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Logo of jphysiolThe Journal of Physiology SiteMembershipSubmissionJ Physiol
J Physiol. May 15, 2007; 581(Pt 1): 75–90.
Published online Mar 1, 2007. doi:  10.1113/jphysiol.2006.127472
PMCID: PMC2075219

The role of synaptotagmin I C2A calcium-binding domain in synaptic vesicle clustering during synapse formation

Abstract

Synaptic vesicles aggregate at the presynaptic terminal during synapse formation via mechanisms that are poorly understood. Here we have investigated the role of the putative calcium sensor synaptotagmin I in vesicle aggregation during the formation of soma–soma synapses between identified partner cells using a simple in vitro synapse model in the mollusc Lymnaea stagnalis. Immunocytochemistry, optical imaging and electrophysiological recording techniques were used to monitor synapse formation and vesicle localization. Within 6 h, contact between appropriate synaptic partner cells up-regulated global synaptotagmin I expression, and induced a localized aggregation of synaptotagmin I at the contact site. Cell contacts between non-synaptic partner cells did not affect synaptotagmin I expression. Application of an human immunodeficiency virus type-1 transactivator (HIV-1 TAT)-tagged peptide corresponding to loop 3 of the synaptotagmin I C2A domain prevented synaptic vesicle aggregation and synapse formation. By contrast, a TAT-tagged peptide containing the calcium-binding motif of the C2B domain did not affect synaptic vesicle aggregation or synapse formation. Calcium imaging with Fura-2 demonstrated that TAT–C2 peptides did not alter either basal or evoked intracellular calcium levels. These results demonstrate that contact with an appropriate target cell is necessary to initiate synaptic vesicle aggregation during nascent synapse formation and that the initial aggregation of synaptic vesicles is dependent on loop 3 of the C2A domain of synaptotagmin I.

At mature synapses, synaptic vesicles (SVs), exocytotic proteins and voltage-dependent calcium (Ca2+) channels (VDCCs) are localized to active zones of the presynaptic site to ensure effective synaptic transmission (see Augustine, 2001; Atwood & Karunanithi, 2002). However, the mechanisms underlying presynaptic protein organization in synapses remain unclear.

Prior to contacting their synaptic partners, both potential presynaptic and postsynaptic neurons possess the synaptic proteins necessary for neurotransmission. However, development of mature synaptic structures requires continuous interaction between the presynaptic and postsynaptic cells (Zoran et al. 1991; Dan & Poo, 1994). In hippocampal glutamatergic synapses, the assembly of presynaptic proteins precedes postsynaptic differentiation (Rao et al. 1998; Ahmari et al. 2000; Friedman et al. 2000). The formation and stabilization of heterogeneous presynaptic structures at the active zone requires contact with an appropriate postsynaptic target (Dan & Poo, 1994; Daly & Ziff, 1997). However, VDCCs (Ahmari et al. 2000) and a number of SV-bound proteins including synaptotagmin (syt) (Littleton et al. 1995; Daly & Ziff, 1997), vesicle associated membrane protein (VAMP) (synaptobrevin, vSNARE) (Ahmari et al. 2000), synapsin (Daly & Ziff, 1997) and synaptophysin (Fletcher et al. 1991), and tSNARE proteins, such as syntaxin and SNAP25 (Littleton et al. 1995), are found in premature synapse-like terminals independent of target cell contact. Our current understanding of how SVs are selectively targeted to presynaptic sites, and how the presynaptic components of the nascent synapses attain their final organizational profiles is limited (see Ziv & Garner, 2004).

Target cell contact in vitro induces an immediate increase in the presynaptic Ca2+ level (Zoran et al. 1991; Dai & Peng, 1993). The in vivo synaptic connections (Syed et al. 1992) made in the great pond snail, Lymnaea stagnalis (L. stagnalis), can reform in culture between the cell somata of individually identified synaptic partner neurons (Feng et al. 1997, 2002). The ability to reform soma–soma synapses between identified neurons has allowed the development of a convenient in vitro model to study synapse formation (Feng et al. 1997, 2002; Magoski & Bulloch, 1998; Hamakawa et al. 1999). The typical somata synapse readily exhibits synaptic transmission, which can be recorded directly from the presynaptic and postsynaptic cells (Feng et al. 1997). In addition, voltage-dependent Ca2+ hotspots in the presynaptic site can be induced by membrane contact with synaptic target cells (Feng et al. 2002). These Ca2+ hotspots are not seen when a cell is paired with a non-target neuron (Feng et al. 2002).

The Ca2+-binding SV protein syt I, a putative Ca2+ sensor (Matthew et al. 1981; Geppert et al. 1994), is highly localized at the presynaptic site in mature synapses. It contains a small glycosylated N-terminal intravesicular domain separated from a palmitoylated cysteine-rich region by a transmembrane domain anchor (Perin et al. 1991; Chapman et al. 1996; Veit et al. 1996). The cytoplasmic segment of this protein is composed of two C2 domains, C2A and C2B. Each C2 domain contains eight β-strands topped with three flexible loops containing five highly conserved acidic residues (Asp) important for Ca2+ binding (Davletov & Sudhof, 1993; Fernandez et al. 2001; Bai et al. 2002). Syt I is required for fusion and recycling of SVs (Fukuda et al. 1995; Llinas et al. 2004). However, the involvement of syt I in SV aggregation during the initial stages of synapse formation has not been tested directly.

