Information travels around the nervous system along cells called neurons, which communicate with each other via connections called synapses. When a signal travelling along one neuron reaches a synapse, it triggers the release of molecules known as neurotransmitters. These molecules are then taken up by the next neuron to pass the signal on. Neurotransmitters are stored in compartments called synaptic vesicles and their release from the first neuron depends on the synaptic vesicles fusing with the membrane that surrounds the cell. This “membrane fusion” process is driven by a group of proteins called the SNARE complex.

Membrane fusion is triggered by a sudden increase in the amount of calcium ions in the cell, which leads to an increase in the activity of a protein called synaptotagmin-1. A region of this protein known as the C2B domain is able to detect calcium ions, and it can also bind to the cell membrane and SNARE complex proteins. However, it is not clear what roles these interactions play in driving the release of neurotransmitters.

Wang, Li et al. have used a variety of biophysical techniques to study these interactions in more detail using purified proteins and other cell components. The experiments show that all three interactions occur at the same time and are all required for synaptotagmin-1 to trigger membrane fusion. Wang, Li et al. propose that these interactions allow synaptotagmin-1 to bend a section of the cell membrane in response to calcium ions.

The experiments also show that the C2B domain interacts more strongly with the SNARE complex than previously thought. A future challenge is to observe whether synaptotagmin-1 works in the same way in living cells.

Ca²⁺-triggered neurotransmitter release by synaptic exocytosis is an exquisitely regulated process for interneuronal communication. The core machinery governing the process includes the SNAREs synaptobrevin, syntaxin-1 and SNAP-25, which form tight SNARE complexes to bridge synaptic vesicles to the plasma membrane and catalyze membrane fusion (Jahn and Scheller, 2006; Sutton et al., 1998; Weber et al., 1998). Syt1, the Ca²⁺ sensor for the fast component of Ca²⁺-triggered release (Chapman, 2008; Fernandez-Chacon et al., 2001; Geppert et al., 1994), confers Ca²⁺ sensitivity to SNARE-dependent synaptic vesicle fusion. The triggering function of Syt1 depends on its interplay with membranes, SNAREs, complexins and other key proteins of the release machinery (Rizo and Xu, 2015).

Syt1 consists of two C2 domains, known as C2A and C2B. The C2A and C2B domains adopt similar structures and bind three and two Ca²⁺ ions, respectively, through their Ca²⁺-binding loops located at the top of the structures (Fernandez et al., 2001; Shao et al., 1998; Sutton et al., 1995). These loops mediate penetration of Syt1 C2 domains to acidic membranes containing phosphatidylserine (PS) in response to Ca²⁺, and this activity is required for the triggering function of Syt1 (Chapman and Davis, 1998; Fernandez-Chacon et al., 2001; Rhee et al., 2005). In addition, Syt1 readily binds to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) via its C2B domain in a Ca²⁺-independent manner, which helps increase the apparent Ca²⁺ affinity of Syt1 and thereby enhances release probability (Bai et al., 2004; Li et al., 2006; Radhakrishnan et al., 2009; van den Bogaart et al., 2011a). Furthermore, Syt1 binds to SNAP-25 and syntaxin-1, and to SNARE complexes mainly through its C2B domain, which is believed to position Syt1 on the pre-fusion SNARE complexes to trigger release in response to Ca²⁺ (Brewer et al., 2015; Zhou et al., 2015; de Wit et al., 2009; Mohrmann et al., 2013).

Although the biochemical properties of Syt1 have been studied in detail, the functional importance of individual properties has remained unclear. It has been found that disrupting Ca²⁺ binding to C2B impairs release much more strongly than disruption of C2A Ca²⁺ binding sites in vivo (Mackler et al., 2002; Nishiki and Augustine, 2004; Robinson et al., 2002).Moreover, previous work showed that isolated C2B, instead of C2A, can promote SNARE-dependent membrane fusion in response to Ca²⁺ in vitro (Gaffaney et al., 2008; Xue et al., 2008). These findings suggested that C2B plays a more preponderant role than C2A in neurotransmitter release. A number of studies have revisited this issue and suggested that the functional importance of C2B arises partly from its ability to deform membranes. For instance, C2B can induce vesicle clustering and/or membrane curvature in response to Ca²⁺ (Arac et al., 2006; Martens et al., 2007; Hui et al., 2009; Xue et al., 2008). However, it is unclear why C2B has such membrane-deformation activity while C2A does not, given the fact that both C2 domains of Syt1 exhibit similar Ca²⁺-dependent membrane-insertion properties in vitro.

