Solution single-vesicle assay reveals PIP 2 -mediated sequential actions of synaptotagmin-1 on SNAREs
2012; Springer Nature; Volume: 31; Issue: 9 Linguagem: Inglês
10.1038/emboj.2012.57
ISSN1460-2075
AutoresJae Yeol Kim, Bong‐Kyu Choi, Mal‐Gi Choi, Sun‐Ae Kim, Ying Lai, Yeon‐Kyun Shin, Nam Ki Lee,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoArticle9 March 2012free access Solution single-vesicle assay reveals PIP2-mediated sequential actions of synaptotagmin-1 on SNAREs Jae-Yeol Kim Jae-Yeol Kim Department of Physics, Pohang University of Science and Technology, Pohang, Korea Search for more papers by this author Bong-Kyu Choi Bong-Kyu Choi School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea Search for more papers by this author Mal-Gi Choi Mal-Gi Choi Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Korea Search for more papers by this author Sun-Ae Kim Sun-Ae Kim Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA Search for more papers by this author Ying Lai Ying Lai Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA Search for more papers by this author Yeon-Kyun Shin Corresponding Author Yeon-Kyun Shin Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Korea Search for more papers by this author Nam Ki Lee Corresponding Author Nam Ki Lee Department of Physics, Pohang University of Science and Technology, Pohang, Korea School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea Search for more papers by this author Jae-Yeol Kim Jae-Yeol Kim Department of Physics, Pohang University of Science and Technology, Pohang, Korea Search for more papers by this author Bong-Kyu Choi Bong-Kyu Choi School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea Search for more papers by this author Mal-Gi Choi Mal-Gi Choi Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Korea Search for more papers by this author Sun-Ae Kim Sun-Ae Kim Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA Search for more papers by this author Ying Lai Ying Lai Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA Search for more papers by this author Yeon-Kyun Shin Corresponding Author Yeon-Kyun Shin Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Korea Search for more papers by this author Nam Ki Lee Corresponding Author Nam Ki Lee Department of Physics, Pohang University of Science and Technology, Pohang, Korea School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea Search for more papers by this author Author Information Jae-Yeol Kim1,‡, Bong-Kyu Choi2,‡, Mal-Gi Choi3, Sun-Ae Kim4, Ying Lai4, Yeon-Kyun Shin 4,5 and Nam Ki Lee 1,2 1Department of Physics, Pohang University of Science and Technology, Pohang, Korea 2School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea 3Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Korea 4Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA 5Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Korea ‡These authors contributed equally to this work *Corresponding authors: Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011-3111, USA. Tel.: +1 515 294 2530; Fax: +1 515 294 0453; E-mail: [email protected] of Physics, Pohang University of Science and Technology, Pohang 790-784, Korea. Tel.: +82 54 279 2097; Fax: +82 54 279 3099; E-mail: [email protected] The EMBO Journal (2012)31:2144-2155https://doi.org/10.1038/emboj.2012.57 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Synaptotagmin-1 (Syt1) is a major Ca2+ sensor for synchronous neurotransmitter release, which requires vesicle fusion mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). Syt1 utilizes its diverse interactions with target membrane (t-) SNARE, SNAREpin, and phospholipids, to regulate vesicle fusion. To dissect the functions of Syt1, we apply a single-molecule technique, alternating-laser excitation (ALEX), which is capable of sorting out subpopulations of fusion intermediates and measuring their kinetics in solution. The results show that Syt1 undergoes at least three distinct steps prior to lipid mixing. First, without Ca2+, Syt1 mediates vesicle docking by directly binding to t-SNARE/phosphatidylinositol 