In this study, we used the in vitro L. stagnalis soma–soma synapse model to investigate the spatiotemporal development of SV aggregation during synapse formation. Using this system, we have determined the spatiotemporal distribution of syt I, the integral membrane protein of SVs, in response to target cell contact, and elucidated the involvement of the loop 3 within C2 Ca2+-binding motifs in SV aggregation in the nascent synapse.

Methods

Animals, cell culture and cell isolation

Animal maintenance, conditioned medium preparation and isolation of individual identified neurons were conducted as previously described (Syed et al. 1990; Feng et al. 1997). The experiments were carried out according to the guidelines of the Animal Care Committee of the University of Toronto.

Animals

Fresh water pond snails, L. stagnalis, obtained from an inbred culture at Free University in Amsterdam, were raised and maintained in aquaria at the University of Toronto. All animals were kept in water at 20°C on a 12 h light–dark cycle, and fed green leaf lettuce twice a week. Two-month-old snails with a shell length of 15–20 mm were used in experiments.

For Western blot analysis, male, Sprague–Dawley rats (250–300 g) were used (Charles River Laboratory).

Primary cell culture and cell isolation

Snails were anaesthetized with 10% (v/v) Listerine. The central ring and the buccal ganglia were dissected in sterile snail saline containing (mm): NaCl 51.3, KCl 1.7, CaCl2 4.1, MgCl2 1.5 and Hepes 2 (pH adjusted to 7.9 with NaOH), as previously described (Syed et al. 1990; Feng et al. 1997), and incubated with 3 mg ml−1 trypsin (Type III, Sigma, Ontario, Canada) for 20 min. The connective tissue sheath surrounding the neurons was removed using fine forceps. Using a fire-polished pipette (2 mm, WPI, 1B200F) coated with Sigmacote (Sigma), gentle suction was applied to isolate individually identified neurons. Respiratory central pattern generator neurons visceral dorsal 4 (VD4) form inhibitory synapses with right pedal dorsal 1 (RPeD1) (Syed et al. 1990; Feng et al. 1997) and excitatory synapses with left pedal dorsal 1 (LPeD1) (Hamakawa et al. 1999). RPeD1 does not form synaptic connection with pedal A (PeA) (Spencer et al. 2000). Individual, non-paired VD4 and LPeD1 were used as the control for the synaptic pairs; individual, non-paired RPeD1 and PeA cells were used as the control for the non-synaptic pairs.

We subsequently plated neurons, either individually or with another cell, with overlapping neurite stumps on a poly-l-lysine-coated culture dish, and maintained the cells in conditioned medium (CM) at room temperature (~21°C). The CM was prepared in advance by incubating the central ring and buccal ganglia in defined medium (DM) for 2–3 days at room temperature. The DM consisted of serum-free 50% (v/v) Liebowitz L-15 medium (without salts or l-glutamine; Gibco, Grand Island, NY, USA), with the addition of (mm): NaCl 51.3, KCl 1.7, CaCl2 4.1, MgCl2 1.5, glucose 10, l-glutamine 1.0 and Hepes 2, and 20 mg ml−1 gentamycin (pH adjusted to 7.9 with NaOH).

Peptide synthesis and treatment

We aligned the protein sequence of L. stagnalis synaptotagmin I (l-syt I, AF484090) (Spafford et al. 2003) with various known invertebrate and vertebrate orthologues using NCBI BLAST. These alignments revealed an approximate 90% homology within the cytoplasmic domain of the orthologues, and conserved loop 3 regions in the C2A and C2B Ca2+-binding regions (Fig. 1). Control and specific peptides were generated by the Advanced Protein Technology Centre (the Hospital for Sick Children, Toronto, Canada): syt I C2A peptide, DFDRFSKH; syt I mC2A peptide, NFNRFSKH; syt I C2B peptide, DYDRIGTS; control peptide, DIDGDGQVNYEEFVQDTLASLSKLAKGLKALPQS. To facilitate peptide uptake by neurons, each peptide was constructed with an HIV-1 TAT internalization signal sequence (YGRKKRRQRRR) at the N-terminus. To evaluate peptide internalization, an Alexa-350 fluorophore (excitation, 346 nm; emission, 440 nm) was conjugated to an N-terminal cysteine on the peptide (Alexa-350 protein labelling kit; Molecular Probes). After cells were plated and left to settle onto the culture dish for approximately 15 min, 2 μl freshly made peptide solution was gently applied into the centre of the dish, resulting in a final peptide concentration of ~0.5 μm. Cells were incubated with peptide for at least 6 h.

Figure 1
Mammalian anti-syt I antibodies specifically detectL. stagnalissyt I

Immunocytochemistry

Cells were fixed using 1% (w/v) paraformaldehyde saline (1.7 mm KCl, 4.1 mm CaCl2 and 1.5 mm MgCl2) for 24 h at 22°C. Cells were permeabilized using 0.3% Triton X-100 solution in skimmed milk, and incubated with specific antibodies for 24 h at 4°C. Mouse monoclonal anti-syt antibody (1: 250) was either obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Department of Biological Sciences, Iowa City, IA, USA; originally developed by L. Reichardt, University of California, San Francisco, CA, USA) or purchased from Chemicon (Temecula, CA, USA). After incubation with the anti-syt I antibody, cells were washed three times with snail saline (5 min each) and then incubated with secondary antibody (1: 500, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse; Chemicon) at room temperature for 2 h. Cells were washed and mounted using Permaflour mounting medium (Fisher Scientific).

