 6 0.07
Cerulean-CtBP1 1 Citrine-CAST 1 12 58 6 2 3.24 6 0.04 42 6 2 1.06 6 0.12 2.33 6 0.11
*Bsn1692-3263
Cerulean-Bsn1692-32631Citrine-CtBP11 10 24 6 1 3.37 6 0.04 49 6 3 1.39 6 0.08 28 6 2 0.24 6 0.07 1.54 6 0.07
Citrine-CAST
The fluorescence lifetimes of Cerulean were not perturbed in the presence of coexpressed Citrine in the same cell. However, significant changes in the properties
of Cerulean were observed depending on the protein to which it was tagged as well as in the presence of energy transfer. The donor mean lifetimes displayed
significant reduction in the presence of FRET. The lifetimes are denoted t1, t 2, and t 3, and their corresponding contributions at the donor emission maxima
are denoted t1%, t 2%, and t3%. tmean represents the mean lifetime at the donor emission maxima. Errors ,1% for the contributions are not shown. The number
of independent measurements is denoted n. All data were recorded using the point detector. *Bsn1692-3263 refers to the untagged Bsn1692-3263.

Biophysical Journal 94(4) 1483–1496

1490 Jose et al.

FIGURE 5 Fluorescence emission of

Cerulean-Bsn1692-3263 in the absence
and presence of Citrine-CtBP1 in living
COS-7 cells. (a) Comparison of fluo-
rescence emission spectra of Cerulean-
Bsn1692-3263 (black) with the FRET
sample (shading). A significant Citrine
enhancement was observed in the pres-
ence of FRET. (b) Fluorescence decay
of Cerulean-Bsn1692-3263 in the
FRET sample (shading) was shorter
compared to the control (black). IRF is
shown in light shading. (c) DAS of
control Cerulean-Bsn1692-3263. The
preexponential factors of lifetimes
t1 (solid), t2 (shading), and t3 (light
shading) were plotted along the wave-
length (Table 1). (d) Mean lifetimes of
each emission band were plotted along
the wavelength for Cerulean-Bsn1692-
3263. The variability between the cells
is represented by error bars, which
shows deviation due to the aggregating
nature of the construct. (e) DAS of the
FRET sample, Cerulean-Bsn1692-
32631Citrine-CtBP1. The preexpo-
nential factors of lifetimes t1 (solid),
t2 (shading), and t3 (light shading) (see
Table 1) were plotted along the wave-
length. Negative amplitudes were ob-
served for t2 at the acceptor emission
maximum in the presence of FRET. (f)
Mean lifetimes of each emission band
were plotted with error bars along the
wavelength for the FRET sample.

energy transfer efficiencies were calculated from the donor sample of Cerulean-CtBP1 with Citrine-CAST showed fluo-
mean lifetimes of the control and the coexpressed sample rescence dynamics very similar to Cerulean (Table 1, Sup-
(Table 1). The results indicated a low energy transfer effi- plementary Material Fig. 5, c and d). The results indicated
ciency of 8%, which could be due to the large distance between that there is no direct interaction between the two proteins in
the fluorophores of the interacting molecules. This was in COS-7 cells, confirming the observation of Fig. 2, e–g, that
accordance with the reduction of the contribution of t 1 with coexpression of CAST does not change the endogenous
a simultaneous increase in the contributions of the short localization of CtBP1.
lifetime components at the donor maxima of the coexpressed
sample (Table 1), compared to the control. The large devi- Interaction studies of Bassoon, CtBP1, and CAST
ations in the lifetimes, particularly in t 1 (Table 1), could be
attributed to the highly oligomerizing nature of Bsn1692- To check whether Bassoon can simultaneously bind CtBP1
3263 and CAST (26), which could also result in the lowering and CAST, the fluorescence dynamics of Cerulean-CtBP1
of acceptor mean lifetimes. and Citrine-CAST were monitored in the presence of un-
tagged Bsn1692-3263 (Supplementary Material Fig. 5, e and f)
as well as the untagged full-length Bsn construct. For the
FLIM studies of CtBP1 and CAST
triple-transfected cells, the mean lifetimes at the donor emis-
COS-7 cells expressing CtBP1 and CAST were measured to sion maxima showed a slight reduction, to 2.33 6 0.11 ns
study interactions between the proteins in the absence of (Table 1). The influence of the aggregating character of
Bassoon. Cerulean-CtBP1 alone as well as the coexpressed Bsn1692-3263 on Cerulean-CtBP1 dynamics was counter-