Another potential reason for the striking functional asymmetry of the Syt1 C2 domains was provided by recent studies showing that interactions between C2B and SNARE complexes are crucial for the function of Syt1 in neurotransmitter release (Zhou et al., 2015; Brewer et al., 2015). However, these studies have yielded conflicting results. For instance, a recently solved Syt1–SNARE complex crystal structure showed a relatively large binding interface between Syt1 and the SNARE complex that involves two basic residues (Arg398 and Arg399, referred to as the R398 R399 region, see Figure 1A) on the bottom of C2B (Zhou et al., 2015), whereas another dynamic Syt1–SNARE complex structure model obtained by nuclear magnetic resonance (NMR) indicated a SNARE binding interface that is located on the polybasic patch (Lys326 and Lys327, referred to as the K326 K327 region, see Figure 1A) at the side of C2B (Brewer et al., 2015). Moreover, both of these basic regions have been previously implicated in binding to acidic membrane lipids, such as PS and PI(4,5)P2 (Xue et al., 2008; van den Bogaart et al., 2011a). Some other reports argued against the interaction between Syt1 and the SNARE complex, and suggested that the triggering function of Syt1 requires specific binding of C2B to acidic membranes rather than binding to SNARE complexes (Honigmann et al., 2013; Park et al., 2015). Taken together, although it seems clear that C2B can interact with either acidic SNARE complexes or acidic membranes due to the abundance of highly positive charges around its surface (Figure 1B), the binding mode of C2B with SNARE complexes and membranes underlying the actual mechanism of Syt1 in release remains elusive.

Figure 1

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To address these conundrums, we systematically investigated the binding properties of the C2B domain with SNARE complexes and membranes using diverse biophysical techniques. Our results showed that, prior to the Ca²⁺ signal, the C2B domain interacts with PI(4,5)P2 and membrane-anchored SNARE complexes through the K326 K327 region and the R398 R399 region, respectively. We also found that the two interactions persist during insertion of the Ca²⁺-binding loops into the membrane upon Ca²⁺ influx. Consistent with a Syt1 triggering model proposed recently by Brunger and colleagues (Zhou et al., 2015), our data suggest that the local membrane deformation driven by the C2B domain constitutes a key step for triggering fusion.

Syt1 acts as a Ca²⁺ sensor and plays key functions in neurotransmitter release through its C2 domains. The C2 domains exhibit similar overall structures and Ca²⁺-induced membrane-insertion properties but differ strikingly in their function during release. Despite the fact that the functional significance of the C2B domain in vivo has been successfully reproduced in SNARE-dependent membrane fusion assays in vitro the mechanism by which C2B acts with the SNAREs and membranes to promote fusion is unclear. In the present study, we suggest a membrane-bending property of the C2B domain that arises from its simultaneous interactions with SNARE complexes and membranes. The relevance of our proposed C2B–SNARE complex–membrane interactions is supported by the present study and many previously reported data (Radhakrishnan et al., 2009; van den Bogaart et al., 2011a; Zhou et al., 2015), concordantly with the increasing realization that Syt1 cooperates with the SNARE complex and membranes in neurotransmitter release.