Confocal imaging and data analysis

Images of syt I staining were acquired using a Leica TCS SL laser confocal microscope (Leica Confocal Software, version 2.5, build 1347, Leica Microsystems, Germany). Z-series scan images of labelled cells were viewed under a 40 × oil immersion lens using laser line wavelengths of 488 nm (via an argon laser). A Z-series scan of the cell(s) was performed at a zoom level that maximized the view of each cell pair. Each plane was averaged three to four times and scans stepped approximately 0.5 μm in the z-axis. The FITC-conjugated fluorophore used in all experiments excites at 483 nm and emits at a peak intensity of 543 nm (green). Background fluorescence (between 1 and 5 intensity units from a scale of 255 units) was determined throughout the entire z-stack from three selected regions not containing cells. All imaging was performed at the same magnification and laser settings.

To quantify antibody staining, the mean amplitude fluorescence intensities (arbitrary units, AU) were averaged from sections of the z-stack for each region of interest. For paired cells, the cell contact and three non-contact regions were measured for both pre- and postsynaptic cells. Signal was measured at the cell contact region arbitrarily defined as 0 or 12 o'clock. Signal was also detected at three non-contact regions, at the 3, 6 and 9 o'clock positions relative to the contact site, and then averaged (see schematic diagram of the measurement in Fig. 2Db). Fluorescence intensities of three equal-sized samples from each region were recorded and averaged. Regions overlapping the nucleus were not measured in this analysis. For the unpaired individual cells, the fluorescence intensities emitted from all four positions were measured and averaged.

Figure 2
Presynaptic Ca2+ hotspots and syt I clustering at the contact site between functional synaptic partners

Western blot analysis

Protein samples were prepared from snail ganglia or rat brain. The rats were anesthetized with halothane and then decapitated followed immediately by brain sample removal. As previously described (Fei et al. 2007), the tissue samples were homogenized with 80–100 μl 1 × lithium dodecyl sulphate (LDS) lysis buffer (Invitrogen, USA) with a motor grinder on ice. The resulting homogenate was centrifuged at 16 000 r.p.m (13200g) at 4°C for 2 min to remove cellular debris. The supernatant was mixed with 1 μl 10 × sample reducing agent and 2.5 μl 4 × LDS lysis buffer (Invitrogen, USA). The protein concentration was determined using bicinchoninic acid method (BCA Protein Assay Kit, Pierce, USA).

Western blots were performed using a NuPAGE Electrophoresis System with Novex Bis-Tris gels (Invitrogen, Ontario, Canada), according to the manufacturer's instructions, as previously described (Fei et al. 2007). Specifically, protein samples (20 μg) and protein marker (Invitrogen Canada) were separated on a NuPAGE Novex Bis-Tris gel at 80 V (60 mA)−1 with Mes running buffer, and transferred onto a polyvinylidene difluoride (PVDF) membrane (0.2 μm pore size; Invitrogen) in NuPAGE transfer buffer (Invitrogen Canada) at 30 V and 170 mA. The membrane was blocked overnight with 3% skimmed milk blocking solution at 4°C. The membrane was incubated with monoclonal mouse anti-syt I antibody (1: 300; L. Reichardt, University of California, San Francisco, CA, and Developmental Studies Hybridoma Bank, Department of Biological Sciences, the University of Iowa, Iowa City, IA, USA) for 2 h at room temperature. After washing, the membrane was incubated with goat anti-mouse secondary antibody HRP (1: 1000, Chemicon) for 2 h at room temperature. The antibody-labelled protein bands were visualized using an enhanced chemiluminescent reagent (Amersham Biosciences, Ontario, Canada), and analysed by scanning densitometry with Kodak 1D image analysis software (Eastman Kodak, USA).

Electrophysiology

Intracellular recordings were performed under current-clamp mode using an Axopatch 700A amplifier (Axon Instruments, Union City, CA, USA) linked to a personal computer equipped with pCLAMP software (Version 9). Microelectrode pipettes (Sutter borosilicate glass, BF 150-117-15) were pulled using a Sutter P-87 microelectrode puller. Pipette resistances were typically ~100 MΩ when the pipettes were filled with internal solution containing (mm): KCl 39.3, ATP 2, Hepes 10, GTP-Tris 0.1 (pH adjusted to 7.4 with KOH); snail saline was used as the bath solution. Simultaneous recordings were made for both the presynaptic and postsynaptic cells in paired neurons. For detecting synaptic connections between cell pairs, action potentials were elicited in the presynaptic cell by current injection and electrical activities were detected in the postsynaptic cell. The data were filtered at 1 kHz (−3 dB) using a four-pole Bessel filter and digitized at a sampling frequency of 2 kHz. Data were analysed using Clampfit (Axon Instruments).