Biophysical Journal 94(4) 1483–1496

Synaptic Interactions by FRET-FLIM 1491

FIGURE 6 COS-7 cells coexpressing Ceru-

lean-Bsn1692-3263 and Citrine-CtBP1 measured
by the imaging detector. (a) The fluorescence
emission was split into Cerulean-Bsn1692-
3263 and Citrine-CtBP1 emission bands as
shown. Selected ROIs are indicated by colored
circles. (b) Fluorescence decays from corre-
sponding ROIs marked in a. In the presence of
energy transfer, Cerulean-Bsn1692-3263 (green)
showed short fluorescence decay with a simul-
taneous rise for Citrine-CtBP1 (red). IRF is
shown in black. (c) The quality of the individ-
ual fits (donor-green, acceptor-red) is shown by
the distribution of the residues. (d) The lifetime
distribution map of the FRET sample. The
lifetimes increased significantly from the donor
(top) to the acceptor (bottom), indicated by the
transition from blue to red in the lifetime scale.
(e) In the corresponding fluorescence images
from the CCD camera, the green and red spots
denote the donor and acceptor channels, re-
spectively.

checked by coexpressing the proteins to exclude any differ- CtBP1 and CAST on Bassoon were competitive, or if dif-
ences in the donor properties. Donor mean lifetimes in this case ferent Bassoon molecules were binding to CtBP1 and CAST,
were very similar (t D ¼ 2.51 6 0.04 ns) to that of Cerulean- an overall decrease in the FRET efficiency between Bassoon
CtBP1 alone. From the donor mean lifetimes of the control and CtBP1 would be expected in the presence of coexpressed
(t D) and of the coexpressed sample (t DA, Table 1), the results CAST. This should result in an increase in the donor mean
indicated a very low energy transfer efficiency, 7%, pointing lifetime of the triple-coexpressed sample close to the control,
to a simultaneous binding of CtBP1 and CAST to Bassoon. in contrast to the FRET pair Bassoon-CtBP1. Interestingly,
A different strategy was adopted to confirm this hypothesis. the fluorescence kinetics (Table 1) and the resulting FRET
COS-7 cells expressing Cerulean-Bsn1692-3263 together with efficiency between the Bassoon-CtBP1 pair remained un-
Citrine-CtBP1 and Citrine-CAST were measured. FLIM mea- changed (E ¼ 26%) in the presence of Citrine-CAST. The
surements between Bsn1692-3263 and CAST have shown a presence of all three proteins in the same complexes was
reduced FRET efficiency, with donor mean lifetimes close to ensured by immunofluorescence stainings of the measured
that of the control (Table 1). Therefore, if the binding of cells with antibodies against CtBP1 and CAST. The results

Biophysical Journal 94(4) 1483–1496

1492 Jose et al.

TABLE 2 Fluorescence dynamics of Cerulean in COS-7 cells measured by the imaging detector in the absence
and presence of FRET
Construct t 1% t1 (ns) t 2% t2 (ns) t 3% t3 (ns) tmean (ns)
Cerulean-Bsn1692-3263 40 6 3 3.14 6 0.09 41 6 2 1.57 6 0.07 19 6 3 0.39 6 0.12 1.98 6 0.11
Cerulean-Bsn1692-3263 1 Citrine-CtBP1 21 6 3 3.3 6 0.23 48 6 4 1.44 6 0.20 31 6 5 0.38 6 0.12 1.49 6 0.18
The fluorescence lifetimes of Cerulean-Bsn1692-3263 as well as their fractional contributions were significantly changed in the presence of FRET, which
resulted in a drastic decrease of donor mean lifetimes. The lifetimes are denoted t1, t2, and t3, and their corresponding contributions are denoted t1%, t2%,
and t 3% in the donor emission band. t mean represents the mean lifetime in the donor channel. The values shown are from five independent measurements by
the imaging detector, with at least six ROIs selected from each cell.

