Localization of synaptic proteins involved in ... - Wiley Online Library

Journal of Neurochemistry, 2007, 100, 664–677

doi:10.1111/j.1471-4159.2006.04225.x

Localization of synaptic proteins involved in neurosecretion in different membrane microdomains Elena Taverna,* Elena Saba,* Anna Linetti,* Renato Longhi, Andreas Jeromin,à Marco Righi,* Francesco Clementi* and Patrizia Rosa* *CNR Institute of Neuroscience, Department of Medical Pharmacology, University of Milan, Milan, Italy CNR Institute of Chemistry and Molecular Recognition, Milan, Italy àCenter for Learning and Memory, University of Texas, Austin, Texas, USA

Abstract A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Cav2.1 (P/Qtype) channels and soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergentresistant membranes (DRMs) containing proteins of the exocytic machinery (Cav2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In

Amongst many other proteins, neurotransmitter secretion is mediated by voltage-gated calcium channels (VGCCs) localized in pre-synaptic membranes (Catterall 2000, and references cited therein). It is known that the pore-forming subunits (a1) of N-type (Cav2.2) and P/Q-type (Cav2.1) VGCCs at nerve terminals interact with components of the soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor (SNARE) family (Jahn et al. 2003, and references cited therein), such as syntaxin1, 25-kDa synaptosome-associated protein (SNAP-25) and the calcium sensor protein synaptotagmin1 (Catterall 1999; Atlas 2001). Disrupting this interaction by injecting inhibitor peptides in the nerve terminals reduces the efficiency of Ca2+ entry during exocytosis, thus suggesting that the co-localization of VGCCs with proteins of the exocytic machinery is crucial in

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line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-b-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery. Keywords: exocytosis, membrane microdomains, neuronal calcium sensor-1, soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors, voltage-dependent calcium channels. J. Neurochem. (2007) 100, 664–677.

Received June 29, 2006; revised manuscript received August 29, 2006; accepted September 7, 2006. Address correspondence and reprint requests to Patrizia Rosa, CNR Institute of Neuroscience, Department of Medical Pharmacology, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy. E-mail: [email protected] Abbreviations used: DRM, detergent-resistant membrane; GPCR, G protein-coupled receptor; HDRM, high-density DRM; LDRM, lowdensity DRM; LP1, lysate pellet 1; LP2, pellet from the LS1 fraction; LS1, lysate supernatant 1; LS2, lysate supernatant 2; MbCD, methyl-bcyclodextrin; NCS-1, neuronal calcium sensor-1; P2, synaptosome; PIP2, phosphatidylinositol-4,5-bisphosphate; NGF, nerve growth factor; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SNAP-25, 25-kDa synaptosome-associated protein; SNARE, soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor; VAMP-2, vesicle-associated membrane protein-2; VGCC, voltage-gated calcium channel.

2006 The Authors Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 664–677

Neurosecretion and membrane microdomains

optimizing stimulus–secretion coupling (Sheng et al. 1998). Furthermore, the binding of SNARE proteins to VGCCs also modulates the channels’ gating properties (Catterall 1999; Atlas 2001). In addition to SNAREs, VGCC activity is modulated by a number of intracellular messenger pathways. Several lines of evidence have demonstrated that the activation of G proteincoupled receptors (GPCRs) affects channel activity via different mechanisms that imply the direct binding of G protein bc subunits to specific domains of the cytoplasmic loop (loop I–II) and the C-terminal region of the a1 subunit of Cav2 channels, or the activation of phosphokinases (phosphokinase C, A and even src) followed by phosphorylation of the a1 subunits (Strock and Diverse-Pierluissi 2004; De Waard et al. 2005; and references cited therein). Recent data have demonstrated that activated GPCRs may also affect calcium channels by modulating phosphatidylinositol-4,5bisphosphate (PIP2), a lipid that is known to interact with the channels and sustain their activity (Wu et al. 2002). The activation of GPCRs, followed by PIP2 hydrolysis, therefore inhibits calcium channels, but some GPCRs do not inhibit, but rather sustain, Cav2 activity via a mechanism that involves neuronal calcium sensor-1 (NCS-1). This myristoylated, calcium-binding protein (frequenin in Drosophila m.) is widely expressed in neurons and neuroendocrine cells, and plays an important role in neurotransmitter release in various ways (Burgoyne and Weiss 2001; Hilfiker 2003; Burgoyne et al. 2004; Jeromin et al. 2006; and references cited therein). It has been shown that NCS-1 increases the levels of polyphosphoinositides in PC12 cells by modulating the membrane recruitment and activity of phosphatidylinositol 4-OH kinaseb (Koizumi et al. 2002; Taverna et al. 2002; de Barry et al. 2006). More recent data have indicated that it may also directly bind PIP2 with high affinity in vitro and in living cells (O’Callaghan et al. 2005). At the pre-synaptic nerve terminals, it can mediate the activity-dependent facilitation of P/Q-type calcium currents, thus mediating activity-dependent synaptic facilitation (Tsujimoto et al. 2002). However, it is still unclear whether NCS-1 acts on neuronal calcium channels directly and/or indirectly (Weiss and Burgoyne 2001; Tsujimoto et al. 2002). The finding that different signalling molecules can modulate Cav2 and, ultimately, exocytosis through different pathways suggests that the characterization of their precise spatial distribution may be important in improving our understanding of the organization of the protein network involved in neurotransmitter release. Recent studies have demonstrated that SNARE proteins, synaptotagmin1 and Cav2.1 are localized in cholesterol-enriched microdomains. Altering the organization of these domains by means of methyl-b-cyclodextrin (MbCD) treatment impairs regulated exocytosis and also affects the co-localization of SNAREs and Cav2.1 (Chamberlain et al. 2001; Lang et al. 2001; Taverna et al. 2004). This suggests that these molecules play

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a role in defining the ‘sites’ for synaptic vesicle release, and have a function in organizing the molecular complexes involved in neurosecretion (Rohrbough and Broadie 2005; Salaun et al. 2004). In order to gain further insights into the organization of these exocytic ‘sites’, we investigated whether heterotrimeric G proteins co-localize with SNAREs and Cav2.1 channels in membrane microdomains, and whether NCS-1 can also be recruited to microdomains with proteins of the exocytic machinery. To this end, we combined the biochemical analysis of detergent-resistant membranes (DRMs) isolated from synaptic membranes with confocal imaging of the lipid microdomains on the inner leaflet of the plasma membrane of neuronal-like phaeochromocytoma (PC12) cells. The data indicate the following: (i) the proteins of the exocytic machinery (SNAREs and Cav2.1) are segregated in lipid microdomains other than those containing NCS-1; (ii) both microdomain subpopulations contain aliquots of Ga subunits and the modulatory molecule PIP2; and (iii) the observed recruitment of NCS-1 to membrane microdomains is significantly enhanced by bradykinin stimulation.