Ratiometric fura-2 Ca2+ imaging

Intracellular Ca2+ level was measured using fura-2 ratiometric Ca2+ imaging as previously described (Feng et al. 2002). Cells were incubated for 30 min with fura-2 AM (2 μm, Molecular Probes), a membrane permeable Ca2+ sensitive dye, and washed out with recording solution three times prior to imaging. These experiments were carried out in the dark to prevent photobleaching of the dye. Fluorescence imaging was performed concurrently with intracellular recordings. The corresponding fura-2 fluorescence signal was determined using alternating 340 and 380 nm excitation wavelengths and a 530 nm long-pass emission filter using a high-speed random monochromater (PTI, Ontario, Canada), controlled by Image Pro 5 software (PTI). The fluorescence was sampled at a frequency of 2 Hz. The fluorescence signal was detected and digitized by an intensified charge-coupled device camera. The intensities of the fluorescence signals detected at the 340 and 380 nm excitation wavelengths were processed to floating point images, and the fluorescence ratio (F340/F380) was analysed using Image Pro 5.

Statistics

All data are presented as the mean value ± s.e.m. Statistical analysis was carried out using SigmaStat 3.0 software (Jandel Scientific, Chicago, IL, USA). Differences between mean values from each experimental group were tested using a one-way analysis of variance (ANOVA) for multiple comparisons. Differences were considered significant if P < 0.05.

Results

SV accumulation at the presynaptic site in the mature synapse requires target cell contact

Following target cell contact in culture, a functional synapse forms between the somata of identified presynaptic and postsynaptic L. stagnalis neurons (Feng et al. 1997, 2002; Magoski & Bulloch, 1998; Hamakawa et al. 1999), and voltage-dependent Ca2+ signals accumulate at the presynaptic sites (Feng et al. 2002). The soma–soma synapse exhibits synaptic activity similar to in vivo activity previously observed in these neurons. Here we tested whether target cell contact induces SV clustering during synapse formation. We first demonstrated that functional synapses formed between somata of identified synaptic partners within 12 h of contact. Consistent with our previous findings of soma–soma synapse formation, we showed that electrical stimulation of a presynaptic neuron in somatic contact with its target cell (Fig. 2Aa) induced excitatory postsynaptic potentials (EPSPs) in its postsynaptic partner 12 h after plating (Feng et al. 2000) (Fig. 2Ab). Concurrent fura-2 imaging revealed Ca2+ accumulation in response to electrical stimulation (evoked Ca2+ hotspots) at the cell contact site of the presynaptic cell (Feng et al. 2002) (Fig. 2B). We then took advantage of the properties of syt I, an integral membrane protein of SVs, to determine the SV distribution pattern. Western blot analysis (Fig. 2C) showed that a single band migrating at 65 kDa was detected in both rat and snail protein samples indicating that l-syt I was labelled by the mouse anti-syt I antibody, consistent with our previous findings (Fei et al. 2007). To study the spatial distribution pattern of l-syt I in somata paired neurons, immunocytochemical staining was carried out using this anti-syt I antibody. Figure 2Da shows high fluorescence intensity for syt I at the cell contact site in the presynaptic neuron of the same cell pair shown in Fig. 2A and B, indicating localization of this SV protein at the functional synapse. Figure 2Db depicts the regions where the measurements were collected. In contrast to the highly localized presynaptic syt I signals, the syt I signal in cultured individual neurons (Fig. 2Ea) was evenly distributed in the subcytoplasmic membrane region of the cell soma (Fig. 2Eb). We performed negative controls using only secondary antibody to show that the background labelling of paired neurons (Fig. 2Fa) was low, and not a significant source of fluorescence (Fig. 2Fb). The mean amplitude fluorescence measured from somata paired cells connected by synapses and from individual cells are summarized in Fig. 2G. The fluorescence signal intensity measured at presynaptic sites (pre-contact, 123.08 ± 10.35 AU, n = 9) was significantly higher than that at other regions in the cell pair (pre-non-contact, 78.04 ± 6.43 AU; post contact, 46.68 ± 8.28 AU; post non-contact, 43.06 ± 6.04 AU; n = 9, P < 0.05), or in individual unpaired cells (57.04 ± 7.69 AU, n = 15, P < 0.05). Signal intensities in postsynaptic cells versus individual unpaired cells were not statistically different (P > 0.05). These observations indicate that (1) cell contact induces mechanisms that regulate syt I distribution, and (2) cell contact induces increased levels of syt I at presynaptic sites. These findings raise two specific questions: (1) When does up-regulation of syt I levels occur? (2) Are the changes in syt I levels and distribution specific to synaptic target cell contact?