4,5-biphosphate (PIP2) complex and increases the docking rate by 103 times. Second, synaptobrevin-2 binding to t-SNARE displaces Syt1 from SNAREpin. Third, with Ca2+, Syt1 rebinds to SNAREpin, which again requires PIP2. Thus without Ca2+, Syt1 may bring vesicles to the plasma membrane in proximity via binding to t-SNARE/PIP2 to help SNAREpin formation and then, upon Ca2+ influx, it may rebind to SNAREpin, which may trigger synchronous fusion. The results show that ALEX is a powerful method to dissect multiple kinetic steps in the vesicle fusion pathway. Introduction Neurons use Ca2+-triggered exocytosis of synaptic vesicles to release neurotransmitters for interneuronal communication. To release neurotransmitters, synaptic vesicles undergo several steps; tethering and docking onto the presynaptic plasma membrane, priming that prepares vesicles to be fusion ready, and membrane fusion triggered by Ca2+ influx (Sudhof, 2004). Vesicle fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: t-SNAREs syntaxin-1A and SNAP-25 on the plasma membrane and v-SNARE VAMP-2 (or synaptobrevin-2) on the synaptic vesicles (Weber et al, 1998; Jahn and Scheller, 2006; Sudhof and Rothman, 2009). SNAREs from two membranes engage and form a parallel four-helix bundle (Poirier et al, 1998; Sutton et al, 1998), which brings membranes into close proximity, facilitating fusion (Sudhof and Rothman, 2009). Synaptotagmin-1 (Syt1), a vesicle protein that has two Ca2+-binding tandem C2 domains (C2A and C2B), is a major Ca2+ sensor for neuroexocytosis (Brose et al, 1992; Fernandez-Chacon et al, 2001; Chapman, 2008). The disruption of Syt1 gene typically abolishes the rapid Ca2+-dependent synchronous exocytosis (DiAntonio et al, 1993; Littleton et al, 1993; Nonet et al, 1993; Geppert et al, 1994). Syt1 is known to bind the SNARE complex (or SNAREpin) as well as phospholipids in the presence of Ca2+ (Schiavo et al, 1996; Zhang et al, 2002; Rickman et al, 2004, 2006; Bai et al, 2004b; Bhalla et al, 2006; Tang et al, 2006; Lynch et al, 2007; Connell et al, 2008; Hui et al, 2011). The SNARE complex binding may play a role in declamping the complexin clamp that holds vesicles at the primed state until the Ca2+ influx or it may just assist SNARE complex formation (Chapman, 2008; Rizo and Rosenmund, 2008; Sudhof and Rothman, 2009). Meanwhile, the phospholipid binding may function to induce a membrane curvature that is favourable for fusion (Martens et al, 2007; Hui et al, 2009) or it simply serves to enhance the Ca2+ sensitivity for C2 domain (Brose et al, 1992; Schiavo et al, 1996). Besides its Ca2+ sensing activity, a new emerging role of Syt1 is its functions prior to fusion (Bai et al, 2004a; Chapman, 2008; de Wit et al, 2009; van den Bogaart et al, 2011b). It has been proposed that this Syt1 function involves again the binding of Syt1 to SNAREs (Loewen et al, 2006; Rickman et al, 2006), although the specifics of this interaction is not clear. Several morphological studies using EM showed that the knockout of Syt1 decreased the docked vesicles on the plasma membrane (Reist et al, 1998; Liu et al, 2009; de Wit et al, 2009), which suggested that Syt1 plays a role in synaptic vesicle docking (or tethering) (Reist et al, 1998; Loewen et al, 2006; Rickman et al, 2006; de Wit et al, 2009). A recent study in adrenal chromaffin cells has revealed that the binding partner of Syt1 is binary t-SNARE and this specific interaction plays an important role in vesicle docking (de Wit et al, 2009). In contrast, an in-vitro study using proteoliposomes showed that Syt1 has strong affinity to phosphatidylinositol 4,5-biphosphate (PIP2) without Ca2+, which led to a proposition that Syt1 acts as a steering factor to bring vesicles closely to the plasma membrane prior to the Ca2+ influx (Bai et al, 2004a). Recent work also showed that Syt1 tethers vesicles without Ca2+, but does not require its specific interaction with t-SNARE (van den Bogaart et al, 2011b). Thus, there is ample evidence that Syt1 is involved in a process upstream of the Ca2+ action, but it is still controversial whether it interacts with t-SNARE or