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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

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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

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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

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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

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-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

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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

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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

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0

0.2

0.4

0.6

0.8

1

Nor

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ized

Ave

rage

Eap

p

+VAMP2

CSYS CSYS-5RK CSYS-5RK/A

+VAMP2 +VAMP2

** **

CSYS-5RK

**** **

+VAMP2+SNAP-25

A B

0

0.2

0.4

0.6

0.8

1

Nor

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ized

Ave

rage

Eap

p

Figure 6Jo

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