Materials and methods Materials and antibodies The ultra-pure Triton X-100 solution (Surfact-Amps X-100) was obtained from Pierce (Rockford, IL, USA), and cholesterol, MbCD, saponin, mouse IgG, rabbit IgG, horseradish peroxidase-coupled cholera toxin subunit B and protease inhibitor cocktail were purchased from Sigma-Aldrich (Milan, Italy). The nerve growth factor (NGF 7S) was obtained from Alomone Laboratories Ltd., Jerusalem, Israel. The sources of the antibodies were as follows: antibodies against syntaxin1, SNAP-25, synaptobrevin/vesicle-associated membrane protein-2 (VAMP-2), pan-cadherin, b-catenin, actin, tubulin and anti-rabbit and anti-mouse IgG or IgM conjugated to peroxidase from Sigma-Aldrich; the anti-transferrin receptor from Zymed Laboratories (San Francisco, CA, USA); the monoclonal antibodies against synaptobrevin/VAMP-2, SNAP-25 and neuroligin from Synaptic Systems (Goettingen, Germany); the anti-bassoon antibody from Stressgen Bioreagents (Victoria, BC, Canada); the anti-flotillin antibody from BD Biosciences (Erembodegem, Belgium); the anti-Gaq, -Gao, -Gai and -Gb antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the anti-PIP2 antibody mouse IgG2b from Assay Designs (Ann Arbor, MI, USA); and the anti-PIP2 antibody mouse IgM (clone 2C11) from Molecular Probes Europe (Leiden, Netherlands). The antibodies against Munc-18-1 and NCS-1 were raised in rabbits and characterized as described previously (Rowe et al. 2001; Taverna et al. 2002). The antibodies against synaptotagmin1 and ribophorin were kind gifts of A. Malgaroli (DIBIT, Milan, Italy) and G. Kreibich (New York University, School of Medicine, New York, USA), respectively. The sources of the secondary antibodies were as follows: the anti-mouse and anti-rabbit IgG conjugated to fluorescein, rhodamine or Cy5 from Jackson Immuno Research Laboratories (West Growe, PA, USA), and the Alexa Fluor 488 goat anti-mouse IgG from Molecular Probes Europe.


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Antibodies against a1A The polyclonal anti-sera against the pore-forming subunit of rat Cav2.1 (Swissprot accession P54282) were raised in rabbits using synthetic peptides corresponding to amino acids PSSPERAPGREGPYGRE(Cys) (a1A865)881) and SEPQQREHAPPREHV(Cys) (a1A882)896) coupled to keyhole-limpet haemocyanin. The antibodies were affinity purified and then tested by western blotting. As shown in Fig. S1 (see ‘Supplementary material’), the antibodies from both sera recognized two prominent bands of about 160–190 kDa and, after longer exposure, additional bands of 205–220 kDa; these polypeptides correspond to isoforms of the a1A subunits in rat brain, as demonstrated previously by Sakurai et al. (1996). Bands of about 95 kDa were detected by anti-a1A865)881, and a polypeptide of similar molecular weight has been identified previously in rat brain and described as a Cav2.1 hemichannel (Scott et al. 1998). Immunolabelling of all of the detected bands was completely abolished when the antibodies were pre-absorbed with the peptides used for immunization; furthermore, no bands were detected when rat liver membrane extracts were immunostained (Fig. S1). In the following experiments, we preferentially used the anti-a1A882)896 antibody. Rat brain subcellular fractionation and synaptosome stimulation The rat brain specimens were fractionated by differential centrifugation, essentially as described previously (Taverna et al. 2002, and references cited therein). Briefly, the post-nuclear supernatants (S1) were centrifuged at 9200 g for 15 min to yield a pellet corresponding to the synaptosomes (P2). This pellet was then subjected to hypo-osmotic lysis (30 min in 7.5 mM HEPES–NaOH, pH 7.4), followed by centrifugation for 20 min at 25 000 g to yield the so-called lysate pellet 1 (LP1), containing large membranebound vesicles, and lysate supernatant 1 (LS1), which was further centrifuged at 165 000 g for 2 h to obtain a second pellet (LP2) enriched in synaptic vesicles. The protein concentrations in the samples were determined using the Bio-Rad Protein Assay (BioRad Laboratories s.r.l., Segrate, Italy). For the stimulation experiments, 6000 lg of P2 was washed in oxygenated Krebs–Ringer buffer, centrifuged and resuspended in 3 mL of Krebs–Ringer buffer. After incubation for 3 min at 37C in the presence or absence of 5 lM bradykinin (Sigma Aldrich), the P2 samples were centrifuged at 10 000 g for 15 min at 4C, and then resuspended in 750 lL ice-cold buffer A (150 mM NaCl, 2 mM EGTA, 50 mM Tris-HCl, pH 7.5, Sigma-Aldrich protease inhibitor cocktail) for Triton X-100 solubilization and gradient analysis. Triton X-100 solubilization, cholesterol depletion and sucrose flotation gradients The LP1 or P2 samples (6000 lg of proteins) were adjusted in buffer A (750 lL) supplemented with 1% (w/v) Triton X-100, and incubated for 30 min on ice. In some experiments, before Triton X-100 solubilization, cholesterol was removed from the LP1 membranes by treatment with 1% saponin or 30 mM MbCD, as described previously (Taverna et al. 2004, and references cited therein). After the detergent treatments, each sample was adjusted to 1.2 M sucrose, placed in a centrifuge tube and overlaid with a linear gradient ranging from 0.30 to 0.14 M sucrose (all prepared in buffer A). The gradients were centrifuged at 190 000 g for 19 h

using an SW 41 Ti rotor (Beckman Instruments, Inc., Fullertone, CA, USA). Fifteen fractions (800 lL each), and the pellets suspended in 800 lL of buffer A, were collected and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The sucrose concentration in each fraction was determined by refractometry, and the results were expressed as the mean values of six gradients from three different experiments. Western blotting and dot blots Aliquots of 20–30 lL were subjected to SDS-PAGE on 6%, 10% or 12% polyacrylamide gels, and were then blotted onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Immunolabelling was carried out as described previously (Taverna et al. 2004). For ganglioside GM1 Tris-buffered saline solution or PIP2 detection, aliquots of 0.2–1 lL of each gradient fraction or immunoprecipitate were spotted directly onto nitrocellulose membranes, which were then probed with peroxidase-coupled cholera toxin subunit B or anti-PIP2 antibodies diluted 1 : 3000 or 1 : 5000 in Tris-buffered saline solution (TBS, 20 mM Tris-Hcl, pH 7.4, 150 mM NaCl) containing 5% milk and 0.3% Tween 20. The primary antibodies were detected using anti-rabbit IgG and antimouse IgG or IgM conjugated to peroxidase (diluted 1 : 50 000). The peroxidase was detected using a chemiluminescent substrate (Pierce). DRM immuno-isolation DRMs and their associated proteins were isolated from the sucrose gradient fractions by immunoprecipitation, as described previously (Taverna et al. 2004). All of the procedures were carried out on ice or at 4C. Aliquots of fractions 6 and 8 were adjusted to 0.3 M sucrose using buffer A without detergent, and incubated with the primary or non-immune Ig pre-bound to protein A or G sepharose beads (Amersham-Pharmacia, Milan, Italy). After overnight incubation at 4C with gentle mixing, the samples were centrifuged at 180 g for 3 min, and the beads were washed three times with buffer A containing 0.3% Tween 20. The immuno-isolated complexes and the aliquots of the samples used for the immuno-isolation (inputs) were analysed by western or dot blotting. Immunoprecipitation The P2 samples were extracted for 1 h at 4C in ice-cold immunoprecipitation buffer [150 mM NaCl, 2 mM EGTA, 0.5% (w/v) Triton X-100, 0.5% (w/v) saponin, 50 mM Tris-HCl, pH 7.5, and protease inhibitor cocktail], and then centrifuged at 20 000 g for 30 min. Aliquots of the supernatants (200 lg of proteins) were incubated for 1 h with protein A or G sepharose beads. The beads were then removed by centrifugation, and the ‘pre-cleared’ supernatants were added to protein A or G beads that had been pre-incubated with the primary antibodies or non-immune IgG for 2 h at 4C. After extensive washes with immunoprecipitation buffer, the immunocomplexes were analysed by western blotting. Cell culture and plasma membrane sheet preparation The PC12 cells (clone 251) were maintained in culture and, when required, transfected with 5–10 lg of pcDNA3-NCS-1 by electroporation, as described previously (Koizumi et al. 2002; Taverna et al. 2004). In order to prepare the membrane sheets, the PC12 cells