The functional importance of C2B arises most likely from its unique structure that contains abundant basic residues on its surface, which endows C2B with the ability to bind acidic SNARE complexes and/or membranes. Contiguously electrostatic potentials created by the bottom R398 R399 residues and the side K326 K327 residues of C2B contribute to the SNARE binding, as observed in Figure 2A,B. However, as demonstrated with our co-flotation and FRET experiments (Figure 2C–G), the presence of PI(4,5)P2-containing membranes shifts the equilibrium towards an energetically favorable binding where the K326 K327–PI(4,5)P2 interaction dominates. The much higher efficiency of the K326 K327 region, compared to the R398 R399 region, in PI(4,5)P2 binding arises most likely because the K326 K327 region contains a much higher positive-charge density, which enables it to bind tightly to lipid head groups with a highly negative-charge density [i.e., PI(4,5)P2-microdomains at active zones] (Honigmann et al., 2013; Joung et al., 2012; Park et al., 2012). Consistent with this, further investigations on the C2B–SNARE complex interaction using a more sensitive bimane-tryptophan quenching assay (Figure 3 and 4) indicate that the R398 R399 region of C2B binds to the SNARE complex or the membrane-anchored SNARE complex in a Ca²⁺-independent manner. Thus, the existence of the K326 K327–PI(4,5)P2 interaction and the R398 R399–SNARE complex interaction prior to Ca²⁺ influx likely recruits Syt1 to the fusion sites, which underlies the docking function of Syt1, as suggested previously (de Wit et al., 2009; Honigmann et al., 2013).

A recent report (Park et al., 2015) argued against the Syt1–SNARE complex interaction because this interaction measured in the study appeared to be completely abolished in the presence of ATP and Mg²⁺. However, Syt1 used in the study was labeled at residue 342, which is close to the polybasic patch, suggesting that this study actually measured the FRET between the K326 K327 region and the SNARE complex, and it is unlikely that it reflects the real Syt1–SNARE complex interaction. Instead, our study found that the R398 R399–SNARE complex interaction persists in the absence and presence of ATP and Mg²⁺ (Figure 3). It is also noteworthy that C2B T285W and SNAP-25 R59C mutations used in our bimane-tryptophan quenching assay seem unlikely to affect the particular interaction between Syt1 and the SNARE complex, because these residues are outside the 'primary' interface (interface area: 720 Ų; including residues Arg398 and Arg399) between Syt1 and the SNARE complex (Zhou et al., 2015). In addition, the C^α–C^α distance between the two labeling sites is measured at ~12 Å based on the Syt1–SNARE complex structures (Zhou et al., 2015), consistent with the relatively large effect on the FRET observed in our experiments. Moreover, we measured a reasonably strong binding K_d [0.86 ± 0.04 μM and 1.53 ± 0.04 μM in the presence and absence of PI(4,5)P2, respectively] between C2B and the SNARE complex in the presence of membranes (Figure 4). Thus, our binding results, together with the observations that both spontaneous and Ca²⁺-evoked release are not affected by the presence of 3 mM ATP (Zhou et al., 2015), strongly suggest that the interaction between Syt1 and the SNARE complex is not affected by ionic shielding and is physiologically relevant.

The finding that the R398 R399 region binds preferentially to the SNARE complex in this study seems to be incompatible with our previous studies (Arac et al., 2006; Xue et al., 2008). This discrepancy may arise from the different experimental conditions used between the present work and our previous studies. Re-examination of liposome clustering in a more stringent condition containing abundant SNARE complexes showed that the liposome-clustering ability of C2B is totally abrogated (Figure 2H). This data suggests that the R398 R399–PS binding might be displaced by the R398 R399–SNARE complex interaction. However, our results could not completely rule out the possibility that a small population of Syt1 molcules act to shorten the distance between membranes via the direct interaction of the R398 R399 region with acidic phospholipids in response to Ca²⁺.

