Post on 15-May-2023
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Tracking Ca2+-Dependent and Ca2+-Independent Conformational Transitions in Syntaxin 1A
During Exocytosis in Neuroendocrine Cells
Dafna Greitzer-Antes*, Noa Barak-Broner*, Shai Berlin, Yoram Oron, Dodo Chikvashvili, and
Ilana Lotan #
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University,
Ramat Aviv 69978, Israel.
*These authors contributed equally to this study
# Corresponding author at the Department of Physiology and Pharmacology, Sackler School of
Medicine, Tel-Aviv University, 69978 Ramat-Aviv. Tel: +972-3-6409863; Fax: +972-3-6409113;
E-mail: ilotan@post.tau.ac.il
Running Title: Tracking syntaxin conformations
Keywords: Syntaxin 1A, exocytosis, SNARE, FRET, PC12 cells.
© 2013. Published by The Company of Biologists Ltd.Jo
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JCS Advance Online Article. Posted on 2 May 2013
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Summary
A key issue for understanding exocytosis is elucidating the various protein interactions and
the associated conformational transitions underlying SNARE protein assembly. To monitor
dynamic changes in syntaxin 1A (Syx) conformation along exocytosis, we constructed a novel
fluorescent Syx - based probe that can be efficiently incorporated within endogenous SNARE
complexes, support exocytosis, and report shifts in Syx between ‘closed’ and ‘open’
conformations by Fluorescence Resonance Energy Transfer analysis. Using this probe we
resolve two distinct Syx conformational transitions during membrane depolarization-induced
exocytosis in PC12 cells: a partial ‘opening' in the absence of Ca2+ entry and an additional
‘opening’ upon Ca2+ entry. The Ca2+ -dependent transition is abolished upon neutralization of the
basic charges in the juxtamembrane regions of Syx, which also impairs exocytosis. These novel
findings provide evidence of two conformational transitions in Syx during exocytosis, which
have not been reported before: one transition directly induced by depolarization and additional
transition that involves the juxtamembrane region of Syx. The superior sensitivity of our probe
also enabled detection of subtle Syx conformational changes upon interaction with VAMP2,
which were absolutely dependent on the basic charges of the juxtamembrane region. Hence, our
results further suggest that the Ca2+ -dependent transition in Syx involves zippering between the
membrane-proximal juxtamemrane regions of Syx and VAMP2 and support the recently implied
existence of this zippering in the final phase of SNARE assembly to catalyze exocytosis.
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Introduction
Syntaxin 1A (Syx), a plasma membrane (PM) neuronal Q-SNARE (soluble N-ethylmeleimide-
sensitive factor attachment protein receptor), is a major protein component of the machinery
involved in the maturation steps through which a vesicle undergoes before it can release a
neurotransmitter (Sorensen, 2004), steps such as docking, priming, and fusion (Wojcik and Brose,
2007). During the priming process, sequential formation of the neuronal trimeric SNARE complex
occurs (Brunger, 2001; Bruns and Jahn, 2002; Chen and Scheller, 2001; Jahn and Sudhof, 1999).
Initially, Syx assembles with PM SNARE, SNAP-25, to form the binary t-SNARE complex (Dun
et al., 2010), which is followed by assembly of the vesicular SNARE, VAMP2, with the complex,
yielding the trimeric SNARE complex, SNAREpin (Fasshauer and Margittai, 2004). The assembly
of SNAREpin is a highly regulated multistep process going through pre-fusion partially zippered
trans-complexes to the post-fusion fully zippered cis-complex, comprising Ca2+-independent and
Ca2+-dependent intermediates (Malsam et al., 2008; Melia, 2007). Importantly, it is known that
Syx undergoes one or more conformational changes upon its interaction with its SNARE partners
and with regulatory proteins throughout the steps leading to secretion. However, the details of
these conformations remain elusive. Indeed, conformational changes in Syx have been the subject
of numerous studies. The majority of the approaches involved in vitro interactions of soluble
protein motifs, studies of purified proteins reconstituted in lyposomes, and use of X-ray
crystallography, all of which provided important, yet limited, knowledge about the conformational
changes occurring in membrane-bound Syx in neuronal or neuroendocrine cells and their relevance
to events occurring during secretion. In particular, examination of the X-ray structure of the
neuronal SNARE complex including the transmembrane regions of Syx and VAMP2 led to the
hypothesis that the juxtamembrane region of Syx may play an important role in SNARE complex
assembly (Stein et al., 2009). In accordance with this hypothesis, by using a reconstituted
membrane fusion system, zippering of this region with the corresponding region in VAMP2 has
recently been implicated in the SNARE complex assembly required for efficient fusion
(Hernandez et al., 2012).
In this study we generated a novel Syx intramolecular Fluorescence Resonance Energy
Transfer (FRET) reporter probe that is incorporated within endogenous SNARE complexes and
reports dynamic conformational changes in Syx, in a neuronal-like cellular environment, during
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exocytosis. This probe enabled us to resolve two discrete secretion-related conformational changes
in Syx in PC12 cells and provided two novel findings. First, Syx undergoes two distinct
conformational transitions during exocytosis: a 'partial opening’ induced by depolarization but in
the absence of Ca2+ entry and further ‘opening’ that occurs upon Ca2+ entry. Second, the
conserved juxtamembrane region of Syx plays a crucial role in the Ca2+-dependent ‘opening’ of
Syx, probably at the final stage of the SNARE complex assembly. Thus, this probe enables one to
test and validate Syx conformational transitions associated with specific interactions already
documented in cell-free studies and to gain insights about novel interactions in vivo.
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Results
Construction of intramolecular Syx-based FRET probes
It is generally accepted that Syx can shift between two conformational states. In its ‘closed’
conformation the Habc domain folds back onto the SNARE motif (H3 domain), which is involved
in forming a coiled-coil SNARE complex with the SNARE motifs of SNAP-25 and VAMP2
(Figure 1A) (Margittai et al., 2003a; Verhage et al., 2000), thus preventing the formation of the
SNARE complex that drives vesicle fusion. The ‘closed’ conformation constitutes a key intrinsic
property of isolated Syx when not assembled into the SNARE complex (Chen et al., 2008),
although a small percentage of Syx may spontaneously open (Margittai et al., 2003b). To enter t-
SNARE and trimeric SNARE complexes, Syx must assume the ‘open’ conformation, subsequently
exposing the H3 domain (Jahn and Scheller, 2006; Sutton et al., 1998).
To better understand Syx conformational changes associated with the exocytotic process in a
living cell, we constructed double-labeled fluorescent Syx probes that may report conformational
changes in Syx by FRET. We explored the recent crystal structure of Syx in order to rationalize
our design of the probes. In the available structure, the N terminus of Syx and its juxtamembrane
region, connecting the transmembrane anchor and the H3 domain, are in proximity in the 'closed'
rather than in the 'open' conformation (Dulubova et al., 1999; Misura et al., 2001). Accordingly,
we fused two fluorescent molecules to Syx via flexible linkers: Cyan Fluorescent Protein (CFP) to
the N terminus and Yellow Fluorescent Protein (YFP) to the juxtamembrane region (Figure 1A).