were grown for 36 h on poly-L-lysine-coated coverslips, and disrupted, as described previously (Lang et al. 2001), using ultrasound treatment in ice-cold sonication buffer (20 mM HEPES, pH 7.2, 120 mM potassium glutamate, 20 mM potassium acetate, 2 mM Mg-ATP and 0.5 mM dithiothreitol). Formaldehyde fixed or unfixed membrane sheets were incubated with the primary antibodies for 90 min at room temperature. The unfixed sheets were then washed, incubated for 30 min in 4% formaldehyde, followed by 30 min in 0.1 M glycine–Tris buffer, pH 7.3, and the primary antibodies were revealed by incubation with the appropriate conjugated secondary antibodies. In order to investigate protein localization in cholesterol-dependent microdomains, unfixed membrane sheets were incubated for 10 min on ice with 0.3% Triton X-100 as described previously (Hering et al. 2003), or treated for 10 min at 37C with 10 mM MbCD (both in sonication buffer). The samples were fixed and then immunostained as described above. After immunolabelling, the coverslips were mounted in VectaShield (Vector Laboratory, Burlingate, CA, USA) and images were collected using an MRC-1024 laser scanning microscope (BioRad) with · 60 objective lenses. To compare the double-stained patterns, the images from the fluorescein, rhodamine and Cy5 channels were acquired separately from the same areas, and superimposed. The images were processed using Photoshop (Adobe Systems, Mountain View, CA, USA). In order to verify the integrity of the membrane sheets after sonication and MbCD treatment, they were incubated for 2 min with 20 lM FM1-43, and the labelled samples were imaged before and after 10 min of treatment with 10 mM MbCD using a Zeiss Axiovert 200 fluorescence microscope (Ziess S. P. A., Arese, Italy) equipped with temperature and CO2 controllers (set at 37C and 0.2%, respectively), and a Roper Micromax CCD 512 · 512 camera (Crisel Instruments s.r.l., Rome, Italy) controlled by the MetaMorph program. Quantitative analyses Autoradiograms from the NCS-1, Gaq, syntaxin1 and SNAP-25 immunoblottings were used for quantitative analyses. In order to obtain linear signals and prevent rapid saturation occurring in the high-density fractions, 30 lL of fractions 4–12 and 6 lL of fractions 13–15 and pellets were loaded in the gels. Unsaturated autoradiograms were acquired using an ARCUS II scanner (AgfaGevaert, Mortsel, Germany), and the density of each band was quantified using NIH Image J (National Technical Information Service, Springfield, VA, USA). The signals in each fraction were then expressed as percentages of the total, taking into account the 1 : 5 dilution of fractions 13–15 and the pellets. The results were expressed as mean values ± SEM, and the significance of the differences was assessed using a two-tailed, non-paired Student’s t-test. In order to assess molecular co-localization in isolated DRMs, we analysed sequentially acquired confocal images stained for SNAP-25 and syntaxin1, or SNAP-25 and NCS-1. The images were processed using the Image J program equipped with a public domain co-localization plug-in (P. Bourdoncle, Institut Jacques Monod, Service Imagerie, Paris, France). After adjusting the signal thresholds to exclude a specific background value, positive co-localization was scored when the intensity ratio of the overlapping signals was greater than 0.75. The results were expressed as the percentage of co-localization between SNAP-25 and either syntaxin1 or NCS-1. The pooled results were expressed as mean values ± SEM.

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Results

NCS-1 and proteins of the exocytic complex are enriched in different DRMs In order to characterize the lipid microdomains of synaptic plasma membranes, rat brain cortices were fractionated using a well-established procedure (Taverna et al. 2002, and references cited therein) that made it possible to obtain fractions enriched in synaptosomes (P2), plasma membrane (LP1), synaptic vesicles (LP2) and synaptic cytosolic proteins (LS2) (Fig. S2, see ‘Supplementary material’). As expected, LP1 contained large amounts of syntaxin1 and SNAP-25, whereas LP2 was enriched in the synaptic vesicle proteins synaptobrevin and synaptophysin (Fig. S2). Proteins known to be concentrated in the synaptic plasma membrane (e.g. cadherin/b-catenin complex, neuroligin) (Song et al. 1999; Bamji 2005) or at the active zone (e.g. bassoon) (Garner et al. 2000; Dresbach et al. 2003; Takao-Rikitsu et al. 2004) were detected in P2 and LP1, but not in LP2. P2 and LP1 also contained significant amounts of heterotrimeric G proteins, and a small but consistent amount of NCS-1 (Fig. S2). DRMs (London and Brown 2000; Pike 2004; and references cited therein) were then isolated from LP1 (Fig. 1) and P2 (Fig. S3, see ‘Supplementary material’) on the basis of their insolubility in weak detergent at 4C, and their ability to float in a density gradient. In order to obtain the efficient resolution of differently buoyant DRMs, we used a linear gradient ranging from 0.3 to 0.14 M sucrose. After centrifugation, two distinct bands were observed in the lowdensity gradient region: one at 0.47 M sucrose (mainly collected in fraction 6, and hereafter referred to as ‘lowdensity DRM’ or LDRM) and a second mainly localized at 0.6 M sucrose (collected in fraction 8, and referred to as ‘high-density DRM’ or HDRM). A total of 15 fractions and pellets was collected, and protein assays confirmed the presence of two protein peaks (fractions 6 and 8) in the lowdensity gradient region (Fig. 1). Analysis of the gradient fractions showed that the lipid microdomain markers, GM1 and flotillin, were mainly localized in fractions 6–10 (sucrose concentrations: 0.47– 0.74 M), and enriched in fractions 8 and 9 (Figs 1 and S3). In contrast, transferrin receptor (a membrane protein excluded from the lipid microdomains) was only detected in the veryhigh-density fractions 13–15 (Fig. 1). Ribophorin, a membrane protein of the endoplasmic reticulum that is also detected in LP1 (Fig. S2), was also found in the very-highdensity fractions, in line with the very low levels of cholesterol found in the endoplasmic reticulum (Fig. 1). When the distribution of the exocytic machinery proteins was investigated, it was found that significant amounts of SNAP-25, syntaxin1 (and its interacting protein Munc-18), synaptobrevin2 and synaptotagmin1 floated in the gradient,