PIP2, or both, to induce vesicle tethering. It is also unknown how Syt1 helps vesicle tethering kinetically and what happens to Syt1 after SNAREpin is formed (Chapman, 2008; Rizo and Rosenmund, 2008). The bulk in-vitro fluorescence assay using proteoliposomes appears to be an ideal tool to dissect interactions of Syt1 with t-SNARE and PIP2 (Weber et al, 1998). The anticipated transition of Syt1's interactions, first with t-SNARE (and/or PIP2) and subsequently with the SNARE complex (and/or PIP2) can be examined effectively through such a well-defined in-vitro setting. However, the fundamental weakness of this technique is its inability to discriminate the earlier steps, happening before lipid mixing from the fusion step. For example, vesicle docking is undetected in this fluorescence-based method, because the docking of a vesicle pair does not give rise to the change in the fluorescence signal. Therefore, this method is not adequate to study Syt1's interactions with t-SNARE or PIP2, and subsequently with the SNARE complex, which will be followed by lipid mixing. Here, we introduce a new single-molecule technique, alternating-laser excitation (ALEX), which is capable of sorting out the subpopulations of fusion products, such as unreacted vesicles, fused vesicles, and fusion intermediate of docked-but-unfused species and measuring the kinetics of each subpopulation in solution (Figure 1A; Supplementary Figure S1; Kapanidis et al, 2004; Lee et al, 2005). The docked and fused vesicles have been discriminated by a single-vesicle assay by immobilizing vesicles on a surface or lipid bilayer (Bowen et al, 2004; Fix et al, 2004; Liu et al, 2005; Yoon et al, 2006; Kyoung et al, 2011). But this method often suffers from non-specific binding of vesicles on surface. Recently, as a method bypassing surface immobilization, fluorescence cross-correlation spectroscopy (FCCS) has been used to monitor docking and fusion kinetics (Cypionka et al, 2009). However, FCCS observes several vesicles at the same time and thereby does not provide direct measurement of full subpopulations of fusion products. In comparison with these methods, ALEX detects vesicles at the single-vesicle level without the need of surface immobilization and directly provides full subpopulations and the kinetics of fusion products. Figure 1.Sorting subpopulations of vesicle mixture by single vesicle assay, alternating-laser excitation (ALEX). (A) Schematic description of single-molecule alternating-laser excitation set-up (Supplementary Figure S1). Donor- and acceptor-excitation lasers are alternated faster (400 μs) than the transit time (∼5 ms) of a vesicle through the confocal excitation volume (∼1 femto-liter). Dilution to ∼100 pM vesicle concentration ensures that one vesicle passes through the excitation volume at a given time. The fluorescent emissions of donor and acceptor are detected separately, which results in fluorescence time traces. (B–E) Typical fluorescence time traces of vesicles. A random event of diffusing in and out of a vesicle generates a fluorescent burst (or spike) in time traces. Each time trace contains three different photon streams: IDD, fluorescent emissions of donor dyes (DiI) excited by donor-excitation laser (green line), IDA, fluorescent emissions of acceptor dyes (DiD) excited by donor-excitation laser, which are FRET signals (orange line), and IAA, fluorescent emissions of acceptor dyes excited by acceptor-excitation laser (red line). (B) Time trace of t-vesicle, reconstituted with t-SNARE and doped with DiI. (C) Time trace of v-vesicle, reconstituted with VAMP-2 and doped with DiD. (D) Time trace of docked-but-unfused vesicle. Both IDD and IAA present significant intensity. (E) Time trace of fused vesicle. Due to FRET, the intensity of IDD is reduced, while that of IDA is increased. (F) Two-dimensional E (FRET efficiency)-S (sorting number) graph. Three fluorescent intensities of a burst are used to calculate S and E (see ‘Definition and calculation of E and S of a single vesicle’). The unreacted, docked, and fused vesicles have characteristic values of S and E, and, as