We predicted that the two fluorophores would reside in proximity when Syx is in the 'closed'
conformation, yielding a high FRET signal. Conversely, the 'open' conformation of Syx should
robustly cause the fluorophores to separate, leading to a decreased FRET signal (Figure 1A). Two
probes were constructed: (1) CSYS (CFPNT-Syx-YFPdisatl-H3-Syx), with YFP inserted in the middle
of the polybasic juxtamembrane region (KARRKK), and (2) CSYS-5RK, with YFP inserted
between the H3 domain and the polybasic sequence (Figure 1B). As control probes, we generated
CSYS-Open and CSYS-5RK -Open, each with two point mutations, L165A and E166A, inserted at
the linker region between the Habc and H3 domains, previously shown to shift the equilibrium of
Syx toward the ‘open’ conformation (Dulubova et al., 1999; Richmond et al., 2001). Although
several intramolecular FRET probes, based on SNAP-25, were previously reported (An and
Almers, 2004; Takahashi et al., 2010; Wang et al., 2008), providing valuable insights about
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SNARE complex formation in living cells, to the best of our knowledge, no such Syx-based FRET
probes have been reported. We reasoned that Syx-based probes will prove more sensitive in
reporting the assembly of SNARE proteins associated with exocytosis in PC12 cells. Unlike the
SNAP-25-based probes, prone to dilution by endogenous SNAP-25 (found in large excess over
Syx in PC12 cells (>10-fold; (Knowles et al., 2010)), exogenously expressed Syx-based probes are
more likely to compete efficiently with the relatively small amounts of endogenous Syx and be
incorporated efficiently into native SNARE complexes.
Next, we performed experiments to determine whether CSYS and CSYS-5RK can form binary
t-SNARE and trimeric SNARE complexes, as does native Syx (Figure 1C-E). Since both probes
showed similar results, they were collectively termed CSYS in these experiments.
Coimmunoprecipitation (IP) analysis performed in Xenopus oocytes co-expressing metabolically
labeled CSYS and SNAP-25, using either anti-Syx or anti-SNAP-25 antibodies, revealed that
CSYS effectively associates with SNAP-25 to form t-SNARE complexes (Figure 1C). In addition,
SDS-resistant SNARE complexes were detected from oocytes coexpressing metabolically labeled
CSYS, SNAP-25, and VAMP2, upon immunoprecipitation with either anti-Syx or anti-SNAP-25
antibodies, confirming the ability of CSYS to form trimeric SNARE complexes (Figure 1D). To
demonstrate that our probes are as effective as native Syx in forming trimeric complexes with
SNAP-25 and VAMP2 as does native Syx, we assessed the SDS-resistant complexes formed in
oocytes by CSYS and compared them to those formed by native Syx (Figure 1E). Indeed, CSYS
readily formed SDS-resistant complexes which contained also SNAP-25 and VAMP2, similarly to
those formed by native Syx (Figure 1E; note the difference in mobility of Syx- and CSYS-
containing trimeric complexes).
CSYS probes can report the conformational ‘opening’ of Syx
Next, we analyzed the conformations adopted by CSYS and CSYS-5RK, using the spectral
FRET technique in Xenopus oocytes (Etzioni et al., 2011; Zheng et al., 2003). The probes were
efficiently targeted to the PM and exhibited a high FRET signal under resting conditions (static
FRET; Figure 2A). As expected, the static FRET signal did not change over a wide range of
expression levels because of a ~1:1 donor-to-acceptor ratio (Supplemental Figure S1; (Berlin et al.,
2010)). Surprisingly, the FRET signals of CSYS and CSYS-5RK were significantly different,
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although the position of the YFP fluorophore was shifted only 4 a.a. within the polybasic
juxtamembrane region (Figure 2A, right panel). This prompted us to investigate the importance of
this highly conserved region and to generate an additional probe, CSYS-5RK/A, in which the 5
positively charged residues in CSYS-5RK were neutralized (Figure 1B). CSYS-5RK/A had a FRET
signal similar to that of CSYS, but it was significantly different from that of CSYS-5RK (Figure
2A, right panel). These results suggest that changes within the polybasic region affect the
conformation of Syx.
We then tested the ability of the probes to report structural rearrangements related to the
‘opening’ of Syx. Co-expressed SNAP-25 significantly and dose-dependently reduced the FRET
signals of CSYS and CSYS-5RK to levels similar to those obtained by the corresponding Open
probes (Figure 2Ba,b), confirming previous observations regarding the ‘opening’ of Syx by SNAP-
25 (Jahn and Scheller, 2006; Sutton et al., 1998). Thus, we concluded that reductions in the FRET
signals of CSYS and CSYS-5RK most probably report the ‘opening’ of Syx.
CSYS-5RK/A also reported a SNAP-25-mediated ‘opening’, similarly to CSYS and CSYS-5RK
(Figure 2Bc). Indeed, concomitant co-immunoprecipitation analysis in oocytes co-expressing
metabolically labeled CSYS-5RK or CSYS-5RK/A with SNAP-25 and VAMP2 revealed that
CSYS-5RK/A is as effective as CSYS-5RK in binding SNAP-25 and VAMP2 (Supplemental Figure
S2). Importantly, these results indicate that, although the neutralization of the juxtamembrane
region of Syx affects the initial probe conformation (see above), it does not affect the ability to
associate with its SNARE partners and to report structural ‘openings’ in vivo.
High K+- depolarization induces conformational transitions in CSYS probes in PC12 cells
Our next aim was to use our FRET probes to investigate conformational changes in Syx
associated with SNARE complex formation in a physiologically relevant setting of secreting PC12
cells. All our results in PC12 cells discussed hereafter (unless otherwise noted) were obtained with
both CSYS and CSYS-5RK probes, which yielded similar results; hence, they are collectively
termed CSYS. Several preliminary analyses were performed. First, we verified that CSYS targeted
properly the PM in PC12 cells. Indeed, 90% of cells transfected with CSYS exhibited a fluorescent
signal at the PM region, indicating PM expression (see the membrane expression in Figure 3B).