To determine the time course of the syt I aggregation, we monitored the spatial distribution of the syt I signal at various times following synaptic cell pairing. As shown in Fig. 3Aa, the syt I signal in both presynaptic (VD4) and postsynaptic (LPeD1) cells was evenly distributed in the somata and neurite stump within the first hour of isolation (Fig. 3Aa). Analysis of syt I average signal intensity (Fig. 3Ab) was not significantly different among presynaptic, postsynaptic, paired or individual cells at the 1 h time point (pre-cell contact, 77.98 ± 7.2 AU; pre-non-contact, 74.05 ± 7.4 AU; post cell contact, 58.21 ± 5.3 AU; post non-contact, 57.17 ± 5.8 AU, n = 7; individual unpaired, 61.5 ± 2.85 AU, n = 5, P > 0.05). By 3 h after cell pairing, the syt I signal intensities detected in presynaptic cells increased relative to postsynaptic cells, as shown in Fig. 3Ba. As summarized in Fig. 3Bb, the signal intensities were significantly higher at the contact site (101.1 ± 8.3 AU, n = 6) in presynaptic cells than in other regions (Pre-non-contact, 88.1 ± 3.6 AU; Post contact, 55.09 ± 3.03 AU; Post non-contact, 60.37 ± 3.3 AU, n = 6); in individual cells maintained under identical conditions the signal intensities were significantly lower than those at the contact site in presynaptic cells (66.56 ± 9.23 AU, n = 5, P < 0.05). After pairing cells in culture, the syt I signal in the cells increased gradually with time. At 6 h after cell contact, syt I signal was highly localized to the cell contact site in presynaptic cells (Fig. 3Ca). As summarized in Fig. 3Cb, syt I signal intensities in presynaptic sites (pre-contact) increased to 119.71 ± 9.15 AU (n = 10), which is significantly higher than signal intensities in other regions of the paired cells (pre-non-contact, 80.52 ± 5.97 AU; post contact, 48.43 ± 5.2 AU; post non-contact, 49.73 ± 6.04 AU, n = 10), or in the individual cells (59.86 ± 9.23 AU, n = 7). These observations indicated that (1) target cell contact induced an up-regulation of syt I, (2) the increase in syt I signal intensity, which began about 3 h after contact, was specific to the presynaptic cell and (3) syt I signal was highly localized to the cell-contact site in presynaptic cells at 6 h after cell pairing. As syt I is an SV protein, these data indicated that SV preferential aggregation at cell contact versus non-cell contact sites in the presynaptic neuron was initiated by target cell contact.

Figure 3
Time-dependent alteration of syt I expression in presynaptic neurons following target cell contact

To determine whether syt I expression was a result of synaptic target cell contact, or alternatively was the result of non-specific cell membrane interactions, two neurons (RPeD1 and PeA) that do not form synaptic connections in vivo or in vitro (Spencer et al. 2000), were placed in a soma–soma configuration. The distribution pattern of syt I in the non-synaptic partner pairs, and individual non-paired neurons was determined at various time points following cell contact. We measured and compared the expression level of syt I at multiple sites in non-partner paired cells (in the same manner as above) from 3 to 12 h after cell contact. Representative images recorded at 3 h or 12 h after cell contact are shown in Fig. 4Aa and 4Ba, respectively. No syt I accumulation was observed at cell contact sites. Summaries of these imaging data are depicted in Fig. 4Ab and Bb. Figure 4Ab shows the average syt I levels in seven paired cells and seven non-paired individual cells after 3 h in culture (cell 1 contact, 61.17 ± 5.13 AU; cell 1 non-contact, 65.35 ± 5.49 AU; cell 2 contact, 54.99 ± 6.87 AU; cell 2 non-contact, 63.18 ± 7.03 AU; single, 66.87 ± 3.12 AU); differences among the groups were not statistically significant. Average syt I levels from nine paired cells and five non-paired individual cells after 6–12 h in culture varied from 47.49 ± 9.94 to 66.69 ± 3.19 AU with no significant difference in syt I levels between cell contact and non-contact sites of the paired cells, or among the individual unpaired cells (Fig. 4Bb). By contrast, pairing RPeD1 with its appropriate target cell induced presynaptic syt I accumulation (data not shown). Taken together, our observations indicate that the up-regulation of the presynaptic syt I requires contact with specific and appropriate target cells.

Figure 4
Lack of syt I clustering in non-synaptic cell pairs

C2A Ca2+-binding motif loop 3 is involved in target cell contact-induced syt I aggregation

Consistent with our previous results (Feng et al. 2002), we found that target cell contact resulted in the appearance of Ca2+ hotspots in presynaptic regions (Fig. 2), indicating the involvement of Ca2+ in synapse formation. Therefore, we hypothesized that syt I C2A and C2B Ca2+-binding motifs may be critical for SV aggregation upon target cell contact. To test this possibility, we produced TAT-conjugated peptides containing amino acid sequences specific to loop 3 of the C2 Ca2+-binding motifs. The HIV-1 TAT protein transduction domain readily translocates across cell membranes (Vives et al. 1997; Nagahara et al. 1998), and therefore should assist C2 peptide uptake into the cells under study. Also, an Alexa-350 fluorophore was conjugated to an N-terminal cysteine residue of these TAT–C2 peptides so that peptide entry into cells could be monitored by confocal imaging. After cells were plated in culture dishes, we added these membrane traversable peptides to the culture medium. With confocal imaging we showed that the peptides effectively entered cells within 10 min after application. Prior to imaging, cells were washed thoroughly to remove any extracellular peptide. We observed fluorescence signal throughout entire cells, and punctate labelling indicated that the peptide may have entered subcellular compartments or vesicles (Fig. 5B). Western blot analyses indicated that the C2 peptides did not bind directly to the anti-syt I antibody, nor inhibit syt I–anti-syt I antibody interactions (data not shown).