Fig. 1 Proteins associated with detergent-resistant membranes (DRMs) isolated from synaptic membranes. The lysate pellet 1 (LP1) fractions underwent Triton extraction on ice, and were then centrifuged in sucrose gradients to isolate the DRMs. Fifteen gradient fractions and pellets were collected and analysed for their protein concentrations (top left panel). As fractions 1–3 did not contain any proteins, the figure only shows the analyses of fractions 4–15 and the pellets (P). Equal volumes (20 lL) of each fraction were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then analysed by western blotting with antibodies against flotillin (1 : 1000), transferrin receptor (TrfR, 1 : 1000), ribophorin (Riboph, 1 : 400), synaptotagmin1 (Syt, 1 : 5000), Munc-18 (1 : 1000), syntaxin1 (Syn1, 1 : 10 000), 25-kDa synaptosome-associated protein (SNAP-25, 1 : 5000), vesicle-associated membrane protein-2 (VAMP, 1 : 10 000), a1A882)896 (a1A, 1 lg/mL), cadherin (Cadh, 1 : 10 000), b-catenin (b-Cat, 1 : 2000), bassoon (Bass, 1 : 3000), Gao (1 : 3000), Gai (1 : 1000), Gaq (1 : 5000), Gb (1 : 4000) or neuronal calcium sensor-1 (NCS-1, 1 : 1000). The distribution of the ganglioside GM1 marker was determined by dot blots using peroxidase-conjugated cholera toxin (1 : 100 000). The sucrose concentrations in the fractions (mean of four gradients) are indicated in the top right panel. The arrows and arrowheads indicate the positions of the low-density and high-density DRMs, respectively. The blots are representative of at least six separate experiments.

and peaked in the HDRMs (fraction 8; Figs 1 and S3). Furthermore, the pore-forming subunit of Cav2.1 was completely recovered in fraction 8 (Figs 1 and S3). We also examined the gradient distributions of bassoon and the cadherin/b-catenin complex, given their interaction with elements of the secretory machinery and their role in organizing the active zones and/or synapses (Garner et al. 2000; Takao-Rikitsu et al. 2004; Bamji 2005; and references cited therein). As shown in Figs 1 and S3, bassoon (immunodetected as a doublet of about 400 kDa) (Dieck et al. 1998) was largely enriched in fraction 8, with a small amount of lower molecular weight polypeptides corresponding to degradation products (Dieck et al. 1998) being

recovered in the high-density fractions. Similarly, about 50% of the cadherin/b-catenin complexes were immunodetected in fractions 8 and 9 (HDRMs), which is consistent with previous data showing them in rafts isolated from myoblasts (Causeret et al. 2005). The heterotrimeric Ga subunits had a particular distribution. In line with previous data (Moffett et al. 2000; Oh and Schnitzer 2001), Gai, Gao and Gaq were detected in DRMs, but their distribution was different from that of the exocytic complex. As shown in Figs 1 and S3, all three proteins were bimodally distributed in the gradients, with one peak in fraction 8 (HDRMs) and a second in the lighter fraction 6 (LDRMs). Interestingly, a small but consistent aliquot of the calcium-binding protein NCS-1 was detected in the DRMs isolated from LP1 or intact synaptosomes (P2), and was largely localized in the LDRM fraction (only traces were identified in fractions 7 and 8 of the P2 samples; Fig. S3). In order to demonstrate further that the examined proteins were localized in cholesterol-enriched microdomains, we analysed the effects of cholesterol depletion on their buoyancy using well-established procedures. As shown in Fig. S4 (see ‘Supplementary material’), saponin treatment completely altered the organization of the lipid microdomains, with all of the proteins being recovered in the highdensity fractions (12–15 and pellets); furthermore, 30 mM MbCD (which is known to reduce the cholesterol content of synaptic membranes by about 50%) (Taverna et al. 2004) also caused the recovery of larger amounts of DRMassociated proteins in the soluble fractions and pellets (Fig. S4). SNAREs and NCS-1 are localized in distinct lipid microdomains of PC12 cell plasma membrane sheets The biochemical results described above suggest that NCS1 and the proteins of the exocytic machinery are localized in different membrane microdomains. Because several lines of evidence suggest that a number of non-physiological rearrangements may occur when Triton X-100 is added to membranes (Munro 2003; Mukherjee and Maxfield 2004; Pike 2004), we used a different approach to analyse whether the results obtained after Triton X-100 solubilization reflect the presence of NCS-1 and the exocytic complexes in different membrane microdomains. To this end, we used a method developed by Lang et al. (2001) to characterize lipid microdomains containing SNARE proteins at the inner leaflet of the plasma membrane of PC12 cells, in which cells plated on coverslips are unroofed by sonication, and the membrane sheets that remain attached to the coverslips can be used for immunolabelling. As this method could not be used to study synaptic boutons, because of the resolution limits imposed by their small size and the impossibility of applying the sonication protocol to mature neurons in cultures (no synaptic bouton membranes were found on the coverslips after sonication), we used the



PC12 cell line, which has been widely used to investigate the protein complexes and mechanisms involved in neurosecretion (see, for examples, Chamberlain et al. 2001; Lang et al. 2001, Aoyagi et al. 2005; Blackmer et al. 2005; Milosevic et al. 2005). The cells were grown on coverslips for 36 h or 4 days in the presence of NGF, and were then fixed with formaldehyde and analysed by immunofluorescence, or underwent a brief sonication protocol. As PC12 cells endogenously express small amounts of NCS-1 (which is partially cytosolic and partially membrane associated as in synaptosomes), the cells in some of the experiments were transfected with a plasmid coding for NCS-1 (Koizumi et al. 2002) in order to compare the distribution of NCS-1 with that of the more abundant syntaxin1 and SNAP-25. The staining of non-sonicated PC12 cells revealed the presence of syntaxin1 and SNAP-25, as well as an aliquot of NCS-1, at the plasma membrane of the transfected cells (Fig. 2a, c, e). When unfixed or formaldehyde-fixed membrane sheets were immunolabelled for SNAP-25 and syntaxin1, both were found in clusters on the inner membrane leaflet (Fig. 2b, d), in line with the data of Lang et al. (2001). NCS-1 had a similar dot-like distribution in both the transfected (Fig. 2f) and non-transfected (Fig. 2f, arrows) cells. Immunostaining for all three proteins was specific, as control experiments showed irrelevant staining when the membrane sheets were labelled with non-immune mouse or rabbit IgG, or irrelevant antibodies, or when the primary antibodies were omitted from the immunolabelling procedure (Fig. S5a and b; see ‘Supplementary material’). In addition, the uniform retention of the FM1-43 lipid dye by the membrane sheets confirmed that sonication did not disturb the integrity of the lipid bilayers (Fig. S5c). We next investigated whether SNAREs and NCS-1 were present in different or similar clusters. Confocal microscopy of membrane sheets that were double immunolabelled for syntaxin1 and SNAP-25, or SNAP-25/syntaxin1 and NCS-1, revealed some co-localization between the two t-SNAREs, as reported previously (Fig. 3a–a¢¢) (Lang et al. 2001), but there was very little overlap between SNAP-25 (and syntaxin1) and NCS-1 (Fig. 3b–b¢¢, and data not shown). Co-localization analysis of the pixels with an intensity of more than 50% maximal fluorescence in the green or red channel was performed for SNAP-25 and syntaxin1, or SNAP-25 and NCS-1; data quantification revealed that approximately 23% (22.92 ± 2.91, n ¼ 13) of the highly labelled pixels of SNAP-25 overlapped with those of syntaxin1, whereas only 5% (4.82 ± 1.02, n ¼ 9) of the SNAP-25 pixels overlapped with NCS-1. A similar small overlap between SNAREs and NCS-1 was also observed in PC12 cells after 4 days of NGF treatment (not shown), and data quantification revealed that approximately 6% (6.02 ± 0.62, n ¼ 9) of the SNAP-25 pixels overlapped with NCS-1, whereas approximately 26%