Second, we evaluated the impact of CSYS on secretion. Briefly, we used an established secretion
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assay in PC12 cells in which fluorescence decline of mRFP-tagged vesicular neuropeptide Y
(NPY-mRFP; dimming of cells as they release the granular marker) is monitored in response to
membrane depolarization induced by perfusion of a high [K+] (hK) solution (Figure 3Aa; (Singer-
Lahat et al., 2007)). More than 70% of the cells displayed a significant amount of secretion that
was practically eliminated when intracellular [Ca2+] elevation was blocked in the presence of
cadmium (Cd) (Figure 3Ab; Supplemental Figure S3Aa). This corroborated the occurrence of the
well-documented dependence of secretion on Ca2+ entry via voltage-gated Ca2+-channels under our
experimental conditions. Importantly, the expression of CSYS in these cells did not change the
depolarization-induced elevation of the cytosolic Ca2+ level (Supplemental Figure S3Ab) and
significantly enhanced secretion (Figure 3Ab), suggesting that CSYS can associate with
endogenous SNARE partners and form functional exocytic complexes. Third, realizing that the
SNARE functionality of CSYS is of utmost importance in serving as a reporter of SNARE-
conformational changes during exocytosis, we sought to rigorously challenge the ability of CSYS
to substitute for native Syx and to support secretion in cells transfected with the light chain of
BoNT–C1, which cleaves Syx and inhibits membrane fusion (Schiavo et al., 1995). To this end,
we generated a CSYS mutant, CSYS(R), bearing a mutation (K253I; (Lam et al., 2008)) in the Syx
sequence that conferred resistance to BoNT-C1 (Figure 3B; Supplemental Figure S3B). Indeed,
whereas secretion triggered by hK was reduced to 15% in cells expressing CSYS and BoNT-C1,
secretion in cells experssing CSYS(R) and BoNT-C1 was rescued to 65% (Figure 3C; the
expression levels of CSYS-5RK and CSYS-5RK(R) were similar; partial SNAP-25 cleavage by
BoNT-C1 could contribute to the incomplete rescue). Thus, we concluded that CSYS could
substitute for endogenous Syx, could be successfully incorporated into endogenous SNARE
complexes, and could support exocytosis. However, the validity of this conclusion is dependent on
the ability of BoNT-C1 to cleave endogenous Syx in the presence of the overexpressed cleavage-
resistant CSYS(R). We verified this by showing that Syx was equally sensitive to BoNT-C1 in the
absence and presence of CSYS or CSYS(R) (Supplemental Figure S3B). Taken together, the results
of the above preliminary analyses validated the suitability of CSYS to serve as a reporter for Syx’s
conformational changes upon depolarization-induced exocytosis in secreting PC12 cells.
Next, conformational changes associated with SNARE complex formation were monitored by
dynamic FRET changes in response to hK depolarization in PC12 cells expressing CSYS. Time
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series images of PC12 cells were acquired before and during hK depolarization and fluorescent
intensities were collected from the PM (Figure 4A), from which the FYFP/FCFP ratio was calculated
(Berlin et al., 2010; Hein et al., 2005). Remarkably, significant reductions in FRET following
exposure to hK solution were evident already in single cells (Figure 4A) and were reproducible in
more than 70% of the CSYS-expressing cells. We verified that these FRET changes exhibited an
intra-molecular interaction with no contribution from an inter-molecular interaction (Supplemetary
Figure 1D), thus reporting conformational changes associated with ‘opening’ of CSYS. Figure 4B
shows a significant decrease of ~ 5% in the average normalized FRET ratio, reporting a
conformational shift of CSYS toward the 'open' state upon hK stimulation. To further substantiate
this conclusion, we stimulated cells expressing CSYS-Open with an hK solution. As predicted, no
significant changes in FRET were observed (Figure 4C).
We next tested the role of Ca2+ in the conformational shift of CSYS. In the presence of Cd,
which completely blocked intracellular [Ca2+] elevation (Supplemental Figure S3A) and secretion
(Figure 3Ab) in response to hK stimulation, CSYS only partially ‘opened’ upon hK stimulation
(Figure 4D; in one of these experiments, BAPTA-AM, a membrane-permeant Ca2+ chelator, was
also included to further rule out any local [Ca2+] rise). This Cd-resistant partial ‘opening’
demonstrates that Syx undergoes, in the absence of intracellular [Ca2+] elevation, a depolarization-
dependent, yet Ca2+-independent, conformational transition that does not support by itself
exocytosis. Notably, no similar conformational change upon hK stimulation was detected in
CSYS-Open (Figure 4C), strongly suggesting that the partial Ca2+-independent ‘opening’ of CSYS
represents a physiologically related transition and not a stimulation-induced non-specific
conformational change in the probe. To better understand the nature of the partial Ca2+-
independent ‘opening’ of CSYS, we investigated whether it represents an intermediate step that
can be transformed into a 'full opening’ in the presence of Ca2+. To address this issue, we
performed a two-step hK stimulation, first, stimulation in a Ca2+-free solution, followed by a
second stimulation in a Ca2+-containing solution (Figure 4E). In the absence of Ca2+, a 'partial
opening’ of CSYS occurred, the extent of which was similar to that observed in the presence of Cd
(compare Figure 4D & E). Upon addition of Ca2+, an additional ‘opening’ occurred, to a level
similar to that of the one-step stimulation in the presence of Ca2+ (compare Figure 4D & E).
Importantly, no such additional ‘opening’ occurred upon prolonged incubation of the cells in hK
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solution with no Ca2+ added (Figure 4F); namely, the addition of Ca2+ is responsible for the further
decrease of the FRET ratio. These results suggest that a two-component conformational transition
takes place during hK stimulation. The Ca2+-independent, but depolarization-dependent, 'partial
opening’ of CSYS may possibly be an intermediate structure along a sequential pathway leading to
a 'full opening’. Notably, we failed to detect conformational changes induced by membrane
depolarization, using a dynamic FRET assay in oocytes expressing CSYS, either clamped to
different depolarized voltages (Supplemental Figure S4A) or subjected to hK solution
(Supplemental Figure S4B). This suggests that the 'partial', as well as the 'full openings’, apparent
in PC12 cells, require a secreting cell environment. This also rules out the possibility of an hK
solution-related artifact.
Taken together, our results indicate the ability of CSYS to resolve conformational transitions in
the process of being incorporated into endogenous SNARE complexes in secreting cells. We
suggest, for the first time, that the ‘full opening’ of Syx during depolarization-induced exocytosis
is mediated by two separate mechanisms related to Ca2+-independent and Ca2+- dependent steps.
The polybasic juxtamembrane region of Syx is important for Ca2+-dependent conformational
transitions in Syx during exocytosis
The results of the static FRET analysis in oocytes suggested that changes in the juxtamembrane
region of Syx affect the conformation of Syx (Figure 2A). Recent X-ray structure implicated this
region of Syx in the assembly of the neuronal SNARE complex (Stein et al., 2009). Taken
together, this led us to hypothesize that the juxtamembrane region of Syx may be an important
component of Syx's depolarization-induced conformational transitions (Figure 4). We tested our
hypothesis by using CSYS-5RK/A (in which all the basic residues of the juxtamembrane region of
CSYS-5RK are neutralized; Figure 1B). Importantly, this probe retains the ability to associate with
its SNARE partners (Supplemental Figure S2) and to report structural ‘openings’ in vivo (Figure
2C). Using the dynamic FRET assay in hK stimulated PC12 cells (as done in Figure 4), we
compared the conformational transitions in CSYS-5RK/A with those in CSYS-5RK. Cells
expressing CSYS-5RK/A exhibited a smaller, but statistically significant (p<0.05), reduction in the
FRET ratio compared with cells expressing CSYS-5RK (Figure 5A). This conformational transition
of CSYS-5RK/A was unaffected by Cd (Figure 5B), suggesting that it may reflect the Ca2+-
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independent transition of CSYS-5RK. Indeed, it was similar to that of CSYS-5RK in the presence of
Cd, which was tested in the same experiment (Figure 5C). These results indicate that neutralization
of the basic residues abolished the Ca2+-dependent hK-induced transition of Syx ‘opening’. The
neutralization also impaired exocytosis as hK-induced secretion from cells expressing CSYS-
5RK/A was smaller than that from cells expressing CSYS-5RK (Figure 5D; the expression levels of
CSYS-5RK and CSYS-5RK/A were similar). Hence, using our reporters, we found that the full
‘opening’ of Syx during the Ca2+-dependent transition absolutely depends on the positive charges
in the juxtamembrane region. Our findings strongly suggest a crucial role for this region in Ca2+-
dependent structural transition of Syx that underlie efficient exocytosis.