Figure 5
The HIV-1 TAT syt I peptides enter neurons

In order to minimize disruption of the folding and function of the Ca2+-binding motifs, and also to prevent potential fluorescence signal interference with anti-syt I antibody staining, non-fluorescent versions of these peptides were used to study the chronic effect of the peptides on presynaptic syt I localization in synaptic partner cells, VD4 (pre) and LPeD1 (post). TAT–C2 peptides (0.5 μm) were added to the culture medium 10 min after cell contact, allowing membrane contact between the paired cells. After 6 h treatment, paired intracellular recordings were made to test for synaptic connections. The cell pairs were then fixed and stained with anti-syt I antibody for confocal imaging of syt I signal. As shown in Fig. 6Aa, electrical stimulation failed to evoke postsynaptic responses in cell pairs treated with the C2A peptide indicating that the C2A domain may be required to form a functional synaptic connection. Immunocytochemical staining showed that syt I did not accumulate at the cell contact site between C2A-treated paired cells (Fig. 6Ab). The average fluorescence intensity at either the cell contact (46.3 ± 4.1 AU, n = 10) or the non-contact site (58.61 ± 3.8 AU, n = 10) in presynaptic cells was not statistically different from the average intensity observed in postsynaptic cells (contact, 41.02 ± 3.78 AU; non-contact, 52.40 ± 3.91 AU, n = 10) or in single non-paired cells (54.86 ± 11.06 AU, n = 4, P > 0.05). After treating cultured paired cells with the TAT–C2A peptide for 24 h, we observed neither presynaptic syt I aggregation nor functional synaptic connection in these cells (data not shown). To confirm that the C2A peptide, but not the TAT translocation sequence, disrupted synapse formation, these experiments were repeated using a control peptide conjugated to TAT. As shown in Fig. 6Ba, at 6 h after cell pairing, electrophysiological recordings indicated formation of functional synaptic connections and the presynaptic syt I signal was up-regulated and localized in the presence of the control peptide (Fig. 6Bb). The average syt I signal intensities for these experiments are summarized in Fig. 6Bc. The syt I signal intensity at presynaptic cell contact sites (123.32 ± 6.83 AU, n = 5) was significantly greater than in other regions of the same cell (pre-non-contact, 82.05 ± 6.16 AU), the paired postsynaptic cells (post contact, 56.9 ± 4.97 AU; post non-contact, 61.86 ± 4.99 AU, n = 5) or in individual non-paired cells (single, 57.98 ± 9.21 AU, n = 4, P < 0.05).

Figure 6
The TAT–C2A loop 3 peptide specifically blocks clustering of syt I and prevents formation of functional synapses

To evaluate the importance of the Ca2+-binding residues in the C2A peptide, we produced a mutant syt I C2A loop 3 peptide (mC2A) in which the Ca2+-binding aspartic acid residues were replaced by asparagines, which do not bind Ca2+ (Fernandez-Chacon et al. 2002). Synaptic partner cells were paired in the presence of TAT–mC2A peptide for 6 h. Electrophysiological recordings indicated that synaptic connections formed between paired cells in the presence of this peptide (n = 6), and immunocytochemical staining revealed that syt I expression increased at cell-contact sites in presynaptic cells (Fig. 6Cb). The average syt I level was significantly higher at presynaptic contact sites (contact, 100.47 ± 4.83 AU, n = 8, P < 0.05) than in other regions of the same cell (pre-non-contact, 76.17 ± 6.28 AU), in paired postsynaptic cells (post contact, 68.08 ± 6.15 AU; post non-contact, 58.29 ± 4.64 AU; n = 8) or in individual non-paired cells (single, 54.83 ± 2.34 AU, n = 4, P < 0.05; Fig. 6C). These data indicate that the C2A Ca2+-binding residues, which may only be partially responsible for syt I accumulation, are required for SV clustering and functional synapse formation.

We next tested the C2B peptide for effects on SV clustering and functional synapse formation. As shown in Fig. 7Aa, electrophysiological recordings revealed synaptic connections between the paired cells and demonstrated that a functional synapse formed in the presence of C2B peptide. The syt I immunofluorescence signal (Fig. 7Ab) increased in presynaptic cells after 6 h of C2B peptide treatment, as seen in control pairs (Fig. 3C). As summarized in Fig. 7Ac, the syt I signals in presynaptic regions (contact, 104.99 ± 11.39 AU; non-contact, 84.77 ± 6.19 AU, n = 10) were significantly greater than in the postsynaptic regions (contact, 48.86 ± 4.99 AU; non-contact, 38.60 ± 3.56 AU, n = 10) or in single cells (53.78 ± 4.58 AU, n = 4, P < 0.05). Syt I expression in postsynaptic cells appeared lower than that of single cells; however, the difference between these groups was not statistically significant. These results indicate that, unlike syt C2A loop 3, the C2B Ca2+-binding motif loop 3 may not be involved in target cell-induced clustering of syt I.

Figure 7
TAT–C2B Ca2+-binding motif peptide did not affect syt I clustering during synapse formation, but inhibited synaptic transmission in mature synapses

Several studies have shown that syt I C2 regions are critical for synaptic transmission (Fukuda et al. 1995; Desai et al. 2000; Mackler et al. 2002). We investigated whether the C2 peptides affected synaptic transmission in mature synapses. As shown in Fig. 7B, acute application of the C2B peptide (0.5 μm) to synapse pairs with fully formed and functional synaptic connections, resulted in a time-dependent inhibition of postsynaptic potentials without altering the electrical activity of the presynaptic cell (Fig. 7Ba). EPSP amplitude measured from four independent synapses showed an average 77% decrease 25 min after C2B peptide application (Fig. 7Bb). In comparison, representative current recordings (Fig. 7Ca) showed that acute application of the C2A peptide (0.5 μm) had no effect on synaptic transmission between mature synapsed neurons. Figure 7Cb shows the mean data obtained from four independent synapses.