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Fig. 2 Soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) and neuronal calcium sensor-1 (NCS-1) show a dot-like distribution in plasma membrane sheets of PC12 cells. (a, c, e) Non-sonicated PC12 cells, untransfected (a, c) or transfected (e) with pcDNA3-NCS-1, were fixed and immunolabelled for syntaxin1 (Syn, 1 : 100, a), 25-kDa synaptosome-associated protein (SNAP-25) (SN-25, 1 : 100, c) or NCS-1 (1 : 100, e). (b, d, f) Fixed plasma membrane sheets from non-transfected (b, d) and pcDNA3-NCS-1transfected (f) cells immunolabelled with anti-syntaxin1 (Syn, b), antiSNAP-25 (SN-25, d) or anti-NCS-1 (f) antibodies. In (f), the arrows indicate untransfected PC12 cells expressing endogenous levels of NCS-1. The images were collected using a CCD camera. Bars, 10 lm.

(26.59 ± 3.43, n ¼ 11) of the highly labelled pixels of SNAP-25 overlapped with those of syntaxin1. In order to further demonstrate the presence of NCS-1 and SNAREs in cholesterol-dependent microdomains, we tested their stability and distribution on the membrane sheets after treatment with Triton X-100 or MbCD. As shown in Fig. 4a–a¢¢, Triton X-100 extraction did not extensively solubilize SNAREs or NCS-1, and the proteins still remained concentrated in small clusters on a significant number of sheets. In contrast, short treatment with MbCD affected the dot-like distribution of SNAP-25 and NCS-1 (Fig. 4b–b¢¢)



Fig. 3 Soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) and neuronal calcium sensor-1 (NCS-1) are widely distributed in different microdomains. Membrane sheets were double immunostained with anti-syntaxin1 monoclonal antibodies (Syn, a) and anti-25-kDa synaptosome-associated protein (antiSNAP-25) polyclonal antibodies (SN-25, a¢), or with anti-SNAP-25 monoclonal antibodies (SN-25, b) and anti-NCS-1 polyclonal antibodies (b¢). The primary antibodies were revealed by incubation with Alexa Fluor 488 anti-mouse IgG and anti-rabbit IgG conjugated to rhodamine. The images were collected using a confocal microscope, and the merges are shown in (a¢¢) and (b¢¢). Note that NCS-1 does not significantly co-localize with SNAP-25 (b–b¢¢). Bars, 5 lm.

without gross changes in the morphology of the sheets, as revealed by FM1-43 staining (Fig. S5d). As a result of cholesterol extraction, there was more diffuse staining for both proteins, followed by some mixing of the two (Fig. 4b–b¢¢). Taken together, these results indicate that NCS-1 and SNAREs are present in distinct membrane microdomains. NCS-1 and proteins of the exocytic machinery co-localize with heterotrimeric G proteins As co-fractionation in the gradients does not correspond to co-localization in DRMs, we further analysed the localization of SNAREs and NCS-1 with heterotrimeric G proteins using a previously described DRM immuno-isolation protocol (Taverna et al. 2004). Evidence that this procedure allows the immuno-isolation of proteins in the DRMs comes from the data shown in Fig. 5, which demonstrates the presence of the GM1 marker in immunocomplexes isolated from fractions 6 (LDRMs) or 8 (HDRMs) with antibodies against NCS-1, syntaxin1, SNAP-25 or a1A. As shown in Fig. 6, the DRMs immuno-isolated from fraction 8 of the P2 gradients using an anti-syntaxin1 antibody contained significant amounts of SNAP-25 and VAMP-2. Interestingly, although particularly enriched in the fraction containing syntaxin1, consistent amounts of bassoon cadherin and flotillin were not immunoisolated with syntaxin1, thus suggesting that they are localized in DRMs other than those containing the proteins of the exocytic machinery. NCS-1, which was present in very small

Fig. 4 The soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor (SNARE) and neuronal calcium sensor-1 (NCS1) microdomains are altered by cholesterol depletion. PC12 cell membrane sheets were incubated for 10 min at 4C with 0.3% Triton X-100 (a–a¢¢), or at 37C with 10 mM methyl-b-cyclodextrin (MbCD) (b–b¢¢), fixed and double immunostained for 25-kDa synaptosomeassociated protein (SNAP-25) (SN-25, a, b) and NCS-1 (a¢, b¢). The primary antibodies were revealed by incubation with anti-mouse IgG conjugated to fluorescein (b) or Cy5 (a), and anti-rabbit IgG conjugated to rhodamine. The images were made using a confocal microscope; the merges are shown in (a¢¢) and (b¢¢). SNAP-25 and NCS-1 retained their separate dot-like distribution in the membrane sheets treated with Triton X-100 (a–a¢¢), but both proteins started to lose their clustered distribution in the samples incubated with MbCD. Bar: (a¢¢) 3.5 lm; (b¢¢) 5 lm.

amounts in fraction 8 of the P2 samples (see Fig. S3), was also not detected in the DRMs isolated using the anti-syntaxin1 antibodies and, similarly, the anti-NCS-1 antibodies did not co-immuno-isolate a significant amount of syntaxin1 from fraction 6 (Fig. 6). When anti-Gaq antibodies were used (Fig. 6b), consistent amounts of synaptotagmin1, a1A and t-SNARE proteins were co-immuno-isolated with the protein from fraction 8, but not from fraction 6, whereas, vice versa, the anti-Gaq antibody immuno-isolated NCS-1 from fraction 6, but not from fraction 8. The G protein b subunits were immunodetected in DRMs containing NCS-1, as well as in those containing the exocytic machinery (Fig. 6b), thus confirming the presence of heterotrimeric G proteins in both DRMs. The non-immune rabbit or mouse IgGs used in the control experiments did not immuno-isolate the proteins examined (Fig. 6a and b) or GM1 (see Fig. 5). In order to reveal possibly stable interactions between the proteins co-localized in DRMs, P2 samples were solubilized with saponin and Triton X-100 in order to dissolve the lipid microdomains completely, and the proteins underwent



Fig. 5 Phosphatidylinositol-4,5-bisphosphate (PIP2) in detergentresistant membranes (DRMs). (a) Ganglioside GM1 and PIP2 were detected in P2 and lysate pellet 1 (LP1) flotation gradients by dot blots using peroxidase-conjugated cholera toxin and an antibody against PIP2. One microlitre of each fraction (undiluted for PIP2 and 1 : 10 diluted for GM1) was spotted onto nitrocellulose filters and incubated with peroxidase-conjugated cholera toxin or anti-PIP2 monoclonal antibodies (1 : 5000) overnight at 4C. The results are representative of four independent experiments. (b, c) Aliquots (250 lL) of fractions 6 (b) or 8 (c) were immunoprecipitated with polyclonal antibodies against neuronal calcium sensor-1 (IP NCS-1), 25-kDa synaptosome-associated protein (SNAP-25) (IP SN-25) and a1A (IP a1A), monoclonal antibodies against syntaxin1 (IP Syn) and non-immune rabbit (IP RIgG) or mouse (IP MIgG) IgG. PIP2 and GM1 were then detected in the input and immunoprecipitates by dot blots. Small aliquots of each immunoprecipitate (5% and 1% for PIP2 and GM1, respectively) and input (10% and 2% for PIP2 and GM1, respectively) were spotted onto the filter and probed with cholera toxin and anti-PIP2 antibodies. The dot blots are representative of two separate experiments.