Next, we tested the possibility that the Ca2+-dependent structural transition may relate to
zippering between the juxtamembrane regions of Syx and VAMP2 during SNARE complex
assembly along the fusion pathway, as suggested in (Hernandez et al., 2012; Stein et al., 2009). To
this end, we tested the prediction that neutralization of the basic residues in the juxtamembrane
region of CSYS should impair interaction between CSYS and VAMP2. Interaction between Syx
and VAMP2 (in the absence of SNAP-25) was previously assayed in vitro and was found to be
very weak (Fasshauer et al., 1997; Hazzard et al., 1999) or undetectable (Gao et al., 2012). Here,
we first tested for this interaction in vivo, using our FRET probes. We found that in oocytes co-
expressing VAMP2 with CSYS or with CSYS-5RK (in the absence of co-expressed SNAP-25) the
FRET of the probes was significantly decreased (by ~20%), establishing that VAMP2 interacts
with both Syx probes and mediates their ‘opening’ (Figure 6A). As expected from a weak
interaction between VAMP2 and Syx, this reduction in FRET was significantly smaller than that
obtained in oocytes coexpressing SNAP-25 with CSYS (Figure 6B). In agreement with our
prediction, the FRET of CSYS-5RK/A remained unaffected (Figure 6A). Namely, neutralization of
the basic residues in the juxtamembrane region abolished the interaction with VAMP2,
strengthening our hypothesis of the role of the juxtamembrane region of Syx when interacting with
VAMP2. Interestingly, concomitant co-immunoprecipitation analysis, using Syx antibody,
performed on the same oocytes, revealed that VAMP2 associates with both CSYS-5RK/A and
CSYS-5RK (albeit the association with CSYS-5RK/A was weaker by ~20%); both associations were
very weak and probably reflect interaction mainly mediated by the corresponding SNARE motifs;
Supplemental Figure S5). Taken together, this data demonstrates the superior sensitivity and
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specificity of our Syx-based FRET reporters, even exceeding those of biochemical analyses, in
capturing conformational changes of Syx arising from subtle changes in its interaction(s) with
protein partners.
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Discussion
In this study we generated novel Syx intramolecular FRET probes, termed CSYS, which are
able to report dynamic changes in Syx structure in the process of being incorporated into
endogenous SNARE complexes formed along the pathway to exocytosis. Using these probes we
resolved, for the first time in vivo, secretion-related conformational transitions in Syx. We showed
that full ‘opening’ of Syx is mediated by two separate mechanisms occurring during Ca2+-
independent and Ca2+-dependent steps of depolarization-induced efficient exocytosis. Notably,
although Ca2+-independent structural changes in Syx have previously been implicated in vesicle
exocytosis (see discussion below), this is the first case showing that such events can be induced by
membrane potential depolarization. Furthermore, we found that the Ca2+-dependent ‘opening’ of
Syx is contingent on positive charges in its juxtamembrane region, neutralization of this region
impairs exocytosis. This finding, obtained in living cells, strongly highlights the crucial role of this
region in structural transition(s) of Syx that underlie efficient exocytosis and validates predictions
made by previous structural (Stein et al., 2009) and membrane fusion-related studies in cell-free
reconstituted systems (Hernandez et al., 2012).
Our results open the question regarding the precise physiological correlates and molecular
events underlying the two conformational transitions assumed by Syx. The Ca2+-independent
partial ‘opening’ of Syx, induced by membrane potential depolarization, may report
conformational changes occurring during vesicle docking, priming, and entry into partially
assembled SNAREpins. Such conformational changes, controlled by SNARE-associated
regulatory factors, are pre-Ca2+ intermediates documented in numerous reconstituted assays in
relation to vesicle docking and priming reactions (Malsam et al., 2008; Melia, 2007; Parisotto et
al., 2012; Sudhof and Rothman, 2009). It is quite likely that Syx itself cannot sense membrane
electric field and trigger these molecular events. We have examined this possibility and failed to
detect any similar conformational changes in CSYS-expressing oocytes that were voltage-clamped
to different depolarized voltages (Supplementary Figure S4). In addition, we demonstrated (Figure
5) that eliminating all charged residues located in the vicinity of the membrane, which could
potentially serve as a voltage sensing module, does not eliminate the voltage sensitivity of our
probe. A more plausible explanation is that the voltage sensitivity arises from a separate voltage-
sensitive protein, serving as a voltage sensor, which is not present in the oocyte. Such a mechanism
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would be similar, for instance, to the well-characterized voltage sensing mechanism of Ca2+ release
from sarcoplasmic reticulum (SR) in skeletal muscle. There, the L-type Ca2+ channel, located in
the plasma membrane, senses depolarization and signals, via direct contact, to the Ca2+ channel
located in the SR (for a review see: (Iino, 1999)). There are several candidate proteins that could
similarly signal to Syx, via direct physical contact, by serving as a voltage sensor: (1) the neuronal
voltage-dependent Ca2+ channels of N- or L-type (Bergsman and Tsien, 2000; Bezprozvanny et al.,
1995; Wiser et al., 1996) (for a review, see (Catterall, 2000)), (2) the voltage-dependent K+
channel Kv2.1, which is the predominant channel in neuroendocrine cells and is thought to interact
directly with Syx to enhance vesicle priming by promoting assembly and/or stabilization of the t-
SNARE complex (Feinshreiber et al., 2010; Michaelevski et al., 2003; Singer-Lahat et al., 2007),
or (3) G protein-coupled receptors (Linial et al., 1997). All these proteins undergo conformational
changes upon depolarization (Ben-Chaim et al., 2006; Hille, 2001).
The Ca2+-dependent full ‘opening’ of Syx reported by CSYS may reflect one or more structural
changes in Syx involved in the final phase of SNARE assembly when cys-complexes are formed.
The CSYS-5RK/A probe with the neutralized juxtamembrane region demonstrates that the
polybasic juxtamembrane region is crucial for these Ca2+-dependent structural changes reported by
CSYS (Figure 5A). According to the X-ray structure analysis of the neuronal SNARE complex
(Stein et al., 2009), the juxtamembrane regions of Syx and VAMP2 form helices continuous with
their SNARE motifs and transmembrane regions. Furthermore, a recent study of membrane fusion
intermediates in a cell-free system, shows that efficient fusion up to an extended form of
hemifusion requires zippering beyond the core SNARE complex to the juxtamembrane regions of
Syx and VAMP2 (Hernandez et al., 2012). Taken together, the Ca2+-dependent full ‘opening’
reported by CSYS most likely reflects zippering between the juxtamembrane regions of Syx and
VAMP2. According to this interpretation, it is predicted that neutralization of basic residues in the
juxtamembrane region of CSYS impairs the interaction between CSYS and VAMP2. In agreement
with our prediction, FRET analysis detected an interaction between VAMP2 and CSYS, but could
not detect similar interaction between VAMP2 and CSYS-5RK/A (Figure 6). Interestingly, CSYS-
5RK/A compromised, but did not entirely eliminate hK-induced secretion (Figure 5D), as had been
demonstrated before for native Syx with a neutralized juxtamembrane region (Lam et al., 2008).