Taken together, these data demonstrate that the C2B peptide effectively inhibits synaptic transmission at functional synapses and is consistent with previous reports (Fukuda et al. 1995; Desai et al. 2000; Mackler et al. 2002). However, this peptide did not affect the process of target cell-induced SV aggregation during earlier stages of synapse formation. By contrast, the C2A peptide specifically exerted an inhibitory effect on the clustering of syt I, and inhibited synapse formation.

The inhibition of syt I clustering or neurotransmission by the TAT–C2 peptides might result from peptide sequestration of free Ca2+. To test this notion, fura-2 ratiometric Ca2+ imaging was used to detect the basal and evoked Ca2+ concentrations in single isolated RPeD1 cells before and after application of the peptide (0.5 μm). Figure 8A shows a representative example of fura-2 imaging and electrophysiological stimuli employed during these experiments. A 3 Hz (5 s) action potential train stimuli elevated intracellular Ca2+ concetration in the cell soma; Ca2+ levels returned to basal levels upon recovery. When cells were treated with TAT–C2A peptide, no significant change in either basal or evoked maximal (Fig. 8B) intracellular Ca2+ levels was detected relative to controls (n = 4). Similarly, the C2B peptide did not alter either basal or evoked Ca2+ signals (data not shown).

Figure 8
TAT–C2A peptide did not alter Ca2+ levels in individual neurons

Discussion

In this study, we have demonstrated that SV aggregation at presynaptic contact sites is induced by target cells at soma–soma synapses between identified adult L. stagnalis neurons. The spatiotemporal distribution of the SV protein syt I was largely dependent upon the identity of the contacted cell. We found a characteristic global increase in the level of syt I in presynaptic cells following cell contact; the aggregation of syt I at the presynaptic site occurred ~6 h after cell pairing. This process occurred in a target cell-specific manner. Loop 3 within the calcium-binding region of C2A in syt I appeared to be necessary for these target cell-dependent events. These results provide a novel insight into the molecular mechanisms necessary for SV aggregation during synapse formation between adult neurons.

In neonatal mouse hippocampal neurons in culture, synapses begin to form at day 3–8 coincident with a distinguishing punctuate SV/syt I labelling at contact sites between neuritic processes (Kraszewski et al. 1995; Zhang et al. 2002). However, these SV hotspots are also seen in the neurites lacking cell contacts; thus, the specificity of such punctate hotspots to synapses remains. Any correlation between morphological characterization and function of such synapses has not been directly tested in these preparations because of the technical difficulty of resolving such small contact points between neurites, and the unknown identity of the neurons involved. In our study, we showed that the target cell not only modulates the expression, but also directs the localization of SV/syt I during development of synaptic connections between identified synaptic partner cells. It is interesting that this did not occur between inappropriate partners, demonstrating that target cell contact is sufficient to induce increased presynaptic syt I expression and discrete localization of the protein in adult neurons.

We have also shown that individual unpaired neurons express syt I levels similar to those seen in postsynaptic neurons. The significant global increase in syt I expression in presynaptic cells was an early process, occurring prior to visible clustering of syt I at presynaptic sites (Figs 2 and and3).3). These findings are consistent with previous reports of increased expression of SV proteins in central (Lou & Bixby, 1993) and motor neurons (Campagna et al. 1997) during development. To broaden these findings, we demonstrated that the increased expression of syt I is specifically induced by synaptic target cells. However, although Syt I mRNA expression in motor neurons is modulated by contact with the target cells (Campagna et al. 1997), the rate of syt I protein synthesis in hippocampal neuronal cultures is relatively stable during development (Daly & Ziff, 1997), suggesting that the increase in syt I protein levels may be a consequence of an increased half-life of the protein possibly regulated by a post-transcriptional mechanism (Daly & Ziff, 1997). The specific molecular mechanisms underlying the target cell contact-induced increase in syt I levels remain unclear.

Synthetic peptides have been very useful tools in attempting to identify specific domains responsible for protein functions (Antz et al. 1997; Tsujimoto et al. 2002; Morgan et al. 2003). In previous studies, injections of either synthetic peptide fragments (Bommert et al. 1993; Desai et al. 2000) or specific antibodies (Fukuda et al. 1995; Mikoshiba et al. 1995) have shown that the Ca2+-binding motifs within the tandem C2 domains of syt I are critical for neurotransmitter release. Whether these Ca2+-binding motifs are involved in the localization of SVs has not been directly tested. Because knock out of the syt I gene did not reveal a dramatic alteration in SV function in either mouse (Geppert et al. 1994; Nishiki & Augustine, 2001) or Drosophila neurons (Yoshihara & Littleton, 2002), the involvement of syt I in synapse formation seems questionable. However, multiple syt isoforms participate in synaptic transmission at presynaptic sites in mice (Geppert et al. 1991; Ullrich et al. 1994; Sugita et al. 2001). Syt II is attached directly to SVs, as is syt I (Geppert et al. 1991; Ullrich et al. 1994), and syt III and VII (Sugita et al. 2001) are located at the presynaptic plasma membrane. Thus, several synaptotagmins may function as complementary Ca2+ sensors on opposing SV and presynaptic plasma membranes during fusion (see Sudhof, 2002). It is possible that the lack of an apparent SV function for syt I in the knockout models is attributable, at least in part, to the compensatory effect of other syt isoforms. Currently, only one syt isoform (syt I) involved in synaptic transmission has been identified in Drosophila. However, the role of syt in synaptic function is complex, and the involvement of other potential syt isoforms in Drosophila remains a possibility. Our current understanding of the consequence of syt I deletion on the presynaptic locus may also be incomplete. In contrast to results obtained from syt I knockout models, when we used exogenous peptides to perturb the function of the endogenous syt I protein, we observed profound effects on synaptogenesis. It may not be surprising that the peptides have a different effect than the syt I deletion on synapse formation because these peptides probably act as dominant negative effectors; dominant negative mutations and effectors often have phenotypes that differ from those of deletion mutants.