immunoprecipitation analysis. As shown in Fig. 7, antisyntaxin1 antibody immunoprecipitated consistent amounts of the SNARE complexes and synaptotagmin1 (in line with previous data), but did not co-immunoprecipitate NCS-1 or Gaq subunits and, consistently, neither NCS-1 nor Gaq antibodies immunoprecipitated SNARE proteins or synaptotagmin1. Furthermore, NCS-1 and Gaq did not seem to form stable complexes, as they were not significantly

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Fig. 6 Immuno-isolation of detergent-resistant membranes (DRMs) containing the exocytic complexes or neuronal calcium sensor-1 (NCS-1). (a) Aliquots (250 lL) of gradient fractions 6 and 8 were incubated with anti-NCS-1 (IP NCS-1), non-immune rabbit IgG (IP RIgG), anti-syntaxin1 (IP Syn) or non-immune mouse IgG (IP MIgG). (b) Aliquots (250 lL) of gradient fractions 6 and 8 were incubated with Gaq (IP Gaq) or non-immune rabbit IgG (IP RIgG). The proteins of each immunocomplex were analysed by western blotting using antibodies against NCS-1, synaptotagmin1 (Syt), syntaxin1 (Syn), 25-kDa synaptosome-associated protein (SNAP-25), vesicle-associated membrane protein-2 (VAMP), bassoon (Bass), cadherin (Cadh), flotillin, Gaq, a1A and Gb subunit. Aliquots (8%) of the samples used for immunoprecipitation (a, b, Input) were also analysed by western blotting. The inputs and immunoprecipitates for each antigen were autoradiographed using the same time of exposure, except for the inputs of Gaq and a1A shown in (b) (see asterisks), which were autoradiographed for longer exposure times than their respective immunoprecipitates.



Fig. 7 Immunoprecipitation assays. Aliquots (200 lg proteins) of P2 samples solubilized in saponin and Triton X-100 were incubated with antibodies against syntaxin1 (IP Syn), neuronal calcium sensor-1 (IP NCS-1), Gaq (IP Gaq) Gb (IP Gb) or non-immune rabbit (IP RIgG) or mouse (IP MIgG) IgG. The immunoprecipitates were analysed by western blotting using antibodies against Gaq, synaptotagmin1 (Syt), syntaxin1 (Syn), 25-kDa synaptosome-associated protein (SNAP-25), NCS-1 or vesicle-associated membrane protein-2 (VAMP). Aliquots (8%) of the samples used for immunoprecipitation (Input) were also analysed by western blotting. The inputs and immunoprecipitates for each antigen were autoradiographed using the same time of exposure, except for the input of NCS-1 (asterisk) which was autoradiographed for a longer exposure time than its immunoprecipitates.

co-immunoprecipitated with anti-NCS-1 or anti-Gaq antibodies. Given that previous cell-free studies of recombinant proteins have shown the interaction of the Gbc subunit with SNAREs (Jarvis et al. 2002; Blackmer et al. 2005; Gerachshenko et al. 2005), we next investigated whether anti-Gb subunit antibodies could co-immunoprecipitate SNARE proteins or NCS-1 from synaptosome extracts. As shown in Fig. 7, a significant amount of Gaq was co-immunoprecipitated with Gb antibodies, but neither SNAREs nor NCS1 were found in the immunocomplexes. Similarly, no b subunit immunoreactivity was detected after immunoprecipitation of the SNARE complexes using the anti-syntaxin1 or anti-SNAP-25 antibodies (Fig. S6, see ‘Supplementary material’). The non-immune rabbit or mouse IgGs used in the control experiments did not co-immuno-isolate any of the examined proteins. PIP2 is associated with DRMs isolated from synaptosomal membranes As previous data have demonstrated that PIP2 plays a role in neurosecretion (Aoyagi et al. 2005; Milosevic et al. 2005;

and references cited therein), we analysed the distribution of PIP2 in the DRMs isolated from LP1 or P2 flotation gradients. Dot blot analyses of aliquots of the fractions revealed the presence of PIP2 in the sucrose low-density fractions (Fig. 5a) and, in order to demonstrate the possible co-localization of PIP2 with NCS-1, DRMs were immunoisolated from fractions 6 or 8 using antibodies directed against NCS-1, SNAP-25, a1A or syntaxin1, as described above (see Fig. 6). Immuno-isolated complexes were then analysed using antibodies against PIP2 or peroxidase-conjugated cholera toxin. The results demonstrated that the antibodies immuno-isolated consistent aliquots of intact DRMs, as revealed by cholera toxin staining, and that PIP2 was detected in the DRMs immuno-isolated with antibodies against the proteins of the exocytic machinery (Fig. 5). Interestingly, PIP2 was also detected in LDRMs immunoisolated with the anti-NCS-1 antibodies (Fig. 5b). No significant labelling for PIP2 or GM1 was detected in the immunoprecipitates carried out using pre-immune rabbit or mouse IgG (Fig. 5b,c). Taken together, these results suggest that PIP2 is also present in membrane microdomains enriched in NCS-1. NCS-1 recruitment in LDRMs following bradykinin stimulation To shed light on the role of these microdomains in organizing the protein complexes during exocytosis and/or calcium channel modulation, we investigated whether it was possible to modify the distribution of the exocytic proteins and NCS-1 in the DRMs by applying specific stimuli. To this end, we tested the action of bradykinin (Fig. 8), because it has been shown previously to modulate Ca2+ currents in sympathetic neurons via a mechanism that involves the activation of Gaqcoupled B2 receptors and NCS-1 (Gamper et al. 2004). It is known that B2 receptors are widely expressed in the central nervous system, and so we incubated the P2 fraction isolated from brain cortices with 5 lM bradykinin for 3 min. Upon stimulation, the synaptosomes were solubilized in Triton X-100 and centrifuged in sucrose gradients, and the fractions collected from the control or bradykinin-treated samples were analysed as described in the ‘Materials and methods’ section. After stimulation with bradykinin, the distributions of bassoon, SNAP-25, syntaxin1 and Gaq were unchanged. Quantitative analyses revealed that similar amounts of SNAP-25 in fraction 8 (control 19.70 ± 6.3% vs. bradykinin 14.00 ± 3.05%), and Gaq in fraction 6 (control 1.73 ± 0.66% vs. bradykinin 3.58 ± 0.82%) and fraction 8 (control 6.33 ± 0.90% vs. bradykinin 6.47 ± 0.83%), were recovered in the control and treated samples. In contrast, larger amounts of NCS-1 were recruited on bradykinin stimulation: quantitative analysis revealed that 44% (44.40 ± 5.69%) of total synaptosomal NCS-1 was recovered in fraction 6 after treatment, in comparison with only 5% (5.50 ± 1.16%) found in the same fraction of the control samples. Subcellular