Indeed, as previously discussed (Stein et al., 2009), structural perturbation of the juxtamembrane
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interaction between VAMP2 and Syx (as was done in earlier studies by manipulating the
juxtamembrane regions’ length and the amino acid composition) should be less disruptive for
fusion than is perturbation of the interaction within the four-helix bundle of the SNARE motifs.
Overall, our results suggest a role for the polybasic juxtamembrane region of Syx in facilitating
exocytosis and validate, in a neuronal-like environment, the notion that this final phase of SNARE
assembly is directly coupled to efficient membrane fusion.
It should be noted that the polybasic juxtamembrane region of Syx has been recognized as a
lipid binding domain. Consequently, it was proposed that Syx–acidic phospholipids interactions
are critical in determining the energetics of the SNARE-mediated fusion event by sequestering
fusogenic lipids to sites of fusion (Lam et al., 2008). Notably, electrostatic interactions of the
polybasic residues with PIP2 (James et al., 2009; Murray and Tamm, 2009) have recently been
shown to underlie clustering and segregation of Syx into distinct microdomains where synaptic
vesicles undergo exocytosis (Van den Bogaart et al., 2011). Thus, it is quite possible that the
conformational transitions linked to the juxtamembrane region of Syx, detected in our study, also
reflect interaction of this region with PIP2, hence, complementing the implication of this region in
the efficient assembly of the complete fusion machinery in spots of exocytosis.
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Materials and Methods
CSYS constructs - Double-labeled Syx (CSYS) cDNA was generated first by fusing enhanced
cyan fluorescent protein (CFP), flanked by EcoRI and XhoI sites to the N terminus of the rat Syx
cDNA in pGEMHJ vector, followed by insertion of NcoI restriction sites after Arg 263 residue,
which lies between the transmembrane anchor and the H3 domain of Syx. Next, the enhanced
yellow fluorescent protein (YFP) was fused into the aforementioned restriction site via Gly-Gly-
Gly-Ser linkers. CSYS-5RK and CSYS-5RK/A were generated based on CSYS cDNA, using
point mutation site-directed mutagenesis. The open forms of the constructs were generated by
introducing two point mutations, L165A and E166A, at the linker region between the Habc and H3
domain of CSYS (Dulubova et al., 1999), using point mutation site-directed mutagenesis (Edelheit
et al., 2009). The BoNT-C1-resistant construct, CSYS(R), was generated by introducing one point
mutation, K253I, at the BoNT-C1 recognizing sequence (Lam et al., 2008). For PC12 transfection,
the constructs were cloned into pcDNA3 vector using EcoRI and XbaI restriction sites.
Immunoprecipitation (IP) and Immunoblotting (IB) – In IP experiments de-folliculated
Xenopus oocytes were metabolically labeled 4 hrs after mRNA injection by incubation in NDE
solution containing 0.3 mCi/ml of [35S] Methionine/Cysteine for 2 days until homogenization was
achieved. Six to eight oocytes were homogenized in 1 ml of medium composed of 20 mM Tris (pH
7.4), 5 mM EDTA, 5 mM EGTA, and 100 mM NaCl (containing protease inhibitor cocktail
(Roche)). Debris was removed by centrifugation for 10 min at 4°C. After the addition of Triton X-
100 to a final concentration of 1%, followed by microcentrifugation for 15 min at 4°C in a desktop
centrifuge, antiserum (Syx 1A antibody (Alomone), SNAP-25 antibody (BD Transduction lab) or
VAMP2 antibody (Abcam)) was added to the supernatant and the homogenate was incubated for
16 hrs. Then, the antibody-antigen complex was incubated for 1 h at 4°C with protein A-Sepharose
and then pelleted by centrifugation for 1 min at 8000xg. Immunoprecipitates were washed four
times with immunowash buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris (pH 7.5), and 0.1%
Triton X-100); the final wash contained no Triton X-100. Samples were boiled in SDS gel loading
buffer and electrophoresed on SDS- 8% or 12% polyacrylamide gel together with standard
molecular mass markers. Digitized scans were derived by PhosphorImager (Amersham
Biosciences), and relative intensities were quantified by ImageQuant.
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In IB experiments proteins expressed in Xenopus oocytes or in PC12 cells were co-
immunoprecipitated by Syx 1A antibody (Alomone), subjected to Western blot analysis and
detected by Syx antibody (Sigma) with the ECL detection system (Pierce protein research
products, Thermo Scientific).
Fluorescence Resonance Energy Transfer (FRET) in Xenopus oocytes
Xenopus oocyte preparation - Xenopus oocytes were prepared and injected with mRNA as
previously described (Dascal and Lotan, 1992; Peleg et al., 2002). Each probe concentration was
determined in a calibration experiment in order to obtain the same expression levels among all
constructs in every experiment. Oocytes were injected with the following mRNA concentrations
(ng/oocytes) of the various probes: 5 CSYS, 5 CSYS-5RK, 15 CSYS-5RK/A, and 15 of various
probes in the 'open' form. Oocytes were injected with various CSYS probes alone or together with
increasing concentrations of SNAP-25 and/or VAMP2 (ranging from 5-25 ng/oocytes), for a dose-
dependent response and in order to detect the concentration that produces the highest effect.
Static FRET assay in oocytes - Oocytes were imaged in ND96 solution in a 0.7-mm glass-bottom
dish. Fluorescence emissions from CFP and/or YFP-tagged proteins were collected from the
animal hemisphere of the oocyte with a Zeiss inverted confocal microscope (Zeiss Axiovert LSM
510META), using a 20x0.75 NA air objective and laser excitations of 405 nm and 514 nm,
respectively. We used a spectrum-based method to remove contamination caused by donor
emission and for direct excitation of the acceptor. The FRET assay was performed as described in
(Zheng and Zagotta, 2004). Briefly, two emission spectra were collected from each oocyte, one
with 405nm excitation and the other with 514nm excitation. A scaled CFP spectrum, collected
from control oocytes expressing CFP-tagged proteins only, were used to normalize the CFP
emissions from the spectrum taken from oocytes expressing both fluorophores at 405nm
excitation. This procedure allows one to dissect the YFP emission spectrum, termed 405F . 405F has
two components: one is due to direct excitation of YFP ( directF405 ) and the other is due to FRET
( FRETF405 ). 405F is normalized to the total YFP emission with 514nm excitation at the same oocyte,
514F . The resulting ratio, termed RatioA, can be expressed as 514
405
514
405
514
405
F
F
F
F
F
FRatioA
FRETdirect
+== . The direct
excitation component in the calculated RatioA, termed RatioA0, is experimentally determined from
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a large population of oocytes expressing only YFP-tagged proteins. This allows precise
calculations of the bleed-through of direct excitation of YFP by the 405nm laser. The difference
between RatioA and RatioA0, (RatioA-RatioA0) is directly proportional to FRET efficiency:
514
4050 F
FRatioARatioA
FRET
=− . The apparent FRET efficiency from an individual cell, Eapp, can be
calculated as D
Aapp
RatioARatioA
Eεε
−= 1
0
, where εD and εA are molar extinction coefficients for the donor
and acceptor, respectively, at the donor excitation wavelength (Gao et al., 2007; Takanishi et al.,
2006).