a result, occupy different areas in E-S graph: t-vesicle only (green box), v-vesicle only (red box), docked vesicle (purple box), and fused vesicle (orange oblique). (G) Comparison of bulk FRET assay with ALEX. For bulk FRET assay, T (reconstituted with t-SNARE) and V (reconstituted with VAMP-2) were mixed to be 50 μM lipid concentration at 35°C (black line). From single-exponential fitting, fusion rates were obtained; kfusion, bulk=1.03±0.02 × 10−3 s−1 and kfusion, ALEX=1.19±0.08 × 10−3 s−1, respectively. As a control, SNAP-25 was not added (grey line). For ALEX, the mixture was incubated at 35°C and diluted to be 3 μM lipid concentration at the selected time points and then measured for 5 min. The fractions of docked and fused vesicles (purple box and orange oblique, respectively) were obtained from three independent measurements. Dotted line denotes the level of random coincidence. (H–J) 2D E-S graphs obtained by ALEX. Each dot denotes a vesicle. (H) T-V mixture after 60 s incubation (blue arrow in G), and (I) after 2400 s incubation (red arrow in G). (J) No SNAP-25 control after 2400 s incubation (green arrow in G). Colour scale bar indicates the number of vesicles. Download figure Download PowerPoint We used ALEX to dissect the interactions of membrane-anchored Syt1 with t-SNARE, PIP2, and the SNARE complex prior to lipid mixing and studied the effect of Syt1 on docking and fusion kinetics. We found that Syt1 interacts with both t-SNARE and PIP2, which gives rise to vesicle tethering without Ca2+. When VAMP-2 binds to t-SNARE to form the SNARE complex, Syt1 dissociates from the complex. However, when Ca2+ is introduced, Syt1 rebinds to the SNARE complex. We find that PIP2 is required in this final step too. Thus, in the absence of Ca2+ Syt1 may function to bring vesicles to the plasma membrane in proximity through t-SNARE/PIP2 interaction, which helps SNAREpin formation, and finally binds to SNAREpin and membrane again in the presence of Ca2+, which may trigger membrane fusion. Kinetically, the docking rate of vesicles containing Syt1 is as much as 103 times higher than that of SNAREs-only vesicles. Moreover, the fusion step is found to be the rate-limiting step for Syt1-containing vesicles in the absence of Ca2+. The results demonstrate that ALEX is a powerful tool for discriminating the subpopulations and measuring the kinetics of docking and fusion of vesicles in solution, which will be useful in studying the functions of SNAREs and regulatory proteins in vesicle fusion. Results ALEX sorts out subpopulations and measures their kinetics in an in-vitro fusion assay The bulk in-vitro fusion assay using fluorescently labelled proteoliposomes has been an important tool for investigating the functions of SNAREs and their regulatory proteins (Weber et al, 1998). In this method, liposomes reconstituted with t-SNAREs (t-vesicles) are mixed with another population of liposomes reconstituted with v-SNAREs (v-vesicles). As fusion progresses, the reaction builds up a mixture of fusion intermediates and products as well as unreacted v- and t-vesicles. However, this bulk method is not effective in dissecting subpopulations corresponding to these individual species, and the results are often ambiguous (Smith and Weisshaar, 2011). In ALEX, we detect and analyse single-vesicle species diffusing in and out of a confocal volume (Figure 1A) and sort them into subpopulations according to their fluorescence characteristics (Figures 1B–F). We dope t-vesicles with fluorescence donor (DiI) and v-vesicles with fluorescence acceptor (DiD). We focus both donor- and acceptor-excitation lasers into a small confocal volume (Figure 1A). The reaction mixture is sufficiently diluted (∼100 pM vesicle concentration) that only a single particle diffuses into the detection volume at a given moment. The donor- and acceptor-excitation lasers alternate sufficiently fast to excite and detect both the donor and acceptor fluorescence before the vesicle diffuses out of the volume. Figures 1B–E show typical time traces, where each fluorescent burst represents a single vesicle, a