indicated that there was no inhibition for the Bassoon-CtBP1 with lifetimes of 3.29 6 0.04 ns (t 1; 25%), 1.29 6 0.11 ns (t 2;
interaction in the presence of CAST, thus confirming the 44%), and 0.16 6 0.04 ns (t 3; 31%) (Table 3, Fig. 8 b),
model of simultaneous binding of CtBP1 and CAST on the resulting in a mean lifetime of 1.44 6 0.16 ns. Due to energy
same Bassoon molecule. The different binding studies and transfer, the mean lifetime increased to 2.89 6 0.14 ns (Fig. 8
the resulting FRET efficiencies are summarized in Fig. 7. f) at the acceptor emission peak; the increase was accompa-
nied by a dramatic change in the contributions of t 1 (84% 6
6%), t 2 (10% 6 4%), and t 3 (,10%), with t 2 displaying
Interaction studies of Bassoon and CtBP1 by negative preexponential factors at the same (Fig. 8 e).
FRET-FLIM in living hippocampal neurons Subsequent to the spectroscopic analysis with the point
Since the maximum FRET efficiency was observed between detector, subcellular compartments of the same neuronal
Bassoon and CtBP1, this interaction was further studied at processes were measured using the imaging detector (Fig. 9).
synaptic complexes of hippocampal neurons. To overrule any The deduced lifetimes from the multiexponential analysis
deviations in the photophysical properties of Cerulean due to showed minor changes from the point detector. Cerulean-
ionic variations in neurons along development (6), neurons Bsn1692-3263 in the colocalized complexes displayed faster
expressing the donor fusion construct Cerulean-Bsn1692- fluorescence decay compared to the donor control (Fig. 9 b).
3263 were imaged at young (DIV 9) as well as at mature (DIV This was accompanied by a rise in the acceptor decay kinetics.
16) stages. Data presented here are from mature cells (DIV The lifetime distribution map also indicated the presence of
16). Irrespective of the maturation stage of neurons, the fluo- energy transfer in the synaptic complexes, with reduced mean
rescence dynamics of Cerulean-Bsn1692-3263 remained un- lifetimes for Cerulean-Bsn1692-3263 increasing significantly
changed, with the emission maximum at 486 nm (Fig. 8 a). In in the acceptor channels (Fig. 9 c). This was confirmed from
contrast to the triexponential decay of this construct in COS-7 the multiexponential analysis, which revealed negative con-
cells, a biexponential fluorescence decay was observed in tributions (,1%) for t 2 in the acceptor channels. The difficulty
neurons. The multiexponential analysis revealed lifetimes of (due to its lower wavelength resolution) in observing negative
3.14 6 0.14 ns (t 1) and 1.29 6 0.16 ns (t 2) with contributions preexponential factors using the imaging detector instead of
of 60% 6 3% and 40% 6 3%, respectively, at its emission the point detector has been discussed (6). The imaged cells
maxima (Table 3, Fig. 8 b). This resulted in a mean lifetime of were fixed and immunostained with antibodies against the
2.39 6 0.15 ns at its emission maxima, which remained postsynaptic marker SAP90/PSD-95; this identified many of
constant over the spectra (Fig. 8 d). DAS displayed positive the colocalized complexes of Bassoon and CtBP1 specifically
values for the preexponential factors of all lifetimes (Fig. 8 c). as synapses (Fig. 9 d).
The unchanging fluorescence dynamics of Cerulean-Bsn1692- FRET efficiency calculations were performed in the same
3263 at different stages of neuronal development (DIV 9 and way as those for COS-7 cells. For interactions in the pro-
16) confirmed the photostability of the probe, proving it to be cesses, using t D of Cerulean-Bsn1692-3263 (Table 3) and
a suitable FRET donor in living neurons. The biexponential t DA of the FRET sample (Table 3), the FRET efficiency was
fluorescence decay of recombinant Bassoon in neurons indi- calculated to be 40% in synapses. Similar FRET efficiencies
cated the lower aggregation probability of the construct when were also observed for Bassoon-CtBP1 complexes in the soma.
expressed in its natural environment (17). The difference in efficiency between neurons and COS-7
Fluorescence emission from processes of mature neurons at cells may be attributed to the variation in the donor prop-
DIV 16 expressing Cerulean-Bsn1692-3263 and Citrine-CtBP1 erties due to the differential aggregation of Bassoon between
was collected to study the interactions at synapses. The area the cell types.
of illumination was restricted by closing an iris in the beam
path (see Materials and Methods). The fluorescence spectra
DISCUSSION
collected from colocalizing complexes showed the donor emis-
sion maximum at 486 nm with an additional Citrine en- Using a novel FLIM approach with simultaneous acquisition
hancement at 529 nm (Fig. 8 a), the latter arising due to energy of donor and acceptor decays and DAS to verify FRET, we
transfer. The fluorescence decays at the donor maxima were fit confirmed a direct physical association of synaptic proteins