Dynamic FRET assay in oocytes- Oocytes were voltage clamped and their fluorescence
measurements were taken simultaneously as described in (Berlin et al., 2010). Oocytes were kept
at a holding potential of -100mV for 20 sec and were gradually depolarized from -80 to +80 with
step increments of 20mV. Fluorescent signals were collected with a Zeiss 510META confocal
microscope using its "channel mode". Oocytes were excited with a 405-nm laser band only and the
emission was filtered through the main beam splitter HFT405/514/633nm and further separated by
a secondary beam splitter, NFT515nm. CFP and YFP fluorescences were collected by a 470-
500nm and 505-550nm band pass filter, respectively, and directed into two separate
photomultipliers. Under these settings, the leak of YFP into the CFP recording window is purely
optical, very low (<1%) (Okamoto et al., 2004), and remained constant regardless of changes in
FRET, thus not requiring any corrections. A region in the oocyte membrane area was sequentially
imaged every second for 120 sec. For each oocyte, CFP and YFP intensities were normalized to
initial CFP and YFP intensities and their FRET ratio (FYFP/FCFP) was calculated. Changes in FRET
are reflected in changes in the FRET ratio.
FRET and secretion procedures in PC12 cells
PC12 cells preparation and transfection - PC12 cells were maintained at 37oC/5% CO2 in
Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (Sigma) supplemented with
10% Bovine serum, 5% L-glutamin, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. For
imaging, cells were replated to a ~50% confluence onto poly-L-Lysine (Sigma) coated 35mm-
glass bottom culture dishes and allowed to adhere overnight. Cells were transfected with 0.5µg
vesicular neuropeptide Y attached to red fluorescence protein (NPY-mRFP) cDNA alone or
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together with 0.3µg of various CSYS probes cDNA, using Lipofectamine 2000 (Invitrogen). In the
BoNT-C1 experiments, cells were transfected with 0.5µg NPY-mRFP, 1.2µg of BoNT-C1 and
with 0.5µg CSYS or 0.7µg CSYS(R) cDNAs. The concentration of each probe was determined
before transfection in a calibration experiment in order to obtain the same expression levels among
all constructs in every experiment. Imaging experiments were conducted at room temperature, 24
hours after transfection. During the experiment the transfected cells were superfused through a 2-
ml bath, with control and 105mM hK stimulation solutions as described in (An and Almers, 2004).
Since Ca+2 currents in PC12 cells are usually small (An and Almers, 2004; Taraska et al., 2003), in
both solutions elevated [Ca2+] was used in order to obtain more reliable exocytosis in our PC12
cells.
Dynamic FRET assay in PC12- PC12 cells were imaged using a C-Apochromat 40x/1.2 NA water
objective and excited with a 405-nm laser every 5 sec for a total of 400 sec. During the sequential
imaging the cells were imaged in control solution for 100 sec before and after 200 sec using
105mM hK stimulation, for a total imaging time of 400 sec. CFP and YFP fluorescences were
collected with the same configuration described in the dynamic FRET assay in oocytes. YFP and
CFP intensities at a region of interest (ROI) on the cell`s plasma membrane were calculated and
background fluorescence was quantified from an ROI in each image defined, in an area containing
no fluorescent cells. Background-subtracted fluorescence intensity at each exposure time point was
normalized to the average of the initial measurements in each cell (before hK solution was added).
The FRET ratio of normalized intensities was denoted as FYFP/FCFP. Changes in FRET are reflected
in changes in the FRET ratio.
Secretion assay in PC12- Cells were imaged every 5 sec for 400 sec using a C-Apochromat
40x/1.2 NA water objective. Cells were excited with a 543nm excitation laser and the fluorescence
signal was collected from an ROI region containing the whole cell, using a 560-615nm bandpass
filter. One hundred sec after beginning the measurement, the control solution was replaced by
stimulation of 105mM hK solution for the remaining experiments. Exocytosis was measured as a
rapid reduction in fluorescence due to release of the NPY peptide in response to hK stimulation. In
the control experiments no stimulation solution was added during the entire 400 sec of
measurement or Ca2+ elevation was diminished by using 2mM Ca2+ and by adding 200 µM
Cadmium (Ca2+ channel blocker) to the control and stimulation solutions.
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Cytosolic Ca2+ imaging- PC12 cells transfected with CSYS were grown to approximately 50%
confluence in eight-well LabTec (Nunck) clusters with a #1.5 coverglass bottom. The cells were
incubated for one hour in Hank's solution with 2 µM of fura-2 acetoxy-methyl ester (AM), at 37°C
in a 6% CO2 atmosphere. Next, the cells were washed with Hank's solution and monitored for
fura-2 fluorescence with a Nikon TMD microscope equipped with a high numerical aperture (1.3)
X40 oil immersion fluorescence objective (Nikon). Successive frame pairs (at 340 and 380 nm
excitation) were acquired at 0.2-s intervals every 0.5-5 s. The Sutter DG-4 light source was used.
Emission was measured at 510 nm. The emission signal was recorded with an Isis Photonic
Science intensified camera using Universal Imaging Metafluor software. The cells were
stimulated by a 105mM hK solution, which was applied directly into the wells. The results are
presented as the emission ratio, obtained by dividing the 340nm image by the 380nm image.
Statistical Analysis- All statistical analyses were performed using SigmaStat Software. Results
are presented as means ± S.E.M. Multiple groups were compared by one-way analysis of variance
(ANOVA), followed by a post hoc Bonferroni test or Dunnett’s Multiple Comparison Test. Two
groups were compared using an unpaired two-tailed t-test. In the dynamic FRET and secretion
experiments, the significance was tested 'within groups' by comparing each average time point
measurement with the average measurements before stimulation, using a two-tailed t-test and
'between groups' by comparing each average time point measurement between two or more groups,
using a t-test or one-way ANOVA, respectively. Asterisks indicate statistically significant
differences as follows: * P<0.05; ** P<0.001.
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Acknowledgements
We thank Prof. Uri Ashery for critically reading the manuscript. This work was supported by
grants from the Israel Academy of Sciences (I.L), the United States-Israel Binational Foundation
(I.L.), and the Joan and Jaime Constantiner Institute of Molecular Genetics (D.G, N.B).