pair/aggregate of docked vesicle, or a fused vesicle, diffusing in and out of the confocal volume. Each single burst contains three types of fluorescence intensities, IDD, IDA, and IAA, where IDD and IDA are the donor and acceptor fluorescence intensity, respectively, when the vesicle is irradiated with the donor-excitation laser, and IAA is the acceptor fluorescence intensity when irradiated with the acceptor-excitation laser. These three intensities are used to calculate two parameters, S, a sorting number, and E, the fluorescence resonance energy transfer (FRET) efficiency of each vesicle (for detail, see Materials and methods) (Kapanidis et al, 2004). Briefly, S becomes 1 for a vesicle responding only to the donor-excitation laser but not to the acceptor-excitation laser, which represents a single t-vesicle, while it is 0 for that responding to the acceptor-excitation laser but not to the donor-excitation laser, which represents a single v-vesicle. For a vesicle pair that contains both t- and v-vesicles, S becomes 0.5 by responding to both lasers. The E value is calculated based on the equation E=IDA/(IDD+IDA), which reports the degree of lipid mixing. In the two-dimensional (2D) display of S and E parameters, subpopulations corresponding to unreacted v- and t-vesicles, docked vesicles, and fused vesicles are resolved into distinct areas (Figure 1F). We prepared v-vesicle with VAMP-2 (V, the lipid-to-protein ratio L/P=300) and t-vesicle with t-SNARE (T, L/P=300). Figure 1G depicts the typical results of the bulk measurement of the T-V mixture at 35°C, which shows fusion between T and V through the increase of FRET (black line), while the control without SNAP-25 shows no FRET increase (grey line). The 2D E-S graph (Figure 1H), taken 1 min after incubating the T-V mixture at 35°C (Figure 1G, blue arrow), shows considerable subpopulation of unreacted vesicles (green and red boxes), while docked and fused vesicles are rare (purple box and orange oblique, respectively). However, after 40 min incubation (Figure 1G, red arrow), the population of fused vesicles was considerably increased, while the populations of unreacted vesicles were decreased and docked vesicles were still scarce (Figure 1I). As a control, when SNAP-25 is not included, no fused vesicles were observed even after 40 min incubation (Figure 1G, green arrow), but unreacted v- and t-vesicles are clearly seen (Figure 1J). The E values of donor-only (T) and acceptor-only (V) vesicles in the 2D graph are determined by fluorescent backgrounds. For example, E of donor-only vesicles (0.13) was generated by the leakage (donor emission detected by acceptor emission detector), and the high E of acceptor-only vesicles was caused by the direct excitation of acceptor dyes by donor-excitation laser (Lee et al, 2005). We then quantified the subpopulations of docked and fused vesicles from the 2D E-S graph. A simple quantitative analysis (Supplementary data) using this E-S graph was used to measure the fractions of fused and docked vesicles. In this analysis, we select bursts using ‘acceptor photon search’ (Supplementary Figure S2; Kapanidis et al, 2006). To select fluorescent bursts from the time traces (Figures 1B–E), a threshold photon count above the background is used (Kapanidis et al, 2004). Typically, all photon counts, that is, the sum of donor and acceptor intensities (IDD+IDA+IAA), are used for burst search to present all fluorescent species in 2D E-S graphs (Figure 1H–J). However, this searching method selects docked and fused vesicles, which contain both donor and acceptor dyes, more efficiently than unreacted vesicles. For this reason, when quantitative analysis of subpopulations is required, IAA is used for burst search (acceptor photon search) (Kapanidis et al, 2006). Because unreacted v-vesicle, docked, and fused vesicles have similar intensities of IAA, this ‘acceptor photon search’ provides unbiased quantitative measurement of the subpopulations (see Supplementary Figure S2 for comparison between two burst searching methods). We found that the fusion kinetics measured from the fractions of fused