Biophysical Journal 94(4) 1483–1496

Synaptic Interactions by FRET-FLIM 1493

The data provided a direct confirmation of the interaction of

Bassoon and CtBP1 at synapses of living hippocampal
neurons.
Though Bassoon, CtBP1, and CAST were recruited to the
same molecular complexes (Figs. 2 and 3), it was essential to
understand whether CtBP1 and CAST could bind simulta-
neously to Bassoon or whether the binding of the proteins
would be competitive. The capability of Bassoon to simul-
taneously bind the two interaction partners was confirmed
from FLIM studies.
Cerulean-Bsn1692-3263, when expressed in COS-7 cells,
exhibited complex behavior and had a shorter lifetime com-
ponent in the fluorescence decay (Table 1, Fig. 5 b). This was
in agreement with previous reports that showed the tendency
of the protein to form clusters in nonneuronal cells (16).
Fluorescence characterization of the different deletion con-
structs of Bassoon traced this property of aggregation to the
second coiled-coil domain of the protein, when overex-
pressed. The comparison of the fluorescence dynamics of
Cerulean alone with its fusion constructs showed how the
properties of a fluorescent probe depend on the protein
component to which it is fused (Table 1). The higher ag-
gregation tendency as well as the largeness of the fused pro-
tein may affect the intrinsic properties of fluorophores. This
may result in a variation in the photophysical properties of
fluorophores of recombinant proteins from those of the con-
trol. This was also in accordance with the biexponential fluo-
rescence decay of the same construct (Cerulean- Bsn1692-3263)
in neurons, where lower aggregation was observed (Fig. 8 c).
In the presence of Citrine-CtBP1, Cerulean-Bsn1692-3263
showed FRET with DAS displaying negative preexponential
factors for t 2, indicating the involvement of this lifetime in
energy transfer (Figs. 5 e and 8 e). The change in sign at the
FIGURE 7 Combinatorial expressions of Bassoon, CAST, and CtBP1 in acceptor emission maxima evolved only due to the presence
COS-7 cells. (a) Scheme illustrating the proposed binding regions for CtBP1 of energy transfer (25). The comparison of contributions of
and CAST on Bassoon (11,14). The white lines indicate the projection of individual lifetimes further affirmed the suitability of the
Bsn1692-3263 on the full-length construct. Zn denotes the zinc fingers and
CC the coiled coil domains. (b) The different binding studies for determining
approach used here, whereby FRET could be distinguished
the association of the molecules involved in the trimeric complex. The even in the presence of additional lifetime components from
maximum FRET efficiency (E) was observed between Bassoon and CtBP1, the donor, as in COS-7 cells (Table 1). Rigorous error analysis
which remained unchanged in the presence of CAST. Bassoon was essential performed on multiple lifetimes confirmed the reliability of
for the formation of a trimeric complex of these proteins, without which no the deviations in fluorescence properties in the presence of
association between CtBP1 and CAST was observed. The data confirmed
the simultaneous association of CtBP1 and CAST with the same Bassoon
energy transfer.
molecule. The white and shaded ellipses represent Cerulean and Citrine, For a detailed analysis of the Bassoon-CtBP1 interaction
respectively. at individual puncta, recordings were done using the imaging
detector. Small ROIs corresponding to groups of these com-
plexes (Figs. 6 and 9) were selected for multiexponential
analysis. The morphological distribution of mean lifetimes
Bassoon and CtBP1 in living COS-7 cells as well as in living was obtained from the FLIM map (Figs. 6 d and 9 c). These
neurons. Despite deviations in the photophysical properties combined approaches, in turn, gave more extensive infor-
of fluorophores depending on the protein component to which mation on the heterogeneity of protein interactions inside a
they were fused, interactions were associated with significant cell at normal conditions and confirmed the presence of en-
changes in the donor and acceptor mean lifetimes as well as ergy transfer.
negative amplitudes for the FRET lifetimes at the acceptor There was no direct way to determine whether CtBP1 and
emission maxima. Bassoon was observed to be essential for CAST can be recruited to the same Bassoon molecule, since
forming a macromolecular complex with CtBP1 and CAST. the coexpression of these proteins with the untagged Bassoon