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Fig. 8 Neuronal calcium sensor-1 (NCS-1) is recruited in low-density detergent-resistant membranes (LDRMs) after bradykinin stimulation. Synaptosomes (P2) were incubated for 3 min at 37C in Krebs–Ringer buffer in the absence (CON) or presence (BK) of 5 lM bradykinin. The synaptosomes were recovered by centrifugation, solubilized on ice-cold Triton X-100, and then centrifuged in sucrose gradients. (a) Equal volumes (30 lL) of gradient fractions 4–15 and pellets were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and analysed by western blotting or dot blots as described in Fig. 1. Note the presence of a larger amount of NCS-1 in fraction 6 of the bradykinin-stimulated samples. (b) Fractions 4–12 (30 lL) and fractions 13–15 and pellets (6 lL) were loaded in the gels for quantitative analysis using unsaturated autoradiograms (an example of a typical autoradiogram used to quantify NCS-1 is shown at the bottom of the graph). The signals in each fraction were expressed as percentages of the total, taking into account the 1 : 5 dilution of fractions 13–15 and pellets. Mean values ± SEM; *p < 0.005. SNAP-25, 25-kDa synaptosome-associated protein; Syn1, syntaxin1.

fractionation of the control and bradykinin-treated synaptosomes, followed by the analysis of NCS-1 in the LP1, LS1, LS2 and LP2 fractions, revealed that an increased amount of NCS-1 (10%, n ¼ 2) was detected in LP1 membranes after stimulation, together with a decrease in protein levels in the LS1 and LS2 fractions. Discussion

A number of studies have demonstrated that cholesteroldependent membrane microdomains play an important role in neurosecretion, as they are involved in the organization of the exocytic sites containing SNARE proteins and Cav2.1 channels (Salaun et al. 2004; Rohrbough and Broadie 2005; and references cited therein). The aim of this study was to acquire additional information concerning the molecular composition of these sites, and the organization of the signalling molecules involved in neurosecretion. Our results show that distinct DRMs containing proteins of the exocytic complex (SNAREs, synaptotagmin and Cav2.1) or the calcium-binding protein NCS-1 can be separated by fractionation on sucrose gradients, thus

indicating their localization in different membrane microdomains. Immunocytochemical data showing NCS-1 and t-SNARE proteins in distinct cholesterol-enriched microdomains in the inner leaflet of PC12 cell plasma membranes further suggest their localization in different microdomains. The data concerning SNARE proteins are in line with the results obtained by other authors who have worked on the characterization of ‘exocytic sites’ using PC12 cells as a model (Lang et al. 2001; Aoyagi et al. 2005). These cells have limitations when studying the distribution of synaptic proteins, but technical reasons prevented us from analysing the presence in microdomains of t-SNAREs and NCS-1 at the nerve terminals of mature neurons after sonication, and further experiments using different methods and morphological system analysis are still required. Our study also shows that DRMs immuno-isolated from fractions 8 or 6 contain aliquots of heterotrimeric G proteins and PIP2, which co-localize with the exocytic machinery or NCS-1, respectively. Although the mixing of different subdomains was observed after the Triton X-100 extraction of total membranes prepared from a post-nuclear fraction of rat brains (Madore et al. 1999), there was apparently no



widespread intermingling of membrane domains in our preparations obtained from partially purified synaptosomes. This conclusion is supported by the observation that, although enriched in fraction 8, cadherin was not coimmuno-isolated with SNARE proteins, which is consistent with the finding that adhesion molecules are localized in synaptic membrane regions outside the ‘exocytic sites’ (Bamji 2005, and references cited therein). Furthermore, flotillin did not co-immuno-isolate with SNAREs, which is in line with results describing it as being clustered in specific membrane microdomains involved in a clathrin-independent endocytic pathway (Glebov et al. 2006). Protein distribution in DRMs may therefore reflect their organization in endogenous membrane microdomains

showed that cholesterol is required for fast, Ca2+-triggered membrane fusion in two ways: (i) it helps the spatial organization of molecular components at the fusion sites; and (ii) as a membrane component of intrinsic negative curvature, it lowers energy barriers to the formation of transient fusion intermediates. However, despite this evidence supporting the role of lipids in organizing the sites of neurosecretion, interactions between proteins (e.g. SNAREs) may also be required to generate these domains. In this context, it is interesting to note that protein–protein interactions (rather than lipids) create microdomains that concentrate or exclude cell surface proteins in order to facilitate cell signalling in T cells (Douglass and Vale 2005).

Co-localization of exocytic machinery complexes with heterotrimeric G proteins Recent studies have demonstrated strong interactions between Gbc complexes and SNAREs or Cav2 channels (Stanley and Mirotznik 1997; Blackmer et al. 2001, 2005; Jarvis et al. 2002; Rousset et al. 2004; Strock and Diverse-Pierluissi 2004; De Waard et al. 2005; Gerachshenko et al. 2005), but we did not find any direct interaction between G protein b subunits and SNARE proteins after complete membrane microdomain solubilization by saponin and Triton X-100. There are a number of reasons for the differences between ours and other recently published results (Blackmer et al. 2001, 2005; Jarvis et al. 2002; Gerachshenko et al. 2005). Firstly, the direct interactions between SNAREs and Gbc found in previous reports were revealed by binding studies using cell-free systems and recombinant fusion proteins (Blackmer et al. 2001, 2005; Jarvis et al. 2002; Gerachshenko et al. 2005), whereas we immuno-isolated the endogenous protein complexes from tissues. It has also been demonstrated that it is specifically the Gb1c2 complex that interacts with high affinity with SNAREs. Furthermore, these interactions are expected to be transient and, in vivo, they may depend on the concentration of the bc complex, which varies on the basis of GPCR activation. It is therefore possible that the amount of endogenous SNARE–Gb1c2 complex is too small to be revealed by co-immunoprecipitation. Although we did not find direct interactions between endogenous proteins, our results demonstrate that G protein subunits are recruited with SNAREs and Cav2.1 in the same membrane microdomains. These data are in line with the finding that G proteins directly interact and regulate the function of the exocytic machinery, and further suggest that the lipid environment may help in organizing the exocytic machinery with signalling molecules in specific cholesterolenriched microdomains (Salaun et al. 2004; Rohrbough and Broadie 2005; and references cited therein). The critical role of cholesterol in exocytosis is also supported by recent findings. Churchward et al. (2005) used a well-established model to analyse the Ca2+-triggered steps of exocytosis, and

A possible role accounting for the presence of NCS-1 in distinct microdomains Our findings also show, for the first time, the localization of NCS-1 in cholesterol-dependent microdomains and, furthermore, that these microdomains seem to be different from those containing the proteins of the exocytic machinery. NCS-1 has a myristoyl moiety at the N-terminus (Burgoyne and Weiss 2001, and references cited therein), which could account for its association with the membrane and, possibly, with lipid microdomains. Previous studies of PC12 cells have shown that NCS-1 is only partially membrane associated, and that the activation of GPCRs (e.g. purinergic receptors or bradykinin receptors) increases its translocation from the cytosol to membrane fractions (Taverna et al. 2002). We have demonstrated that NCS-1 is significantly increased in DRMs isolated from synaptic terminals after stimulation with bradykinin, whereas the DRM levels of proteins of the exocytic complex and molecules of the active zone remain largely unchanged. Furthermore, after stimulation, NCS-1 is still recruited in the LDRMs (fraction 6), thus further supporting the conclusion that NCS-1 domains are different from those containing the exocytic machinery and involved in secretory vesicle fusion (Lang et al. 2001; Aoyagi et al. 2005). However, these results do not demonstrate whether the increased amount of NCS-1 in DRMs is a result of protein movement from the cytosolic pool or from a membrane-bound but detergent-soluble fraction. In fact, after bradykinin stimulation, the DRMs were prepared from crude synaptosomes and not from LP1 membranes as the hypo-osmotic treatment used to open the synaptic boutons partially affected the membrane-bound NCS-1. Nevertheless, taking into account the results of previous studies, as well as recent data showing a direct and rapid translocation of NCS-1 from the cytosol to the plasma membrane during stimulation (Taverna et al. 2002; de Barry et al. 2006), it is likely that a portion of cytosolic NCS-1 (rather than a previously membrane-bound aliquot of the protein) is recruited in DRMs. This hypothesis is also supported by