vesicles agrees well with the bulk measurement (Figure 1G). We estimates that ∼5% of docked vesicles is the background, which stems from two vesicles entering into the detection volume simultaneously without docking. The subpopulation of docked vesicles was not seen, indicative of a significantly slower rate of docking than the rate of fusion for the T-V reaction (Smith and Weisshaar, 2011). Both binary t-SNARE and PIP2 are required for Syt1-mediated docking Previously, using a surface single-vesicle assay, it has been demonstrated that membrane-anchored Syt1 is capable of promoting vesicle docking and fusion in a Ca2+-independent manner (Lee et al, 2010). However, because both Syt1 and VAMP-2 were present in v-vesicle, it was unclear whether such an effect was due to the direct interaction between Syt1 and the target membrane components (t-SNARE and PIP2) or due to its catalytic role in helping VAMP-2 interaction with t-SNARE. To investigate the possible direct interaction between Syt1 and t-SNARE in trans, we prepared v-vesicle without VAMP-2 but containing Syt1 (S in Figure 2A, L/P=600). We included 1% PIP2 in t-vesicle (T) (L/P=500 for t-SNARE). We mixed S with T to induce docking at room temperature for 5 min at 10 μM lipid concentration and analysed the mixture using ALEX at 3 μM lipid concentration (∼100 pM vesicle concentration). The 2D E–S graph of the T-S mixture (Figure 2B) shows a considerable docking population (purple box). A simple quantitative analysis (Supplementary data; Supplementary Figure S2) using this E–S graph shows that 40% of S are paired (or docked) with T (Figure 2K). To confirm that vesicle docking occurred through Syt1, we prepared an Syt1 mutant (Syt1 Y311N) that has a single point mutation in the C2B domain, which is known to reduce Syt1's binding to t-SNARE (Yoshihara and Littleton, 2002; Rickman et al, 2006). Indeed, with Syt1 Y311N vesicle docking was significantly impaired nearly to the background level of 7% (Figures 2C and K). This result shows that Syt1 is responsible for vesicle docking observed for T-S (Figure 2B). Figure 2.Syt1 is able to mediate vesicle docking by interacting with both t-SNARE and PIP2. (A) Schematic description of vesicle docking measurement. S, v-vesicle without VAMP-2 but containing Syt1. T, t-vesicle with t-SNARE and 1% PIP2. After mixing two vesicles, the mixture was analysed by ALEX. (B–E) 2D E-S graphs for mixture of t- and v-vesicles. (B) Mixture of S and T. Significant amounts of docked vesicles were observed (purple box). (C) Mixture of SY311N (v-vesicles incorporated with Syt1 Y311N mutant) and T. (D) Mixture of S and Tno PIP2 (t-vesicle incorporated with t-SNARE but without PIP2). (E) Mixture of S and Tsyx-only (t-vesicle containing only syntaxin-1A but containing 1% PIP2). (F) Positions of SNAP-25 mutations in SNARE complex. (G–I) Three SNAP-25 mutants of SNAP-25 D51K/E52K, D179K/D186K, and BoNT/A were used to reconstitute T (TD51K/E52K, TD179K/E186K, and TBoNT/A, respectively), and each one of them was mixed with S. (G) TD51K/E52K-S; (I) TD179K/E186K-S; (H) TBoNT/A-S. (J) Time-dependent T-S docking. For detailed method, see ‘ALEX measurements for the docking and fusion kinetics’ in Supplementary data. The error bars within 5 min were obtained from 10 independent measurements, while those after 5 min from three measurements. (K) Bar graph of the fraction of the docking subpopulations. Error bars (standard deviation) were obtained from more than three independent experiments, and >1000 vesicles were analysed in each measurement (*P<0.005). (L) PIP2 only control. We mixed protein-free liposome, containing 1% PIP2, and S. (M) The t-SNARE/PIP2 molar ratio determines docking efficiency of TD51K/E52K-S. We kept 1% PIP2, but varied the amounts of t-SNARE constructed with SNAP-25 D51K/E52K mutant, and then measured the docking efficiency of TD51K/E52K-S. We used initial input molar ratio of t-SNARE/PIP2 and the incorporation efficiency of t-SNARE into vesicle (60±11%; Supplementary Figure S5) to determine the t-SNARE/PIP2 ratio. Error bars were obtained from more than three independent