Biophysical Journal 94(4) 1483–1496

1494 Jose et al.

FIGURE 8 Fluorescence emission

dynamics of Cerulean-Bsn1692-3263
in the absence (n ¼ 8) and presence
(n ¼ 5) of Citrine-CtBP1 at synapses of
living neurons at DIV 16. (a) Compar-
ison of fluorescence emission spectra of
Cerulean-Bsn1692-3263 (black) with
the FRET sample (shading). A signif-
icant Citrine enhancement was ob-
served in the presence of FRET. (b)
Fluorescence decay of Cerulean-
Bsn1692-3263 in the FRET sample
(shading) was shorter than the control
(black). IRF is shown in light shading.
(c) DAS of Cerulean-Bsn1692-3263 in
living neurons. The preexponential fac-
tors of lifetimes t1 (solid) and t2
(shading) (see Table 3) were plotted
along the wavelength. The preexponen-
tial factors showed only positive values.
(d) Mean lifetimes of each emission
band of Cerulean-Bsn1692-3263 was
plotted along the wavelength. No devi-
ations along the spectra were observed.
(e) DAS of the FRET sample, Cerulean-
Bsn1692-32631Citrine-CtBP1. The pre-
exponential factors of lifetimes t1 (solid),
t2 (shading), and t3 (light shading)
(Table 3) were plotted along the wave-
length. Negative amplitudes were ob-
served for t2 at the acceptor emission
maximum in the presence of FRET. (f)
The plot of mean lifetimes showed
significant deviations along the wave-
length in the presence of FRET.

did not result in a strong energy transfer. Therefore, an al- and CAST and potentially other molecules of the CAZ
ternative strategy for confirming this model was applied in network inside a cell.
which all three proteins with fluorescent tags were coex- The CAZ proteins known to date, including CAST, RIM1,
pressed. The results indicated no difference from the co- Munc13-1, Bassoon, and Piccolo, have been proposed to
expression of Bassoon and CtBP1, thus confirming that the form a large dynamic multimolecular complex in vivo (12).
proposed trimeric complex can be formed. The different Here, a direct physical interaction between Bassoon and
binding strategies proved Bassoon to be an essential linker in CtBP1 was shown in synaptic complexes of living hippo-
the formation of such multiprotein complexes linking CtBP1 campal neurons (Figs. 8 and 9). Although the physiological

TABLE 3 Fluorescence dynamics of Cerulean in living hippocampal neurons in the absence and presence of FRET
Construct n t 1% t1 (ns) t 2% t 2 (ns) t 3% t3 (ns) tmean (ns)
Cerulean-Bsn1692-3263 8 60 6 3 3.14 6 0.14 44 6 3 1.29 6 0.16 - - 2.39 6 0.15
Cerulean-Bsn1692-32631Citrine-CtBP1 5 25 6 6 3.29 6 0.04 44 6 5 1.29 6 0.11 31 6 3 0.16 6 0.04 1.44 6 0.16
Similar to the results observed in COS-7 cells, the contributions of the lifetimes of Cerulean-Bsn1692-3263 was significantly affected in the presence of
Citrine-CtBP1. The lifetimes are denoted t1, t2, and t3, and their corresponding contributions at the donor emission maxima are denoted t1%, t2%, and t3%.
tmean is the mean lifetime at the donor emission maxima. The number of independent measurements is denoted n. The data were taken from the synaptic
complexes in axonal processes of hippocampal neurons, using the point detector.