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Figure legends:
Figure 1: The CSYS probes are effective in forming SNARE complexes. (A) Domain
structure of Syx and the various CSYS constructs. (B) Schematic diagrams of CSYS in 'closed' and
'open' states, modified from (Neshatian et al., 2007). (C) The CSYS probes form t-SNARE
complexes with overexpressed SNAP-25. SDS-PAGE analyses of CSYS and SNAP-25 proteins
co-precipitated by either anti-Syx (IP by Syx; a) or anti-SNAP-25 (IP by SNAP-25; b) antibodies
from oocytes injected with CSYS or SNAP-25 mRNAs, together or alone. (D) The CSYS probes
form SDS-resistant complexes with overexpressed SNAP-25 and VAMP2. CSYS, SNAP-25, or
VAMP2, expressed together or alone, were co-precipitated by anti-SNAP-25 (a) or anti-Syx (b)
antibodies and incubated in SDS sample buffer at 37°C (a and b) or 95°C (c) for 10 min. The SDS-
resistant complexes (SNARE complex band), apparent at 37 °C, disappeared at 95°C. (E) The
CSYS probes form SDS-resistant complexes with SNAP-25 and VAMP2, as efficiently as native
Syx. Shown are proteins co-precipitated by Syx (a), SNAP-25 (b) or VAMP2 (c) antibodies and
incubated in SDS sample buffer at 37°C (upper panels) or 95°C (lower panels) for 10 min, as
indicated. Note the difference in mobility of native Syx- (*) and CSYS (o)- containing SNARE
complexes (apparent at 37 °C, disappeared at 95°C).
Figure 2: Reduction of the apparent FRET efficiency (Eapp) of the CSYS probes by co-
expressed SNAP-25 in oocytes. (A) Left, confocal images taken with 514nm (a) and 405nm (b)
laser excitations of plasma membrane of oocytes expressing CSYS or CFP-Syx, taken with a
405nm (c) laser excitation. White bar = 20 µm. Right, CSYS and CSYS-5RK/A have similar Eapp
values, whereas that of CSYS-5RK is significantly higher. Shown are the average Eapp values (see
Methods) of oocytes expressing CSYS, CSYS-5RK and CSYS-5RK/A in a single representative
experiment (11-17 oocytes per group). (B) Normalized average Eapp values of the probes are
reduced upon co-expression with SNAP-25 to the Eapp value of the corresponding ‘open’ probe. (a)
Oocytes expressing CSYS without or with increasing concentrations of SNAP-25 (5, 15 and 25
ng/oocyte; n=36-200 per group; 7 experiments). Inset; CFP emission spectra of CFP-Syx (black),
CSYS (blue), CSYS + SNAP-25 (orange), and CSYS Open (yellow), taken with a 405nm
excitation laser. The dichroic mirror is marked by a light gray area. FL, fluorescent. (b and c)
Oocytes expressing CSYS-5RK or CSYS-5RK/A, with or without 15-25 ng/oocyte of SNAP-25
mRNA (n=15-56 per group; 4 experiments). **,p< 0.001.
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Figure 3: CSYS can be substituted for native Syx and can support secretion. (A) CSYS
elevates secretion. (a) Confocal images of PC12 cells co-expressing CSYS and NPY-mRFP before
(0 sec) and after (300 sec) hK depolarization. White bar = 10 µm. (b) Secretion is elevated in cells
expressing NPY-mRFP and CSYS compared with cells expressing NPY-mRFP alone (p<0.001).
There was no significant secretion in the presence of 200µM Cd or in the absence of stimulation.
(B) CSYS(R) is resistant to cleavage by BoNT-C1. Top, Confocal images of PC12 cells
demonstrating that CSYS(R) expression with (iv) or without (iii) BoNT-C1 is distributed to the PM,
in contrast to the cytosolic expression of CSYS in the presence of BoNT-C1 (compare ii with i).
White bar = 5 µm Bottom, normalized fluorescence intensity profiles of the above cells indicating
PM or cytosolic expression. The fluorescence profiles were determined from line scans (red lines;
top) taken from the outside to the middle of each cell. (C) CSYS can be substituted for native Syx
and can support secretion. Whereas secretion induced by hK is almost eliminated in cells co-
expressing CSYS with BoNT-C1 (n=39; p<0.001), secretion from cells expressing CSYS(R) is
prominent in the presence of BoNT-C1 (n=39; p<0.001), albeit statistically smaller than in cells
expressing CSYS (p<0.001). Random cells exhibiting double fluorescence of CSYS and mRFP were
assayed for each group.
Figure 4: ‘Opening’ of the CSYS probes induced by hK depolarization of PC12 cells is
only partly dependent on Ca2+ entry. (A) Magnified region of the cell plasma membrane,
expressing CSYS, used for analysis. White bar = 10 µm. Below; Changes in the normalized FRET
ratio in response to hK depolarization (shaded area) from a single representative cell expressing
CSYS. (B and C) Changes in the average normalized FRET ratio in response to hK. Cells
expressing CSYS (B, n=24) exhibited a significant decrease in FRET ratio (p<0.001) in
comparison with CSYS-Open (C, n=19). Cells expressing CSYS and CSYS-5RK exhibited similar
results (B, inset), thus they were collectively termed CSYS. (D) Smaller decrease in the FRET
ratio in the presence of 200µM Cd. The response in the presence of Cd is significant (green, n=56,
three experiments; p<0.05,) and is different from the response in the absence of Cd (blue, n=59,
three experiments, p<0.05). (E and F) A sequential decrease in FRET ratio in transition from low
(0mM) to high (50mM) Ca2+ depolarization. Cells expressing CSYS were imaged in the same
protocol as in D but without the addition of Cd. Following 200sec (100-300sec) of hK
depolarization in 0mM Ca2+, the cells were depolarized for another 200sec in a 50mM Ca2+ hK
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solution (E, n=19) or in 0mM Ca2+ hK solution (F, n=6; superimposed on the cells from E). Note
that in the absence of Ca2+ the FRET ratio reached a plateau at about 180ms from the onset of
depolarization (F), whereas in the presence of 50mM Ca2+ there was a further decrease in the
FRET ratio (E).
Figure 5: The Ca2+-dependent ‘opening’ of CSYS is abolished upon neutralization of the
juxtamembrane region charge. (A) Changes in the average normalized FRET Ratio in PC12
cells in response to hK in cells expressing CSYS (filled circles, n=41) and cells expressing CSYS-
5RK/A (open circles, n=28), from two experiments. CSYS-5RK/A exhibited a smaller, but
significant (p< 0.05), decrease in the FRET ratio upon hK depolarization than did CSYS. The
difference between the two groups is statistically significant (p< 0.05). (B) There was no
significant difference between cells expressing CSYS-5RK/A in the presence (filled circles, n=23)
and absence (open circles, n=28) of 200µM Cd. (C) There was no significant difference between
cells expressing CSYS-5RK/A (open circles, n=28) and cells expressing CSYS in the presence of
200µM Cd (filled circles, n=36). (D) Secretion was impaired by neutralization of the
juxtamembrane region charge. Shown is secretion from cells expressing CSYS (n=21) or CSYS-
5RK/A (n=8). The difference between the two groups is statistically significant (**,p< 0.001).
Figure 6: VAMP2 reduces the FRET efficiency of the CSYS probes; neutralization of the
juxtamembrane region charge abolishes the reduction. Normalized average Eapp values of
oocytes expressing (A) the various CSYS probes with or without VAMP2 (n=26-32 per group; 2
experiments; **, P<0.001) and (B) CSYS-5RK co-expressed with SNAP-25 or VAMP2. The
reduction in the normalized average Eapp values of CSYS-5RK by VAMP2 is significantly smaller
than that of SNAP-25 (39-48 oocytes per group from 3 experiments; **, p< 0.001).