experiments. Download figure Download PowerPoint In T, there are two potential binding partners for Syt1, t-SNARE and PIP2 (Tucker et al, 2003; Bai et al, 2004a). To find which interaction is dominant for vesicle docking, we prepared two types of T; one without PIP2 (Tno PIP2) and the other without SNAP-25 (Tsyx-only) while keeping other components the same. The absence of PIP2 significantly reduced the subpopulation of docked vesicles (Figure 2D), indicating that PIP2 is essential for Syt1-induced vesicle docking. Likewise, when SNAP-25 is absent docking population was reduced to the similar level in the case without PIP2 (Figure 2E). This shows that the docking by Syt1 required both PIP2 and binary t-SNARE. To further confirm the need of binary t-SNARE for Syt1 docking, we reconstituted t-vesicle with the SNAP-25 D51K/E52K mutant (Figure 2F), which is known to disrupt the interaction between Syt1 and t-SNARE without interfering with formation of SNARE complexes (Rickman et al, 2006; Supplementary Figure S3). As expected, the subpopulation of docked vesicles was considerably reduced for this mutant (Figure 2G). Thus, this result shows that Syt1 directly interacts with t-SNARE for docking. On the other hand, when two other SNAP-25 mutants D179K/D186K in which two aspartic acids in the middle were changed to lysines and BoNT/A in which the C-terminal 9 residues are cleaved were used (Figure 2F), the subpopulation of docked vesicles were similar to that for wild type (Figures 2H and I). Thus, the results suggest that Syt1 interacts with the central region of the SNAP-25 SNARE motif, where positions 51 and 52 are located rather than the C-terminal region or the face where 179 and 186 resides are located (Figure 2F), consistent with the previous single FRET result (Choi et al, 2010). Then, we tested if PIP2 offers a specific binding for Syt1 or it is simply to add the surface negative charge in the target membrane. We increased the PS concentration from 15 to 25% in T. This increased the docked population marginally from 7 to 10% (Figure 2K; Supplementary Figure S4). In sharp contrast, adding just 1% PIP2 increased the docking population to 40%. Therefore, the results show that the stimulation of docking by Syt1 is highly specific to PIP2. Overall, the ALEX results support that Syt1 is capable of inducing vesicle docking in the absence of VAMP-2 and Syt1 uses its interaction with both t-SNARE and PIP2, consistent with the in-vivo observation in chromaffin cells (de Wit et al, 2009). The kinetics of Syt1-mediated vesicle docking Next, we investigated how fast the docking between T and S occurs (Figure 2J). To measure the docking kinetics, we collected the ALEX data for 5 min soon after mixing T and S with 3 μM final lipid concentration. Then, the data were divided into five time bins and the fractions of docked vesicles were obtained for each time bin. For later times than 5 min, the mixture was incubated at room temperature for a desired duration and then measured for 5 min as described above. Because of the delay between mixing and measurement, the earliest time point we obtained was 35 s. We found that the docking became saturated to 40% even at 35 s, the earliest time point that we obtained. Thus, the low limit of the docking rate (kdock, T-S) between T and S was 1.1 × 108 M−1 s−1 (Supplementary data). This is close to the diffusion-limit rate constant (klimit) of 3.1 × 109 M−1 s−1 for vesicles (Supplementary data). The average collision number (klimit/kdock, T−S) before docking is only 30. This collision number is much smaller than 104–105 collisions of T-V for docking, a number previously reported (Cypionka et al, 2009; Smith and Weisshaar, 2011) and also confirmed in this work (see below). Overall, we found that Syt1 enhances the rate of vesicle docking by ∼103 times when compared with that of SNARE-only vesicles. The t-SNARE/PIP2 ratio determines Syt1 binding to either PIP2 or t-SNARE/PIP2 complex How does PIP2 cooperate with t-SNARE to promote Syt1-mediated docking? PIP2 itself is known to inter
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