Biophysical Journal 94(4) 1483–1496

Synaptic Interactions by FRET-FLIM 1495

FIGURE 9 Processes of hippocampal neurons at DIV 16

coexpressing Cerulean-Bsn1692-3263 and Citrine-CtBP1,
as measured by the imaging detector. (a) The fluores-
cence emission was split into Cerulean-Bsn1692-3263 and
Citrine-CtBP1 emission bands as shown. (b) In the presence
of energy transfer, Cerulean-Bsn1692-3263 (green) showed
a short fluorescence decay with a simultaneous rise for
Citrine-CtBP1 (red) with negative amplitudes for t2. IRF is
shown in black. (c) The lifetime distribution map of the
FRET sample. The lifetimes increased significantly from
the donor (top) to the acceptor (bottom), indicated by the
transition from blue to red in the lifetime scale. (d) The
imaged cells were fixed and immunostained with antibodies
against the postsynaptic marker protein Sap90/PSD-95,
which identified the colocalized complexes of Bassoon and
CtBP1 to be presynapses. Confocal images were obtained
from similar regions as measured by FLIM. In the overlay
image, Cerulean-Bsn1692-3263, Citrine-CtBP1, and PSD-
95 are shown in green, red, and blue, respectively.

significance of this interaction is not yet known, the interac- mation of such networks of proteins could be the basic
tion of Bassoon and CAST has been shown to be implicated mechanism by which Bassoon serves its scaffolding role in
in synaptic transmission (11,27). The interaction of these the molecular organization of the CAZ. The studies were
proteins could play a role in the molecular arrangement of the extended to confirm direct physical association of Bassoon
CAZ. Due to its capacity to form oligomers (26), CAST could and CtBP1 in synaptic complexes of living hippocampal
serve as a cross-linker of other CAZ molecules, including neurons. The association and dissociation of such presynap-
Bassoon and its sister molecule Piccolo. CtBP1 is a multi- tic complexes could, in turn, affect the synapse formation,
functional protein involved in the fission of vesicles from the maintenance, and plasticity. Further studies are necessary to
trans-Golgi network (28). It was originally identified as a better understand the molecular mechanisms that spatially
transcription corepressor localized in the nucleus (15). More- and temporally regulate the coupling of the CAZ protein com-
over, the paralog CtBP2 is part of a larger molecule called plex and the neurotransmitter release machinery. Though
Ribeye, which is a major structural component of synaptic FRET-FLIM studies in living neurons were highly chal-
ribbons—a specialization of the CAZ at ribbon synapses in lenging due to the different cellular influences involved,
retinal photoreceptors or inner ear hair cells (29,30). Data time-resolved imaging under optimal conditions with proper
presented here suggest recruitment of CtBP1 to the CAZ characterization resulted in a wealth of information regarding
network via its association with Bassoon. synaptic scaffolding mechanisms that, to our knowledge, are
not available by any other existing method.

CONCLUSIONS
SUPPLEMENTARY MATERIAL
The nanoscale organization of the CAZ is thought to be vital
To view all of the supplemental files associated with this
for the efficient functioning of presynapses. The potential for
article, visit www.biophysj.org.
the formation of a trimeric complex with the simultaneous
binding of CtBP1 and CAST on the same Bassoon molecule The authors thank Prof. D. W. Piston, Vanderbilt University, Tennessee,
was proven by FRET using time domain FLIM. The for- and Prof. R. Y. Tsien, University of California, San Diego, for kindly