the results showing some translocation of NCS-1 from the cytosol to the LP1 membrane during bradykinin stimulation. We cannot completely exclude the possibility that an aliquot of the protein is first recruited in the membranes and then translocated to the DRMs, but this process could not be revealed by our biochemical approach. The observation that NCS-1 microdomains contain aliquots of heterotrimeric G protein subunits and PIP2 may also be consistent with recent work by Gamper et al. (2004), who found that the stimulation of B2-bradykinin receptors coupled to the Gq subunit in sympathetic neurons does not inhibit Cav2.2 (as has been reported in the case of other Gq-coupled receptors), but rather increases the Ca2+ current. One possible explanation is that B2 receptors stimulate the synthesis of PIP2 via the pathway that involves the membrane recruitment of NCS-1 and the activation of a phosphatidylinositol 4-OH kinase (Gamper et al. 2004, and references cited therein). It is thought that the increased level of PIP2 compensates for the lipid consumption caused by phospholipase C activation, and thus sustains the activity of the calcium channels. In the light of these results, it has been speculated that certain GPCRs (e.g. B2 and purinergic receptors) may be linked to different signalling pathways which, in order to function, may be sequestered in different microdomains (Delmas et al. 2005). Our data showing the localization of NCS-1 in distinct membrane microdomains and, more interestingly, its significant recruitment in these microdomains after stimulation with bradykinin may support this hypothesis. Furthermore, they indicate that NCS-1 affects Cav2.1 activity indirectly via second messenger(s) or the activation of other signalling molecule(s). These different membrane microdomains may therefore play a crucial role in organizing this complex network of intracellular signals converging on neurosecretory processes. It is possible that specific microdomain subpopulations organize a pool of G proteins involved in the direct modulation of the exocytic complex via interactions with the bc dimers (De Waard et al. 1997). These interactions occur rapidly (less than 1 s) after receptor stimulation, and this is consistent with the close distribution of G proteins, SNAREs and Cav2.1 in the same microdomains. In contrast, different domains may be involved in the signalling pathway modulated by NCS-1, which is slower and appears to involve a pool of PIP2. This lipid is present in NCS-1-enriched DRMs, and our preliminary results suggest that about 20% of PIP2 co-localizes with NCS-1 in membrane sheets (Taverna E., Saba E., Rosa P., unpublished data). It would be interesting to investigate whether GPCR activation may increase the amount of PIP2 localized in microdomains. We were unable to detect significant differences in the distribution of PIP2 in DRMs after bradykinin treatment by dot blot analysis. Imaging techniques with high resolution will be required to allow a quantitative analysis of PIP2 in distinct microdomains.

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Acknowledgements We would like to thank Drs G. Kreibich and A. Malgaroli for their kind gifts of antibodies. We are indebted to Professor N. Borgese for her helpful discussions and comments. ET was supported in part by a Philip Morris postdoctoral fellowship. This work was supported by grants from the Italian Consiglio Nazionale delle Ricerche and Ministero Istruzione Universita` e Ricerca (MIUR and BNEO1RHZM), and from Fondazione Cariplo (Ref. 2004.1600 to PR).

Supplementary material The following material is available for this paper online Fig. 1S. Specificity of anti-a1A antibodies. Equal amounts of proteins (20 lg) from rat brain synaptosomes (Brain) and rat liver membranes (Liver) were separated on 6% SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with 2 lg/ml of anti-a1A antibodies (Abs) raised against peptides 865–881 (A) and 882–896 (B). Specificity was demonstrated by preincubating the antibodies (4 lg in 2 ml) with 5 lg of the corresponding antigens (Abs + Pep). Fig. 2S. Distribution of synaptic proteins in rat brain subcellular fractions. Rat brain cortices were homogenised (S1), and the synaptosomes (P2) obtained by differential centrifugation. The fractions enriched in small vesicles (LP2), plasma membrane (LP1) or cytosolic proteins (LS2) were obtained by hypo-osmotic lysis of the synaptosomes followed by differential centrifugation. Equal amounts of proteins (20 lg) from each fraction were analysed by SDS-PAGE and Western blotting. The blots were probed with antibodies against VAMP-2 (VAMP, 1:10,000), synaptophysin (1:2,000), syntaxin1 (Syn1, 1:10,000), ribophorin (Riboph, 1:400), cadherin (N-Cadh, 1:10,000), b-catenin (1:2,000), neuroligin (1:1,000), bassoon (1:3,000), actin (1:1,000), tubulin (1:5,000), NCS-1 (1:1,000) and Gao (1:3,000). The data are representative of three separate experiments. Fig. 3S. Analysis of DRMs isolated from P2 samples. The rat brain synaptosomes were extracted with 1% Triton X-100, and then centrifuged to isolate the DRMs. Equal volumes of fractions 4–15 and pellets (P) were analysed by Western blotting or dot blots as described in the legend to Figure 1. The data are representative of three separate experiments. Fig. 4S. Cholesterol depletion affects the floatation properties of DRM-associated proteins. The LP1 fractions were treated with 1% saponin or 30 mM MbCD before Triton X-100 extraction. After centrifugation, equal volumes of fractions 4–15 and the pellets (P) were separated by SDS-PAGE and then analysed by Western blotting using a panel of antibodies as described in the legend to Figure 1. The distribution of the GM1 marker was determined by dot blots. The data are representative of three separate experiments. Fig. 5S. Specificity of immunostaining for t-SNARE proteins and NCS-1. Fixed membrane sheets were labelled with rabbit IgG (A) or mouse IgG (B) followed by anti-rabbit- or anti-mouse-IgG conjugated to rhodamine. Images were collected using a CCD camera. Bar ¼ 5 lm. In C and D, unfixed membrane sheets were stained for 1 min with 10 lM FM1-43 and immediately acquired using a Zeiss Axiovert 200 fluorescence microscope equipped with temperature and CO2 controllers, and a Roper Micromax CCD camera (C, FMCon). The same area was imaged after 10 min incubation of the FM1-43 labelled sheets with 10 mM MbCD (D, FM-MbCD). The times of exposure were 300 msec in C and 1200 msec in D. Note



that after MbCD the morphology of the membrane sheets was largely conserved. Bars ¼ 5 lm. Fig. 6S. Immunoprecipitation assays. Aliquots (200 lg proteins) of P2 samples solubilised in saponin and Triton X-100 were incubated with antibodies against syntaxin1 (IP Syn), SNAP-25 (IP SN-25) or mouse IgG (IP MIgG). The immunoprecipitates were analysed by Western blotting using antibodies against Gb, syntaxin1 (Syn), SNAP-25 (SN-25), or VAMP-2 (VAMP). Aliquots (8%) of the samples used for immunoprecipitation (Input) were also analysed by Western blotting. This material is available as part of the online article from http:// www.blackwell-synergy.com

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