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H3Domain
Cell membrane
‘Open’ CSYS‘Closed’ CSYSHabc Domain
CSYSCSYS
SNAP-25 SNAP-25
100 100
75 75
25 25
CSYS + SNAP-25
CSYS + SNAP-25
CSYS SNAP-25
CSYS SNAP-25
IP Syx IP SNAP-25
A
B
C a b
CFP Ha Hb Hc H3 Domain TMKARR KK
CFP Ha Hb Hc H3 Domain TMKARRKK
CFP Ha Hb Hc H3 Domain TMAAAAAA
CFP Ha Hb Hc H3 Domain TM
CFP Ha Hb Hc H3 Domain TM
CFP Ha Hb Hc H3 Domain TM
CFP Ha Hb Hc H3 Domain TM
CFP Ha Hb Hc H3 Domain TM
CFP Ha Hb Hc H3 Domain TM
CFP Ha Hb Hc H3 Domain TMCFP Ha Hb Hc H3 DomainCFP Ha Hb Hc H3 DomainCFP Ha Hb HcCFP Ha Hb Hc H3 Domain TMTMTM
CFP Ha Hb Hc H3 Domain TMCFP Ha Hb Hc H3 DomainCFP Ha Hb Hc H3 DomainCFP Ha Hb HcCFP Ha Hb Hc H3 Domain TMTMTM
CFP Ha Hb Hc H3 Domain TMCFP Ha Hb Hc H3 DomainCFP Ha Hb Hc H3 DomainCFP Ha Hb HcCFP Ha Hb Hc H3 Domain TMTMTM
CSYS CSYS-5RK
CSYS-5RK/A
Ha Hb Hc H3 DomainHa Hb Hc H3 DomainHa Hb Hc H3 DomainHa Hb Hc H3 DomainHa Hb Hc H3 DomainHa Hb Hc H3 DomainHa Hb HcHa Hb Hc H3 Domain TMKARRKK TMTMTMTMTMTMSyx
SNAP-25 + VAMP2
CSYSCSYS
CSYS + SNAP-25 + VAMP2
CSYS
CSYS
SNARE complex
SNAP-25
VAMP2
IP Syx
CSYS + SNAP-25 + VAMP2
CSYS
SNAP-25
VAMP2
IP SNAP-25
CSYS + SNAP-25 + VAMP2
CSYS
SNAP-25
VAMP2
IP Syx
a b c370c 370c
a b c
950c
75100
25
15
75100
25
15
75
25
15
(28-146) (192-254) (266-288)
C
CY Y
L LYFP
L LYFP
L LYFP
SNARE complex
D
E
100150 150 150
CSYS + SNAP-25 + VAMP2
CSYS
CSYS
IP Syx
75
100
250
150
Syx + SNAP-25 + VAMP2
Syx
CSYS
75
100
250
150
370c
950c
CSYS + SNAP-25 + VAMP2
Syx + SNAP-25 + VAMP2
75
100
250
150
75
100
250
150
370c
950c
IP SNAP-25
Syx
CSYS + SNAP-25 + VAMP2
Syx + SNAP-25 + VAMP2
75
100
250
150
370c
75
100
250
150
IP VAMP2
950c
CSYS
CSYS
CSYS
CSYS
*O
*O
*O
Figure 1Jo
urna
l of C
ell S
cien
ceA
ccep
ted
man
uscr
ipt
0
500
1000
1500
440 490 540 590
FL. i
nten
sity
(a.u
)
Emission wavelength (nm)
0
0.2
0.4
0.6
0.8
1
Nor
mal
ized
Ave
rage
Eap
p
Open+SNAP-25 5
+SNAP-25 15-25
**** **
10
15
20
Ave
rage
Eap
p(%
)
CSYS CSYS5RK
CSYS 5RK/A
**
Open
CSYS CSYS-5RK CSYS-5RK/A
5
0
**
******
+SNAP-25 15-25
+SNAP-25 15-25
A
B a b c
a b c
514 405 405
CSYS CFP-Syx
dich
roic
mirr
or
Figure 2Jo
urna
l of C
ell S
cien
ceA
ccep
ted
man
uscr
ipt
A
b
0 s 300 s
aNPY-mRFP NPY-mRFP
B
I
0
0.25
0.5
0.75
1
BoNT-C1
CSYS
Nor
mal
ized
Tot
al S
ecre
tion
CSYS(R)
****
**
- +-+ -+
+ +-
+ BoNT-C1- BoNT-C1
CSYS
CSYS(R)
0.5 1.5 2.5 3.5 4.5 0.5 1.5 2.5
ii
iii iv
i
ii
iii iv
C
1
0.50.75
0.250
1
0.50.75
0.250N
orm
aliz
ed In
tens
ityDistance (µm)
0
0.25
0.5
0.75
1
Nor
mal
ized
Tot
al S
ecre
tion
**** **
1.2
CdHigh K+
CSYS - +-
- -+- --
+
+-
3.5 4.5-
-+
**
Figure 3Jo
urna
l of C
ell S
cien
ceA
ccep
ted
man
uscr
ipt
-100 0 100 200 300
High K +
-0.94
1
0.98
0.96
-
BA
CSYS-Open
CSYS
0.94
1
0.98
0.96
Time (Sec)100 0 100 200 300-
High K+
0.94
1
0.98
0.96
-100 0 100 200 300--Time (Sec)
High K+
Ave
rage
N
orm
aliz
ed F
RET
Rat
io
C
CSYS
CSYS+Cd
0.94
1
0.98
0.96
100 0 100 200-Time (Sec)
Nor
mal
ized
FR
ET
Rat
io
High K+
Time (Sec)
D
Ave
rage
N
orm
aliz
ed F
RET
Rat
io
Ave
rage
N
orm
aliz
ed F
RET
Rat
io
E
CSYS0.94
1
0.98
0.96
-100 0 100 200 300
CSYS-5RK
0.94
1
0.98
0.96
Time (Sec)70 0 100 200 300
High K+No Ca2+
Ave
rage
N
orm
aliz
ed F
RET
Rat
io
High K++ Ca2+
400
CSYS
-0.94
1
0.98
0.96
Time (Sec)70 0 100 200 300
High K+No Ca2+
Ave
rage
N
orm
aliz
ed F
RET
Rat
io
-
F
Figure 4Jo
urna
l of C
ell S
cien
ceA
ccep
ted
man
uscr
ipt
Time (Sec)
CSYS-5RK
CSYS-5RK/A
0.94
1
0.98
0.96
-100 0 100 200 300
Time (Sec)
0.94
1
0.98
0.96
-100 0 100 200 300
A B
C
High K+
High K+
Ave
rage
N
orm
aliz
ed F
RET
Rat
ioA
vera
ge
Nor
mal
ized
FR
ET R
atio
CSYS- 5RK/A
+ Cd
High K+
Time (Sec)-100 0 100 200 300
0.94
1
0.98
0.96
Ave
rage
N
orm
aliz
ed F
RET
Rat
io
D
CSYS-5RK/A
**
+--+
CSYS-5RK/A
1
0.75
0.5
0.25
0
Nor
mal
ized
To
tal S
ecre
tion
CSYS-5RK + Cd
CSYS-5RK
Figure 5Jo
urna
l of C
ell S
cien
ceA
ccep
ted
man
uscr
ipt