Biophysical Journal 94(4) 1483–1496

1496 Jose et al.

providing the p-Cerulean and p-Citrine cDNAs, respectively. We are 15. Corda, D., A. Colanzi, and A. Luini. 2006. The multiple activities of
grateful to Kathrin Gruss for her expert technical assistance. CtBP/BARS proteins: the Golgi view. Trends Cell Biol. 16:167–
173.
This work was supported by the Deutsche Forschungsgemeinschaft (ZU59/
5-1/2 and FOR 521-HA3498/1) to W.Z. and (AL 1115/1-1) to W.D.A.; the 16. Dresbach, T., A. Hempelmann, C. Spilker, S. tom Dieck, W. D.
Altrock, W. Zuschratter, C. C. Garner, and E. D. Gundelfinger. 2003.
European Commission NMP4-CT-2005-013880 and MRTN-CT-2005-
Functional regions of the presynaptic cytomatrix protein bassoon:
0198481 to W.Z. and SynScaff to E.D.G.; as well as the Land Saxony significance for synaptic targeting and cytomatrix anchoring. Mol. Cell.
Anhalt (N2), the Fonds der Chemischen Industrie and Max Planck Award Neurosci. 23:279–291.
of the Humboldt Foundation and the Max Planck Society to E.D.G.
17. Dresbach, T., V. Torres, N. Wittenmayer, W. D. Altrock, P. Zamorano,
W. Zuschratter, R. Nawrotzki, N. E. Ziv, C. C. Garner, and E. D.
Gundelfinger. 2006. Assembly of active zone precursor vesicles:
REFERENCES obligatory trafficking of presynaptic cytomatrix proteins Bassoon and
Piccolo via a trans-Golgi compartment. J. Biol. Chem. 281:6038–6047.
1. Ballestrem, C., and B. Geiger. 2005. Application of microscope-based 18. Kuner, T., and G. J. Augustine. 2000. A genetically encoded ratio-
FRET to study molecular interactions in focal adhesions of live cells. metric indicator for chloride: capturing chloride transients in cultured
Methods Mol. Biol. 294:321–334. hippocampal neurons. Neuron. 27:447–459.
2. Jares-Erijman, E. A., and T. M. Jovin. 2003. FRET imaging. Nat. 19. Rizzo, M. A., G. H. Springer, B. Granada, and D. W. Piston. 2004. An
Biotechnol. 21:1387–1395. improved cyan fluorescent protein variant useful for FRET. Nat.
3. Chen, Y., J. D. Mills, and A. Periasamy. 2003. Protein localization in living Biotechnol. 22:445–449.
cells and tissues using FRET and FLIM. Differentiation. 71:528–541. 20. Griesbeck, O., G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y.
4. van Munster, E. B., and T. W. Gadella. 2005. Fluorescence lifetime im- Tsien. 2001. Reducing the environmental sensitivity of yellow fluores-
aging microscopy (FLIM). Adv. Biochem. Eng. Biotechnol. 95:143–175. cent protein. Mechanism and applications. J. Biol. Chem. 276:29188–
5. Nair, D. K., M. Jose, T. Kuner, W. Zuschratter, and R. Hartig. 2006. 29194.
FRET-FLIM at nanometer spectral resolution from living cells. Opt. 21. Lippincott-Schwartz, J., and G. H. Patterson. 2003. Development and
Express. 14:12217–12229. use of fluorescent protein markers in living cells. Science. 300:87–90.
6. Jose, M., D. K. Nair, C. Reissner, R. Hartig, and W. Zuschratter. 2007. 22. Kemnitz, K., L. Pfeifer, R. Paul, and M. Coppey-Moisan. 1997. Novel
Photophysics of Clomeleon by FLIM: discriminating excited state detectors for fluorescence lifetime imaging on the picosecond time
reactions along neuronal development. Biophys. J. 92:2237–2254. scale. J Fluoresc. 7:93–98.
7. Dresbach, T., B. Qualmann, M. M. Kessels, C. C. Garner, and E. D. 23. Kapusta, P., R. Erdmann, U. Ortmann, and M. Wahl. 2003. Time-
Gundelfinger. 2001. The presynaptic cytomatrix of brain synapses. resolved fluorescence anisotropy measurements made simple. J Fluoresc.