In this study, we used non-invasive means to deliver peptides into neurons. The use of the HIV-1 TAT protein transduction domain is a well-established method for delivering peptides into cells; however, this method is not always successful (Vives et al. 1997; Green et al. 2003). In our case, the TAT-conjugated peptides rapidly entered the cells (Fig. 5) and efficiently induced peptide-specific responses (Figs 6 and and7).7). Using circular dichroism spectroscopy analysis, we recently showed that conjugation to TAT did not alter the secondary structure of a peptide inhibitor of neuronal Ca2+ sensor 1 (Hui et al. 2005). Therefore, we anticipate that the conformation of the C2 loop 3 peptide is similar to the homologous region in the full-length syt I protein. We found that the peptide specific to the loop 3 C2A Ca2+-binding site, but not the mutant C2A, the control TAT peptide or the wild-type C2B peptide, prevented syt I aggregation or formation of functional synapses, indicating that the Ca2+-binding region of the syt I C2A domain is essential for presynaptic syt I clustering. Our findings that the C2B peptide blocks synaptic transmission in mature synapses (Fig. 7B) indicates that the peptide is functional. Therefore, failure of the peptide to block synapse formation after 6 h incubation could indicate that the newly formed synapse is resistant to the peptide or that the concentration of the peptide is below the effective concentration for interrupting transmission. In contrast, we failed to record synaptic transmission in cell pairs incubated with the C2A loop 3 peptide for 6 h, indicating that under the same conditions, the C2A loop 3 peptide plays an important role in synapse formation and synaptic transmission compared to the C2B loop 3 peptide.

Consistent with results obtained in other studies, we previously reported that target cell contact induces increased evoked intracellular Ca2+ concentrations at presynaptic sites (Dai & Peng, 1993; Zoran et al. 1993; Feng et al. 2002). This raises the possibility that the TAT–C2A loop 3 peptide interferes with syt I clustering as a result of interference with Ca2+-dependent mechanisms; perhaps the C2A loop 3 peptide competes with endogenous syt I for Ca2+ binding, and thus prevents Ca2+-induced conformational changes in syt I that are required for localization. However, we found that the peptide had only minor effects on basal or evoked Ca2+ levels, which is not consistent with the notion that the C2A peptide inhibits syt I clustering by competing for free cytoplasmic Ca2+. The C2A loop 3 peptide-induced inhibition of syt I clustering probably arises from a Ca2+ binding-dependent competition for interactions with effector molecules because the mC2A loop 3 peptide, which lacks functional Ca2+-binding residues, failed to interfere with syt I clustering or synapse formation. Both loops 1 and 3 in the C2A domain facilitate the coordination of Ca2+ ions. Loop 1 (partially) and loop 3 (fully) interact with Ca2+. Loop 3 of the C2A domain fully stabilizes a single Ca2+ ion and almost fully coordinates the binding of a second Ca2+ ion (Ubach et al. 1998). The TAT–C2A loop 3 peptide is probably capable of stabilizing and coordinating Ca2+ ions to form a structure that is very similar to the native loop in syt I (Desai et al. 2000). Thus, Ca2+-bound peptide could interact with possible effectors of loop 3 that are necessary for targeting syt I to the presynaptic site. Failure of the TAT–C2B loop 3 peptide to inhibit clustering of syt I suggests that the C2B loop 3 region does not play an important role in target cell-induced syt I clustering.

Although SV accumulation at the presynaptic contact site during development is dependent on a vesicle recycling mechanism (see Ziv & Garner, 2004), it is not known how SVs get to the presynaptic site during early stages of synapse formation. Our results demonstrate that syt I clustering is directly correlated with formation of synaptic connections between adult synaptic partner cells and that the initial aggregation of SVs requires target cell contact. The clustering of syt I to the target site is necessary for synapse formation, and is also required for asymmetric vesicle recycling. The C2A loop 3 domain of syt I may be crucial in trafficking and/or clustering SVs for immature synapse formation following initial cell contact, and syt I may be intimately involved in facilitating the formation of functional synapses prior to the onset of vesicle recycling.

Acknowledgments

This work was supported by an operating grant to Z-P.F. from the Canadian Institutes of Health Research (CIHR, MOP 62738), start up funds from the Department of Physiology, the Faculty of Medicine and the University of Toronto, and equipment grants from the Canada Foundation for Innovation (CFI) and the Ontario Innovation Trust (OIT). Z-P.F. holds a New Investigator Award from the CIHR, and a Premier's Research Excellence Award (PREA) of Ontario. P.G. and D.W.K.L. are recipients of Ontario Student Opportunity Trust Funds (OSOTF) Crothers fellowship awards. G.J.H. received an NSERC summer studentship award. We thank Jiang Su for her technical assistance. We thank Drs H.L. Atwood, M. Charlton and J.F. MacDonald for critical reading of an early version of the manuscript.

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