By detecting penetration of C2B into membranes (with PS) as well as binding of C2B to the SNARE complex and PI(4,5)P2 at the same time (Figures 5 and 6), our results provides acceptable proof for the persistence of the K326 K327–PI(4,5)P2 interaction and the R398 R399–SNARE complex interaction during Ca²⁺ influx. The presence of the simultaneous SNARE-containing membrane binding of the top Ca²⁺-binding loops, of the side K326 K327 region, and of the bottom R398 R399 region, leads to a possible membrane-deformation mechanism of Syt1 (Figure 9): before Ca²⁺ influx, both the K326 K327–PI(4,5)P2 interaction and the R398 R399–SNARE complex interaction fasten C2B in an orientation parallel with the plasma membrane, leaving the Ca²⁺-binding loops held back from membrane insertion (Figure 9A); Ca²⁺ influx strongly induces fast insertion of the Ca²⁺-binding loops into membranes, rearranging C2B to an orientation vertical to the plasma membrane. The persistent binding of C2B to PI(4,5)P2 and the membrane-anchored SNARE complex would thus exert a membrane bending force to buck the local membrane outward in response to Ca²⁺ (Figure 9B). Furthermore, it is likely that binding of C2B to PI(4,5)P2 and SNARE complexes before Ca²⁺ influx would lead to a ring-like arrangement of C2B molecules around the fusion pore (Wang et al., 2014), which would then facilitate bucking local membranes upon collective bending forces in response to Ca²⁺. This possible membrane-deformation mechanism is in good agreement with the Syt1 working model proposed by Brunger and colleagues (Zhou et al., 2015), and is supported by a recent observation that local membrane protrusions (5 nm in height, similar to the size of one C2B molecule) bucked on the surface of GUV (giant unilamellar vesicles) require the presence of Syt1 and assembled SNARE complexes (Bharat et al., 2014).

Figure 9

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The proposed membrane-deformation mechanism is supported by the finding that all three interactions of C2B are required for the triggering function of the Syt1 in liposome fusion (Figure 7). This membrane-deformation mechanism explains very well the recent observation that isolated C2B readily bends membranes (Hui et al., 2009), and suggests that membrane insertion of the Ca²⁺-binding loops alone is not sufficient to drive membrane deformation without the assist of the R398 R399 and the K326 K327 regions. Thus, the functional importance of the C2B Ca²⁺-binding sites observed in previous in vivo studies and our present liposome fusion experiments (Figures 7 and 8) can be explained: disruption of the C2B Ca²⁺-binding sites abrogates the simultaneous interaction of C2B with acidic membrane lipids [i.e., PI(4,5)P2 and PS] and the membrane-anchored SNARE complex, so that the ability of C2B to bend membranes is absolutely abolished. Our data reinforce the idea that local membrane deformation by Ca²⁺–Syt1 is key for the triggering function of Syt1 in release (Hui et al., 2009; Zhou et al., 2015). Our data also reinforce the notion that the coordinated efforts of two or more interactions from one protein can induce membrane deformation (McMahon and Boucrot, 2015).

The liposome fusion results support the notion that SNARE complexes are already partially assembled before Ca²⁺ influx (Figure 7 and Figure 7—figure supplement 2B), which enables complexin-1 binding and thereby strains such a complex in a stage ready for fusion (Rizo and Xu, 2015). In response to Ca²⁺, C2B-induced membrane bucking would reduce the distance between two apposed membranes, which might cooperate with the action of C2B in displacing inhibitory complexin-1, to facilitate the continuous helical SNARE complex assembly that propagates through the linker region into the transmembrane domains (Figure 9C). Thus, high curvature stresses induced by C2B and the energy released from the C-terminal SNARE complex assembly might be coupled together in response to Ca²⁺ to overcome the energy barrier for membrane fusion. It is noteworthy that C2A would have an ancillary role by binding to one membrane and helping to dictate the apparent Ca²⁺ affinity of Syt1 (Robinson et al., 2002; Zhou et al., 2015). Altogether, our results add increasing evidence for the triggering mechanism by which Syt1 acts in concert with the SNARE complex and membranes to promote membrane fusion.

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Author details

1. Shen Wang

   Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China

   Contribution

   SW, Conception and design, Acquisition of data, Analysis and interpretation of data

   Contributed equally with

   Yun Li

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-5013-1039

2. Yun Li

   Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China

   Contribution

   YL, Acquisition of data, Analysis and interpretation of data

   Contributed equally with

   Shen Wang

   Competing interests

   The authors declare that no competing interests exist.

3. Cong Ma

   Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China

   Contribution

   CM, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   cong.ma@hust.edu.cn

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-7814-0500

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