Cell. Mol. Life Sci. 58:94–116. 13:179–183.
8. Gundelfinger, E. D., M. M. Kessels, and B. Qualmann. 2003. Temporal 24. Beechem, J. M., and E. Haas. 1989. Simultaneous determination of
and spatial coordination of exocytosis and endocytosis. Nat. Rev. Mol. intramolecular distance distributions and conformational dynamics by
Cell Biol. 4:127–139. global analysis of energy transfer measurements. Biophys. J. 55:1225–
9. tom Dieck, S., L. Sanmarti-Vila, K. Langnaese, K. Richter, S. Kindler, 1236.
A. Soyke, H. Wex, K. H. Smalla, U. Kampf, J. T. Franzer, M. Stumm, 25. Lakowicz, J. R. 1999. Principles of Fluorescence Spectroscopy.
C. C. Garner, and E. D. Gundelfinger. 1998. Bassoon, a novel zinc- Kluwer Academic/Plenum Publishers, New York.
finger CAG/glutamine-repeat protein selectively localized at the active 26. Deguchi-Tawarada, M., E. Inoue, E. Takao-Rikitsu, M. Inoue, T.
zone of presynaptic nerve terminals. J. Cell Biol. 142:499–509. Ohtsuka, and Y. Takai. 2004. CAST2: identification and characteri-
10. Fenster, S. D., W. J. Chung, R. Zhai, C. Cases-Langhoff, B. Voss, zation of a protein structurally related to the presynaptic cytomatrix
A. M. Garner, U. Kaempf, S. Kindler, E. D. Gundelfinger, and C. C. protein CAST. Genes Cells. 9:15–23.
Garner. 2000. Piccolo, a presynaptic zinc finger protein structurally 27. Altrock, W. D., S. tom Dieck, M. Sokolov, A. C. Meyer, A. Sigler,
related to bassoon. Neuron. 25:203–214. C. Brakebusch, R. Fassler, K. Richter, T. M. Boeckers, H. Potschka,
11. Takao-Rikitsu, E., S. Mochida, E. Inoue, M. Deguchi-Tawarada, M. C. Brandt, W. Loscher, D. Grimberg, T. Dresbach, A. Hempelmann,
Inoue, T. Ohtsuka, and Y. Takai. 2004. Physical and functional inter- H. Hassan, D. Balschun, J. U. Frey, J. H. Brandstatter, C. C. Garner,
action of the active zone proteins, CAST, RIM1, and Bassoon, in neu- C. Rosenmund, and E. D. Gundelfinger. 2003. Functional inactivation
rotransmitter release. J. Cell Biol. 164:301–311. of a fraction of excitatory synapses in mice deficient for the active zone
12. Schoch, S., and E. D. Gundelfinger. 2006. Molecular organization of protein bassoon. Neuron. 37:787–800.
the presynaptic active zone. Cell Tissue Res. 326:379–391. 28. Nardini, M., S. Spano, C. Cericola, A. Pesce, A. Massaro, E. Millo, A.
13. Wang, Y., X. Liu, T. Biederer, and T. C. Sudhof. 2002. A family of Luini, D. Corda, and M. Bolognesi. 2003. CtBP/BARS: a dual-function
RIM-binding proteins regulated by alternative splicing: implications for protein involved in transcription co-repression and Golgi membrane
the genesis of synaptic active zones. Proc. Natl. Acad. Sci. USA. fission. EMBO J. 22:3122–3130.
99:14464–14469. 29. Schmitz, F., A. Konigstorfer, and T. C. Sudhof. 2000. RIBEYE, a
14. tom Dieck, S., W. D. Altrock, M. M. Kessels, B. Qualmann, H. Regus, component of synaptic ribbons: a protein’s journey through evolution
D. Brauner, A. Fejtova, O. Bracko, E. D. Gundelfinger, and J. H. provides insight into synaptic ribbon function. Neuron. 28:857–872.
Brandstatter. 2005. Molecular dissection of the photoreceptor ribbon 30. Khimich, D., R. Nouvian, R. Pujol, S. Tom Dieck, A. Egner, E. D.
synapse: physical interaction of Bassoon and RIBEYE is essential for Gundelfinger, and T. Moser. 2005. Hair cell synaptic ribbons are es-
the assembly of the ribbon complex. J. Cell Biol. 168:825–836. sential for synchronous auditory signalling. Nature. 434:889–894.

Biophysical Journal 94(4) 1483–1496